Yucatan Ethnobotany

Diss. ETH No. 13555

Yucatec Mayan Medicinal Plants:

Ethnobotany, Biological Evaluation

and

Phytochemical Study of Crossopetalum gaumeri

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH

For the degree of

Doctor of Natural Sciences

Presented by

ANITA SABINE ANKLI

Eidg. dipl. Apothekerin

born June 25, 1967

Zullwil/Meltingen (SO)

Accepted on the recommendation of

Prof. Dr. 0. Sticher, examiner

Prof. Dr. M. Heinrich, co-examiner

Dr. J. Heilmann, co-examiner

Zürich 2000

Acknowledgement

Acknowledgements

I wish to express my sincerest thanks to the following people:

- Prof. Dr. Otto Sticher, my supervisor, for giving me the chance to carry out a

ethnobotanical-phytochemical project in his research group, for providing

extraordinary working facilities as well as for the open door to discuss problems. I

am most grateful to Prof. Dr. Michael Heinrich, my co-supervisor, for introducing

me into the fascinating field of ethnobotany, for the encouraging discussions and

for the valuable support during the field study in Mexico. Special thanks go to Dr.

Jörg Heilmann for helping me with the structure elucidation of the isolated

compounds and accepting to support this thesis as a referee. -

- the healers and midwives of Chikindzonot, Ekpedz and Xcocmil for their

openness and patience in teaching me the usage of the medicinal plants and for

the opportunity to participate in their healing sessions and ceremonies. Many

thanks for answering my repeated questions. Only the knowledge of the healers

and midwives made it possible to carry out this thesis.

- the family of Don Abundio Chan Kauil and Dona Claudia Uc Cahun for their

hospitality, allowing me to stay in their home, for the delicious food and for the

introduction in the Mayan cosmovision. I would like to express my warmest thank

to Miriam and Gregoria Chan Uc for their courage to sleep in my house, thus

protecting me and for passing a good time together. I also like to thank Cresencio

Chan Uc for the uncountable discussions on any topic. (I like to apologize for the

mistakes I made due to cultural differences or lack of understanding).

- Marciana Poot Kauil for helping me as a Maya-Spanish translator and friend and

for making it easier to come in contact with the healers and midwives of Ekpedz

and for her help to collect plants in the forest and in hardly passable regions. I am

Acknowledgement

grateful to her father Don Silvestre Poot Poot and her mother for their hospitality

and the introduction in the actual Mayan beliefs and mystic tales.

- children of Chikindzonot who filled my house with great happiness and who

followed, not yet, the social roles and taboos.

- Dr. Ingrid Olmsted for the botanical support in the CICY (Centro de

Investigaciön Cientifica de Yucatan) and for the invitations to several botanical

excursions on the Peninsula of Yucatan. For the help of the botanical identification

I am very much indebted to the biologists and co-workers of the CICY, especially to

Jorge Carlos Trejo, Paulino Sima and Dr. Rafael Duran. I also like to thank the

botanists and specialists of the MEXU (Herbario Nacional de Mexico, Mexico D.F.):

Dr. Mario Sousa, Dr. Oswaldo Tellez, Dr. Rafael Lira, Dr. Jose-Luis Villasenor,

Dr. Fernando Chan and Dr. M. Martinez Gordilla.

- Dr. Ignacio Tuz and Dona Aurora (INI, Instituto Nacional Indigenista, Valladolid)

for supporting the project and inviting me to the meetings of the healers.

- Gisel Vargas, Roxana Chavarrfa and Don Julio Chavarrfa for their friendship

and hospitality in Mérida and Valladolid and for telling me stories about the

Mexican way of life.

- Dr. Barbara Pfeiler (Universidad Autönoma de Yucatan, Mérida) and Dr. Carlos

Viesca (UNAM, Universidad Nacional Autönoma de Mexico, Mexico D. F.) for the

linguistical and anthropological discussions and the invitations in their wonderful

homes. I am grateful to Dr. Ramon Arzâpalo (UNAM) and Jolanda Arzapâlo for

their fruitful discussions on Maya culture and for their hospitality in the biggest city

of the world.

Acknowledgement

- Dr. Carlos Zolla (INI, Mexico D.F.), Dr. Arturo Argueta and Dr. Gonzalo Solis

(INI, Mérida) for their advice to find a good place of study and for their friendliness

to accept and respect me as a broken-Spanish speaking person (at that time).

- Dr. Matthias Baltisberger (ETH) for sending the valuable equipment to dry

plants, Prof. Dr. Daniel Moerman (University of Michigan-Dearborn) for the

productive discussions about medicinal and non-medicinal plants.

- Dr. Oliver Zerbe for teaching me in the interpretation of NMR spectra and for the

untiring and fruitful discussions about NMR problems. I also wish to thank Dr.

Engelberg Zass for performing search on chemical compounds and Dr. Walter

Amrein, Oswaldo Greter and Rolf Häflinger for recording mass spectra.

- Dr. Hongmei Liu and Dr. Jimmy Orjala for their support and stimulating

discussions about chromatography and Micheal Wasescha for the determination

of KB cell cytotoxicity of plant extracts and pure compounds. My warm thanks go to

Dr. Barbara Frei Haller for the encouraging ethnobotanical discussions, the

literature search on plants and for her excellent pioneer work in ethnobotany in our

phytochemistry group.

- Prof. Dr. Horst Rimpler for the good co-operation of the Freiburg-Zürich group

(Albert-Ludwigs-University, Freiburg). I especially would like to thank Dr. Peter

Bork, Dr. Bilkis Heneka and Dr. Elke Beha.

- Dr. Lutz Wolfram, Dr. Peter Bauerfeind (University Hospital, Zurich), Dr.

Claudia Weiss, PD. Dr. Reto Brun, Cécile Schmid (Swiss Tropical Institute,

Basel), Regina Bruggisser (University, Basel), Jürg Gertsch (ETH) and Dr.

Helmut Wiedenfeld (University, Bonn) for testing extracts of different plants. I

would like to thank Dr. Christoph Schachtele and Dr. Frank Totzke for

determining protein kinase activity (Klinik für Tumorbiologie, Freiburg). I am

Acknowledgement

grateful to Prof. Dr. Marcus Schaub (University, Zurich) for proposing the

hypothesis about the effect of cardenolides in this treatment for snake bites.

- David McLaughlin and Anna Jen for the Engl.sh correction of this thesis.

- all my colleagues and staff at the Institute of Pharmaceutical Sciences, ETH

Zurich for the great time we had together, especially I like to thank my laboratory

colleagues (17L80).

- all my friends who visited me in my home in Yucatan and those who critically

supported my ideas and dreams.

Last but not least I express my deepest thank to my parents and Julian

Granados for their private support and patienc3, which gave me great motivation

to overcome various problems during this PhD-thesis.

Financial support during the work of this thesis was obtained from:

- Swiss Agency of Development Cooperation (SDC), Berne

- Swiss Academy of Natural Sciences (SANW), Zurich

- Barth Fonds of ETH, Zurich

Contents

Abbreviations

Summary

Resumen

Zusammenfassung

1

4

6

8

Part I Medicinal Ethnobotany

1 Introduction 12

1.1 Goal and objectives 16

2 Yucatan and the Mayas 17

2.1 Background 17

2.1.1 Geology and fauna 17

2.1.2 Ancient Maya history 18

2.1.3 From the Spanish conquest to the Caste War 21

2.1.4 The Yucatec Maya today 23

2.1.5 Manuscripts on Maya medicinal plants 28

2.1.6 Medicinal ethnobotany of Yucatan 30

2.2 Methods in the field 33

2.3 Abbreviations mentioned in the plant list 37

2.4 Plant list 41

2.5 Informants 60

2.6 Gardens of medicinal plants 61

2.7 Selection of plant species for their biological evaluations 62

3 Publication I: Medical Ethnobotany of the Yucatec Maya:

Healer's consensus as a quantitative criterion 63

4 Publication II: Yucatec Maya medicinal plants versus non-

medicinal plants: indigenous characterization

and selection 97

Part II Plant Evaluation

5 Publication III: Yucatec Mayan medicinal plants:

Evaluation based on indigenous uses 134

6 Additional results 160

6.1 Antimicrobial activity 160

6.2 Comparison of disk method and TLC method 161

6.3 Protein kinase activity; Method and results 162

6.4 Other activities 167

6.5 Crossopetalum gaumeri- the plant species for the

phytochemical study 168

Part III Phytochemistry of Crossopetalum gaumeri

7 Celastraceae family and the genus Crossopetalum 170

7.1 Botanical taxonomy 170

7.1.1 Crossopetalum gaumeri 171

7.2 Phytochemistry of the Celastraceae 172

7.2.1 Terpenoids (terpenes, isoprenoids) 172

7.2.2 Alkaloids 176

7.2.3 Flavonoids and other phenolic compounds 176

7.3 Phytochemistry of Crossopetalum species 176

7.4 Biosynthesis of terpenoids (terpenes, isoprenoids) 177

7.5 Chemosystematic and phylogenetic relationships 180

7.6 Biological activities among the Celastraceae 182

7.7 Popular medicinal use 185

7.7.1 Yucatec Maya medicinal use of C gaumeri 185

7.7.2 Medicinal application of other Crossopetalum species 186

7.7.3 Global medicinal use of Celastraceae species 186

8 Methods (isolation procedure) 188

8.1 Thin layer chromatography (TLC) 188

8.2 Vacuum liquid chromatography (VLC) 188

8.3 Middle pressure liquid chromatography (MPLC) 188

8.4 High performance liquid chromatography (HPLC) 189

8.5 Open column chromatography 189

8.6 Liquid-liquid partition (LLP) 189

9 Methods of structure elucidation 190

9.1 Nuclear magnetic resonance spectroscopy (NMR) 191

9.2 Mass spectrometry (MS) 193

9.3 UV spectroscopy (UV) 194

9.4 Optical rotation 194

9.5 Acidic hydrolysis 194

10 Plant extraction 195

10.1 Small scale plant extraction 195

10.2 Large scale plant extraction 195

10.3 Fractionation of the methanol extract 196

10.4 Fractionation of the dichloromethane extract 197

11 Structure elucidation of the isolated compounds 200

11.1 Cardenolides 200

11.2 Ourateacatechin 213

11.3 Triterpenes 219

11.3.1 Pristimerin 219

11.3.2 Friedelane-3-on-29-ol 221

11.3.3 2,3,7-Trihydroxy-6-oxo-1,3,5(10),7-tetraene-24-nor-

friedelane-29-oic acid methylester 224

11.3.4 Celastrol 228

11.4 3,15-Dihydroxy-18-norabieta-3,8,11,13-tetraene 231

12 Biological activities of isolated compounds 233

12.1 Cytotoxicity 233

12.2 Other activities 235

13 Biomedicine, a way to explain the medicinal use

of C. gaumeri ? 236

13.1 Gastrointestinal problems 236

13.2 Snakebites 237

13.2.1 C. gaumeri used in the treatment for snake bites 238

13.2.2 What have the cardenolides to do with snake bites ? 240

14 Publication IV: Cytotoxic cardenolides and antibacterial

terpenoids from Crossopetalum gaumeri 243

15 Conclusion 261

References 264

List of publications 278

List of poster presentations 278

Oral presentations 279

Curriculum Vitae 280

Abbreviations

Abbreviations

A

AT

[a]D

B

C

ATCC

CDCI3

CHCI3

CH2CI2

Ô

d

dd

2D

DEPT

DER

DQF-COSY

EI-MS

ESI

eV

EYE

FAB-MS

FEM

Gl

H20

HMBC

HSQC

HPLC

non-polar extract (dichloromethane-methanol 2:1)

bites and stings of venomous animals

specific optical rotation

polar extract (1-butanol)

ethanol extract

American type cultures collection

deuterated chloroform

chloroform

dichloromethane

chemical shift

doublet

double doublet

two-dimensional

distortionless enhancement by polarization transfer

dermatological conditions

double quantum filter correlation spectroscopy

electron impact-mass spectrometry

electronspray ionization

electron Volt

illnesses of the eyes

fast atom bombardment-mass spectrometry

women's medicine

gastrointestinal disorders

water

heteronuclear multiple bond correlation

heteronuclear single quantum correlation

high performance liquid chromatography

1

Abbreviations

Hz

'Cgo

INADEQUATE

J

KB cell line

m

MeOD

MeOH

MIC

MHz

MS

MPLC

m/z

Mr

n-BuOH

NCI

NF-kB

NMR

3-NOBA

NOE

OTH

PK

PFE

ppm

q

RES

ROESY

Rf

Hertz

50 % inhibition concentration

incredible natural abundance double quantum transfer

experiment

coupling constant

human nasopharyngeal carcinoma cell line

multiplet

deuterated methanol

methanol

minimum inhibition concentration

Megahertz

mass spectrometry

medium pressure liquid chromatography

mass to charge ratio (MS)

relative mass

1-butanol

National Cancer Institute

nuclear factor kB

nuclear magnetic resonance

3-nitrobenzyl alcohol

nuclear Overhauser effect

other uses

protein kinase

illnesses associated with pain and fever

parts per million

quartet

respiratory illnesses

rotating frame Overhauser enhancement spectroscopy

retention factor (TLC analysis)

RP reversed phase

RT room temperature

s singulet

Si gel silica gel

sp. species

spp. species (plural)

ssp. subspecies

t triplet

TLC thin layer chromatography

TOCSY total correlation spectroscopy

UR urological problems

UV ultraviolet spectroscopy

VLC vacuum liquid chromatography

Summary

Summary

The use of medicinal plants played an important role in the lives of the Ancient

Maya. Also today, more than 450 years after the conquest of the New World,

medicinal plants are an essential part of the medical system of the lowland Maya of

Yucatan.

During 18 months of field work in three Yucatec Mayan communities (Mexico)

information about medicinal plants, the concepts of disease and methods of

treatment were collected. Based on the knowledge of 40 healers and midwives,

360 medicinal plants and 1828 single- use reports could be documented. In a

quantitative approach, the most frequent illnesses of this region were evaluated.

Gastrointestinal problems (32 %) and dermatological conditions (19 %) were the

most important medical problems, followed by illnesses associated with pain and/or

fever (13 %), respiratory illnesses (11 %), '"women's medicine" (8 %), other uses (5

%), bites and stings of venomous animals (5 %), urological problems (4 %) and

eye disorders (3 %). To better understand the selection criteria for medicinal

plants, 12 healers and midwives were interviewed about ten plants that in their

opinion have no medicinal value. The characteristics of these non-medicinal plants

were compared with those of the medicinal ones. The results showed that odor and

taste are essential criteria for plant characterization. Also humoral classification

plays an important role. In general, illnesses are classified as hot or cold and the

medicinal plants ought to have the opposite humoral classification. Color, form and

texture are also important criteria in the selection of medicinal plants.

In the second part of the study, 48 medicinal plants were evaluated in several

bioassays. All plant extracts were tested for their antibacterial (gram-negative and

gram-positive bacteria), cytotoxic (KB cells) and anti-inflammatory (NF-kB) activity.

In addition, they were tested in further bioassays based on their indigenous uses.

Plant species used against gastrointestinal problems were evaluated for

antiparasitic (Giardia duodenalis) and additional antibacterial (Helicobacter pylori

4

Summary

and Campylobacter jejuni) activity. The plants of the group used for skin conditions

were also tested for their anti-fungal effects (Candida albicans). For the plants

traditionally used against pain and fever the antimalarial activity (Plasmodium

falciparum) was examined. Plants used in the treatment of type II diabetes were

tested for a-amylase inhibitory effect and the dopamine D2 receptor test was

applied for the taxa used in the group "women's medicine". Different activities were

evaluated that substantiate the traditional use of the herbal remedies.

In a third step, one plant species -Crossopetalum gaumeri (Celastraceae)- was

investigated phytochemically. The roots of this plant were chosen due to their oral

and local use against diarrhea and snake bites, and on the basis of the positive

results obtained in the above mentioned bioassays. From the methanol extract one

known and four new highly cytotoxic cardenolides and the known ourateacatechin

were isolated. The dichloromethane extract afforded a new diterpene of the

abietane type and a new pentacyclic triterpene. Three known triterpenes

(pristimerin, celastrol and friedelane-3-on-29-ol) were also isolated and examined

in different bioassays. Pristimerin and celastrol showed high antibacterial activity

and remarkable cytotoxicity against KB cells. In some respects, the activities of the

isolated compounds substantiate the indigenous uses of C. gaumeri. However, the

plant should be used with caution due to its high cytotoxicity.

5

Resumen

Resumen

El uso de las plantas médicinales por las sociedades mayas prehispanicas

representaba parte importante del sistema medicinal. Aün hoy en dia, después de

450 ahos de la conquista, las plantas médicinales son una parte esencial del

conocimiento médico-farmacolôgico de las sociedades mayas en la Peninsula de

Yucatan.

Se trabajö durante 18 meses en très comunidades mayas, colectando informacion

sobre el uso de las plantas médicinales, tratamiento de las enfermedades y la

conceptualizacion de lo que se denomina enfermedad. Basado en el conocimiento

de 40 curanderos y comadronas se documentaron 360 especies de plantas

médicinales y 1828 usos médicinales. Usando un criterio cuantitativo se

clasificaron las enfermedades mâs frecuentes de la region en que se trabajö. Los

problemas gastrointestinales (32 %) y las enfermedades de la piel (19 %) son los

principales problemas de salud. Seguidamente se encuentran las enfermedades

relacionadas con problemas de dolor y/o fiebre (13 %), problemas respiratorios (11

%), medicina de las mujeres (8 %), otras indicationes (5 %), mordedura y picadura

de animales venenoso (5 %), problemas urologicos (4 %) y enfermedades de la

vîsta (3 %). Para entender cuâl es el criterio que se usa para decidir si una planta

es medicinal o no, se encuestö a 12 curanderos y comadronas, a los cuales se les

pidio que seleccionasen 10 especies de plantas no médicinales. Basandose en

dichas especies se les preguntö sobre el criterio de selecciön para dichas plantas.

Las respuestas acerca de las plantas no médicinales se compararon con las

obtenidas para las plantas médicinales. Los resultados muestran que el sabor y el

olor son caracteristicas para las selecciön de una planta medicinal. Las

caracteristicas humorales (frîo o caliente) son también otro criterio de importancia

para la selecciön de las plantas médicinales. En general las enfermedades se

clasifican en frias y calientes y este criterio se considéra en la clasificacion

humoral de las plantas. Color, forma y textura son también criteriorios de

selecciön de gran significado para la selecciön de una planta medicinal.

6

Resumen

La segunda parte del trabajö consistiö en la evaluaciön de 48 especies de plantas,

usando diferentes bioensayos. Todas los extractos de plantas fuero evaluados con

bioensayos antibacteriales (Bacterias gram-positivas/negativas), citotixicidad

(Celulas KB) y efecto antiinflamatorio (NF-kB). Ademâs, se testaron los usos

indigenas, usando otros bioensayos. Plantas que se usan contra enfermedades

gastrointestinales, se probaron contra usando bioensayos antiparasitales {Giardia

duodenalis) y antibacteriales {Helicobacter pylori, Campylobacter jejuni ). Las

plantas clasificadas en el grupo de enfermedades de la piel, fueron testadas contra

actividad antifungicida {Candida albicans). Las plantas clasificadas como

antifebriles, fueron testadas contra malaria, usando Plasmodium falciparum .Las

plantas clasificadas como Diabetes II se testaron usando el test a-Amylase contra

hiperglucemia. Se encontraron respuestas activas en las plantas, que

corresponden a los usos indigenas en que se clasificaron las plantas.

En la tercera parte del estudio se analisö fitoquimicamente la especie

Crossopetalum gaumeri. Las rai'ces de esta especie son de uso antidiarréico y

antiviperino, tal y como lo confirman los resultados del bioensayo en que se testé

dicha actividad. Del extracto obtenido de la planta con metanol, se encontraron

cuatro nuevos glucösidos cardîacos, altamente citotöxicos; asi como también el

conocido compuesto Ourateacatequina. A partir del extracto con diclorometanol,

se encontaron nuevos abitandipertenos y nuevos pentacicloterpenos. Asi mismo,

se aislaron très terpenos ya conocidos (Pristimerin, Celastrol und Friedelane-3-on-

29-oI) y fueron testados usando los bioensayos anteriormente mencionados. La

Pristimerina y el Celastrol muestran una gran capacidad antibacterial y citotöxica.

Desde una amplia perspectiva se puede decir, que el uso de las plantas

médicinales aqui estudiadas, dada su alta citotoxicidad, debe recomendarse de

una forma cautelosa.

7

Zusammenfassung

Zusammenfassung

Der Gebrauch von Medizinalpflanzen spielte im Leben der alten Maya eine

wichtige Rolle. Auch heute, nach über 450 Jahren der Eroberung der Neuen Welt,

sind Medizinalpflanzen ein wesentlicher Bestandteil des Gesundheitswesens der

Tiefland-Maya von Yukatan.

Während eines 18-monatigen ethnobotanischen Feldaufenthaltes in drei

yukatekischen Maya-Dörfern (Mexiko) wurden Informationen über Medizinal¬

pflanzen, Krankheitskonzepte und Behandlungsmethoden gesammelt. Auf das

Wissen von 40 Heilem und Hebammen basierend konnten 360 Medizinalpflanzen

und 1828 einzelne Anwendungen dokumentiert werden. In einer quantitativen

Auswertung wurden die häufigsten Erkrankungen dieser Region eruiert.

Gastrointestinale Beschwerden (32 %) und Hauterkrankungen (19 %) waren die

wichtigsten medizinischen Probleme, gefolgt von Krankheiten verbunden mit

Schmerz und/oder Fieber (13 %), respiratorischen Beschwerden (11 %),

„Frauenmedizin" (8 %), andere Indikationen (5 %), Bisse und Stiche von Gifttieren

(5 %), urologische Probleme (4 %) und Augenkrankheiten (3 %). Zum besseren

Verständnis der Auswahlkriterien von Medizinalpflanzen wurden 12 Heiler und

Hebammen über zehn Pflanzen, welche ihrer Meinung nach keinen medizinischen

Wert haben, befragt. Die Eigenschaften dieser Nicht-medizinalpflanzen wurden mit

denjenigen der Medizinalpflanzen verglichen. Die Resultate zeigten, dass Geruch

und Geschmack wichtige Parameter zur Charakterisierung der Pflanzen sind. Auch

die humorale Einteilung spielt eine wichtige Rolle. Im allgemeinen werden die

Krankheiten in heiss und kalt eingeteilt und die verwendeten Medizinalpflanzen

erhalten die entgegengesetzte humorale Bezeichnung. Farbe, Form und Textur

sind bei der Auswahl von Medizinalpflanzen ebenfalls bedeutende Kriterien.

Im zweiten Teil der Arbeit, wurden 48 Pflanzenarten in verschiedenen biologischen

Testsystemen evaluiert. Alle Pflanzenextrakte wurden auf antibakterielle (gram¬

negative und gram-positive Bakterien), zytotoxische (KB Zellen) und entzündungs¬

hemmende (NF-kB) Wirkung untersucht. Zusätzlich wurden sie bezüglich der

8

Zusammenfassung

indigenen Anwendung in weiteren Testsystemen geprüft. Pflanzen, die gegen

gastrointestinale Beschwerden eingesetzt werden, wurden auf antiparasitäre

(Giardia duodenalis) und zusätzliche antibakterielle (Helicobacter pylori,

Campylobacterjejuni) Aktivität untersucht. Pflanzen der Gruppe Hauterkrankungen

wurden ferner auf antifungale Wirkung (Candida albicans) getestet. Die

Antimalaria-Aktivität (Plasmodium falciparum) wurde für die Pflanzen, die gegen

Fieber angewendet werden, ermittelt. Pflanzen zur Behandlung von Diabetes II

wurden in einem a-Amylase Test auf antihyperglykämische Wirkung geprüft und

der D2 Dopamin-Rezeptor Test wurde für die Spezies, die im Gebiet der

Frauenmedizin eingesetzt werden, verwendet. Es resultierten verschiedene

Wirkungen, welche die traditionelle Verwendung der pflanzlichen Heilmittel

begründen können.

Im dritten Teil der Studie, wurde eine Pflanzenart -Crossopetalum gaumeri

(Celastraceae)- phytochemisch untersucht. Die Wurzeln dieser Pflanze wurden

auf Grund der oralen und lokalen Anwendung gegen Diarrhöe und Schlangenbisse

sowie der positiven Resultate in den oben genannten Testsystemen ausgewählt.

Aus dem Methanolextrakt konnten ein bekanntes und vier neue, sehr zytotoxische

Cardenolide sowie das bekannte Ourateacatechin isoliert werden. Die

Untersuchung des Dichlormethanextraktes führte zu einem neuen Abietanditerpen

und zu einem neuen pentazyklischen Triterpen. Zusätzlich wurden drei bekannte

Terpene (Pristimerin, Celastrol und Friedelan-3-on-29-ol) isoliert, und in den

verschiedenen biologischen Testsystemen geprüft. Pristimerin und Celastrol

zeigten starke antibakterielle Wirkung sowie beachtliche Zytotoxizität in KB Zellen.

In mancher Hinsicht belegen die Aktivitäten der isolierten Substanzen die

traditionelle Verwendung, doch sollte die Pflanze auf Grund der starken

Zytotoxizität mit Vorsicht eingesetzt werden.

9

Part I Medicinal Ethnobotany

Introduction

1 Introduction

The main goal of this interdisciplinary study is the detailed documentation of the

medicinal plants of the Yucatec Maya of three communities and their botanical

identification. The understanding of the healing methods and the concepts of

diseases were further purposes of the field study of 18 months. In the second

stage, the most important plant species were evaluated in various bioassays based

on indigenous uses. Additionally, one of the species was investigated

phytochemically and the isolated compounds were examined in different

bioassays.

Medicinal plants are an important element of the medical system of the Yucatec

Maya (Mexico). An impressive number of healers and midwives apply the empirical

medicine, which has been developed over hundreds of years. Some plant uses are

supported by documentary evidence in old manuscripts written by the Maya or

Spaniards in the 16th century (Arzapälo Marin, 1995; Diego de Landa, 1992). Also

the performance of ceremonies addressing the rain-god to ask for rain and the

ceremonies for protecting milpas are examples of cultural heritages which go back

to the time of the Ancient Maya (Diego de Landa, 1992). The Yucatec Maya are

one group of the direct descendents of the Ancient Maya, which were a highly

advanced civilization. Herbal remedies must have been very important to the them.

Since no historic records survived from this it is of particular interest to study the

use of medicinal plants by the modern Maya. Another important point for studying

the medical system of the Yucatec Maya is the lack of modern studies of medicinal

plants and their scientific identification. There are several interesting books and

studies about medicinal plants of Yucatan. However, their information is often

based on secondary sources with data documented at earlier times or with plants

not scientifically identified.

12

Introduction

Ethnobotany is an interdisciplinary specialty, which studies plant-human

interrelationships, which occurs in different aspects of the lives of human beings,

such as medicine, nutrition, and ecology. It also includes various fields such as

ethnology, botany, medicine, linguistics and pharmacy. Knowledge about useful

plants must go back to the beginning of human existence. Humans certainly had to

differentiate between plants without any use, plants from which they could obtain

nourishment or stimulation, plants which could alleviate ailments or even cure

sicknesses, plants with psychoactive properties, and plants which could be used to

kill (Schultes and Siri von Reis, 1995).

Medical ethnobotany is an interdisciplinary science, which studies the use of

medicinal plants among cultures. There are several ways to examine this topic.

One is the anthropological approach to the way indigenous people interpret and

treat their useful plants. Another goal concerns the documentation of the medicinal

plants and their evaluation in subsequent bioassays. The discovery of natural

products for the development of new drugs is a further aim in this field of study.

The term "medical ethnobotany" is closely allied with ethnopharmacology, however

it does not necessarily include the examination of the physiological and clinical

impact of plant use on human health.

One of the objectives of medical ethnobotany is to document the uses of medicinal

plants and thus to rescue an important cultural heritage. The quantification of

documented medicinal plant usages determines which plants are most frequently

used in a culture. Their evaluation in biological test systems and their detailed

study are of high importance for the safety and efficacy of plants used in primary

health care. The WHO (World Health Organization) has estimated that about 80 %

of the people living in less developed countries rely almost exclusively on

traditional medicine for their primary health care needs. Thus, there is a need for

the study of these plants concerning safety and efficacy and to develop galenical

preparations that are standardized and stable (Farnsworth, 1980). The goals of

WHO are: (1) To strengthen research on the safety and efficacy of herbal

13

Introduction

medicines. (2) To strengthen and promote the rational use of herbal medicines

(WHO, 1993).

The phytochemical study of medicinal plants based on an ethnobotanical

approach led to the development of several drugs used in modem medicine. One

of the best examples is curare (from Chondrodendron spp., Menispermaceae). The

paralyzing effect of the plant extract is used as an arrow-poison among some tribes

in South America. One component of this plant extract, tubocurarine, is used as a

muscle-relaxant in modern medicine. Another example is foxglove {Digitalis

purpurea, Scrophulariaceae) which was used in England to treat dropsy and

epilepsy. Today the cardiac glycosides, digitoxin and digoxin, as the most

important compounds of the species are used in the treatment of chronic heart

insufficiency. One of the most recent and promising medicines is artemisinin,

isolated from Artemisia annua (Asteraceae), a species which has been used in

traditional Chinese medicine against fever and malaria for two thousand years.

This antimalarial drug is of great interest due to the development of resistance

against the known remedies and the great health risk in tropical areas. Farnsworth

et al. (1985) mentioned a total of 119 pure compounds, isolated from plants which

are currently used in medicine.

The phytochemical investigation of plants, the isolation of compounds and their

examination in biological test systems not only is essential for the study of safety

and efficacy of plants but can also help to find potent and selective drugs. There is

considerable need to obtain new, active compounds particularly in the field of

cancer, infections and tropical diseases. The plant world with about 250,000

species of flowering plants is an immense source of chemical compounds with a

vast array of unusual chemical structures that display a variety of biological

activities. Until now only a small part of this resource has been explored.

The anthropological approach of medicinal ethnobotany investigates, among

other things, the selection criteria for medicinal plants and the classification system

14

Introduction

of plants used among indigenous people. Some aspects of this key field of study

are reflected in the following questions. Why is a plant medicinal? What are the

criteria for medicinal plant selection? How do indigenous people classify plants?

Are non-medicinal plants also classified? What are the differences between

medicinal and non-medicinal plants?

Several studies have systematically investigated the hot-cold concept and its role

in indigenous medical systems especially for classifying plants and illnesses

(Foster, 1988; Ingham, 1970). Some authors mentioned the hot-cold categorization

of humoral medicine as the "basic cognitive principle" of traditional medicine in

Latin America (Tedlock, 1987). Others criticized the hot-cold system as too narrow

to explain plant choices and showed that taste and odor are important parameters

for the characterization of medicinal plants (Brett, 1992; Heinrich, 1989). To the

author's knowledge, there has been no study that focused on the comparison of

non-medicinal plants with medicinal ones and on the ways people perceive such

plants. Thus, the study of the non-medicinal plants could shed new light on

indigenous selection criteria. Furthermore it helps us to better understand the

classification system of a culture. Hence, one of our studies focused on the

Yucatec Maya medicinal plants in comparison with the non-medicinal ones

concerning taste and smell perception as well as the hot-cold concept.

15

Introduction

1.1 Goal and objectives

The goal of the studies presented in this thesis was to document the medicinal

plants of the Yucatec Maya; to study their perception and classification of the

medicinal plants; to evaluate the most important species in different bioassays; to

phytochemically study one species and to examine the isolated compounds for

biological activity.

Specific Objectives

To understand the Mayan healing practice, the illnesses, their cause, symptoms

and prevention; the medicinal plants and the preparation of the remedies, the

combination with other plants, the popular plant name, the plant part used, the

dosage of the remedies and side effects as well as their classification.

To obtain knowledge about the history of becoming a healer, the way they select

medicinal plants, and how they diagnose illnesses and perform healing

ceremonies.

To evaluate important medicinal plants in different bioassays based on the

indigenous use.

To select a medicinal plant which is used against the most important health

problem for phytochemical investigation.

To study a plant species phytochemically using bioactivity-guided isolation and

isolate pure compounds, to identify the chemical nature of the compounds, to

examine their activity and cytotoxicity as well as to correlate them with the

indigenous use of this plant.

To return the documented data and the results of this thesis to the informants of

the study region and the people and organizations which supported this project

as well as to the libraries of Mérida so that the thesis is accessible to people

who have an interest in this subject.

16

Medicinal Ethnobotany

2 Yucatan and the Mayas

2.1 Background

2.1.1 Geology and fauna

The Maya territory occupies the northwestern half of the Central American Isthmus

and is divided into highlands and lowlands. The proximity of two coasts, the

contrast of rainfall due to the tropical climate, and the varied relief result in

considerable differences in the two environments.

The Yucatan Peninsula consists of the Mexican states of Yucatan, Campeche,

Quintana Roo, as well as small portions of the states of Chiapas (the Lacandon

Jungle and Marquez de Comillas) and Tabasco (Balancan region), the north of

Belize and the Peten region of Guatemala. The Maya Mountains constitute the

southeastern limit of the Peninsula, and the mountains to the north of Chiapas form

the southwestern limits. The Mexican part of the Peninsula is formed by a porous

limestone platform with altitudes of less than 350 m above sea level. Due to the

porous limestone the rainwater sinks immediately below the surface, where it forms

underground reservoirs in great caves. Due to the absence of surface streams the

cenotes (open sinkholes, which are connected to the water-bearing bed) have

always been the main sources of drinking water for the inhabitants. Rivers only

exist in the extreme southwest and southeast.

The Yucatan Peninsula maintains a very characteristic flora. Of the 2,300 species

of vascular plants, which build the flora of the Mexican part of the Peninsula, 168

are endemic taxa (7.3 %) (Düren et al., 1998). The vegetation shows floristic

elements of the neighboring areas such as the Antillean region, Central America,

and the southeast of Mexico (Standley, 1930; Rzedowski, 1988). According to

Estrada-Loera (1991) the most important floristic elements are those of Central

America, however the endemic species and the floristic similarities with the Antilles

are of special interest (Standley, 1930).

17


Not only the geological and floristic aspect, but also by its physiographic feature

and human inhabitants, the Yucatan Peninsula is sharply differentiated from the

rest of Mexico.

Most of the Peninsula shows an Aw climate (hot with long dry period, rainy season

in summer), however a narrow northern coastal strip is of BS type (dry and hot).

More information of the geographical, floristic and climatic conditions is provided, in

publication I.

2.1.2 Ancient Maya history

Origins of agriculture

12,000 years ago human population subsisted by various forms of hunting and

gathering. In Mesoamerica, a shift toward plant and animal domestication occurred

9,000 years ago. In the Fertile Crescent of the Near East it happened 10,000 years

ago and in Southeast Asia 7,000 years ago (Lewin, 1999). Social and political

complexity was a consequence of the adoption of agriculture.

The Preclassic Period (2,000 B.C.-A.D. 250)

By about 1,500 B.C. settled farming life was established in most parts of Meso¬

america, including the Maya world. Before 1,000 B.C., a new kind of society was

emerging along the Gulf Coast: the Olmec centers. The society stood out for the

sharp contrast and status, marked with centralized political power reflected in

monumental architecture and sculpture. Although some parts of the Maya world,

mostly in the highlands, were tied into the economic networks of the Olmec world.

The early Maya communities in the lowlands were small and simple villages. After

500 B.C. some communities were beginning to reflect a new development. Jewelry

and other goods made from exotic raw material indicated increasing prosperity,

and sharper differences in wealth and social status. Decorated public buildings

reflected the emergence of powerful permanent leader, chiefs or kings (Henderson,

1997).

18

xvï r???

Figure 2.1. Landscape of the Yucatan Peninsula with Uxmal

Figure 2.2. Cenote (natural sinkhole)

19


The Classic Maya Civilization (A.D. 250-1000)

Especially during the Early Classic period, the concentration of the political and

economic power in the hands of elite grew. Several regions experienced intensified

population growth with well-developed hierarchies of communities. Many cities

enjoyed a boom in building. Relationships with distant societies intensified. Nobles

acquired greater political and religious authority. Specialist multiplied in every field:

architecture, arts, crafts, writing and in the intellectual sphere generally.

Interchanges took place in the aristocratic as well as in the intellectual sphere

among priests devoted to astronomical and astrological investigations. The Classic

period was a time of cultural florescence throughout the Maya world (Henderson,

1997).

Transformation. In the ninth century, new processes, involving internal strains as

well as external pressures disrupted long-standing patterns of growth and

expansion. This episode, which was once conceptualized as a sudden and

universal collapse of all facets of the Maya civilization, now appears to have been a

series of processes that operated over several centuries. An extraordinary and

unusual aspect of the transformation was that most regions went through a stage

of deep decline in the cultural development at about the same time (Henderson,

1997). By the middle of the tenth century quite every southern city was an

abandoned ruin. The state institutions declined with consequent transformations of

aristocratic economies. In most regions, village and household life went on, but in

much-simplified political and economic systems. The cities in northern Yucatan

generally continued to flourish during the tenth century (Chichén Itzâ, Uxmal),

although a process of decline may have begun in a few places as external

pressures intensified (Henderson, 1997).

The Postclassic Maya (A.D. 1000-1525)

During the Postclassic period long-distance exchange increased and agriculture

was less central to economic systems. Political systems shifted away from a near-

exclusive focus on one person as the ruler, to new, more flexible forms of

20


organization that involved a much broader distribution of power. The public roles of

religion were reduced but ritual maintained a central place in domestic life

(Henderson, 1997).

The Lowlands. When Chichén Itzâ fell into decline as a political and economic

center, Mayapän, an important political center, replaced it. The Mayapân's sphere

disintegrated into different provinces at least a century before the arrival of the

Spaniards. At that time, several families controlled the provinces in Yucatan: for

example Canul (West of Yucatan), Cupul (eastcentral Yucatan), Tutul Xiu (Mam)

and Cocom (Sotuta) (Henderson, 1997).

2.1.3 From the Spanish conquest to the Caste War

The Spanish conquest (16th-17th centuries)

In 1517 and 1518 two Spanish expeditions in search of gold, new territory and

slaves were started from Cuba. In 1519 Hernân Cortés landed in Veracruz from

where the Spaniards conquered the Aztecs, situated in the center of Mexico, in a

year. It took another 20 years to conquer the numerous Maya provinces of

Yucatan. In Yucatan and Guatemala the colonial regime was firmly established

with Montejo (about 1546) and Alvarado (about 1525) , respectively. However, until

about 1697, vast areas of jungle between the mountains of Guatemala and

northern Yucatan remained unconquered.

The colonial regime

Under Spanish law the Indians were considered subjects of the Spanish crown and

slavery was outlawed. As well as being slaughtered during the Conquest itself, the

Indians also fell victim to viral and other infections transmitted by the Spaniards,

which decimated the population. The increase of the power of the Catholic Church

and the suppression of ancient beliefs provoked rebellions among the Mayas,

including those of Canek (Yucatan) in 1671, Chiapas (1692), and the Tzeltal

rebellion at Cancuc in 1713. Fray Diego de Landa ordered an auto-da-fé during

21


which Indians were tortured and executed and hundreds of idols and more than

twenty Mayan codices (books) were burnt (Baudez and Picasso, 1990).

Only four codices of the pre-conquest Maya culture written in hieroglyphs could be

rescued and are known today. All of them are made of amate (bark of Ficus sp.)

and have 12-56 pages (9-25 x 12-20 cm). The Codex Dresden conserved in the

Sächsische Landesbibliothek of Dresden (Germany) mentions mythology,

astrology, ceremonies and Gods. The content of the Codex Madrid or Tro-

Cortesianus, in the Museo de Americas of Madrid, refers principally to the

prophecy with the following themes: hunting, agriculture, cloth and rituals asking for

rain. The Codex Paris can be found in the Bibliothèque Nacional de Paris contains

sequences of ceremonies and rites. Codex Grolier is conserved in the Instituto

Nacional de Antropologia e Historia (Franch, 1992).

After independence

The Mexican priest Miguel Hidalgo initiated the call for independence from Spain.

After a long struggle, Mexico declared independence in 1821, followed immediately

by Yucatan, Chiapas and Gutemala. In 1841 Yucatan declared independence from

Mexico.

Caste War (1847-1904)

During the second half of the 19th century the Yucatan Peninsula was shaken by a

violent conflict between the Maya and Europeans. The Indians, heavily taxed by

the government, saw their ancestral lands being taken from them. The Maya,

armed by English settlers in Belize, regained 90 % of their lands. At this time the

Mayas inexplicably withdrew to their villages. It is said that the Mayas went home,

because of the beginning of the rainy season, thus they had to plant their milpa.

The Europeans regrouped with the help of military force of Mexico and the United

States. Hence, the Maya were driven back to Chan Santa Cruz (now Felipe Carrillo

Puerto), where they founded the cult of the Talking Cross. Inspired by the oracle

that promised them victory, they resisted vigorously for several years. In 1904, the

22


eastern part of the Peninsula (Quintana Roo) converted to Mexican national

territory. After 1920, the chicle production and chicle gum, as its main product

{Manilkara zapota L. Van Royen), as well as the export of mahagony {Swietenia

macrophylla King) in the forest of Quintana Roo attracted people from different

parts of the country. The rebelling Maya had their own territory and did not permit

the entry to strangers. In 1936, this Maya region was accepted by the Government

as a Maya zone.

2.1.4 The Yucatec Maya today

Today more than three million people speak one of the twenty-six Mayan

languages and dialects, which are divided into ten large groups. Most of these

different languages are spoken in Guatemala, just a few ones are spoken in

Mexico. The languages are classified into the following groups (Castaheda, 1988;

Coe, 1975): (1) Huastec, Chicomuceltec, (2) Choi, Chontal, Chorti, Mopan, (3)

Tzeltal, Tzozil, Tojolabal, (4) Chuj, (5) Kanjobal, Jacaltec, Solomec, (6)

Motozintlec, (7) Mam, Tec, Aguatecpec, Ixil, (8) Quiche, Rabinalachi, Uspantec,

Cakchiquel, Tzutuhil, (9) Kekchi, Pocomchi, Pocoman, (10) Yucatec (dialects:

Lacandon, Itza)

On the Peninsula one-third of the population (1.1 million) have Yucatec Maya as

their mother tongue (Wilhelmy, 1990). Even though the Maya population increased

since 1950 this group has suffered a decline in the relative number of the native

speakers (Olivera et al., 1982). Bilingual programs at schools have always had the

goal of assimilation and resulted in a move towards learning Spanish, but this goal

seems to have come under attack because of ideological changes in education in

Mexico and autonomous movements of indigenous people throughout the

Americas (Burns, 1998). Land problems in the Maya area are still not resolved.

Two insurrections, one in Guatemala (1963-1993) and one in Chiapas (1994-...)

are evidence of the multitude of problems that still persist.

23


Figure 2.3. Groups of Mayan languages (Coe, 1975)

Chikindzonot, Ekpedz, Xcocmil - the study area

According to oral tradition Chikindzonot (= west of the cenote; sinkhole) was found

in the 14th century. However, until the end of the 16th century a stable encomienda

(place of forced labor) was known. Chikindzonot and the neighboring villages

Ekpedz and Xcocmil were abandoned during the Caste War and repopulated at

around 1915. The inhabitants of the villages still tell stories about the outbreak of

the war in Thiosuco, a nearby village, and the recovery and rebuilding of the

villages afterwards. Oversized churches dominate Chikindzonot and Ekpedz, this is

a common picture in most villages in Yucatan. The walls of the catholic churches

and the baptismal font are ornamented with indigenous figures. This kind of fusion

of Maya culture with Christian and Spanish elements can be seen in practically

every part of daily life. For example, the inhabitants of Yucatan celebrate two

24


baptisms, a Christian one and hekmetz, which is a heritage of the Ancient Maya

(Diego de Landa, 1990). Chocolate (formerly cacao) is an engagement gift, which

the fiancé gives to the family of his future wife. As opposed to this, the bride at the

official wedding celebration in the church wears a white wedding dress of

European style. The architecture of the houses made of planks, branches and

palm leaves looks the same as 500 years ago. However, the building of stone

houses is getting more popular. Food consists mainly of corn products, beans,

marrow and chili. To enrich the meal, animals are hunted in the forest or domestic

animals like chicken, pigs, and cows, introduced by the Spaniards, are kept. The

work of the women generally takes place in the house and the extensive home

garden. The most important site in the kitchen is the fireplace which is always built

using three stones. A small table and chairs are used as working place for the

women, to make tortillas, and as the dining-table for the whole family. The metate

(millstone), formerly used to turn the corn into flour, has been replaced by a hand

or motor-operated mill. Today the metate is used for grinding herbs to enrich meals

or for the preparation of medicine. Gourds and bowls made of clay or wood as well

as metal and plastic bowls are common utensils in the kitchen. Men principally

dedicate their time to the work in the milpa, the culture of bees and the breeding of

cattle. For sowing corn in the milpa, a traditional digging stick, called a choul, is

used and has undergone only one development since prehistoric times: the point,

originally hardened by the fire, has been replaced by a steel tip. Bullfights are the

central part of the feasts celebrated in honor of the Saint of the villages. Baseball,

introduced from the United States, seems to be the national sport of the Yucatec

Maya (Redfield and Villa Rojas, 1990 [orig. 1934]).

The fusion of the Mayan culture with aspects of the Christian faith are also present

in the incantations and prayers used for the treatment of patients or in the

numerous ceremonies. The modern ethnopharmacopoea of the Mayas consist

mostly of plants species, which originated on the Peninsula. But also introduced

ones are used, for instance Mentha spp. (Europe), Aloe spp. (Africa), Citrus fruits

(Asia) and are important medicinal plants.

25

Figure 2.4. Houses, new style and old style (poles, palm-leaf thatch)

Figure 2 5. House interior, kitchen

26

Figure 2.6. H-men (shaman) performing a

santiguar

Figure 2 7 Curandera (healer)

preparing herbal medicine

27


Since about 1990, a tarred street connects the three villages with Peto and

Valladolid, two nearby towns. Electricity and thereupon TV changed the rhythm of

daily live (telephone since 1995). An increasing number of young people finish

secondary school in Chikindzonot and look for a job outside the villages,

particularly in the cities of Valladolid, Mérida and Cancun. Even though the

influence of the outside forces has been enormous, the Yucatec Maya still retain a

large number of ancient traditions.

2.1.5 Manuscripts on Maya medicinal plants

Medicinal plants played an important part in Ancient Maya culture. Evidence of this

is provided by old books and documents with medicinal characters. Two of them

are by indigenous writers, namely the Ritual de los Bacabes and Libros de Chilam

Balam, whereas the others are written by Spaniards (Gubler, 1997).

In the Ritual de los Bacabes (ritual of the spokesman/interpreter) incantations

and prayers for the treatment of disease are recorded. They reflect the concept

of Mayan illnesses and illustrate medicine to be closely associated with religion.

This unique Mayan work was written in the 18th century. The main author is

unknown, two pages were written by Joan Camul (Arzapâlo, 1987).

The Books of Chilam Balam are the sacred books of the Maya of Yucatan and

were named after their greatest prophet Chilam {Balam: Jaguar, priest).

Conventionally they are generally named after the towns in which they were

found. Three of the Books namely, Ixil, Kaua, and Nah include information on

medicinal therapy and mention several remedies against a variety of illnesses

(Gubler, 1997).

After the auto-da-fé in 1558, Diego de Landa wrote a history of the life of the

Maya of Yucatan. In the Relacion de las cosas de Yucatan (1565) and the

Relaciones de Yucatan (1580-1585), he explicitly mentioned that the Maya had

specialists for curing illnesses. He listed some medicinal plants and celebrated

28


the beauty of the flora of Yucatan. His words indicate little confidence in

indigenous people (Diego de Landa, 1990 and 1992).

"There is in this land a great quantity of medicinal plants of various

properties, and if there were any person here who possessed a

knowledge of them, it would be most useful and effective. There is no

disease to which the native Indians do not apply the plants. But when

they are asked for an account of their properties, they can give none

other than that they are cold or hot, and that they are accustomed to

employ them to obtain the effect for which they apply them. However, as

a matter of fact, there are many of great virtue for every sort of illness

and as antidotes. On the other hand there are those which are

poisonous and deadly" (Roys, 1976).

In the Calepino de MotuI names of indigenous plants and some diseases as well

as some of the specialists who treat them are registered. The work was written

in the 16th century, when the Maya medicine was still largely unaffected by

Spanish influence (Roys, 1931). The book mentioned for instance: ac haban, a

herb with bad smell, which is used against cold and pechuguera (Gubler, 1997;

Arzâpalo, 1995).

El Libro del Judio is one of the most important studies of the medicinal plants of

Yucatan. The main part of the book gives a version, which is attributed to Juan

Francisco Mayoli, a roman physician who lived in Valladolid during the 18th

century and who used the pseudonym of Ricardo Ossado and the surname "El

Judio". The book contains a long list of indigenous uses and medicinal plants as

well as a few introduced ones. Furthermore it gives information on diseases.

The original work probably was written in the 16th or 17th century. Barrera and

Barrera Väsquez (1983) note that this is not just one book, but that several

copies and different versions of copies exist.

29


An important source, which is not published, carries the title Libro de medicinas

muy seguro para curar varias dolencias con yerbas muy experimentadas y

provechosas de esta provincia de Yu[ca]than. It is a copy of an old manuscript

of 1751 and contains a list of medicinal plants and their prescriptions. Some of

the remedies incorporated European plants and elements. These documents

are preserved in the Bibliotheca Crescendo Carrillo y Ancona, Mérida (Gubler,

1997).

Another unpublished source, which can be found in the same library, is the

Relaciôn de las cosas y sus nombres de la provincia del YucalPeten written in

1710. Different themes are discussed and including two pages with a list of

indigenous medicinal plant (Gubler, 1997).

Two further books, Cuademo de Teabo and Cödice Perez, are mentioned as

containing medicinal texts (Gubler, 1997; Edmonson, 1986).

2.1.6 Medicinal ethnobotany of Yucatan

As part of a Mexican national evaluation, a list of medicinal plants was generated

based on publications, state inventories and student theses. This review includes

3,352 vascular plant species distributed in 1,214 genera and 166 families. Although

the vascular plant flora in Mexico has not been thoroughly explored, it is estimated

that it consists of at least 21,600 species. Hence, 15 % of the Mexican flora has

been employed for medical purposes. In order to produce catalogs of regional

medicinal plants, many Mexican institutions have compiled state inventories. The

states with highest percentage of locally documented medicinal plants are:

Quintana Roo (99 % of 373 species), Yucatan (60 % of 623 species), Veracruz (28

% of 548 species), Durango (26 % of 255 species), and Sonora (18 % of 548

species) (Bye etal., 1995; Argueta et al., 1994).

The flora of the Mexican part of the Yucatan Peninsula as mentioned before has

about 2,300 species of vascular plants and is therefore not very rich in comparison

with other regions in Mexico. Thus, Bye et al. (1995) points out that a greater

30


degree of species richness does not necessarily indicate higher ethnobotanical

diversity. He suggests that the more phytochemically interesting plant-human

interaction is found in environmentally marginal or stressful areas than in species-

dense regions.

The botanical exploration of the Yucatan Peninsula began 1893. Several botanists

among them Gaumer, Millspaugh, Standley and Steyermark were dedicated to the

taxonomic investigation of the flora of Yucatan. Georg F. Gaumer, a practicing

physician who spent forty-five years in Yucatan, was keenly interested in medicinal

properties attributed to the plants by the indigenous people and in their Maya plant

names. Most of these voucher specimens are deposited in the Field Museum of

Natural History in Chicago (Standley, 1930).

In The Ethno-Botany of the Maya, Roys (1931) describes remedies, which are

essentially herbal but also contain animal and mineral materia medica. The texts

written in Maya and English were collected from several old books like El Judio,

and organized according to illnesses. The medicinal plants are identified based on

the works of Gaumer, Loesener, Millspaugh and Standley.

Mendieta and del Arno (1981) reviewed information about medicinal plants of

Yucatan and published this information in the Plantas Médicinales del Estado de

Yucatan. They also used secondary sources which are mentioned above. Others

are the Catalogos de Nombres Vulgares y Cientificos de Plantas Mexicanas

(Martinez, 1979) and Nomenclatura Etnobotanica Maya (Barrera et al., 1976). In

the latter book, the Mayan names of plants, their scientific identification, the origin

of the popular names and the etymological significance are discussed. Also here,

the information is based on secondary sources.

Alfonso Villa Rojas, born in Mérida, took charge of the Chan Kom school in 1927, a

village about 27 km from the study area of this thesis. He wrote several books and

publications about different aspects of the life of the Yucatec Maya. Together with

31


Redfield, an anthropologist from the University of Chicago, he published the

ethnography Chan Kom - A Maya village, in which they included one chapter on

diseases, and their treatment as well as the meaning of nature (Redfield and Villa

Rojas, 1990 [orig. 1934].

One of the most important works on medicinal plants of the Yucatan, which

includes phytochemistry and toxicity, is the Atlas of Medicinal Plants of Middle

America - Bahamas to Yucatan of Morton (1981). The data here are also based on

secondary sources.

Until now no modern detailed ethnobotanical study on the Yucatec Maya has been

available. Thus, one of the main points of this study was to document the actual

use of medicinal plants of Yucatan and the scientifically identify the plant species.

The lack of pharmacological, toxicological and phytochemial studies of Mexican

plants provided a further point of the thesis. In the most recent work listing 2,049

medicinal plants of Mexico carried out by the Institute Nacional Indigenista (INI)

394 species were studied chemically, 280 species were studied chemically and

pharmacologically, 177 species were investigated chemically, pharmacologically

and toxicologically and from 69 species active principles were studied (Aguilar et

al., 1994; Argueta and Zolla, 1994).

32


2.2 Methods in the field

Ethnobotanical data was collected during a total of 18 months, from February 1994

to June 1995 and from September 1996 to October 1996.

Qualitative and quantitative methods were used for the ethnobotanical evaluation

(Martin, 1996; Russell, 1988). Participant observations were carried out as one

of the methods used. The author lived with the Maya people and shared with them

many facets of their life: e.g. plant use in daily life, healing sessions, ceremonies,

cooking and marriages. Subjects like dreams, omen and witchcraft were discussed

in semi-structured or open-ended interviews. By means of questionnaires and

lists of questions and topics structured and semi-structured interviews were

held with 40 healers and midwives (Figure 2.8). They were interviewed

independently from each other. Only a few persons with non-specialist knowledge

concerning medicinal plants were interviewed. Excursions were made with the

specialists to their home gardens, the forests and along the borders of the paths in

the villages. The species were collected, pressed and dried in a field dryer

(wooden box of 70 x 120 x 80 cm, with a metal wire-lattice at the bottom and

underneath two electric bulbs to maintain a temperature of about 40 °C, Figure

2.9). Some interviews, prayers and songs during ceremonies were recorded on

tape with the permission of the healers. After becoming familiar with the medicinal

plants a portable herbarium was made and presented in some interviews. The

form of the interviews as well as the data obtained concerning medicinal plants

were documented and transferred to a database (FileMaker Pro ).

At the beginning of the field work, the ethnobotanical project was presented in a

meeting of the healers and midwives of Chikindzonot and Ekpedz. They were

asked to participate in the project. Initially, interviews with bilingual specialists were

carried out. Later on the interviews were held in Maya and translated to Spanish

with the help of a young woman of Ekpedz.

33

Figure 2.8. Questionnaire (in English and Spanish)

Popular name:

nombre popular

Scientific name:

Nombre cientifico

Number. Numero

Collection: Herbano

Picture- Foto

Informant, profession: Family.Flower:

Informador, profesion Familia

Floi

Seed, fruit:

Semilla, fruta

Uses: Collection' Leaf:

Usos Recolection Hoja

Identification Root:

Identificacion Raîz

Plant part used:

Partes utelisadas

Uses in other locations.

Utilizacion diferente que en el lugar de investigacion

Preparation:Preparacion

Description of the plant

Description

Doses, application (form,

time):

Habitat (vegetation type):

Dosis, aplicacion (forma,

tiempo) Habitat (tipo de veqetacion)

Effect: Observations:

Efecto Observaciones

Side effect, contra¬ Illustration of the plant:indication:

Efecto secundano, contra-

indicacion

Classification-

Clasificacîon Dibujode la planta

Description of the plant

(informant):

Importance of the plant (informant).

Description de la planta

(infomador) Importance de la planta (infomador)

34


The author participated and supported the projects of the organization OMIMPY

(Organizaciön de Medicos Indigenas traditionales de la Peninsula de Yucatan)

during their meetings organized by the INI (Institute Nacional Indigenista). In order

to get a broader knowledge about the medical system of different regions of

Yucatan, the participating specialists in the meetings of this association were

interviewed and discussions were initiated. The ethnobotanical project was

presented at the secondary school of Chikindzonot and a small excursion was

carried out with the pupils. They were asked to collect one medicinal plant, which is

used in their homes, and to describe their medicinal effects. These species, dried,

pressed, labelled and covered with plastic, were deposited in the secondary school

at Chikindzonot. Other voucher specimens were deposited as described in

publications I, II and III.

Figure 2.9. Field dryer

The voucher specimens were identified with the help of botanists and specialists at

the CICY (Centro de Investigacion Cientifi'ca de Yucatan), Mérida and MEXU

(Herbario Nacional at the Universidad Nacional Autönoma de Mexico, D. F.). For

the plant evaluation of 48 species 100 - 200 g of plant material were collected. In

35


the cases of the species which were of phytochemical interest, 1 - 2 kg of the plant

part used as part of a remedy were collected and dried in the shade.

The plant material was collected and exported with official permission of the

Secretana de Medio Ambiente, Recursos Naturales y Pesca - Instituto Nacional

de Ecologia, Mérida (22. April 1994 [No. 01245]; 12.April 1995 [No. 01105]; 21.

February 1996 [No. 686]) and Secretana de Agricultura, Ganaderfa y Desarrollo

Rural, Dirrecciôn General de Sanidad Vegetal, Mexico (Certificado Fitosanitario

Internacional, 12. May 1995 [No. 24075]; 23. October 1996 [No. 186]).

36


2.3 Abbreviations mentioned in the plant list

Table 2.1. Plant use

No llnesses/conditions Enfermedades/condiciones

AT

1

counteract bites and stringsof venomous animals

snake bites

mordeduras y picaduras de

animales venenosas

mordedura de vibora

DER

1

dermatological conditions

inflammation

enfermedades

dermatologicasinflamacion

2 pimples granos

3 abscess absceso

4

5

inflammation of the throat

chickenpox

inflamacion de garganta

(mumps)viruella

6 measles sarampion

7 dermatomycosis hongos

8 pellagra pellagra

9 warts verrugas

10 psoriasis psoriasis

11 discoloration of the skin mal de pinto

12 scabies sarna

13 infection infecion

14 burning quemadura

EYE illnesses of the eyes enfermedades oculares

1 pain dolor

2 pimples granos

3 eye complaint problemas oftalmologicas

FEM women's medicine medicina para mujeres

1 spasm pasmo

2 problems of the vagina problemas vaginales

3 infertility infentilidad

4

5

pain of menstruation, disorder

of menstrual cyclechildbirth

dolor menstural, menstruacion

irregularinduccion del parto

6 "to induce" abortion induccion del aborto

7 inflammation of the vagina inflamacion vaginal

8 prevention of abortion prevencion del aborto

9 vomiting and fever duringconfinement

vomito and fiebre durante

alumbramiento (jobenal jolol)Gl gastrointestinal disorders afecciones gastrointestinales

1 diarrhea diarrea

2 dysentery dientena

3 mal de ojo mal de ojo

4 vomiting vomito

37


5 spasm pasmo

6 constipation constipacion

7 bad air in the stomach mal aire en el estomago

8 parasites parasitas

9 stomachache dolor de estomago

10 mal viento mal viento

11 problems of the bile problemas de la billis

12 cirro cirro

OTH different uses otros usos

1 dandruff caspa

2 toothache dolor dental

3 pimples in the mouth herbes bucal

4 antidote antidoto

5 fractures of bone fracturas de huesos

6 hemorrhage hemorragia

7 vitamin deficiency deficiencia vitammica

8 earache doloren la oreja

9 ceremony ceremonia

10 splitting hair

Illnesses associated with

pelo con orquilla

PFE enfermedades asociadas con

pain and/or fever dolor y/o fiebre

1 rheumatism reumatismo

2 sweat during night, cold body sudacion nocturna, cuerpo fno

3 fever fiebre

4 headache dolor de cabeza

5 insomnia (lover's grief) insomnio (mal de amor)

6 trembling of babies tetano de bebes (dolor de

ombligo)7 invigorate the muscle tonifica los musculos

RES respiratory illnesses problemas respiratorias

1 catarrh catarro

2 bronchitis bronquitis

3 respiratory problems problemas respiratorias

4 cough tos

5 asthma asma

UR

1

2

3

4

urogenital problems

kidney trouble

diabetes II

anuresis

pain of the urogenital system

problemas urogenitales

problemas del nnon

diabetes II

anuresis

problemas urologicas


Table 2.2. Plant part used

Abbreviation of Plant part used Parte usada de la planta

plant part used

ap aerial part hierba

ba bark corteza

bu bulb bulbo

fl flower flor

fr fruit fruta

gp green part hojas y tall os verdes

ju juice (watery) agua, jugo

la latex (milky) latex

Iv leaf hoja

pu pulp pulpa

re resin résina

rh rhizome rizoma

ro root rai'z

se seed semilla

st stalk tallo

tr trunk tronco

tu tuber tuberculo

wh whole plant planta entera

wo wood madera

so shoot retoho

Table 2.3. Application

Abbreviation for mode of

application

Mode of application Modo de aplicaciôn

con

lOG

nas

ora

pulrec

spi

vag

conjunctivallocal

nasal

oral

pulmonalrectal

spiritual

vaginal

conjuntivallocal

nasal

oral

pulmonal-rectal

espiritual

vaginal

39


Table 2.4. Preparation

Abbreviation Mode of preparation Forma de preparaciön

M: combination with other plants combinaciön con otras plantas

bath bath baho

dec decoction decocto

drops drops gotas

empl emplastrum emplastruminf infusion infusion

lini liniment liniment

mac maceration rnaceracion

oint ointment pomada

pow powder polvo

soap soap jabon

syrup syrup jarabe

cer ceremony ceremonia

Table 2.5. Plant classification among the Mayas

Abbreviation Plant classification Clasificaciön de plantas

Tb bitter amargo

Ta astringent astringenteTe sweet dulce

Ts spicy picanteTi acid agrioTn no taste sin sabor

Sa aromatic, good smell buen olor

Ss strong smell olorfuerte, apestosoSb bad smell mal olor

Sn no smell sin olor

Sf little smell poco olor

Hh hot caliente

He cold frio

HI lukewarm tibio

Ho cool freso

2.4

Plant

list

Plantname(AANK#voucher)

Popularname

Use

Part

Applica-

Preparation

Ciassifica-

No.

of

used

tion

tion

resp.

ACANTHACEAE

Blechumbrownei(Kunth)(4

14)

ElytrariaimbricataPers.(310,449)

Ruellianudiflora(Engelm.&Gray)

Urb.

;

Syn.

:R.yucatanaTharp.

&Barkley

(115)

Ruelliasp.(528)

AGAVACEAE

Agaveamericana

L.(312)

Agave

maculata;

Syn.

:Manfredamaculata

(Reg

el)Rose

(197

)

Agave

aff.

fourcroydesLemaire(6

54)

YuccaelephantipesRegel

(416

)

ALLIACEAE

Alliumcepa

L.(6

41,642,643)

Alliumschoenoprasum

L.(4

63)

AMARANTHACEAE

Achyranthes

aff.

indica

Mill

.(5

37)

AMARYLLIDACEAE

(LILIACEAE)

Crinum

aff.americanum

(223

)

Crinumerubescens

Ait.

(408

)ANACARDIACEAE

AstroniumgraveolensJacq.(4

73)

Mangiferaindica

L.(4

09)

Spondiaspurpurea

L.(2

86)

ANNONACEAE

Annonamuricata

L.(630,631)

Annonapurpurea

Moc.&Sessé

(618

)

Annona

reticulata

L.(2

58)

AnnonasquamosaL

(261

)

Ak'abxiw

GI3PFE2

Ivfl

loc

bath

1

Kabalxa'an,Kambaxa'an

GI34

5,FEM1

2ap

ro

ora

loc

decbath

Hh

7

Kaba

lya'

axni

k,UR1

EYE1

ap

ora

loc

decM:bath

2

Sb

Maguey

FEM3

Ivora

M:

inf

Hh

Pets'k'im,

Pets'k'inil

PFE4

leloc

emp

He

Henequen

NI

IvTb

Tuk

PFE5

Iv

Ajo

FEM3

4PFE4

bu

ora

M:macdec

He

Cebollina

PFE4

bu

ora

mac

Bayche'

Gl6

ora

Sb

2 2 1 1 4 1

Pets'kini,

Pets'kinil

PFE4

Ivloc

empl

He

2

Xts'ulam

DER4

AT1

bu

loc

empi

1

-

RES3

Ivloc

1

Mango

FEM5PFE3

Ivloc

empl

Tn

1

Abal,Ciruela

DER5

6Iv

loc

M:

bath

iempl

SaTa

5

Guanâbana

RES4

Ivora

M:decsyrup

sweet

2

Poox

NI

fr1

Oop,Anona

DER1

PFE6

Ivloc

M:emp

ilbath

He

5

Saramuyo,Ts'almuy

GI4RES4

Ivora

loc

dec,bath

HeHhTa

9

ro


Popularname

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

Malmeadepressa

(Bai

ll.)

R.

E.

Fr.;

Syn.:

Guatteria

leio

phyl

la(F.D.Sm.)

Saff.Ex

Standi.(161

)

Sapranthuscampechianus

(Kunth)

Standley

(291,509)

APIACEAE(UMBELLIFERAE)

CoriandrumsativumL

(005

)

Pirnpinellaanisum

L.(2

55)

APOCYNACEAE

Catharanthusroseus

(L.)

G.Don

f.(2

71)

Echitesyucatanensis

Millsp.exSt

andl

ey(5

19)

Plumeriasp.(472,562,628)

Rauvolfiatetraphylla

L.(0

95)

Tabernaemontanaam

ygda

lifo

liaJacq.

(190229)

ThevetiagaumeriHemsl.

(324)

Urechites

andrieuxiiMuell.Ar

g.(4

41,446)

ARACEAE

Anthurium

schlechtendaliiKunth

ssp.

schlechtendalii(2

43)

Philodendronhederaceum

(Jacq.)Schott

(586,505)

Syngoniumpodophyllum

Schott(5

06)

ARISTOLOCHIACEAE

AristolochiaanguicidaJacq.(1

39)

AristolochiamaximaJacq.

(350

)

AristolochiapentandraJacq.(315)

Elemuy

UR1

2ro

Chuyuchajum,Sak-

UR1

NI

elemuy

Cilantro

GI4

Anisengranos

FEM5

GI1

Vicaria

FEM2

Sak-

vipe

rol,

Vipe

rol

rojo

DER1

AT1

Nikte'ch'om,

Flordemayo

DER7

Kambamuk

EYE2

DER8

NI

Uts'

upek

'DER1

9NI

Akits,

Cojönde

perro

DER1

AT1

Vipe

rolverde,Vi

pero

lAT

bejuco

Bobtun

FEM1

-

DER2

NI

-

NI

Wahk'oh-ak',Wahk'ohde

GI7FEM1

5

pato,Guaco

Wahk'oh

cast

illo

,Guaco

GI7FEM1

6

castillo

Wahk'oh

cast

illo

,Guaco

GH

2

castillo

se

se

Ivse

ro

re

Iv

ju-f

rre

re

re

ro

Iv

wh

ro

ro

ro

ora

ora

ora

ora

vag

loc

loc

loc

loc

loc

loc

ora

loc

ora

ora

ora

dec

dec

TbTaSs

Te

14

dec

Sa

11

powdec

SaTb

He

4freshbath

dec

M:decempl

Tb

2

drops

3

drops

Iv:TnTaSn

7

Sb

drops

Iv:TnSnSb

7

He

M:drops

2

freshempl

TbHe

5

M:dec

1

TnSn

2

TnSn

2

M:pow

inf

Sa

3

M:powdec

HhTbSa

1£

M:macdec

HhTsTb

3


Popularname

ASCLEPIADACEAE

(158)

Asclepias

curassavica

L.(0

70)

Cynanchum

schlechtendalii(Decne.)

Standi.&

Stey

erm.

(390

)Matelea

vindifloraWoodson;

Syn.

:Gonolobus

vindiflorusReom.

(022,

192,365,450)

Mateleayucatanensis(S

tand

i)Woodson

(345)

ASPHODELACEAE(LILIACEAE)

Aloevera

(L.)

Burm.f.;Syn..Aloe

barbadensis

Mill

.(065)

ASTERACEAE(COMPOSITAE)

AgeratumgaumeriRob.

(052

)

Artemisialudoviciana

Nutt.ssp.mexicana

(Willd.)

Keck(0

26)

Bidens

sp.(062

)Bidenssquarrosa

Less.

(121,

170,187,

404)

Galea

urti

cifo

liaMillsp.var.yucatanensis

Wussow,

Urb.&

Sullivan(1

35,153)

EupatonumodoratumL;Chromolaena

odorataR.M.King&

H.Rob.

(339

)Millenaquinqueflora

L.(3

69,413)

Montanoa

atri

phcifoliaC.Koch

(382)

Partheniumhysterophorus

L.(4

48)

Plucheasymphytifoha

(Mil

l.)Gillis

(009

,

109)

PorophyllumpunctatumBlake(152)

Tagetes

sp.(494

)

Tithoniadiversifoha(Hemsl

)Gray

(017

)

TrixisinulaCrantz(3

55)

VerbesinagiganteaJacq.(288)

Kalakts'u'um

Anal,

Ik'a

bal,

Polkuts

Xîum-ak'

Piin

-k'a

k',Kuyuch-ak',

Xp'okini,Emtsul

Ensul,Emtsul

Sâbila

,Petk'inki

Xpasmarxiw

Si'i

sim,

Artemisia

Sahun,Saksahun

Ya'axk'an-ak'

Xka'xikin,

Xikinkaax

Tok'aban

Xontolok

Xtankas-

ak',

Xuxtankas

Altamisa

Chalche',SantaMaria

Xuk'ii

Tempula

Arnica,Chaksu'um

Fluxionxiw,Xtankas-ak'

Chulkeeh

Use

Part

used

DER4

ro

OTH2

re

UR2

3re

Applica-

Preparation

Classifica-

No.

of

tion

tion

reso.

locora

empldrops

ora

drops

HI

ora

drops(d

ec)

OTH3

re

loc

drops

Hh

PFE6

loc

fresh

RES1

Ivora

M:dec

He

7

DER1

23

OTH1

loc

ointsoap

GI58

ap

ora

dec

Hh

6

GI48

Ivora

dec

inf

HhHcSaTb

26

PFE3

RES2

5Iv

ora

dec

2

GI3

Ivloc

M:

bath

8

DER2

10

Ivloc

M:bath

TB

10

Gl4

nas

inn

UR1

23

ro

ora

M"dec

TnSn

6

DER2

Ivloc

drops

3

GS10

ap

loc

bath

2

OTH4

FEM2

wh

loc

freshdec

2

FEM5

6Iv

ora

M:dec

inf

Hh

12

OTH

Ivloc

dec

Ss

4

FEM7

ap

loc

1

PFE

1Iv

loc

oint

5

PFE4

Ivro

loc

empl

2

RES2

Ivloc

empl

hot

1

ÊPlantname(AANK#voucher)

Popularname

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

_usecf

tion

tion

resp.

Wedelia

fertilisMcVaugh

(217

)

BASELLACEAE

AnrederavesicariaC.

F.Gaertner(116,

196)

BIGNONIACEAE

AmphilophiumpaniculatumKunth

var.

molle

(Schltdl.&Cham.)

(118

,172)

AmphilophiumpaniculatumKunth

var.

paniculatum

(144

)ArrabidaeafloribundaLoes.(2

07)

Ceratophytumtetragonolobum

(Jacq.)

Sprague&Sandw.

(180

)Crescentiacujete

Veil.(2

39)

Cydista

diversifoliaMiers.

(201,550)

Godmania

aesculifolia(Kunth)Standi.

(412

)Parmentieraaculeata(Kunth)Seem.;

Syn.:

P.edulisDC.

(107,096)

Parmentieramillspaughiana

(L.)

Williams

(135)

Pithecocteniumcrucigerum

(L.)

A.Ge

ntry

;Syn.:Pithecocteniumechinatum

Schum.

(002)

Stizophyllumri

pari

umSandw.

(156

)Tecoma

stansJuss.(0

04)

BIXACEAE

Bixaorellana

L.(2

33)

BOMBACACEAE

Ceiba

aesculifolia(Kunth)

Britton&Baker

(497

)CeibapentandraGaertn.

(456,614)

PachiraaquaticaAublet(4

37)

Sahun

Kaxichel

Sit'macho,

Petak'

Sosk

i-ak

',Xdunt'-ak'

Sak-ak'

Bakchiwoh

Luch.Jicara

Ek'k'ixil,

Soski-ak'

Xo'k'ab

Pepino

cat,

Kat

Katche'

Xache'ma'ax,Xache'xnuuk

AT1

DER2

DER1

OTH5

GI1

3

G12DER2

13

DER11

AT1

RES2

5

RES5

DER1

2NI

FEM5

7

UR1

23

UR1

IV

loc

1:bath

Xa'bach

RES2

5

K'anlol,

Tonadora

UR2

NI

Ki'wi',Achiote,K'uxub

GI9PFE3

PFE6

Pi'im

EYE1

DER10

Ya'axche'

PFE7

K'unche',K'

uych

e',Bonete

PFE3

Ivfr

ju-b

a

Iv ba

ora

loc

con

loc

loc

ora

pow

oint

drops

bath

drops

1

tu

loc

M:empl

HcTnSn

11

Ivloc

M:bath

Sa

4

Lv

loc

bath

2

Ivloc

M:bath

1

Ivpow

inf

ora

1

pu-fr

ora

dec

5

Ivf!

loc

M:empl

bath

SnSbTn

7

Ivfr

ora

M:dec

1

flfr

ro

ora

M:dec

SnTn

fr:Te

4

Ivora

M:dec

1

roseca

loc

M:pow

Sb

1

rofr

ora

dec

Te

1

ro

Ivfl

ora

dec

4


Popularname

Pseudobombax

elli

ptic

um(Kunth)

Dugand;

Syn.

:Bombax

elli

ptic

umKunth

(275)

BORAGINACEAE

CordiadodecandraA.DC.

(634,635,636)

Ehretia

tinifoli

aL.

(021

)

Amabola

bianco,

Sikl

ite,

Xk'uxche'

Cirricote,

Kop'te'

Beeb,

Roble,Xi'mche'

Hehotropiumangiospermum

Murr.(053,

131)

Toumefortia

volubilis

L.(126,165)

BRASSICACEAE(CRUCIFERAE)

Raphanus

sativus

L.(0

06)

BROMELIACEAE

Aechmea

bracteatavar.bracteataGnseb.

(167

)Bromeliakaratas

L.(3

73)

TillandsiabalbisianaSchuttes&Schultes

f.(625)

Tillandsiabrachycaulos

Schltdl.(4

96)

TillandsiaelongataScheudel

(479

)TillandsiaschiedeanaSteud.(6

26)

Tillandsiasp.(6

24)

BURSERACEAE

Burserasimaruba

Sarg

.(0

42)

CACTACEAE

Hylocereusundatus

(L)Britton&

Rose;

Syn.:CereusundatusHaw.

(427

)

Nopalea

cochenillifera

(L.)

Salm-Dyck

(313)

Selenicereusdonkelaarii

Britton&

Rose;

Syn.:CereusdonkelaniSalm-Dyck

(284)

Xnema'ax

Xulk

'ini

,Sal

Râbano

Ch'uk,Cinta

k'uk'

Ch'om,Ch'am,Pinuela

Ch'u

Ch'u

Ch'u

Ch'u

Ch'u

Chakah

Pitahaya

Nopal,Pak'am

Tsaran-ak'

en

Use

Part

Applica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

RES1

25

Ivba

ora

M:dec

Te

PFE3

RES25

ba

ora

dec

3

RES1

Ivba

locrec

M:dec

TeSa

15

PFE3

NI

ora

empl

GH268

ap

locrec

bathdec

HhTnSn

7

DER3

ap

loc

M.powempl

2

RES1

tu

ora

dec

2

FEM8

FEM5OTH6

FEM9

FEM4

FEM4

FEM9

FEM4

PFE2

3

GI2

RES2

UR4

OTH3

6

Iv Iv wh

wh

wh

wh

wh

Ivre

pu-l

v

Iv Iv

ora

Mmac

oraloc

decpow

loc

bath

ora

M.dec

ora

M:dec

loc

bath

ora

M:dec

loc

Hh

mac

bath

HeSs

ora

mac

HeTnSn

17

oraloc

macempl

Hh

3

ora

drops

5

èPlantname(AANK#voucher)

Popularname

CAESALPINIACEAE

Bauhiniadivancata

L.(0

07)

Bauhmia

herrerae

(Britton

&Rose)

Standi.

SSteyerm.

(124,148,442)

CaesalpmiagaumeriGreenman

(155,

344)

ChamaecristaglandulosaGreene

var.

flavicoma(Kunth)

H.

S.

Irwin&

Barneby;

Syn.:Cassia

g.(362)

Sennaatomana

(L.)

H.S.Irwin&Barneby;

Syn.

:Cassiaatomana

L.(2

73,337)

Senna

fruticosa

(Mil

l.)H.

S.

Irwin&

Barneby

(035

)Senna

obtusifolia

(L.)

H.S.Irwtn&Barneby

(361)

Sennaracemosa

(Mil

ler)

M.

Irwin&

Barneby

Senna

sp.(525)

Senna

uniflora

(P.

Mill

er)H.

Irwin&

Barneby

Senna

villosa

(Mil

l.)H.S.

Irwin&

Barneby;

Syn.:Cassia

villosa

Mill.(0

84)

Tamarmdus

indica

L.(2

69)

CAPPARACEAE

Cieomegynandra

L.(4

93)

CARICACEAE

Caricapapaya

L.(6

46)

CELASTRACEAE

Crossopetalumgaumeri

(Loes.)Lundell;

Syn.

:Mygindagaumeri

Loes.ex

Millsp.;Rhacoma

g.(0

38,128)

HippocrateaexcelsaKunth

(174

)

Mayvaca,Patadevaca

Kibix,

Mayvaca

rojo

,

Ts'ulubtok'

Kitamche'

Salatxiw,

Salat-ik'

Tu'ha'abin

K'anchik'in-ak'

Mehenbu'ul-xiw

Chululdzu

Xpahpul

Saalche',Boxsaal

Tamanndo

Papaya,

Put

Vipe

rolnegro

Sak-bo'ob.Xooknom

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

RES14UR12

ap

ora

Mdec

GI3

loc

Mbath

Gl3

Ivro

loc

Mbath

RES4

ora

Mdec

PFE4

Ivloc

pow

lini

EYE1

con

drops

Sa

10

7 4 3

PFE1

NI

Iv

GI3

Ivse

GH

PFE1

Iv

NI

Iv

NI

Iv

NI

Iv

DER2

Iv

GI1

ju-f

r

NI

Iv

AT1

re

AT1

ro

iv

GI1

loc

loc

loc

loc

ora

M-bath

M:

bath

bath

Sb

Tb

bath

decfresh

TbSb

Sf

TnSn

5 3 1 1 1 10

3

loc

drops

Tb

1

loc

ori

empl

fresh

TaTbHc

20

ora

M:dec

RES1

2Iv

ora

pow

inf

HhHc

Plantname(AANK#

voucher)

Popularname

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

•vl

CHENOPODIACEAE

ChenopodiumambrosioidesBertex

Steud.;Syn.:Teloxysambrosioides

(L.)

W.A.Weber

(028

)COMBRETACEAE

Terminaliacatappa

L.(3

26)

COMMELINACEAE

CommelinaelegansKunth

(093,106)

Rhoeo

discolorHance

(306

)CONVOLVULACEAE

Ipomoeabatatas

(L.)

Poir.(6

37)

IpomoeaheterodoxaStandi.&Steyerm.

(218

,248)

Turbinacorymbosa

(L.)

Raf.(0

46)

CRASSULACEAE

Kalanchoë

blossfeldiana

Poelln.(0

68)

Kalanchoë

intégraKuntze

(016

)Kalanchoëpinnata

Pers

.;Syn.:

BryophyllumpinnatumKurz(4

39)

CUCURBITACEAE

CayaponiaracemosaCogn.

(431

)

Cionosicyosexcisus(Griseb.)C.Jeffrey

(387

)Ibervilleamillspaughii

(Cogn.)C.

Jeff

rey;

Syn.:Co

ralo

carp

usmillspaughii

Cogn.

(094

)

Lagenaria

siceraria(M

olin

a)Standi.(3

83)

Luffaaegyptiaca

Mill.;

Syn.:

Luffa

cylindrica

Roem.

(149)

Momordicacharantia

L.(2

68)

Epazote,Lukumxiw

Almendra

G185

UR1

GI68

ro

Ivora

ora

dec

dec

Hh

after

25

cooking:

Sa

Ta

Uk'

ak'ah

ko'l

ebil

,EYE2

ju-fl

con

loc

3

Ya'axha'xiw

Chakts'am

DER78

11

Ivloc

empl

bath

3

Is,Camote

DER2

Ivloc

bath

Sb

1

Chiwohk'ax,Cancerxiw

DER12

PFE6

ro

loc

empl

bath

Hc

3

Xtabentun

PFE2

RES25

NI

Ivloc

M:bath

SbTb

3

FEM5

ro

ora

M:empi

Belladonna

DER1

Ivloc

empl

1

Belladonna

DER12OTH5

Ivloc

M:emp:

oint

Hc

11

Siempreviva

DER1

ivloc

1

Takeeyl

DER8

Ivloc

bath

-4

I

Kasam

DER8

Ivloc

empl

1

K'umkanul

DER1

PFE1

tu

loc

freshempl

SnSb

17

Lek

PFE1

pu-fr

loc

empl

1

Limpion

UR3

GI11

se

Ivora

dec

1

Morax

DER2

NI

Ivloc

bath

HITb

Sf

4

œPlantname(AANK#

voucher)

Popularname

SicydiumtamnifoliumCogn.

(036,336)

CYPERACEAE

Cyperus

articulatus

L.(0

15)

Sclerialithosperma

L.(1

75)

DIOSCOREACEAE

Dioscorea

spiculiflora

Hemsl.

(213

)

EBENACEAE

DiospyrosanisandraBlake(1

34)

Diospyroscuneata

Standi.

(431,424)

ERYTHROXYLACEAE

Erythroxyium

rotundifoliumLunan

(206,

379)

EUPHORBIACEAE

AcalyphaalopecuroidesJacq.(3

59)

Acalypha

sp.(4

32)

AcalyphaunibracteataMuell.Ar

g.(4

33,

507,581,593)

Astrocasiatremula(Griseb.)Webster

(455,504)

Cnidoscolus

aconitifoliusspp.

aconitifolius

(cultivated:

chayamansaMcVaugh)

(037)

CnidoscolussouzaeMcVaugh

(384

)Crotonchichenensis

Lundell(216,374)

Croton

humilis

L.(4

38)

Crotonlobatus

Forssk.;

Syn.:Jatropha

iobatusMuell.Ar

g.(1

38)

Croton

lundelli

iStandi.8040,

127,216,

374)

Chakmots-ak',Hoykep,

Saloli-ak',Cbikimu-ik'

Tupux

Xoknoon

Cancer-ak',

Wil-ak'

Xkakalche'

Sibi

l

Xik'iche'

Xmis

bil,

Mehenmis,Cola

degato

Sakpasmarxiw

Ch'ilibtux

il

P'ixt'onk'ax

Chaya,Chay

Chayademonte

Xebalam,Butsumukuy,

Xikinch'omak

lk-aban

Cruzoj

oxiw

Kokche'

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

EYE1

2AT1

Ivcon

loc

dropsempi

Hc

11

DER1

RES24

tu

ora

dec

Sa

4

RES24

ap

ora

M:pow

inf

1

AT1

Ivro

loc

dec

Hc

2

DER2

12

Ivloc

bath

Ss

13

DER1

NI

Ivloc

bath

Tb

4

DER2

Ivloc

bath

GI3

ap

loc

dec

TnSn

7

UR2

NI

ora

DER2

Ivloc

bath

1

Gl3

NI

Ivloc

bath

SnTb

7

NI

Ss

2

FEM1OTH67

re

Ivora

dec

Hh

2

PFE1

Ivloc

empl

1

DER2

12

Ivre

loc

bathemip

lHc

3

DER9

re

loc

drops

1

GI3

Ivloc

M:

bath

3

RES1

4Iv

ora

M:decinf

Hc

t


Popularname

CrotonperaeruginosusCroizat(1

32,205.

231,289)

Croton

reflexifoiiusKunth

(143

,377,399)

CrotonyucatanensisLundell(1

27)

Euphorbiaarmourrii

Millsp.(111,305)

Euphorbiahete

rophylla

Desf.(1

81,221)

Euphorbia

hirta

L.(3

64)

Euphorbia

aff.ocymoides

(112

)

EuphorbiaptercineuraA.Berger(0

34)

JatrophacurcasWall.(225)

JatrophagaumeriGreenman

(419)

ManihotesculentaCrantz(081)

Pedilanthusitzaeus

Millsp.(2

19)

Pedilanthusnodiflorus

Millsp.

(297

)

PhyllanthusacuminatusVahl(3

57)

Ek'balam,Xikm

burro,

Xikinch'omak

Pets'k'uts

Sakpokche',Ik-haab

Sakchakah,

Sibik'

Hobonk'ak'

Xanabmukuy

Kambalchakah

Much'kok

Sikl

ite'

Pomolche',Pinon

Yuca,Ts'nm

Ya'axhalalche'

Nabalche',Nahualte

Xulimil

4^CD

Phyllanthusglauscecens

Schltr.&Cham.

(228,261,526)

Phyllanthusmicrandrus

Muell.Ar

g.(0

88,

117,309,421)

Phyllanthussp

(202

)

Ricinuscommunis

L.(353)

Tragia

aff.

yucatanensis

Mill

.(459)

FLACOURTIACEAE

CaseanacorymbosaJacq.

(150,237)

SamydayucatanensisStandi.

(146,212)

XylosmaflexuosumHemsl.

(168

,209)

Zuelaniaguidoma(S

w.)

Bntt.&

Millsp.

(357,522,585)

ILLIACEAE

liliciumverumHook.

f.(255a)

LAMIACEAE(LABIATAE)

Hypt

issp.(4

75)

P'ix'ton-ak'

Pets'k'mi,

Kambaikiche'

P'ix'tonche'

Xk'ooch,

Higuerilla

P'op

'ox

Xi'mche'

Naranjache'

Puts'ukche',Xchaknif

Tamay,

Bot'ox

Anis

estrella

Xta'ulum,OreganoKaX

Use

Part

used

Appl

ica¬

tion

Preparation

Classifica¬

tion

N re

DER2OTH3

Ni

re

loc

decdrops

HcToTs

9

DER2OTH3

re

loc

drops

Hc

7

PFE3

Ivloc

inf

1

DER4

AT1

re

leloc

dropsempl

Hc

2

EYE1

re

con

drops

2

DER2

re

loc

drops

3

DER4

AT1

Ivloc

empl

1

RES1

24

reap

ora

decdrops

HhTb

8

DER2OTH3

re

loc

drops

TbTa

2

GI2

roju-s

tora

dec

TiTa

6

PFE2

DER212

Ivloc

decbath

Sb

2

AT1

wh

locora

empldec

Hc

1

OTH2

3re

ora

drops

Hc

2

DER1

2NI

Ivloc

decempl

Hh

6

bath

UR1

NI

Ivba

ora

dec

SsTb

4

DER1

OTH26

Ivap

locora

empldec

9

DER1

Ivloc

empl

1

GI6PFE34

Ivse

ora

loc

decempl

4

PFE1

Ivloc

empl

lini

2

PFE3

DER12

NI

Ivro

loc

bath

TbSa

6

UR23

ro

ora

dec

4

AT1

DER3

Ivloc

empl

2

FEM1

ro

ora

syrupdec

Hh

5

GI7

frora

M:dec

1

GI3

Ivloc

M:

infbath

Sb

1

en


Popularname

Use

Part

Appl

ica¬

Preparation

Classifica¬

No.

of

used

tion

tion

resp.

Leonotisnepetaefolia

(L.)

R.

Br.(4

05)

-PFE4

Ivloc

mac

1

Mentha

aff.

arvensis

L.(029)

Hierbabuena

GI459

ap

ro

ora

M:dec

SaTe

13

Mentha

aff.

citrataEhrh.(0

30)

Toronjil,Balsamo

GI45OTH2

Ivro

ora

M.dec

Sa

14

Mentha

aff.

pipe

rita

L.(0

31,045,054)

Menta,Balsamoxiw

GI49PFE4

ap

Ivora

M:mac

inf

dec

SaTaHh

23

Ocimum

basilicum

L.(0

71)

Albahaca

PFE4OTH2

ap

locspi

M:decdrops

16

EYE1

se

con

fresh

Ocimummicranthum

Willd.(033,147)

Xkakaltun,Albahacade

GI2

ju-l

vap

ora

Mdrops

HcSaTa

13

monte

DER2

ap

loc

Mbath

oint

Origanumvulgare

L.(6

38)

Oregano

FEM6

Ivora

dec

1

Plectranthusamboinicus(Lour

)Spreng;

Oregano

cast

illo

,Oregano

OTH8

ju-l

vloc

drops

Hc

8

Syn..Coleusamboinicus

Lour.(0

13)

chino

Rosmarinus

officinalis

L.(589)

Romero

FEM5

Ivora

Mdec

Hh

1

SalviacoccineaJuss.exMurray

(110

)Pezuhade

caballo,Unas

de

caballo,Chaktsits

GI2

NI

ro

ora

mac

Ta

3

SalviamicranthaDesf.(0

25)

Xiax-k'ax,

Chi-k'ak',

DER12

13

ap

loc

Mbath

SaTeHcTb

14

Contrahierba

RES1

GI10

Ni

ora

dec

Satureja

brownei(S

w.)

Briq

.;Sy

n.:

Poieo

PFE4

Ivora

loc

M:decmac

SaHe

15

MicromenabrowneiBenth.

(044

)Scutellaria

aff.gaumenLeonard

(338

)Orozuz,Balsamoxiw

GH

4ap

ora

Mdec

1

LAURACEAE

Perseaamencana

Mill

.(4

53)

On,Aguacate

UR1

2RES4

Ivora

M-decsyrup

TeTa

4

LOASACEAE

Gronoviascandens

L.(3

58)

Laalmuch

PFE1

Ivloc

empl

1

LOGANIACEAE

Spigelia

sp.(5

35)

Lombnzero

GI8

wh

ora

dec

1

MALPIGHIACEAE

BunchosiaswartzianaGnseb.

(142,

173,

Sipche'

GI3PFE4

NI

wh

spi

purifie

6

552)

bath

Byrsonima

crassifoliaKunth

(426

)Chi1,Nance

GI1

DER2

6Iv

ba

ora

Mdecbath

5

MALVACEAE

(024)

Taman-ka'an

PFE4

GI10

se

locora

powempl

2

AbelmoschusesculentusMoench;

Syn.:

Caféchino

AT1

se

loc

powempl

1

Hibiscusesculentus

L.(0

19)

Plantname(AANK#

voucher)

Popularname

Abutilonpermolle

Sweet(0

91)

Gaya

calyptrata

Kunth

(356

)

Gossypium

hirsutumCav.

(086

)

Gossypium

sp.(632,647)

Hibiscusrosa-sinensis

L.(0

67)

Hibiscustubiflorus(Moç&Sessé)DC.

(120,474)

MalvaviscusarboreusCav.

var.arboreus

(140,189,597)

SidaacutaBurm.

f.(3

66)

Sida

aff.

rhombifolia

L.(4

42,588)

MARANTACEAE

Marantaarundmacea

L.(4

64,558)

MARTYNIACEAE

Martyniaannua

L.(4

95)

MELIACEAE

Cedrelaodorata

L.(3

01)

TnchiliaarboreaC.DC.

(503

)Tnchilia

hirta

L.(5

14)

MENISPERMACEAE

Cissampelos

pareira

L.(0

08)

MIMOSACEAE

Acaciaangustissima

Mill

.(378)

Acacia

collinsn

Saff.(0

39)

Sakmisbil,Sakpetmis

Xpup

ul-i

k'

Taman,Algodôn

gris

Chuy-taman

Tulipan

Bisil,Xcampana

ka'ax

Bisi

l-ch

e',

Bisil,

Holol

Chichibe

Pasmarxiw

Chaak

Unade

gato

,Carnavalia,

Sarsapanlla

Cedro

Chobenche'

Chobenche'

Peteltun,Orejade

ratön

Waxim

Subinche'

Acaciasp.(432

,640)

Desmanthus

aff.

depressusHumb.&

Bonpl.ex

Willd.(1

84,287,298)

EnterolobiumcyclocarpumGnseb.

(018

)

Leucaenaleucocephala

(L.)

deWit

(481)

Lysiloma

latisi

liqu

aBenth.(3

80)

Mimosabahamensis

Benth.(3

42)

Ch'imay

Sala

t-ik

',Sibik'xiw,

Sib-

ik",

Sik'ink'ax

Pich

Waxim

Tsalam

Sak-katsim,Katsim

Use

Part

Appl

ica¬

Preparation

Classifica-

No.

of

used

tion

tion

resp.

OTH3

NI

Ivloc

fresh

TnSn

Sb

8

GI3DER2

Ivro

ora

loc

decbath

2

RES

young

frora

M:syrupdec

Te

2

RES1

Ivora

RES4

5Iv

ora

M:dec

2

GI3

Ivfl

loc

M:bath

2

GI2

Nl

ro

ora

macdec

HcSnTn

13

GI3OTH3

re

loc

decbath

2

GI510

9P

ora

Mdec

Hh

8

GI23

rh

ora

ju

2

UR3

trora

dec

1

OTH6

Ivnas

fresh

Ss

6

NI

IvTb

Sn

3

NI

IvTnSn

1

GI3

loc

M-

oath

10

DER2

Ivloc

bath

HITb

1

AT1

DER12

Ivloc

powempl

dec

HhHc

4

GI6DER2

ro

Ivora

loc

dec

2

EYE1

ivcon

drops

4

GI310

NI

Ivloc

Mbath

3

DER1

ivloc

dec

Hc

2

OTH2

ba

loc

empldec

Hh

1

RES4

roba

ora

dec

3

r5Plantname(AANK#voucher)

Popularname

PitheceliobiumalbicansBenth

;Sy

n.:

Havardiaalbicans

Britton&Rose

(166

)

Pitheceliobiumdulce(Roxb.)Benth.(4

99)

Pitheceliobiummangense.;

Syn.

:Chloro-

leuconm.

Britton&Rose(163)

MORACEAE

Brosimum

alicastrumSw

(092

)

Cecropia

obtusifoliaBertol.(0

41)

Chlorophoratinctona(L

)Gaud.(515)

DorsteniacontrajervaL

(330

)

Ficus

cotinifoliaKunth

(020

)

MUSACEAE

Musasp

(539

,541,542,543.544)

MYRSINACEAE

Ardisia

aff.

escallonioidesSchltr.&Cham.

(436)

MYRTACEAE

Calyptranthes

millspaughii

Urban.

(517

)

Eucalyptussp

(613,629)

Eugenia

aff.

capuli

(Schlecht&Cham

)

Berg

(479)

Eugenia

buxifolia(Sw

)Willd.(1

33)

Pimenta

dioica

(L)Merr.;Sy

n..Pimenta

officinalisLmdl.

(023

)Psidiumguajava

L.(2

35)

Chukum

Ts'iuche'

Xiax-ek'

Ox,Ramon

Xk'oochle',Guarumo

Morax

Xkambalhaw

Kopo

',Alamo

Plâtano,Ha'as,Platano

manzano

Xook'num

Eucalyptus

Chaknii

Xhilnich',

Sakloobche'

Nohochpol,Pimientade

Tabasco

Pich

i',Guajaba

Psidiumsartonanum

(Ber

g)Nied

(211

)

NYCTAGINACEAE

Boerhaviasp.

(471

)

Neeapsychotnoides

F.D.Sm.

(274,512)

Pisoniaacuieata

L.(154,249,407)

OLACACEAE

Ximeniaamencana

L.(4

18)

Pichiche'

Chakle'

Xtatsim

Beeb,Unadegato

Nabche',Tsu'tsup

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

DER1

79

ba

loc

bath

HhHc

7

NI

IvSnTb

1

AT1

DER3

Ivloc

M:powempl

1

RES4

5re

ora

drops

9

UR

Ivora

Mdec

5

NI

IvTa

1

GI7FEM3

rh

ora

pow

infdec

HhSaTb

27

RES4

NI

re

ora

drops

4

GI1

ora

Mfreshdec

Hh

RES45

loc

empl

NI

IvSaTi

2

RES45

Ivora

Mpow

inf

2

DER12

EYE3

ba

Ivloc

1

DER2

Ivloc

bath

Tb

3

FEM2

34

frora

M.dec

Hh

14

GI4

Ivloc

GH

frora

dec

fresh

Iv:HhTaTb

34

DER2

6Iv

loc

bath

Safr:Hc

DER2

Ivloc

bath

TaSa

HI

12

DER2

12

loc

Mbath

H

DER2

13

NI

frIv

loc

bath

2

FEM5

so

ora

dec

Tb

8

GI1

2ro

ora

Mdecmac


Popularname

ORCHIDACEAE

CatasetumintegerrimumHook.

(188,302)

Ch'itku'uk

Encyclia

aff.

belizensis(Reichb.

f.)Sc

hltn

.;Xkananikte'

(425

)Oncidiumascendens

Lind

ley(244)

Puts'ubche',Bac

chivo:

Puts'maskab

Oncidiumcarthagenense(J

acq.

)Swartz

U'tsumpek

(245)

Spirantessp.(2

41)

Chiwohk'aak'

OXALIDACEAE

Oxalis

lati

foliaKunth(352,612)

Elel

PALMAE

Cocos

nucifera

L.(650)

Coco

Sabal

sp.(633,648,649)

Guano,

Ka'nal-xa'an

PAPAVERACEAE

Argemonemexicana

L.(026)

Carmesanta

PAPILIONACEAE

(465)

Chaksaal

(345

)Saklooche'

Abrus

precator

ius

L.(001)

Oxo

Aeschynomene

fascicularisCham.&

Salat-ik'

Schlt.(0

63)

Canavaliaensiformis

(L.)

DC.

(513

)Canavalia

Centrosema

sagittatum(Humb.&

Bonp

l.Buy-ak'

ex

Willd.)Malme;

Syn.

:Glycine

sagi

ttat

aHumb.&

Bonpl.ex

Willd.

(128

,137)

Daleacarthagenensis

var.barbata

Azüfrexi

w,Suyk'ak'

(Oer

st.)

Barneby

(125

,151,178)

Desmodium

aff.canumSchinz&

Thell.

Pak'umpak'

(367

)Desmodium

sp.(523)

-

mDiphysacarthagenensisJacq.

(435

)co

Susup,Ts'us'uk

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

DER1

13OTH5

so

loc

M.empi

Hc

7

DER1

bu

loc

1

DER9

Ivloc

empl

1

FEM3

ora

M:dec

1

DER2

7ro

loc

pow

Tb

2

PFE3DER2

Ivora

dec

Ta

2

FEM8

ju-f

rora

fresh

1

FEM349

NI

so

ora

dec

Hh

3

UR3

PFE3

RES2

Ivse

ora

dec

DER2

Ivloc

bath

Hh

1

DER2

Ivloc

dec

1

GI3

Ivloc

bath

Hc

12

DER1

Ivloc

M.powempl

1

NI

IvTbSn

EYE1

AT1

ro

Ivcon

loc

2

DER2

Iv

DER1

Iv

Nl

Iv

ATI

G13

Iv

loc

loc

dropspow

4

empl

1

SnTn

1

M:empl

bath

Te

5

$5Plantname(AANK#voucher)

Popularname

ErythnnastandleyanaKrukoff(3

79)

Chakmolonche'

Indigofera

jamaicensisSpreng.

(360

)Xoxo-ak'

Indigofera

suffruticosa

Mill

.(2

08)

Sujuxiw

Lonchocarpuspunctatusssp.

longistylus

Balche'

(351)

LonchocarpusxuulLundell(1

77)

Xuul

Mucunaprunens

(L.)DC;Syn/

Stizo-

Xpica

lobium

prunens

(L.)

Medic.

(518,549)

NissohafructicosaJacq.(3

43)

Xk'ant'uul

Pachyrnzuserosus

(L.)

Urban

var.

Kup,Jicama

palmatilobus(D

.C.)

Clausen

(346

)Piscidiapiscipul

a(L

.)Sarg.

(123,320)

Ha'abm

PASSIFLORACEAE

PassifloraconaceaeJuss.

(246

)

Passiflorafoetida

L.(4

52)

PHYTOLACCACEAE

PhytolaccaicosandraSims

(388)

Rivinahumilis

L.(089,308)

PIPERACEAE

PiperamalagoL

(098

,105)

POACEAE(GRAMINEAE)

Cymbopogon

citratus(Nees)

Stap

f;Syn.:

Andropogon

citratusHortexDC.

(061

)Lasiacis

ruscifoliaHitchc.&Chase

var.

ruscifoiia(3

33)

Zeamays

L,(363

,575)

POLYGONACEAE

AntigononleptopusHook.&

Arn.

;Syn.:A.

cordatum

Mart.&

Gal

(237

)CoccolobaspicataLundell(5

10)

Coccolobauvifera

L.(5

45)

Xik'sots'

Xpoch

T'eikox

Colario,

Ikiche*

Xpeheche'

Zacatede

limon

Sut

Maïs,Cabellode

elote

Atol

e,Pinole

SanPedro

Bob

Uvademar

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

tion

resp.

EYE2

se

con

pow

3

RES5

UR3

ora

M:dec

Gl3

Ivloc

bath

1

GI2

Ivloc

bath

1

RES5

PFE3

ba

ora

dec

HhTnSs

3

OTH9

NI

cer

PFE134

ro

loc

bath

Jim

5

GI6

frora

mac

2

AT1

ro

Ivloc

empl

1

RES1

24

frro

ora

freshdec

TeHc

3

GI2

RES4

OTH8

Ivlocdrops

1

DER12

frlocfresh

2

DER6

froralocdecbathTe

9

AT1

DER8

rolv

loc

empl

oint

HcSb

Tb3

Ivora

mac

syrup

lb

IvlocdropsfrlocfreshfroralocdecbathTe

ro

Iv

loc

empl

oint

HcSb

IvlocfreshsoapSsIvora

dec

HhTeIvlocorafreshempl

TnSn

froraM:dec

OTH10

NI

IvlocfreshsoapSs

10

RES1

4GI9

Ivora

dec

HhTe

12

DER9

NI

ivlocorafreshempl

TnSn5

UR1

2GI14

froraM:dec

7

RES4

5rofl

ora

dec

1

OTH3

DER2

NI

re

Ivloc

dnoos

TnSn

3

UR1

Ivora

dec

1


Popularname

Gymnopodium

floribundumRolfe(3

72)

Ts'i

ts'i

lche

'

NeomillspaughiaemarginataS.

F.Blake

Sakitsa',Xtastabin

(203)

POLYPODIACEAE

Microgramma

nitida

(J.Sm.)

A.ReedSm.

Tipte'-ak'

(183,303)

PRIMULACEAE

Samolus

ebracteatusKunth

(032

)Tsunya'hi

PUNICACEAE

Punicagranatum

L.(2

72)

Granada

RANUNCULACEAE

Clematis

dioica

L.(520)

Xmexmexib

en

cn

RHAMNACEAE

Colubrinagreg

giS.Watson

var.

yucatanensis(5

00)

Gouania

lupuloidesUrban

(376,417)

KrugiodendronferreumUrban

(498

,571,

578,623)

ROSACEAE

Rosachinenesis

L.(1

30)

RUBIACEAE

Borreriasp.

(470

)Borreria

vert

icil

lata

G.Meyer

(276,608)

ChiococcaalbaHitchc;

Syn.

:C.

racemosaL.

(215,240)

Coffeaarabica

L.(2

70)

HameliapatensJacq.

(199

)MorindayucatanensisGreenman

(113

)

PsychotriamicrodonUrban

(329

)

PsychotriapubescensSw.

(400

)Randia

longil

obaHemsl.

(415

)

Pujuche'

X-om-ak'

Chintok'

Rosa

ychina

Haway

Haway,Haway-k'ak'

Chimes-kas,

Xiax-al'

Café

Ele'kabi,K'anan

Piha

ak',

Pihakam

Xbakalik'

Tschul-keeh

K'ax

Use

Part

Appl

ica-

Prep

arat

ion

Classifica¬

No.

of

used

tion

tion

resp.

RES1

5flro

ora

dec

Ss

3

DER11

AT1

NI

RES1

so

loc

bath

SnTb

3

GI12

wh

ora

dec

HcSnTb

17

DER1

4OTH5

Ivloc

M:empl

GI1

frora

M:fresh

DER2

Ivloc

M:

bath

TaTi

12

12

DER2

Ivloc

1

NI

IvSbTb

4

OTH3

NI

ro

ora

mac

HcTbSn

5

UR1

ba

ora

dec

4

OTH2

wo

loc

empl

RES1

45

Ivora

dec

8

DER12

ap

loc

M:

bath

1

DER2

12

ap

loc

bath

Hh

10

DER2

Ivro

loc

pow

bath

Tb

9

FEM5

se

ora

inf

Tb

1

DER1

2Iv

loc

emplbath

Hh

5

DER9AT1

frlv

loc

empl

HcSb

9

PFE4

Ivloc

M:empl

bath

1

PFE34

Ivloc

mpi

bath

3

OTH9

frsp

ifresh

3

en

CD

Plantname(AANK#

voucher)

Popularname

Use

Part

Appl

ica¬

Preparation

Classifica¬

No.

of

used

tion

tion

resp.

Yuy,Sihun

G19RES1

PFE12

NI

Ivro

loc

decbath

9

Limön

pals,Limönagna

RES4

5ju

ora

drops

HcSaTbTi

27

GI5

Ivdec

dec

Pak'aal,Naranja

agri

aGI47

Ivora

dec

HhSa

18

Lima

GI3

Ivloc

dec

1

Mandarine

Gl7

Ivora

M:dec

1

Naranjadulce,China

GI7

Ivfr

ora

M:decdropsSaTb

13

Cajera

GI1

ro

ora

powmac

Hh

1

Limonaria

RES2

5fl

loc

pow

oint

4

Tamkasche',

Siische'

GI910RES5

ro

ora

linidec

Hh

11

Ruda

PFE4GI1

37

ap

Ivloc

M:macdec

Hc

24

Sinanche',Matade

PFE24

GI10

ro

Ivju

loc

pow

lini

HISb

16

escorpiôn

-

DER2

NI

Ivloc

SaSnTn

3

Sihum

NI

IvTbTnSn

5

Chi'keeh,Caimito

silvestre

GI127

ro

ora

M.powmac

2

Ya',

Zapote,Chicle-zapote

G12

ba

ora

M:dec

HcTa

Ti

32

Zapote

bianco,Sakya'

G11

frora

dec

3

RUTACEAE

CasimiroatetrameriaMillsp.(049)

Citrusaurantiifolia(Christm.)

Swingle

(257

)Citrusaurantium

L.(2

36,253)

Citruslemon

(L.)

Burm.

f.(6

51)

Citrus

reticulataBlanco(2

52)

Citrussinensis

(L.)Osbeck

(247,251)

Citrussp.(236

)

Murrayapaniculata

(L.)

Jack.

(099

)

PilocarpusracemosusVahl(4

54)

Rutachalepensis

L.(0

55)

ZanthoxylumcaribaeumLam.

(368

)

SAPINDACEAE

Allophyloscominia(L)Swartz(389,511)

Sapindussaponaria

L.(5

08)

SAPOTACEAE

ChrysophyllummexicanumBrandegee

(386

)Manilkarazapota

(L.)

Royen;

Syn.:Achras

zapota

L.;Sapotaachras

Mill

.(2

34)

Pouteriacampechiana

(Kunth)Baehni;

Syn.:LucumacampechianaKunth

(108)

Pouteriasapota

H.

E.MooreandSteam;

Syn.:Calocarpummammosum

Pierre

(540

,547)

Pouteriauniloculars(Donn.Smith)Baehni

(619

)SCROPHULARIACEAE

Capraria

biflora

L.(0

97)

Mamey

Zapote

amarillo

Claudiosa,Sak

clav

iosa

,

Chokwil-xiw

GI1

NI

rose

se

ora

dec

GI5OTH8

NI

loc

drops

Hc

Plantname(AANK#

voucher)

Popularname

RusseliasarmentosaJacq.(1

60,340)

SELAGINELLACEAE

Sela

gine

llalo

ngis

pica

taUnderw.

(214

)

SIMAROUBACEAE

AlvaradoaamorphoidesLiebm.

(136

)

SOLANACEAE

CapsicumchinenseJacq.

(265

)

Cestrumnoctumum

L.(0

50)

Datura

aff.

inoxia

Miller(4

58)

Nicotianatabacum

L.(0

82)

Physaliscinerascens(Dunal)

Hitch.(5

02)

Siik

'xiw

,Oxletk'ax

Mooch-tut,

Flordepiedra

Belsinikche',Palode

hormigas

Habanero

Juandenoche

Chaniko,Chamisa

K'uts,

Tabaco

Xpurusi

Solanum

aff.

armentalis

J.Gentry(4

47)

Xsikli-much

en

-vl

Solanumcandidum

Lind

ley(s

pec,

related

toS.hirtumVahl.)

(440

)SolanumerianthumG.Don

f.(334,530)

Solanum

hirtumVahl.(0

90)

Solanumnigrum

L.(2

67,420)

SolanumrudepannumDunal(s

pec.

relatedtoS.torvum)(4

69)

Solanum

sp.(5

83)

Solanum

sp.(5

27)

STERCULIACEAE

Guazuma

ulmifoliaLam.

(250

)

HelicteresbaruensisJacq.

(176

,553)

THEOPHRASTACEAE

Jacquiniaaurantiaca

Ait.

(371)

TILIACEAE

Lueheaspeciosa

Willd.(3

47)

TriumfettasemitrilobaJacq.

(230

)

Triumfetta

aff.

ulmifolia(2

48)

Papera

Xpahhux,Ukuch

kax

Putbalam

Hierbamorax

Xsikli-much

Tomate,

p'ak

'

Xpuh-hi

Pixo

y,Nohoch-pixoy

Tsutup,Suput

Sink'inche'

K'askat

Mul-och

Kambapixoy

Use

Part

Appl

ica¬

Preparation

Classifica¬

No.

of

used

tion

tion

resp.

AT1

DER2

ap

loc

empl

bath

6

UR1

ap

ora

powdec

2

DER2

710

NI

Ivloc

bath

HhSnTe

9

PFE6

frloc

powemp

2

DER1

PFE6

Ivro

loc

M:bathempl

Hc

8

DER2

Ivloc

ointempl

3

DER2

12

Ivloc

M:dec

lini

Hc

5

NI

IvTbSb

2

DER2

Ivloc

M:dec

oint

empl

2

DER2

frloc

empl

2

DER13

NI

Ivloc

emlp

2

AT1

frloc

empl

1

DER113

Ivloc

empl

Hc

5

DER2

Ivloc

bath

1

DER14

ju-l

vloc

drops

1

NI

IvSb

1

FEM5

ba

ora

dec

HhSa

12

OTH11

frsp

ifresh

10

OTH9

spi

amu

Hh

AT1

DER2

Ivloc

empl

2

GI2FEM5

ro

ora

decmac

HhHcSnTn

26

FEM4

5Ivba

ora

dec

HhSa

8

öSPlantname(AANK#voucher)

Popularname

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

TURNERACEAE

Turneradiffusa(Willd.ex

Schu

lt.)

(193

)

URTICACEAE

UreracaracasanaGaud,ex

Griseb.(3

25)

VERBENACEAE

Callicarpa

acuminataRoxb.

(169

)

CornutiapyramidataAiton(0

48)

Durantarepens

L.(222)

Lantanacamara

L.(2

82)

Lippia

albaN.

E.

Br.Ex

Britton&Wilson

(011)

Lippia

dulcisTrev.;

Syn.

:Phyla

scaberrima

(Juss.

ex

Pers.)

Moldenke;

ZapaniascaberrimaJussex

Pers.

(059)

Lipp

iaaf

f.graveolensKunth

(014

,554,

611)

Lipp

iastoechadifoliaKunth

(010

)

Petrea

volubilis

Veil.(003,119)

PrivalappulaceaePers.(4

30)

StachytarphetajamaicensisGardner(027,

521)

Stachytarpheta

sp.(5

36)

VitexgaumeriGreenman

(047

)

VIOLACEAE

Hybanthusthiemei

(F.Donn.Sm.)Morton;

Syn.

:Indiumthiemei

F.Donn.Sm.

(266)

Hybanthusyucatanensis

Millsp.(533,534)

VITACEAE

Cissus

trif

oliata

Lour.(0

43)

Oregano

k'ax

,Oreganode

RES4

Iv

monte

Laal,Or

tiga

,Pica--pica

GI9

PFE1

4Iv

Xpuk

'in,

Puk'im

GH

2so

Xolte'xnuk

RES1

PFE1

3Iv

K'anpokolche'

DER9

NI

fr

Tédemonte

GI4

Iv

Tédelimön

Orozuz

GI4

PFE3GI4

Iv Iv

Oregano

FEM6DER8GI4

ora

loc

ora

loc

loc

ora

ora

ora

ora

dec

Sa

dropsmac

Hh

dec

7

mac

bath

bath

drops

inf

HcTbSa

HcHc

SnTa

HhTn

12

9 2 1

mac

SaHc

10

dec

2

Tédechina

GI4

Ivora

dec

HcHhSa

29

Yochop'tsimin,Comidade

GI3

Ivfl

loc

bath

3

caballo

Xpak'umpak'

AT1

Gi7

Ivloc

empl

bath

2

Verbena,

Iben-xiw

PFE1FEM4

Ivloc

M:

lini

5

Malva,Verbenaxiw

NI

IvSs

1

Ya'axnik

PFE2

Ivloc

bath

Sa

1

Xpluxionxiw

OTH2

PFE4

ap

locora

dec

Hc

5

Sakbakelkam

AT1

NI

ro

loc

empl

TnSn

1

Cruzoj

oxiw

GI3

Ivloc

bath

14


Popularname

Vitistr

ifol

iataHumb.&

Bonp

l.exRoemer

Tabkanih

&Schultes(5

24,531)

WINTERACEAE

Drimys

winteriForster&

Forster

f.(2

56)

Canela

ycuyo

ZINGIBERACEAE

Zingiber

officinaleRoscoe

(370

)Em

ojib

le,(J

engi

bre)

(566)

Katku'ut

(574)

Muela

(476)

Oxo

k'ax

(157)

Ts'u

ts'u

pche'

(570)

Tusik'

(563)

Xiek'

in

enCD

Use

Part

Appl

ica-

Preparation

Classifica-

No.

of

used

tion

tion

resp.

NI

ivTnSn

G!

ba

ora

po'w

inf

HhSaTb

1

GI7

rh

ora

M:powmac

HhSaSs

8

UR2

ro

ora

M:dec

1

DER2

Ivloc

M:bath

1

GI3

frloc

M:

bath

1

EYE2

Ivcon

drops

1

RES4

5ap

ora

M:INF

1

DER2

34

Ivloc

empl

Hc

1


2.5 Informants

Table 2 6 List of healers and midwives participating the ethnobotanical project

Name Specialisation Location

Claudia Uc Cahun Parfera hie>rbatera Chikindzonot

Abundio Chan Kauil H-men, hiei'batero partero Chikindzonot

Gregono Cen Uc H men, hier•batero Chikindzonot

Florencio Hoi Chan Hierbatero Chikindzonot

Juanita Pech Balam Parfera Chikindzonot

Maria Pastora Kauil Pech Hieratera Chikindzonot

Juventina Kauil Diaz Parlera Chikindzonot

Claudia Naidelfia Noh Pech Parfera Chikindzonot

Fehpa Moo Kahun Fartera Chikindzonot

Jose Carlos Chan Kauil Curandera Chikindzonot

Vicente de Paul Moo Pat Hierbatero, sobadoro EkpedzWilfndo Poot Moo Herbatero Ekpedz

Regulo Moo Dzib Hierbatero EkpedzRosa Maria Dzib Kauil Hierbatera EkpedzGumersindo Cocom Chan Hierbatero EkpedzAntolina Poot Kauil Parfera EkpedzCeliana Moo Pat Parfera EkpedzJustino Dzib Kauil Hierbatero EkpedzNarcisa Poot Poot Hierbatera EkpedzVicente de Paul Dzib Dzul Curandero, hierbatero EkpedzJuana Paula Moo Dzib Parfera EkpedzSinla Pat Cocom Hierbatera EkpedzJuan Santos Dzib Moo Hierbatero EkpedzNarcisa Poot Poot Hierbatera EkpedzJuana Paula Moo Dzib Parfera EkpedzValentina Pat Hue Parfera Ekpedz

Eligio Pat Carnal, H-men EkpedzAndrea Moo Pat Parfera EkpedzVicente Zim Poot Huesero, curandero EkpedzVicente de Paul Dzib Dzul Curandero, hierbatero EkpedzVacunda Dzib Dzul Parfera, curandera Ekpedz

Felipe Pat Chan Hierbatero EkpedzJustino Dzib Kauil Hierbatero EkpedzPedro Acantara Pat Cocom Hierbatero EkpedzJuan Santos Dzib Moo Hierbatero EkpedzPorfina Cocom Pech Parfera EkpedzJose Jsabel May Poot Curandero Xcocmil

Rajelia Poot Poot Hierbatera Xcocmil

Anselmo Chulm Chi Curandero Thiosuco

Emihana Parfera, Hierbatera Chichimila

Age of the informants 27 - 96 years

60


2.6 Gardens of medicinal plants

The development of a medicinal plant garden in Chikindzonot and Ekpedz was

planned as a mark of gratitude towards the healers and midwives for their co¬

operation in the project. It was also meant as a sign of support of the use of

medicinal plants. These intentions were successful in some cases in others not.

For instance, the building of two medicinal gardens, one close to the clinic run by

the SSA (Secretana de Salud y Asistencia) in Chikindzonot and the other one in

the garden of the clinic of the traditional healers in Ekpedz, was not continuously

cared for due to organizational and political problems. On the other hand, the

translator, influenced by our work together, created a small medicinal plant garden

close to her home and she was (and still is) invited to the healer organization of the

INI (Instituto Nacional Indigenista). The species of medicinal garden in the CICY

(Centra de Investigacion Cientifica de Yucatan, Mérida) were labeled based on the

information of the healers and midwives of the study region.

Species of the botanical garden in Chikindzonot and Ekpedz:

Aloe vera; Bidens squarrosa; Capraria biflora; Chenopodium ambrosioides;

Cnidoscolus aconitifolius spp. aconitifolius; Crossopetalum gaumeri; Cymbopogon

citratus; Dorstenia contrajerva; Guazuma ulmifolia; Hamelia patens; Malmea

depressa; Morinda yucatanensis; Ocimum micranthum; Piscidia piscipula; Ruellia

nudiflora; Tecoma stans.

Species of the botanical garden in Mérida:

Aloe vera; Anredera vesicaria; Bursera simaruba; Cecropia obtusifolia; Citrus

aurantiifolia; Cymbopogon citratus; Hamelia patens; Malmea depressa; Mentha aff.

piperita; Pilocarpus racemosus; Psidium guajava; Punica granatum; Selaginella

longispicata; Tamarindus indica; Tithonia diversifolia.

61


2.7 Selection of plant species for their biological evaluations

Several plant species were screened in various bioassays. The goal of this

evaluation was to better understand the use of the medicinal plants and their

pharmacological effects. The selection of the species was based on:

their importance as a remedy among the Yucatec Maya of the study region (the

numbers of the documented use reports)

the healers consensus concerning the plant use and preparation

the endemic occurrence of the species on the Yucatan Peninsula or the original

occurrence of the species in America

the lack of phytochemical and/or pharmacological investigations of the species

the analysis of ethnobotanical literature regarding the species

The results of the biological and pharmacological evaluations are shown in chapter

5 and 6.

62

Publication I

Medical ethnobotany of the Yucatec Maya:

Healers' consensus as a quantitative criterion

Anita Ankli1, Otto Sticher1 and Michael Heinrich2

1) Department of Pharmacy, Swiss Federal Institute of Technology (ETH) Zurich,

Winterthurerstr. 190, CH-8057 Zürich, Switzerland

2) Institute of Pharmaceutical Biology, Schänzlestr. 1, Albert-Ludwigs-University,

D-79104 Freiburg, Germany

Published in

Economic Botany 53 (1999) 144-160

Publication i

Abstract

There is an urgent need to obtain information on the relative importance of a taxon

used medicinally as compared to others within a culture. This was achieved

through a documentation of the current indigenous medical uses of 320 species in

three Yucatec Maya communities during 18 months of fieldwork. The 1,549

indivdual reports documented were divided into nine groups, which classify

indigenous uses. The frequency of usage of the individual plants reported was

employed in the analysis of the ethnobotanical importance of the respective taxa.

Species cited more frequently in a group of indigenous uses are regarded to be of

greater ethnobotanical importance than those cited only by a few informants. In

order to obtain information on possible biological, pharmacological and

toxicological effects of some particularly important species, the scientific literature

on these taxa was evaluated systematically.The study is the basis for

phytochemical and pharmacological evaluations of the traditional uses.

Key Words: Yucatec Maya traditional medicine, indigenous medicinal plants,

ethnobotany, evaluation of indigenous uses, quantitative method, Yucatan

(Mexico).

64

Publication I

Etnobotanica medica de los Mayas Yucatecos: consenso de curanderos

como criterio cuantitativo

Se considéra esencial la documentacion de la importancia relativa que un taxon de

uso medicinal tiene, en comparaciön con otros taxones dentro de una misma

cultura. Con este propösito se realize un estudio etnobotânico de 18 meses,

investigando el uso de 320 especies en très comunidades Mayas del Estado de

Yucatan (Mexico). Se documentaron 1,549 usos indigenas, que se clasificaron en

9 grupos. Se utilize el numéro de usos indigenas para determinar la importancia

relativa de cada especie; asi, las especies médicinales que fueron citadas con

mayor frecuencia se consideran las de mayor importancia, mientras que las

especies citadas con menor frecuencia son las de menor importancia. Para

evaluar los usos indigenas se obtuvo informacion sobre efectos biolögicos,

farmacologicos y toxicologicos de las especies, através de una revision

sistemâtica de la literatura cientifica. Este estudio es la base para la selecciön de

plantas que se evaluaran en estudios fitoqui'micos y farmacologicos.

65

Publication I

Introduction

In recent years we and others called attention to the lack of information on the

relative importance of a medicinal plant (or other useful plant) within a culture and

the need for comparing the uses of plants interculturally (Heinrich, Rimpler and

Antonio B. 1992, cf. Etkin 1994, Moerman 1996). A constructive method to obtain

such information is the quantification of indigenous uses (Phillips 1996) which is

appropriate when the relative importance of each use is similar, as with

pharmaceutical preparations such as medicinal plants used for different types of

illnesses. Accordingly, this paper is the third in a series on Mexican indigenous

medicinal plants (Frei, Sticher and Heinrich 1998 on the Isthmus Sierra Zapotecs,

Oaxaca; Weimann and Heinrich 1997 on the Nahua of the Sierra de Zongolica,

Veracruz). An additional goal of these studies has been the selection of plants for

phytochemical and biological/pharmacological studies (Bork et al. 1997, Kato et al.

1996).

Therefore all three of our ethnobotanical studies use similar methodologies (see

also Methods): (1) Specialists in medicinal plants (for example, healers, midwives,

herbalists) were interviewed during 14-18 months of fieldwork and the use-reports

of each informant recorded. (2) The use of the plants is grouped into 9-10

categories.

The principal groups are similar in all three studies: gastrointestinal disorders,

illnesses of the skin (mostly infections and subsequent inflammatory reactions),

respiratory disorders, gynecological (and andrological) conditions. Since there exist

ethnobotanical differences between the three ethnic groups, 5 - 6 additional groups

were formed, which are only used in one or two of the studies, for example, plants

used for bites and stings of poisonous animals (only Maya), opthalmological

illnesses (Maya and Zapotec), and culture bound syndromes (Nahua and

Zapotecs).

This comparative method facilitates the selection of medicinal plants for phyto¬

chemical and biological/pharmacological studies, and is useful in determining the

ethnobotanical importance of a particular plant in contrast with others in the same

use category (Heinrich et al., 1998). There have been several other approaches to

66

Publication I

establish quantitative criteria for the relative ethnobotanical importance of plants

(Berlin and Berlin 1996, Friedman et al. 1986, Johns, Kokwar and Kimanani 1990,

Phillips 1996). The method of Berlin and Berlin is of special relevance to ours. We

both used a similar approach, however they interviewed the general population and

thus recorded and evaluated an enormous set of positive responses (30,000,

Berlin and Berlin 1996: 81-82). Their method requires a considerable investment in

research funds and personnel. The method presented here is tailored to allow for

the assessment of the relative cultural, medical and, consequently, also the socio-

economical importance of plants employed by medical specialists in an ethnic

group and is feasible with a lower input of resources.

A detailed study of Yucatec Mayan medicinal plant use seemed to be of particular

relevance. Traditional forms of treating illness among the Yucatec Maya of Mexico

who still use locally available plant resources are of considerable importance. Their

medical system and knowledge is without doubt a vital part of their culture

(Redfield and Villa R. 1990 [orig. 1934], Roys 1931, Standley 1930, Steggerda and

Korsch 1943). Although many aspects of Mayan ethnobotany have been

addressed in detail, such as ethnoecology (Herrera 1994, Humphries 1993, Terän

and Rasmussen 1994) and plant nomenclature (Barrera M., Barrera V. and Lopez

F. 1976, Sosa V. et al. 1985), only a few reports exist on the medicinal plants

currently used. For example, Arnason et al. (1980) studied a Mayan community in

Belize, Comerford (1996) a Mayan community in lowland Guatemala, and Alcorn

(1984) the Huastec Maya. In addition, some theses have been written on the topic

and booklets are distributed locally or regionally (Cardeha V. 1985, Pulido S. and

Serralta P. 1993; cf. Mendita and Arno 198, Morton 1981, Argueta V. 1994).

Our study thus has a twofold purpose: (1) to systematically record the use of

medicinal plants in communities of the Yucatec Maya in Mexico, and (2) to form the

basis for comparative studies on Mexican Indian medicinal plant use.

67

Publication I

Background

Yucatan and the Maya

The peninsula of Yucatan forms the easternmost part of Mexico which is divided

politically into three states; Yucatan, Quintana Roo and Campeche. The northern

parts of Guatemala and Belize are also part of the peninsula and have a high

percentage of Lowland Mayan speakers (Fig. 1). The peninsula is an enormous

plateau, made of limestone, with an average altitude of less than 100 m a s I. No

surface rivers exist in the northern part of the peninsula. The most important water

sources are cenotes (natural sink holes formed by the collapse of the limestone

surface over the ground). The annual rainfall is highest in the southeast (1,300 -

1,400 mm) and diminishes towards the north and northwest (400 mm). The natural

vegetation in the southeast is tropical rainforest, while in the extreme northeast it is

low tropical deciduous forest. Due to the low latitude, the climate is warm and in

the southeast, humid.

For more than a millennium the civilizations of the Ancient Maya flourished on the

peninsula and adjoining regions (Koehler 1990). Currently 600,000 persons, or 36

% of the total population of the peninsula, are mono- or bilingual speakers of Maya

(Pfeiler 1995). Even though the influence of outside forces has been enormous, the

Yucatec Maya still retain a large number of ancient traditions and have opposed

the cultural dominance of the Spaniards and the surrounding national Mexican

culture. The so-called "caste war" of the last century which involved the Maya, is

just one example of the resulting conflicts (Orosa D. 1991).

Yucatec Maya language belongs to the mayance (or mayoide) subfamily of

Macropenutian. Maya vowels and consonants are generally pronounced as in

Spanish. A glottal stop ['] is known and glottalized consonants are frequent. In this

article, Maya words are transcribed after Barrera M., Barrera V. and Lopez F.

(1976).

68

Publication I

The Communities

This study was conducted in the communities of Chikindzonot (20° 20' degrees

latitude north, 88° 29' degrees west, 30 - 40 m a s I) and the neighboring Ekpedz

and Xcocmil south of the city of Valladolid in the southeastern part of the state of

Yucatan. These communities were selected because of a high rate of speakers of

Yucatec Maya, the well known cultural conservatism in this region and the high

number of healers known (Orosa D. 1991, Tuz, Valladolid, pers. comm.)

Figure 1. Region of fieldwork

Average annual temperature in the area is 25.7 °C and it has an average annual

precipitation of 1,220 mm. According to Duch (1988) the climatic subtype is Aw

1"(x')(i')g, i.e., a hot subhumid climate with rain from May to October (980 mm) and

little thermal oscillation throughout the year. The vegetation is characterized as a

median semideciduous forest with an average height of 10 - 20 m. 50 to 75 % of

the species remain deciduous during the dry season. Typical tree species include

Acacia pennatula (Schlecht. & Cham.) Benth., Bursera simaruba Sarg.,

Caesalpinia gaumeri Greenman, Cochlospermum vitifolium Willd. ex Spreng.,

Enterlobium cyclocarpum Griseb, Guazuma ulmifolia Lam., Gymnopodium

floribundum Rolfe, Mimosa bahamensis Benth. and Vitex gaumeri Greenman

(Salvador F. and Espejel C.1994).

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Publication I

The communities of Chikindzonot and Ekpedz have 1,500 and 800 inhabitants,

respectively (INEGI 1990). The whole municipio of Chikindzonot has 2,750

inhabitants. 56% of the persons older than 15 yrs. are literate and one third of

those older than 5 yrs. are monolingual speakers of Maya and the remainder

bilinguals. The economy is based on subsistence agriculture (mostly maize, beans

and squash) and on the production of honey, fruit (watermelon and citrus fruits)

and cattle breeding. Hunting is still practiced regularly, especially by younger men.

Handicraft articles (hammocks and huipiles - female dress) are sold in the market

of Valladolid. No detailed anthropological monograph on the Maya of this area is

available, but the community of Chan Kom, which was first studied by and Villa R.

1990 [orig. 1934], is only 27 km to the north.

Health and healing

According to the unpublished data of the local health authorities and our surveys it

is apparent that gastrointestinal disorders (frequently diarrhea and - in younger

children - as a result thereof, dehydration) and respiratory illnesses are major

health problems. Infected wounds and other inflammatory skin diseases are also

common. Bites from poisonous snakes (e.g., tsab - cascabel - Crotalus durissus L.)

are feared, yet only a few cases have been recorded in recent years. Chronic and

infectious eye diseases are frequent. Diabetes is now considered an important

health problem by the local health authorities, and informants often claim to suffer

from this illness. Most of the medicinal plants sold in the markets of Valladolid and

Mérida promise to alleviate diabetes or to function as diuretics.

The best known group of healers are h-men (Maya for knowledgable healer or

prayer maker), who are not only healers but also specialists in religious rites and

who perform ceremonies addressing the rain-god to ask for protection of the milpa

or the community. He or she is the owner of a sastun, a stone used for divining.

Midwives and herbateros (specialist in medicinal plants) form another group of

healers. Those of the latter group are generally proficient in treating broken bones

and thus work as hueseros. Massages are given by another group of healers - the

sobadores - and by midwives. All these groups of healers make extensive use of

70

Publication I

medicinal plants. Some groups largely use these plants as part of empirical

medications, while others, in particular, the h-men, also use the plants for ritual

purposes.

An outpatient clinic run by the SSA (Secretaria de Salud y Asistencia) and staffed

with a pasante (a medical student in her or his last year of training) and a mestizo

nurse provides biomedical health care, but for most conditions people still prefer

the Mayan healers. In 1993/1994 the pasante (a woman) was called only once to

help during a delivery.

Methods

Ethnomedical and ethnobotanical data were collected mostly in the two

communities of Chikindzonot and Ekpedz from February 1994 until May 1995 and

September/October 1996, however the data from the second stay are not included

here. Specialists in medicinal plants and/or healers of the different regions were

interviewed. This paper is based on structured and unstructured interviews with 40

healers. Twelve healers aged between 29 and 71, who represent all the groups of

specialists mentioned above were interviewed frequently and contributed a large

share of the information presented here. Together with informants we collected

plant materials that were cited as medicinal. During meetings of groups of

indigenous healers, we conducted unstructured interviews on the medicinal plants

and methods of treatment. We thus obtained information on the use(s),

preparation(s), plant parts used, application(s) and properties of the plants as well

as descriptions of illnesses and treatments, which we compiled into ethnobotanical

data sheets.

Voucher specimens were collected and are deposited at the Herbarium of the

"Centra de Investigacion Cientifica de Yucatan" (CICY) in Mérida, the National

Herbarium of Mexico (MEXU), the Instituto Nacional Indigenista (INI) in Valladolid

(both Yucatan), the ETH Zurich (ZT) and the Institut für Pharmazeutische Biologie

in Freiburg, Germany (collection numbers A. Ankli, AANK1 - 540). Plants were

identified by comparison with authentic specimens and in some cases with the

assistance of specialists at CICY and MEXU.

71

Publication I

Reports of ethnobotanical uses were documented for each plant. The healers were

asked to demonstrate the plants which they currently use or which they had used.

In order to analyze the data, the plants were arranged into nine classes of

indigenous use and for each class the data were quantified by adding up the

individual reports on the uses of each plant. Species were then ranked according

to the number of reports of use. Plants in these groups which were cited as

medicinal by four (in case of the gastrointestinal illnesses by five) or more

informants are presented here, A literature search in BIOSIS PREVIEW (and in

selected cases, also in NAPRALERT and Chemical Abstracts) was performed for

the plants most frequently cited in order to obtain data on phytochemistry and on

biological and pharmacological effects of the respective taxa.

Results and discussion

Illness according to the Yucatec Maya may be classified as being humorally "hot"

or "cold" (Redfield and Villa R. 1990 [orig. 1934]). imbalance of the body, for

example, caused by consuming something "cold'' when a person is in a "hot" state

may lead to illness. Diarrhea, for example, is considered to be a "cold" illness,

while dysentery (bloody diarrhea) is classified as "hot". This classificatory system is

also central during and after childbirth. After giving birth, a woman is considered to

be in a "cold" state and therefore is not allowed to eat certain food such as pork,

beef and foods which contain much grease. The "hot/cold" classification of the

Yucatec Maya was recorded by Redfield and Villa R. 1990 [orig. 1934] and was

reported in the sixteenth century (Lopez A. and Viesca T. 1984, Villa R. 1981).

Nonetheless, the humoral system does not encompass the entire medical system

of the people we worked with. Many uses of plants are based on personal

experience or oral transmission within the family.

Illness may be caused by "wind" (i.e., by bad air which enters a weak person's

body), witchcraft or nocturnal birds and bats. Younger informants now refer to

microbes as another causative agent of disease. When a person is treated, ritual

and empirical plant use are closely connected. If the illness requires it, the healer

72

Publication

includes a ritual cleansing ceremony (santigua or limpia). The healer asks God for

help and for the permission to cure.

In this paper we concentrate on the medicinal use of plants among the Yucatec

Maya. We document a total of 1,549 individual responses to queries concerning

320 plant species. These responses were grouped into nine categories of

indigenous use (Fig. 2). We constructed all major and most of the lesser categories

to coincide with indigenous classifications (cf. Berlin and Berlin 1996). A few of the

smaller categories are combinations in order to accommodate, for example, the

various forms of chronic and acute "pain" and illnesses associated with a rise of

body temperature. One residual group ("other uses") is also included. Yucatec

Maya healers are well acquainted with the causes and detectable signs

(symptoms) of illness. It is no surprise then that the groups relate to the organs

affected during a certain illness (gastrointestinal tract, skin, respiratory system).

Figure 2. Quantitative ethnobotanical analysis of the nine groups which classify the

indigenous use reports (total number of species: 320; n = 1549 use reports; AT =

counteract bites and strings of venomous animals; DER = dermatological

conditions; EYE = illnesses of the eyes; FEM = women's medicines; Gl =

gastrointestinal disorders; PFE = illnesses associated with pain and/or fever; OTH

= other uses; RES = respiratory illnesses; UR = urological problems).

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Publication I

The largest number of species and individual use-reports (Fig. 2) were obtained for

gastrointestinal disorders. This group of disorders was second only to

dermatological illnesses in the studies of the Isthmus Sierra Zapotecs and Nahua

of the Sierra de Zongolica (Frei, Sticher and Heinrich 1998., Weimann and Heinrich

1997) in terms of the number of use-reports and among the Zapotecs in terms of

the number of species used. Among the Nahua it was the group with the largest

number of species recorded. Dermatological problems are the second largest

group among the Yucatec Maya. It is noteworthy that these two groups of uses are

prominent in all studies mentioned. Also, respiratory illnesses yield a large number

of use reports and 87 taxa - the third largest number. Of particular importance

according to the Yucatec Maya are plants used in the treatment of bites from

venomous animals, especially snakes (4.8% of all use-reports). In the following,

the principal species used by the Yucatec Maya for each of the 9 groups are

discussed. The ethnobotanical data are summarized in Tables 1 - 7.

Gastrointestinal disorders In this group 147 species with 476 use reports were

documented. Diarrhea is a frequent condition, especially in children. Other

illnesses, included in this group, are gastrointestinal cramps, vomiting and mal de

ojo (evil eye, no Yucatec Maya term used). The latter is an illness caused by a

person, who looks at a child with a so called "strong glance". A drunken person, a

women during menstruation and people born on Tuesday or Friday are particularly

likely to cause this illness. Its signs are various gastrointestinal symptoms,

particularly "green" diarrhea, gastrointestinal cramps and vomiting. To cure the ill

person, the one who has caused the illness has to embrace the patient or to show

her/his care in another way. Additionally herbal preparations are used. Another

important illness is tip'te' (cirro). It is an "organ" reported to sit below the umbilicus.

If a person has eaten inadequate food or after having carried something heavy the

tip'te' palpitates and is dislocated (cf. Berlin and Jara 1993 on a similar concept in

Highland Chiapas). Treatment consists mostly in circular massages around the

navel and in drinking a decoction of tip'te' ak {Microgramma nitida).

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Publication I

The three members of the genus Mentha - M. piperita, M. citrata (leaves/aerial

parts) and M. arvensis (roots and leaves), which are widely used to treat vomiting

and to a lesser degree diarrhea (Table 1), are widely known and effective

antispasmodics of European origin (Haensel et al. 1993). Abrus precatorius is used

only externally for diarrhea and the evil eye in the form of baths and therefore is not

discussed further. Manilkara zapota (mostly bark, also roots and fruits) is employed

for diarrhea and is best known for yielding chicle - a latex rich in polyisoprenes.

The bark is also known to be rich in tannins (Hegnauer 1973:296), but no data on

their structure have been published (Hegnauer 1990: 498). While a huge number

of publications on the production and use of the latex are available, no

pharmacological information relating to the indigenous uses is available. The

relevant reports on the genus Aristolochia were recently summarized (Weimann

and Heinrich 1997). No data are available to substantiate the Mayan uses for

diarrhea. Callicarpa acuminata (shoots) is a Yucatec Maya remedy for mal de ojo

associated with green diarrhea and dysentery. The genus Callicarpa is one of the

few in the Verbenaceae (s.l.) in which no iridoids could be found (Falk 1992).

Instead diterpenoids as well as triterpenoids and flavonoids were reported from the

genus (Glasby 1991). Whether these exert any relevant pharmacological effects is

unknown. Lippia alba, L. stochadifolia (leaves) and related species are used in

many different cultures for gastrointestinal disorders, the Yucatec Maya value the

species to induce vomiting. Many species of this genus are rich in essential oil

{Lippia alba > 1 %; Hegnauer 1973:668) and thus may act as carminatives.

Roots of Dorstenia contrajerva are used for a variety of gastrointestinal conditions,

chiefly for stomach ache, "air in the stomach" and gastrointestinal cramps. Similar

uses of D. drako are reported from the Istmo Sierra Zapotecs (Frei, Sticher and

Heinrich, 1998). Furanocoumarins are known from D. contrajerva (Terreaux et al.

1994), but no data are available on its pharmacological effects. Pimenta dioica is

rich in essential oil (with approx. 35 % eugenol and 40 - 45 % eugenol

methylether), has a wide use for unspecified gastrointestinal problems (List and

Hoerhammer 1977) and may well be an adequate treatment of "air in the stomach".

A central nervous depressant effect of the aqueous (and to a lesser degree of the

75

Publication I

ethanolic) leaf extract applied i.v. was recently shown as part of a hippocratic

screening (Suârez et al. 1997). Psidium guajava (leaves and sometimes also root)

is used for diarrhea and dysentery. These uses are also described by Aguilar et al.

(1994), Weimann and Heinrich (1997) and Giron et al. (1991). The leaves are

mentioned in Farmacopea Mexicana as astringents. Pharmacological tests on the

leaves showed activity against E. coli and spasmolytic effects (Berlin and Berlin,

1994, Weimann and Heinrich 1997) which reportedly are due to a calcium-

antagonist effect of quercetin glycosides (Morales et al., 1994). All plant parts are

rich in phenols, especially hydrolyzable tannins and proanthocyanidins (Okuda et

al. 1987). Teloxys ambrosioides (syn.: Chenopodium ambrosioides) is another

widely used Mesoamerican species employed as anti-emetic, antiparasitic and

antidiarrhetic. Ascaridol is a well-known monoterpene with antiparasitic effects, but

also with undesirable side effects (Hegnauer 1964:421).

Artemisia ludoviciana ssp. mexicana (leaves) has a long tradition of use in

Mesoamerica. The Yucatec Maya value this plant for treating vomiting. It is rich in

sesquiterpene lactones, but no data on anti-emetic effects are available (Bork et al.

1996, Heinrich 1996). Ruta chalepensis (leaves) is known from many cultures and

widely used as antispasmodic. It is prominent for its content of a large number of

different alkaloids and furanocoumarins, is a common abortifacient and has severe

side effects (Haensel et al. 1993, 1994, Heinrich 1989).

76

TABLE

1SPECIESUSEDFORGASTROINTESTINALDISORDERS

(lis

ted

inorderofdecLrvngfrequency

ofuse)

Number

Vouche-'

Species

Family

PlantPart

MayaName

MainUses

of

AANK

Uses

Mentha

aff

piperita

L

indet

AbrusprecatonusL

Manilkarazapota

(L)vRoyen

AristolochiamaximaJacq

Call

icar

paacuminata

Kunth

Lippia

stoechadifolia

(L)Kunth

Teloxysambrosioides

(L)WA

Weber


Mentha

aff

citrataEhrh

Pimentadioica(L

)Merr

PsidiumguajavaL

Artemisia

ludoviciana

Nutt

ssp

mexicana

(Wil

ld)Keck

Cissampelos

pareiraL

Citrusaurant

i um

L

Lipp

iaalba

(Mil

l)N

EBrex

Britton&

Wilson

MalvaviscusarboreusCav

var

arboreus

RutachalepensisL

BidenssquarrosaLess

Cissus

trifol

iata

(L)Lour

Citrussinensis

(L)Osbeck

Labiatae

Iv,tw,ap

ba,wo

Legummosae

se,

Iv,

fl

Sapotaceae

ba,

rt,fr

Anstolochiaceae

rt,wp

Menta,Balsamoxiw

Canelaycuyo

Oxo

Ya

,Zapote

Chiclezapote

Wahkohak

,Guaco

cast

illo

Verbenaceae

sh

Puk

in

Verbenaceae

kTedechina

Chenopodiaceae

Iv,tw,

rtEpazote,Apazote

Moraceae

rhKambalhaw

Labiatae

VTo

ronj

il

Myrtaceae

Iv,se

PimientadeTabasco

Myrtaceae

rt,

fr,

IvPichi

,Guayava

Compositae

kSi

isim

Menispermaceae

Iv

Rutaceae

W

Peteltun,Orejade

raton

Pak

al,Naranjaagna

Verbenaceae

IvTedelimon

Malvaceae

rt,iv

Bisil,

Holol

Rutaceae

Compositae

Iv,ap

V

Ruda

Ya'xkan-ak'

Vitaceae

Rutaceae

k Iv,se,

pe

fr,

-fr

C^uz

ojo

China,Naranjadulce

vomiting,

parasites

inthestomach

diar

rhea

,dysentery,

a,r

inthestomach

malde

ojo,greendiarrhea

diarrhea,dysentery

diarrhea,dysentery,

air

>nthestomach,

pasma*

malde

ojo,greendiarrhei

dysentery

vomiting

para

site

s,pasma,

vomiting

air

inthestomach

vomitingpasma

air

inthestomach

diarrhea

vomiting,diarrhea

malde

ojo,greendiarrhea

yellowdiarrnca,

air

inthestomach

of

youngerchildren

vomiting

vomiting

dysentery

malde

ojo,mal

viento,diarrhea

malde

ojo,

green

diar

rhea

,vomiting

malde

ojo,

greendiarrhea

air

inthestomach

ofyoungerchildren

18

031

12

256

12

001

12

234

11

350

11

169

11

010

10

025

10

330

10

030

10

023

10

235

9012

9008

9236

9011

9140

9055

8121

8043

8247

-J

-si

co

TriumfettasemitrilobaJacq

ZingiberofficinaleRoscoea

Mentha

aff

arvensisL

OcimummicranthumWilld

PunicagranatumL

mdet

Hylocereus

undatusjxL

)Britton&Rose

Microgramma

nitida

(JSm

)A

Reed

Sm

Piscidiapi

scipula(L

)Sarg

Bauhiniaherrerae

(Britton

&Rose)

Standley&Steyerm

Citrusaurantiifolia(Chnstm

)Swingle

Crossopetalumgaumeri(Loes)L

unde

lI

HeliotropiumangiospermumMurray

PimpmellaanisumL

ZanthoxylumcanbaeumLam

Tihaceae

rtMul-och

dysentery

8230

Zingiberaceae

rhGengibre

indi

gest

ion,

air

inthestomach

8370

Labiatae

rt,ap

Hierbabuena

vomiting,parasites

7029

Labiatae

Iv,ju

-lv

Kakalîun

dysentery

7033

Punicaceae

fr,

IvGranada

yellow

diarrhea

7272

-

Iv,ap

Pasmarxiw

stomachache,cramps,pasma

6422

Cactaceae

kPitahaya

dysentery

6427

Polypodiaceae

wp

Tip'

te'-ak

cirro

tip

te

,stomachache

6183

Leguminosae

sh

Ha

abin

malde

ojo,dysentery

6123

Leguminosae

kKibix,

Ts'ulubtok

malde

ojo,

whitedysentery

5124

Rutaceae

kLimon

airinthestomach

5257

Celastraceae

rtViperolnegro

diarrhea,dysentery

5038

Boraginaceae

IvNema

ax

dysentery,malde

ojo

5053

Apiaceae

sc

Anisengranos

air

inthestomach

5255

Rutaceae

rt,

ivSinanche

mal

vien

to,diarrhea

5368

*

pasma(r)

Oftenmentionedasacause

forgastrointestinal

illnessesand

isus

uall

yassociatedwith

drinksheorshe

gets

ill,

ap=

aerial

part

,ba^bark,

fl=flowers,

fr=

fruits,

ju-fr=juiceof

fruits,

ju-l

=peel

of

frui

t,pet=

peta

ls,

re=

resin,

rh=rhizome,

rt=

root

,se=seeds,

sh=

shoot,

tw=

twig,

stomchacheWhen

apersonma

hotstatetakescold

j=juiceofle

aves

,la=

latex(milky),

Iv=

leav

es,

pe-f

rwo=wood,

wp=whole

plan

t

Publication I

Dermatological Conditions In this group 302 use reports referre to 150 species.

Small cuts, infections and other skin problems are frequent and generally treated at

once with plants (150 species with 302 use reports) that are readily available.

Other frequent health problems are pimples (saa sak' winkli), warts and scabies.

The rootstock of Anredera vesicaria is used as a wound dressing and for infections

(Table 2). Practically no chemical information is available on the genus. Only a

retrochalcone was isolated form another species of this genus (Calzado et al.

1990). Calea urticifolia, another remedy for pimples, contains sesquiterpene

lactones and is an effective inhibitor of the transcription factor NF-kB, which

controls genes responsible for the inflammatory responses of the body (Borges de

C. et al. 1981, Bork et al. 1997), Crossopetalum gaumeri is known to have

antibacterial and cytotoxic effects (see Plants Used to Counteract Venomous

Animal Bites and Stings). Diospyros anisandra is employed in the treatment of

pimples and scabies. Of the approximately 240 species in this genus, only D. kaki

and D. mollis were investigated in greater detail by various groups. Triterpenes

(lupeol, betulin, betulinic acid) and naphtoquinones are widely distributed in the

genus. D. anisandra has yet to be studied (Hegnauer 1989:403-407). The leaves of

Ocimum micranthum and Salvia micrantha are both used for infections and

inflammation of the skin. Neither species has been studied in detail, but O.

micranthum especially merits further study (Heinrich 1992). Species of both genera

are rich in essential oil; in Salvia spp. neo-clerodane diterpenoids were frequently

reported as bioactive compounds (Rodriguez-Hahn et al. 1995). The use of

Kalanchoë intégra (leaves) as a topical anti-inflammatory seems to be based on

the succulent nature of the leaves. Whether the plant exerts any specific anti¬

inflammatory effect is unknown. Leaves of Psidium sartorianum are used for the

same conditions. While no specific data for this taxon are available, species of this

genus are rich in hydrolyzable tannins and proanthocyanidins in all plant parts

(Okuda et al. 1987), and thus may have some anti-infective effects. No

pharmacological data are available to substantiate the claims in the case of

Samolus ebracteatus.

79

coo

TABLE

2SPECIESUSEDFORDERMATOLOGICALCONDITIONS

Species

Family

Plant

Part

MayaName

Pnmulaceae

kTs

unya

iSBasellaceae

rtKaa'xicheel

Crassulaceae

kBelladona

Compositae

kKaxikin

Celastraceae

rt.l

vVi

pero

lnegro

Ebenaceae

kKakalche',Kanan

Labiatae

Iv,ap

Kakaltun

Myrtaceae

IvPichiche1

Labiatae

Iv,ap

Ya'xkax

Rubiaceae

ap

Haway

Orchidaceae

kCh

itkuuk

Euphorbiaceae

la.l

vEk'balam,Xikmburro

Rubiaceae

kElelkabil,

Kanan

Euphorbiaceae

kXul-im-il

Euphorbiaceae

kXilkin,Kambaikiche'

Apocynaceae

laUtsupek'

Simaroubaceae

kBelsinikche'

Leguminosae

kAzufre-xiw

Rubiaceae

fr,

IvPiha

ak'

Phytolaccaceae

frT'elkox

Myrtaceae

kPi

chi'

,Guayaba

Punicaceae

kGranada

Leguminosae

kSaal,Saalche'

Cucurbitaceae

kChakmotz-ak',Hoyke

MainUses

Number

Voucher

of

AANK

Uses

9032

8196

8016

6153

6038

6134

6033

6211

6025

5276

5188

5205

5199

5357

5088

5190

4136

4125

4113

4388

4235

4272

4084

4036

Samolus

ebracteatusKunth

Anredera

vesicariaGaertner

f

Kalanchoë

inté

graKuntze

Calea

urti

cifoliaMi

llsp

varyucatanensis

Wussow,Urb&

Sullivan

Crossopetalum

gaumeri(Loes

)Lundell

DiospyrosanisandraBlake

OcimummicranthumWilld

Psidiumsartonanum

(Ber

gius

)Nied

Salvia

micranthaVahl

Borreria

verticillata

(L)G

Mey

CatasetumintegernmumHook

CrotonperaeruginosusCroizat

HameliapatensJacq

PhyllanthusacuminatusVahl

PhyllanthusmicrandrusMeuII

Arg

Tabernaemontana

amyg

dali

foli

aJacq

AlvaradoaamorphoidesLiebm

Daleaca

rthagenensis

var

barbata

(Oerst

'

Barneby

MonndayucatanensisGreenman

Phytolacca

icosandra(L

)Sims

PsidiumguajavaL

PunicagranatumL

Senna

villosa

(Mil

l)Irwin&Barneby

Sicydiumtamnifolium(Kunth)Cogn

wounds,

inflammation

inflammation

tumor,

inflammation

litt

le,redpimples

x-onob

inflammation

oftheneck

itch

ingpimples

usan

pimples,whitespots

siklikmuch'

measles

k'aa'ahko

lebill

scabies

pimples

x-onob

smal

l,whitepimple

bigpimples

chukum

pimples

inflammation,redpimples,

wounds

inflammation

oftheni

pple

s

wound,

infe

ctio

n,inflammation

of

theneck

warts

itch

ingpimples

pimples

wounds

measles

pimples

saasak

wmkli

measleskaaah

kolebil

hard

,li

ttlep'mples

AbbreviationseeTable

1.

Publication I

Illnesses Associated with Pain and/or Fever This category (see Table 3) is

rather diverse and includes plants (112 species 204 with use reports) that are used

for chronic or acute pain and fever. These ailments are commonly treated with

external appplications of species known to be rich in essential oils (e.g. Ruta

chalepensis, leaves, aerial parts, locally known as an external remedy for

headache, see above). Illnesses associated with fevers are generally treated with

baths. Pain is generally treated with plasters and other external applications.

Satureja brownei [syn.: Micromeria brownei {Sw.) Benth.; leaves] is used in the

form of baths for headaches and has a diverse range of uses among many other

peoples in Central America, the Caribbean and Mexico (Heinrich 1992). Essential

oil and methylated flavones are found in taxa from this genus (Tomas B., Husein

and Gil 1988). The ethanolic leaf extract showed inhibitory activity against

Staphylococcus aureus and Streptococcus pyogenes in a screening of 68

Guatemalan plants (Caceres et al. 1991), but no relevant pharmacological data are

available on the species used and therefore it is not possible to evaluate the

Mayan use. Zanthoxylum caribaeum (roots) is widely known among the Yucatec

Maya as a cure to rheumatism, headache, and "mal viento". Many species of this

large genus with approximately 200 species have been studied phytochemically

and/or pharmacologcially in detail. Characteristic are benzyl isochinolin alkaloids,

lignans, coumarins and unusual amides (Hegnauer 1990: 449-455). Since the plant

is employed externally in ritual cleaning ceremonies, an evaluation of the

indigenous claims goes beyond the scope of this study.

Ocimum basilicum is a well known species rich in essential oil (0.5 - 1.5 %)

introduced into numerous indigenous medical systems of the Americas (Heinrich

1992). Analgesic or antibacterial effects would be of relevance for the Yucatec

Maya use in case of toothache, and there is some information on antibacterial

effects of the essential oil (Heinrich 1989, List and Hoerhammer 1977). Leaves of

Bursera simaruba are exclusively employed externally for fever (baths). The genus

is rich in essential oils, Triterpenes, lignans and procyanidies are also found

(Khalid 1983), but are probably of no relevance to the use discussed here. Its

81

Publication I

external use for fever is known from other parts of Mexico and seems to be

partially based on the aromatic smell of the leaves, resin and bark (Heinrich 1989).

Respiratory Illnesses The species in this group (87 with 177 use reports) are

locally used for cough, bronchitis and "asthma" (Table 4). The latter illness is

described as a longlasting wheezing, caused by winds called tus ik'. In

phytotherapy in many European countries plants rich in essential oils or in

polysaccharides are regarded as useful therapies for the first two conditions (Ph.

Helv. 8). Only Cymbopogon citratus (leaves) is known to be rich in essential oil

(usually employed as a spasmolytic). The genus Ehretia is rich in rosmarinic acid,

which has antiviral, antihistamine release and anti-inflammatory effects (Kuhnt et

al. 1995, Simpol et al. 1994). Other important Mayan medicinal taxa are Croton

lundellii (leaves), Euphorbia ptercineura (latex), Rosa chinensis (leaves) and

Turnera diffusa (leaves). Some species might have moderate antibiotic effects, but

there are no data to further substantiate the Mayan claims. No chemical data on

these taxa are available that would substantiate the indigenous claims, but some

species {Croton lundellii, Euphorbia ptercineura) might have severe side effects,

because of the presence of phorbole esters.

82

TABLE

3.SPECIESUSED

INTHETREATMENTOFILLNESSESASSOCIATEDWITHPAINORFEVER

NumberVoucher

Species

Family

Plant

Part

MayaName

MainUses

ofAANK

Uses

Satureja

brownei(S

w.)

Briq.

Labiatae

Rutachalepensis

L.

Rutaceae

ZanthoxylumcaribaeumLam.

Rutaceae

Ocimumbasilicum

L.Labiatae

Burserasimaruba

(L.)

Sarg

.Burseraceae

Blxaorellana

L.Bixaceae

BunchosiaswartzianaGriseb.

Malpighiaceae

Cestrumnocturnum

L.

Solanaceae

Ehretia

tini

foli

aL.

Boraginaceae

Hybanthusthiemei

(F.Donn.Sm.)Morton

Violaceae

UreracaracasanaGriseb.

Urticaceae

Iv,ap

Poleo

headache

Iv,ap

Ruda

headache,

trembling

rt,Iv

Sinanche'

ma!

vien

to,feverrheumatism

ap

Albahaca

headache,toothache

Iv,re

Chakah

feverwithacoldbody:

k'ilkab

sh

Kiwi,K'uxub,Achiote

feve

r,trembling

ofbabies

ap,tw

Sipche'

headache,malde

ojo,

fever

kJuandenoche

feverwithacoldbody:

k'ilkab

Iv,ba

Beek,

Roble

fever

ap

Fluxion'xiw

toothache

kLaal,Or

tiga

rheumatism

8044

8055

8368

7071

6042

4233

4142

4050

4021

4266

4325

TABLE

4.SPECIESUSEDFORRESPIRATORYILLNESSES

NumberVoucher

Species

Family

Plant

Part

MayaName

MainUses

of

AANK

Uses

CDco

Croton

lundelliiStandley

Cymbopogon

citratus(Nees)Stapf

Ehretia

tini

foli

aL.

EuphorbiaptercineuraA.Berger

Rosachinensis

L.

Turnera

diffusa

(Willd.exSchultes)

Bauhiniadivahcata

L.

Citrusaurantiifolia(Christm.)

Swingle

Gossypiumhlrsutum

L..

Pseudobombax

ellipticum

(Kunth)Dugand

Piscidiapisc

ipul

a(L.)

Sarg

.Cornutiapyramidata

L.

Murrayapa

nicu

lata

Jacq

.

Euphorbiaceae

Iv

Gramineae

Iv

Boraginaceae

ba,

Iv

Euphorbiaceae

la

Rosaceae

Iv

Turneraceae

k

Leguminosae

Iv,

fl

Rutaceae

Iv,ju

-fr

Malvaceae

Iv,

fr,

fl

Bombacaceae

Iv,ba

Leguminosae

sh

Verbenaceae

Iv

Rutaceae

fl,tw

Kok-ché

Zacatede

limön

Beeb,

Roble

Xmuch

kok

Rosa

Oreganodemonte

Mayvaca,Patadevaca

Limön

pais

Chuytaman,Algodon

Amabola,

Xk'unche'

Ha'abin

Xolte'xnuk

Limonaria

asthma:

tus

ik',

bron

chit

is,cough

asthma,cough,

catarrh

cough,asthma

asthma,cough

cough

cough,

bronchitis

asthma,cough,

bronchitis

cough,asthma,

bronchitis

cough,asthma:

tus

ik'_

asthma,cough,

bronchitis

cough,asthma

feve

r,asthma

asthma,

bronchitis

7040

7061

7021

7034

7130

6193

5007

5257

5086

5275

4123

4048

4099


1.

Publication I

Gynecological Uses Table 5 shows the most frequently mentioned species out of

74 species with 129 use reports. Plants used during delivery are the most

prominent group in this category. As described above the categorization into "hot"

and "cold" illness and remedies is culturally important. Infertility of the women is

regarded as a cold illness and consequently "hot" remedies (e.g. Pluchea

symphytifolia) are prescribed. Infertility is considered an important problem by the

healers.

Bark and leaves of Guazuma ulmifolia are used to relieve the pain of childbirth. It is

one of the best known plants in the two communities and has practically no other

uses. Among the Lowland Mixe it is used as a remedy for diarrhea and also for

pain in the uterus and vaginal hemorrhages (Heinrich 1989), among the Isthmus

Sierra Zapotecs for diarrhea and fever (Frei, Sticher and Heinrich, 1998).

Polymeric proanthocyanidins are common and antisecretoric effects on cholera

induced colonic secretion were reported (Hoer, Rimpler and Heinrich 1995). No

data are available to validate the therapeutic claims of the Yucatec Maya. Pluchea

symphytifolia (leaves) is mostly used to "warm up the womb", for "irregular

menstruation" and uterine spasms. According to the Yucatec Maya the preparation

of the remedy is essential for the therapeutic effect. If an abortive effect is desired,

the remedy has to be drunk while it is still hot. For the other therapeutic uses the

remedy is drunk when it has cooled down. While some data on the biological

effects of the plants and on biologically active caffeoylquinic acids are available

(Scholz, Heinrich and Hunkler 1994), no information validates the traditional Mayan

uses. No data are available which support the reported use for relieving pain of

Pisonia aculeata.

Plants Used to Counteract Venomous Animal Bites and Stings In this group 44

species with 76 use reports were documented. Most of the plants are applied

topically on the wound caused by a snake or scorpion. Many persons state that the

treatment of snake bites requires 16 species, but no informant listed that many

taxa. Crossopetalum gaumeri is the most frequently mentioned species in this

group. All informants report that immediately after the bite of a snake one should

84

Publication I

chew a piece of the drug. The powder drug is also applied externally and/or orally

as a decoction. Triterpenes of the oleanan, lupan amd friedelan type are frequently

reported in the Celastraceae and also in the genus. Characteristic for the family are

chinoid pigments - the celastroids - which are derived from friedelanes. These

compounds are reported to have antibacterial and cytotoxic effects (Hegnauer

1989:223). Anredera vesicaria and Urechitis andrieuxii are wound dressings. No

data on antivenomous effects of any one of these species are available.

Urological Problems In Table 7, 44 species with 66 use reports are summarized.

"Kidney trouble" is mentioned most frequently, yet it does not represent a specific

condition. Many of the plants may act as diuretics, the Yucatec Maya term for the

illness - k'aluix - is generally explained as "the patient can't pass the urine". Also

included in this group is "diabetes". The Yucatec Maya consider plants to be

effective in the treatment of the latter illness if the plants act as diuretics. Malmea

depressa - the most popular treatment of "kidney trouble" among the Yucatec

Maya - was recently investigated phytochemically in detail for the first time and

revealed the presence of phenylpropanoids (Jimenez A. et al.1996). No data to

substantiate the indigenous claims are available.

Eye Remedies In this group 39 use reports referred to 27 species. Inflammations

and disturbing, long lasting spots on the surface of the eye (buy) are frequently

treated with herbal remedies. Often drops prepared from the leaf-sap of various

plants are used and applied topically. No plant stands out as particularly more

important than the others: Ocimum basilicum L, Chamaechsta glandulosa Greene

and Desmanthus spp. are mentioned three times each, Euphorbia hirta L. and E.

heterophylla Desf. two times. In the case of 0. basilicum the mucilaginous seeds

are used for the treatment. No attempts were made to validate the indigenous

uses.

85

Co

CD

TABLE

5.WOMEN'SMEDICINES

NumberVoucher

Species

Fami

lyPlant

Part

MayaName

MainUses

of

AANK

Uses

Guazuma

ulmifoliaLam.

Sterculiaceae

ba,

ivNohoch

pixoy

Pisoniaaculeata

L.Nyctaginaceae

sh

Beeb,Unadegato

Plucheasymp

hyti

foli

a(M

ill.)

Gillis

Compositae

kChalche'

Pimenta

dioica

(L.)

Merr.

Myrtaceae

Iv,

frNukuch

pool

,Pimientade

Tabasco


Aristolochiaceae

itWahk'oh

ak'

indet.

Ulmaceae

ba

Kamba

pixoy

Plmpinella

anisum

L.Apiaceae

se

Anisengranos

Zuelanlaguidonia

(Sw.

)Britton&

Flacourtiaceae

itBotox,Tamay

Millsp.

childbirth,abortion*

childbirth,abortion

desire

ofhaving

achild,

pasmo0,

abortion

pain

,co

lic,

problems

ofmenstruation,pasmo,

bath

ofvagina

problems

ofmenstruation,pasmo

childbirth,abortion

childbirth

desire

ofhavinga

child,pasmo

12

250

7154

7009

6023

5350

5248

4255

4375

*

The

inductionorprevention

ofabortiondependson

thedosage

(high,

low)

and

ofthepreparation

(hot,co

ld);

"painduring

menstruation,darkblood,

infe

rtil

ity.


1.

TABLE

6.SPECIESUSEDFORINJURIESCAUSEDBYVENOMOUSANIMALS

Species

Family

PlantPart

MayaName

MainUses

Number

of

Uses

Voucher

AANK

Crossopetalumgaumeri(Loes.)Lundell

Anredera

vesicaria_Gaertner

f.

UrechitesandrieuxiiMuell.Arg.

Celastraceae

Basellaceae

Apocynaceae

rt,iv

rt rt,Iv

Vipe

rolnegro

Kaa'xiche'el

Vipe

rolbejuco

snakebite

snakebite,

snakebite,

wound

inflammation

12 6 5

038

196

466


1.

TABLE

7.SPECIESUSEDFORUROLOGICALPROBLEMS

NumberVoucher

Species

Family

Plant

Part

MayaName

MainUses

of

AANK

Uses

Malmeadepressa

(Bâillon)R.E.Fries

Annonaceae

Chromolaenaodorata

(L.)

R.M.King&

H.Rob.

Compositae

Bauhiniadivaricata

L.Leguminosae

Cecropia

obtusifolia

Bertol.

Moraceae

Parmentieraaculeata(Kunth)Seemann

Bignoniaceae

rtElemuy

diur

etic

,kidneytr

oubl

e,kidney

stones

rtTok'aban

diabetes,kidney

trou

ble,

urine

does

notpass:

k'alwix

Iv,

rtMayvaca

kindney

trouble

Iv,

rtK'

ooch

le',

Guarumbo

diabetes

rt,fr

Kat

diabetes,

urinedoes

notpass:

k'alwix

8161

6339

4007

4041

4135


1.

c»

Publication I

Other Uses The uses summarized in this group (53 species with 80 use reports)

are very diverse. But only two plants stand out as being of some importance. In

most cases it is not possible or meaningful to evaluate the therapeutic claims.

Piper amalago (6 use reports) is popularly used to fight dandruff and split hair tips.

No attempt was made to ascertain the therapeutic value. The rootstock of

Anredera vesicaria is used for broken bones as a hard gypsum like plaster

bandage.

Conclusion

This study clearly demonstrates that medicinal plants still are an important natural

resource for the Yucatec Maya of Chikindzonot, Ekpedz and Xcocmil. However,

biomedical pharmaceuticals are becoming increasingly popular. Such

pharmaceuticals are of less importance in the area we studied as compared to

other Yucatec Mayan communities. No detailed study on these forms of treatment

in the Yucatec Maya area exists (van der Geest, Reynolds and Harden 1996).

Most of the species recorded in this study have been reported as useful medicinals

in other regions of Central America, Mexico and the Caribbean, but contrary to

these studies we have presented data that allow the evaluation of the relative

importance of a particular species in the medical system of the Yucatec Maya. The

method also is useful for other culturally important plant products (e.g. dyes). It is

based on interviews conducted by a single researcher, with the help of a field

assistant or translator. In our previous studies with the Zapotecs and Nahua, we

documented 3,600 and 800 individual use-reports, respectively. (Frei, Sticher and

Heinrich 1998, Weimann and Heinrich 1997). These use reports yield relevant

information on the intracultural importance of a specific plant as compared to other

taxa. In a subsequent analysis the three ethnobotanical studies conducted with a

similar methodology will be compared.

The quantitative data have to be seen in comparison to other types of

ethnobotanical information. Garden plants are generally more important in the

medical system of the Yucatec Maya than plants collected outside the community.

Many informants also proudly show (newly) introduced taxa (esp. Mentha spp.,

88

Publication I

also Citrus spp., Lippia alba). Consequently, in order to foment the indigenous use

of medicinal plants a medicinal plant garden is being built up in cooperation with

two of the three communities. Other types of information relating to the cultural

importance of a plant can be drawn from the classification of plants in the

indigenous system (the humoral system or systems based on organoleptic

properties of plants; Heinrich n.d). Such a discussion goes beyond the scope of

this paper.

Based on estimates by Bye (1993:707) 15 % or 5000 species of the Mexican flora

are used medicinally. The method presented here contributes to the selection of

the most important ones, which then can be studied pharmacologically,

toxicologically and phytochemically in order to evaluate the indigenous claims

(Robineau and Soejarto 1996). These studies will also help to revalue the

indigenous uses of medicinal plants. Plants which seem to be of particular

relevance for such studies are, for example, Anredera vesicaria, Dorstenia

contrajerva, Diospyros anisandra, Guazuma ulmifolia and Zanthoxylum caribaeum.

Studies on some of these plants are consequently under way.

An earlier version of this paper focusing on plants used in gastrointestinal medicine

was presented at the symposium "Plants for Food and Medicine", July 1 - 6, 1996,

Imperial College, London at the joint meeting of the Society for Economic Botany

and the International Society for Ethnopharmacology.

Acknowledgments

This research would not have been possible without the collaboration of the

healers, midwives and other inhabitants of the communities we worked in, who are

the traditional keepers of this knowledge. The botanical identification at CICY and

MEXU was performed in collaboration with the numerous specialists of this

institution. Particularly we would like to thank Dra. I. Olmsted, J. Granados, P.

Sirna and J.C. Tejön of CICY as well as 0. Tellez, R. Lira and Dr. M. Sousa of

MEXU. This research owes a lot to the continued support of Prof. H. Rimpler

(Freiburg), and to the help of Dra. B. Pfeiler (UADY, Mérida), Dr. Tuz (INI,

Valladolid) and Dr. Baltisberger (Zürich). We are very grateful to Dr. John Plant

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(Freiburg) for critically revising the manuscript. Financial support by SDC (Swiss

Agency for Development and Cooperation, Berne, Switzerland) and the SANW

(Swiss Academy of Natural Sciences) is gratefully acknowledged.

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Yucatec Maya medicinal plants versus nonmedicinal

plants: Indigenous characterization and selection

Anita Ankli1, Otto Sticher1 and Michael Heinrich1,2

1) Department of Pharmacy, Swiss Federal Institute of Technology (ETH) Zurich,

Winterthurerstr. 190, CH-8057 Zürich, Switzerland

2) On leave from: Institut für Pharmazeutische Biologie, Albert-Ludwigs-Universität,

Schänzlestr. 1, D-79104 Freiburg, Germany,

Fax.:+49-761-203-2803

Published in

Human Ecology 27 (1999) 557-580

Publication II

Abstract

Medicinal plants are an important part of the environment as it is perceived by

Mexican indigenous groups. The aim of this study, which was conducted over a

period of 18 months in three Yucatec Mayan communities, is to better understand

the selection criteria for medicinal plants. An important group of selection criteria

are the flavor and aroma of plants. The absence of smell or taste indicates that the

taxon has no potential medical value. Medicinal plants are more often considered

to be sweet or aromatic (to smell good) or astringent, while a similar percentage of

medicinal and nonmedicinal plants are considered bitter, spicy, acidic, or bad

smelling. The relationship between the ethnobotanical data obtained for the

individual plants and the secondary plant products (natural products) prominent in

each species is specifically addressed in this paper. It shows that an understanding

of the indigenous concepts used to distinguish medicinal from nonmedicinal

species has considerable heuristic value.

KEY WORDS: Indigenous knowledge; medicinal plants; nonmedicinal plants;

traditional medicine; ethnobotany; plant selection criteria; taste; smell; hot-cold

classification; Yucatec Maya; Yucatan (Mexico).

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Desde el punto de vista de los grupos indigenas Mexicanos, las plantas

médicinales son una parte relevante del entorno natural donde estos se

desarrollan. El objetivo de este estudio se centra en el entendimiento del o los

criterios que el grupo indigena Maya Yucateco utiiiza para seleccionar una planta

como medicinal. El trabajo se realize durante 18 meses en très comunidades

Mayas Yucatecas. Cuando se compararon las plantas médicinales contra las

plantas no-medicinales se encontre que las caracteristicas de sabor y olor son un

criterio para la seleccion de una planta como medicinal. Asi, la ausencia de sabor

o olor indica un potencial valor medicinal. Se encontre que las plantas médicinales

son consideradas frecuentemente como dulces, aromâticas (con olor agradable) 6

astringentes, siendo para el caso de las plantas no-medicinales las caracteristicas

de amargo, picante, acido y con olor agradable los aspectos que constituyen el

criterio de selecciön.

Se analiza de manera especi'fica la relaciön entre los datos etnobotânicos

obtenidos para cada planta y los productos naturales secundarios de las mismas;

ésto, con el objetivo de mostrar que el entendimiento de los conceptos indigenas

usados para distinguir las especies de plantas médicinales de las no-medicinales

es de un considerable valor heristico.

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Introduction

Impressive collections of documented plants used in indigenous medical systems

are available (for the Americas see, for example, Aguilar et al., 1994; Argueta V.,

1994; Heinrich, 1996; Moerman, 1998; Morton, 1981), but the ethnobotanical study

of medicinal plants has largely remained descriptive, and the process and rationale

for the use of plants as medicine has not been explored in detail (Brett and

Heinrich, 1998). Researchers in the field have generally been more interested

towards the practical application of ethnobotanical information in Western

biomedicine or its uses in primary health care1 (Balick and Cox, 1996; Robineau

and Soejarto, 1996). Among the most noteworthy exceptions are the work of Etkin

(e.g. 1994), Moerman (1996), and Johns (1990).

On the other hand, theoretically informed ethnobotanical studies have made major

contributions to related fields such as ethnoecology (Alcorn, 1984; Balée, 1994;

Ellen and Fukui 1996), cognitive anthropology, ethnoscience (Berlin 1992), and the

study of humoral classification of such disparate phenomena like food, types of

illnesses and plants (Foster, 1994). Foster's study is particularly relevant in the

context of this paper. Some authors have systematically explored the hot-cold

concept and its role in indigenous medicine and diet (Foster, 1994), but little

information is available on botanically identified species and their classification in

this system (cf. Messer, 1991). Also, we have recently criticized the hot-cold

system as too narrow to explain plant use (Brett and Heinrich, 1998). The selection

of plants as medicine based on their taste and smell seems to be important, but

hitherto little explored in many cultures (Brett and Heinrich, 1998 and references

therein; Crellin and Philpott, 1997). That taste and smell are particularly important

criteria for characterizing medicinal plants has been shown in ethnobotanical

1 Such approaches are exemplified by many of the articles published in journals such as the Journal of Ethnopharmacology,

Fitoterapia, Economic Botany and Pharmaceutical Biology (formerly International Journal of Pharmacology).

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studies with the Tzeltal and Mixe (Brett, 1998; Heinrich, 1998). Those two studies

did not, however, look at the differences in the people's taste and smell perceptions

of medicinal versus nonmedicinal plants, the specific focus of this paper. The

cultural reasons for selecting a plant as a medicinal one, the cultural processes

which allow the selection of new medicinal plants, the forms of transmission of this

knowledge, and the management of culturally important plants in the environment

(e.g. by growing them in the house yard or sparing a plant during the cleaning of an

area; Heinrich, 1997) are pertinent topics for ethnobotanical research on medicinal

plants.

The Maya of the Yucatan peninsula (Mexico) are a particularly relevant example,

since plants are an integral and important part of their indigenous culture and since

they have strongly resisted outside influences. Traditional treatments for illness

among the Maya of the Yucatan peninsula, who still use locally available plant, are

of considerable importance. Detailed studies of their medical system and

knowledge (Redfield and Villa R., 1990) and of many aspects of their ethnobotany,

including ethnoecology (Herrera O, 1994; Terân and Rasmussen, 1994) and plant

nomenclature (Barrera et al., 1976; Sosa et al., 1985) are available. Only a few

reports address currently used medicinal plants (cf. references cited in Ankli et el.,

1999). In this paper we analyze the Yucatec Mayan2 criteria for distinguishing

between medicinal and nonmedicinal plants. The wealth of ethnobotanical

information of the Yucatec Mayan healers as well as the intra- and intercultural

variations in medicinal plant knowledge has been described and analyzed before

(Ankli et al., 1999; Heinrich étal., 1998). In this paper, the hot-cold classification

and the classification system based on taste and smell among Yucatec Maya are

2Unless stated otherwise the term "Yucatec May a" is used throughout this paper to refer to the inhabitants of three

communities south of Valladolid (see Background), since no common denominator exists for these three communities. If we

make reference to the Yucatec Maya in general, we use the term Maya of the Yucatan peninsula.

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examined to discover how the Maya distinguish between medicinal and

nonmedicinal plants.

The Study Area and the people

Yucatan and the Maya

The land of the lowland Maya stretches over most of the peninsula of Yucatan,

which contains the easternmost part of Mexico and the northern parts of

Guatemala and Belize. The peninsula is an enormous limestone plateau; its

highest altitude barely reaches 400 ms above sea level (Hernandez, 1985). No

surface rivers run through the northern part of the peninsula; the most important

water sources are cenotes (natural sinkholes formed by the collapse of the

limestone surface over the ground). The annual rainfall is greatest in the southeast

(1,300 - 1,400 mm) and diminishes towards the north and northwest to 400 mm.

The southeast portion is tropical rainforest, and in the extreme northeast low

tropical deciduous forest. The latitude and the adjoining warm sea make the

climate warm and humid.

Yucatec Maya language belongs to the Mayance (or Mayoide) subfamily of

Macropenutian. Maya vowels and consonants are generally pronounced as in

Spanish. A glottal stop ['] is used, and glottalized consonants are frequent.

Currently 600,000 persons, or 36 % of the total population of the peninsula, are

mono- or bilingual speakers of Maya (Pfeiler, 1995). In this article, Maya words are

transcribed after Barrera et al. (1991).

This study was conducted, in the communities of Chikindzonot and the neigh¬

boring Ekpedz and Xcocmil, south of the city of Valladolid in the southeastern part

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of the state of Yucatan. Average annual temperature in the area is 25.7 °C and the

average annual precipitation 1,220 mm. It is a hot, subhumid climate that has rain

from May to October (980 mm) and little temperature variation throughout the year

(Duch, 1988). The vegetation is characterized as a median semideciduous forest

with an average height of 10 - 20 m. Some 50 % to 75 % of the species remain

deciduous during the dry season (Salvador and Espejel, 1994). The communities

of Chikindzonot and Ekpedz have 1,500 and 800 inhabitants, respectively (INEGI,

1990) and the whole municipio of Chikindzonot has 2,750 inhabitants (Fig.1). Fifty-

six percent of the people over 15 years of age are literate and a third of those over

five years are monolingual speakers of Maya, the rest being bilingual. The

economy is based on subsistence agriculture (maize, beans, and squash) and on

the raising of honey, citrus fruits, watermelons and cattle.

Fig. 1. Place of field study.

The most important group of healers are h-men, who are not only healers but also

specialists in religious rites and who perform ceremonies asking the rain god for

protection for the milpa (com field) or the community. He or she is the owner of a

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sastun, a stone used for divining. Midwives {ah-k'ax tuch ahal: person who cuts

the navel string) and herbalists {ts'a ts'ak xiw: person who gives medicinal plants)

form another group who are generally proficient in treating broken bones.

Massages are given by another group of healers - the masseur {yet)- and

midwives3. While all these groups of healers make extensive use of medicinal

plants, some use them largely as part of phytotherapeutic preparations while

others, in particular, the h-men, also use the plants for ritual purposes.

No detailed anthropological monograph on the Maya of this area is available, but

the community of Chan Kom, which was first studied in the 1930s by Redfield and

Villa R. (1990) is only 27 km to the north.

Methods

Ethnomedical and ethnobotanical data were collected from February 1994 until

May 1995 and in September/October 1996, mainly in the two villages of

Chikindzonot and Ekpedz. The information was gathered from structured and

unstructured interviews with 40 traditional healers and midwives. Twelve healers

aged 40 - 71 years were interviewed frequently and contributed much of the

information presented here. Ten of these were between 40 and 50 years of age,

and all actively practice their healing art. All twelve consider themselves herbalists,

and most of them also work as h-men (2), masseurs (5), midwives (4) or bone

setters (3). Generally, the midwives also work as masseurs. Six healers are men

and six women.

Together with informants, we collected voucher specimens and additional material

of medicinal taxa. During meetings of groups of indigenous healers, we conducted

unstructured interviews on the medicinal plants and methods of treatment. We

3The Maya terms for the various groups of empirical healers generally are descriptive.

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thus obtained information on the use(s), preparation(s), plant parts used,

application(s) and properties of the plants as well as descriptions of illnesses and

treatments, which were compiled into ethnobotanical data sheets.

Reports of ethnobotanical uses were documented for each plant. The healers were

asked to indicate which taxa they were currently using or which ones they had

used. For each species, information on the plant part(s) used, their preparation and

application, and the Maya uses, plus data on the indigenous classification of the

species, were recorded. To analyze the data, the use reports for a species were

arranged into nine groups based on symptoms or application techniques

mentioned by the healers (Ankli et ah, 1999; Heinrich et al., 1998). For each group,

the data were quantified, adding up the individual reports on the uses of each

plant. They were then ranked according to the number of reports of use (see Ankli

et ai, 1999).

To distinguish between medicinal and nonmedicinal plants we looked at plants

considered by a single informant (generally a healer) to be medicinal and

compared them to those plants the same person considered not useful as

medicine. After having completed the documentation of medicinal plants of the

communities, individual healers were asked to select a maximum of 10

ethnobotanical taxa that in their opinion had no medical value. The healers were

next asked about the taste and smell, the humoral, and other properties of the

taxa. The informants generally were unfamilier with the taste and smell of taxa they

did not use. Therefore, they were tasted by the informant, the translator, and the

key investigator. The healer then gave his opinion on the taste and smell properties

of each taxon. Voucher specimens were collected and are deposited at the

National Herbarium of Mexico (MEXU), the Herbarium of the Centra de

Investigacion Cientifica de Yucatan (CICY) in Mérida, the Instituto Nacional

Indigenista (INI) in Valladolid, Yucatan, the ETH Zurich (ZT), and the Institut für

105

Publication II

Pharmazeutische Biologie in Freiburg, Germany (collection numbers A. Ankli 1 -

540). They were identified by comparison with authentic specimens and in some

cases with the assistance of specialists at CICY and MEXU.

The selection of medicinal plants

The informants gave various reasons why a plant was considered to be a

medicine. For many informants, the traditional knowledge passed on from one

generation to the next was a key to deciding why they used a certain plant. Most

of them were taught by an experienced healer {h-men) or by elderly relatives. In

the initial interviews on medicinal plants, a traditional healer reported that good-

smelling plants were useful against stomach ache, bitter-tasting ones against skin

problems, and sweet plants to strengthening the body and the blood. The

reasoning of other healers was not always that a plant was a medicine because it

has a certain taste or smell property but often that the plant was used for an illness

and that it was bitter, astringent or aromatic, etc. The informants did not recall

whether the information on the plants' characteristics was also transmitted to them,

but it seems likely that they learned both what plants to used and, implicitly the

reasons for their use. Other information was obtained through dreaming about

useful plants and subsequently testing the effect of these plants in treatment.

Some plants were selected because they showed similarities to a certain illness or

the diseased body or organ. The flower of Matelea yucatanensis (Standi.)

Woodson (Asclepiadaceae) resembles the navel. Therefore, a single flower is used

against pain of the navel of the babies and for trembling of babies. Because of its

spines, which "hold back the fetus", the aerial part of the cactus pitaya4

4 To facilitate the reading of the text, common English terms for species (following Morton, 1981) have been added in the

main body of the paper if possible (but not in the tables) along with their botanical names. Indigenous names were recorded

and can be found, for example, on the voucher specimens.

106

Publication II

[Hylocereus undatus (L.) Britton & Rose, Cactaceae] are used for preventing

abortion. The fruit of Godmania aesculifolia (Kunth) Standi. (Bignoniaceae)

resembles the umbilical cord and is therefore used to speed up labor and to expel

the placenta.

Based on the work by Redford and Villa Rojas (1990), we had anticipated that

humoral concepts would be important in the selection of medicinal plants. Our

informants never mentioned hot or cold properties of a plant as a reason for its

medicinal use. When asked about these properties they first made reference to the

disease and its humoral characteristics, which this remedy would be used to treat,

and then volunteered data on a plant's property. Additionally, in explaining the

causes of an illness, thermal concepts5 were frequently mentioned by the healers6.

Plants are not chosen at random for treating a certain illness. Sweet plants are

clearly preferred for treating respiratory illnesses; 65 % of all use reports in the

group "respiratory illnesses" concerning taste properties are about plants

commonly considered to be sweet (Table I). Another important characteristic of the

plants in this group is that they typically have a strong odor (65% of all taxa with

ascribed smell properties). Plants for the treatment of bites of venomous animals,

especially snakes, are frequently mentioned (n=14 answers) and generally are

classified as bitter (n=10; 71%). Smell is not an important criterion in this group (n

= 1). Women's medicine are considered to have no smell (40%) or no taste (57%)

or to be aromatic (55 %). For skin conditions, bitter (64%) and astringent (33 %),

5There is a methodological problem in distinguishing between the thermal and humoral properties of a species or an illness.

Frequently the informant refer to a "hot" or "cold'1 item without it becoming clear immediately which of the two types they were

referring to. The data in the following discussion are therefore based on systematic inquiries into the meaning of the terms of

the infoimant.

6Water of the cenotes is considered to be cold. Cool water should not be drunk or used for washing or bathing. Caution must

also be observed with cold winds coming from the North (Maya; cascac ik). On the other hand, the hot sun is feared as a

cause of illness.

107

Publication II

and aromatic (47%) taxa are preferred. The various gastrointestinal illnesses were

divided into three subgroups: dysentery, diarrhea and vomiting. For vomiting,

aromatic plants (61%) or bitter (58%) ones are preferred. Diarrhea generally is

treated with astringent (50%), aromatic (65%), or bitter (33%) plants, whereas the

most popular type of plants for dysentery are those that have only minimal smell, if

any, (67%) or a bitter taste (35%). Pain and fever are treated mostly with aromatic

(52%) or bad-smelling plants (43%).

Responses were sought on the classification of the plants in the various groups of

indigenous uses according to the humoral system (Table II). Disorders specific to

women are treated with remedies considered to be hot (88 % of all responses).

Pain and fever, on the other hand, call for cold ones (74 %). A particularly

interesting group is gastrointestinal disorders. Roughly half of all reports in this

group classify the taxa as hot or cold, respectively. But if one looks, for example, at

dysentery, it is clear that it requires remedies that are cold (96 %). On the other

hand diarrhea and vomiting call for hot remedies. Dysentery is considered to be a

hot illness because of the blood in the feces. Diarrhea and vomiting are caused by

cold winds, cloudy skies, or by rain during the rainy season. Consequently, these

two illnesses are cold and require hot remedies. Inflammatory skin diseases and

bites of venomous animals produce a localized reddening of the skin and

temperature elevation and thus require remedies that are cold.

The main groups of skin conditions distinguished are inflammation and pimples.

Inflammation is considered a hot illness; pimples a cold one. For respiratory

illnesses, the classification of the plants is not clear-cut: 53% of the plants are said

to be hot and 47% cold.

108

Table

I.Classification

ofMedicinal

PlantsbySensory

CharacteristicsandGroup

ofuse:

Taste(%)

Smell(%)

Group

ofuse"

Bitter

Astr

ing¬

Sweet

Tasteless

Spicy

Acid

Aromatic

Odorless

Strong-

Bad-

ent

"tot

smelling

smelling

"tot

Gastrointestinal

42

29

715

43

73

56

26

11

788

disorders

Dysentery

35

17

926

49

23

17

67

-6

15

Diarrhea

33

50

13

4-

-24

65

24

66

17

Vomiting

58

19

-

15

8-

26

61

16

16

756

Dermatological

64

33

3-

--

33

47

720

27

15

conditions

Women's

medicine

29

--

57

14

-

14

55

40

-

520

Painandfever

25

-

25

25

25

-

452

-5

43

21

Respiratoryillnesses

917

65

--

923

24

665

617

Bitesbyvenomous

71

21

7-

--

14

-_

-

(1)

1

animals

a

Figu

reswithshadedbackground:>30%,>3

responses.

bUrological

problems^

tot=

1°I

otherusesn^

=2;

conditionsoftheeyesn

^0^

:

Publication II

Table II. Hot-Cold Classification of Medicinal Plants in the Groupsof Uses3

Hot Cold

Group of usea (%) (%) /7tot

Gastrointestinal disorders 65 45 101

Dysentery 4 96 27

Diarrhea 67 33 18

Vomiting 77 23 56

Dermatological conditions 29 71 35

Infection 4 96 24

Pimples 82 18 11

Women's medicine 88 12 25

Pain and fever 26 74 23

Respiratory illnesses 53 47 19

Bites by venomous animals - 100 13

a

Figures with shaded background: >70°'o of the total number of

individual use reports; two responses: Urological problems; conditions

of the eyes; other uses.

Differences between medicinal and nonmedicinal plants

Individual healers were asked about differences between medicinal and

nonmedicinal plants. Practically all medicinal plants are named folk taxa. For 28 %

of the nonmedicinal plants, the informants did not know the names of plants that

they did not personally use.

Most of the nonmedicinal taxa are trees and herbs, and they usually are rather

inconspicuous plants, without showy flowers and good-tasting fruit, for example.

When selecting a plant to be discussed as a nonmedicinal, the informants in most

cases described and tasted the leaves. Since the Maya clearly distinguish between

bok (smell) and kii (taste) (Barrera et al., 1991), the answers were recorded in two

groups (Fig. 2 and 3). There is a significant difference between the two groups with

respect to taste and smell properties (%2 = 56, 99.9% CI of %2 is 20 with 5 degrees

of freedom).

Smell was generally described as being good, bad, or absent. In explaining the

smell properties, the informants additionally compared the odor with common ones,

such as the smell of a person, of a lemon, or honey, or the strong, aromatic smell

of adrue {Cyperus articulatus L, Cyperaceae). According to the Maya, a strong

110

^

CE

60

50

40

30

20

10

n169 Responses on

Medicinal Taxa

m 100 Responses on

Non-Medicinal Taxa

Aromatic Odorless Strong smell Bad smell

Quality of Smell

Fig. 2. Smell of medicinal and nonmedicinal plants of the Yucatec Maya.

50

45

40

35

30

25

20

15

10

5

o *•-•

166 Responses on

Medicinal Taxai

m 100 Responses on I

Non-Medicinal Taxa1

I—WM

itter Astringent Sweet Tasteless

Quality of Taste

Spicy Acid

Fig. 3. Taste of medicinal and nonmedicinal plants of the Yucatec Maya.

m

Publication II

smell can be pleasant or unpleasant, but it is distinguished from other groups of

smell by being unusually strong. The ways in which the Maya characterize taste

and smell of a plant were used in the analysis (i.e., we accepted the criteria as they

were stated by the Maya [Table III], including somewhat ambiguous categories

such as "strong" for bad smell).

We have documented a total of 335 responses about taste and smell properties

and 222 about the humoral classification for 329 medicinal taxa. For the 69

nonmedicinal ones, 200 responses concerning sensory perception and just two

responses for humorally hot were recorded (Figs. 2 and 3).

Nonmedicinal plants were more often reported to have no smell (Fig. 2) or no taste

(Fig. 3). Medicinal plants, on the other hand, are more often aromatic (good smell),

and there is practically no difference in the frequencies of responses about bad or

strong smell (Fig. 2). A good odor was mentioned in 50 % of the cases as a

characteristic of a medicinal plant and thus is a sign for medicinal use, whereas the

absence of smell indicates that the taxon has no potential medicinal value.

With respect to taste, a larger percentage of medicinal plants were reported to be

astringent or sweet, but there are no differences in the qualities bitter, spicy, and

acid (Fig. 3). It is noteworthy that the informants considered 44 % and 42% of the

nonmedicinal plants and medicinal plants, respectively, to be bitter. This

characteristic is therefore attributed to a rather large segment of the surrounding

flora but seems to be of no direct relevance to the selection of medicinal plants.

It is worth repeating that, for the Maya, it is not the case that medicinal plants are

characterized as unusually bitter. There is no difference in the percentages of

medicinal and of nonmedicinal plants that are considered to be bitter. This

contradicts reports in other ethnobotanical studies that bitterness is a particular

characteristic of many medicinal plants (e.g., Heinrich et al., 1992).

112

Table

III.

Qualities

ofMedicinalandNonmedicinal

Plants

oftheLowlandMaya

Maya

Spanish

English

Example

Taste

k'a

Amargo

Bitter

suts'

Astringente

Astringent

ch'uhuk

Dulce

Sweet

pap

Picante

Spicy

pu

pah

Agrio

Acid

kl'ubok

Buen

olor

Aromatic,goodsme

chee

ol

Olor

tuerie,apestoso

Strong

smell

tu'ubok

Malolor

Bad

smell

:-cold

chokô

Caliente

Hot

sis

Frio

Cold


(Loe

s.)Standi,

Psidiumguajava

L.

Pachyrhizuserosus

Urb.

var.palma

Piperamalago

L.

Citrusaurantiifolia

Swingle

Psidiumguajava

L.

Chenopodiumambrosioides

L.

Zanthoxylumcaribaeum

Lam.

Dorsteniaco

ntra

jerv

aL.

Citrusaurantiifolia

Swingle

Publication II

The humoral properties of the plants were also elucidated in the interviews. The

informants sometimes did not clearly distinguish between thermal and humoral

characteristics. Questions about the humoral properties of a plant made no sense

to some of the informants, who proceeded to describ the process of preparing the

remedy (i. e., hot or cold extraction). Frequently, the humoral connotations of hot

and cold describes the illness. The medicinal plant to be used must have the

opposite property (i.e., cold for a hot illness). This does not refer to the selection

criteria for a plant, but to its classification. It therefore comes as no surprise that

nonmedicinal plants are generally not classified humorally (Table IV). In only two

cases was a nonmedicinal one considered to be humorally hot: one {Senna sp.)

because of the Mayan name chak sal (red; little pimples), since red is considered

humorally hot, and the other {Sabal sp.), because the healer was told to use this

species as a women's medicine, nearly all of which are hot.

Table IV. Hot-Cold Classification of Medicinal and Nonmedicinal

Plants of the Lowland Mayas

Responses on Responses on

medicinal taxa nonmedicinal

Classification ntot (%) taxa ntot

Hot 104(46.8) 2

Cold 101 (45.5) _

Cool 12(5.4) __

Lukewarm 5 (2.2) -

Perception and chemical constituents of the plants

The documented sensory perceptions of the medicinal and nonmedicinal plants

were further analyzed using published information on the known chemical

constituents of the species. With this analysis, we intend to reach a better

understanding of the relationship between the indigenous perception of a plant's

taste or odor and the chemical constituents. Tables V and VI show all the plants

114

Publication II

with three or more reports on taste or smell properties and the known (groups of)

constituents. (To facilitate the reading of the text, the plant families and the authors

are listed in the tables).

Taste. It comes as no surprise that species considered to be astringent contain

polyphenols (hydrolyzable tannins and/or proanthocyanidins). The astringent and

disinfecting properties of the polyphenols makes plausible their use for infections

like diarrhea and skin disease.

Bitter-tasting plants are quite common, and bitterness may be attributable to

various groups of compounds such as cardenolides {Dorstenia contrajerva,

Urechites andrieuxii). Other bitter-tasting compounds are terpenes, like the neo-

clerodane-type diterpenes of Salvia spp. or quassin, a quassinoid found in many

taxa of the Simaroubaceae {Alvaradoa amorphoides), and the sesquiterpene

lactones found in many Compositae, such as Calea urticifolia. The genus

Callicarpa is one of the few in the Verbenaceae (s.l.) in which no bitter iridoids

have been found. Instead, bitter diterpenoids, triterpenoids, and flavonoids are

known from this genus. Crossopetalum gaumeri has a bitter taste due to

cardenolides (Ankli et ah, submitted). Naseberry {Manilkara zapota) has latex rich

in polyisoprenes, but no information is available on compounds, which may

produce the bitter taste. Alkaloids are responsible for the bitter taste {Casearia

corymbosa). For soap tree {Sapindus saponaria) saponins or flavonoids are the

relevant compounds.

The yam bean tuber {Pachyrhizus erosus) has a sweet taste probably attributable

to glycoproteins or the flavonoid pachyrhizin. The acid amides of Jamaika black

pepper {Piper amalago) are responsible for its typical spicy taste, like that of black

pepper {Piper nigrum). A typical acid-tasting plant is acid lime (in leaves and fruit

of Citrus aurantiifolia), which contains acidic derivatives of limonoids and acidic

phenols in

115

TableV.MedicinalandNonmedicinal

Plants

ClassifiedbyTasteandSmell

Prop

erti

esAccording

toYucatecMayan

Healersandthe

Constituents

oftheseTaxa3,

Plantname

(familyf

TA

EQ(%)

SL

DT

AK

CM

Other

Ref

Astringent

plant

PsidiumguajavaL,MRT

Manilkarazapota

(L.)

vanRoyen,SPT

Crossopetalumgaumeri(L

oes.

)Standi.,CEL

Punicagranatum

L.,PUN

Bitter

plant

Crossopetalumgaumeri(Loes.)Standi.,CEL

Manilkarazapota

(L.)

vanRoyen,SPT

Dorsteniacontra

jerv

aL.

,MOR

AlvaradoaamorphoidesLiebm.,SMR

Galea

urti

cifo

liaMillsp.var.yucatanensis,

CMP

Callicarpa

acuminataRoxb.,VRB

CaseariacorymbosaJacq.,

FLC

Salviamicrantha

Desf

.,LAB

Sapindussaponaria

L.SAP

+H,

P

+ + +Iv

,fr

+ +

+ +

UrechitesandrieuxiiMuell.

Arg.

,APO

Sweet

plant

PachyrhizuserosusUrb.var.palma,LEG

Spicy

plant

Piperamalago

L.,PIP

+rt

Acid

plant

CitrusaurantiifoliaSwingle,

RUT

+fr

JatrophagaumeriGreenman,EUP

+

Manilkarazapota

(L.)

vanRoyen,SPT

+

PsidiumguajavaL,MRT

+H,

P+

+ +

Lv,

fr

Polyisoprenes

Triterpenes,

sugaralcohols

Lv:tr

iter

pene

s,st

eroi

ds,

fr:sugars,

phenolicacids

Triterpenes,sugaralcohols

Polyisoprene,

fr:sugar

+FU

Cardenolides

Anthraquinones,

trit

erpe

nes(quassinoids)

Phloroglucins

Trit

erpe

noid

s,flavonoids

+Saponins

Terpenoids,

flavonoids

Saponins,

flavonoids,

lipids

Cardenolides

Rt:gl

ycop

rote

ins,

flavonoids(p

achy

rhiz

in)

Piperamides,

sesquiterpenes

+Iv

Terpenes

(lim

onoi

ds),

organicacids

Saponins,

cyanogeniccompounds

Polyisoprene

Lv.fr

12

14,

9

89

39

39,

8

79

79,

3

69,

23

39,

5,

20

39,2

35

39

317,

21

39,

11,

12,25

39

36

49,1

29

29

29

214

,9

Table

V.

(continued)

Plantname

Aromatic

smelling

plant

Psidiumguajava

L.,MRT


L.(T

eloxys

ambrosioides),CHN

Dorsteniaco

ntra

jerv

aL.

,MOR

Artemisialudovicianassp.mexicana

(Wil

ld.)

Keck,CMP


RUT

Lippia

stoechadifoliaKunth,VRB

Lippia

albaN.

E.

Br.ex

Britton&

Wilson,VRB

Mentha

spp.,LAB

Satureja

brownei(Micromeriabrownei(S

w.)

Benth),LAB

OcimummicranthumWùld.,LAB

Strong-smelling

plant


L.(Teloxys

ambrosioides),CHN


RUT

Piperamalago

L.,PIP

AlvaradoaamorphoidesLiebm.,SMR

Bad-smelling

plant

ZanthoxylumcaribaeumLam.,RUT

Colubrinagr

eggi

var.yucatanensis,RHM

Senna

uniflora(P.

Mill

er)H.

Irwin&

Barneby,

LEG

Bitter

plant

without

smell

AcalyphaunibracteataMuell.Ar

g.,EUP

TA

EO

SL

DT

AK

CM

Other

Ref.

+P

+(-

2)

+(f

r:0.

4)+

(>1)

+(>1)

+H)

+(-

4)

+(-

2)

+(f

r:0.

4)+

+IA

Lv,

fr

Saponins,

trit

erpe

nes,

organic

acid

s,

flavonoids

FU

Cardenolides

Triterpenes,xanthophylls,flavonoids

IvTerpenes

(lim

onoi

ds),

organicacids

Phenolics

(ros

mari

nic

acid

),flavonoids

Phenolics,

iridoidgl

ycos

ides

,flavonoids

Phenolics,flavonoids

Saponins,

trit

erpe

nes,

organic

acid

s,

flavonoids

+Iv

Terpenes,organicacids

Piperamides

Anthraquinones,

trit

erpe

nes(quassinoids)

Lignans,unusualamids

Saponins,flavonoids

Anthraquinons,

prot

eins

,aromatic

compounds,

flavonoids

Phenolics,cyanogeniccompounds

sugars

12

14,9,

10

99

89,23

59,

18

57,

9

59

49

421,

8

424,

9

37

49

39,

7

39

39,

5,

20

59,

4

39

39

Table

V.

(continued)

Plantname

TA

EO

SL

DT

AK

CM

Other

Ref:

Plants

without

specific

tasteans

smell

AcalyphaalopecuroidesJacq.,EUP

AbutilonpermolleSweet,MLV

TriumfettasemitrilobaJacq.,

TIL

Hylocereusundatus

(L.)

Britton&Rose,CAC

SapindussaponariaL,SAP

+

Microgramma

nitida

(J.Sm.)A.Reed,PLG

+

Phenolics,cyanogeniccompounds

sugars

5/5

Mucilage,

trit

erpe

nes,

fatty

oil,

phenolic

acids

Mucilage,cyanogeniccompounds,

flavonoids

Flavonoids,betalain

Saponins,

flavonoids

Triterpenes,

cyanogeniccompounds,

sugars,

resin

4/4

9

4/4

9

3/4

9

3/4

9

3/3

9

aTA:

tannins;EO,

essential

oils

;SL,sesquiterpene

lactones;DT,diterpenes;AK,

alkaloids;

CM,

coumarins;

H:hydrolyzabletannins;

P,proanthocyanidins;

Bl

benzylisoquino

line

alka

loid

s;1A

,indolealka

loid

s;FU,furanocumarins;

Iv,leaves;

fr,fruits;

rt,roots.

bAbbreviations

ofplantfamiliesfollowthecode

ofWeber,W.

A.(1

982)

.cReferences:1Achenbach

et

al.(1984).2

Borges

delCastilloet

al.(1981).3

Chung

et

al.(1997).4

DeliaCasaandSojo

(1967).

5

Glasby

(1991).cGomes

et

al.

1997

).7Guenther(1949).8

Hansel

et

al.(1993).9

Hegnauer

(1962-1996).10Ji

et

al.(1991

).11Lemos

et

al.

(1992).VlLemos

etal

(1994).nMollonbeck

et

al.

1997).

uOkuda

et

al.(1

987)

.15

Patitucciet

al.(1

995)

.16Rodn'guez-Hahn

et

al.(1

995)

.1/Ruiz-Cancino

et

al.

(1993).

!aScholz

et

al.

(1994).nSchratz

(196

6).2

0SteineggerandHansel

(199

2).2

1Tanabe

et

al.(1

992)

.22Terreaux

et

al.(1995).23Tomas

et

al.

(1988).24WahabandSelim

(198

5).

Publication II

the fruit. Jatropha gaumeri contains caustic latex, which consist of di- and

triterpenes. Especially the unripe fruit of the guava {Psidium guajava) contain little

sugar and are rich in fruit acids. No data are available on naseberry (M. zapotä)

which is also said to be sour.

Smell. All species mentioned as aromatic plants contain relatively large amounts

of essential oils, which are volatile, odoriferous mixtures of compounds that are

largely insoluble in water. The Labiatae (e.g., Mentha, Satureja, Ocimum spp.) and

Myrtaceae {Psidium) are typical representatives of the essential oil-containing plant

families. Two species of the genus Lippia (Verbenaceae) were mentioned several

times by Maya as being aromatic. This genus is one of the few in the Verbenaceae

whose species are rich in essential oil. American wormseed {Teloxys

ambrosioides), Citrus spp., and also estafiate {Artemisia ludoviciana ssp.

mexicana) are mentioned several times as having a good smell, and are all rich in

essential oil (Table V). Dorstenia contrajerva is reported to contain coumarins, but

no data on its essential oil content are available.

The strong-smelling medicinal plants are a puzzling group. American wormseed

{Teloxys ambrosioides) and acid lime {Citrus aurantiifolia) have a strong smell

because they contain much essential oil. Piper amalago and Alvaradoa

amorphoides, on the other hand, are not remarkably fragrant, but their strong and

very characteristic taste seems to be culturally interpreted as strong smell. In this

group a judgment as to the sensory perception seems to be made by the

informants when tasting or smelling the plant. Accordingly, no specific group of

natural products is responsible for a "strong odor".

Among bad-smelling plants, no special group of constituents are responsible for

the unpleasant smell. Scorpion tree {Zanthoxylum caribaeum) is said to smell like

pork, a feature that can probably be attributed to the essential oil or the unusual

amides present in this genus. Senna uniflora is particularly prominent for being rich

119

Table

VI.TypicalHumorallyColdandHotPlantsAcoordmg

toYucatecMayan

Healersand

theConstituents

ofTheseTaxa3

Plantname

TA

EO

SL

DT

AK

CM

Other

Ref

Cold

plant


RUT

+f

r

Hylocereusundatus

(L)Britton&Rose,CAC

Triumfettasemitnloba

Jacq

,TIL

MalvaviscusarboreusCav

var

arboreus,MLV

Crossopetalumgaumeri(Loes

)Standi,CEL

f

Manilkarazapota

(L)vanRoyen,SPT

+

Psidiumguajava

L,MRT

+H,P

+

Artemisialudovicianassp

mexicana

(Willd

)+

+

Keck,CMP

Microgramma

nitida

(JSm

)A

Reed,PLG

++

Satureja

brownei(Micromenabrownei(Sw

)+

Benth),LAB

Hot

plant


,MOR

ChenopodiumambrosioidesL

(Teloxys

+

ambrosioides),CHN

Triumfettasemitnloba

Jacq

,TIL

Plucheasymp

hyti

foli

a(M

ill

)Gills,CMP

+

Psidiumguajava

L,MRT

+H,P

+

Artemisialudovicianassp

mexicana

(Willd

)+

+

Keck,CMP

Aristolochiaspp

,ARS


RUT

-r

fr

Microgramma

nitida

(JSm

)A

Reed,PLG

++

Pimenta

dioica(L

)Merr,MRT

+

UreracaracasanaGriseb

,URT

+

ZingiberofficinaleRoscoe,Z1N

+

+lv

Terpenes

(lim

onoi

ds),

organicacids

Flavonoids,betalames


flavonoids

Mucilage,fl

avon

oids

,phenolic

acids

Trit

erpe

nes,

sugaralcohols

Polyisoprenes

Lv.fr

Triterpenes,xanthophylls,flavono'ds

Triterpenes,


sugars,

resin

Phenolics(rosmannic

acid

),indo'd

glycosides,flavonoids

+FU

Cardenolides

Saponins,

trit

erpe

nes,

organicacids,

flavonoids


flavonoids

Caffeoylquinic

acd,

flavonoids,simple

phenols,

phenolic

acids

Triterpenes,

xanthophylls,f'avonoids

Anstolochicac

id,anstoloiactame,

lign

anods

+lv

Terpenes

(lim

onoi

ds),

organicacids

Triterpenes,


sugars,

resin

Mucilage

Rt

galano'actons

pheno'ic

acid

s,resin

10

9

89

79

59

49

49

414,9

49

39

324,9

12

9,23

79

79

619

,9

514

,9

49

39

39

39,

15

316

39

313

,22

aTA

tannins,EO,

essential

oils

,SL,sesquiterpene

lactones,

idin

s,Bl

,benzylisoquinoline

alka

loid

s,IA,indolealkaloids,

FU

bAbbreviations

ofplantfamiliesfollowthecode

ofWeber,W

DT,di

terp

enes

,AK,

alka

loid

s,CM,

coumarins,H

hydrolyzabletannins,P

proanthocyn-

,furanocumanns,

Iv,leaves,

fr,fr

uits

,rt

,roots

A(1982)

References-seeTableV

Publication 11

in anthraquinones, but an aromatic smell is not a prominent characteristic

according to the perception of the principal investigator. No essential oil has yet

been reported from Colubrina greggi. Since these three species are not necessarily

very rich in essential oil, the "bad" property seems to be an unpleasant feeling

when tasting the plant. Nearly all plants said to have no smell are also said to be

tasteless. The only exception is Acalypha unibracteata, which has no smell but

reportedly tastes bitter. Practically all plants in this group are known to contain

flavonoids, and some contain cyanogenic compounds and/or tannins.

Based on these data, several clear-cut correlations becomes apparent between

indigenous perceptions and groups of natural products (e.g., aromatic and

astringent compounds). In other cases, no such correlation is noticed. Plants

considered to be aromatic generally contain essential oil, while, for example, plants

described as astringent contain hydrolysable tannins and/or proanthocyanidins.

Hot-cold classification. We were unable to identify any specific group(s) of

compounds associated with the alleged hot or cold properties of a plant (Table VI).

Interestingly, five of 17 species were described by some informants as hot and by

others cold. Thus, agreement among the informants is much lower for the hot-cold

classification.

Conclusions

The aim of this study is to better understand the selection criteria of medicinal

plants used by the lowland Maya. As far as we know, no study comparing

medicinal and nonmedicinal plants nor any that deals specifically with

nonmedicinal plants exists. Our study shows that an understanding of the concepts

indigenous people used to distinguish medicinal from nonmedicinal species has

considerable heuristic value. Sampling the secondary plant products by tasting and

smelling them yields culturally defined clues about a species' potential value and

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helps them to distinguish between used and non-used plants. The approach

employed in this paper thus sheds new light on the selection criteria of the Maya of

the Yucatan peninsula. Plants are not selected at random, nor are they selected

purely by abstract criteria, like hot and cold.

The taste and odor of medicinal plants and the labels applied to them (e.g.,

astringent, bitter, aromatic) include, or encode, considerable information about the

groups of illnesses a particular phytomedicine is best used to treat. Examples

among Yucatec Maya and other cultures are plant remedies classified as

astringent or aromatic. It is, on the other hand, noteworthy that in this study, there

was no difference in the percentage of species classified as bitter in the medicinal

and nonmedicinal plant groups. This contradicts the assumption common in our

culture that medicine has to be bitter. Therefore, no particular sensory property

definitively characterizes a medicinal plant. Taste and smell are very important

selection criteria, but they are not the central unifying principle of indigenous Maya

medicinal plant classification. Indeed, such a unifying concept does not exist (cf.

Worsley, 1997).

In the Yucatec Maya understanding of medicinal plants, specific sensory

properties, like smell, taste, color, form, and texture are the first criteria for

selecting a medicinal plant. These characteristics presumably also serve as

mnemonic aids for identifying medicinal plants that are in regular use. If a plant is

to continue being used as a medicine, it must show a "positive health effect", as

that is interpreted culturally. Therefore, not all plants that share a certain property

serve the same purpose.

Because smell and taste can characterize typical groups of natural products

another key interest here is the relationship between the ethnobotanical data

obtained for the individual taxa and the secondary plant products (natural products)

prominent in each species. This is not only essential for the recognition and

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selection of medicines but can also help to explain the pharmacological effects of a

species (Ankli et al., 1999). Examples are astringent plants, which are widely used,

also among Yucatec Maya, against gastrointestinal disorders and dermatological

problems. The pharmacological effects of these plants, which contain polyphenols,

can partly be explained by the natural products characteristic of the species. Such

a clear-cut connection exists for a few classes of compounds (especially astringent

compounds). Bitter-tasting compounds, on the other hand, are distributed among

a large variety of groups of natural products, so no specific pharmacological

conclusions can be drawn from the fact that a given plant tastes bitter.

Nevertheless, such information is an additional criterion for selecting plants for

phytochemical and pharmacological analysis. Examples are the two species of

Acalypha. The informants consider one of these to be bitter {A. unibracteata),

whereas another {A. alopecuroides) is said to have neither taste nor smell. A

detailed phytochemial comparison of the two would thus be of interest. Since the

data in the scientific literature do not allow quantification of the relevant groups of

natural products, no definite conclusions on the physiological effects of the species

used medicinally can be drawn. In future phytochemical studies, it may be of

interest, not to search for the active principle(s) with an assay for a certain

pharmacological (e.g., anti-inflammatory) activity as a lead but to identify what

compounds are responsible for the taste or smell properties reported by the Maya,

Shifting the focus from the valuable to the "non-valuable" plants gives new

information on the hot-cold dichotomy. In Yucatec Mayan culture, this parameter is

not very important. For our informants, it is a mnemonic aid and an explanation of

plant use and selection for certain illnesses, when the therapy is already known.

Generally, what a species is used for is primary, and, once this is known, the

humoral characteristics are assigned. People who have had regular and more

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Publication II

intensive contact with Mestizo culture also seem to rely more heavily on this

system than informants who have had less contact.

The analysis presented here also raises a large number of methodological and

conceptual questions. Comparative data on the classification by taste and smell

from other cultures, especially from South America, are urgently needed. Thus, it is

essential that future fieldwork in South America and Mesoamerica not focus on the

hot-cold system or purely on botanical documentation of indigenous medicinal

plants. It will be also interesting to look at the development of classificatory

systems over time to detect changes in the systems. Changing the perspective

from the medicinal to the nonmedicinal plants raises serious questions about our

definitions of medicinal plants. Is a medicinal plant one that is widely used in a

culture and that has been shown to have the pharmacological effects desired by

the informants? What about plants used by very few people or ones that have no

known pharmacological effects? Are these species also medicinal? What

distinguishes a medicinal from a nonmedicinal one? A better understanding of the

multiple ways medicinal plants are classified and thus distinguished form

nonmedicinal will shed light on the differences between the two groups.

Acknowledgments

We are very grateful to the healers, midwives and the inhabitants of Chikindzonot,

Ekpedz and Xcocmil, Yucatan, for their collaboration, for their friendship and

hospitality. This manuscript has profited much from electronic and personal

discussions and other input from Prof. Dr. D. Moerman (Dearborn, Mi). The

botanical identification at CICY and MEXU was performed in collaboration with the

numerous specialists of these institutions. Particularly, we would like to thank Dra.

I. Olmsted, J. Granados, P. Simâ, J.C. Trejo, Dr. R. Durân of CICY as well as O.

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Tellez, Dr. R. Lira, Dr. J. Villaserior and Dr. M. Sousa of MEXU. This research

owes a lot to the help of Dr. B. Frei (Zürich), Prof. H. Rimpler (Freiburg), Dra. B.

Pfeiler (UADY, Mérida), Dr. Tuz (INI, Valladolid), Dr. C. Viesca (UNAM) and Dr.

Baltisberger (Zürich). We are very grateful to S. Ritt for the English revision of the

manuscript and to R. Fisullo and Y. Fang for their help in statistical analysis.

Financial support by SDC (Swiss Agency for Development and Cooperation,

Berne, Switzerland) and the SANW (Swiss Academy of Natural Sciences) is

gratefully acknowledged.

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131

Part II Plant Evaluation

Publication II

Yucatec Mayan medicinal plants:

Evaluation based on indigenous uses

Ankli Anita3, Heinrich Michael11'5, Bork Peterb, Wolfram Lutzc, Bauerfeind Peter0,

Brun Retod, ScHMiDCéciled, Weiss Claudia0, Bruggisser Regina', GERTSCHJürg1,

Wasescha Michael8, Sticher Ottoa*

a Department of Applied BioSciences, Institute of Pharmaceutical Sciences, Swiss

Federal Institute of Technology (ETH) Zurich, Winterthurerstr. 190, CH-8057

Zürich, Switzerland

b Institute of Pharmaceutical Biology, Albert-Ludwigs-University, Schänzlestr. 1, D-

79104 Freiburg, Germany

c Department of Internal Medicine, Division of Gastroenterology, University

Hospital Zurich, Rämistr. 100, 8091 Zürich, Switzerland

d Department of Medical Parasitology, Swiss Tropical Institute, Socinstr. 57, CH-

4002 Basel, Switzerland

e Department of Infectious and Tropical Diseases, London School of Hygiene and

Tropical Medicine, Keppel Street, London, UK

f Department of Pharmaceutical Biology, University of Basel, Totengässlein 3, CH-

4051 Basel, Switzerland

g current address: Centre for Pharmacognosy and Phytotherapy, The School of

Pharmacy. 29/39 Brunswick Sq., London WC1N 1 AX, UK

Submitted to

Journal of Ethnopharmacology

Publication III

Abstract

As part of an ethnobotanical field study bulk samples of 48 medicinal plants were

collected in three Yucatec Mayan communities. Based on their documented uses

the plants were evaluated using several biological assays.

Three species - Piscidia piscipula, Jatropha gaumeri, Casimiroa tetrameria -

employed to treat gastrointestinal disorders showed remarkable activity against

Helicobacter pylori. Crossopetalum gaumeri showed activity against Giardia

duodenalis. In the group of plants used for dermatological conditions several

species were active against gram-positive bacteria and Candida albicans.

Cytotoxic effects against KB cells were found for C. gaumeri, Diospyros anisandra,

Jatropha gaumeri, Croton reflexifolium, Dalea carthagenensis and Alvaradoa

amorphoides. D. carthagenensis and Luehea speciosa, were found to be active in

the NF-kB test. Oestrum nocturnum, applied in cases of pain and fever, showed a

weak activity against Plasmodium falciparum. Bauhinia divaricata exhibited

antihyperglycemic activity. None of the six species of the group of "women' s

medicine" showed relevant affinity to the D2 dopamine receptor.

Based on this evaluation, plants with strong activities should be further investigated

phytochemically and pharmacologically in order to better understand their potential

in the treatment of illnesses.

Keywords: Yucatec Maya, ethnopharmacological evaluation, medicinal plants,

traditional medicine, antibacterial, anti-inflammatory, antihyperglycemic,

antiparasitic, D2 receptor binding.

135

Publication III

Introduction

One of the numerous objectives of medical ethnobotany is the selection of

culturally important plant species in order to further evaluate them for

pharmacological activity (Browneret al., 1988; Etkin, 1994; Famsworth, 1988; Frei

et al., 1998a, Messer, 1978). For the evaluation of ethnopharmacopoeias,

bioassays should be chosen based on indigenous uses (Frei et al., 1998b; Heinrich

et al., 1992a, b; Lewis and Elvin-Lewis, 1994).

The knowledge of medicinal plants was a part of the ancient Maya culture and they

are still utilized by the Yucatec Mayan inhabitants on the peninsula of Yucatan,

Mexico (Roys, 1933, De Landa, 1992). During an ethnobotanical study in three

Mayan villages (Feb. 1994-June 1995; Sept. 1996-Oct. 1996), 360 medicinal plants

and 1828 reports on their uses were documented. The uses of the plants were

divided into nine therapeutical groups (Ankli et al. 1999, Heinrich et al. 1998).

Forty-eight species were chosen and evaluated in bioassays relevant to the

following groups of illnesses: gastrointestinal disorders, dermatological conditions,

women's medicines as well as pain and/or fever. Plants used to treat "diabetes"

were also tested (Table 2).

Materials and methods

Plant material

The plants were collected in the villages and surroundings of Chikindzonot, Ekpedz

and Xcocmil, Yucatan (Mexico). Authenticated voucher specimens were deposited

at the Herbarium of the Centro de Investigacion Cientffica de Yucatan (CICY) in

Mérida, the National Herbarium of Mexico (MEXU), the Instituto Nacional

Indigenista (INI) in Valladolid, Yucatan, the ETH Zurich (ZT) and the Institut für

Pharmazeutische Biologie in Freiburg, Germany (AANK1-654). They were

identified by comparison with authentic specimens and in some cases with the

assistance of specialists at CICY, MEXU and K (Kew, Royal Botanical Garden,

UK).

136

Publication III

Extract preparation

Shade-dried and powdered plant material (20g) was extracted by maceration with

100 ml dichloromethane/methanol 2:1, repeating the process 2 times during 36 h.

The filtered solvents were combined and evaporated under vacuum to give the

non-polar extract A. The residue of the dichloromethane/methanol mixture was

dissolved in 100 ml methanol/water 7: 3 and macerated 2 times during 24 h. The

solvents were combined, most of the methanol was evaporated and the resulting

water extract was partitioned between n-butanol (3 x 30-50 ml) and water. The n-

butanol fractions were evaporated to obtain the polar fraction B.

In case of S. aureus and Y. enterocolitica extract C was used. To get extract C,

plant material (10 g) was extracted once with ethanol 96% and twice with ethanol

70%.

Bioassays

Antibacterial and antifungal activity

The organisms used to test biological activities are listed in Table 1. Antimicrobial

tests were performed by the disc diffusion technique (Rios et al., 1988; DIN, 1992).

An aliquot (30 ul) of strain stock suspension was transferred into 5 ml broth

(Nutrient Broth for bacteria, Sabouraud Liquid Medium Oxoid for fungi). 10 ml of

Mueller-Hinton agar or malt extract agar (for C. albicans) was inoculated with 50 til

of overnight culture of the test organisms and poured over the agar base. Paper

discs (6 mm Blanc Discs, Oxoid) were impregnated with 200 u.g and 600 u.g plant

extracts, respectively, and placed on the inoculated agar. After 16 h incubation for

bacteria/fungi at 37 °C the plates were sprayed with MTT (methylthiazolyl-

tetrazolium chloride, Fluka). The inhibition zones were measured in mm.

Susceptibility tests with H. pylori and C. jejuni were carried out on Wilkins Chalgren

agar plates supplemented with 5% defibrinated sheep blood and the following

antibiotics: 2 ug amphotericin B/ml, 6 ug vancomycin/ml, 5 ug cefsulodin/ml, and 5

ug trimethoprim/ml. 100 ul of a thick H. pylori suspension (yield from one agar

137

Publication II

plate, resuspended in 1 ml PBS: phosphate buffered saline) were applied onto the

plates, the discs soaked with plant extracts (600 pig) and placed on the plates.

They were then incubated under a water-saturated, micro-aerophilic atmosphere at

37 °C for one day {C. jejuni), or three to five days {H. pylon) and the growth was

controlled regularly.

The MIC determination for H. pylori was carried out in a modified minimal medium

according to Nedenskov (1994). Different concentrations (in the range from 0.3 to

200 jig/ml) of the plant extracts were added to the uninoculated medium (5 ml in a

25 ml Erlenmeyer flask). A fresh H. pylori culture was used as a 1 % inoculum and

grown under micro-aerophilic conditions in a water saturated atmosphere at 37 °C

in a rotary shaker (G25; New Brunswick Scientific, New Jersey, USA). The

incubation was continued for two days at 175 rpm. After two days the optical

density of the cultures were photometrically determined at 600 nm (DU-64

spectrophotometer, Beckman, UK). The MIC value was defined as the extract

concentration not allowing visible growth (less than 0.03 in comparison with 1.2 for

the control).

Table 1. Test organisms for antibacterial and antifungal activity

Microorganism Origin Clinical picture'"

gastrointestia! problems dermatologicalconditions

Bacillus cereus ATCC 10702 diarrhea

Campylobacterjejuni*

enteritis, diarrhea

Candida albicans H29 ATCC

26790

mycosis

Escherichia coli ATCC 25922 diarrhea, dysentery infected wounds

Helicobacter pylori ATCC 43504 gastritis, peptic ulcer

Pseudomonas aeruginosa r\ \ \j\j ^oy^c infected wounds

Staphylococcus aureus ATCC 25933 diarrhea (food

intoxication)

topical infection

Staphylococcus epidermidis ATCC 12228 infection (septicemia)Yersinia enterocolitica 03 enterocolitis

* Obtained from the Department of Internal Medicine, Division of Gastroenterology, University

Hospital Zurich0(Kayseretal., 1993)

138

Table

2.Ethnomedicaldataon

plan

tsstudiedandchosen

testsystems

Family


Group

ofuse

Plant

Tested

for

part

Acanthaceae

Ruellianudiflora(Engelm.&Gray)

Urb.(1

15)

UR

ap

1-7,14

Annonaceae

Malmeadepressa

(Baill.)

R.

E.

Fr.(161)

UR

rt1-7,14

Apocynaceae

Tabernaemontana

amyg

dali

foli

aJacq.(190)

DER

Iv1-5

Araceae

AnthuriumschlechtendaliiKunthssp.schlechtendalii(2

43)

FEM

Iv1-5,15

Aristolochiaceae


(350

)Gl

,FEM

rt1-10,15

Asteraceae

VerbesinagiganteaJacq.

(288

)RES

Iv1-5

BidenssquarrosaLess.

(121

)Gl

ap

1-12

Basellaceae

Anredera

vesicariaC.FGaertner(196)

DER

Iv,tu

1-7

Bignoniaceae

Parmentieramillspaughiana

(L.)

Williams(1

35)

UR

Iv1-5,14

Parmentieraaculeata

(Kunth)Seem.

(096)

UR

Iv,

rt1-5

Bombacaceae

Pseudobombax

elli

ptic

um(Kunth)Dugand

(275

)RES

Iv1-5

Boraginaceae

Ehretia

tinifolia

L.(0

21)

RES,UR,PFE

Iv1-5,13

Bromeliaceae

Aechmea

bracteata

var.bracteataGriseb.

(167

)DER

Iv1-7

Cactaceae

Hylocereusundatus

(L.)

Britton&Rose

(427

)Gl

,UR

Iv1-12,14

Caesalpiniaceae

Bauhinia

divaricata

L.(0

07)

RES,UR,Gl

Iv1-10,14

CaesalpiniagaumeriGreenman

(155)

PFE

Iv1-7,13

Celastraceae

Crossopetalumgaumeri(L

oes.

)Lundell

(038

)Gl

,DER

Iv1-12

Cucurbitaceae

Ibervilleami

llsp

augh

ii(Cogn.)

C.Je

ffre

y(0

94)

DER

tu

1-7

Ebenaceae

DiospyrosanisandraBlake(1

34)

DER

Iv1-5

DiospyroscuneataStandi.

(341

)DER

iv1-5

Euphorbiaceae

Croton

reflexifoliusKunth

(143

)DER

Iv1-5

datrophagaumeriGreenman

(419

)Gl

,DER

rt1-12

Flacourtiaceae

CaseariacorymbosaJacq.

(150

)DER,PFE

iv1-7,13

Lamiaceae

Salviamicrantha

Desf.(0

25)

DER,FEM

ap

1-7,15

Metiaceae

Cedrelamexicana

L.(3

01)

RES

Iv1-5

Moraceae

BrosimumalicastrumSw.

(092

)RES

Iv1-5

Dorsteniaco

ntra

jerv

aL.

(330

)Gl

,FEM

rh

1-12,15

Myrtaceae

Psidiumsarîorianum(Berg)

Nied.(211

)DER,

Gl

Iv1-10

Nyctaginaceae

Neeaps

ychotrioides

F.D.Sm.

(274)

DER

Iv1-7

Pisoniaaculeata

L.(154)

FEM

Iv1-7,15

Papilionaceae

Daleacarthagenensis

var.barbata

(Oer

st.)

Barneby

(125

)DER

Iv1-5

Piscidiapi

scip

ula

(L.)

Sarg.

(123

)Gl

,RES

iv1-10

Phytolaccaceae

PhytolaccaicosandraSims

(388

)DER

fr1-5

Rivinahumiiis

L.(0

89)

DER

ap

1-5

Polygonaceae

Neomillspaughiaemarginata

S.

F.Blake(2

03)

DER,RES

Iv1-5

Polypodiaceae

Microgramma

nitida

(J.Sm.)

A.ReedSm.

(183

)Gl

wp

1-10

Rubiaceae

Rutaceae

Sapotaceae

Sela

gine

llac

eae

Simaroubaceae

Solanaceae

Sterculiaceae

Tiliaceae

Borreria

vert

icil

lata

G.Meyer

(276

)MorindayucatanensisGreenman

(113

)Casimiroa

tetrameriaMillsp.(0

49)

ChrysophyllummexlcanumBrandegee

(386

)Manilkarazapota

(L.)

Royen,Achraszapota

L.(2

34)

Sela

ginell

alo

ngis

pica

taUnderw.

(214

)AlvaradoaamorphoidesLiebm.

(136

)Cestrumnocturnum

L.(050)

SolanumerianthumG.Don

f.(3

34)

Solanumnigrum

L.(2

67)

HelicteresbaruensisJacq.

(176

)Lueheaspeciosa

Wiild.(3

47)

DER

ap

1-7

DER

fr1-7

Gl,PFE

Iv1-12

Gl

rt1-10

Gl,PFE

ba

1-10,13

UR,RES

ap

1-5

DER

Iv1-7

DER,PFE

Iv1-5,13

DER

iv1-5

DER

Iv1-5

FEM

Iv1-5,15

DER

Iv1-

7,11,12

UR:

urological

problems

(includi

ng"d

iabe

tes"

),DER:

dermatologicalconditionsin

clud

ing

inju

ries

causedbyvenomous

animals;

FEM:women's

medicines;

Gl:ga

stro

inte

stin

aldisorders;PFE:

illnessesassociated

withpa

inand/orfe

ver;

RES:

resp

irat

ory

illn

esse

s;ap:

aerialpa

rts;

ba:ba

rk;

fr:fr

uits

;Iv:leaves;

rh:rhizome;

rt:ro

ot;

tu:tuber;wh:whole

plan

t;1:

B.cereus;

2:

E.

coli;3:

C.

albicans;

4:KB-cell

line;

5:NF-kB;

6:

P.aeruginosa,

7:

S.ep

ider

midl

s;8:H.

pylori;

9:

C.jejuni;10:

G.duodenalis;

11:

S.aureus;

12:

Y.enterocolitis;

13:

P.fa

lcip

arum

;14:cx-amylase;

15:D2-receptorbindingassay

Publication III

Cytotoxicity study using KB cell culture

The cytotoxicity of the plant extracts was assessed using the KB cell line (ATCC

CCL 17; human nasopharyngeal carcinoma). The test was carried out with some

modifications according to the screening technique of Swanson and Pezzuto

(1990). The assay was performed in 96-well plates (Falcon) with an inoculum of

2.5 x 104 cells/ml. Total volume was 150 ul. The dried extracts were dissolved in

ethanol. Water was added to dilute the solution five fold. Concentration of 50 Lig/ml

with max. 1% ethanol was tested. These solutions were diluted 20 fold by mixing it

with culture medium. For active extracts, IC50 values were determined. The

quantification was performed by adding 15 ul of a solution of MTT with 5 mg/ml in

PBS (Mosmann, 1983). After incubation at 37 °C for 4 h, the metabolically active

cells produced an insoluble formazan dye. The medium was drawn off and the

formazan dye was dissolved using 150 ul of 10 % SDS (sodium dodecylsulfate) in

water. After 24 h of incubation at room temperature, the optical density was

measured at 540 nm using a microplate reader (MRX, Dynex Technologies).

Inhibitory activity on NF-kB (Nuclear Factor kB)

Anti-inflammatory activity of the plant extracts was assayed in EMSA shift

experiments (Electrophoretic Mobility Shift Assay) using the inhibition of NF-kB

binding to a radioactive labeled oligonucleotide as a molecular target. The

bioassay was carried out as described in Bork et al. (1996).

Antimalarial activity

Antimalarial activity was assessed for the chloroquine resistant K1 strain and the

chloroquine sensitive T9-96 clone of P. falciparum. The parasites were maintained

in continuous culture of infected A+ human red blood cells in RPMI 1640

supplemented with 6.9 mg/ml HEPES, 2 mg/ml glucose, 2.33 mg/ml NaHC03, 50

urg/ml hypoxanthine, 40 ug/ml gentamicin (all Sigma) and 10% A+ serum (North

London Blood Transfusion Center) (Fairlamb et al., 1985). Antimalarial IC50 values

were assessed using the modified in vitro lactate dehydrogenase assay (Makler et

141

Publication III

al., 1993). Extracts were tested in concentrations from 1000 to 4.12 u.g /ml (3-fold

dilution). Fifty jlx! of a 1% parasitemia blood suspension (predominantly ring form)

were added to 50 pi of drug solution in RPMI 1640 (final hematocrit 2%). The 96-

well microtiter plates were incubated for 48 h at 37 °C in an atmosphere of 1% 0?

and 3% C02 in balanced N2. After the incubation period, 20 ul of the parasite

suspension was added to 100 ul of Malstat reagent (Flow Incorporated, USA)

and incubated at room temperature (RT) for 15 minutes before adding 20 ul of

freshly made 1:1 NBT/PES-mixture (2 mg/ml and 0.2 mg/ml respectively) to each

well. The plates were reincubated for 20 min at RT in the dark and subsequently

read at 650 nm. All compounds were tested twice in triplicate.

Giardia duodenalis

G. duodenalis trophozoites were cultivated in Diamondi's modified TYI-S-33

medium (Keister, 1983) supplemented with 10 % heat inactivated fetal calf serum.

The in vitro assay was performed as described for the Alamar Blue® assay for

trypanosomes by Raez et al. (1997) with modifications for G. duodenalis WB strain

(isolated 1982 from a human in Afghanistan). Briefly, 200 uJ of a trophozoite

suspension were inoculated into 96-well microtiter plates (Costar, USA) at a

density of 4 x 105 trophozoites/ml culture medium. The trophozoites were

incubated in the presence of serial 3-fold dilutions of extracts for 72 hours at 37 °C.

Wells without drug served as controls. Minimum inhibitory concentration (MIC) was

determined microscopically after 70 hours of incubation (the lowest drug

concentration at which no trophozoite with normal morphology could be observed).

Ten uJ Alamar Blue® were added to each well and after 2 hours of incubation the

fluorescence determined using a fluorescence measuring instrument (Cytofluor,

Millipore; excitation wavelength at 530 nm, emission at 590 nm). IC50 values were

calculated by linear interpolation selecting values above and below the 50 % mark.

142

Publication III

Dopamin D2 receptor binding assay

Two concentrations of extracts (100 ug/ml and 10 j-tg/ml) were tested in the

dopamine receptor binding assay. The affinity of the extracts to the dopamine

receptor was assessed according to Berger (1998).

a-Amylase assay

Plant extracts (1, 3, 6 mg/ml in 5 ul solvent) were mixed with 45 ul of amylase

reagent (ET-G7 PNP 1,0 mmol/l, magnesium chloride 10 mmol/l, sodium chloride

50 mmol/l, cx-glucosidase 25.000U/I, buffer pH 7.0, sodium azide 0.05%) obtained

from Sigma Diagnostics and incubated at 37 °C for 2-10 min. The absorbance was

recorded at 405 nm versus water as a reference. The incubation was continued

and the absorbance was read after exactly 1 and 2 min. The amylase activity was

calculated according to Pierre et al. (1976).

Results

All polar and non-polar extracts of the 48 plants were screened for cytotoxic activity

against KB cell culture, B. cereus, E. coli, C. Candida and in the NF-kB test.

Extracts were additionally evaluated in selected test systems, which are of direct

relevance to the indigenous uses of the species (Table 2). The microorganisms

chosen cause gastrointestinal problems and/or dermatological illnesses, and are of

direct relevance for these conditions (Table 1). The extracts, which showed

noteworthy positive activities, are listed in Tables 3-7.

Active plants for gastrointestinal problems

Gram-positive and gram-negative bacteria as well as protozoa (G. duodenalis),

known as causes of gastrointestinal problems, were used to determine activities of

species used for gastrointestinal disorders (Table 3). Six species showed at least

some activity against G. duodenalis with IC50 values <100 ug/ml, three of them with

MIC values <100 jig/ml. The most active extract was the non-polar extract (A) of

Crossopetalum gaumeri (IC50: 2.1 ug/ml, MIC: 6.3 ug/ml), whereas the polar extract

143

Publication III

(B) showed very weak antiprotozoal activity. The non-polar and polar extracts of

Psidium sartorianum, Piscidia piscipula, Bidens squarrosa, Casimiroa tetrameria

and the non-polar fraction of Bauhinia divahcata showed a weak activity with IC50

values between 20 and 90 ug/ml. Several plants were active against H. pylori,

which is considered to play a critical role in the pathogenesis of gastritis and peptic

ulcer (Eaton et al, 1991). The non-polar extract of P. piscipula showed the highest

activity (MIC: 0.7 jig/ml) followed by the polar one of the same species (MIC: 3

(.ig/ml) (Fabry et al., 1996; Cellini et al, 1996). The non-polar extracts of Casimiroa

tetrameria (MIC: 3 ug/ml) and Jatropha gaumeri (MIC: 5 ug/ml) were active against

H. pylori. Other active extracts were Dorstenia contrajerva (A and B) and the polar

extracts of Psidium sartorianum, Microgramma nitida, Chrysophyilum mexicanum.

The non-polar root extract of J. gaumeri was found to be the most active plant

tested against gram-positive B. cereus. Extract A of C. gaumeri as well as extracts

A and B of P. sartorianum showed weak activities against this strain. The ethanol

extract of G tetrameria weakly inhibited the growth of S. aureus. There was no

significant activity against the gram-negative bacterium C. jejuni. The butanol

fraction of C. gaumeri and the dichloromethane/methanol extract of Dorstenia

contrajerva weakly inhibited the growth of this strain. None of the tested plant

species used for gastrointestinal problems showed activity against E. coli and V.

enterocolitica.

Active plants for dermatological conditions

Selected plants, documented as medicines for dermatological conditions, were

evaluated for anti-inflammatory, antibacterial and antifungal activities (Table 4). To

determine the cytotoxicity of the plant extracts, KB cell line and the HeLa cell line

were used (Table 4).

Crossopetalum gaumeri showed the most potent effect in the NF-kB test with an

inhibitory activity down to a concentration of 25 jig/ml. Already at 100 jig/ml the

polar fraction was cytotoxic to the cell line during the period of the test. The non-

144

Table

3.Evaluation

ofplantspeciesused

forga

stro

inte

stin

aldisordersbytheYucatecMaya

Ext.

KB

IC*

G.duodenalis

H.

pylori

B.cereus

S.aureus

Plantname

MIC

ICso

MIC

600ug

200ug

600ug

200ug

[ug/

ml]

[ug/

ml]

[ug/

ml]

tug/

mi]

[mm]

[mm]

[mm]

[mm]

Bidenssquarrosa

B_

-

70

nt

.-

.-

Bauhiniadivaricata

A-

-

51

nt

--

-nt


A0.7

6.3

2.1

nt

-

12

-

B10.2

-

90

nt

--

-

nt

Jatrophagaumeri

A7.8

--

58

23

-

Dorsteniaco

ntra

jerv

aA

--

-

10

4-

--

B-

--

nt

3-

-nt

Psidiumsartorianum

A-

69.2

51

nt

-<1

1nt

B-

-

65

nt

4-

1nt

Piscidiapisc

ipul

aA

-41

27

0.7

11

--

nt

B-

-

70

312

--

nt

Microgramma

nitida

B-

--

nt

3-

-

nt

Casimiroatetrameria

A-

-72

34

--

C:1

Chrysophyllummexicanum

B-

--

nt

3-

-nt

Metronidazole

8.5

5

Ornidazole

1.1

0.5

Tetracycline

20

(10ug)

Ampicillin

23

(100ug

)Kanamycin

19(40ug

)Streptomycin

19

(50ug

)Chloramphenicol

22

(25ug)

8(10

ug)

8(10ug)

Cipr

oflo

xacin

1(0

.01)

KB:

cytotoxicity,

(-):

1C50

>50

ug/ml);

Gl:Giardiaduodenalis,

(-):

IC50>100

ug/m

l;H.

pylo

ri:

(-):<2mm;

Antibacterial

activitywasmeasuredas

inhibitionzone

in[mm];

(-):

no

activity;A:non-polar

extract,

B:po

larextract,

C:ethanol

extract;

nt:nottested.

Table

4.Evaluation

ofpl

antspeciesused

fordermatological

conditionsbytheYucatecMaya

Plantname

Ext.

KB,

IC50

NF-kB

S.epidermidis

200

ug

600ug

[ug/

ml]

[ug/

ml]

[mm]

[mm]

Aechmea

bracteata

B_

_3

5


A0.7

25

23

B10.2

*-

-

Dios

pyro

sanisandra

A14

100

nt

nt

Diospyroscuneata

A-

75

nt

nt

Jatrophagaumeri

A B A

7,8

--

Croton

reflexifolium

39

_

nt

nt

Caseariacorymbosa

A-

-

11

Psidiumsartorianum

A-

--

1B

--

1.5

2Morindayucatanensis

A-

-

24

Daleacarthagenensis

A31

150

--

Alvaradoaamorphoides

A10

--

-

B14

--

-

Lueheaspeciosa

A-

100

--

Podophyllotoxin

0.006

Parthenolide

10uM

PDTL

100'uM

Chloramphenicol

8(1

0ug)

8(1

0ug)

Tetracycline

Miconazole

E.

coli

C.albicans

600ug

600ug

[mm]

[mm]

6(10ug)

5(1

ug)

KB:

Cyto

toxi

city

,{-

):IC

>50

ug/m

l;NF-kB(HeLa

cell

line),

(-):>150

ug/ml;*cy

toto

xicat100ug/mldu

ring

theperiod

ofthe

test

;Antimicrobial

acti

vitywasmeasuredas

inhibition

zone

in[mm];

A:non-po

larex

trac

t,B:po

larex

trac

t,nt

:not

tested.

Publication HI

polar extracts of Diospyros anisandra, D. cuneata and Jatropha gaumeri inhibited

NF-kB. These effects are probably due to the potent cytotoxicity of the extracts.

The non-polar fractions of Dalea carthagenensis and Luehea speciosa elicited

remarkable inhibition of NF-kB binding at 150 ug/ml and 100 ug/ml, respectively.

The former one showed cytotoxic activity on KB cells (IC50; 31 ug/ml), whereas no

such effect could be detected for L. speciosa.

Several extracts showed antibacterial effects against gram-positive S. epidermidls

and gram-negative E. coli. The most active extract against the former strain was

the butanol extract of Aechmea bracteata. The non-polar fruit extract of Morinda

yucatanensis was active against S. epidermidls. Other active ones were: the non-

polar fraction of C. gaumeri and Casearia corymbosa as well as both extracts of P.

sartorianum. The growth of E. coli was inhibited by the dichloromethane/methanol

extract of D. anisandra at 600 jig of the tested plant species used for

dermatological conditions showed activity against S, aureus and P. aeruginosa.

Antimalaria activities

Plants used against fever and/or pain were screened in vitro for antimalaria activity

against P. falciparum (Table 5). The non-polar extract of Oestrum nocturnum

showed some antimalaria activity without exhibiting overt cytotoxicity.

D2-receptor binding affinities

Dopamine-agonists are used in the treatment of the premenstrual syndrome (PMS)

(Steiner, 1997). For the dopamine D2 receptor binding assay five plant species of

the group of "women's medicine" were chosen (Table 2). The affinities of the

extracts on the dopamine receptor, which inhibit the secretion of prolactin, were

tested. No significant activity could be observed. The non-polar extracts of Salvia

micrantha and Aristolochia maxima showed weak receptor affinities.

147

Publication III

Table 5. Anti-malaria activity of plant species used against fever and pain by the Yucatec

Mayai

Plant name Ext. IC50 against HB3 (CS) IC

50 against K1 (CR)[ug/ml] [ug/ml]

Ehretia tinifolia A

B

A

B

A

B

A

--

Caesalpinia gaumeri --

Casearia corymbosa 441.40(2.26) 494.49 (3.07)

Manilkara zapota --

Cestrum nocturnum

B

A

B

172.40(2.80) 283.31 (0.48)

Chloroquine 7.96 ng (0.03) 193.96 ng (0.81)

K1: chloroquine-resistant (CR) strain of Plasmodium falciparum;HB3: chloroquine-sensitive (CS) clone;

(- ): >500 ug/ml; no extract showed cytotoxicity in KB cell line (IC 50>50 ug/ml

Hypoglycemic effects

The polar extracts of five plant species, used against "diabetes" were tested for

their ability to inhibit a-amylase. The inhibition of the enzyme leads to a reduced

splitting of poly- and disaccharides of food in the colon. It comes to a delayed

resorption and to a balance of the value of blood sugar (Keller and Berger, 1983).

Bauhinia divaricata was the most potent inhibitor of the enzyme, followed by

Hylocereus undatus (Cactaceae) (Table 6). Polyphenols, known to interfere with

enzymes, don't occur very freguently in these genera or families (Hegnauer, 1989;

Hegnauer and Hegnauer, 1994).

148

Publication III

Table 6. Anti-diabetic plants tested for a-amylase inhibition effect

Extract: Extract: Extract:

Plant name Ext. 1 mg/ml 3 mg/ml 6 mg/ml

[U/l] [U/l] [U/l]

Ruellia nudiflora B 435 403 378

Malmea depressa B 400 288 79

Parmentiera millspaughiana B 424 205 93

Hylocereus undatus B 255 0 0

Bauhinia divaricata B 0 0 0

Negative control: 620 U/l; control with methanol: 548 U/l

Other activities

Some extracts showed activities in assays which are not directly related to the

indigenous uses (Table 7). The antibiotic effects of the polar and non-polar

fractions of Caesalpinia gaumeri used for pain of the body and headache are

particularly noteworthy.

Discussion

The species evaluated in this paper were selected because of their cultural

importance with the Yucatec Maya and because of their use(s) for specific

syndromes. In this section the relevance of these findings for interpreting the

indigenous uses is discussed.

The roots of Crossopetalum gaumeri are used orally for diarrhea and snake bites

and topically to prevent inflammation after a snake bite. In a detailed

phytochemical study of C. gaumeri the non-polar fraction showed the presence of

terpenoids, whereas the butanol extract consisted of several cardenolides (Ankli et

al.; 2000, accepted). The study exhibited that the terpenoids are responsible for

antibacterial activities, which support the therapeutical value for diarrhea. The

potent antiprotozoal effect as well as the potent inhibitory activity on NF-kB is

probably due to the high non-specific cytotoxicity of the extracts.

149

Table

7.

Activities

inthebiological

assayswhichcould

notdirectly

becorrelated

totheYucatecMayanuses

Plantname

(group

ofuse)

Ext.

NF-kB

[ug/

ml]

C.albicans

S.epidermidis

E.

coli

B.cereus

600ug

200ug

600

ug

600

ug

200

ug

600ug

[mm]

[mm]

[mm]

[mm]

[mm]

[mm]

Y.enterocolitica

600

ug

mm

Caesalpiniagaumeri(PFE)

A B

Caseariacorymbosa(DER,NEU)

Casimiroatetrameria(Gl)

A

Diospyrosanisandra(DER)

A

Lueheaspinosa(DER)

C

Piscidiapisc

ipula

(Gl)

A

Verbesinagigantea(RES)

B

100

2 1

3 2

2 1 nt

3 2 5

Antibacterial

acti

vitywasmeasuredas

inhibitionzone

in[mm];

nt:nottested.

Positivecontrol:

s.Table

3-4.

Publication III

The leaves of Piscidia piscipula are applied as a medicine for gastrointestinal

disorders (especially diarrhea and cramps) and for cough. The remarkable

activities of this species against H. pylori and to some extent against G. duodenalis

may be a reason why Yucatec Maya value this plant for treating gastrointestinal

problems. In a study by Caceres et al. (1991) P. piscipula is shown to have

antimycotic effects. Another species, P. erythrina L., has been widely investigated

and isoflavones were found as spasmolytic principles (Delia Loggia et al., 1994).

Among other ethnic groups, P. piscipula is used as fish poison (Acevedo-

Rodriguez, 1990). The determined activity in the NF-kB assay is of no direct

relevance to the traditional use of this plant (Table 7).

Bauhinia divaricata is used for a variety of illnesses like gastrointestinal problems

but more frequently for "diabetes" and respiratory problems. Its activity against G.

duodenalis may be one of the reasons for the indigenous use. The leaf extract of

B. purpurea L. was reported to have significant antidiarrheal activity in vivo

(Mukherjee et al., 1998). A possible hypoglycemic activity was also found in our a-

amylase test which supports the reported hypoglycemic effects in a previous study

by Roman R. et al. (1992).

The roots of Jatropha gaumeri are used for diarrhea and the resin is used as a

medicine for herpes (labialis). The therapeutic value against diarrhea may partially

be based on the antibacterial activity against B, cereus. Different pharmacological

effects are mentioned in the literature concerning the genus Jatropha, among

others antimicrobial effects (Odebiyi, 1980). Many taxa of the Euphorbiaceae are

known to have cytotoxic phorbol esters. Cytotoxic effects of J. curcas L. using the

brine shrimp assay were described (Gupta et al., 1996).

Psidium sartorianum is employed internally (diarrhea) and externally (mostly for

measles or any kind of pimples). The antibacterial activities {H. pylori, B. cereus

and S. epidermidis) might be of interest for the antidiarrheal therapy and the use

for skin problems. To evaluate the use against measles, antiviral tests will have to

be performed. No pharmacological data for P. sartorianum are available, but P.

151

Publication III

guajava L. was shown to have antibacterial effects, especially against the

enterobacterium Shigella.

In the "Ethno-Botany of the Maya" (Roys, 1976 [orig. 1931]) Casimiroa tetrameria

is mentioned as a medicine for gastrointestinal problems including diarrhea. Today

it is still used in this way but also for rheumatism and fever. The antiprotozoal effect

as well as the antibacterial activities against H. pylori and S. aureus may be of

relevance for the internal use. The antifungal activity is of no direct relevance for

the traditional use (Table 7). No pharmacological data are available, but when C.

edulis Llave and Lex. was tested against enterobacteria, no activity was detected

(Câceres et al., 1990). The antidiarrheal and antispasmodic effects are currently

investigated (Heneka et al, in preparation).

Diospyros anisandra and D. cuneata are used for dermatological problems. The

antibiotic and the antifungal activities explain the species' use in a rational way.

The NF-kB inhibiting activities is probably a result of the cytotoxic effects.

Antimicrobial activity of Diospyros lycioides Desf. (Li et al., 1998), as well as in vivo

anti-inflammatory activity of Diospyros leucomelas Poir. (Recio et al., 1995) and

antitumor promoting effects (Kapadia et al., 1997) were reported.

Various species of the genus Croton are used in the medical system of the

Yucatec Maya for dermatological problems but also for fever and respiratory

illnesses. The resin of C. reflexifolius is used for pimples in the mouth (herpes) and

eye problems. The possible occurrence of phorbol esters in this species

(Euphorbiaceae) may express the medicinal value of the use as an antiviral drug.

The application of the resin into the eyes might have severe side effects because

of the cytotoxicity.

The leaf decoction of Alvaradoa amorphoides is applied externally for skin

problems. Cytotoxic effects were found in the KB cell line, which is a common

effect in the Simaroubaceae family due to quassinoids (Phillipson, 1995).

Luehea speciosa is an effective inhibitor of the transcription factor NF-kB, which

controls genes responsible for the inflammatory responses of the body. The

Yucatec Maya value this plant for treating skin diseases and toothache, and apply

152

Publication III

the medicine in form of plasters. The inhibitory effect on NF-kB partially explains

the use of this plant. The weak antibacterial activity against Y. enterocolitica can

not substantiate the traditional use of the plant. To our knowledge no

pharmacological or phytochemical data have been published yet.

Aechmea bracteata (Bromeliaceae) and Morinda yucatanensis are applied in form

of plasters against snake bites and infected wounds and showed antibacterial

potency against S. epidermidls. This confirms the indigenous uses. M.

yucatanensis is also employed in the treatment of warts. No data on the antiviral

effect of this species are available, but many studies on Morinda spp, concerning

antitumor or antimalaria activities are available (Awe & Makinde, 1998).

Several reports on plants applied for fever are obtained, but only one of the healer

explicitly mentioned malaria. Cestrum nocturnum is applied for children with night

fever and cold bodies. Even though the drug was not mentioned specifically for

malaria by the Maya, further testing of fractions of this plant would be of interest.

Phytochemically the species is well investigated but no data have been found for

antimalarial effect (Ahmad et al. 1991),

The therapeutical group of "women's medicine" contains a lot of plants used to

relieve pain of delivery, induce labor or help "fulfill the desire for a child" (infertility).

The symptoms of PMS were never mentioned explicitly by the Mayan healers and

midwives but reports of menstrual pain are documented. A possible explanation of

the negative results of the screening may be that the bioassay is specific for PMS

and of no immediate relevance to the indigenous uses.

Conclusion

One goal of this evaluation is to better understand the use of Yucatec Mayan plant

and their healing concepts. In this paper we show some correlations between uses

of the medicinal plants and the activities obtained in the selected bioassays. Other

indigenous uses currently cannot be explained in a bioscientific manner because

the bioassays applied are not appropriate. In other cases the symbolic aspects

might be more important to the Maya. The combination of a detailed

153

Publication IN

documentation of ethnomedical uses and bioassays, which are of immediate

relevance for these uses, are of great importance and lead to a better

understanding of the ethnopharmacopoeia of the Yucatec Maya (and other

indigenous groups).

Evaluations of plants as well as the phytochemical and further pharmacological

studies are important tasks. Organizations like WHO (World Health Organization)

and TRAMIL (Central America; popular pharmacopoeia) encourage the use of

remedies that have been proven to be safe and effective. Herbal medicines are a

valuable and readily available resource for primary health care and complementary

health care systems. Hopefully this study contributes to strengthen and promote

the use of traditional medicine.

Acknowledgements

The authors wish to thank all persons who have helped in the field study and

especially the healers, midwives and the inhabitants of Chikindzonot, Ekpedz and

Xcocmil, Yucatan, for their collaboration, for their friendship and hospitality. The

botanical identification at CICY {Centro de Investigacion Cientffica de Yucatan ),

and MEXU (National Herbarium of Mexico) was performed in collaboration with the

numerous specialists of these institutions. Particularly we would like to thank Dra. I.

Olmsted, J. Granados, P. Sirna, J.C. Trejo, Dr. R. Duran of CICY as well as O.

Tellez, Dr. R. Lira, Dr. J. Villaserior and Dr. M. Sousa of MEXU. This research

owes a lot to the help of Dr. J. Heilmann (Zürich), Dr. J, Orjala (Davis), Dr. B. Frei

Haller (Zernez), Prof, Dr. W. Schaffner (Basel) and Prof. Dr. H. Rimpler (Freiburg).

Financial support by SDC (Swiss Agency for Development and Cooperation,

Berne, Switzerland) and the SANW (Swiss Academy of Natural Sciences) is

gratefully acknowledged.

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159

Plant Evaluation

6 Additional results

6.1 Antimicrobial activity

In addition to the antimicrobial tests reported in publication III, two further strains

were used to evaluate the antimicrobial activity of the 96 plant extracts, namely

Micrococcus luteus (ATCC 9341) and Mycobacterium fortuitum (isolated from a

patient). M. luteus is a gram-positive coccus whereas M. fortuitum belongs to the

gram-negative, acid fast asporogenous rods. M. fortuitum causes dermatological

problems (local abscess) or less often illnesses of the bones and junctions. The

genus Mycobacterium, which causes important human infections such as

tuberculosis and leprosy, is of high interest due to these diseases and the problem

of resistance against known antibiotics.

The antimicrobial tests were performed with all extracts mentioned in Table 2 of

publication III using the disc diffusion technique as described in the same

publication. M. fortuitum is a slow growing bacterium, thus the cultivation takes

several weeks. Because of contamination with faster growing bacteria, the results

obtained with M. fortuitum must be interpreted with great caution.

Table 6.1. Activitiy of plant extracts against M. luteus and M. fortuitum

Plant name (family) Ext. M. luteus M. fortuitum''

200 ug [mm] [mm]

Aechmea bracteata (Bromeliaceae) B - 2

Caesalpinia gaumeri (Caesalpiniaceae)

Casearia corymbosa (Flacourtiaceae)

Crossopetalum gaumeri (Celastraceae)

Jatropha gaumeri (Euphorbiaceae)

Psidium sartorianum (Myrtaceae)

Chloramphenicol 1 ug

Tetracycline 5 ug

A 1

A -

A 1

A -

B 1

B 1

- 3.5

1.5

A: non-polar extract; B: polar extract; spat|ent material; obtained from the Institute of

Medical Microbiology, University of Zurich; -: not active:

160

Plant Evaluation

The water extracts of the 48 plants (Table 2, publication III), obtained from the

liquid-liquid partition between 1-butanol and water, were also examined against

gram positive and gram-negative bacteria, the fungus C. albicans as well as for

KB-cell cytotoxicity. In two cases the water extract were active: Psidium

sartorianum was active against S. epidermidls and M. fortuitum, and Crosso¬

petalum gaumeri showed KB cell cytotoxicity.

6.2 Comparison of disk method and TLC method

The non-polar and the polar extracts of seven species were not only tested with the

disc method but also using a TLC method. By means of micropipettes the same

quantity of extract was applied directly onto a TLC plate (9x9 cm) and 10 ml of the

overnight culture was poured over it (TLC in petri plates; 10x10 cm). The test

organisms used were B. cereus, S. epidermidls and E. coli. The inhibition zones of

the extracts using the TLC method were compared with those of the disc method

(Table 6.2).

Results: The inhibition zones of the extracts obtained from the TLC method were

generally larger than the inhibition zones of the same extracts in the disc method.

The only exception was the non-polar extract (A) of C. gaumeri tested against S.

epidermidls. Here the inhibition zone obtained from the disc method is larger than

the one from the TLC method. Generally, the differences between the two methods

were more pronounced among the polar extracts (B) and less important among the

non-polar extracts (A). This phenomenon seems to be caused by the

disadvantageous diffusion of the non-polar extracts (A) from the discs into the polar

agar medium. Furthermore, the contact of the extracts with the agar is limited in the

disc method. As a consequence, the zone of inhibition in the disk method was

generally smaller than the one of the TLC method. In order to compare results

standardized methods and standard positive controls should be used. However, in

this case, the TLC technique is preferred due to decreased diffusion problem and

minimized error.

161

Plant Evaluation

Table 6.2. Comparison of inhibition zones obtained from disc - and TLC method

Plant name Ext. B. cereus S. epidermidis t. coli

TLC Disc TLC Disc TLC Disc

Caesalpinia gaumeri A 2 2 2 2 -

(Caesapliniaceae) B 3 - 3 1 -

Casearia corymbosa A 1.5 1.5 1 1 .

(Flacourtiaceae) B 2 - 2 -.

Crossopetalum gaumeri A 1 1 0.5 2 -

(Celastraceae) B 2 - 1.5 - .

Dalea carthaginensis A 2 < 1 3 - 1

(Papilionaceae) B 2 - 1 - .

Diospyros anisandra A 7 <5 15 nt 2 < 1

(Ebenaceae) B 3 - 5 nt -

Jatropha gaumeri A 3 2 4 < 1 .

(Euphorbiaceae) B - - - - -

Psidium sartorianum A 2 < 1 2 - .

(Myrtaceae) B 2 - 2 1.5 -

Chloramphenicol 1 ug - 10 2.5 - - -

Tetracycline 1 jig - - - 2 - 6 4

Quantity of extract tested: 200 ug; the inhibition zone is given in [mm]; nt = not tested; - : not active.

6.3 Protein kinase activity: Method and results

Protein kinases (PK) are enzymes that transmit phosphate to proteins. With the

induced change of conformation in proteins, they regulate different physiological

processes. Thus, they are important in the control of growth, division and

differentiation of cells. Based on today's knowledge, overexpression and lack of

control of PK activities are involved in the creation and development of tumors.

Therefore, they are targets for the development of new drugs in tumor therapy, as

well as in treatment of atherosclerosis, psoriasis and inflammatory processes.

Method: The non-polar (A) and the polar (B) extracts of 33 medicinal plants used in

the treatment of respiratory illnesses (RES), dermatological conditions (DER),

illnesses associated with pain or fever (PFE) and women's health (FEM) were

evaluated in a high throughput screening with PKs. The catalytic activity of the

162

Plant Evaluation

enzymes was measured in 96-well microtiter plates with recombinantly produced

human PKs. By means of 33P-marked phosphate, their incorporation into the

substrate was measured. The reaction volume used was 50 ul and the incubation

time was 80 minutes at 30 °C (Schachtele and Totzke, 1999). The concentration

tested of the non-polar (A) and polar (B) extracts were 1 and 10 ug/ml. The results

in Table 6.3 refer only to the lower concentration (1 ug/ml) in order to focus on the

extracts with pronounced activity. The plant family and the authors of the species

are given in Table 2 of publication III. The following kinases were employed in the

screening:

CDK4/CyclinD1 * TIE-2 » PKB (AKT)

CDK2/CyclinE ErbB2 FLT-4

EGFR Kinase IGF-R1

Results: The results are compared with those of the KB cell test and the NF-kB test

(publication III). Of the 66 tested plant fractions tested with 8 different PK, 66

positive results on the PKs were obtained (13 %) (Table 6.3). Extract B of A.

vesicaria showed 53 % specific inhibition of CDK2/CyclinE kinase. The same

extract did not show any activity in the KB cell test and the NF-kB test. However,

the non-polar extract (A) exhibited weak cytotoxic effect at 50 ppm. The polar

extracts (B) of A. bracteata var. bracteata (58 %), H. baruensis (93 %), P. piscipula

(72 %) and S. micrantha (91 %) blocked the PKB and did not show any inhibition of

other PKs. Of these four species the lipophilic extract (A) of P. piscipula were

active in the NF-kB test, down to a concentration of 100 pig/ml and the lipophilic

extract (A) of S. micrantha showed weak KB cell cytotoxicity at 50 ppm. The

hydrophilic extracts (B) of B. verticillata, C. corymbosa and E. tinifolia were active

against IGF-R1 kinase and PKB, respectively. From these plants, the non-polar

extract (A) of E. tinifolia was weakly cytotoxic in the KB cells system at 50 ppm.

163

Table

6.3.

Inhibition

ofdifferentprotei

nkinasesby

plan

textracts

at

1ug/ml

Plantname(GROUPOFUSE)

%effect

Ext.

CDK4/

CDK2/

EGFR

1GF-R1

CycIinDI

CyclinE

Kinase

TIE-2

PKB

(AKT)

FLT-4

Aechmea

bracteata

var.bracteata,

DER,FEM

Alvaradoaamorphoides,DER

Anredera

vesi

cari

a,DER

Anthunum

schlechtendaliissp.schlechtendalii,

FEM

Bauhinia

diva

rica

ta,RES

Borreria

verticiIlata,DER

Caesalpiniagaumeri,PFE

Caseariacorymbosa,DER

Croton

refl

exif

oliu

s,DER

Daleacarthaginensis,DER

Ehretia

tini

foli

a,PFE,RES

Helicteresbaruensis,FEM

Neomillspaughiaemarginata,DER

Piscidiapiscipula,RES

Psidiumsartorianum,RES

Salviamicrantha,DER,RES,FEM

B A A A A B B B B A B B

-70

-62

-57

-5£

-53

-71

-53

-68

-53

-56

-58

-55

-65

-82

-82

-86

-86

58

-51

-63

-69

54

-83

-89

-87

68

-65

-82

55

-57

-80

-56

-55

-63

-67

-80

-75

-89

-86

-58

-97

-101

-57

-96

-65

-83

-83

-80

-92

-84

-99

-98

-97

-93

-102

-72

-82

-100

-91

-80

-72

-69

-64

-54

-95

Plant Evaluation

Quite unspecific PK inhibition was caused by the polar extracts (B) of B. divaricata,

C. reflexifolius, D. carthaginensis and N. emarginata. They were active against

three to six different PKs. From B. divaricata not only the polar (B), but also the

non-polar (A) extract inhibited the PKB. Furthermore, extract A was weakly active

in the KB cell test at 50 ppm. The genus Croton of the Euphorbiaceae family is

known to have PK activity and to be cytotoxic due to phorbol esters. The

cytotoxicity of the non-polar extract (A) of C. reflexifolius (Euphorbiaceae) was

determined as IC50= 39 ug/ml. The non-polar extract of D. carthaginensis showed

cytotoxicity in the KB cell system (IC50= 31 ug/ml) as well as NF-kB activity down

to a concentration of 150 ug/ml.

The most unspecific activities in the PK screening were obtained from A.

amorphoides, A. schlechtendalii spp. schlechtendalii, C. gaumeri and P.

sartorianum. Both extracts of A. amorphoides showed PK inhibition and

additionally strong KB cytotoxicity (A: IC53= 10 ug/ml, B: IC50= 14 ug/ml). Extract A

of P. sartorianum exhibited PK inhibition as well as weak KB cytotoxicity at 50 ppm.

With respect of the search for new and specific kinase inhibitors, these four

species are less important due to the inhibition of all PKs tested.

Comparing the results of the PK tests, it is significant that polar extracts (B) were

particularly active, whereas the non-polar ones were less active or did not show

any PK inhibition. In contrast, only non-polar extracts (A) showed activity in the KB-

cell and NF-kB tests, with the exception of extracts of A. amorphoides (publication

111). However, cytotoxicity and PK activity do not necessarily correlate, this may

partially be due to the differences in the methods used. Explanation of these

phenomena presumably can be found in the different methods of the test systems.

The kinases are enzymes produced by recombinant technology and are not

incorporated into cells, whereas the KB cell test and the NF-kB test are performed

with human cell lines. To inhibit PKs located inside the cells, the compounds first

have to pass the lipophilic cell membrane. Non-polar extracts are able to pass the

membranes more easily than polar ones and, therefore, are advantageous to

165

Plant Evaluation

inhibit the PKs. Thus, particularly lipophilic extracts showed positive effect in the

cell line tests.

Another problem, in the case of crude extracts, especially in polar ones, is the rate

of false positive results. Highly purified enzyme-based targets and cell membranes

can be affected by polyphenols and saponins, respectively (O'Neill and Lewis,

1993). This is most likely reason why a higher amount of polar extracts (B) in

comparison to non-polar extracts (A) showed inhibition in the PK test system.

Most of the crude extracts tested have the capacity to inhibit PKB. Accordingly, this

kinase seems an ineffective target for providing a useful basis for specific drug

development. However, tyrosine kinase of the ErbB2 receptor was not effected by

any of the plant products. Compound or extracts that inhibit this type of PK would

probably be more interesting for further investigations.

Is there a link between the results of the PK test and traditional phytotherapy?

The initial idea was to screen the ethnobotanically pre-evaluated drugs used in the

treatment of respiratory illnesses (RES), dermatological conditions (DER) and

illnesses associated with pain or fever (PFE) for their anti-inflammatory potency.

The results of the screening showed a relative high number of active samples as

well as promising results. The ethnobotanical approach may be a reason for these

effects. Another reason may be the false positive results based on the presence of

polyphenols (e.g. P. sartorianum). It has to be pointed out, that polyphenols are

important compounds in popular phytotherapy, however they are less important in

the search for new drugs. Their ability to unspecifically inhibit enzymes, to act as

antioxidants as well as further effects, may have positive influences in the healing

process. Generally, the PK test is too specific for evaluating plant extracts and for

explaining the mechanism of healing. Therefore, further bioassays relevant for the

species use have to be conducted. In addition, more detailed phytochemical

investigation of the extracts and subsequent PK tests with isolated compounds

have to be performed. Also biopharmaceutical aspect (resorption, distribution,

metabolism) of the compounds must be incorporated into a detailed study of the

plant remedies.

166

Plant Evaluation

6.4 Other activities

Casimiroa tetrameria:

Casimiroa tetrameria Millsp. (Rutaceae), Hylocereus undatus (L.) Britton & Rose

(Cactaceae) and Bidens squarrosa Less. (Asteraceae) are used against diarrhea.

Hence, they were tested for their antisecretory activity in the USSING chamber

model (Greger et al., 1991). The crude extract of the leaves of C. tetrameria

reversed the Prostaglandine E2-induced CT-secretion significantly. Thus, this

species was chosen for further phytochemical investigations. Among others, four

fractions each containing a different polymethoxyflavone as the main compound,

showed the highest antisecretory activity. The same polymethoxyflavone

containing fractions showed significant antispasmodic activity when tested on the

isolated guinea pig ileal smooth muscle (Samuelsson, 1991). (This work was

carried out by Bilkis Heneka at the Institute für Pharmazeutische Biologie, Albert-

Ludwigs Universität Freiburg, Germany).

Malmea depressa:

The root of Malmea depressa (Baill.) R.E.Fr. [syn.: Guatteria leiophylla (Donn. Sm.)

Saff. Ex. Standi.,- Guatteria gaumeri Greenmann (Fries, 1939)] (Annonaceae) is the

most important remedy for the treatment of diabetes type II in Yucatan. Based on

ethnobotanical information, an aqueous root extract was prepared and tested on

male Whistar rats. The test medium, dissolved in physiological NaCI-solution, was

given orally to rats after an intraperitoneal injection of 50 mg/kg streptozotozin and

waiting 7 to 15 days. In addition, the test was performed with pure NaCI-solution

and glibenclamide (positive control). The application of the aqueous root extract of

M. depressa led to promising results.

Hernandez et al. (1993) studied the effect of ce-asarone, a hypolipidemic active

principle of M. depressa. Jimenez-Arellanes and Mata (1996) phytochemically

investigated this plant. They isolated five phenylpropanoids from the CHCI3 extract

of the stem bark. It might be possible that also these compounds are responsible

167

Plant Evaluation

for the hypoglycemic effect of the remedy since several similar compounds were

found to show hypoglycemic activity (Hernandez et al., 1993; Sarges et al., 1996).

6.5 Crossopetalum gaumeri -the plant species for the phytochemical study

Crossopetalum gaumeri was chosen for the detailed phytochemical study and

further biological investigations. The following points led to this selection:

its importance as a remedy among the Yucatec Maya of the study region

the healers consensus concerning the plant use and preparation

the use in a decoction against diarrhea, which is a major health problem among

Yucatec Maya

the oral use of the fresh root against snake bites

the endemic occurrence of the species on the Yucatan Peninsula

the cytotoxic and antibacterial effects detected in the in-house bioassays

4 the lack of the phytochemical investigation of C. gaumeri

the few phytochemical studies, which have been carried out with the genus

Crossopetalum

» the importance of the Celastraceae family in the search for new, effective

compounds in the field of antibiotics and cancer therapy (Kupchan et al., 1972b).

C. gaumeri was chosen for the phytochemical investigation with the aim isolating

the antibacterial and cytotoxic principles. Furthermore, the research was carried

out to clarify the empirical phytotherapy of C. gaumeri.

168

Part III Phytochemistry of Crossopetalum gaumeri

Phytochemistry

7 Celastraceae family and the genus Crossopetalum

7.1 Botanical taxonomy

Species: Crossopetalum gaumeri (Loes.) Lundell

Genus: Crossopetalum

Family: Celastraceae (Bittersweet family)

Order: Celastrales

Subclass: Rosidae

Class: Rosopsida (former; Dicotyledoneae)

Subdivision: Magnoliophytina (Angiospermae)

Division: Spermatophyta

(Frohne and Jensen, 1998)

The family Celastraceae including the Hippocrateaceae encompasses 94 genera

with approximately 1,300 species. It is a widely distributed family, which is native to

the tropics. Fewer species are native to warmer temperate regions. The species of

this family, usually glabrous with laticifers, are trees, shrubs and lianas. The chief

genera are Cassine, Celastrus, Crossopetalum, Hippocratea, Maytenus, and

Salacia (Mabberley, 1996).

The genus Crossopetalum is represented by 36 species in tropical America. They

are little trees and shrubs with indeciduous, opposite leaves. They are basically

distributed in Central America and the Antilles, only one species is reported from

South America, namely Crossopetalum rhacoma Crantz (Gentry 1993). The same

species, which is the most widely distributed one of the genus, is also native to

Florida. On the Yucatan Peninsula two species of the genus Crossopetalum are

known, namely C, gaumeri and C. rhacoma.

170

Phytochemistry

7.1.1 Crossopetalum gaumeri

C. gaumeri has different synonyms, these include Rhacoma gaumeri (Loes.)

Standi, and Myginda gaumeri Loes (Figure 7.1). The habitat of the plant is moist,

wet thickets or thin forests, mostly on limestone, higher than 200 m above sea

level. The species is widely distributed on the Yucatan Peninsula, which includes

the Mexican states of Yucatan, Campeche and Quintana Roo, as well as in Belize

and the northern part of Guatemala. The Mayan name of the plant in Yucatan is

recorded as camba-och-lob (Standley and Steyermark, 1949). Today the Maya in

the area of the place of this study call the plant viperol.

Figure 7.1. Crossopetalum gaumeri (Loes.) Lundel

171

Phytochemistry

Botanical description of C. gaumeri (Standley and Steyermark, 1949):

"A shrub, commonly about a meter high, glabrous except in the inflorescence, the

young branches green, tetragonous, sometimes sparsely hirtellous; leaves on

rather stout petioles 12 mm long or shorter, ovate to elliptic or obovate-elliptic, 5-11

cm long, 2-5.5 cm wide, obtuse to acuminate, usually bright green when dried,

coriaceous, lustrous, narrowly long-attenuate to the base, appressed-serrate, often

conspicuously so, the nerves and vines elevated and very conspicuous on both

surfaces, the veins closely reticulate; inflorescences mostly 4 cm long or shorter,

few-many-flowered, the branches stout, the pedicels filiform, much longer than the

flowers, puberulent; flowers densely puberulent or minutely pilose; calyx 1.5 mm

broad, the lobes short, rounded; petals maroon-red, orbicular, more than 1 mm

long; stamens very short; style almost none; fruit obovoid, somewhat asymmetric,

almost or fully 1 cm long, somewhat narrowed at the base."

7.2 Phytochemistry of the Celastraceae

The family of Celastraceae and the Hippocrateaceae are discussed together due to

the similarities in chemosystematics (chapter 7.5). The members of Celastraceae

family generally contain dulcit, terpenoids, polyisoprenes, polyphenols and

alkaloids (Brüning and Wagner, 1978).

7.2.1 Terpenoids (terpenes, isoprenoids)

Cardiotonic steroids (cardenolides)

Euonymus europaea L. is the best known species of Celastraceae family in Middle

Europe. The seeds of a brilliant orange (Carotinoids) are toxic due to the presence

of cardiotonic steroids and alkaloids. The cardiac glycosides of the Celastraceae

belong without exception to the cardenolide type. The different mono-, di- and

triglycosides of the species of Euonymus are generally derived from digitoxigenin

and cannogenol.

172

Phytochemistry

Digitoxigenin: R^R^R^H, R3=CH3

Cannogenol: R1=R2=R4=H. R3=CH2OH

Strophanthidin: R1= R4=H, R2=OH, R3=CHO

Antiarigenin: R1= H, R2=R,;=OH, R3=CHO

The aqueous bark extract of Lophopetalum toxicum Loher was used as arrow

poison among Philippinan indigenous people. Cardiac glycosides from the

strophanthidin and antiarigenin type were isolated from the bark, which was shown

to be cytotoxic and to have positive inotropic activity (Habermeier, 1980; Wagner et

al. 1984). Indigenous people of the Philippiness mentioned that during the

preparation of the arrow poison contact with acidic substances must be avoided

not to decrease or neutralize the effect of the poison. This may be due to the

aldehyde function on position C-10 of the aglycon, which is easily autoxidated in

water (~> oxidation to carboxyl group -» decarboxylation) (Hansel et al., 1999).

An unusual type of cardiac glycoside was isolated from seeds of Elaeodendron

glaucum Pers. by Kupchan et al. (1977). The elaeodendrosids show double bonds

on position C-4/5 and contain double binding sugars like some cardenolides of the

family Asclepiadaceae.

0\ ^O

* Elaeodendroside

O O

173

Phytochemistry

Thus, cardenolides were found in three genera (Euonymus, Lophopetalum and

Elaeondron) of the Celastraceae (Hegnauer, 1989). With the isolation of cardiac

glycosides from Crossopetalum gaumeri a fourth genus is added.

Quinone triterpene (celastroloid, quinone methide)

The orange-red pigments of the root bark of several species of the family

Celastraceae are pentacyclic triterpenes. They have a conjugated lOrt-system with

a quinone methide part of ring A and B. These compounds are characteristic

structural types for the Celastraceae. Pristimerin and celastrol are two prototypes

of these compounds. Tingenon is another member of the quinone triterpenes.

Several celastroids showed interesting activity like high cytotoxicity and

antibacterial activity.

yRi

» Pristimerin: R^COOCHg

Celastol: R, = COOH

4 Tingenon: H^ = H, C-21 : keto group

Non-quinone triterpene

Triterpenes with friedelane and oleanan skeleton are common in the Celastraceae

as in many other taxa. Friedelane triterpenes with different oxidation levels were

isolated from several species of the Celastraceae. Some of them are precursors of

the characteristic quinone methides like the maytenon acid with antileukemic

activity (Abraham et al., 1971). Besides these triterpenes, phytosterines, lupan-

and taraxeran triterpenes have been isolated from the Celastraceae.

174

Phytochemistry

* Friedelane: R1 = H

* Maytenon acid: R^ = COOH

Diterpenes and sesquiterpenes

Several diterpenes with an abietan skeleton and their derivatives are known from

members of the Celastraceae. Interesting tri-epoxide diterpenes, particularly

triptolid and tripdiolid, with antileukemic activities were isolated from Tripterygium

wilfordii Hook f. (Kupchan et a!., 1972a). Other characteristic terpenoids are

sesquiterpenes (dihydroagarofuran) and sesquiterpene-polyalkaloids. Compounds

of both of these structure types are described to have insecticidal and insectifuge

effects (Delle Monache et al., 1984).

4 Triptolid, R,= H

* Tripdiolid, R,= OH

Dihydroagarofuran

175

Phytochemistry

7.2.2 Alkaloids

A great variety of alkaloids have been found in the Celastraceae but most of them

were isolated in very small amounts. Phenylalkylamines are well known from the

fresh leaves of Catha edulis (Vahl) Endl. (Cathin = norpseudo-ephedrin). Other

isolated alkaloids are tetrahydroisochinolin and tetrahydrochinolin alkaloids,

pyrrolizidin, peptid, spermidin, xanthin and macrocyclic alkaloids. The antitumor

active maytansinoids were isolated for the first time from Maytenus species

(Kupchan etal., 1972b).

4 General structure of

maytansinoids

7.2.3 Flavonoids and other phenolic compounds

Most of the flavonoids are kaempferol and quercetin glycosides. Catechins,

particularly ourateacatechines, are characteristic of Celastraceae. Proanthocyani-

dines, anthocyanidines and tannins have been confirmed in several Celastraceae.

7.3 Phytochemistry of Crossopetalum species

The genus Crossopetalum has not been well investigated phytochemically. From

Crossopetalum tonduzii (Loes.) Lund, sesquiterpenes (Tincusi et al., 1998) and

from Crossopetalum uragoga 0. Ktze triterpenes have been isolated (Dommguez

etal. 1984).

176

Phytochemistry

7.4 Biosynthesis of terpenoids (terpenes, isoprenoids)

At present more than 22,000 natural isoprenoids are known (Zamponi et al. 1998).

They occur in all organisms, but a great variety of them were found in higher

plants, however, they are not randomly distributed over the plant kingdom. Certain

isoprenoids or combinations of them are characteristic of plant species and are

helpful in the chemosystematic. Most terpenoids are considered "secondary"

metabolites, because they lack an apparent role in the basic processes of growth

and development. Many are thought to have ecological functions, serving as

defense against herbivores and pathogens, as attractant for pollinators and fruit-

dispersing animals, or as allelopathics.

Terpenoids are biosynthesized from isoprene moieties. Squalene, or in higher

plants oxidosqualene (C30), is the biosynthetic intermediate for triterpenoids and

steroids. During phytosterol biosynthesis, it is enzymatically cyclized to

cycloartenol. After an oxidative separation of 3 methyl groups cholesterol is built,

which is an intermediate for all steroids (Figure 7.2).

Cyclization to pentacyclic triterpenes such as ß-amyrin proceeds via the chair-

chair-chair conformation of oxidosqualene. Abe et al. (1993) demonstrated that the

proton-initiated cyclization produces first the tetracyclic dammarenyl cation. The

subsequent rearrangement leads to the baccharenyl and lupenyl cationic

intermediates and to the pentacyclic oleanyl cation. Finally a series of 1,2-hydride

shifts with elimination of the H-12 proton gives ß-amyrin. Friedelane triterpenes

belong to the oleanen triterpene group and are biosynthesized by different methyl

and hydrid shifts, particularly the 4,5-methyl shift of one of the methyl group on C-4

(Brieskom, 1987). Friedelane is supposed to be the precursor of the natural

triterpene quinone methides. Itokawa et al. (1991) suggested a biogenetic route

involving the oxidative elimination of the methyl group on position 24 from

friedelane to give pristimerin-type triterpenes (Figure 7.3).

177

^ 2,3-Oxidosqualene

enzymatic cyclization

chair-chair-boat conformation

HO'

Protosteryl cation

Cycloartenol, C30 -3C

1

chair-chair-chair conformation

Tetracyclic dammarenyl cation

Y

Baccherenyl cation

Lupenyl cation

Oleanyl cation

I

HO

Cholesterol, C27

Side chain shortening

Addition of acetyl-CoA

Figure 7.2. Biosynthesis scheme of steroids and terpenoids

Cardenolides, C23

intermediate stage:

ß-Amyrin/Friedelin

Friedelane-3-on-29-ol

CH2OH

O.

HO'

AH

A

Pristimerin

o.'^r

CHO

Cangoronine A

HO/;,..

Salaspermic acid

COOH

Maytenoic acid

Figure 7.3. Proposed biogenetic pathway forcelastroloids

£OOH

fKH

COOH

Phytochemistry

Tricyclic diterpenes, particularly abietan diterpenes, are built by an enzymatic

cyclization of geranylgeraniol (C20) to a dicyclic diterpene, namely labdadien.

Abietan is then built by ring closure of labdadien to pimaran and subsequently it is

modified by methyl shifts (Hanson, 1971) (Figure 7.4).

H^

V.*H >

Geranylgeraniol

-4$f!L-_

Labdadien

\ H

Abietan Pimaran

Figure 7.4. Biosynthesis scheme of abietan diterpenes

7.5 Chemosystematic and phylogenetic relationships

Celastraceae and Hippocrateaceae contain several common compounds like

dulcit, guttapercha, proanthocyanidines, phenolcarbon acids and celastroloids.

The very close chemotaxonomical relations between these two families as well as

other criteria justify their fusion, however, no sesquiterpene polyesters and

polyesteralkaloids have been found in the Hippocrateaceae. In recent literature like

Mabberley (1996) or Frohne and Jensen (1998) only the Celastraceae are listed

whereas the Hippocrateaceae are not mentioned as a separate family. The

180

Phytochemistry

chemical similarities of the Celastraceae with other families within and beyond the

order Celastrales is shown in Figure 7 5

Figure 7.5. Chemical relationship of the Celastraceae with other families (Bruning

and Wagner, 1978)

The phytogeny of the order Celastrales is not clear. The definition of this order

varies according to different authors. The term Celastrales was first introduced by

Bentham and Hooker (1862) and was defined, among other characters, by "ovula

erecta, raphe ventralis". They included the families Celastraceae, Stack-

housiaceae, Rhamnaceae and Vitaceae. In the Engler system of Melchior (1964),

other families, particularly Aquifoliaceae and Icacinaceae, were added to

Celastrales due to some vegetative features. They were also placed in this order in

the classification of Takhtajan (1980). However, Thome (1983) excluded

Aquifoliaceae and Icacinaceae from Celastrales, placing them in Theales and

181

Phytochemistry

Cornales, respectively. Savolainen et al. (1994) suggest that the majority of the

morphological characters used to separate families of the Celastrales are

insufficient. Therefore, they carried out molecular systematic investigations with

chloroplast genes of 19 species of angiosperms. According to their results, the

order Celstrales is polyphyletic. The lineage of Celastrales should be restricted to

Celastraceae including Hippocrateaceae. The Aquifoliaceae and Icacinaceae do

not belong to this order, but form a separate lineage. Furthermore, the authors

mentioned that Euphorbiaceae (Euphorbia, Mercurialis) comprises a sister group of

the Celastrales (Euonymus, Salacia. Hippocratea). Together, they form a

monophyletic group with the Rosaceae (Rosa, Geum, Malus) and the

Rhamnaceae (Rhamnus) (compare with Figure 7.5).

Takhtajan (1980) indicated a common ancestor for the Celastrales and Santalales,

being derived from the Saxifragales. According to Frohne and Jensen (1998),

Celastrales and Santalales are listed in the same group of order and relationships

with Urticales, Saxifragales and Caryophyllidae are mentioned.

7.6 Biological activities among the Celastraceae

Many compounds isolated from the Celastraceae exhibited biological activities,

some of them showed promising pharmacological effects (Table 7,1).

In recent years, an impressive number of phytochemical studies on Trypterygium

wiifordii Hook f. (Thunder God vine) were carried out, particularly by the Takaishi

group. The plant has been used for several illnesses in Chinese folk medicine

(Table 7.2). Due to in vitro and in vivo antileukemic activity of the alcoholic extract

a detailed phytochemical investigation was made and led to the isolation of

antileukemic diterpenoid triepoxides (Kupchan et al. 1972a). The evaluation of

triptolide showed relatively potent but non-specific cytotoxicity and modest

antitumor activity (Shamon et al., 1997). In other studies the plant species was

found to show anti-inflammatory, anti-tumor, anti-immunosuppressive and anti-

AIDS activity (Shishido et al. 1994; Morota et al. 1995; Ujita et al., 1993; Chen et

182

Phytochemistry

al. 1992). Salsaspermic acid (Figure 7.3) also isolated from Thpterygium wilfordii

is reported to show promising anti-HIV activity.

The maytansinoid alkaloids (chapter 7.2.2) isolated from several species of

Celastraceae showed high cytotoxicity (KB cells: ED5010"4-10"6 ug/ml). Maytansine

was found to have the greatest potency and has been studied extensively as an

anti-tumor agent. The compound binds to tubulin and leads to arrest of cell division

in metaphase (antimitotic). Maytansine was tested in several clinical phase I and II

studies. Beside some positive clinical observations, several toxic effects were

reported, particularly neurological and gastrointestinal toxicity, and therefore, the

clinical study was stopped (Smith and Powell, 1984). In recent years the interest in

maytansinoids has increased again due to success in binding to mice-antibodies.

These carriers bring the drug to the tumor cells where they bind to receptors and

are concentrated. Thus, the toxic effects can be avoided. This effect must be

investigated further (Chari et al., 1992).

183

Table7

1Biological

activities

ofcompounds

ofCelastraceae

Activity

Responsible

compounds

Plantname

Literature

posi

tiv-

inot

rop,

cardiotonic,

cyto

toxi

c

antitumor,anti-AIDS

antitumor,antileukemic

antitumor

antitumor

antileukemic,antitumor

cytotoxic

cytotoxic

antibacterial

antimicrobial

anti

vira

l,antimicrobial

antimalana

immunosuppressive,

antiin¬

flammatory,an

tipe

roxi

dati

on

sedativ

psychoactive

insecticidal,antifeedant

cardiacglycosides

trit

erpe

nes

maytansinoides

sesquiterpenes

dulotol

celastroloide

elaedendroside

diterpeneepoxides

celastroloide

trit

erpe

nedimers

sesquiterpenes

celastroloide

celastro1

Euonymus

sp

,

LophopetalumtoxicumLoher

CelastrushindsuBenth

Maytenussp

andothers

Celastrusst

epha

notu

foli

usMakino

Maytenus

ebenifoliaReiss

Celastrussp

andothers

ElaedendronglaucumPers

Tnpterygium

wilf

ordi

iHook

f

Maytenussp

andothers

Maytenusumbellata(R

Br)Mabb

GlyptopetalumsclerocarpumLaws

Salaciakraussu(Hary

)Harv

,

Maytenussenegalensis(Lam

)Exel1

Tnpterygium

wilfordiiHook

f,others

esteralkaloids

Celastrus

sp

,Cassmesp

'phenylalkylamines

Catha

edulis(V

ahl)

End1

sesquiterpenes

Maytenus

sp

,Celastrussp

Bishayand

Kowaiejvski

1973

Habermeier1980,Kitanakaetal,1996

KuoandKuo,

1997

Kupchanetal

1971,Kuoetal

1990

Takaishietal

,1993

Shirotaetal

1998

Abraham

et

al

,1971,Itokawa

etal

,1991

Kupchan

et

al

,1977

Kupchan

et

al

,1972a

Gonzalezetal,1996

Gonzalez

et

al1992

Sotanaphun

etal

,1999

Figueiredo

et

al

,1998,E

Tahir

et

ai

1999

Li,1993

Sassaetal

,1994

Snethetal

,1963

Hofmann

e*

ai

1955

Gonzalez

et

al1997

<//j

etai,1992

Phytochemistry

7.7 Popular medicinal use

7.7.1 Yucatec Maya medicinal use of C. gaumeri

According to various h-men (shamans), healers and midwives of the villages of

Chikindzonot, Ekpedz and Xcocmil (Yucatan) a two-cm piece of root is used as a

treatment for snake bites. It is chewed as soon as possible after the bite. The

powdered root mixed with water is put on the wound in the form of a plaster.

Sometimes the decoction is combined with other medicinal plants like Morinda

yucatanensis Greenman, Samolus ebracteatus Kunth and Anredera vesicaria C. F.

Gaertner. The rootstock of latter plant which is rich in polysaccharide is responsible

for the galenic form of the plaster. Diarrhea is a further usage of C. gaumeri. A

decoction of the root, mixed with other species, is orally used against diarrhea.

In the study region C. gaumeri is named viperol. Formerly it was called camba-och-

lob (Standley and Steyermark, 1949). The species is also listed in the Diccionario

Maya as kambaochdob, but no medicinal use is mentioned (Barrera et al., 1991).

The change of the plant name seems to come from the medicine named viperol,

which in the past was produced from the roots of C. gaumeri. According to oral

history, the medicine in the form of ampoules was used orally as an antidote

against venomous snake bites. The use of the medicine, under the name viperol, is

still known but detailed information about ingredients, preparation and place of

production could not be obtained (oral information from healers, employee of the

Instituto Nacional Indigenista and other inhabitants of the Yucatan Peninsula).

According to the healers of the study region two other species named viperol are

known. These are Urechites andrieuxii Muell. Arg. and Echites yucatanensis Millsp.

ex Standley. Both species are members of the Apocynaceae family and are also

used against snake bites.

185

Phytochemistry

7.7.2 Medicinal application of other Crossopetalum species

The Huastec Maya, situated in the northeastern part of Mexico, use

Crossopetalum uragoga O. Ktze not only as an anti-diarrheal medicine, but

also for burning eyes, hemorrhage and as a dermatological medicine

(Dominguez, 1984; Alcorn, 1984).

4 Crossopetalum parvifolium (Hemsley) Lundell is applied for dysentery among

the Tzotzil of Zinacantan in Chiapas, Mexico (Breedlove and Laughlin, 1993).

In the West Indies and Cuba, the roots of Crossopetalum rhacoma Crantz are

used in the treatment of kidney stones and the leaves are applied as diuretic

and for kidney inflammation (Morton, 1981 ).

Two species from Panama, namely Crossopetalum tonduzii (Loes.) Lund,

and C. lobatum Lundell were screened for antimicrobial and cytotoxic activity.

Both showed antibacterial activity and C. lobatum exhibited cytotoxic activity

against HeLa cells (Gonzalez et a!., 1994). Unfortunately no data about local

medicinal use of these species could be found.

7.7.3 Global medicinal use of Celastraceae species

The Celastraceae are widespread throughout the world and have a long history of

use at the folk level both for medicinal and agricultural purposes. Some of the

medicinal uses are listed in Table 7.2.

186

Table 7 2 Medicinal use of Celastraceae species

Plant species Uses (plant part used)' Source Location

* Hippocratea

- cclastroides Kunth

- excelsa Kunth

Maytenus

- amazonica C

Martius

- buxifolia (A. Rich)

Griseb

- laevis Reissek

- senegalensis

(Lamarck) Exell

* Celastrus

- angulatus Max

-scandens

Euonymus

- atropurpurea Jacq

- europaea L

tranquilizer (p), Morton 1981 Yucatan

asthma, bronchitis, cough (i) Ankli see appendix Yucatan

rheumatism flu, gastrointestinal diseases, Peru

antitumoral, (various parts), Chavez et al 1998

Stomach ache, diarrhea colds fever, menstrual Brhamas

hemorrhage (I) Morton 1981

painkiller rheumatism stimulant d'uretic(b), Western

Schultes and Rauffauf, 1990 Amazonia

arrow poison (b) gastrointestinal problems Africa,

fever, yellow fever snake bite (r) Neuwinger, Tanzania

1996, Gessler 1995

insecticide, Wang et al,1997 China

diarrhea, sores to regulate menstruation, (I), New Jersey

diuretic, laxative cancer cough (r) Still, 1998

laxative (p), eye bath, uterine problems (b) New Jersey

Still, 1998

cardiotonic, emetic purgative, insecticide and Europe

vermin, Bishay and Kowalewski 1973

Cassine matabelica skin cancer, diarrhea, stimulant (r), Bruning, Afrca, India

(Loes)Steedm 1978

bilharziosis, dysentery (r), Figueiredo et al, Mozambique

1998

dermatitis, rheumatoid arthritis, nephritis China

insecticide (r) stimulant others Chen et al

1992, Shishido etal,1994

* Salacia kraussn

(Harv ) Harv.

Tnpterygium sp

(I) leaf, (r) root, (b) bark, (p) whole plant

187

Phytochemistry

8 Methods (isolation procedure)

8.1 Thin layer chromatography (TLC)

Thin layer chromatography (TLC) was used to optimize the mobile phase for VLC,

MPLC, HPLC and open-column chromatography. The eluents were optimized

based on the eight proposed solvent groups of the PRISMA model and with

modified mobile phases for special structure types reported in literature (Nyiredy

et al., 1988; Wagner et al., 1983). TLC was further employed for chromatographic

examinations of fractions and extracts (two-dimensional TLC), for preparative

chromatography, for monitoring chemical derivation (hydrolysis) and for locating

biological activity on TLC plates. To enhance good separation, the TLC mobile

phase was transferred to off-line systems by reducing the solvent strength

(Kowalska, 1996).

8.2 Vacuum liquid chromatography (VLC)

Vacuum liquid chromatography (VLC) was initially used as separation procedure

of the crude extracts. Furthermore some small fractions were separated by RP-

VLC. The columns were dry-packed under vacuum. All extracts were adsorbed on

Celite 535 and applied to the column in order to avoid obstruction. The fractions

were dissolved in the starting mobile phase and the separation was forced by a

water suction pump producing a vacuum (Hostettmann et al., 1998).

8.3 Middle pressure liquid chromatography (MPLC)

Medium pressure liquid chromatography (MPLC) was employed to separate the

complex fractions after the application of the VLC technique. The column and

pre-column were slurry-packed under pressure (21 bar, flow speed: 8 ml/min).

After packing, the pre-column was removed. The sample (1.2 g), dissolved in a

little solvent (< 5 ml), was injected via a sample loop (Hostettmann et al., 1998).

188

Phytochemistry

8.4 High pressure liquid chromatography (HPLC)

The semipreparative high-pressure liquid chromatography (HPLC) was used as

one of the final steps of the isolation procedure. Samples were applied in the

amount of 5 mg for one injection. Further parameters see publication III. For semi¬

preparative HPLC 1-100 mg probe can be applied, depending on the column size

and particle size of the mobile phase (Unger, 1995).

8.5 Open column chromatography

The classical open column chromatography was applied for the separation of

complex fractions and for small fractions. The size of the column was chosen

according to the quantity of the sample (sample:stationary phase = approx. 1:100)

(m:m). The columns were slurry packed either with normal phase material (silica

gel) or reversed phase material (octadecyl phase) (Müller and Keese, 1988).

8.6 Liquid-liquid partition (LLP)

Liquid-liquid partition was used tor the first separation step of the methanolic

crude extract. The separation is based on the dipole interaction properties of the

solvents. This separation technique takes place under mild conditions and

without any loss of material (Müller and Keese, 1988).

189

Phytochemistry

9 Methods of structure elucidation

Several spectroscopic and spectrometric instruments exist for the process of

structure determination of natural products. The combination of physical data e.g.

UV- spectroscopy, optical rotation, MS-spectrometry and NMR-spectroscopy,

together with information of chemical methods is used to determine the structure

of compounds.

Isolate

1H NMR

13C NMR

DEPT

Inverse gated

number of carbons and hydrogens

functional groups

reliable 13C integrals

M S molecular weight

UV chromophore

Molecular formula; double bond equivalent functionalities

HSQC

DQF-COSY

TOCSY

HMBC

T INADEQUATE

one-bond correlation (C-H)

vicinal protons (CH-CH) and geminal protons (CH2)

protons in a spin system

long-range correlation (C-C-C-H), (C-C-H)

C-C coupling

Planar structure

X-Ray n

ROESY

[a]D f relative/absolute stereochemistry, optical rotation

y dérivât.J

Complete structure with relative/absolute stereochemistry

Figure 9.1. Scheme of methods for structure elucidation

190

Phytochemistry

9.1 Nuclear magnetic resonance spectroscopy (NMR)

Nuclear magnetic resonance spectroscopy (NMR) is a method which is applied

for nuclei that have a nonzero spin quantum number (e.g. 1H, 13C, 15N). When

these types of nuclei are brought in a static, homogenous magnetic field, they

interact with it. Depending on the surrounding electron density, the observed

nuclei are able to absorb energy, when they are irradiated with the appropriate

radio frequency.

The pulsed Fourier transform NMR spectroscopy (FT-NMR) is used as the

particular NMR technique in which all of the nuclei of one isotope are activated

simultaneously by an appropriate pulse. The absorbed energy is subsequently

lost to the surroundings or to other nuclei over a period of time (relaxation time).

The response of the system is monitored as interferogram (intensity as a function

of time). The resulting emission signal from the excited nuclei is known as the free

induction decay (FID) and the Fourier transformation of this decay yields the NMR

spectrum.

One dimensional NMR methods

The frequency, in which a nucleus is able to absorb energy, depends on the

environment of the particular nucleus and is proportional to the strength of the

magnetic field. It is expressed as the chemical shift ô and given in parts per

million. Functional groups are identified by characteristic 1H and 13C chemical shift

values. The number of signals gives information about the number of nuclei of the

molecule under investigation. Furthermore the multiplicity and the coupling

pattern of a signal is important. Many signals are split into several lines due to the

effect of neighboring nuclei. The frequency differences between such multiple

lines are given as coupling constants J (Hz) and depend on the number and

nature of bonds and the angular relationships of the coupled nuclei (Byrne, 1993;

Kalinowski et al., 1984).

By means of DEPT (Distortionless Enhancement by Polarisation Transfer), a

further one-dimensional experiment, the number of hydrogens bonded to the

191

Phytochemistry

particular carbon atoms can be determined. DEPT 135 distinguishes positive

signals for CH3 and CH and negative signals for CH2 resonances.

The inverse gated experiment is used as a method to get reliable integrals of

13C spectra. In order to obtain comparable integrals, it is necessary to avoid

building up of NOEs (Nuclear Overhauser Effect) in the sample; it is usually

reached by making them all equal to zero. This can be achieved by inverse

gated decoupling in which the decoupler is on during pulse and acquisition,

but is off during a relaxation delay. At the moment of pulse, none of the carbon

signals is enhanced and all have the same integrals (Sanders and Hunter,

1988). The different relaxation times, which also leads to unreliable integrals

can be avoided by increasing the relaxation delay, so that nuclei with long

relaxation times can turn back to the basic state. The inverse gated

experiment was used for detecting doubled intensities of carbons of the

symmetrical unit of compound 1 (ourateacatechin).

Two dimensional NMR methods

Two dimensional (2D) NMR techniques give more information on the interaction

between nuclei. The homonuclear and heteronuclear scalar coupling techniques

provide the direct connectivities between the atoms and enable the configuration

of a molecule to be determined (DQF-COSY, TOCSY, HMQC, HMBC,

INADEQUATE). The homonuclear dipolar coupling techniques give the

internuclear distances. Thus, the relative stereochemistry of the substance can be

determined (ROESY).

Homonuclear 2D NMR methods

The DOF-COSY (Double Quantum Filter Correlation SpectroscopY)

experiment is the standard 2D experiment for homonuclear shift correlation of

vicinal and geminal protons.

The TOCSY (TOtal Correlation SpectroscopY) experiment is employed to

define all protons within a spin-system.

192

Phytochemistry

* The ROESY (Rotating frame nuclear Overhauser Effect SpectroscopY)

experiment helps identifying pairs of protons close enough to interact through

space.

* The INADEQUATE (Incredible Natural Abundance Double Quantum Transfer

Experiment) detects signals due to 13C,13C coupling. It is used to trace the

entire C skeleton of a molecule. The experiment shows extremely low

sensitivity, therefore a high amount of material is used depending on the

molecular weight (~ 1/5 of M/1000 ).

Heteronuclear 2D NMR methods

* The HSQC (Heteronuclear Single Quantum Correlation) experiment is

employed for one-bond heteronuclear chemical shift correlation.

The HMBC (Heteronuclear Multiple Bond Correlation) is a method for

determining connectivities between 1H and 13C atoms, separated by two or

three bonds.

9.2 Mass spectrometry (MS)

Mass spectrometry involves the generation of positive or negative ions from

organic molecules that are in gas-phase. Subsequent separation and detection of

fragment ions provide information about the molecular formula and also about

structure elements of a compound (Baldwin, 1995).

Electron impact (El) MS: In this standard method, the vaporized sample

molecules are bombarded with electrons emitted from a heated filament (70

eV). Due to the energy, electrons are ejected with the formation of positive

ions.

Fast atom bombardment (FAB) MS: The compound is suspended in a viscous

liquid with low volatility (3-nitrobenzylalcohol) and bombarded by fast atoms

193

Phytochemistry

(argon or xenon) or ions. Molecular information yielded [X+Hj+ ions or [X+Na]+,

[X+K]+.

Electronspray ionization (ESI) MS: The involatile sample is injected in a

flowing liquid stream of methanol and ammonium acetate in order to achieve

better ionization ([X+H]+, [X+NHJ/, [X+Na]+; apparatus: Finnigan 7000 TSQ).

9.3 UV-Spectroscopy (UV)

Due to the electronic structure of a molecule, the characteristic absorption of a

chromophore enables the identification and classification of structural elements.

UV- Spectroscopy normally refers to the absorption in the ultraviolet (200-380

nm) and visible (380-800 nm) light.

9.4 Optical rotation

A compound is optically active, if it is able to change the direction of the linearly

polarized light. The specific rotation [ct]D, measurable angle to the plane of the

incident light, is a characteristic property of chiral compounds using the sodium D-

line (589.5 nm).

9.5 Acidic hydrolysis

In order to identify monosaccharides of sugar chains, acidic hydrolysis was

performed. Subsequent analysis of the sugar moiety by TLC in comparison with

defined sugar references allowed identifying the monosaccharides.

194

Phytochemistry

10 Plant extraction

10.1 Small scale plant extraction

Air-dried and powdered roots of Crossopetalum gaumeri (20 g) were macerated

in 70 ml petroleum ether for 4 hrs at room temperature. The subsequent extraction

was carried out in a percolator (2.5 x 30 cm) with a series of solvents of increasing

polarity, namely, petroleum ether, dichloromethane, methanol, a mixture of

methanol-water 1:1 and water. The percolation took place over a 43 h period, i.e

over 4 (petroleum ether), 8 (CH2C!2), 4 (MeOH), 18 (MeOH-H20) and 9 hrs (H20),

respectively. Chromatographic examinations of the five extracts by TLC showed

the similarity of the petroleum ether and the dichloromethane extract. Both

extracts exhibited NF-kB activity and antibacterial potency against different gram-

positive bacteria, namely, B. cereus, S. epidermidls and M. luteus. As a result of

these preliminary experiments, the process for the main extraction was

accordingly modified. Instead of petroleum ether and dichloromethane, only the

latter one was used for the non-polar extraction.

All five extracts exhibited cytotoxic activities down to a concentration of 10 ppm

based on KB cell culture. The extracts showed neither antifungal activity nor any

growth inhibition of E. coli.

10.2 Large scale plant extraction

Powdered roots (sieve 6 mm, then 1 mm) of C. gaumeri (2.26 kg) were

successively extracted in a percolator (18 x 45 cm) with distilled dichloromethane,

dichloromethane/methanol, methanol, 70% aqueous methanol mixture and de-

ionized water. From time to time the drug was manually stirred with a spatula (I =

60 cm). The CH2CI2 extraction took place over a 168 h period, the CHgCL/MeOH

and the MeOH extraction over a 120 h period. The MeOH/H20 and the H20

extractions went on for 72 h, each. The change in the polarity of the solvents was

indicated by the change in the color of the solvents. By removing the solvents in

195

Phytochemistry

vacuo, five extracts were obtained and stored in a freezer at -20°C. The

extraction scheme is shown in Figure 10.1.

Roots of Crossopetalum gaumeri

(2.26 kg)

Percolation at ambient temperature

Evaporation in vacuo

Extract: CH2CI2 CH2CI2/MeOHMeOH MeOH/H20 H20

Quantity: 94 g 23 g95 g 35 g

27 g

Color: brown-red red-orange brown red-orange light-brownI J v J v~.„ ^s

Figure 10.1. Extraction scheme

The cytotoxic potential of the CH2CI2, MeOH and H20 extracts were assessed

based on KB cell culture. The MeOH extract showed activity down to 1.25 ppm,

the brown-red CH2CI2 extract was still active at 2.5 ppm and the water fraction at 5

ppm. Furthermore the CH2CI2 extract showed antibacterial activity {B. cereus, S.

epidermidis and M. luteus).

10.3 Fractionation of the methanol extract

As a crude separation of the methanol extract, liquid-liquid partition was carried

out twice for each phase. Further fractionation was done using VLC, open column

chromatography and HPLC. UV-spectra of relatively pure fractions were obtained

on a spectrophotometer in order to optimize the wavelengths for the UV-detection

and HPLC separation of the compounds. Thus, the isolated cardenolides were

detected by X = 226 nm. The fractions obtained were collected and combined

196

Phytochemistry

The detailed isolation procedure is shown in Figure 10.2 and described in

publication IV. The numbers of the isolates in this thesis do not correspond to the

numbering system in the publication.

10.4 Fractionation of the dichloromethane extract

For preliminary fractionation, the CH2CI2 extract was separated by VLC on silica

gel. The fractions obtained were then chromatographed using MPLC and RP-

VLC. The final purification steps were carried out with semi-preparative RP18-

HPLC, C18 cartridges (Sep-Pak®) or by liquid-liquid partition. The isolation

process was guided by the antibiotic activity against S.epidermidis and with the

help of 1H NMR spectroscopy. Detailed fractionation and isolation is shown in the

flow chart (Figure 10.3) and discussed in publication IV.

197

Methanolextract(42g)

Liquid-liquid

part

itio

n60%aqueousCH3OH-CHCI3

1:1

CH3OH/H20

extract

Liquid-liquid

partition

H20-n-BuOH

1:1

CHCI3

extract

(3g)

H20

extract

(17

g)

n-BuOH

extract

(22

g)VLC

silicagel

CHCI3

-

>CH3OH

stepgr

adie

nt

Fr.9

130mg

Fr.2(24mg),

HPLCRP18

ACN-H20

20:80

Fr.5(155

mg),

HPLCRP18

ACN-H20

20:80

Fr.13-15

(6.9

g),

Opencolumnchromatographyon

silicage

lCHCl3-CH3OH-H?0

->89:10:1

->45:50:5

Fr.7

(72mg),

HPLCRP18

ACN-H20

20:80

Fr.9(250mg).

HPLCRP18

ACN-HpO

15:85

Fr.11

(640mg),

HPLCRP18

ACN-H20

15:85

5

39mg

6

2.4mgJ

Figure

10.2.

Isolationscheme

ofthemethanol

extract

Dichloromethaneextract(33

g)

VLC

silicage

ln-hexane—>EtOAc

Fr.8

7

1.8g

Fr.3

Fr.6(63mg)

Liqu

id-l

iqui

dpartition

8

14mg

Fr.11&

12

(3.6

g)MPLC

silicage

ln-hexane-EtOAc-CH3OH

15:3:0.5

10:10:5

Fr.8

(1.3

g)VLCRP18

CH3OH-H20

6:4

Fr.13(11mg)

RP18

CartridgeSe

p-Pa

k®CH3OH-H20

1:1

->CH3OH

CH3OH

Fr.24&25

(147mg)

VLC

silicage

ln-hexane-»CHCI3

Fr.10-15(3

4mg)

HPLCRP18

ACN-H20

9:1

9

5.3mg

10

2.1mg

Figure

10.3.

Isolationscheme

ofthedichloromethaneextract

Phytochemistry

11 Structure elucidation of the isolated compounds

In this chapter, additional information on the isolated compounds is discussed in

publication IV. Interesting data of the isolates are compared for each group of

natural compounds. The discussion of the isolates follows the numbers as shown

in the isolation protocols (Figure 10.2 and 10.3).

11.1 Cardenolides

Five cardenolides were isolated from the methanol extract. Four different types of

aglycones (securigenin, sarmentosigenin, sarmentogenin, and 19-

hydroxysarmentogenin) and three different types of sugar moieties were

identified, namely ß-6-deoxygulose, ß-6-deoxyallose and a-allose.

\—Q

HO—/ V"*-

HO tDH

RiO

B

HOH2C

rilOl:b-OH HO OH

Name of the isolated cardiac glycoside R1 R2 R3

Securigenin-3ß-0-ß-6-deoxyguloside (2)

Sarmentosigenin-3ß-0-ß-6-deoxyguloside (3)

19-Hydroxy-sarmentogenin-3ß-0-ß-6-deoxyguloside (4)

Sarmentogenin-3ß-0-[a-allosyl-(1 -^4)-ß-6-deoxyallose] (5)

Securigenin-3ß-0-[cc-aIlosyl-(1 -^4)-ß-6-deoxyallose] (6)

A H CHO

A OH CHO

A H CH2OH

B H CH3

B H CHO

200

Phytochemistry

Color reaction on planar chromatography (TLC)

The cardiac glycosides showed characteristic color reactions on TLC after

detection with vanillin-H2S04 reagent and heated to 110 °C for 5-10 minutes. The

color pattern included greenish to intense blue colors, depending on the

substitution pattern of the genin and the sugar moieties. Compound 5 with the

methyl group on C-10 resulted in an intensely blue spot. This phenomenon

supports the assumption that only cardenolides. which are not oxidized on C-19,

respond with a blue color (Pauli and Junior, 1990).

(1 ) orange spot; ourateacatechin

(2) greenish spot, fluorescence under UV-366 nm

(3) green-yellow spot, fluorescence under UV-366 nm

(4) green spot, fluorescence under UV-366 nm

(5) blue-green spot, fluorescence under UV-366 nm

(6) brown-greenish spot, fluorescence under UV-

366 nm

Figure 11.1. Thin layer chromatography (TLC) of the isolated compounds 1-6.

Mobile phase: ethyl acetate-methanol-water 81:11:8; stationary phase: silica gel

60 F254; detection: sprayed with vanillin-H2S04 reagent and heated to 110 °C for

5-10 min, evaluation under visible and under UV light 366 nm.

201

Phytochemistry

The vanillin-H2S04 reagent reacts with the aglycone and the sugar moiety (Stahl

and Glatz, 1982). The color reaction under visible light depends partly on the

sugar moiety while fluorescence on UV 366 nm is attributed to the aglycones

(Pauli, 1993). The transformation with Kedde reagent (NaOH, dinitrobenzoic

acid), which has not been used for the detection in this study, takes place on the

butenolide ring and formation of a Meisenheimer-complex occur (Jork et al.,

1993).

1H NMR comparison of the cardenolides

The cardenolides showed complex 1H NMR spectra due to the excessive signal

overlap in the 1.0-2.5 ppm chemical shift area. The signals at 5 4.91-4.92 {dd, J =

18.2-18.5, 1.5, H?-21) and 5 5.90-5.94 (s, H-22) are characteristic signals for the

butenolide ring of the cardenolides. The sugar moieties showed shift ranges

between 5 3.28 and 5 4.74 with a methyl group between 51.21-1.29 {d, J- 6.1-

6.7, H-6') each. The aldehyde group on C-19 of compound 3 at 5 9.97 is shifted

downfield compared to those of 2 and 5 (5 9.41 and 9.42). This phenomenon is

caused by the hydroxyl group at position C-5 of compound 3. The genin of

compound 4 is characterized by an alcohol function at C-19, replacing the

aldehyde function of 2, 3, 6 (s. 9.41-9.97). The two doublets were shifted to

higher fields and resonated at 5 3.86 and 3.71 (each d, J = 10.8 Hz). The

chemical shift of H-11 (5 3.72-4.42) changed due to different substituents at C-5

and C-19. Further characteristic signals among the five cardiac glycosides are the

triplet signals for H-17 at 5 2.93 (r, J = 6.9-7.1 Hz). The signals of H2-21 are not

clearly expressed because of water suppression at approx. 4.80 ppm. The MeOD

peak at 5 3.31 was always accompanied by a MeOH peak at lower field (Figure

11.3).

202

Phytochemistry

13C NMR comparison of the cardenolides

Two quaternary carbon signals in the range of 5 175.7-177.7 showed different

intensities due to different relaxation times. The weaker signal in higher field was

assigned to the carbonyl carbon C-20 of the butenolide ring. Due to HMBC

correlations the stronger signal was assigned to C-23. Different substituents on C-

5 and C-19 indicated differences in the 13C NMR shifts between the isolated

cardenolides. Thus, the vicinal carbons C-4 (5 29.1-34.0), C-6 (5 27.7-37,1) C-10

(5 37.5-55.1) and also C-1 (5 19.7-33.8) shifted downfield or upfield. The C-4' (5

84.0-84.1) and C-6' (5 18.2) signals of the disaccharides 5 and 6 in comparison

with the monosaccharides 2, 3 and 4 (C-4!: 72.2-73.7; C-6': 14.9-16.4) are shifted

to lower field due to the O-linkage of the second sugar moiety (Figure 11.4),

COSY and HMBC correlations of the cardenolides

The assignment of the signals of the cardenolides and their sugar moieties was

verified using COSY, TOCSY, HSQC and HMBC measurements, particularly.

With the aid of COSY and TOCSY spectra the saccharides were identified (Figure

11.5, 11.6).

Relative and absolute stereochemistry of the cardenolides

The relative stereochemistry was elucidated by ROESY experiments, the 1H,1H

coupling constants and the application of steric arguments. The results were

verified by comparing the data with those of the literature.

A/B ring junction:

Due to the 1H,1H ROESY correlations between H-5 and H-19 A/B eis

configurations were suggested for the aglycones (Figure 10.2). The comparison

with the 13C NMR data for C-5, C-1, C-7 and C-9 of all five compounds with those

of several A/B eis configuration and also with uzarigenin (A/B trans system)

verified the geometry of the A/B ring junction as eis (Drakenberg, 1990; Cheung et

al., 1981). The chemical shifts of C-5 of uzarigenin (H-a) are in lower field

203

Phytochemistry

compared to the same carbon atoms in A/B c/s-fused systems. These differences

are due to steric compression shifts induced in the latter systems by forcing the

ring system into the c/s-configuration (Wehrli, 1979).

Figure 11.2. ROESY correlations of compound 6 (600 MHz, MeOD)

* B/C ring junction:

The B/C frans-fusion was indicated by the observation of correlations in the

ROESY spectrum between H-8/H-19 (2, 3, 4, 5), H-11/H-19 (2, 5, 6), H-8/H-11

(5, 6) and the lack of any correlations between H-8/H-9 (Figurel 1.2).

C/D ring junction and H-17 orientation:

The C/D ring junction was assigned as eis, with the butenolide ring attached at

the C-17ß position. In support of this configuration, the protons of H3-18ß gave

rise to strong synaxial NOE correlations with the proton of H-11ß and H-8ß, H2-21

and H-22 of the free rotating butenolide ring showed correlations to the H3-18ß,

which indicated a ß position of the lactone ring. In addition, the chemical shift of

C-12 of the aglycones lie in the range of 48.5-50.6 ppm, whereas a butenolide

ring in C-17a position is characterized by an upfield shift of approx. 8 ppm to

approx. 5 42 for C-12 (Kawaguchi et al., 1993). Due to this evidence and in

204

Phytochemistry

comparison with the 13C and 1H shifts of digitoxigenin, C/D ring junction as eis and

C-17ß orientation were verified (Drakenberg, 1990).

Relative stereochemistry of the saccharides and H-3 orientation:

The relative stereochemistry of the sugar moieties is described in detail in

publication IV.

Mass spectrometry of the cardenolides

For the cardiac glycosides positive FAB-MS were measured due to the soft

ionization method of FAB technique. A fragment of m/z 146 indicates the

presence of a deoxyhexose (2, 3), m/z 162 substantiates the presence of a

hexose (5, 6) (Figure 11.7). For the disaccharides, 5 and 6, no molecular peaks

were obtained. Instead, peaks were obtained which indicated the loss of a

hexose [M+Na-sugar]+, [M+K-sugar]\ Based on an ESI-MS the molecular ion of

the disaccharide cardenolides was detected.

205

ce

II

O

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cd

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roo CD

HOHoC.

6"

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HqC.

6'

HO

-o

1">-""0|'

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Qr

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OH

HO

OH

HO—<4' \

=Ri

HO

OH

23 20

Rt0^3

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11.4.13CNMR

ofcompounds

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6,2,

3inMeOD

(75.

5MHz)

20

ppm

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COs

co c\T_""^v

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315

30

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ppm

Figure

11.5.1H1HDQF-COSYspectrum

ofcompound6

inMeOD

(600MHz);dashed

line

:de

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entire

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11.7

PositiveFABmassspectrum

ofcompound2

Phytochemistry

11.2 Ourateacatechin

(-)-4'-0-methyl-epigallocatechin (Drewes and Mashimbye, 1993), 3,3',5,5',7-

pentahydroxy-4'-methoxy-2,3-c/"s-flavane (Weeratunga et al., 1985)

OCH,

2 ^

'%

OH

1

M,: 320

C16Hl607

Ourateacatechin (1) was isolated as a brown-orange amorphous solid. The 13C

NMR and the DEPT experiment detected 14 carbons. In the 1H NMR a double

intensity of the aromatic H-2' {6 6.55) was observed (Figure 11.8). The inverse-

gated 13C (1H) NMR experiment verified the double intensity of the methine group

at 107.12 ppm and showed the same phenomena for the quaternary carbon at

151.15 ppm (Figure 11.9). This indicated some type of symmetry in the molecule

leading to at least eight quaternary carbons, six methine groups, one methylene

and one methyl group. The combination of these results with the EI-MS, which

showed a pseudo-molecular peak at m/z 322.0 [M+2H]+, allowed the

establishment of the molecular formula C16H160T. Furthermore, the mass spectrum

revealed a pair of important fragment peaks at m/z 183 and 140 that are typical for

retro-Diels-Alder fragmentations (Garcia et al., 1993) (Figure 11.10). The 13C-

INADEQUATE NMR allowed the formulation of the carbon skeleton C4-C3-C2-

CT-C2'-C3'-C4' and C-4 to C-10 (Figure 11.11). The two fragments were

composed with the help of HMBC experiments, especially with the correlation

between H-2 and C-9 and the correlation of H-6 to C-5/8/10 and H-8 to C-6/7/10.

Thus, 4'-0~methyl-epigallocatechin (1) was identified. The stereochemistry was

213

Phytochemistry

elucidated by ROESY experiments and coupling constants. The intensive NOE

correlation between the protons of H-2 and H-3 indicated a eis relationship of the

B ring and the hydroxyl group. This was confirmed due to the lack of a 7 Hz vicinal

H-H coupling constant, which would be consistent with a trans relationship

(Palazzo de Mello et al., 1996). The *H and 13C NMR data showed proton and

carbon shifts, which are in accordance with literature values (Delle Monache et al.

1992) (Table 11.1).

Table 11.1. 1H, 13C NMR data and long-range correlations (HMBC) of

ourateacatechin (1) in MeOD

No. ,3cr, 1H. 5 ppm, HMBC

8 ppm (Jm Hz) correlations

2 79 46 d 4.77 br s 3.4, 9, 1s, 2-

3 67.24 d 4.19 br s 10

4 a 29 03 t 2.74 dd (16.8, 2.6) 2,3, 5,9, 10

ß 2.87 dd (16.8, 4.4)5 157.77 s - -

6 96.40 d 5.97 d (2.2) 5, 8, 10

7 157.42 S - -

8 95.86 d 5.93 d (2.2) 6, 7. 10

9 157.00 s - -

10 100.03 s - -

r 136.45 s - -

276' 107.12 d 6.55 s 2, 3', 4', 27=6'

3',5' 151.15 s - -

4' 135.94 s - -

OMe 60.77 q 3.79 s 4!

*

Multiplicities determined by DEPT sequences

Physical data of ourateacatechin (1). Brown-orange amorphous powder: [a]D -

47.3 ° (MeOH, c 1.00, 24 CC); UV Anax (MeOH): 271, 209 nm; positive EI-MS (70

eV) m/z : 322 [M+2H]+, 183, 168, 154, 140,33.

214

OCH,

2/6'

K„_7

2

OCH3

3

/A.

4ß

4a

65

60

55

-1

1—

50

45

40

35

30

ppm

ro

cn

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re11

81HNMR

spectrum

ofourateacatechin

(1)

inMeOD

(300

MHz)

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276'

10

Ü

4

150

140

130

120

110

100

90

30

70

60

50

40

ppm

Figure

11.11.13CINADEQUATEspectrum

ofourateacatechin

(1)

inMeOD/pyridine-d5(125MHz)

Phytochemistry

11.3 Triterpenes

11.3.1 Pristimerin

(20a)-3-Hydroxy-2-oxo-24-nor-friedelane-1(10),3,5,7-tetraen-carbonacid-(29)-

methylester (Bruning and Wagner, 1978)

= 464

C30H40O4

Compound 7 was found to have the molecular formula C3ûH40O4 by a combination

of mass spectrometry and 13C NMR experiments including DEPT, The 1H NMR

showed three olefinic protons (<5 6.35, 6.55 7.03) and seven methyl groups,

among them one vinyl methyl (5 2.21, C-23) and a methoxy group (5 3.66, C-31)

(Figure 11.12). Based on these data and the isolation of a remarkable quantity of

the compound (ca. 20% of the CH2CI2 extract) with an intensive orange color

suggested the presence of pristimerin (7). Pristimerin is a well-known pigment in

the root bark of plants of the Celastraceae (Grant and Johnson, 1957;

Habermeier, 1980). The comparison with the 1D NMR data with those of the

literature (Gunatilaka et al., 1989) confirmed the isolation of pristimerin (7), a

quinone-methide triterpene. The 1H and 13C chemical shifts are listed in Table

11.2 and 11.3, respectively.

219

co

CM

CO

WCD-CM

LOCM

CO

CM

CM c>CM b

1»

CD -A

Q.

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r "xt

CD

co

//r- CM>- CM

CO —

"\

J

CO LO 7?

"-^ CO

CM CD -

S

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co

'~\

(X"xf

LO

CO

C0-

CL

loCO

o

ro

oreg

o

N =>-' o-CD

o

CO-

CO •r*-

CM

CM

220

Phytochemistry

Physical data of pristimerin (7). Orange amorphous solid: [a]D - 179.0 °

(CHCI3, c

4.67, 24 °C); UV Amax (MeOH): 213, 423 nm; positive EI-MS (70 eV) m/z : 464, 267,

253,241,201,27.

11.3.2 Friedelane-3-on-29-ol

D:A-friedooleanan-29-ol-3-one (Itokawa et al., 1991), 29-hydroxyfriedelane-3-one

(Betancor et al., 1980), 3-oxofriedelane-29-ol (Patra and Chaudhuri, 1987)

31

Mr = 442

C30H50U2

8

Compound 8 was isolated as a white amorphous powder. The molecular formula

was obtained by EI-MS (M+ at m/z 442) as well as 13C NMR and formulated as

C30H50O2. The 1H NMR showed seven methyl groups attached to quaternary

carbons (Table 10.2 and 10.3). The partial spin systems obtained by COSY

spectra (H-1/H-2, H-1/H-10, H-4/H-23, H-6/H-7, H-18/H-19) were connected using

the aid of long-range correlations* and led to the identification of the known

friedelane triterpene 8 (Figure 11.14).

*Long-range correlations of HMBC measurements: H-4 to C-1/2/5, H-23 to C-

3/4/5, H-24 to C-4/5/6/10, H-25 to C-8/9/10/11, H-26 to C-8/13/14/15, H-27 to C-

12/13/14/18, H-28 to C-16/17/18/22, H-29 to C-19/20/21/30, H-30 to C-

19/20/21/29.

221

Phytochemistry

The stereochemistry was identified by ROESY experiments. The following

relevant correlations were observed: H-4/H-10, H3-23/H3-24, H3-24/H-8, H3-25/H-

8, H3-25/H3-26, H3-26/H-18, H,-26/Hr28, H„-27/H,-29, (Figure 1113)

Figure 11.13. ROESY correlations of compound 8 (300 MHz, CDCI3)

Physical data of fnedelane-3-on-29-ol (8). White amorphous solid [a]D ~ 25.2 °

(CHCI3, c 1.15, 24 °C); UV A^dX (MeOH) 212 nm, positive EI-MS (70 eV) m/z ; 442

[M*], 301, 273, 258, 246, 231, 216, 205, 188, 177, 163, 149, 83, 48

222

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CD (/ \ \ LOc^ i n -xt

l \v 1 1 °1 y

y77-7

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79

coCM

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oco

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7

Kir

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oo

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Phytochemistry

11.3.3 2,3,7-Trihydroxy-6-oxo-1,3,5(10),7-tetraene-24-nor-

friedelane-29-oîc acid methylester

= 496

C3cH40O6

The main problem in the structure elucidation of the new compound 9 was the

identification of the weak signal C-7 of the quaternary carbon at 147.4 ppm in the

13C NMR. In addition, it did not show any long-range correlation in the HMBC

experiment due to the absence of the protons at C-5, C-9 and C-14 (Figure 11.

16). The 1H and 13C NMR data showed similar chemical shifts as those of the C, D

and E-rings of pristimerin (7) and were assigned with the aid of the HMBC and

COSY experiments (Figure 11.17; Tables 11.2 and 11.3). The stereochemistry

was identified by ROESY measurements (Figure 11.15). Comparing the 13C NMR

and 1H NMR data with a similar compound regeol C, the structure of compound 9

could be confirmed as 2,3,7-trihydroxy-6-oxo-1,3,5(10),7-tetraene-24-nor-

friedelane-29-oic acid methylester (Takaishi et al. 1997).

224

Phytochemistry

Figure 11.15. ROESY correlations of compound 9 (300 MHz, MeOD)

225

ppm

-110

-120

1-130

M40

-150

-160

M70

-180

5/1

4/1

3/1

62/1

'

10/1

,6/1

*1/23

$5/23

o4/23

$3/23

,2/23

*10/23

8/25$

»8/26

10/11

§10/25

29/30

429/31

•«29

/19

*»

=»

<>•

6/23.

Wy

29/19

29/21

710

;

6:0

'

5:0

'

4'.0

'

3!0

Figure11.16.

PartofHMBC

spectrum

ofcompound9

inMeOD

(H/C:300/75.5MHz)

..-—

2.0

-r-^

ppm

ECL

CL

-O CM.

co

o

1.

CM •* co

)

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CD

LO

CM

is* t—

00

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CM

"CM

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PCM

CO

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sm-

N

X

OO

Q

OCD

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=3O

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v*^pn)r\^ ~>~*^^yvwvw—*ya/ inf ~W- ""co CD

227

Phytochemistry

11.3.4 Celastrol

(20a)-3-Hydroxy-2-oxo-24-nor-friedelane-1(10),3,5,7-tetraen~carbonacid

= 450

^29''38^4

The comparison of 1H and 13C NMR data with those of pristimerin 7 showed a

close similarity between these two molecules (Table 11.2 and 11.3). The only

difference the methoxy group (5 3.55, C-31), which not present in compound 10.

The mass spectra exhibited the molecular peak at m/z 450 and confirmed the

molecular formula C29H3804 as well as the absence of the methyl group. The

relative stereochemistry was determined by a ROESY experiment. The

correlations between H-18/H3-28, H3-25/H3-26 and H3-26/H-18 indicated diaxial

relationships of these protons. Therefore ring fusions C/D and D/E were trans and

eis, respectively. Compound 10 was identified as celastrol, a pigment common to

many genera of the Celastraceae (Gisvold, 1939; Kutney et al. 1981: Morota et

al., 1995). In comparison with pristimerin (7) (6 g), Celastrol (10) was isolated in

very small quantity (2.1 mg).

Physical data of celastrol (10): Orange-yellow amporphous powder: [a]D - 23.0 °

(MeOH, c 1.00, 24 °C); UV Amax (MeOH): 213, 423 nm; positive EI-MS (70 eV) m/z.

450,436,253,241,202, 149, 83.

228

Phytochemistry

Table 11.2.1H NMR data of compounds 7, 8,10 in CDCI3 9 in MeOD (J in Hz)

H 7 (300 MHz) 8 (300 MHz) 9 (300 MHz) 10 (500 MHz)S ppm

1 6.55 s 1.97 m, 1.69 6.86 s 6.50 s

2 - 2.38 dd (3.3, 1.9)2.42 dd (3.2, 1.9)

- -

4 - 2.25 m - -

6 7.03 d (6.9) 1.777

1.31

- 7.06 d (7.0)

7 6.35 d (7.1) 1.50\1.42'

~ 6.33 d (7.2)

8 - 1.40} - -

10 - 1.53' - -

11 1.86°, 1.477 2.16°, 2.14 m,

2.17° 1.34J 1.97 dt (4.2, 14.2) 1.81e

12 1,81°, 1.35' 1.82 m, 1.87:,1.68* 1.66 dt (4.1, 14.1) 1.65"

15 1.65°, 1.56\ 2.88 m, 1.59",1.57° 1.32° 1.85° 1.54°

16 1.52°, 1.60°, 1.91', 1.81*.

1.89° 1.34-' 1.45' 1.47*

18 1.59° 1.62' 1.59 d (7.8) 1.58'

19 2.42 d (15.6) 1.50', 2.47 brd (15.5), 2.50 d (2.5),1.223 1.71 dd (7.4, 14.7) 1.75'

21 2.20°, 1.37' 2.17,

2.25',

1.37° 1.43' 1.36'

22 2.05°, 1.395, 2.12 dt (4.1, 13.8), 0.94 m.

0.97° 1.00' 0.96 m 2.10m

23 2.21 s 0.89 d (6.3) 2.57 s 2.21 s

24 - 0.73 s - -

25 1.45 s 0.88 s 1.49 s 1.44 S

26 1.26 s 1.04 s 1.37 s 1.26 s

27 0.53 s 1.05 s 0.69 s 0.59 s

28 1.09 s 1.22 s 1.08 s 1.10 s

29 - 3.29 d (10.7)3.25 d (10.7)

- -

30 1.17 s 1.03 s 1.15s 1.28 s

OCH, 3.55 s - 3.55 s

1H chemical shifts were assigned on the basis of HSQC experiments

0

Signal multiplicity was not described due to signal overlap.

229

Phytochemistry

1 able 113 13C NMR data of compounds 7, 8 10 in CDCI3 and

9 in MeOD (75 5 MHz, <Sppm)

Ca 7 8 9 10

1 1195 d 22 3 t 109 0 d 120 5 d

2 178 2 s 41 5 t 151 7 s 178 3 s

3 146 0 s 213 2 s 143 5 s 146 9 s

4 1170 s 58 2 d 127 9 s 1183 s

5 127 3 s 42 1 s 119 9s 127 5 s

6 133 9 d 41 3 t 182 8 S 135 2 d

7 1180 d 182 t 147 4 s 120 1 d

8 169 9 s 53 4 d 139 1 S 172 4 s

9 42 8 s 37 4 s 40 7 s 43 0 s

10 164 6 s 59 4 d 152 8 s 165 0 s

11 33 5 t 35 6 t 34 2 t 33 7 t

12 29 6 t 30 6 t 30 5 t 29 3 t

13 39 3 s 39 9 s 40 0 S 39 3 s

14 44 9 s 38 2 s 47 2 s 45 3 s

15 28 5 t 32 7 t 29 6 t 28 7 t

16 36 3 t 35 8 t 37 8 t 36 3 t

17 30 4 s 30 5 s 31 1 s 30 6 s

18 44 2 d 41 8 d 45 4 d 44 3 d

19 30 8 t 29 7 t 31 9 t 31 Ot

20 40 3 s 33 1 s 41 7 s 39 9 s

21 29 8 t 27 8 t 30 9 t 29 5 t

22 34 7 t 39 5 t 36 2 t 34 5 t

23 101 q 68q 140 q 104 q

24 - 146q . -

25 38.2 q 17.9 q 41 6 q 38 3 q

26 21 5 q 20 7 q 196 q 21 5 q

27 182 q 184 q 20 6 q 187 q

28 31 5 q 32 1 q 32 1 q 31 5 q

29 178 6 s 74 8 t 180 7 s 182 2 s

30 32 6 q 25 8 q 32 9 q 32 4 q

OCH, 51 4 q — 52 2 q —

1 Multiplicities determined by DEPT sequences

230

Phytochemistry

11.4 3,15-Dihydroxy-18-norabieta-3,8,11,13-tetraene

M =302

C19H2603

11

The structure elucidation is described in publication IV. 1H NMR and 13C NMR are

shown in Figure 11.18, The stereochemistry was elucidated by a ROESY

experiment. Due to the lack of correlation between H-5 and the protons of C-20,

A/B trans configuration was indicated and confirmed by comparison with literature

data (Nakano etal., 1997). Compound 11 showed an enol-form on position C-

3/4, but no keto function was detected in the 13C NMR spectrum (Kalinowski et al.,

1984). However, the "enolising" of the carbonyl group is generally a slow

reaction, the isolated compound 11 was stabile. The new triterpene 9 also

showed an enol-form on position C-7/8. In this case, the enol-form was favored

due to an expanded conjugated system (keto group on C-6).

231

oCM

h-

CD

CO

Q.

CL

| CM

JQ )CM

*>-CO ~^>

"7 O

\ Ie0

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232

Phytochemistry

12 Biological activities of isolated compounds

12.1 Cytotoxicity

Cardiac glycosides are known to be cytotoxic. The isolated cardenolides (2-6)

showed KB-cytotoxicity in a range of 0.074-0.199 iimol/ml. Our structure-activity

comparison indicated that the difference of one sugar in the chain resulted in a

significant difference in KB-cell cytotoxicity. Furthermore, the introduction of a C-

5ß hydroxyl group increased the cytotoxic activity(publication IV). The

cardenolides isolated from Euonymus alata (Thunb.) Sieb, exhibited KB-cell

activity in a range of 0.186-1.812 umol/ml. The genins of these compounds

showed a methyl group on C-19, a proton on C-5 and a hydroxyl group on C-1

instead of C-11. By a comparison of these cardenolides with different numbers of

sugars moieties, the cytotoxic activities decreased when the numbers of sugars

increase (Kitanaka et al., 1996).

Cassady and Suffness (1980) discussed the anti-tumor activities of the

cardenolides and structure-activity relationships in the KB cell cytotoxicity system

screened at the National Cancer Institute (NCI). The conclusions about structural

requirements for cytotoxicity in the KB system are the following:

There is some influence of the C-3 substituent on cytotoxicity with glycosides

being slightly more active than aglycones.

There is no substantial difference between a hydrogen and hydroxyl group at

C-5ß with regard to cytotoxicity (contradictory to our results, see above).

Those compounds with a hydrogens at C-5 retain activity but are notably less

cytotoxic than those with ß hydrogens at C-5.

Compounds with C-19 hydroxyl groups retain activity but are somewhat less

active than the C-19 aldehydes, while a C-19 acid is inactive.

233

Phytochemistry

* Substituents, which are tolerated without large changes in cytotoxicity, are 1ß-

OH, 11a-OH, and 12ß-OH groups.

4 C-14 requires a ß hydroxy group for maximum cytotoxicity.

Cardenolides are lacking therapeutically relevant cytotoxic activity in in vivo

systems. The activity is not easily reproducible and in the range of the toxic dose.

Consequently, these compound can not be used therapeutically. From the

volume of negative or extremely marginal in vivo data on cardenolides and the

large number of compounds tested at the NCI, it is apparent that there is little if

any prospect of developing therapeutically useful anti-cancer compounds from

this group (Cassady and Suffness, 1980).

Pristimerin (7) and celastrol (10) showed cytotoxicity in KB ceil (IC50 = 0.28, 0.72

fig/ml). According to Schwenk (1962) cytotoxicity may be dependent on the

quinoid structures. The reduction of the quinoid substances in the cells to

hydroquinones, which by autoxidation give hydrogen peroxide, may destroy

tumor cells by inhibition of glycolysis. Campanelli et al. (1980) studied the binding

of tingenone (quinone methine) to DNA. They suggest a possible mode of cellular

interaction, which may elicit anti-tumor activity. These two propositions show that

mechanism of the cytotoxic potential of quinone methides is still unclear.

Cytotoxicity of pristimerin (7) and celastrol (10) was also noted in HeLa cells

used for the NF-KB-test. Forty minutes after adding pristimerin (7), the cells

changed their morphology. After adding celastrol (10) the same phenomenon

was observed after 60 minutes. Gonzales et al. (1987) reported the ID50 value of

pristimerin (7) in HeLa cells as 0.6 ^g/rnl, and therefore it is similar to the value of

the cytotoxicity against KB cells.

Friedelane-3-on-29-ol (8) was not cytotoxic in KB cell (IC50 > 20 (ig/ml), but similar

compounds were reported to have cytotoxicity: 3-oxo-friedelane-29-oic acid (A-

234

Phytochemistry

549 lung carcinoma cells: ED50 = 3.7 uq/ml) and 3-oxo-friedelane-28-oic acid (KB

cells: ED50 = 3.7 ug/ml; L-1210: EDS0 = 2.95 ug/ml) (Mahato and Sen, 1997).

12.2 Other activities

Antibacterial and antifungal activity: The isolated compounds were tested against

several gram-positive and gram-negative bacteria, namely Bacillus cereus,

Staphylococcus epidermidls, Micrococcus luteus, Pseudomonas aeruginosa and

Escherichia coli. Candida albicans was chosen as a model for testing the

compounds against fungi. The results are summarized in publication IV. No

activity was detected agains gram-negative bacteria and against C. albicans.

NF-kB activity. At a concentration of 0.125 jig none of the isolated compounds

were active in the NF-kB assay. As previously mentioned, pristimerin (7) and

celastrol (10) showed cytotoxic activity in HeLa cells. The cytotoxic effect of

pristimerin (7) was probably responsible for the "positive" NF-kB test of the non-

polar extracts seen in the EMSA shift experiments (chapter 10.1).

Other activity. Pristimerin (7) was further tested against the following gram-

negative strains or parasites (methods: publication III).

Microorganism Result

Helicobacter pylori

Campylobacter jejuni

Plasmodium falciparum K1

Plasmodium falciparum T9/96

Trypanosoma brucei

Trypanosoma cruzi (epimastigotes)

Giardia duodenalis

1 mm, inhibition zone (600 ug tested)

not active (600 ug tested)

IC50 = 0.41 ug/ml

IC50 = < 0.41 |jg/ml

MIC = 0.12 ug/ml

MIC = < 3 jig/ml

MIC = 6.3 ug/ml; IC50 = 2.1 ,ug

Pristimerin (7) showed good anti-parasitic activity in all anti-parasitic assays. This

phenomenon is probably due to the unspecific and high KB cell cytotoxicity. The

activity against H. pylori and C. jejuni is very weak.

235

Phytochemistry

Ourateacatechin (1) was tested against H. pylori, C. jejuni, G. duodenalis and P.

falciparum but no activity was found. In the protein kinase screening, the catechin

did not show any inhibition of the kinases tested (chapter 6.3).

Friedelane-3-on-29-ol (8) showed no activity against G. duodenalis.

The cardiac glycoside (2-6) did not show any activity against P. falciparum.

13 Biomedicine, a way to explain the popular use of C, gaumeri ?

13.1 Gastrointestinal problems

Gastrointestinal problems are one of the major health problems among Yucatec

Maya. Especially children are affected by diarrhea (mal de ojo), flatulence and

worms. Adults are frequently medicated for diarrhea, dysentery, vomiting,

flatulence, cramps, indigestion, stomachache and tip'te' (publication I). The most

frequent pathogenic organisms, which cause gastrointestinal problems, are

bacteria, viruses and parasites. Some of the microorganisms causing

gastrointestinal problems are listed in Table 1 of publication III. In addition, Vibrio

cholerae induces cholera, which often leads to death due to dehydration.

Shigella dysenteriae and Entamoeba histolytica causes dysentery and Ascaris

lumbricoides (eelworm) are responsible for flatulence, diarrhea, lost of weight as

well as obstipation.

The antibacterial activity of pristimerin (7) seems to be an explanation of the use

of C. gaumeri as an anti-diarrheal medicine. Also the growth inhibition of G.

duodenalis by pristimerin (7) is of relevance for understanding the indigenous

use in treating diarrhea, pain, and indigestion. The spasmolytic effect of

ourateacatechin (1) supports the use of this species as a remedy for

gastrointesinal problems.

236

Phytochemistry

13.2 Snake bites

Venomous snakes are common in the

lowland Maya area. Three snakes of the

family of the Crotalidae, Crotalus

durissus (rattlesnake, tzabcan), Bothrops

asper (Fer-de-lance, taxinchan) and

Agkistrodon bilineatus {uolpoch) are the

most feared and respected snakes

among the Maya today. Their poisons

contain multi-protein complexes,

enzymes that support the distribution of

the venom, as well as digestive

enzymes. The toxins of the snakes can

be classified in:

a-toxins (postsynaptic inhibitory activity): Peptides with S-S bridges

%-toxins (neuronal inhibitory activity)

* ß-toxins (presynaptic inhibitory activity): Heterogeneous proteins and oligo¬

peptides, similar to phopholipases

* toxins which support presynaptic activity: Built of 57-60 amino acids

Na+-chanal-activator: Built of 39-43 amino acids

Hemorrhagines (metallo-proteases): Mr 20,000-100,000

For the three species of snakes, discussed here the effects of ß-toxins, Na+ -

chanal-activator and hemorrhagines are important. The ß-toxins destroy the

structure of the membranes and change the permeability of ions. As a result,

phospholipase A2 is able to pass the membranes. Depolarisation leads to the

contraction of the heart, skeletal and smooth muscle cells. In addition, some of the

ß-toxins are able to destroy the membranes of muscle cells. As a consequence,

Figure 13.1. Maya hunterbitten

by a rattlesnake (Lee, 1996),

237

Phytochemistry

myoglobinuria occurs. Hemorrhagines (metallo-proteases) destroy the

membranes of the capillaries and/or the inhibitors of the blood plasma.

Consequently blood extravasates to the tissues. Thus, the symptoms are

ecchymosis at the bite, paresthesia around the mouth, twitches, weakness,

syncope, sweating and vomiting. In case of death, shock, and collapse of the

cardiovascular system are observed (Teuscher and Lindequist, 1994).

13.2.1 C, gaumeri used in the treatment for snake bites

A two-cm piece of fresh root is chewed after a venomous snake bite. A piece of

this length weights about 0.8-1.6 g (o = 0.5-1 cm). Thus, 8-16 mg (1 % of dry

weight) of pristimerin (7) 0.16-0.32 mg (0.02 %) of ourateacatechin (1) and 0.08-

0.16 mg (0.01 %) of cardiac glycosides (2-6) are consumed. The other isolated

compounds (8, 9, and 10) are not discussed further because they occur in little

amounts and no strong and relevant activity is expectable.

Also the powdered root mixed with water is used topically. The healers mentioned

that the remedy takes away the pain, reduces the inflammation and "adsorbs

venom". From a biomedical perspective no clear answers can be found, why C.

gaumeri is traditionally used to treat bites. To get an answer, further

pharmacological studies with the plant and their main principles must be carried

out. However some hints about active compounds against snake bites are found

in literature (Houghton and Osibogun, 1993; Mors, 1991).

Persimmon tannin from Diospyros kaki L. f. (Ebenaceae) neutralized

neurotoxic and haemorrhagic venoms. The results were very promising.

Consequently the authors recommended the extract as a washing agent in

the emergency treatment of snake bite wounds. The structure of the D. kaki

tannin was reported as being made up of catechin and gallocatechin units.

Protocatechuic acid from Cryptolepis sinensis Merr. (Asclepiadaceae)

was isolated. It deactivated venom and

238

Phytochemistry

gymnemic acids of the leaves of Gymnema sylvestre R. Br. (Asclepiadaceae)

inhibited ATPase.

* The roots of Aristolochia spp. (Aristolochiaceae) rich in aristolochic acid

deactivate various snake venoms.

The leaves of Strophanthus hispidus (Apocynaceae) prolonged the time

taken to clot for blood treated with the venom of Echis cahnatus, which

causes intra-arterial clotting of blood.

# Cabeca-de-negro (negro's head) is supposed to be the main ingredient of the

oral anti-snake-venom remedy Especffico Pessoa, which is manufactured in

north-east Brazil. Two prenylated pterocarpans were isolated from the

remedy and shown to be active against the toxic cardiovascular effects of

Bothrops venom (Hostettmann et al., 1997).

Features which could possibly be of importance in the treatment of snake bites

are the acidic nature and/or catechol grouping, Polyphenolic substances are well

known to bind on proteins and to inhibit enzymes. Ourateacatechin (1), a

polyphenolic substance, presumably has the ability to inhibit the toxic enzymes of

snake venom by topical application. Generally, catechines show anti-edematic

activities and act as diuretics (Hansel et al., 1999). In case of ourateacatechin,

this effect could help to treat the local and expanded tumescence after a bite of C.

durissus and B. asper. Furthermore, the treatment of the edema could help to

avoid or to diminish necrosis and ecchymos. The mechanisms of the catechins

are assigned to the anti-oxidative property and the inhibition of enzymes (Hansel

etal., 1999).

When the plant remedy is used in the form of a plaster it probably prevents

secondary infections of the bite. This could well be due to pristimerin (7), which

showed high antibiotic activity.

Anti-inflammatory activity is a property common to many plants claimed to be

active against snake bites. But at the test concentration used no compound

isolated from C. gaumeri showed any activity in the NF-kB assay.

239

Phytochemistry

13,2.2 What have the cardenolides to do with snake bites?

Cardiotonic steroids and their activity: Cardenolides are known for their positive

inotropic effect on cardiac muscle by increasing contractility and decreasing the

frequency of heart beat via Na7K+-ATPase inhibition and increasing delivery of

Ca2" to cardiac muscle cells. Cardiotonic steroids have a very narrow therapeutic

window. The emetic dose of cardenolides is approximately 50 % of the lethal

dose. Fatal poisoning after oral ingestion of plants is rare due to the bitter taste

and the subsequent vomiting. The resorption rate of ouabain (g-strophanthin) by

peroral application is less than 5 % due to the substance's polarity (five hydroxyl

groups). Digitoxin with one hydroxyl group is resorbed practically completely (100

%). The isolated compounds (2-6) have two to three hydroxyl groups and it can

be supported that their resorption rate most likely lie between those of ouabain

and digitoxin. The resorption rate furthermore depends on the sugar moieties,

which can be optimized by increasing the lipophilic character (separation of

sugars, etherification and esterification of hydroyl groups). The study on the

structure-activity relationships (SAR) of the cardenolides on the Na7K+-ATPase is

summarized in the following list and show some interesting correlations to the

SAR of cytotoxicity (Malcolm, 1991; Thomas 1992) (see chapter 12.1).

A lactone function (C-17) and sugar side chains (attached to C~3) are not

necessary, but contribute considerably to the selectivity and activity of

cardenolides. The compounds containing 6-deoxy sugars are the most potent

of all cardiotonic steroids. The addition of an extra sugar unit to a monoside

leads to a decrease in activity.

14ß is a prerequisite for receptor recognition and high potency (C/D eis

junction). Trans configuration prevents recognition by Na7Kr-ATPase.

* 5a (A/B trans) is more active than 5ß (A/B eis)

Polarity (e.g. OH-groups on C-1, C-5, C-11, C-12) influences the absorption

from the intestine, but not necessarily the activity.

240

Phytochemistry

Cardenolides in the therapy of snake bites: As mentioned before, a two-cm piece

of root of C. gaumeri contains approximately 0.08-0.16 mg cardiac glycosides. A

standard initial dosage for treating heart insufficiency with digitoxin lies between

1.0-1.5 mg, afterwards a dosage of 0.25 mg every 6 h is prescribed. Thus, the

herbal medicine reaches the therapeutic dosage.

Cardiac insufficiency and cardiac arrhythmia caused by snake venom are not

described in Mexico and Middle America. However failure of cardiovascular

system occurs due to hypovolemic and hemorrhagic shock (Junghanss and

Bodio, 1996). The cardenolides have a positive influence on the cardiovascular

system. The stimulation of the Nervus vagus by the cardiac glycosides decreases

the frequency of the heart and the velocity of the atrioventricular conduction (AV).

This effect is due to an increased KT permeability and thus stabilizes the

membrane potential. As it is well known, cardenolides also exert a positive

inotropic effect on the heart muscle. In consequence, they improve the circulation

of a patient with an insufficient heart and they probably can help to avoid

hypovolemic shock. The plant remedy and the cardenolides are certainly not a

specific antidote against snake venom, but influence the cardiovascular system in

a positive way. Caffeine or coffee is given to patients bitten by a venomous snake

to stimulate the central nervous system (CNS) and in particular, the respiration

(Falbe and Regitz, 1992). Could it be that the cardiac glycosides also have a

general stimulatory effect on the CNS? The mechanism may be a similar one as

observed with local anesthetics or alcohol. These latter agents stabilize the cell

membranes in general and restrain the inhibitory pathways in the CNS resulting

in a predominance of the excitatory neurons. Should the cardenolides have a

similar general stabilizing effect on the membranes in the CNS, one can

hypothesize that the inhibitory pathway may be affected first and this would also

lead to a predominance of the excitatory systems, including stimulation of

respiration. It is well known that the cardiac glycosides accumulate strongly in the

CNS. Indeed, cardenolides often induce side effects in the CNS such as general

241

Phytochemistry

excitement, xanthochromia and hallucinosis in heart patients. In order to further

test this hypothesis in vivo tests should be carried out.

It is of interest that the Yucatec Maya use two other important plant species for the

treatment of snake bites. These are Urechites andrieuxii and Echites

yucatanensis, both belonging to the Apocynaceae. The Apocynaceae are known

to contain cardenolides and therefore strengthen the hypothesis that these

compounds could potentially be useful in the treatment of snake bites.

242

Publication IV

Cytotoxic cardenolides and antibacterial terpenoids from

Crossopetalum gaumeri

Anita Ankli3, Jörg Heilmann3, Michael Heinrich5 and Otto Sticher1

a

Department of Applied BioSciences, Institute of Pharmaceutical Sciences, Swiss

Federal Institute of Technology (ETH) Zurich, Winterthurerstr. 190, CH-8057

Zürich, Switzerland

b Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, 29/39

Brunswick Sq., London WC1N 1 AX, UK

Accepted in

Phytochemistry 2000

Publication IV

Abstract

From the methanol extract of the roots of Crossopetalum gaumeri four new highly

cytotoxic cardenolides, securigenin-3ß-0-ß-6-deoxyguloside (2), 19-hydroxy-

sarmentogenin-3ß-0-ß-6-deoxyguloside (4), sarmentogenin-3ß-0-[a-allosyl-(1 ->4)-

ß-6-deoxyalloside] (5), securigenin-3ß-0-[a-allosyl-(1->4)-ß-6-deoxyalloside] (6)

were isolated. The dichloromethane extract afforded the new diterpene 3,15-

dihydroxy-18-norabieta-3,8,11,13-tetraene (7) as well as the new triterpene 2,3,7-

trihydroxy-6-oxo-1,3,5(10),7-tetraene-24-nor-friedeIane-29-oic acid methylester

(11). The new terpenoids lack cytotoxcity and the antibacterial activity is moderate

to low.

Keywords

Crossopetalum gaumeri; Celastraceae; cardenolides; triterpenes; diterpene;

cytotoxic activity; antibacterial activity; Yucatec Maya; traditional medicine.

244

Publication IV

Introduction

Based on an ethnobotanical field study with the Yucatec Maya and an evaluation of

their medicinal plants, the roots of Crossopetalum gaumeri (Loes.) Lundell were

chosen for a detailed phytochemical study (Ankli et al., 1999; Ankli et al.,

submitted). A piece of root is chewed after a person has been bitten by a snake,

the pulverized root is mixed with water and is put on the wound in form of a plaster.

Furthermore, the decoction is used orally for diarrhea. Crossopetalum is a genus in

the family Celastraceae with 36 species in tropical America (Mabberly, 1987).

Especially due to the discovery of the antitumor effect of maytansinoides and other

novel structure types isolated from Celastraceae, this family is of great interest for

phytochemical investigation (Bruning & Wagner, 1978).

However, the genus Crossopetalum is not well investigated. From C. tonduzii

sesquiterpenes (Tincusi et al., 1998) and from C. uragoga used by the Huastec

Maya as an anti-diarrheal medicine triterpenes have been isolated (Dommguez et

al., 1984). This report describes the isolation, structure elucidation, as well as

cytotoxic and antibacterial activity of five cardiac glycosides (2-6), four terpenoids

(7-11), and one catechin derivative (1) from the methanol and dichloromethane

extracts of the roots of C. gaumeri.

Results and discussion

The methanol extract was fractionated using cytotoxicity against a KB cell line as a

lead. This led to the isolation of the well known ourateacatechin (1) (Drewes &

Mashimbye, 1993) and five cardenolide glycosides (2-6). All cardenolides were

isolated as white amorphous powder and showed greenish to blue spots on TLC

after spraying with vanillin-H2S04 as well as an intense fluorescence under UV 366

nm.

245

Publication IV

2 ß-6-deoxygulosyl H CHO

3 ß-6-deoxygulosyl OH CHO

4 ß-6-deoxygulosyl H CH2OH5 a-allosyl-(1-^4)-ß-6-deoxyallosyl H CH36 <x-allosyl-(1->4)-ß-6-deoxyallosyl H CHO

The 1H NMR spectrum of compound 2 showed characteristic signals of a

butenolactone ring at 8 5.03, 4.92 (each dd, J ~ 18.3, 1.5 Hz, H-21a and b) and

5.92 (s, H-22), as well as a singlet proton at 5 9.41 indicating a cardenolide with an

aldehyde function. A doublet at <5 4.67 (J= 8.2 Hz, H-1'), four additional protons

between 3.99 - 3.46 ppm and a signal at S 1.21 (d, J = 6.7 Hz, H3-6') pointed to the

presence of a ß-linked deoxyhexose (see Table 1). 13C NMR experiments,

including DEPT 135, sorted 29 carbons into two methyl, nine methylene, 13

methine and five quaternary carbons which is consistent with the molecular formula

C29H42O10 (see Table 2). FAB-MS spectrum showed pseudomolecular peaks at m/z

551 [M+H]+and at m/z 573 [M+Na]+. Fragments at m/z 413 and 146 confirmed the

sugar to be a deoxyhexose (Ferth & Kopp, 1992; Habermeier, 1980). In

accordance with the proposed molecular formula positive HRESI-MS revealed a

pseudomoleculare ion at 551.2849 [M+H]+. Based on COSY, HSQC, HMBC and

ROESY experiments and in comparison to literature data the aglycon was

identified as securigenin (Kawaguchi et al., 1993; Ferth et al., 1992). Structure

elucidation of the sugar moiety was performed on the basis of coupling constant

analysis and of ROESY and COSY experiments. H-2' resonated at 3.60 ppm as

246

Publication IV

double doublet indicating an axial-axial relationship to H-1' (J = 8.2 Hz) and an

axial-equatorial to H-3' (J = 3.5 Hz). Due to the small coupling constants of H-4'

(dd, J = 3.5 and 1.0 Hz) and a strong NOE between H-1' and H-5', H-4' is in

equatorial and H-5' in axial position. Based on these data the sugar moiety was

identified as ß-6-deoxygulose, which was confirmed by TLC after hydrolysis of 2

and comparison to authentic ß-6-deoxygulose. Consequently, compound 2 was

identified as the new securigenin-3ß-0-ß-6-deoxyguloside (2). COSY, HMBC and

HSQC experiments showed unambiguously that C-2' resonates at <5 68.1 and C-4'

at 8 72.3 (Table 2). Therefore, the 13C chemical shift assignment for ß-6-

deoxygulose in literature must be corrected (see Table 2).

The 13C NMR spectrum of compound 3 showed close similarities to compound 2

with the exception of the signal of C-5 which resonated as quaternary carbon at c5

74.0 and downfield shifts of C-4 and C-6 pointing to an additional substitution with

a hydroxyl group. In accordance the positive FAB-MS spectrum exhibited a [M+H]+

peak at m/z 567 and a fragment at 421 [M+H-deoxyhexosyl]\ After extensive 1D

and 2D NMR analysis compound 3 was identified as sarmentosigenin-3ß-0-ß-6-

deoxyguloside. It was described for the first time as canescein from the genus

Erysimum (Maslennikova et al., 1967; Makarevich & Kovalev, 1968).

The molecular formula of compound 4 was established as C29H44O10, obtained from

the 13C NMR spectrum and the positive FAB-MS, showing a pseudomolecular peak

at m/z 553 [M+H]+. In contrast to securigenin-3ß-0-ß-6-deoxyguloside (2), in the 1H

NMR spectrum of 4 the aldehyde proton is replaced by two protons resonating at 8

3.86 and 3.71 (each d, J = 10.8) pointing to a hydroxymethyl group. As a result of

MS and NMR analysis, the genin was identified as 19-hydroxysarmentogenin

(Kopp & Kubelka, 1982). The sugar rest was identified as ß-6-deoxygulose as

described for compound 2, Thus, compound 4 is the hitherto unknown 19-hydroxy-

sarmentogenin-3ß-0-ß-6-deoxyguloside.

The 1H and 13C NMR of compound 5 indicated the presence of a cardenolide with

two sugar moieties and a C-19 methyl group (see Tables 1 and 2). The DEPT

experiments sorted 35 carbons into three methyl, ten methylene, 17 methine and

247

Publication IV

five quaternary carbons. The positive ESI-MS spectrum gave a molecular ion at

m/z 698. Therefore, the molecular formula of 5 was determined as C35H54014 and

the aglycon was identified as sarmentogenin (Hanada et al., 1992). The identity

and connectivities of the sugar moiety was deduced from a combination of 1D and

2D NMR experiments (1H,1H COSY, HSQC, HMBC and ROESY) and confirmed by

TLC hydrolysis and comparison with authentic compounds. Therefore, 5 was

established as the new sarmentogenin-3ß-0-[a-allosyl-(1->4)-ß-6-deoxyallosideJ.

The aglycon of compound 6 showed nearly the same chemical shifts and

correlations in the 1D and 2D NMR spectra as compound 2 and was therefore

identified as securigenin. The chemical shifts and connectivities of the sugar

moieties were identical with those of compound 5. Therefore, 6 was identified as

the new securigenin-3ß-0-[a-allosyl-(1-»4)-ß-6-deoxyalloside].

All isolated cardiac glycosides 2-6 showed high cytotoxic activity against a KB cell

line (see Table 3). It is worthy of note that the cytotoxicity is not correlated with the

oxidation status of the C-19 methyl group. Cytotoxicity of compounds 2 and 4 (IC50

0.164 umol 2 vs. 0.199 jimol 4) as well as 5 and 6 (IC50 0.075 jimol 5 vs. 0.104

umol 6) were in the same range. Comparison between compounds with identical

genin and different sugar chain revealed significant difference (IC500.164 umol 2

and IC50 0.104|imol 6). Introduction of a hydroxyl group at C-5 doubled cytotoxicity

(IC500.164 umol 2 vs. 0.074 umol 3).

248

Table

1

'HNMR

data

ofcompounds2-6(CD3OD,

8ppm,J

inHz,500MHz;compound

6,600MHz

)*

H2

34

56

12.

15*,

1.82*

2.47m,2.24m

2.25°,

1.84

*2.30brd(13

4),1.

50*

2.18

*,1.

82*

21.81

51.88*

1.82

*,1.

74*

1.82

*,1.

66*

1.80

*

3 4

4.06

brs

2.15°,

1.73°

4.21brs

2.09°,

1.73*

4.05brs

1.81*,

1.60

*

4.01brs

1.83

*,1.54°

4.06m

1.78

*,1.

70°

52.37m

-

2.11m

1.79°

2.36m

61.76°,

1.46°

1.88

*,1.

66*

1.78*,

1.27

°1.86

*,1.

27*

1.46*

71.82

°2.

07°,

1.29

*1.83*,

1.33

°1.

80*

1.82

*,1.

26*

81.82°

2.01*

1.72*

1.66*

1.84*

91.

82°

1.74

*1.

89*

1.80*

1.82*

r4.42m

3.95*

3.82

td(1

0.5

4.3)

3.72

td(1

0.4

4.3)

4.42m

12

1.75

°,1.50°

15

2.18e,

1.69°

1.68

*,1.50dd

(13.7,

10.7

)

2.16*,

1.69*

1.69m,

1.56brt(1

2.1)

2.25*,

1.78

°

1.68

*,1.

56*

2.21

*,1.75*

1.76

*,1.53brt

(11

2.18

°,1.70*

16

2.18°,

1.91°

2.15°,

1.90

*2.22*,

1.91

°2.19

*,1.

90*

2.17

°,1.

90*

17

2.931(7.1)

18

0.98s

2.931(7.1)

0.90s

2.93m

0.93s

2.91m

0.90s

2.93

t(6

.9)

0.98

s

2"

2.1 1 2

)9.41

s

5.03dd

(18.2

4.92dd

(18.2

Z5.92s

4.67d

(8.2

)3.60dd

(8.2

,

,1.5)

,1.5)

3.5)

9.97s

5.03dd

(18.4,

1.5)

4.92dd

(18.4,

1.5)

5.92s

4.70d

(8.1)

3.59dd

(8.2

,3.3)

3.86d

(10.8),

5.03dd

(18.

24.92dd

(18.

25.94d(1.0)

4.67d

(8.2)

3.62dd

(8.1,

v

3.71

d(10.8)

1.5)

1.5)

3.4)

1.07s

5.02d

(18.

3)4.91d(18.3)

5.91

s

4.67d

(7.9

)3.35°

9.42

s

5.02dd

(18.

5,1.5)

4.91dd

(18.

5,1.5)

5.90s

4.70d

(8.1

)3.37dd

(8.1

,3.1

)3 4 5

3.96°

3.46dd

(3.8

,3.99°

1.0)

3.96*

3.46d

(3.4)

4.01

q(7.2,

1.0)

3.98*

3.47d

(3.5)

3.99

*

4.33

t(2

.9)

3.27dd

(9.3,

3.85°

2.7)

4.341(

2.6)

3.28dd

(9.5,2.

6)3.84*

6 1 2

1.21d(6.7)

1.22

dd(6.6)

1.23d

(6.7)

1.28d

(6.1

)4.73d

(7.9

)3.

34°

1.29d(6.2)

4.74d

(7.7

)3.35°

3 4 5

,

4.05t(2.9)

3.54dd

(9.0,

3.69°

2.7)

4.05m

3.53dd

(8.8,2.

8)3.70°

6'

3.82

°,3.70*

3.82

°,3.70*

ro

10

"Ass

ignm

ents

areconfirmedbyCOSY,HSQCandHMBC,

*Multiplicities areuncleardue

tooverlapping

Table 2

13C NMR data of compounds 2-7,11 (CD3OD, 75.5 MHz, ö ppm)

Ca 2 3 4 5 6 Ca 7 1 1

1 23.5 t 19.7 1 27.5 t 33.8 t 24.9 t 1 34.6 t 109.0 d

2 21.5 t 25.5 t 28.0 t 28.3 t 26.9 t 2 26.0 t 151.7 s

3 72.8 d 73.6 d 74.4 d 75.9 d 74.4 d 3 163.1 S 143.5 s

4 29.1 t 34.0 t 31.4 t 31.6 t 30.4 t 4 119.0 s 127.9 s

5 30.6 d 74.0 s 32.0 d 39.1 d 32.0 d 5 46.4 d 119,9 s

6 28.8 t 37.1 t 27.7 t 28.1 t 30.1 t 6 21.1 t 182.8 s

7 25.5 1 23.9 t 22.2 t 22.7 t 22.9 t 7 24.6 t 147.4 S

8 41.4 d 40.6 d 41.7 d 41.9 d 42.8 d 8 124.1 s 139.1 s

9 41.2 d 44.5 d 42.7 d 43.0 d 42.6 d 9 148.3 s 40.7 s

10 52.1 S 55.1 S 41.1 s 37.5 s 53.5 S 10 36.8 s 152.8 S

11 66.3 d 66.9 d 69.0 d 68.9 d 67.7 d 11 115.7 d 34.2 t

12 48.8 t 48.5 t 50.6 t 50.5 t 50.2 t 12 123.5 d 30.5 t

13 49.6 s 49.5S 51.1 S 51.0 s 51.0 s 13 129.4 s 40.0 s

14 83.9 s 83.8 s 85.8 S 85.6 s 85.3 s 14 154.6 s 47.2 s

15 31.7 t 31.5 t 33.5 t 33.6 t 33.1 t 15 75.7 s 29.6 t

16 26.4 t 26.4 t 27.9 t 27.9 î 27.8 t 16 30.6 q 37.8 t

17 50.2 d 49.9 d 51.8 d 51.8 d 51.6 d 17 30.7 q 31.1 S

18 15.9 q 16.0 q 17.6 q 17.5 q 17.3 q 18 18.5 q 45.4 d

19 207.1 d 209.6 d 66.7 t 24.3 q 208.5 d 19 - 31.9 t

20 175.7 s 175.7 s 177.1 s 177.1 s 177.1 s 20 22.9 q 41.7 s

21 73.9 t 73.9 t 75.3 t 75.3 1 75.3 t 21 30.9 t

22 116.5 d 116.6 d 118.0 d 118.0 d 118.0 d 22 36.2 t

23 176.2 s 176.0 s 177.7 s 177.7 s 177.6 s 23 14.0 q

1' 99.1 d 98.5 d 100.0 d 99.9 d 99.8 d 24 -

2' 68.1 d 68.1 d 69.5 d 72.0 d 72.0 d 25 41.6 q

3' 72.2 d 72.1 d 73.5 d 72.4 d 72.4 d 26 19.6 q

4' 72.3 d 72.2 d 73.7 d 84.1 d 84.0 d 27 20.6 q

5' 68.6 d 68.8 d 69.9 d 69.4 d 69.5 d 28 32.1 q

6' 15.0 q 14.9 q 16.4 q 18.2 q 18.2 q 29 180.7 s

1" 103.5 d 103.5 d 30 32.9 q

2" 72.3 d 72.3 d 31 52.2 q

3" 73.1 d 73.1 d

4" 68.4 d 68.4 d

5" 75.2 d 75.2 d

6" 62.6 1 62.6 t

Multiplicities determined by DEPT sequences.

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Table 3

Cytotoxicity of compounds 1-11 against a KB cell line (IC50 in umol)

Compound IC50 standard error n

1 5.938 ±0.1 4

2 0.164 ±0.015 4

3 0.074 ±0.009 4

4 0.199 ±0.008 6

5 0.075 ± 0.004 8

6 0.104 ± 0.005 6

7 > 66 - 4

8 >45 - 4

9 0.603 ±0.01 4

10 1.6 ±0.14 4

11 9.476 ±0.35 4

podophyllotoxin 0.014 - »4

Bioactivity-guided fractionation of the dichloromethane extract, using antibiotic

activity against Bacillus cereus, Staphylococcus epidermidls and Micrococcus

luteus led to the isolation of the new diterpene 3,15-dihydroxy~18-norabieta-

3,8,11,13-tetraene (7), the three known triterpenes friedelane-3-on-29-ol (8),

pristimerin (9) (Gunatilaka et al.. 1989), celastrol (10) (Kutney et al., 1981; Patra &

Chaudhuri, 1987), and the new triterpene 2,3,7-trihydroxy-6-oxo-1,3,5(10),7-

tetraene-24-nor-friedelane-29-oic acid methylester (11). Compound 7 was isolated

as a pale yellow amorphous powder. The 13C NMR spectrum showed signals of 19

carbons, which could be assigned with DEPT experiments into four methyl, four

methylene, three methine and eight quaternary carbons. The positive EI-MS,

showing a pseudomolecular peak at m/z 301 [M-H]~, in combination with the 13C

NMR spectra allowed the establishment of the molecular formula C19H2603. The

1H,1H TOCSY and COSY spectra revealed three spin systems. Spin system A with

two coupling protons at 8 6.79 and 6.91 (each d, J- 8.3, H-11 and H-12) indicated

the presence of a 1,2,3,4-tetra-substituted aromatic ring. Spin systems B (H-5/H-

6/H-7) and C (H-1/H-2) belong to a CH-CH2-CH2 and a CH2-CH2 moiety,

respectively. The linkage between the spin systems was established by a HMBC

experiment and led to a norabietan skeleton with a C-3/C-4 double bond (Takaishi

et al, 1997a). HSQC, HMBC and ROESY were utilized to clarify the 1H and 13C

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NMR assignments and the stereochemistry. Taken together the data established

the structure 3,15-dihydroxy-18-norabieta-3,8,11,13-tetraene (7). 13C NMR data

clearly revealed that we isolated only the enol form 7 whereas the tautomeric

keton is completely absent.

7 11

The molecular formula of compound 11, a yellow amorphous powder, was

established as C30H40O6 obtained from the 13C NMR spectrum and from positive EI-

MS, showing the molecular peak at m/z 496 [M]+. The 13C NMR and the DEPT

revealed the presence of seven methyl, seven methylene, two methine and 14

quaternary carbons. The UV spectrum contained absorption maxima at 248 and

321 nm due to a phenolic ring system. The 1H and 13C NMR data showed similar

chemical shifts to those of the C, D and E-ring of pristimerin (9) (Gunatilaka et al.,

1989). For ring A and B, long range correlations of the proton H-1 with the carbons

C-2, C-3, C-4, C-5, C-6, C-10 and the protons H3-23 with C-1, C-2, C-3, C-4, C-5,

C-6 and C-10 were observed. These data and comparison with literature data of

regeol C (Takaishi et al., 1997b) led to the identification of 2,3,7-trihydroxy-6-oxo-

1,3,5(10),7-tetraene-24-nor-friedelane-29-oic acid methylester (11).

Confirming earlier published data the triterpenoids 9 and 1 0 showed high

antibacterial activity particularly against S. epidermidls as well as a remarkable

cytotoxicity against KB cells (Kutney et al. 1981; Gonzalez et al., 1998). Both new

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terpenoids 7 and 11 lack cytotoxicity and the antibacterial activity is moderate to

low (see Tables 3 and 4).

The antibacterial activity of pristimerin (9) seems to be an explanation of the use of

C. gaumeri as an anti-diarrheal medicine. Due to its cytotoxic potential C. gaumeri

and preparations thereof should be used with great caution.

Table 4

Antibacterial activities of compounds 7-11 (MIC in umol)

Minimum inhibition concentration (MIC) in broth

Compound ——-

B. cereus S. epidermidls M. luteus

7 423.84

8

9 8.62 0.54 8.62

10 4.44 1.11 4.44

11 129.03 129.03 32.26

chloramphenicol 6.19 12.38 6.19

Experimental

General experimental procedures

Optical rotations were measured in MeOH or CHCI3 on a Perkin-Elmer model 241

Polarimeter. UV spectra were obtained on a Kontron-Uvikon 930

spectrophotometer, using MeOH as solvent. EI-MS were measured on a Hitachi-

Perkin Elmer RMUGM mass spectrometer at 70 eV. FAB-MS were obtained in the

positive mode on a ZAB 2-SEO spectrometer, using 3-nitrobenzylalcohol as matrix.

ESI-MS (positive mode) were measured on a TSQ 7000 mass spectrometer.

Applying HRESI-MS (positive mode) to the cardiac glycosides only compound 2

expressed a detectable [M+H]+ pseudomolecular peak. 1H and 13C NMR spectra

were recorded using Bruker AMX-300, DRX-500 and DRX-600 spectrometers. The

spectra were measured in CD3OD or CDCI3 and the residual CH3OH and CHCI3

resonances were used as internal references. For VLC, silica gel (60F254, 40-60

u,m, Merck) and RP-18 material (40-63 [im, CU Chemie Uetikon AG) was used.

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MPLC was carried out using a Büchi chromatography pump B-688 and a 3.5 x 80

cm Büchi column packed with silica gel (60HF254, 15 j.im, Merck). HPLC

separations were performed with a Merck-Hitachi L-6200 intelligent pump

connected to a Merck-Hitachi L-4000 UV detector or a Waters model 590 pump

connected to a Pharmacia Biotech Uvicord Sil detector. HPLC columns were from

Knauer (Spherisorb S5 ODSII, 250 x 16 mm, EOT Chemie AG, 5 |im - for the

MeOH extract and Spherisorb ODSII, 250-20 mm. Waters, 10 |iin - for the CH2CI2

extract).

Plant material

C. gaumeri was collected in the villages and surroundings of Chikindzonot, Ekpedz

and Xcocmil, Yucatan, Mexico (1994-1995). Authenticated voucher specimens

were deposited at the Herbarium of the Centra de Investigacion Cientifica de

Yucatan (CICY) in Mérida, the National Herbarium of Mexico (MEXU), the Instituto

Nacional Indigenista (INI) in Valladolid, Yucatan, the ETH Zurich (ZT) and the

Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, London,

UK.

Extraction and isolation

Air-dried and powdered roots of C. gaumeri (2.26 kg) were successively extracted

with CH2CI2, MeOH, 70% aqueous MeOH and H20. Extraction with CH2CI2 yielded

42 g extract and from the methanol extraction 95 g were obtained. An aliquot of the

latter extract (42 g) was partitioned between CHCI3 and 60% aqueous MeOH (1:1).

The polar fraction was further partitioned between n-butanol and H20 (1:1). The

butanol fraction was combined with the CHCI3 one and applied to VLC, as two

separate portions, 10 g each. Elution with CHCI3 containing increasing amounts of

methanol yielded 18 fractions. Fraction 9 (CHCI3-MeOH, 9:1) yielded compound 1.

Fractions 13-15 (6.9 g, CHCI3-MeOH, 9:1 to 6:4) were combined and subjected to

an open column chromatography using silica gel 60 (35-70 |±m) as stationary

phase and increasing amounts of aqueous MeOH (MeOH-H20, 99:1) in CHCI3 as

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Publication IV

eluent (100 % CHCI3 to 45 % CHCI3) to give 17 subfractions. The subfractions 2

(83.5:16.5), 5 (78:22), 7 (70:30), 11 (67:33) and 14 (50:50) were purified by HPLC

on RP-18 using ACN-H20 (20:80) as mobile phase for the subfractions 2, 5 and 7

and ACN~H20 (15:85) for the subfractions 11 and 14 yielding the compounds 2-6.

An aliquot (33 g, 35 %) of the CH 2CI2 extract was fractionated over silica gel

(VLC), using n-hexane-EtOAc mixtures with increasing polarity as eluent to afford

22 frs. Fraction 8 (80:20) contained compound 9. Frs 11 (30:70) and 12 (30:70)

were combined and subjected to normal-phase MPLC employing n-

hexane-EtOAc-MeOH (15:3:0.5 to 10:10:5) mixtures as mobile phase. Based on

TLC control the subfractions were combined to give 21 fractions. Fraction 3

(15:3:1) contained compound 9, after purification of fraction 6 (12:8:1) based on

MeOH-solubility, compound 8 was isolated. Fr 13 (10:10:3) was purified on a C18

Sep-Pak® Cartridge with 50% aqueous MeOH increasing the methanol proportion

to give 7. Fr 8 (10:10:1) was separated by RP-VLC using increasing amount of

MeOH in water as eluent to give 38 fractions. Of these, frs 24 and 25 (MeOH-H20,

9:1) were combined and refractionated by a silica VLC using n-hexane and n-

hexane-CHCI3 mixtures with increasing polarity which afforded 19 frs. Frs 10-15

(20:80) were combined and purified by RP-HPLC (ACN~H20, 9:1) to yield 10 and

11.

Detection of cardiac glycosides and hydrolysis of the cardiac glycosides on

TLC plates

As mobile phase for TLC analysis of the cardenolides EtOAc-MeOH-H20

(81:11:8) was used. For detection vanillin-H2S04 was sprayed on the TLC plates

(silica gel 60 F254) and heated at 110 °C for 5-10 min. The evaluation was carried

out under vis and under UV light 366 nm.

The TLC hydrolysis of the cardiac glycosides was realized following Kartnig &

Wegschaider (1971) with some modifications. The tank was not saturated with

36% HCl and the TLC plate was exposed to HCl vapor for 5 min (100 °C). The

plate was then dried for 2 hours in the air and 30 min on a heating plate (80 °C).

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Development was carried out with CHCI3-MeOH-H20 (64:36:8) as mobile phase

and it was sprayed with 0.5 g thymol in 95 ml EtOH and 5 ml H2S04 (cone).

Cytotoxicity study using KB cell culture

The cytotoxicity of the compounds was determined using a KB cell line (ATCC CCL

17; human nasopharyngeal carcinoma). The test was carried out with some

modifications according to the screening technique of Swanson & Pezzuto (1990)

in 96-well plates (Falcon) with an inoculum of 2.5 x 104 cells/ml. Test solutions

were made as stocks in 20% ethanol in water. Before testing, the solutions were

diluted 20 fold and final ethanol concentration was 1% (v/v) or less. Total assay

volume was 150 uJ. For quantification of cytotoxicity 15 ul of an aqueous solution of

methylthiazolyltetrazolium chloride (MTT, Fluka) with 5 mg/ml PBS was added

(Mosmann, 1983). After incubation at 37 °C for 4 h, the metabolically active cells

produced an insoluble formazan dye. The medium was drawn off and the formazan

dye was dissolved using 150 ul of 10 % SDS (sodium dodecylsulfate) in water.

After 24 h of incubation at room temperature, the optical density was measured at

540 nm using a microplate reader (MRX, Dynex Technologies). For determination

of the IC50 values, the optical density was plotted against the log concentration.

The test was performed at least in duplicates.

Antibacterial activity

Antibacterial activity against B. cereus (ATCC 10702), S. epidermidls (ATCC

12228), M. luteus (ATCC 9341) and E. coli (ATCC 25922) were assessed using

the doubling dilution method (Liu et al., 1999).

Securigenin-3ß-0-ß-6-deoxyguloside (2)

White powder (11.1 mg); [a]24 -61.0° (MeOH, d.O); UV Amax (MeOH): 217 nm;

positive FAB-MS m/z 573 [M+Na]+, 551 [M+H]+, 443 [M+K-deoxyhexosyl]+, 369

[M+H-desoxyhexosyl-2H20]+; 1H NMR (500 MHz, CD3OD) Table 1; 13C NMR (75.5

MHz, CD3OD) Table 2.

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Sarmentosigenin-3ß-0-ß-6-deoxyguloside (3)

White powder (9.1 mg); [a]24- 26.0° (MeOH, c 2.3); UV Amax (MeOH): 214 nm;

positive FAB-MS m/z 567 [M+H]+, 421 [M+H-deoxyhexosyl]+; 1H NMR (500 MHz,

CD3OD) Table 1; 13C NMR (75.5 MHz, CD3OD) Table 2.

19-Hydroxy-sarmentogenin-3ß-0-ß-6-deoxy-guloside (4)

White powder (23.4 mg); [a]24 - 36.0° (MeOH, c 1.0); UV Amax (MeOH): 214 nm;

positive FAB-MS m/z 553 [M+Hif; 1H NMR (500 MHz, CD3OD) Table 1; 13C NMR

(75.5 MHz, CD3OD) Table 2.

Sarmentogenin-3ß-0-[a-allosyl-(1 ->4)-ß-6-deoxyallosidel (5)

White-brown powder (39 mg); [a]24 - 5.2° (MeOH, c 2.3); UV Amax (MeOH):

214 nm; positive FAB-MS m/z 573 [M+K-H-hexosyl]+; 1H NMR (500 MHz, CD3OD)

Table 1 ; 13C NMR (75.5 MHz, CD3OD) Table 2.

Securigenin-3ß-0-[a-allosyl-(1 ->4)-ß-6-deoxyalloside] (6)

White-yellow powder (2.4 mg); [a]24 - 28.7° (MeOH, c 2.2); UV Amax (MeOH): 214

nm; positive FAB-MS m/z 573 [M+Na+H-hexosyl]*; 1H NMR (600 MHz, CD3OD)

Table 1 ; 13C NMR (75.5 MHz, CD3OD) Table 2.

3,15-Dihydroxy-18-norabieta-3,8,11,13-tetraene (7)

Yellow-brown powder (6.9 mg); [a]24 + 23.5° (MeOH, c 2.9); UV Amax (MeOH): 273

nm; positive EI-MS m/z 301 [M-H]T, 258, 216, 202, 188, 173, 149, 85, 83, 49; 1H

NMR (300 MHz, CD3OD) 8:1.04 (3H, s, H-20), 1.53 (1H, m, H-1b), 1.56 (3H, s, H~

17), 1.58 (3H,s, H-16), 1.62 (1H, m, H-6b), 1.93 (3H, brs, H-18), 2.23 (1H, m, H-5),

2.29 (1H, m, H-6a), 2.36 (1H, m, H-1a), 2.48 (2H, brd, J = 3.7 Hz, H-2), 2.70 (1H,

dd, J = 9.8, 18.9 Hz, H-7), 2.90 (1H, dd, J = 7.2, 18.4 Hz, H-7a), 6.79 (1H, d, J =

8.3, H-11), 6.91 (1H, d, J=8.3, H-12); 13C NMR (75.5 MHz, CD3OD) Table 2.

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Publication IV

2,3,7-Trihydroxy-6-oxo-1,3,5(10),7-tetraene-24-nor-friedelane-29-oic acid

methylester (11)

Yellow powder (5.3 mg); [a]24 - 45.5° (MeOH, c 3.03); UV Amax (MeOH): 284, 321

nm; positive EI-MS m/z 496, 263, 248, 234, 203, 44; 1H NMR (300 MHz, CD3OD) cS:

0.69 (3H, s, H-27), 0.96 (1H, m, H-22b), 1.08 (3H, s, H-28), 1.15 (3H, s, H-30),

1.37 (3H, s, H-26), 1.43 (1H, m, H-21 b), 1.45 (1H, m, H-16b), 1.49 (3H, s, H-25),

1.59 (1H,d, J=7.8Hz, H-18), 1.66 (1 H, dt, J=4.1, 14.1 Hz, H-12b), 1.71 (1H, dd,

J =7.4, 14.7 Hz, H-19b), 1.82 (1H, m, H-12a), 1.85 (1H, m, H-15b), 1.91 (1H, m,

H-16a), 1.97 (1H, dt, J = 4.2, 14.2 Hz, H-11b), 2.12 (1H, dt, J = 4.1, 13.8 Hz, H-

22a), 2.16 (1H, m, H-11a), 2.17 (1H. m, H-21a), 2.47 (1H, brd, J = 15.5 Hz, H-

19a), 2.57 (3H, s, H-23), 2.88 (1H, m, H-15a), 3.55 (3H, s, H-31), 6.86 (1H, s, H-

1a); 13C NMR (75.5 MHz, CD3OD) Table 2.

Acknowledgements

The authors are very grateful to the healers, midwives and the inhabitants of

Chikindzonot, Ekpedz and Xcocmil, Yucatan (Mexico) for their collaboration, for

their friendship and hospitality during the fieldwork. The botanical identification was

performed in collaboration with the numerous specialists of the Centra de

Investigacion Cientifica de Yucatan (CICY) and the National Herbarium of Mexico

(MEXU). Particularly, we would like to thank Dr. I. Olmsted, Mr. J. Granados, Mr. P.

Simâ, Mr. J.C. Trejo, Dr. R. Durân of CICY as well as Dr. O. Tellez, Dr. R. Lira, Dr.

J. Villasehor and Dr. M. Sousa of MEXU. We are grateful to Prof. Dr. Brigitte Kopp,

Institute of Pharmacognosy, University of Vienna for the reference substances,

desglucocheirotoxol and strophanolosid. The authors thank Dr. O. Zerbe (ETH,

Department of Applied BioSciences) for assistance in NMR measurements, M.

Wasescha (ETH, Department of Applied BioSciences) for performing KB cell

assays, Dr. E. Zass (ETH, Department of Chemistry) for literature search, O.

Greter, R. Häfliger and Dr. W. Amrein (ETH, Department of Chemistry, MS-service)

for recording mass spectra. We are grateful to Prof. H. Budzikiewicz (University of

Cologne, Institute of Organic Chemistry) for performing the HRESI-MS spectra.

This research owes a lot to the help of Dr. Hongmei Liu (ETH, Department of

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Applied BioSciences) and Dr. J. Orjala (Agra Quest Inc. Davis, USA). Financial

support by SDC (Swiss Agency for Development and Cooperation, Berne,

Switzerland) and the SANW (Swiss Academy of Natural Sciences) is gratefully

acknowledged.

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260

Conclusion

15 Conclusion

The healers and midwives of the three Yucatec Mayan communities, Chikindzonot,

Ekpedz and Xcocmil, have an extensive and deeply rooted knowledge about

medicinal plants. Forty informants reported a total of 360 plant species as

medicinal and 1828 individual-use reports were documented. The arrangement of

these responses into nine groups of uses formed the basis for an analysis of the

indigenous uses. The gastrointestinal problems are the most frequently mentioned

health problem of the study region, followed by skin problems. Illnesses according

to the Maya of Yucatan are classified as being humorally "hot" or "cold". However,

these characteristics were never spontaneously mentioned in the case of medicinal

plants. The healers first made references to the disease and its humoral

characteristic, and then referred to the herbal medicine indicated and its property.

In general, the humoral system is only one of the explanatory models used in this

medical system. Also form, texture or color of a plant and dreams about the healing

potency of a plant are important selection criteria.

The study of medicinal plants in comparison with non-medicinal ones showed that

taste and odor characteristics include considerable information about their use in

the treatment of illnesses. Aromatic plants are, for example, indicated to treat

diarrhea and vomiting. Taste and odor further help to distinguish between used and

non-used plants and play an important role in plant selection. Another interesting

point is that there was no difference in the percentage of species classified as bitter

between the medicinal plants and the non-medicinal ones. Thus bitterness is not a

specific critérium for plant remedies.

The evaluation of 48 important medicinal plants in various bioassays had the aim

to better understand the use of Yucatec Maya phytomedicines and their

pharmacological effects. Some correlations between the activities obtained in the

bioassays and the indigenous plant use could be shown. Other indigenous uses

261

Conclusion

cannot be explained based on the results of the pharmacological models. The

evaluation of these plants with respect to activity and cytotoxicity is a contribution

in their study of safety and efficacy. However further investigations should be made

this field.

For the detailed phytochemical study Crossopetalum gaumeri (Celastraceae) was

chosen due to its oral use against gastrointestinal problems, which is the most

frequent health problem in the study region. In addition, it is orally and locally used

in the treatment of snake bites. Furthermore, the extracts showed positive results

in different bioassays. The polar and nonpolar extracts of the roots of

Crossopetalum yielded six new compounds, four cardenolides and two terpenes.

Even though the cardenolide glycosides showed high cytotoxicity, they are not of

interest in the development of new anticancer drugs due to their strong effects on

the human heart and their small therapeutic window. The roots of C. gaumeri

showed good antibiotic and antiparasitic activity. However its oral use as a remedy

in gastrointestinal disorders must be viewed at with caution because of its high

cytotoxicity.

There is considerable knowledge about plant use by indigenous peoples and there

are established methods for testing the safety and efficacy of medicinal plants. The

aim of this ethnobotanical - phytochemical study was to forge a link between these

two areas. The study achieved this by investigating the activity of plants commonly

used by Yucatec Mayan healers and midwives. It is hoped that this collaborative

work between indigenous people and Western researchers has been mutually

beneficial and that it has helped to support the use of medicinal plants in the

region. Similarly, investigation of the phytochemistry and activity of these plants is

a further step towards the development of safer and efficient phytomedicine.

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277

List of publications

Ankli, A., Sticher, O., Heinrich, M., 1999. Medical Ethnobotany of the Yucatec

Maya: Healers' consensus as a quantitative criterion. Economic Botany 53,

144-160.

Ankli, A., Sticher, O., Heinrich, M., 1999. Yucatec Maya medicinal plants versus

nonmedicinal plants: Indigenous characterization and selection. Human

Ecology 27, 557-580.

Ankli, A., Heinrich, M., Bork, P., Wolfram, L., Bauerfeind, P., Brun, R., Schmid, C,

Weiss, C, Bruggisser, R., Gertsch, J., Wasescha, M., Sticher, O., (submitted).

Yucatec Mayan medicinal plants: Evaluation based on indigenous uses.

Journal of Ethnopharmacology.

Ankli, A., Heilmann, J., Sticher, 0., Heinrich, M., (accepted 2000). Cytotoxic

cardenolides and antibacterial terpenoids from Crossopetalum gaumeri.

Phytochemistry.

Heinrich, M., Ankli, A., Frei, B., Weimann, C, Sticher, O., 1998. Medicinal plants in

Mexico: Healers' consensus and cultural importance. Social Science and

Medicine 47, 1859-1871.

List of poster presentations

Ankli, A., Heneka, B., Orjala, J., Sticher, O., Heinrich, M., 1996. Plants in the

treatment of gastrointestinal disorders in two Yucatec Maya communities

(Mexico). Joint Meeting of the Society for Economic Botany and International

Society for Ethnopharmacology: Plants for food and medicine, London, UK, 1-

6 July. [Honorable mentioned]

Ankli, A., Heilmann, J., Sticher, O., Heinrich, M., 1999. New terpenoids from

Crossopetalum gaumeri- A Yucatec Maya medicinal plant. Joint Meeting of:

American Society of Pharmacognosy; Association Française pour

l'Enseignement et la Recherche en Pharmacognosie; Gesellschaft für

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Arzneipflanzenforschung; Phytochemical Society of Europe. Amsterdam, The

Netherlands, 26-30 July.

Heinrich, M., Ankli, A., Frei, B., Weimann, C, Sticher, O., 1999. Arzneipflanzen in

Mexiko: Intra- und interkultureller Vergleich ethnnobotanischer Daten.

Deutsche Gesellschaft für Tropenökologie. Ulm, Germany, 17 February.

Oral presentations

Ankli, A., Sticher, O., Heinrich, M., 1997. Medicinal plants versus nonmedicinal

plants - Yucatec Maya selection criteria. II Congreso Intemacional

Etnobotânica '97. Mérida, Yucatan, Mexico. 12-17 October.

Ankli, A., Frei, B., Weiss, C, Heinrich, M., Sticher, O., 1997. Feldforschung:

Grenzen und Möglichkeiten. Arbeitstagung: "Rechte an biogenetischen

Ressourcen". Berne, Switzerland, 17 June.

Ankli, A., 1999. Charakterisierung der Medizinalpflanzen gegenüber den Nicht-

Medizinalpflanzen durch die Maya in Yucatan (Mexiko). Ethnologisches

Seminar der Universität Zürich. Ethnobotanik und Ethnomedizin in der

Basisgesundheitsversorgung. Zurich, Switzerland, 21 June.

279

Curriculum Vitae

1967 Born on June 25, Laufenburg, Switzerland

1974-1979 Primary school, Stein AG

1979-1983 Secondary school, Rheinfelden

1983-1987 Grammar school of Mathematics and Natural Science, Basel

Summer 1987 Language school in Saffron Waiden, UK

1987-1992 Study of Pharmacy at ETH Zurich

Diploma in Pharmacy (Eidgenössisches Staatsexamen)

1993-1994 Community pharmacist in Kreuz-Apotheke, Winterthur;

Language school in Cuemavaca, Mexico

1994-1995 PhD study: Ethnobotanical fieldwork in Yucatan, Mexico

1995-2000 PhD study under the supervision of Prof. Dr. 0. Sticher, at

section Pharmacognosy and Phytochemistry, Institute of

Pharmaceutical Sciences, ETH Zurich

Teaching in practical courses Pharmacognosy and

Phytochemistry I and II

1996-1999 Part-time employment as community pharmacist in Carmen-

Apotheke, Zurich

January 2000 Final examination to obtain the degree of Doctor of Natural

Sciences, ETH Zurich

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Yucatan Ethnobotany

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