UNIVERSIDADE FEDERAL DE JUIZ DE FORA

DEPARTAMENTO DE PARASITOLOGIA, MICROBIOLOGIA E IMUNOLOGIA

TRABALHO DE CONCLUSÃO DE CURSO DE GRADUAÇÃO

Curso de Ciências Biológicas

Nicolli Bellotti de Souza

Juiz de Fora

2009

The antimalarial activity of Zanthoxylum chiloperone methanolic extract in vivo in

Plasmodium berghei infected mice

ABSTRACT

Malaria is one of the most important diseases nowadays. Therefore, much effort has

been done to obtain new drugs from different sources and plants have shown promising

results in several reports. Some plant species belonging to the Zanthoxylum genus are

widely used as antimalarials or antipyretics in traditional medicine. A decoction of Z.

chiloperone root bark is used traditionally in Paraguay for its antimalaric,

emmenagogue and antirheumatic properties. Besides, because of its also known

antiparasitic properties, we decided to assess the bioactivity of methanolic extracts of

Zanthoxylum chiloperone on Plasmodium berghei-infected mice. It was tested in vivo at

the doses of 50mg/Kg, 100mg/Kg and 200mg/Kg using the 4-day suppressive test. The

inhibition of parasite multiplication (ipm) was almost 40% for both 50mg/Kg and

200mg/Kg treated groups and almost 52% for 100mg/Kg. Besides, mice survived for a

few days longer than the untreated group. Cytotoxicity in mice peritoneal macrophage

was 7.27% for 50mg/Kg, 1.82% for 100mg/Kg and 0% for 200mg/Kg. These results

may be explored for the study of new antimalarial drugs, since the inhibition for

100mg/Kg dose was higher than 50%.

Keywords: antimalarial activity; Plasmodium berghei; Zanthoxylum chiloperone

1. Introduction

Human malaria is caused by species of Plasmodium: P. falciparum, P. vivax,

P. malariae and P. ovale. Malaria, besides being the main cause of high morbity and

economic loss in the world nowadays (Alecrim et al., 2007), attacking 41% of world

population (Witkowski et al., 2009). It takes almost one million lives per year and

afflicts as many as half a billion people in 109 countries in Africa, Asia and Latin

America (WHO, 2009). The control of the disease is difficult due to the lack of an

effective vaccine (Alecrim et al., 2007) and the emergence of the parasite resistance to

drugs, specially Plasmodium falciparum and also P. vivax (Cao et al., 2007; Fidock et

al., 2008; Anstey et al., 2009; Witkowski et al., 2009), and of their vectors to

insecticides (Bourdy et al., 2008). Because of this, the research for new antimalarial

drugs has been emphasized (Fidock et al., 2008) and plants consist in a very promising

and low-cost strategy – mainly in the case of malaria, when the new drug discovery

must be affordable in all ways because of many limitations (Njommang and Benoit-

Vical, 2007; Pagnoni, 2009) – also being promoted by the World Health Organization

(WHO), which recognizes that the centuries-old use of certain plants as therapeutic

resources should be taken into account of their efficacy (Chhabra et al., 2007; Gertsch,

2009).

It is estimated that 80% of the world’s population depends on herbal

remedies for treatment of diseases, mainly because the pharmaceutical medicines are

not available and either because people can not afford it (Dolabela et al., 2008). The

accumulated knowledge of the malaria endemic regions inhabitants about the curing

capacity of plants (Alecrim et al., 2007) ends up providing natural products, as was the

case of not only two of the most important currently available drugs to treat malaria,

quinine and artemisinin (Dolabela et al., 2008; Pillay et al., 2008) but also 30 drugs

originated from plants from the 250 delined as essential by the WHO (Bourdy et al.,

2008). In addition, natural products and derivates represent more than 30% of the

current pharmaceutical market, consisting in a big source of drugs (Newman et al.,

2003; Njomnang et al., 2007; Boyom et al., 2009). Therefore, it should be safe to say

that ethnopharmacological approaches could provide new antimalarial agents

(Phillipson and Wright, 1991).

In many tropical and sub-tropical countries, plants have traditionally been

used to treat fever and other symptoms associated with malaria (Noster and Kraus,

1990; Bickii et al., 2000), the single most important condition treated with herbal

remedies (Chhabra et al., 2007). Indeed, in malaria endemic areas, plant remedies are

still widely used without assurance of their efficacy, what can be proved by clinical

trials in order to validate the traditionally used plants. Knowledge of active compounds

of a medicinal plant is important for development of standardized preparations for pre-

clinical and clinical assays (Dolabela et al., 2008) so as to provide scientific

understanding (Chhabra et al., 2007), what has encouraged the researchers to screen

plant extracts as well as their naturally occurring compounds (Koumaglo et al., 1991;

Gessler et al., 1994; Munõz et al., 2000).

Zanthoxylum is the largest genus in the family Rutaceae and comprises

about 200 species of trees and shrubs, distributed worldwide, but predominantly found

in tropical and temperate regions (Ross et al., 2004) and in Brazil it is found in all

regions, including the Amazon forest (Silva et al., 2007).

Zanthoxylum species are reported to have many medicinal properties and

phytochemical studies on the genus have shown it to be a rich source of coumarins,

lignans, and alkaloids, with febrifuge, sudorific, and diuretic properties (Talapatra et al.,

1973; Ross et al., 2004; Ross et al., 2005; Diehl et al., 2000). This genus embraces

species used for different medical purposes, and some attention has been given to the

toxic effects of its different species. The absence of clinical toxicity is reported by

several studies from which it is possible to confirm several potential therapeutical uses

of plants from the genus Zanthoxylum (Silva et al., 2007). Some plant species belonging

to the Zanthoxylum genus (Z. usambarense, Z. tsihanimposa, Z. rhoifolium, Z. rubescens

and Z. chiloperone) are widely used as antimalarials or antipyretics in traditional

medicine (Milliken, 1997; Radrianarivelojosia et al., 2003; Kirira et al., 2006; Jullian et

al., 2006; Penali et al., 2007).

Zanthoxylum chiloperone Mart. ex Engl. is a dioic tree growing in South

America. A decoction of Z. chiloperone root bark is used traditionally in Paraguay for

its antimalaric, emmenagogue and antirheumatic properties (Miliken, 1997; Guy et al.,

2001). Previous studies reported that three canthin-6-one-related alkaloids, isolated

from the Paraguayan medicinal plant Zanthoxylum chiloperone, were active against 13

fungi (Thouvenel et al., 2003), effective in vivo against Leishmania amazonensis

(Ferreira et al., 2002) and also effective in vivo against Trypanosoma cruzi (Ferreira et

al., 2007). Due to its also known antiparasitic properties, we evaluated herein the

bioactivity of methanolic extracts of Zanthoxylum chiloperone on Plasmodium berghei-

infected mice. This model was recommended by the World Health Organization for

primary screening of potential blood schizonticidal agents (Carvalho et al., 1991).

2. Materials and methods

2.1. Plant materials

The leaves of Zanthoxylum chiloperone were collected and dried at room

temperature. Plant samples were collected in Concepción, Paraguay, in January 2000.

The plant was identified by Dr. F. R. G. Salimena in the Herbarium Leopoldo Krieger, of

the Department of Botany, Federal University of Juiz de Fora, where voucher specimen

(CESJ – 41. 683) was deposited.

2.2. Methanol extracts preparation

Plant samples (100 g) were pulverized, and the respective plant’s powder

was subjected to Soxhlet extraction of 48h using methanol. The methanolic extract was

concentrated in a rotary evaporator and reconstituted into 100 mg/mL stock solution.

The respective stock solution was stored at -18ºC until use. This part was performed a

Faculty of Pharmacy, by Felipe Villela Gomes and Profa. Dra. Magda Narciso Leite.

2.3. Phytochemical screening

Standard screening tests of the extract were carried out for various

constituents (Matos, 1989). The extract was screened for the presence of alkaloids,

flavonoids, triterpenes/steroids, leucoantocianidines, anthraquinones, coumarins,

saponins, tannins and volatile oils using standard procedures. This part was performed a

Faculty of Pharmacy, by Felipe Villela Gomes and Profa. Dra. Magda Narciso Leite.

2.4. Animals

For the in vivo tests, Swiss albino adult mice, 6-8 weeks of age and weighting

20-22g, were infected with 1 x 106 Plasmodium berghei infected red blood cells (NK65

strain) by the intraperitoneal route, randomly distributed in groups of five mice each.

These animals were obtained from Centro de Biologia da Reprodução, in Federal

University of Juiz de Fora (process in Ethic Committee number 063/2007-CEEA).

2.5. “In vivo” antimalarial activity evaluation

Using the 4-day supressive test (Peters, 1965), 5 animals per group were

inoculated intraperitoneally with 0,2ml of that solution (day 1) and divided into 5

groups. These groups were treated orally with 0,1ml of water or drugs, as following:

Group 1 (G1) was untreated (control untreated group); Group 2 (G2) was treated with

200mg/Kg of chloroquine (control treated group); Group 3 (G3), with 50mg/Kg of the

extract; Group 4 (G4), with 100mg/Kg of the extract; and Group 5 (G5), with

200mg/Kg of the extract. Groups 3, 4 and 5 were test groups. These treatments were

performed for 4 days, starting on the day of infection. Blood smears were made on days

1, 3, 5, 7, 11 and 14 after treatment, Giemsa stained and examined microscopically to

assess the percentage parasitaemia. At the day 14 after treatment all mice died, except

the chloroquine treated group.

2.6. Inhibition of parasite multiplication (ipm) percentage

The antimalarial activity of the drugs was established on the basis of average

parasitaemia of each group. The percent inhibition of parasite multiplication was

calculated using treated group compared with untreated group, by means of the

following formula (Carvalho et al., 1991):

[(percent average parasitaemia in the control group (pcg) – percent average parasitaemia in the test

group)/pcg] x 100.

2.7. Cytotoxicity evaluation

The cytotoxicity effect against mammalian cells expressed as cell viability was

assayed on mice’s peritoneal macrophages. Swiss albino mice received thyoglicolate

4%. After 72h, the cells were isolated from peritoneal cavity mice using cold Hank

solution. The 1 × 106 cells per well were cultivated on a 96-well plate with RPMI 1640

supplemented with 10% FBS and incubated at 37 °C in 5% CO2 atmosphere for 24h, so

that the macrophages could adhere. After this, cells not adhered were washed with

phosphate buffered saline (PBS), then adhered cells were covered with RPMI 1640

supplemented with 10% FBS and the extract was added in the doses equivalent to those

used in the in vivo test. Control wells without extract were included. Then, the plate was

incubated at 37 °C in 5% CO2 atmosphere for 48h. After this, the viability of the

macrophages was determined with the colorimetric 3-(4,5 dimethylthiazol-2-yl)-2,5-

diphenyl-tetrazolium bromide (MTT; Sigma) method based on tetrazolium salt

reduction by mitochondrial dehydrogenases. After 2h, the reaction was interrupted by the

addition of isopropanol acid and absorbance at 570 nm was measured in an ELISA

reader. Each assay was performed in triplicate.

3. Results

The evaluation of parasitemia is shown by means of average per group (Fig.1 and

Table 1) and separately per mouse (Fig. 2).

Fig. 1 – Percent average of parasitaemia per group, after the end of treatment until the

death of mice.

Table 1 – Average parasitemia per group, on days after treatment

Treated groups Days after

treatment

Untreated

group 50 mg/Kg 100 mg/Kg 200 mg/Kg

Day 1 8,47 7,16 8,80 8,88

Day 3 11,98 13,99 13,92 17,02

Day 5 14,60 11,48 9,25 11,61

Day 7 17,80 8,46 20,84 14,91

Day 11 27,13 26,74 32,45 23,42

Day 14 45,63 42,16 31,56 33,55

Fig. 2 – Percentage parasitemia for each group. � mouse I, � mouse II, � mouse III,

� mouse IV, � mouse V.

The inhibition of parasite multiplication took place mostly on day 5 after

treatment (Table 2), but only the dose of 100mg/Kg presented a relevant value, higher

than 50% of inhibition. The control treated group (chloroquine 200mg/kg) presented an

inhibition of parasite multiplication of 100% on all days.

Table 2 – Percentage of inhibition of parasite multiplication on days 1, 3 and 5 after

treatment

Group Day 1 Day 3 Day 5

50 mg/Kg 15,47% 0% 39,99%

100 mg/Kg 0% 0% 51,65%

200 mg/Kg 0% 0% 39,31%

Mice treated with chloroquine 200mg/Kg survived for all days. Mice treated with

Z. chiloperone 50mg/Kg, 100mg/Kg and 200mg/Kg survived a few days longer than the

untreated group (Fig.3). The survival can also be seen separately in Figure 4.

Fig 3 – Survival of mice from day 1 of infection to death. G1 – untreated group; G2 –

chloroquine 200mg/Kg; G3 – Z. chiloperone 50mg/Kg; G4 – Z. chiloperone 100mg/Kg;

G5 – Z. chiloperone 200mg/Kg.

Fig 4 – Survival of mice separated by group from day 1 of infection to death. G1 –

untreated group; G2 – chloroquine 200mg/Kg; G3 – Z. chiloperone 50mg/Kg; G4 – Z.

chiloperone 100mg/Kg; G5 – Z. chiloperone 200mg/Kg.

Concerning cytotoxicity, Z. chiloperone methanolic extract was not considered

cytotoxic for mouse peritoneal macrophage. Data shown in Table 3.

Table 3 - Cytotoxicity of the methanolic extract for mouse peritoneal

macrophage

50 mg/Kg 100 mg/Kg 200 mg/Kg

7,27% 1,82% NC* * Not Cytotoxic

4. Discussion

Our main result was that 100mg/Kg dose of Z. chiloperone displays a good

antimalarial activity, inhibiting more than 50% of the parasite multiplication. The

present work is in accordance with a large variety of others (Jonville et al., 2008; Céline

et al., 2008; Boyom et al., 2009; Esmaeili et al., 2009; Bero et al., 2009; Rukunga et al.,

2009; Okokon and Nwafor et al., 2009), supporting the relevance of using natural

compounds in attempt to provide antimalarials.

Some plant species belonging to the Zanthoxylum genus (Z. usambarense, Z.

tsihanimposa, Z. rhoifolium, Z. rubescens and Z. chiloperone) are widely used as

antimalarials or antipyretics in traditional medicine (Miliken, 1997;

Randrianarivelojosia et al., 2003; Kirira et al., 2006; Jullian et al., 2006; Penali et al.,

2007;). Z. rubesces was tested against Plasmodium falciparum exhibiting low activity

(Penali et al., 2007), in contrast to Z. chiloperone trypanocidal in vivo (Ferreira et al.,

2007), leishmanicidal in vitro (Ferreira et al., 2002) and antifungal in vitro (Soriano-

Agatón et al., 2005). In order to confirm activity based on the traditional usage, using

ethnopharmacological approach, we selected Z. chiloperone species to be tested against

Plasmodium berghei in mice. Models can help the comprehension of questions that are

too complex, diverse and expensive to be approached by other means. They can offer

research a more realistic expectation, which is extremely important to the optimization

of intervention programs (McKenzie, 2000). Moreover, in vivo tests provide the

evaluation of metabolic transformations by the host and the late action of the metabolic

compound after digestion. They are reproducible, give helpful information on structure-

activity relationships (Loiseau and Xuong, 1996), and are easy and simple to be

maintained (Carvalho and Krettli, 1991).

The inhibition of parasite multiplication takes place mostly on day five after

treatment. It was near 40% for the 50mg/Kg and 200mg/Kg doses, and higher than 50%

for 100mg/Kg dose. This late activity suggests a matter of drug bioavailability. Actually,

herbal products are composed by combinations of biologically active compounds with

chemicals and inherent pharmacological activities. Thus, they may suffer some

transformations during metabolism altering the consequent action (Venkataramanan et

al., 2006).

Bioavailability is defined by the fraction of administered drug that reaches

systemic circulation and many physiological/pharmacological parameters are involved,

besides the ones concerning the compound itself (Agoram et al., 2001). Its related

factors have received marked attention (Martin, 2005) since potential drug candidates

are in urgent need of discovery due to the rise of world-wide malaria drug resistance,

mainly to Plasmodium falciparum. Taking it in account, studies on the relationship

between chemical and biological properties have been conducted in order to reach

optimization, as the case of halofantrine (Khoo et al., 1998; Khoo et al., 2000) and

dihydroartemisinin (Kongpatanakula et al., 2007).

Besides, it is an important issue concerning the development of bioactive

molecules as therapeutic agents, since it depends on many factors not only of the host –

as variability of metabolizing enzymes, membrane permeation, water complexation,

passive or active processes – but also of the drug properties as well, which includes

structure features, lipophilicity, molecular weight, ease of formulation and chemical

stability (Veber et al., 2002). Structure-activity may also play a role, as demonstrated by

the evaluation of canthin-6-one analogues extracted from Zanthoxylum chiloperone,

whose changes in the structure incited changes in the antifungal ability (Soriano-Agáton

et al., 2005). Indeed, concepts related to drugs can optimize pharmacokinetic and

pharmaceutical properties, as defended by Vistoli et al. (2008) who demonstrated that

they are not “frozen statues but dancing ballerinas”.

The result for the dose of 200mg/Kg (high dose versus low efficacy) brought us to

an intriguing question, which has already been demonstrated by others (Adesomoju et

al., 2007), whose report shows that the suppression due to drug of cytokines is

correlated with exacerbation of early phase of malaria infection in mice. This may be

the case and we have looked forward to clarifying it. It is important to highlight that,

herein, it cannot be due to toxicity, since the methanolic extract demonstrated none at

any doses tested (Table 1). Confirming the absence of clinical toxicity, reported by

others (Silva et al., 2007), Z. chiloperone methanolic extract was not cytotoxic for

mouse peritoneal macrophages.

The higher ipm for the intermediate dose leads us to the supposition that the lower

dose was not enough to provide a considerably higher ipm. It may be due to matters

related to the compound, such as the ones already discussed. Another supposition is

about the fact that the inhibition of 100mg/Kg dose (51,65%) was higher than

200mg/Kg dose (39,31%). This phenomenon may be a matter of hormesis, which is

defined in a general way as benefit at low doses and harm at high ones (Calabrese and

Blain, 2005). Indeed, it is already clear that xenobiotics can modify endogenous

substrates, altering the metabolic equilibrium, as the example of phenorbital (Kitano et

al., 1998), and the suspicious presence of other variables must be taken into

consideration, since biphasic responses may derive from very diverse mechanisms

(Murado and Vazquez, 2007).

Comparing to untreated group, the survival time of all doses of Z. chiloperone

treated mice was longer. Although the parasitaemia of untreated group was lower than

in some Z. chiloperone treated groups, as seen on day 7 and 11 after treatment, these

mice survived longer than untreated mice. This may confirm the antimalarial activity of

methanolic extract of Z. chiloperone.

This work revealed antiplasmodial activity in vivo of the Z. chiloperone

methanolic extract, whose data is very scarce. It also brought up the intriguing question

about high dose versus low efficacy. Furthermore, it represents an addition on Z.

chiloperone range of bioactivity, as it was trypanocidal in vivo (Ferreira et al., 2007),

leishmanicidal in vitro (Ferreira et al., 2002) and antifungal in vitro (Soriano-Agatón,

2005). Thus, these results may be objects of further researches for new antimalarial

agents, since the inhibition of parasite multiplication for 100mg/Kg group was almost

52%, besides the results - almost 40% - for the other doses. Last but not least, essential

to remember is the fact that natural products represent a potential source of

antimalarials, as mentioned before.

Concluding, this report represents a contribution to a better understanding of the

matters related to the attempts of controlling malaria. Therefore, more efforts on these

shall be held.

Aknowledgements

We thank Federal University of Juiz de Fora for financial support to student’s

fellowship and we are grateful to Dr. Erika Martins Braga (Department of Parasitology,

UFMG) and Dr. Kézia Scopel for kindly providing Plasmodium berghei NK65 strain.

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