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PHARMACOGNOSY, PHYTOCHEMISTRY AND
PHARMACOLOGICAL STUDIES ON
Tricalysia sphaerocarpa (Dalzell ex Hook. F,) Gamble
Thesis submitted to the
Pondicherry University
In partial fulfillment of the requirements
for the award of the Degree of
Doctor of Philosophy in Botany
By
G. ANANDHI
Under the guidance of
Dr. A. PRAGASAM
DEPARTMENT OF BOTANY
KANCHI MAMUNIVAR CENTRE FOR POST-GRADUATE STUDIES
PUDUCHERRY-605 008
August - 2014
PHARMACOGNOSY, PHYTOCHEMISTRY AND
PHARMACOLOGICAL STUDIES ON
Tricalysia sphaerocarpa (Dalzell ex Hook. F,) Gamble
Thesis submitted to the
Pondicherry University
In partial fulfillment of the requirements
for the award of the Degree of
Doctor of Philosophy in Botany
Submitted by
G. ANANDHI
Under the guidance of
Dr. A. PRAGASAM
DEPARTMENT OF BOTANY
KANCHI MAMUNIVAR CENTRE FOR POST-GRADUATE STUDIES
PUDUCHERRY-605 008
August - 2014
Dr. A. PRAGASAM
Research Supervisor
Department of Botany
Kanchi Mamunivar Centre for PG Studies
Puducherry-605 008
CERTIFICATE
This is to certify that the PhD research work entitled “Pharmacognosy,
Phytochemistry and Pharmacological studies on Tricalysia sphaerocarpa (Dalzell ex
Hook. f,) Gamble” is based on the original work done by Mrs. G. ANANDHI,
Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Puducherry
and this has not previously formed the basis for the award of any degree, diploma,
associateship, fellowship or any other similar title and it represents entirely an
independent work on the part of the candidate.
I further state that the entire thesis represents the independent work of G.
Anandhi and all the experimental techniques employed in this work were actually
undertaken by the candidate herself under my guidance.
Place: Puducherry (A. PRAGASAM)
Date:
G. ANANDHI
Ph.D. Research Scholar
Department of Botany
Kanchi Mamunivar Centre for Post Graduate Studies
Puducherry- 605 008
DECLARATION
I, Mrs. G. ANANDHI hereby declare that the research work entitled
“Pharmacognosy, Phytochemistry and Pharmacological studies on Tricalysia
sphaerocarpa (Dalzell ex Hook. f,) Gamble” submitted for the award of the Degree of
Doctor of Philosophy in Botany is my original work and has not previously formed the
basis for the award of any degree, diploma, associateship, fellowship or any other similar
title.
(G. ANANDHI)
Place: Puducherry
Date:
ACKNOWLEDGEMENT
I deeply express my sincere gratitude to my guide and research supervisor,
Dr. A. Pragasam, Department of Botany, Kanchi Mamunivar Centre for Post
Graduate Studies, Puducherry for his excellent guidance, inspiration, continued
support and critical perusal of thesis.
I would like to thank Dr.V.Ananthan the present Director,
Dr.R.Swaminathan, Dr.V.Ramassamy, Dr.E.M.Rajan and Dr.O.P.Shyma the
former Directors, Kanchi Mamunivar Centre for PG Studies, Puducherry for
providing necessary facilities to carry out my project successfully.
I extend my thanks to my doctoral committee members, Dr. D.
Ramamoorthy, Associate Professor, Department of Ecology & Environmental
Sciences, Pondicherry University, and Dr. B. K. Nayak, Associate Professor,
Department of Botany, Kanchi Mamunivar Centre for PG Studies, Puducherry, for
providing valuable suggestions during the doctoral committee meetings.
I am grateful to Dr.V. Jayachandran the present Head of the Department,
Dr.D.Kadamban and Dr.S.Nadanakunjidam former Heads of the Department of
Botany, Kanchi Mamunivar Centre for PG Studies, Puducherry, for their
encouragement.
I express my sincere thanks to the faculty member of Botany, Dr. K.
Rajendiran for giving constant support in completing my research programme.
My heartfelt thanks to my husband R. Sachithanantha Kumar and my
beloved parents for their constant encouragement and continued support to complete
the research work successfully.
Mrs. G. ANANDHI
CONTENT TITLE Page No.
1. Introduction 1-11
1.1. Pharmacognosy
1.2. Phytochemistry
1.2.1. Phytochemical Revolution
1.3. Pharmacology
1.3.1. Antioxidant Activity
1.3.2. Anti - Depressant Activity
1.3.3. Anti - Diabetic Activity
2. Review of Literature 11-18
2.1. Family Rubiaceae
2.2. Objectives of the Present Study
3. Materials and Methods 19-50
3.1. Collection of Plant material
3.2. Taxonomy of the Species
3.3. Morphological Features
3.4. Ecology
3.5. Medicinal Uses
3.6. Chemical constituents isolated from the different species of Tricalysia
3.7. Pharmacognostical Studies
3.7.1. Anatomical studies
3.7.2. Histochemical Colour Reactions
3.7.3. Fluorescence Analysis
3.8. Phytochemistry
3.8.1. Physio - Chemical Constants
3.8.2. Preparation of the Extracts
3.8.3. Extractive Values
3.8.4. PH Determination of Powdered Drug
3.8.5. Preliminary Phytochemical Screening
3.8.6. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
3.9. Pharmacology
3.9.1. In vitro Antioxidant activity
3.9.1.1. Inhibitiory effects on DPPH Radical Assay
3.9.1.2. Hydrogen peroxide Assay
3.9.1.3. Superoxide dismutase (L-methionine and NBT) assay
3.9.1.4. Iron Chelating Activity (FRAP)
3.9.2. In vivo Pharmacological Studies
3.9.2.1. Acute Toxicity Studies
3.9.2.2. Anti-Depressant Activity
3.9.2.2.1. Forced Swimming Test (FST)
3.9.2.2.2. Tail Suspension Test (TST)
3.9.2.2.3. Hole Board Test (HBT)
3.9.2.3. Anti Diabetic Activity
3.9.2.3.1. Screening of Hypoglycemic Activity in Normal Rats
3.9.2.3.2. Anti-Diabetic Activity in Experimentally Induced Diabetic Rats
3.9.2.3.2.1. Single-Dose Short Term Study
3.9.2.3.2.2. Multi- Dose Long Term Study
3.9.2.3.3. Effect of Formulation Test Extract on Body Weight in Normal and
Alloxan Induced Diabetic Rats
3.9.2.3.4. Biochemical Parameters Determinations
3.9.2.3.5. Histopathological Studies
4. Results 51-76
4.1. Pharmacognosy
4.1.1. Anatomy
4.1.1.1. Leaf peeling
4.1.1.2. Venation Pattern
4.1.1.3. Quantitative Values of Foliar Epidermis
4.1.1.4. Stem Peeling
4.1.1.5. Maceration
4.1.1.6. Transverse Section of Leaf
4.1.1.7. Transverse Section of Stem
4.1.1.8. Transverse Section of Root
4.1.2. Histochemical Colour Reactions
4.1.3. Fluorescence Analysis
4.2. Phytochemistry
4.2.1. Physico-Chemical Parameters of Various Parts
4.2.2. Extractive Values
4.2.2.1. Batch Process
4.2.2.2. Successive Process
4.2.3. PH Determination of Powdered Drug
4.2.4. Preliminary Phytochemical screening
4.2.5. GC-MS Analysis
4.2.5.1. GC-MS Analysis of Methanolic Extract of Leaf
4.2.5.2. GC-MS Analysis of Methanolic Extract of Stem
4.2.5.3. GC-MS Analysis of Methanolic Extract of Root
4.2.5.4. GC-MS Analysis of Methanolic Extract of Fruit
4.2.5.5. Comparative Analysis of Compounds Identified by GC-MS Analysis
4.3. Pharmacology
4.3.1. Invitro Antioxidant Activity
4.3.1.1. DPPH Scavenging Activity
4.3.1.2. Iron Chelating Activity (FRAP)
4.3.1.3. Hydrogen peroxide Assay
4.3.1.4. Superoxide dismutase (L-methionine and NBT) assay
4.3.2. In vivo Pharmacological Studies
4.3.2.1. Acute Toxicity Studies
4.3.2.2. Anti-Depressant Activity
4.3.2.2.1. Forced Swimming Test (FST)
4.3.2.2.2. Tail Suspension Test (TST)
4.3.2.2.3. Hole Board Test (HBT)
4.3.2.3. Anti Diabetic Activity
4.3.3.1. Screening of Hypoglycemic Activity in Normal Rats
4.3.3.2. Anti-Diabetic Activity in Experimentally Induced Diabetic Rats
4.3.3.2.1. Single-Dose Short Term Study
4.3.3.2.2. Multi- Dose Long Term Study
4.3.3.3. Effect of Formulation Test Extract on Body Weight in Normal and
Alloxan Induced Diabetic Rats
4.3.3.4. Biochemical Parameters Determinations
4.3.3.5. Histopathological Studies
5. Discussion 77-93
5.1. Pharmacognosy
5.2. Phytochemistry
5.2.1. Phytochemical Screening
5.2.2. GC-MS Analysis
5.3. Pharmacognosy
5.3.1. Antioxidant Activity
5.3.2. Anti - Depressant Activity
5.3.3. Anti - Diabetic Activity
6. Summary and Conclusions 94-98
7. Bibliography 99-112
8. Publications
List of Tables
Table 1: Quantitative values of foliar epidermis of Tricalysia sphaerocarpa
Table 2: Histochemical colour reactions of various parts of Tricalysia sphaerocarpa
Table 3 : Fluorescence analysis of Leaf powder of Tricalysia sphaerocarpa
Table 4 : Fluorescence analysis of Stem powder of Tricalysia sphaerocarpa
Table 5: Fluorescence analysis of Root powder of Tricalysia sphaerocarpa
Table 6: Fluorescence analysis of Fruit powder of Tricalysia sphaerocarpa
Table 7: Proximate analysis of various parts of Tricalysia sphaerocarpa
Table 8: Extractive values of various parts of Tricalysia sphaerocarpa by batch process Table 9: Extractive values of various parts of Tricalysia sphaerocarpa by successive
process Table 10: PH Determination of water extract of Tricalysia sphaerocarpa
Table 11: Phytochemical colour reactions of various extracts of Leaf of Tricalysia sphaerocarpa
Table 12: Phytochemical colour reactions of various extracts of Stem of Tricalysia
sphaerocarpa Table 13: Phytochemical colour reactions of various extracts of Root of Tricalysia
sphaerocarpa Table 14: Phytochemical colour reactions of various extracts of Fruit of Tricalysia
sphaerocarpa Table 15: GC-MS Analysis of Methanol extract of Leaf of Tricalysia sphaerocarpa
Table 16: GC-MS Analysis of Methanol extract of Stem of Tricalysia sphaerocarpa
Table 17: GC-MS Analysis of Methanol extract of Root of Tricalysia sphaerocarpa
Table 18: GC-MS Analysis of Methanol extract of Fruit of T. sphaerocarpa
Table 19: Combined table for GC-MS Analysis of Methanol extract of Tricalysia sphaerocarpa
Table 20: Chemical groups obtained from GC-MS Analysis of Methanol extract of
Tricalysia sphaerocarpa Table 21: Antioxidant activity of various extracts using DPPH assay
Table 22: Antioxidant activity of various extracts using Iron chelating activity
Table 23: Antioxidant activity of various extracts using Hydrogen peroxide assay
Table 24: Antioxidant activity of various extracts using Superoxide dismutase assay
Table 25 : Effect of Methanolic extract of Tricalysia sphaerocarpa on Immobility time in FST
Table 26: Effect of Methanolic extract of Tricalysia sphaerocarpa in Immobility time in
TST Table 27: Effect of Methanolic extract of Tricalysia sphaerocarpa. in Hole Board Test
(HBT) Table 28: Effect of Test extract of Tricalysia sphaerocarpa on blood glucose level in
normal fasted rats Table 29: Effect of Test extract of Tricalysia sphaerocarpa on blood glucose level in
Alloxan-induced diabetic rats (Single-dose short term study) Table 30: Effect of multidose administration of Test extract of Tricalysia sphaerocarpa
on blood glucose level in Alloxan-induced diabetic rats (long term study of 15 days daily once)
Table 31: Effect of formulation Test extract of Tricalysia sphaerocarpa on body weight
in Normal and Alloxan induced diabetic rats Table 32: Effect of formulation Test extract of Tricalysia sphaerocarpa on Biochemical
parameters in Alloxan induced diabetic rats.
List of Figures
Figure 1: Extractive values of various parts of Tricalysia sphaerocarpa by Batch process Figure 2: Extractive values of various parts of Tricalysia sphaerocarpa by Successive process Figure 3: GC-MS chromatogram of Methanolic Leaf extract of Tricalysia sphaerocarpa
Figure 4: GC-MS chromatogram of Methanolic Stem extract of Tricalysia sphaerocarpa
Figure 5: GC-MS chromatogram of Methanolic Root extract of Tricalysia sphaerocarpa
Figure 6: GC-MS chromatogram of Methanolic Fruit extract of Tricalysia sphaerocarpa
Figure 7: (a-f) Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa
Figure 8: Anti oxidant activity - DPPH Scavenging assay
Figure 9: Anti oxidant activity - Iron chelating activity (FRAP)
Figure 10: Anti oxidant activity - Hydrogen peroxide assay
Figure 11: Anti oxidant activity - Superoxide dismutase (L-methionine and NBT assay)
List of Plates
Plate 1: Morphology of Tricalysia sphaerocarpa
Plate 2: Leaves of Tricalysia sphaerocarpa
Plate 3: Size and orientation of stomata in Tricalysia sphaerocarpa
Plate 4: Vein islet and Veinlet termination of Tricalysia sphaerocarpa
Plate 5: T. S of Stem of Tricalysia sphaerocarpa
Plate 6: T. S of Root of Tricalysia sphaerocarpa
Plate 7: Macerated elements of stem of Tricalysia sphaerocarpa
Plate 8: Effect of Methanolic stem extract of Tricalysia sphaerocarpa –Anti-Depressant Activity
Plate 9: Effect of Methanolic stem extract of Tricalysia sphaerocarpa - Anti-Diabetic Activity -
Histopathological Studies
1
CHAPTER 1
INTRODUCTION
Human beings came on this earth as guests of plants is a monumental
ancient aphorism. Nature is the supreme creation and man has completely been
dependent on plants. As population increased, he has learnt to implicit natural
resources and to make use of every bit of it. Man since creation has depended on
plants for food, drinks, shelter, clothing, equipment, dental care and medicine
(Gbile, 1986). In fact from the start of life to the last breath, almost every aspect
of human life is deeply associated with plants. Primitive man tried to cure
diseases from plants growing abundantly around him. His experience through trial
taught him a lot about the medicinal properties of different plants. India is
endowed with vast resources of medicinal and aromatic plants. These plants have
been used in Indian health systems. The great interest in the use and importance
of Indian medicinal plants by world health organization in many developing
countries has let to intensify efforts on the documentation of ethno medicinal data
of medicinal plants (Perumalsamy and Ignacimuthu, 2000).
Our forefathers were depending on plants for treatment of various diseases
before the introduction of orthodox medicine. Ancient literatures of world on
medicines suggest that the primitive people of antiquity and those of earlier
centuries have been using several kinds of medicinal plants for combating
diseases. China used drug plants as early as 5000 to 4000 BC. India has over
3000 year-old medicinal heritage based on herbs. The sacred Vedas and other
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ancient Indian treatises give many references of these medicinal plants. The
ancient Indian treatise ‘Rig veda’ deals with medicinal plants. Indians classified
plants into three groups on the basis of their usage as ‘Ubhdida’ (botanical),
‘Annapanandi’ (dietic) and ‘Virechandi’ (medicinal). Parashara
wrote’Virkshayerveda’ describing medicinal plants much before the beginning of
Christian era (Saxena, 1989). There are references of miracle herbs and wonder
drugs in the ancient Indian literatures which had magical properties and were used
to cure some of the incurable diseases from tip to the toe, to increase longevity
and even to bring the dead back to life. The charak and sushrut samhitras were
written between 700-200 BC, and include accounts of the discovery of medicinal
plants (Pandey and Verma, 2005). The Assyrians, Babylonians and Ancient
Hebrews were all familiar with the usage of plants. The Greeks were familiar with
many of the present day drugs, as evidenced by the works of Aristotle,
Hippocrates (Father of medicine), Pythagoras, Theophrastus, Pleny and Galen. In
77 BC Dioscorides wrote his great treastise, “De Materia Medica” which dealt
with the nature and properties of all the medicinal substances known at that time.
The foremost classical work in botany of medicinal flora in the world ‘Hortus
Malabaricus’ was written by Heinrich Van Rheede in Kerala, India. India is now
beginning to search her roots in the past and revive her lost glory of the traditional
system of medicine which flourished here for several centuries and contributed
much to the development of medicinal science to the world. From this crude
beginning the study of drugs and drug plants has progressed until now as
pharmacognosy and pharmacology which are the essential branches of medicine.
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The most valuable of the drug and drug plant has been standardized as a result of
the Pure Food and Drug act of 1906 (Hill, 1972).
Herbal medicine is known in every village and communes of India and
every village has elders both men and women who have acquired knowledge
about the medicinal properties of plants through long tradition and experience. In
the past, sickness was viewed as a punishment of the God and hence was treated
with prayers and rituals that included what may have been considered “magic
portion” prepared from local herbs (Sandhya et al., 2010). Plants produce a wide
variety of compounds that can act on different systems of the body and have high
therapeutic activity. More than 2,40,000 plants are considered to be growing in
different parts of the world. Only about 5-10 percent of them have been screened
for chemical or biological activity. Herbal medicine cures the root cause of a
disease and not merely providing symptomatic relief, as does the modern
synthetic medicine. Thus, traditional medicine not only cures but also rejuvenates
the body’s defense system. The medicine and aromatic plants sector has
traditionally occupied an important position in the socio-cultural, spiritual and
medicinal arena of rural and tribal lives of India (Battacharrya et al., 2005).
Nature keeps ready within its ‘green bag’ substances which would
promptly act to neutralize the effect of any such substance proving unsuitable and
non-compatible to the human body. Chemical investigations of wild medicinal
plants used by the indigenous people of world shows unknown compounds with
promising biological activity. Indigenous culture has provided several ‘miracle
plants’ of immense food and medicinal value to the modern civilization. Seventy
4
four percent of 119 plant derived drugs were discovered as a result of chemical
studies to isolate the active substances responsible for their traditional use
(Farnsworth and Soejarto, 1991). So, plants, especially the higher plants contain a
variety of substances, which are useful as food additives, perfumes and in
treatment of various diseases as medicines due to their versatile therapeutic
potential (Mukherjee and Wahile, 2006). The active secondary metabolites
possess various medicinal applications as drugs or as model compounds for drug
synthesis. Large scale evaluation of the local flora exploited in traditional
medicine for various biological activities is a necessary first step in the isolation
and characterization of the active principle and further leading to drug
development. The identification of drug yielding plants, crude drugs obtained
from them, identification of crude drugs, extraction of the principle drugs, study
of their antimicrobial activities and their potential use as antioxidants are essential
to evolve new natural curatives instead of antibiotics. The worldwide experiments
in these fields are related to pharmacognosy, phytochemistry, and
pharmacological investigations.
1.1. Pharmacognosy:
Pharmacognosy is defined as the scientific and systematic study of
structural, physical, chemical and sensory characters of crude drugs along with
their history, method of cultivation, collection and preparation for the market
(Evans, 1996). Identification of drugs can be done by morphological, histological
and chemical testing. There are five methods of evaluation crude drugs namely
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Morphlolgical or Organoleptic, Microscopical or Histological, Physical, Chemical
and Biological.
Organoleptic evaluation means the study of morphological characteristics
by which the drugs are identified. It also includes those of colour, odour, taste,
consistency of powdered drug, size, shape etc. Micorscopical evaluation is useful
for organized drugs. If the drug is in entire form which we can take transverse or
longitudinal sections and study the cellular structures. Surface preparations can be
studied for stomata or trichomes. If the drug is in powder form, microscopic
identification is done to identify the parts of the crude drug. The measurement of
length, diameter of structures also helps in identification. Physical standards are
studied as under refractive index, moisture content, viscosity, melting point,
optical rotation and solubility of crude drugs. The evaluation of drug can be done
by chemical method such as assays, extractive values, volatile oil content, ash
content and drugs standardized by chemical tests. In the biological method of
estimation of potency of a crude drug is done by means of its effect on living
organisms such as other plants, animals, microbes etc.
1.2. Phytochemistry:
Phytochemistry includes drug development from natural origin,
establishment of botanical identity of herbs, phytochemical isolation and
identification, screening of herbal formulations and isolated compounds.
Phytochemicals (or) secondary metabolites are a wide range of low molecular
weight chemical compounds that are produced and accumulated by the plants.
These include alkaloids, phenolic acids, flavanoids, steroids, terpenoids and
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saponins. Phytochemical analysis of plants, used in folklore has yielded a number
of compounds with various pharmacological activities. Hence medicinal plants
are important substances for the study of their traditional uses through the
verification of pharmacological effects and can be natural composite sources that
act as a disease curing agents. About 3000 materials from 2764 plant species have
been screened for their pharmacological and chemotherapeutic properties (Anon,
1988). Traditionally used medicinal plants produce a variety of compounds of
known therapeutic properties (Iyengar, 1976; Harbone, 1989; Chopra et al.,
1992).
1.2.1. Phytochemical Revolution:
Even modern medicines and some very valuable drugs such as morphine,
digitoxin, reserpine, vinblastine, quinine etc. are obtained from the plants.
Cocaine derived from Erythroxylum cacao lead to the synthesis of procaine and
other related anesthetics. Salicin obtained from Salix purpurea, lead to the
synthesis of acetyl salicylic acid (aspirin). Morphine and codeine from Papaver
somniferum and P. bracteatum lead to the synthesis of pain killer. Anti cancerous
drug taxol is obtainted from Taxus wallichiana and T. buccata. Synthetic anti
cholinergic drugs like atropine and scopolamine are obtained from Atropa
belladonna and A. acuminata. Tinospora cordifolia has been reported to stimulate
indigenous insulin secretion by the pancreas (Gupta, 1967).
1.3. Pharmacology
Parmacology is the study of the relevant forms of knowledge, practice and
cultures implementing them the role of natural products, herbal medicines, tribal
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and traditional medicines is being increasingly appreciated during the recent years
for the prevention and cure of human ailments (Janardhanan et al., 2006)
1.3.1. Antioxidant Activity
Antioxidation compounds in food play an important role as a health
protecting factor. Scientific evidence suggests that antioxidants reduce the risk for
chronic diseases including cancer and heart disease. Primary sources of naturally
occurring antioxidants are whole grains, fruits and vegetables. Plant sourced food
antioxidants like vitamin C, vitamin E, carotenes, phenolic acids, phytate and
phytoestrogens have been recognized as having the potential to reduce disease
risk. Most of the antioxidants in a typical diet are derived from plant sources and
belong to various classes of compounds with a wide variety of physical and
chemical properties. Some compounds, such as gallates, have strong antioxidant
activity, while others, such as the mono-phenols are weak antioxidants. The main
characteristic of an antioxidant is its ability to trap free radicals.
In recent years, there has been great interest in screening various plant
extracts for natural antioxidants because of their great free radical sacvenging
properties (Jia et al., 2007). Antioxidants neutralize reactive oxygen which cause
stress, diseases of our cells and inflict damage to biomolecules, resulting in aging
and genetic changes that lead to cancer. Common sources of antioxidants are
fruits, vegetables and medicinal plants. Therefore, a great number of different
spices and aromatic herbs have been investigated for antioxidant activity
(Erdemoglu et al., 2006). Antioxidants are widely used as food additives to
8
provide protection against oxidative degradation of foods by free radicals (Gulcin
et al., 2002).
1.3.2. Anti - Depressant Activity
According to the World Health report (WHO, 2001), approximately 450
million people suffer from mental or behavioral disorder, yet only a small
minority of them receive even the most basic treatment. This amounts to 12.3% of
the global burden of disease, and will rise to 15% by 2020 (Reynolds, 2003).
Major depression, a debilitating psychiatric disorder, is predicted to be the second
most prevalent human illness by the year 2020. Various antidepressants, ranging
from monoaminoxidase inhibitors to recently developed dual reuptake inhibitors,
are prescribed for alleviating the symptoms of depression. The common
symptoms of major depression include depressed or irritable mood, decreased
interest in pleasurable activities, significant weight loss or gain, insomnia or
hypersomnia, psychomotor agitation or retardation, fatigue or loss of energy,
feeling of worthlessness or excessive guilt, decreased concentrating power, and
increase in suicidal tendencies. Earlier, major depression was considered to be an
old-age disease. However, current trends reveal an increased percentage of
younger populations being affected from its consequences. Major depression is
relatively common among patients with a diagnosis of dementia (Ballard et al.,
1996, Stepaniuk et al., 2008) and also may pose a risk factor for development of
dementia (Kokmen et al., 1996). Despite the availability of these blockbuster
molecules, approximately 30% of depressed patients do not respond to the
existing drug therapies and the remaining 70% fail to achieve complete remission
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(Kulkarni et al., 2009). Herbal drug used in depression are Centella asiatica ,
Hypericum perforatum, Rhodiola rosea, Pfaffia paniculata, Rauwolfia serpentina,
Rhododendron molle, Schizandra chinesis, Thea sinensis, Uncaria tomentosa,
Valeriana officinalis and Withania somnifera (Mamedov, 2005). Moreover,
antidepressants are associated with a plethora of side effects and drug-drug/drug-
food interactions. In this context, novel approaches are being tried to find more
efficacious and safer drugs for the treatment of major depression.
1.3.3. Anti - Diabetic Activity
In India, the prevalence of diabetes mellitus is on increase and needs to be
addressed appropriately. A study of ancient literature indicates that diabetes
(madhumeha) was fairly well known and well conceived as an entity in India. The
knowledge of the system of diabetes mellitus, as the history reveals, existed with
the Indians since prehistoric age. 'Madhumeha' is a disease in which a patient
passes sweet urine and exhibits sweetness all over the body, i.e. in sweat, mucus,
breathe, blood, etc. Diabetes mellitus is a serious complex chronic condition that
is a major source of ill health worldwide. This metabolic disorder is characterized
by hyperglycemia and disturbances of carbohydrate, protein, and fat metabolisms,
secondary to an absolute or relative lack of the hormone insulin. Besides
hyperglycemia, several other factors including dislipidemia or hyperlipidemia are
involved in the development of micro and macrovascular complications of
diabetes that are the major causes of morbidity and death (Kameswararao, 2003).
According to WHO projections, the prevalence of diabetes is likely to increase by
35%. Currently, there are over 150 million diabetic patients worldwide and this is
10
likely to increase to 300 million or more by the year 2025. Statistical projection
about India suggests that the number of diabetics will rise from 15 million in 1995
to 57 million in the year 2025, the highest number of diabetics in the world
(Satyanarayana, 2006). Reasons for this rise include increase in sedentary
lifestyle, consumption of energy-rich diet, obesity, higher life span, etc. Other
regions with greatest number of diabetics are Asia and Africa, where diabetes
mellitus rates could rise to twofold to threefold than the present rates (Eidi, 2006).
Evaluation of plant products to treat diabetes mellitus is of growing interest as
they contain many bioactive substances with therapeutic potential. In recent years,
several authors evaluated and identified the antidiabetic potential of traditionally
used Indian medicinal plants using experimental animals. Previous studies
confirmed the efficacy of several medicinal plants in diabetes mellitus. Although
a large number of medicinal plants have been already tested for their antidiabetic
effects, these effects remain to be investigated in several other Indian medicinal
plants(Sharma, 1994). Herbal remedies are considered convenient for
management of type 2 diabetes with postprandial hyperglycemia due to their
traditional acceptability and availability, low costs and lesser side effects.
The application of systems biology technologies and approaches, that is,
genomics, proteomics and metabolomics, to phytomedicine research may greatly
assist evidence-based phytotherapeutics, and such research may also lead to a
change of paradigm in the development and application of complex plant/
phytochemical compound mixtures in modern medicine (Ulrich et al., 2007). So
11
the present work has been taken up to evaluate the medicinal potential of
Tricalysia spherocarpa (Dalzell ex Hook. F.) Gamble
12
CHAPTER 2
REVIEW OF LITERATURE
Consumption of fruits and vegetables is shown to lower the risk for
chronic diseases such as cancer, cardiovascular diseases and stroke. The positive
health effects may be due to high contents of certain phenolic compounds in
plant-derived foods. Recently, phytochemicals and their effects on human health
have been intensively studied. In particular, a search for antioxidants,
hypoglycemic, and anticancer agents in vegetables, fruits, teas, spices and
medicinal herbs has attracted great attention.
A rich literature is available on the studies of Indian medicinal plants. The
studies of Chaudhuri (1965) on Calophyllum inophylum, Kannabiran and
Krishnamurthy (1972) on Anisomeles malabarica, Nayar et al., (1976) on
Aristalochia tagala, Krishnamurthy and Kannabiran (1982) on Caesalpinia crista,
Santha et al., (1988) on Nilgirianthus heyneanus, Ahmad (1994) on Jatropha
curcas, Nambiar et al., (1996) on Hemidesmus indicus, Seetharam et al., (1999)
on Eclipta alba, Annamalai et al., (2000) on Phyllanthus amarus, Srivastava
(2001) on Curcuma amada, Amerjothy (2003) on Spilanthes calva, Suseela and
Prema (2007) on Lagascea mollis, Devika and Sajitha (2007) on Phyllanthus
niruri, Jushi Singh (2011) on many endangered medicinal plants are some of the
examples to be mentioned.
13
This chapter encompasses the published literature on medicinal uses and
various studies on the genus Tricalysia a member of the family Rubiaceae which
is chosen for the present study.
2.1. Family Rubiaceae
Rubiaceae are a family of flowering plants, variously called the coffee
family, madder family or bedstraw family. Members of the coffee family tend to
be concentrated in warmer and tropical climates around the world. Currently,
about 611 genera and more than 13,000 species are placed in Rubiaceae. This
makes it the fourth-largest family of flowering plants by number of species, and
fifth largest by number of genera. The group contains many commonly known
plants, including the economically important coffee (Coffea), quinine (Cinchona),
and gambier (Uncaria), the medicinal ipecacuanha (Carapichea ipecacuanha),
and the horticulturally valuable madder (Rubia), west indian jasmine (Ixora),
partridgeberry (Mitchella), Morinda, Gardenia, and Pentas.
During the survey of Rubiaceous taxa, it was investigated that most of the
plants of this family are of great medicinal value. Several ailments like ulcers,
dysentery, athlete’s foot, diabetes, whooping cough, bronchitis, asthma, migraine
etc. are successfully cured by the use of the plants. Some plants of family
Rubiaceae are of miraculous importance which are used in the treatment of snake
bite, scorpion sting, regulation of menses and securing the birth of male child. A
very poor attention has still been paid on family Rubiaceae regarding its
medicinal properties.
14
The genus Tricalysia comprises of about 50 species in subtropical and
tropical regions of Asia and Africa (Xiao et al., 1987, He et al., 2002), 2 species
were reported from Western Ghats and Courtallum in Tinnevelly District of Tamil
Nadu state (Gamble, 1986). The International Plant Names Index (IPNI) includes
187 species of Tricalysia (http://www. Plant systematics. org). Tricalysia A. Rich.
comprises of about 10 spp. in Tropical Africa, Madagascar and few in Indomalaya
(George Usher, 1984). It is also found in central and south Maharashtra Sahyadris
(Almeida, 2001). Tricalysia sphaerocarpa (Dalzell ex Hook. F.) Gamble is not
recorded from the tropical dry ever green forest. This species is known only from
Western Ghats and its occurrence is uncommon for the entire east coast and could
be considered to be the relict of the past wetter regimes of the Cuddalore district
(Israel Oliver King, 2004).
Parthasarathy et al., (2008) reported that Tricalysia sphaerocarpa and
Lepisanthes tetraphylla are the dominant evergreen trees in Thirumanikuzhi
sacred grove (Cuddalore, Tamil Nadu). Tricalysia sphaerocarpa is the most
abundant species in Kuzhanthaikuppam (Cuddalore, Tamil Nadu), probably due
to past disturbance (Mani and Parthasarathy, 2006). The wild coffee Tricalysia
sphaerocarpa contributed 50 % of multistemmed individuals in Arasadikuppam
and the site was dominated by 33% of the stand (Venkateswaran and
Parthasarathy, 2003). Anbarashan and Parthasarathy (2013) have reported that
Tricalysia sphaerocarpa formed 72% of the forest stand density at S.Pudhoor.
The studies of Mike O. Soladoye et al., (2010) on ethnobotanical survey
of anti-cancer plants in Ogun state, Nigeria, revealed that the bark of Tricalysia
15
macrophylla along with some other plants and the fruit juice of Citrus medica is
used to cure cancer. Chris Long (2005) studied the ethnobotanical uses of
Tricalysia capensis and Tricalysia lanceolata. Moshi et al., (2009) studied the
ethnobotanical uses of Tricalysia coriacea and Tricalysia coriacea sbsp.
Nyassae. George usher (1984) studied the ethnobotanical uses of Tricalysia
sphaerocarpa. Prajapat and Kumar (2005) studied the ethnobotanical uses of
Tricalysia sphaerocarpa and Tricalysia singularis.
The bioassay-guided fractionation scheme identified the triterpenoids
ursolic and oleanolic acids from Tricalysia niamniamensis Hiern, demonstrated
DNA ligase inhibition profiles to other triterpenes such as aleuritolic acid.
Protolichesterinic acid, swertifrancheside and fulvoplumierin represent three
additional natural-product structural classes that inhibit hLI (human Ligase I).
Fagaronine chloride and certain flavonoids are also among the pure natural
products that were found to disrupt the activity of the enzyme, consistent with
their nucleic acid intercalative properties. Further analysis revealed the step of the
ligation reaction, indicating a direct interaction with the enzyme protein(Tan et
al., 1996).
He et al., (2002) isolated seven rearranged ent-Kaurane glycosides, named
tricalysiosides A-G(1-7) from the leaves of Tricalysia dubia collected from
Okinawa island. Their C-18 and 19 methyls were found to have rearranged to
form and alpha, beta- unsaturated gamma-lactone ring, with other functional
groups remotely located only on C-15,-16, and -17 of the five membered ring.
Using X-ray crystallographic analysis, the structure of tricalysioside A(1) was
16
determined. On the basis of the crystal structure of 1, the structures of the other
tricalysiosides (2-7) were also established.
He et al., (2005) isolated eight ent-kaurane glucoside from the leaves of
Tricalysia dubia. The structure of tricalysioside H (1) was established by X-ray
crystallography and those of tricalysiosides I-O (2-8) were elucidated by analysis
of spectroscopic evidence.
Four rearranged ent-kaurane diterpenoid alkaloids, tricalysiamides A-D
(1-4) having a cafestol-type carbon framework were isolated from the wood of
Tricalysia dubia. Their absolute structures were determined on the basis of 2D
NMR spectroscopy, X-ray crystallographic analysis and chemical methods
(Nishimura et al., 2007).
Tamaki et al., (2008) isolated 2 new rearranged ent-kaurane derivatives
namely tricalysiolides H and I from the EtOAc-soluble fraction of an MeOH
extract of the stem of Tricalysia dubia, together with 5 known rearranged ent-
kauranes, i.e. tricalysiolides A-E, stigmast-4-en-6beta-ol-3-one, (+)-pinoresinol,
scopoletin and syringaldehyde. Their structure were elucidated from the
spectroscopic evidence, and their cytotoxicity toward KB cells and P-gp
inhibitory activity were assayed.
Shitomoto et al., (2010) isolated one new megastigmane gentiobioside
namely tricalysionoside A (1) and 3 sulfates, named sulfatricalysines A-C (2-4)
from the water-soluble fraction of a MeOH extract. Sulfatricalysines D-F(5-7)
and 3 new ent-kaurane glucosides namely tricalysiosides X-Z(8-10) from the 1-
BuOH –soluble fraction of a MeOH extract of leaves of Tricalysia dubia.
17
Xu et al., (2010) isolated two new ent-kaurane glycosides namely
tricalysiosides V and W, with an acylated diasaccharide moiety at the C-3
position from the roots of Tricalysia okelensis and their structures established by
spectroscopic and chemical methods.
The studies of Anandhi and Pragasam (2013a) on pharmacognostical and
preliminary phytochemical studies on leaf extracts of Tricalysia sphaerocarpa
revealed the marked presence of carbohydrate, glycosides, alkaloids, tannin,
flavanoids, moderate presence of protein, phenol, terpenoids and saponin and
absence of triterpenoids, anthraquiones, catachins, coumarins. Anandhi and
Pragasam (2013b) identified 17 phytochemicals from the stem of Tricalysia
sphaerocarpa through GC-MS analysis. They have concluded that the plant is
highly valuable in medicinal usage for the treatment of various human ailments
along with the chemical constituents present in it. The compounds need further
research on toxicological aspects to develop safe drug. Anandhi et al., (2014)
identified 30 phytochemicals from the methanolic extract of leaves of Tricalysia
sphaerocarpa through GC-MS analysis among which fatty acid was the major
group consists of 9 compounds. Eicosanoic acid was found to be present as the
major compound with peak area 35.77% and retention time 21.865 min, followed
by octadecanoic acid (18.81%).
2.2. Objectives of the Present Study
Keeping the view of significances of traditional medicine in the field of
plant-based drug discovery, the important Indian medicinal plant, Tricalysia
sphaerocarpa was selected to carry out the following tasks.
18
1. Pharmacognosy
• Anatomical studies
• Histochemical localization
• Fluorescence analysis
2. Phytochemistry
• Physico-chemical parameters of various parts
• Successive extractive and Batch extractive values
• Ph Determination of aqueous extract
• Preliminary Phytochemical Screening
• GC-MS analysis of methanolic extract of leaf, stem, root and fruit
3. Pharmacology
• In-vitro Antioxidant activity
• In-vivo studies such as Acute toxicity, Anti-depressant activity and Anti-diabetic
activities to develop new plant-based drug that may lead to therapeutic
significance.
19
CHAPTER 3
MATERIALS AND METHODS
3.1. Collection of Plant material:
The plant of Tricalysia sphaerocarpa was collected from the sacred grove
of Thirumanikuzhi, of Cuddalore district, Tamil Nadu. The collected plant
material was botanically identified. The species identity conformation was
engaged at French Institute Herbarium (HIFP), Puducherry. The herbarium
specimen was prepared and deposited at the Department of Botany, Kanchi
Mamunivar Centre for Post Graduate Studies, Lawspet, Puducherry, for future
reference.
3.2. Taxonomy of the Species
Tricalysia sphaerocarpa ( Dalzell ex Hook. F,) Gamble
Basianym :- Discospermum sphaerocarpum Dalzell ex Hook. F.
Common English name : Wild Coffee
Kingdom :- Plantae
Phylum Trachiophyta
Class Magnoliopsida
Order Gentianales
Family Rubiaceae
Genus Tricalysia
Species Tricalysia sphaerocarpa
20
Synonyms:
Diplospora dalzellii (Thwaites) Hook. F.
Diplospora sphaerocarpa (Dalzell ex Hook. F.) Hook. F.
Discospermum dalzellii Thwaites
Tricalysia dalzellii (Thwaites) Alston
Vernacular names:
Tamil : irrukulimaram
Sri Lankans : vella
Kannadam : kaadukafibija.
3.3. Morphological Features (Plate 1)
Habit : Trees up to 15m tall
Trunk /bark : trunk flutted, bark whitish, smooth, fissured when matured; balze
yellowish. The tree outer bark is often attacked by termites giving creamish
appearance.
Branchlets : young branchlets angular to compressed, glabrous, apical bud usually
exudes yellow resin.
Leaves : Leaves dark green, simple, opposite decussate, stipules interpetiolar,
narrow triangular to 0.7 cm long, glabrous, petiole 0.6-1.5 cm long, slightly
canaliculated above, glabrous.
Lamina : 10-13 X 4-7 cm, elliptic to elliptic-ovate, apex acuminate with blunt tip
or obtuse, base attenuate, margin entire, coriaceous, glabrous, midrib raised above
21
secondary nerves 5-8 pairs, hairy domatia present at axils, tertiary nerves broadly
reticulate.
Flowers : inflorescence axillary fascicles, flowers polygamodioecious, white,
scented, minute, calyx lobes oblong – Orbicular, Coralla lobes orbicular, stamens
sessile.
Fruits and Seeds : greenish yellow, berry globose upto 6 in. in diameter; the seeds
flat, smooth, much compressed, with membranous partitions, dispersed by
mammals and birds (Gamble 1921, Gamble 1993, Sasidharan 2004, Almeida
2001, Cook 1903).
3.4. Ecology
Trees in the evergreen forests up to 1000 m.
3.5. Medicinal Uses
The root decoction of Tricalysis pallens Hiern is drunk against malaria.
The root decoction of Tricalysia sp. Aff. mixed with leaf juice of Tricalysia
coricea sbsp. Nyassae is drank, and the body bathed with a root decoction for
malaria. The leaves/roots of Tricalysia coriacea (Benth.) Hiern are boiled and the
decoction drank for skin diseases and malaria/yellow fever (jaundice)(Moshi et
al., 2009). The bark of Tricalysia macrophylla K. Schum is used for the
management of cancer (Mike O. Soladoye et al., 2010). The roots of Tricalysia
capensis (rock jackal coffee) and T. lanceolata (jackal coffee) is used as an emetic
(Chris Long, 2005). The roasted seeds of T. coffeoides Good. Congo. are used
locally as a coffee substitute(George Usher, 1984). The roasted seeds of
Tricalysia sphaerocarpa taste and smell like coffee and the infusion of leaves of
22
T.singularis (Korth.) K.Schum. is used as a beverage in Andala (Sumatra)
(Prajapat and Kumar, 2005). T.singularis (Korth.) K. Schum (alleopathically
enriched) + wild KaliMusli Curculigo orchioides is used as a traditional medicine
for sleep. Tricalysia Sphaerocarpa (Hook. F.) Gamble Null (alleopathically
enriched) + Tinospora giloy type 31 + white flowered Argemone satyanashi is
used traditionally used for sleep(Pankaj Oudhia’s Research documents).
3.6. Chemical constituents isolated from the different species of Tricalysia:
Tricalysia dubia
Leaf : tricalysiosides A-G(1-7), 8 ent-kaurane glucoside, tricalysionoside A (1)
and sulfatricalysines A-C (2-4)
Stem : 4 rearranged ent-kaurane diterpenoid alkaloids, tricalysiamides A-D (1-4),
tricalysiolides H – I, tricalysiolides A-E
Tricalysia okelensis
Root : tricalysiosides V and W
3.7. Pharmacognostical Studies:
The plant is collected from the wild growing in the natural environment of
Cuddalore district and identified using Flora of the Presidency of Madras
(Gamble, 1921). The fresh plant materials were collected and the morphological
features of the specimen were studied directly in the field and were photographed.
Leaf, stem and root were cut into small pieces and fixed in FAA (Formalin,
Acetic acid, and 70% ethyl alcohol in the ratio of 5ml:5ml:90ml, Johansen, 1940)
immediately after collection. Fresh parts of the plant mainly leaves, stem, root and
fruits were collected and kept in polythene bags. The materials collected were
23
dried under shade in the laboratory for 3 to 4 days and the dried materials were
stored in dry polythene bags for carrying out pharmacognostical, phytochemical
and pharmacological investigations.
3.7.1. Anatomical Studies:
Free-hand sections of leaf, stem and root were also employed in the
present study. They were stained in 1% safranin, mounted in 50% glycerine and
sealed with DPX mountant.
To study the foliar epidermal morphology, peels were obtained form the
fresh leaf as well as fixed materials with the help of forceps or a razor. In addition
fixed as well as fresh young and mature leaves were cleared in 5% NaOH solution
for a period of 24-48 hours. They were washed in distilled water thoroughly and
allowed to remain in saturated chloral hydrate solution again for 24 to 48 hours.
They were further washed thoroughly with distilled water, stained with safranin
and mounted in 50% glycerine and sealed with DPX mountant.
Maceration was carried out with the stem and root materials following
Jeffrey’s method (Johansen, 1940). This method involves, cutting the material
(either fresh or dry) into slices of about 300 µm in thick and boiling repeatedly
until free from air. Then macerated in a solution of equal parts of 10% aqueous
nitric acid and 10% aqueous chromic acid. The time varies according to the
material, and cells begin to separating in about 24 hours. A thick glass rod with
rounded end was used to crush the material very gently. Washed very thoroughly
with water to remove the acids. The use of a centrifuge is advisable in order to
speed up the process. The material was stained with safranin (1%). The macerated
24
materials were kept in 1% safranin for about 6 hours and rinsed thoroughly in
water. From this macerated material, a few drops of the stained macerate were
taken, mounted in glycerine and sealed with DPX mountant.
The following parameters were studied:
Epidermal cell number:
Epidermal cell number is the average number of epidermal cells/sq. mm.
For calculation the number of epidermal cells were counted in both the surfaces.
Stomatal number:
Stomatal number is the average number of stomata/sq. mm of epidermis of
the leaf (Evans, 1996). For calculation the number of stomata at different region
of the lower surface of leaf (hypostomatic) were counted.
Stomatal index :
Stomatal index is the percentage, with the number of stomata to the total
number of epidermal cell, each stoma being counted as one cell. Stomatal index is
calculated by using the following equation,
Where,
S.I=S/E+SX100
S.I= Stomatal index
S=Number of stomata per unit area
E=Number of epidermal cells in the same unit area.
Stomatal types:
The distribution of various stomatal types were studied at different regions
of abaxial and adaxial surfaces of leaves.
25
Palisade Ratio:
Palisade ratio is defined as the average number of palisade calls beneath
each upper epidermal cell (Evans, 1996). The semi permanent mounts of cleared
leaves were employed for this study.
Vein-islet number:
Vein islet number is the number of vein-islets/ sq. mm of the leaf surface
midway between the midrib and the margin. This is constant for a given species
of the plant and used as a characteristic for the identification of the allied species.
This number is independent of the size of the leaf and does not alter with the age
of the plant (Wallis, 1985, Evans, 1996).
Veinlet- termination number:
Veinlet-termination number is defined as the number of veinlet
termination/ sq. mm of the leaf surface midway between midrib and margin. To
study the veinlet termination number the method of Khandelwal (2008) was
adopted. The cleared leaves were used for calculating vein-islet number and
veinlet-termination number.
Photomicrography:
Photomicrography of peels and cleared leaves, free-hand section of leaf,
stem and root were taken using Olympic Nikon (Japan) Automatic Camera
attached to the microscope.
3.7.2. Histochemical Colour Reactions:
The histochemical colour reactions of leaves, stem and root of Tricalysia
sphaerocarpa were performed for the identification of major cell components
26
(Johansen, 1940). For testing as far as possible, clear transparent solution were
used. Free-hand sections of plant materials were taken and treated with various
chemicals/ reagents to identify alkaloid, lignin, tannins, mucilage, starch and
proteins. The colour and results are recorded . The tests were as follows:
Lignin:
Fresh free-hand sections were mounted in 1 % neutral aqueous potassium
permanganate and allowed to stand for 15 min, washed thoroughly with water and
placed in 2 % HCl for 2 min, removed and washed with distilled water. Dilute
ammonia solution was added and covered with coverslip. Change to deep red
colour indicates the presence of lignin.
Tannin:
Fresh free-hand sections were placed in 1 % solution of Ferric chloride.
Change of blue to black colour indicates the presence of tannins.
Mucilage:
Fresh free-hand sections were treated with methylene blue reagent.
Change to blue colour indicates the presence of mucilage.
Starch:
Fresh free-hand sections were mounted in 1 % Iodine solution. Change to
blue colour, indicates the presence of starch.
Alkaloid:
Fresh free-hand section were mounted in Meyer’s reagent (36 g of
mercuric chloride was dissolved in 60 ml of water and added to a solution of 5 g
potassium iodide in 20 ml of water and made up to 100 ml).
27
Proteins:
Fresh free-hand sections were stained in aqueous solution of picric acid,
covered and allowed to stand for 24 hours. Change to yellow colour indicates the
presence of proteins.
3.7.3. Fluorescence Analysis:
Quantitative fluorescence analysis utilizes the fluorescence produced by a
compound in day light and ultraviolet light for quantitative evaluation (Evans,
1996). Fluorescence analysis of the drug (dried leaves, fruits, and stem) was
observed in daylight and UV light (365 nm) using drug powder and various
solvent extracts of the drug (Pratt and Chase, 1949) as follows.
The drug powders were treated with the solvents like Benzene,
Chloroform, Acetone, Alcohol, and acid like 1N HCl, H2SO4, NaOH. Then they
were subjected to fluorescence analysis in day light and UV light. The results
were tabulated.
3.8. Phytochemistry:
3.8.1. Physio-Chemical Constants:
The authenticity of a crude drug is established with reference to the
descriptions of the pharmacopoeia or other official publications (BPC; USP) of
the country concerned. The quality and purity required is achieved by standards
(numerical values) also given in the official work of reference. The powdered
plant materials were morphologically and organoleptically screened and subjected
to physio-chemical analysis. The various parameters considered were:
Ash values (Anonymous, 1996; Khandelwal, 2008):
28
Determination of Total Ash:
About 2 g of the crude drug powder is accurately weighed in a silica
crucible which is previously ignited and weighed. The powdered drug is spread in
a fine layer at the bottom of the crucible. The crucible is incinerated at a
temperature not exceeding 450 C until free from carbon. The crucible is cooled
and weighed. The procedure is repeated to a constant weight. The percentage of
the total ash is calculated with reference to the air-dried drug.
Determination of Acid Insoluble Ash:
The total ash values were determined by the ash obtained form leaf and
stem. When it is boiled separately with 25 ml of hydrochloric acid for a few
minutes the insoluble ash is collected on an ashless filter paper and washed with
hot water. The insoluble ash is transferred to the pre-weighed silica crucible,
ignited, cooled, and weighted. The procedure is repeated to the constant weight.
The percentage of acid insoluble ash was calculated with reference to the air-dried
drug. The results were tabulated.
Determination of water soluble ash:
The ash obtained as described in the determination of total ash is boiled
for five minutes with 25 ml of water. The insoluble matter was collected on an
ashless filter paper ignited, cooled and weighed. The weight of the insoluble
matter is subtracted from the weight of total ash. The difference in weight was
considered as the water soluble ash. The percentage of water soluble ash is
calculated with reference to air-dried drug. The results were recorded.
29
Moisture content
The moisture content was determined by using the method of Anonymous
(1996) and Khandelwal (2008).
3.8.2. Preparation of the Extracts:
The collected materials were chopped into small pieces separately, shade-
dried, and coarsely powdered using a pulverizor. The coarse powder were
subjected to successive extraction with chloroform, diethylether, ethylacetate and
methanol by Soxhlet method. The extracts were collected and distilled off on a
water bath at atmospheric pressure and the last trace of the solvents was removed
in vacuo and stored at 4ºC. The resulted extracts were subjected to preliminary
phytochemical screening and GC-MS analysis.
3.8.3. Extractive Values :
Extractive values by (i) Batch process (Kokate, 1986) and (ii) Successive
process (Harborne, 1998) were calculated
3.8.4. PH Determination of Powdered Drug
One gram of the accurately weighed powdered drug was dissolved in
water and filtered. PH of the filtrate was determined by using digital PH meter.
3.8.5. Preliminary Phytochemical Screening
All the extracts were subjected to preliminary phytochemical tests
following the method of Harborne (1998) and Trease and Evans (1983).
Test for Alkaloid (Evans, 1996):
The substance was mixed with little amount of dilute hydrochloric acid
and Meyer’s reagent (36 g of mercuric chloride was dissolved in 60 ml of water
30
and added to a solution of 5 g potassium iodide in 20 ml of water and made up to
100 ml). Formation of white precipitate is the indication for the presence of
alkaloid.
Test for Anthraquinone (Modified Berntrager’s test):
To 5 ml extract, 5 ml of 5 % FeCl3 and 5 ml dilute HCl were added and
heated for 5 minutes in boiling water bath. Then it was cooled and benzene or
any organic solvent was added and shook well. Organic layers were separated and
equal volume of dilute ammonia was added. Ammonical layer shows pinkish red
colour and indicates the presence of anthraquinone.
Test for Aminoacids (Ninhydrin test):
Three ml test solution was heated and 3 drops of 5% Ninhydrin solution
was added in boiling water bath for 10 minutes. Purple or bluish colour indicates
the presence of amino acids.
Test for Catechins:
To the substance, a drop of Ehrlich’ reagent (para- dimethyl amino
benzaldehyde) was added which turns into pink colour indicating the presence of
catechins.
Test for Cardiac glycosides (Keller-Killiani test):
To 2 ml extract glacial acetic acid, one drop of 5% ferric chloride and
concentrated Sulphuric acid were added. Reddish brown colour appears at
junction of the two liquid layers and upper layer appears bluish green.
31
Test for Coumarins:
To the substance, a drop of sodium sulphate was added which turns into
yellow colour, indicating the presence of Coumarins.
Test for flavonoids:
To a little of the substance or powder in alcohol, 10 % sodium hydroxide
solution or ammonia was added. Dark Yellow colouration is the indication of
presence of flavonoids.
Test for Glycosides:
A small amount of the drug is mixed with a little anthrone on a watch
glass. One drop of concentrated sulphuric acid was added to that and a paste is
prepared when warmed gently over water bath. The appearance of dark green
colouration indicates the presence of glycosides.
Test for Gums, Oils, and Resins :
The test solution was applied on filter paper which develops a transparent
appearance on the filter paper indicating the presence of oils, gums and resins.
Test for Phenol:
To the powder substance, a few drops of alcohol and ferric chloride
solution were added. Bluish green or red colour is the indication of the presence
of phenol.
Test for Proteins (Biuret test):
To 3 ml of test solution, 4 % NaOH and few drops of 1% CuSO4 solution
were added. Violet or pink colour indicates the presence of proteins.
32
Test for Phlobotannins:
Deposition of a red precipitate when an aqueous extract of plant sample
boiled with 1 % aqueous HCl is the evidence for the presence of phlobotannins.
Test for Saponins:
A little of the substance is shaken with water and copious lather formation
is the indication for the presence of saponins.
Test for Steroids (Liebermann’s reaction):
Three ml extract was mixed with 3 ml of acetic anhydride then heated and
cooled. A few drops of concentrated sulphuric acid was added. The appearance of
blue colour indicates the presence of steroids.
Test for Reducing Sugars (Fehling’s test):
The substance is mixed with Fehling’s solution A and B. Formation of a
red colouration is the indication for the presence of redusing sugars.
Test for Non - Reducing Polysaccharides (Starch)(Iodine test):
To 3ml test solution a few drops of dilute iodine solution was added. The
appearance of blue colour and disappearance on boiling and reappears on
cooling indicates the presence of non-reducing sugars.
Test for Tannins (Mace, 1963):
The substance is mixed with basic lead acetate solution. Formation of a
white precipitate is the indication for the presence of tannins.
33
Test for Terpenoids (Salkowski test):
To 5 ml test solution, 2 ml of chloroform and 3 ml of concentrated
sulphuric acid were carefully added to form a layer. A reddish brown colour
formation indicates the presence of Terpenoids.
Test for Triterpenoids:
Two or three granules of tin metal was dissolved in 2 ml of thionyl
chloride solution. Then one ml of extract was added to it. The formation of pink
colour indicates the presence of triterpenoids.
3.8.6. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis:
GC-MS analysis was performed with GC Clarus 500 Perkin Elmer
equipment. Compounds were separated on Elite-1 capillary column (100%
Dimethylpolysiloxane). Oven temperature was programmed as follows:
isothermal temperature at 50ºC for 2 minutes, then increased to 200ºC at the rate
of 10ºC/minutes, then increased up to 280ºC at the rate of 5ºC/minutes held for 9
minutes. Ionization of the sample components was performed in the El mode (70
eV). The carrier gas was helium (1ml/minutes) and the sample injected was 2μl.
The detector was Mass detector turbo mass gold-Perkin Elmer. The total running
time for GC was 36 minutes and software used was Turbomass 5.2. Using
computer searches on a NIST Ver.2.1 MS data library and comparing the
spectrum obtained through GC – MS compounds present in the plant samples
were identified.
34
Identification of Compounds:
The individual compounds were identified from methanol extracts based
on direct comparison of the retention times and their mass spectra with the spectra
of known compounds stored in the spectral database, NIST (version year 2005).
3.9. Pharmacology
3.9.1. In Vitro Antioxidant Activity
3.9.1.1. Inhibitiory Effects on DPPH Radical Assay:
DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay was
selected due to its straight forwardness, quickness, sensitivity and reproducibility
(Sanja et al., 2008).DPPH is a nitrogen centered free radical scavenger that shows
strong absorbance at 517 nm. Deep violet coloured methanolic DPPH solution
changes to yellow colour in the presence of DPPH radical scavengers. DPPH
radical accepts an electron or hydrogen radical to become a stable diamagnetic
molecule. Extent of DPPH radical scavenged was determined by the decrease in
absorbance at 517 nm induced by antioxidants, due to the reaction between
antioxidant molecules and free radicals, which results in the scavenging of free
radicals by hydrogen donation (Chang et al., 2002). DPPH radical scavenging
activity of extract was determined according to the method reported by Blois
(1958). An aliquot of 0.5 ml of sample solution in methanol was mixed with 2.5
ml of 0.5 mM methanolic solution of DPPH. The mixture was shaken vigorously
and incubated for 37 minutes in the dark at room temperature. The absorbance
was measured at 517 nm using UV spectrophotometer. Butyl Hydroxyl Toluene
35
(BHT) was used as a positive control. DPPH free radical scavenging ability (%)
was calculated by using the formula.
absorbance of control – absorbance of sample % of inhibition = --------------------------------------------------------
absorbance of control ×100.
3.9.1.2. Hydrogen peroxide Assay
Hydrogen peroxide content was determined by measuring the absorbance
of titanium-hydroperoxide complex. The extract was reacted with titanium
reagent and ammonium to form hydroperoxide-titanium complex. The complex
was dissolved in 1 M sulfuric acid and absorbance of the supernatant was
measured at 415 nm against blank. Concentration of hydrogen peroxide was
determined using the standard curve plotted with known concentration of
hydrogen peroxide.
3.9.1.3. Superoxide dismutase (L-methionine and NBT) Assay (SOD)
SOD activity was assayed by determining the inhibition rate of nitro blue
tetrazolium reduction with xanthine oxidase as a hydrogen peroxide generating
agent. The rate of NBT reduction is directly proportional to SOD levels. The
absorbance at 360 nm was noted and expressed the value as percentage of SOD
levels.
3.9.1.4. Iron Chelating Activity (FRAP)
The method of Benzie and strain (1996) was adopted for the assay. The
principle is based on the formation of O-Phenanthroline-Fe2+ complex and its
disruption in the presence of chelating agents. The reaction mixture containing 1
ml of 0.05% O-Phenanthroline in methanol, 2 ml ferric chloride (200μM) and 2
36
ml of various concentrations ranging from 50 to 500μg were incubated at room
temperature for 10 minutes and the absorbance of the same was measured at 510
nm. Ethylene diamine tetra acetic acid (EDTA 10mM) was used as a classical
metal chelator. The experiment was performed in triplicates.
3.10. In vivo Pharmacological Studies
Selection of extracts
The extractive values is high in methanol solvent, hence the methanol
extract of the stem of T. sphaerocarpa was selected for in vivo pharmacological
studies.
Animals
Young adult male Wister rats, 8 weeks old were used as experimental
model. The weight of each of the animals on the first day of experiment was 120-
180 grams. They were randomly housed 6 per gauge and maintained in 10:14
light: dark cycle and given access to food and water ad libitum. All injections in
this study were performed once daily between 8.00 AM and 9.00 AM. The
experimental protocals were carried out at K.M.C.H. College of Pharmacy,
Coimbatore, Tamil Nadu, India, approved by the Institutional Animals Ethics
Committee (IAEC. NO: 793/ 03/ C/ CP CSEA/112-2013).
Drugs and chemicals
Imipramine hydrochloride (Sigma-Aldrich, St Louis, USA) was used as
reference standard for antidepressant activity.
37
Administration of the extracts
Suspensions of methanolic extract was prepared in distilled water using
Tween-80 (0.2% v/v) as the suspending agent. The extract was administered in
different doses of 100 and 200 mg/kg of body weight to rats by oral route, 45
minutes before the test procedures for pre-pharmacological screening as per
Organization for the Economic Cooperation Development (OECD) 423
guidelines. Control groups were given only the vehicle (0.2% v/v Tween-80
solution) in volume equivalent to that of the plant extracts.
3.10.1. Acute Toxicity Studies
Methanolic extract in the doses of 500, 1000 and 2000 mg/kg were given
orally for the assessment of acute toxicological studies to different groups of mice
(18-25g) and observed for signs of behavioral, Neurological toxicity and mortality
after 14 days. All the parameters were thoroughly checked and dose for the
further studies was calculated as per the Organization for the Economic
Cooperation Development 423 guidelines (OECD). After the conduct of acute
toxicological studies the dose of methanolic extract was decided i.e. 100mg/kg,
200mg/ kg. Oral route was selected for the administration of drugs. The procedure
was followed as per OECD 423 guidelines.
3.10.2. Anti-Depressant Activity
3.10.2.1. Forced Swimming Test (FST)
Rats of either sex (120-150g) were individually forced to swim in an open
cylindrical container (diameter 10 cm, height 25 cm), containing 19 cm of water
at 25±1 °C. All the rats of either sex were divided in to four different groups of 6
38
in each group (n=6). The first group assigned as control received only vehicle
(Distilled water, 5ml/kg). The other two groups received acute oral dose of
methanolic extract (100 and 200mg/kg). The fourth group received standard drug
Imipramine (30 mg/kg). The total duration of immobility was recorded during the
last 6 minutes of the 10-minutes period. Each rat was judged to be immobile when
it ceased struggling and remained floating motionless in the water, making only
those movements necessary to keep its head above water. A decrease in the
duration of immobility is indicative of an antidepressant like effect.
3.10.2.2. Tail Suspension Test (TST)
All the rats of either sex (120-150g) were divided into five different
groups. The first group assigned as control received only vehicle (Distilled water,
5ml/kg). The other two groups received acute doses of methanolic extract (100
and 200 mg/kg). The fourth group received standard drug Imipramine (30 mg/kg).
The total duration of immobility induced by tail suspension was measured
according to the methods described by Steru et al., (1985). Briefly, rat both
acoustically and visually isolated were suspended 50 cm above the floor by
adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time
was recorded during a 6-minutes period. Rats were considered immobile only
when they hung passively and were motionless.
3.10.2.3. Hole Board Test (HBT)
Experiment was conducted, 30minutes after injection of control vehicle,
methanolic extract 100 and 200mg/kg and diazepam (3mg/kg) by placing mouse
39
on a wooden board. The number of head dips and time spent in each dip was
recorded during 3minutes trial.
Statistical analysis
The immobility time in tail suspension test and forced swimming test was
analyzed with ANOVA (Analysis of Variance), further comparisons between
vehicle and drug-treatment groups were performed using the Dunnett's t-test.
Results are expressed as the mean ± SEM. Analyses were performed using the
software SPSS (Statistical program for Social Sciences) version 13 for windows.
The level of statistical significance adopted was **P<0.01, when compared with
the control group.
3.10.3. Anti Diabetic Activity
3.10.3.1. Screening of Hypoglycemic Activity in Normal Rats
Normal fasted rats: Normal albino rats (150-180 g) were first used for the
screening of the herbal drug for hypoglycemic activity. Overnight fasted normal
rats were randomly divided into 5 groups of 6 rats each. The group I served as
control, which received vehicle i.e. 1% Gum acacia solution (1ml/kg, orally).
Group II, III and IV were treated orally with Test extract 125, 250 and 500 mg/kg,
respectively. Group V received Glibenclamide 5 mg/kg orally.
Experimental Design
Group 1 - Treated orally with 1% Gum acacia solution, 1ml/kg
Group 2 - Treated orally with Test extract, 125mg/kg
Group 3 - Treated orally with Test extract, 250mg/kg
Group 4 - Treated orally with Test extract, 500mg/kg
40
Group 5 - Treated orally with Glibenclamide 5mg/kg
Blood samples were collected from tail vein prior and 1, 2, 4 and 6 hours
after treatment. Fasting blood glucose (FBG) was determined by the glucose
oxidase method using CONTOURTMTS Blood Glucose Meter with same test
strips. The percentage (%) fall in blood glucose level was also calculated at peak
hour of effect.
3.10.3.2. Anti-Diabetic Activity in Experimentally Induced Diabetic Rats:
Induction of experimental diabetes
Overnight fasted albino rats (150-180 g) were made diabetic by injecting
Alloxan monohydrate (in the ice cold normal saline) intra peritonially (i.p) at a
dose of 150 mg/kg body weight. Diabetes was confirmed in Alloxan injected rats
by measuring the fasting blood glucose concentration, 72 hours after the
Alloxanization. Rats with blood glucose level above 250 mg/dl were considered
to be diabetic and were used in this study.
Experimental design
The diabetic rats were divided into 5 groups of 6 rats each.
Group 1 - Normal control and received vehicle i.e. 1% Gum acacia Solution,
1ml/kg/BW
Group 2 - Diabetic control and received 1% Gum acacia solution, 1ml/kg/BW
Group 3 - Treated orally with Test extract, 125mg/kg/BW
Group 4 - Treated orally with Test extract, 250mg/kg/BW
Group 5 - Treated orally with Test extract, 500mg/kg/BW
41
Group 6 - Received Glibenclamide 5mg/kg/BW, orally on 3rd day after
alloxanation (i.e. 1st day of treatment)
Group 1 and 2 served as normal and diabetic control respectively and received
vehicle (1ml/kg, po). Group 3, 4 and 5 were treated orally with Test extract, 125,
250 and 500 mg/kg BW respectively. Group 6 received Glibenclamide 5 mg/kg,
orally on 3rd day after alloxanization (i.e. 1st day of treatment).
3.10.3.2.1. In single-dose, short term study :
Fasting Blood Glucose was estimated from the tail vein prior and 1, 3 and
6 hr after administration of test drugs and vehicle.
3.10.3.2.2. In multi dose long term study:
The same animals were continued with the same dose of vehicle, test
extract and Glibenclamide once daily for 15 days. Fasting Blood Glucose in the
blood was collected and measured at 24 hours after the previous dose on 3, 6, 9,
12 and 16th day.
3.10.3.3. Effect of extract in Body Weight in Normal and Alloxan Induced
Diabetic Rats
3.10.3.4. Biochemical Parameters Determinations
After 15 days of treatment, overnight fasted rats were sacrificed and blood
was collected. The serum was separated and analyzed for lysosomal enzymes
such as transaminases (Serum Glutamate Oxaloacetate Transaminase, SGOT and
Serum Glutamate Pyruvate Transaminase, SGPT), and Alkaline Phosphatase
(ALP), by colorimetric method.
42
The pancreas were dissected out and washed with ice-cold saline
immediately. A portion of pancreatic tissue was homogenized and the extract was
used for the estimation of enzymatic antioxidants (Catalase, CAT and Glutathione
Peroxidase, GPX) activities including Lipid Peroxidation (LPO) process to see the
effect of 15 days treatment with test extract.
Determination of Blood Glucose:
The test provides a quantitative measurement of glucose in blood
from 10 to 600 mg/dl as described in the manual of manufacturer Bayer polychem
(India) Limited (CONTOURTMTS Blood Glucose Meter with same Test Strips )
as follows:
Principle:
The CONTOUR TS blood glucose test is based on measurement of
electrical current caused by the reaction of glucose with the reagents on the
electrode of the strip. The blood sample was drawn into the tip of the test strip
through capillary action. Glucose in the sample reacts with FAD glucose
dehydrogenase (FAD-GDH) and potassium ferricyanide. Electrons were
generated, producing a current that is proportional to the glucose in the sample.
After the reaction time, the glucose concentration in the sample is displayed. No
calculation is required.
Chemical Composition: FAD glucose dehydrogenase (Aspergillus sp., 2.0 U/test
strip), 6%; potassium ferricyanide 56%; Non-reactive ingredients 38%.
Determination of Serum glutamate oxaloacetate transaminase (SGOT)
Method using SGOT kit.
43
Principle
SGOT catalyses the following reaction
SGOT α -Keto glutarate + L-asparate L -glutamate + Oxalacetate pH 7.4 Oxaloacetate Alkaline 2,4 dinitrophenyl + hydrazone 2,4 DNPH Medium (Brown coloured)
Oxaloacetate formed in the reaction is spontaneously converted into
pyruvic acid. Rate of reaction is then determined by the estimation of pyruvic acid
using dinitrophenyl hydrazine. Dinitrophenyl hydrazine (DNPH) formed was
estimated at 505 nm. The unreacted α-keto glutarate also gives coloured product
with color reagent but the intensity was much less than that of pyruvate and hence
it was negligible.
Reagents
Reagent 1: Buffered alanine α-KG substrate, pH 7.4
Reagent 2: DNPH colour reagent
Reagent 3: Sodium hydroxide 4 N
Reagent 4: Working pyruvate standard, 2mM
44
Procedure
Tube No. 1 2 3 4 5
Enzyme activity (units/ml) 0 24 61 114 190
Reagent 1 0.5 0.45 0.4 0.35 0.3
Reagent 4 - 0.05 0.1 0.15 0.2
Purified water (ml) 0.1 0.1 0.1 0.1 0.1
Reagent 2 0.5 0.5 0.5 0.5 0.5
Mix well and allow to stand at room temperature for 20 minutes.
Solution I (ml) 5.0 5.0 5.0 5.0 5.0
Mixed well by inversion. Allowed to stand at room temperature for 20 minutes.
and measured the absorbance of all the five tubes against purified water on a
colorimeter using a green filter.
Test procedure
Reagents Blank Test
Reagent 1: Buffered alanine, pH 7.4 0.5ml 0.5ml
Incubate at 37˚C for 5 minutes.
Serum 0.1 ml
Mix well and incubate at 37˚C for 60 minutes.
Reagent 2: DNPH colour reagent 0.5ml 0.5ml
Mix well and allow to stand at room temp. for 20 minutes.
Distilled water 0.1 ml 0.1 ml
Working sodium hydroxide 5.0 ml 5.0 ml
45
Mixed well and allowed to stand at room temperature for 10 minutes. Estimated
with the help of spectrophotometer at 505nm and expressed as IU/l.
Determination of Serum glutamic pyruvic transaminase (SGPT)
Method using SGPT kit.
Principle
SGPT (ALT) catalyses the following reaction
SGPT α -Keto glutarate + L-alanine L -glutamate + pyruvate pH 7.4
Pyurate Alkaline 2,4 dinitrophenyl + hydrazone 2,4 DNPH Medium (Brown coloured)
Pyruvate was coupled with 2,4-dinitrophenyl hydrazine (2,4-DNPH) to give the
corresponding hydrazone, which gives the brown color in alkaline medium and
this can be measured colorimetrically.
Reagents
Reagent 1: Buffered alanine α-KG substrate, pH 7.4
Reagent 2: DNPH colour reagent
Reagent 3: Sodium hydroxide 4 N
Reagent 4: Working pyruvate standard, 2mM
Preparation of working solutions
Solution I: Dilute 1 ml of reagent 3 to 10 ml with purified water.
46
Procedure
Tube No. 1 2 3 4 5
Enzyme activity (units/ml) 0 28 57 97 100
Reagent 1 0.5 0.45 0.4 0.35 0.3
Reagent 4 - 0.05 0.1 0.15 0.2
Purified water (ml) 0.1 0.1 0.1 0.1 0.1
Reagent 2 0.5 0.5 0.5 0.5 0.5
Mix well and allow to stand at room temperature for 20 minutes
Solution I (ml) 5.0 5.0 5.0 5.0 5.0
Mixed well by inversion. Allowed to stand at room temperature for 20 minutes
and measured the absorbance of all the five tubes against purified water on a
colorimeter using a green filter.
Test procedure
Reagents Blank Test
Reagent 1: Buffered alanine, pH 7.4 0.5ml 0.5ml
Incubate at 37˚C for 5 minutes.
Serum 0.1 ml
Mix well and incubate at 37˚C for 60 minutes.
Reagent 2: DNPH colour reagent 0.5ml 0.5ml
Mix well and allow to stand at room temp. for 20 minutes.
Distilled water 0.1 ml 0.1 ml
Working sodium hydroxide 5.0 ml 5.0 ml
Mix well and allow to stand at room temperature for 10 minutes. Estimated with
the help of spectrophotometer at 505nm and expressed as IU/L
47
Determination of Serum alkaline phosphatase (SALP)
The alkaline phosphates level was estimated by p-Nitrophenyl
phosphate (PNPP) method.
Principle
The determination of the activity of alkaline phosphatase in serum based
on the hydrolysis of p- nitrophenyl phosphate (PNPP) by the enzyme with the
formation of free p- nitrophenol.This compound was yellow in alkaline solution.
The formation of yellow colour can be spectrophotometrically readapt 405 nm,
which was directly proportional to the enzymatic activity of alkaline phosphatase
in serum / plasma.
Alkaline Phosphatase PNPP + H2O P- nitrophenol + Phosphate
The method has been recommended by the German Society of Clinical Chemistry
and by the committee on enzyme of the Scandinavian Society of Clinical
Chemistry and Clinical Physiology.
Reagents
Reagents 1: Substrate
Reagents 2: Buffer
Preparation of working solution
Dissolve each vial content (Reagent 1) of dry substance with 3.0 ml of
buffer (Reagent 2). Mixed to dissolve by slow stirring to ensure uniform mixing.
48
Procedure
Test(T) Blank(B)
Working reagent 1.0 ml Distilled water
Sample 20 µl Distilled water
Mix well and read the absorbance at 60, 90, 120 and 150 seconds at 405 nm.
Determine the A /minutes from the linear part of the assay.
Calculation
IU /L of Alkaline phosphatase = A /minutes × 2713
Where F=2713was calculated on the basis of molar extinction coefficient for p-
nitrophenol and total assay volume to sample volume.
We can also measure the change of optical density directly from Bio Chemical
analyzer at 405 nm.
Measurement of Lipid Peroxidation (LPO)
The concentration of thiobarbituric acid reactive substances (TBARS) was
measured (lipid peroxidation product maondialdehyde (MDA) was estimated) in
liver using the method of Okhawa et al., (1979). One ml of the sample was mixed
with 0.2 ml 4 % (w/v) sodium dodecyl sulfate, 1.5 ml 20% acetic acid in 0.27 M
hydrochloric acid (pH 3.5) and 15 ml of 0.8% thiobarbituric acid (TBA, pH 7.4).
The mixture was heated in a hot water bath at 85˚C for 1 hour. The intensity of
the pink colour developed was read against a reagent blank at 532 nm following
centrifugation at 1200 g for 10 minutes. The concentration was expressed as n
moles of MDA per mg of protein using 1,1,3,3,-tetra-ethoxypropane as the
standard.
49
Determination of Glutathione- Peroxidase activity:
The reaction mixture contained 0.1 M reduced glutathione, 10 U/ml of
glutathione reductase, 2 mM nicotinamide adenine dinucleotide phosphate
reduced (NADPH), 0.05 M phosphate buffer (pH 7.0) and 7 Mm t-butyl
hydroperoxide. Decrease in absorbance of NADPH was measured as GPx activity
at 340 nm. One unit of GPx is equal to the number of nano moles of NADPH
oxidized/utilized per minutes at 25˚C.
Measurement of Catalase (CAT)
In animals, catalase was present in all major body organs, especially being
concentrated in liver and erythrocyte. During β-oxidation of fatty acids by
flavoprotien dehydrogenase, hydrogen peroxidewas generated, which was
accepted upon by catalase present in peroxisomes. (Nichollas and Schonbaum,
1963).
The catalase activity was assayed by the method of catalase catalyses the
rapid decomposition of hydrogen peroxide to water.
2H2O2 2H2O + O2
The decomposition of hydrogen peroxide by catalase proceeds at one of the
highest rates known for enzymatic reactions.
Reagents
Dichromate-acetic acid reagent: Five percent of potassium dichromate was
prepared with acetic acid (1:3 v/v in distilled water).
50
Phosphate buffer - 0.01M, pH 7.0: 173 mg of disodium hydrogen phosphate and
122 mg of sodium dihydrogen phosphate were dissolved in 61 ml and 39 ml of
distilled water respectively and made up to 200 ml with distilled water.
Hydrogen peroxide – 0.2M: 2.27 ml h hydrogen peroxide was made upto 100 ml
with distilled water.
Procedure
To 0.1 ml of liver homogenate 1.0 ml of each phosphate buffer and
hydrogen peroxide were added and a timer started. The reaction was arrested by
the addition of 0.2 ml dichromate acetic acid reagent. Standard hydrogen peroxide
in the range of 4 to 20 µm were taken and treated similarly. The tubes were heated
in a boiling water bath for 10 minutes. The green color developed was read at 570
nm in a Double beam UV-VIS spectrometer (Perkin Elmer), Germany. Catalase
activity was expressed as IU/L.
3.10.3.5. Histopathological Studies:
Pancreas were isolated and preserved in 10% formalin. Section of
the pancreas tissues were made, stained with Haematoxylin and Eosin reagent and
observed under low and high power objective for histopathological changes. The
alteration and changes in the histology of pancreas were shown in vide plate and
the results with photomicrograph were given in the result section.
51
CHAPTER 4
RESULTS
4.1. Pharmacognosy
4.1.1. Anatomy
The anatomical studies of Tricalysia sphaerocarpa includes the epidermal
peels of the leaf and stem, clearing of leaf, maceration of stem and transverse
section of leaf, stem and root.
4.1.1.1. Leaf peeling:
Epidermal cell number, stomatal number, stomatal index and palisade
ratio were calculated and presented in the table 1.
Adaxial epidermis: Cells larger than those on the abaxial epidermis, irregularly
shaped, walls thick, sinous, stomata absent, trichomes absent (Plate 2).
Abaxial epidermis: Cells smaller and irregularly shaped in the intercostal region,
elongated in the costal region, walls thick, deeply sinous, stomata more frequent,
irregularly distributed, variously oriented, occur in various sizes in the intercostal
region, less frequent and large in size in the costal region, slightly elongated,
rubiaceous type, edges very thick (Plate 2). Giant stomata, blind stomata, half
stomata, medium size stomata, small size stomata and degenerated stomata are
also encountered. Rare occurrence of unicellular, conical, straight trichomes are
observed (Plate 3).
4.1.1.2. Venation Pattern:
In cleared lamina, the venation system was studied. The veins are thin and
straight. The islets are variable in shape and size. Veins reticulate, showing lateral
52
branches, vein-islet faily large, each islet containing 3-4 termination points. The
vein islet number and veinlet-termination number were 93.8 ± 6.85 and 57.4 ±
3.78 respectively (Table 1) (Plate 4).
4.1.1.3. Quantitative Values of Foliar Epidermis
The mean number of epidermal cells/mm2 is 1122.8 ± 2.84 in adaxial
surface whereas it is 1414 ± 3.76 in abaxial surface. The stomata were
encountered only in the abxial surface. The number of stomata /mm2 was 336 ±
8.43. The stomatal index was 26.9 ± 4.37. The palisade to spongy ratio was 6.2 ±
2.82.
4.1.1.4. Stem Peeling:
Cells larger, axially enlongated, arranged in longitudinal rows, septate,
walls very thick, septa thin, stomata rare, small, rubiaceous, trichomes absent
(Plate 4).
4.1.1.5. Maceration:
Fibres
The fibres are libriform type, with thick lignified walls and fairly wide
lumen. The fibre is uniform in thickness and they become tapering at the ends
(Plate 7).
Vessel Elements
The vessel elements are narrow, long and cylindrical. The end wall
perforation is simple, circular, mostly oblique or horizontal. Vessel elements are
tailed at one end or at both ends.(Plate 7).
Table 1: Quantitative values of foliar epidermis of Tricalysia sphaerocarpa
Quantitative values Abaxial Adaxial Epidermal cell/mm²² 1122.8±2.84 1414±3.76 Stomata/mm²² 336±8.43 - Stomatal index 26.9±4.37 - Palisade ratio 6.2±2.82 Vein islet number 93.8±3.85 Veinlet termination number 57.4±3.78
Note : All the values are expressed as mean ± SEM(n=6)
Table 2: Histochemical colour reactions of various parts of Tricalysia sphaerocarpa
Test for Chemicals/reagents used Status of the substance leaf stem root
Starch Iodine solution + + + alkaloid Meyer’s reagent + + + Proteins Aqueous picric acid solution + + + Tannin Dilute ferric chloride + + - Lignin 1% potassiumpermanganate,
2% HCl, dil. Ammonia - - +
Mucilage Methylene blue reagent - - - Note : + = present; - = absent.
53
4.1.1.6. Transverse Section of Leaf:
Leaf in T. S. is dorsiventral, upper epidermis single layered, cells tabular
in the leaf blade region, hemispherical in the midrib region, cuticle thick
continous in the blade region and arched in the midrib region. Cuticle covers the
radial walls also to some extent. Stomata absent. Lower epidermis single layered,
epidermal cells tabular in the blade region, outer tangential walls deeply arched in
the midrib region. Midrib region raised on the abaxial side into a simple hump.
Stomata sunken. Mesophyll differentiated into upper single layered palisade and
lower spongy parenchymatous ground tissue, group of scleroids and fibres are
observed in the midrib region. Xylem occurs in the form of an arch in the midrib
region surrounded by phloem on the abaxial side. The ground cells are rich in
starch grains, groups of tannineferous idioblasts are evident (Plate 2).
4.1.1.7. Transverse Section of Stem:
The young stem in cross section is mostly dumble shaped. Epidermis is
unilayered, epidermal cells hemispherical surrounded by a thick cuticle. Cuticle
covers the radial walls also to some extent. Hypodermis collenchymatous in
discontinous patches. Cortex parenchymatous, followed by a layer of
sclerenchyma cells (scleroids). Secondary phloem continuous with patches of
sclerenchyma fibres. Secondary xylem continuous with elliptical or oval shaped
vessels. Vessels discrete arranged in radial rows. Vessel members narrow, pitted,
simple perforation plate with long tail. Pith parenchymatous with abundant starch
grains. Tannineferous idioblasts are distributed in cortex, phloem and pith. The
idioblast in the pith are larger than the other (Plate 5).
54
4.1.1.8. Transverse Section of Root:
The root in cross section is circular. The rhizodermis is peeled off. The 8-
10 layers of parenchymatous cells are seen beneath the rhizodermis. Secondary
xylem and phloem are present. Rays are clearly seen. Xylem seen in the centre
and the phloem towards the periphery. The secondary xylem occupies wide major
portion of the root. It includes densely crowded, diffusely distributed wide,
circular, thick walled, narrow xylem fibres. The vessels include both wide and
narrow elements (Plate 6).
4.1.2. Histochemical Colour Reactions:
The histochemical localization tests revealed the presence of starch,
alkaloid and protein in all the plant parts studied. Tannin is present in leaf and
stem, lignin is present only in root and the mucilage is absent in all the plant parts
studied (Table 2).
4.1.3. Fluorescence Analysis
The fluorescence analysis of various parts of plant in different solvents
and chemical reagents observed under ordinary day light and UV light are given
in Table 3-6.
Leaf
Powdered leaf material was green in day light and yellow under UV light.
Similarly, it was dark green in acetone, benzene and chloroform, green in ethanol
and water, yellowish green in n-butyl alcohol. Under UV light, orange colour in
acetone, benzene, chloroform, ethanol and n-butyl alcohol whereas dark brown in
water.
55
Under day light, reddish brown was developed in 10% ferric chloride,
50% sulphuric acid, 50% nitric acid, dark green in 10% aqueous NaOH, green in
1N HCl, 5% ammonia and 1% thionyl chloride. Under UV light, black colour was
observed in 10% ferric chloride and 50% nitric acid, dark brown in 50% sulphuric
acid, brown in 10% aqueous NaOH, green in 1N HCl and 5% ammonia, dark
green in 1% thionyl chloride.
Stem
Powdered stem material was creamy white in day light and pale yellow
under UV light. Similarly, it was yellow in acetone, n-butyl alcohol and ethanol,
pale yellow in benzene, dark yellow in chloroform, creamy white in water. Under
UV light, stem powder was orange colour in acetone, creamy white in benzene
and n-butyl alcohol, pale yellow in chloroform, dark yellow in ethanol, whereas
pale yellow in water.
Under day light, orange colour was developed in 10% ferric chloride,
reddish brown 50% sulphuric acid and 50% nitric acid, yellow in 10% aqueous
NaOH, creamy white in 1N HCl, pale yellow in 5% ammonia and light yellow in
1% thionyl chloride. Under UV light, black colour was observed in 10% ferric
chloride, 50% nitric acid and 50% sulphuric acid, creamy white in 10% aqueous
NaOH, yellow in 1N HCl and 5% ammonia, brown in 1% thionyl chloride.
Root
Powdered root material was creamy white in day light, and pale yellow
under UV light. Similarly, in day light, it was yellow in acetone, benzene,
chloroform, ethanol and n-butyl alcohol, creamy white in water. Under UV light,
Table 3 : Fluorescence analysis of Leaf powder of Tricalysia sphaerocarpa
Chemicals Leaf Day light UVlight
Powder as such Green Yellow Solvent
Acetone Dark green Orange Benzene Dark green Orange Chloroform Dark green Orange Ethanol Green Orange n-butyl alcohol Yellowish green Orange Water Green Dark brown green
Reagents 10% FerricChloride Reddish brown Black 50% Sulphuric Acid Reddish brown Dark brown 50%Nitric acid Reddish brown Black 10%aq. NaOH Dark green Brown 1 NHCl Green Green 5% Ammonia Green Green 1% Thionyl Chloride Green Dark green
Table 4 : Fluorescence analysis of Stem powder of Tricalysia sphaerocarpa
Chemicals Stem Day light UVlight
Powder as such Creamy white Pale yellow Solvent Acetone Yellow Orange Benzene Pale yellow Creamy white Chloroform Dark yellow Pale yellow Ethanol Yellow Dark yellow n-butyl alcohol Yellow Creamy white Water Creamy white Pale yellow
Reagents 10% FerricChloride Orange Black 50% Sulphuric Acid Reddish brown Black 50%Nitric acid Reddish brown Black 10%aq. NaOH Yellow Creamy white 1 NHCl Creamy white Yellow 5% Ammonia Pale yellow Yellow 1% Thionyl Chloride Light yellow Brown
56
stem powder was orange colour in acetone, yellow in benzene and water, pale
yellow in chloroform, dark yellow in ethanol and n-butyl alcohol.
Under day light, reddish brown colour was developed in 10% ferric
chloride and 50% sulphuric acid, orange in 50% nitric acid, yellow in 10%
aqueous NaOH and 5% ammonia and greenish yellow in 1N HCl, and light
yellow in 1% thionyl chloride. Under UV light, black colour was observed in
10% ferric chloride and 50% sulphuric acid, brown in 50% nitric acid and 1%
thionyl chloride, dark yellow in 10% aqueous NaOH, pale yellow in 1N HCl and
green in 5% ammonia.
Fruit
Fruit powder was light brown in day light, and creamy white under UV
light. Similarly, in day light, it was yellow in acetone, benzene and chloroform,
pale yellow in ethanol, light brown in n-butyl alcohol and water. Under UV light,
stem powder was green colour in acetone, creamy white in benzene, chloroform,
ethanol and n-butyl alcohol and yellow in water.
Under day light, orange colour was developed in 10% ferric chloride and
50% nitric acid, reddish brown in 50% sulphuric acid, light brown in 10%
aqueous NaOH, pale yellow in 5% ammonia and 1N HCl and yellow in 1%
thionyl chloride. Under UV light, brown colour was observed in 10% ferric
chloride and 50% sulphuric acid, dark brown in 50% nitric acid, creamy white in
10% aqueous NaOH, yellow in 1N HCl and 5% ammonia and black in 1% thionyl
chloride.
Table 5: Fluorescence analysis of Root powder of Tricalysia sphaerocarpa
Chemicals Root Day light UVlight
Powder as such Creamy white Pale yellow Solvent
Acetone Yellow Orange Benzene Yellow Yellow Chloroform Yellow Pale yellow Ethanol Yellow Dark yellow n-butyl alcohol Yellow Dark yellow Water Creamy white Yellow
Reagents 10% FerricChloride Reddish brown Black 50% Sulphuric Acid Reddish brown Black 50%Nitric acid Orange Brown 10%aq. NaOH Yellow Dark yellow 1 NHCl Greenish yellow Pale yellow 5% Ammonia Yellow Green 1% Thionyl Chloride Light yellow Brown
Table 6: Fluorescence analysis of Fruit powder of Tricalysia sphaerocarpa Chemicals Fruit
Day light UVlight Powder as such Light Brown Creamy white
Solvent Acetone Yellow Green Benzene Yellow Creamy white Chloroform Yellow Creamy white Ethanol Pale yellow Creamy white n-butyl alcohol Light Brown Creamy white Water Light Brown Yellow
Reagents 10%FerricChloride Orange Brown 50% Sulphuric Acid Reddish brown Brown 50%Nitric acid Orange Dark brown 10%aq. NaOH Light Brown Creamy white 1 NHCl Pale yellow Yellow 5% Ammonia Pale yellow Yellow 1% Thionyl Chloride Yellow Black
57
4.2. Phytochemistry
4.2.1. Physico-Chemical Parameters of Various Parts
The physico-chemical parameters such as moisture content, total ash, acid
insoluble ash and water soluble ash of leaf, stem, root and fruit of Tricalysia
sphaerocarpa were analysed and presented in the Table 7.
Leaf
In leaf the moisture content, total ash, acid insoluble ash and water soluble
ash were 15 %, 4 %, 0.84 % and 3.24 % respectively.
Stem
In stem the moisture content, total ash, acid insoluble ash and water
soluble ash were 20 %, 5.16 %, 1.06 %, and 3.90 % respectively .
Root
In root the values for similar parameters were 18 %, 3.26 %, 0.38 %, and
2.28 % respectively .
Fruit
In fruit the moisture content, total ash, acid insoluble ash and water
soluble ash were 17.8 %, 1.5 %, 0.5 %, and 1.1 % respectively.
4.2.2. Extractive Values
The results of extractive values are given in Table 8 and 9 and figure 1 and 2.
Table 7: Proximate analysis of various parts of Tricalysia sphaerocarpa: Parameter Results %
Leaf Stem Root Fruit Loss of drying 15 20 18 17.8 Total ash 4 5.16 3.26 1.5
Acid insoluble ash 0.84 1.06 0.38 0.5 Water soluble ash 3.24 3.90 2.28 1.1 Table 8: Extractive values of various parts of Tricalysia sphaerocarpa by batch
process. Solvent Values in %
Leaf Stem Root Fruit Acetone 15 18 17 13.8 Benzene 11.5 11 8 5 Chloroform 11 9 8 6 Diethyl ether 9 7 5 3 Ethanol 26 20 15 10 n-butyl alcohol 20 10 11.4 9 Methanol 32 30 20 26 Water 30 20 19 20
Table 9: Extractive values of various parts of Tricalysia sphaerocarpa by successive
process. Solvent Values in %
Leaf Stem Root Fruit Chloroform 11.3 14 8.7 5 Diethyl ether 11 9 7.8 4.6 Ethyl acetate 18.2 20 15.5 12 Methanol 20 22.9 17.5 17.9
Table 10: PH Determination of water extract of Tricalysia sphaerocarpa
Various parts PH Leaf 6.4 Stem 6.7 Root 6.8 Fruit 6.8
58
4.2.2.1. Batch Process
Leaf
In leaf, the highest extractive value was recorded in methanol (32%)
followed by aqueous extract (30%), ethanol (26%), n-butanol(20%),
acetone(15%), benzene(11.5%), chloroform(11%) and the lowest extractive value
was found in diethylether (9%).
Stem
In stem, the highest value was seen in methanol (30%), followed by
aqueous extract (20%) ethanol (20%) acetone (18%), benzene (11%), n-butanol
(10%), chloroform (9%) and the lowest in diethylether (7%).
Root
In root, the highest extractive value was recorded in methonal (20%),
followed by aqueous extract (19%), when compared to other solvents like
acetone(17%), ethanol(15%), n-butanol(11.4%), benzene(8%), chloroform(8%)
and diethylether(5%).
Fruit
In fruit, the highest extractive value was recorded in methonal (26%),
followed by aqueous extract (20%), acetone(13.8%), ethanol (10%), n-butanol
(9%), chloroform (6%), benzene(5%) and the lowest value was found in
diethylether(3%).
59
4.2.2.2. Successive Process
Leaf
In leaf, the highest extractive value was recorded in methonal (20%)
followed by ethyl acetate (18.2%), chloroform (11.3%), and diethylether (11%).
Stem
In stem, the highest value was seen in methanol extract (22.9%) when
compared to other solvents like ethyl acetate (20%), chloroform (14%), and
diethylether (9%).
Root
In root, the highest value was recorded in methanol (17.5%), when
compared to other solvents like ethyl acetate (15.5%), chloroform (8.7%), and
diethylether (7.8%).
Fruit
In fruit, the highest value was seen in methanol (17.9%), when compared
to other solvents like ethyl acetate (12%), chloroform (5%), and diethylether
(4.6%).
4.2.3. PH Determination of Powdered Drug
The water extract of the powdered drug of various parts like leaf, stem,
root and fruit were slightly acidic in nature. It showed that the aqueous extract
contains more number of acidic compounds (Table 10).
4.2.4. Preliminary Phytochemical Screening
Preliminary phytochemical screenings of various extracts are given in
Table 11-14.
60
Leaf
The preliminary phytochemical studies in methanol, aqueous and powder
drug showed similar results. It revealed the presence of cardiac glycosides,
glycosides, alkaloids, protein, phenolic group, steroid, saponins, reducing sugar,
non-reducing polysaccharide, flavanoids and terpenoids in methanol, aqueous and
the powder drug. Aminoacids, quinones, phlobatannins, triterpenoids,
anthraquiones, catachins, coumarins and tannins were absent. In diethylether
extract, only the alkaloids were present and others were absent. In chloroform
extract, alkaloids, cardiac glycosides, flavonoids, reducing sugar, non-reducing
polysaccharide (starch), glycosides, protein, phenolic group, saponins, terpenoids
were present and the other phytocompounds were absent. In ethylacetate extract,
only cardiac glycosides, glycosides, reducing sugar, non-reducing polysaccharide
(starch) and steroids were present and the others absent. Gums, oils and resins are
absent in all the extracts.
Stem
The preliminary phytochemical studies in methanol, aqueous and powder
drug showed similar results. It revealed the presence of cardiac glycosides,
glycosides, alkaloids, protein, phenolic group, steroid, saponins, flavanoids,
reducing sugar, non-reducing polysaccharide (starch) and terpenoids in methanol,
aqueous and the powder drug and absence of aminoacids, quinones,
phlobatannins, triterpenoids, anthraquiones, catachins, coumarins and tannins. In
diethylether extract, only the alkaloids were present and other phytocompounds
were absent. In chloroform extract, alkaloids, cardiac glycosides, flavonoids,
Table 11: Phytochemical colour reactions of various extracts of stem of Tricalysia sphaerocarpa
Phytochemicals Diethylether extract
Chloroform extract
Ethylacetate extract
Methanol extract
Aqueous extract
Powder as such
Alkaloids ++ ++ - ++ ++ ++ Anthraquinones - - - - - - Amino acids - - - - - - Cardiac glycosides
- ++ ++ ++ ++ ++
Catechins - - - - - - Coumarins - - - - - - Flavonoids - ++ - ++ ++ ++ Gums, oils and resins
- - - - - -
Glycosides - ++ ++ ++ ++ ++ Non reducing polysaccharides
- ++ ++ ++ ++ ++
Proteins - + - ++ ++ + Pholobatannins - - - - - - Phenolic group - + - + + + Quinones - - - - - - Reducing sugars ++ ++ ++ ++ ++ Saponins - + - + ++ ++ Steroids - - ++ ++ + + Tannins - - - - - - Terpenoids - + - + + + Triterpenoids - - - - - - Note : ++ = marked present; + = moderate present; - = absent.
Table 12: Phytochemical colour reactions of various extracts of leaf of Tricalysia sphaerocarpa
Phytochemicals Diethylether extract
Chloroform extract
Ethylacetate extract
Methanol extract
Aqueous extract
Powder as such
Alkaloids ++ ++ - ++ ++ ++ Anthraquinones - - - - - - Amino acids - - - - - - Cardiac glycosides
- ++ ++ ++ ++ ++
Catechins - - - - - - Coumarins - - - - - - Flavonoids - ++ - ++ ++ ++ Gums, oils and resins
- - - - - -
Glycosides - ++ ++ ++ ++ ++ Non reducing polysaccharides
- ++ ++ ++ ++ ++
Proteins - + - ++ ++ + Pholobatannins - - - - - - Phenolic group - + - + + + Quinones - - - - - - Reducing sugars ++ ++ ++ ++ ++ Saponins - + - + ++ ++ Steroids - - ++ ++ + + Tannins - - - - - - Terpenoids - + - + + + Triterpenoids - - - - - - Note : ++ = marked present; + = moderate present; - = absent.
61
glycosides, protein, phenolic group, reducing sugar, non-reducing polysaccharide
(starch), saponins, terpenoids are present and the others chemical compounds
were absent. In ethylacetate extract, only cardiac glycosides, glycosides, reducing
sugar, non-reducing polysaccharide (starch) and steroids were present and the
others were absent. Gums, oils and resins are absent in all the extracts.
Root
The preliminary phytochemical studies in methanol, aqueous and powder
drug showed similar results. It revealed the presence of cardiac glycosides,
glycosides, reducing sugar, non-reducing polysaccharide (starch), alkaloids,
protein, phenolic group and saponins in methanol, aqueous and the powder drug
and absence of terpenoids, steroids, triterpenoids, anthraquiones, catachins,
coumarins, aminoacids, quinones, phlobatannins and tannins. In diethylether
extract, the phytoconstituents were absent. In chloroform extract, alkaloids,
cardiac glycosides, flavonoids, reducing sugar, non-reducing polysaccharide
(starch), glycosides, protein, phenolic group, saponins were present and the others
were absent. In ethylacetate extract, only cardiac glycosides, reducing sugar, non-
reducing polysaccharide (starch) and glycosides were present and the others were
absent. Gums, oils and resins are absent in all the extracts.
Fruit
The preliminary phytochemical studies in methanol, aqueous showed
similar results. It revealed the presence of cardiac glycosides, glycosides,
reducing sugar, non-reducing polysaccharide (starch), alkaloids, protein, phenolic
group, saponins, steroids and flavanoids in methanol and aqueous extract and the
Table 13: Phytochemical colour reactions of various extracts of root of Tricalysia sphaerocarpa
Phytochemicals Diethylether extract
Chloroform extract
Ethylacetate extract
Methanol extract
Aqueous extract
Powder as such
Alkaloids - + - + + + Anthraquinones - - - - - - Amino acids - - - - - - Cardiac glycosides - ++ + ++ ++ + Catechins - - - - - - Coumarins - - - - - - Flavonoids - - - - - - Gums, oils and resins
- - - - - -
Glycosides - ++ + ++ ++ ++ Non reducing polysaccharides
- ++ ++ ++ ++ ++
Proteins - + - + + + Pholobatannins - - - - - - Phenolic group - + - + + + Quinones - - - - - - Reducing sugars ++ ++ ++ ++ ++ Saponins - + - + ++ ++ Steroids - - - - - - Tannins - - - - - - Terpenoids - - - - - - Triterpenoids - - - - - - Note : ++ = marked present; + = moderate present; - = absent.
Table 14: Phytochemical colour reactions of various extracts of fruit of Tricalysia sphaerocarpa
Phytochemicals Diethylether extract
Chloroform extract
Ethylacetate extract
Methanol extract
Aqueous extract
Powder as such
Alkaloids - + - + ++ + Anthraquinones - - - - - - Amino acids - - - + - - Cardiac glycosides
- ++ + + ++ ++
Catechins - - - - - - Coumarins - - - - - - Flavonoids - + - + ++ + Gums, oils and resins
- - - - - -
Glycosides - ++ ++ ++ ++ ++ Non reducing polysaccharides
- ++ ++ ++ ++ ++
Proteins - ++ - ++ ++ + Pholobatannins - - - - - - Phenolic group - + - + + - Quinones - - - - - - Reducing sugars ++ ++ ++ ++ ++ Saponins - + - + ++ ++ Steroids - - - + + - Tannins - - - - - - Terpenoids - - - - - - Triterpenoids - - - - - - Note : ++ = marked presence; + = moderate presence; - = absent.
62
other phytocompounds were absent. The powder drug showed the presence of
cardiac glycosides, glycosides, alkaloids, reducing sugar, non-reducing
polysaccharide (starch), protein, phenolic group, saponins, and flavanoids and
absence of aminoacids, quinones, phlobatannins, steroids, triterpenoids,
anthraquiones, catachins, coumarins and tannins. In diethylether extract, all the
phytocompounds were absent. In chloroform extract, alkaloids, reducing sugar,
non-reducing polysaccharide (starch), cardiac glycosides, flavonoids, glycosides,
protein, phenolic group, saponins were present and the others were absent. In
ethylacetate extract, only cardiac glycosides, reducing sugar, non-reducing
polysaccharide (starch) and glycosides were present and the others were absent.
Gums, oils and resins are absent in all the extracts.
4.2.5. GC-MS Analysis
Phytochemicals were best extracted in methanol because of its high
polarity. Hence, the methanol extract of leaves, stem, root and fruit were
subjected to GC-MS analysis to detect the possible compounds present in the
active fraction.
4.2.5.1. GC-MS Analysis of Methanolic Extract of Leaf
Totally 30 chemical compounds were identified from the methanolic
extract of leaf. Of which 9 belong to fatty acids (Oleic acid, Octadecanoic acid,
Nonadecanoic acid, n-Hexadecanoic acid, Tetradecanoic acid, 9,12,15-
Octadecatrienic acid, (Z,Z,Z)-, 9,12-Octadecadienoic acid (Z,Z)-, Eicosanoic acid,
Docosanoic acid), four to aliphatic and aromatic bicyclics
(Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-,[1R-1α,2β,5α]-,2,2-Dimethyl indene, 2,3-
63
dihydro-, Bicyclo[3.1.1]heptane,2,6,6-trimethyl-, 2AH-Cyclobut[a]indene-2a-
carboxylic acid, 1,2,7,7a-tetrahydro, methyl ester), two each to aromatic
hydrocarbons groups(2-(2-Hydroxyphenyl)buta-1,3-diene, Phenanthro [3,2-
b]furan-7,11-dione, 1,2,3,4,8,9-hexahydro-4,4,8-trimethyl-,(+)-, (2-
Methoxyphenyl) carbamic acid, naphthalene-2-yl ester), aromatic nitrile groups
(-Ethylbenzonitrile, 2,5-Dimethylbenzonitrile), aromatic dicarboxylic esters
groups (Bis(2-ethylhexyl) phthalate, Phthalic acid, di(2-propylpentyl ester)). Of
which one compound belonged to each of the class terpenoids (Squalene),
barbiturates (cyclobarbital), aromatic alcohols group(Dibenzo[a,c]phenazin-10-
ol), aliphatic aldehydes group (9,17-Octadecadienal, (Z)-, 1H-Benzimidazole, 5,6-
dimethyl-), aromatic ketones group (Chrysene-1,7(2H,8H)-dione, 3,4,9,10-
tetrahydro-2,8-dimethyl-, tert-Butyl (5-isoproply-2-methylphenoxy)
dimethylsilane), aromatic ethers (2,3,5,6-Tetrafluoroanisole), phenolic group(4,6-
Bis(1,1-dimethylethyl)-4´-methyl-1-1´-biphenyl-2-ol) and to pyrimidinedione
group (2,4(1H,3H)-Pyrimidinedione, 5(trifluoromethyl)-). Among this, eicosanoic
acid was found to be present as major constituent with the peak area 35.77% and
retention time 21.86 minutes, followed by octadecanoic acid with the peak area
18.81 % and retention time 20.09 minutes, and followed by 9,12-octadecatrienoic
acid,(z,z)- and 9,17-octadecadienal, (z)- with the peak area 11.54 % and retention
time 19.97 minutes. 1H-Benzimidazole, 5,6-dimethyl-, 2,2-Dimethylindene,2,3,-
dihydro- and 2-(2-Hydroxyphenyl) buta-1,3-diene was found to be as least
quantity with the peak area 0.51 % and retention time 22.8 minutes (Table 15 and
Figure 3).
Table 15: GC-MS Analysis of Methanol Extract of Leaf of Tricalysia sphaerocarpa
No. Name of the compound Molecular formula
Molecular weight
RT Peak area %
Aliphatic & Aromatic bicyclics 1 Bicyclo[3.1.1]heptane, 2,6,6-
trimethyl-, [1R-1α,2β,5α]- C10H18 138 16.942 0.91
2 2,2-Dimethylindene,2,3-dihydro-
C11H14 146 22.882 0.51
3 Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-
C10H18 138 16.942 0.91
4 2AH-Cyclobut[a]indene-2a-carboxylic acid, 1,2,7,7a-tetrahydro, methyl ester
C13H14O2 202 13.456 2.89
Aromatic nitriles 5 -Ethylbenzonitrile C9H9N 131 13.456 2.89 6 2,5-Dimethylbenzonitrile C9H9N 131 13.456 2.89 Aromatic ethers 7 2,3,5,6-Tetrafluoroanisole C7H4F4O 180 13.863 1.05 Pyrimidinedione 8 2,4(1H,3H)-
Pyrimidinedione, 5(trifluoromethyl)-
C5H3F3N2O2 180 13.863 1.05
Fatty acids 9 Oleic acid C18H34O2 282 20.979 1.10 10 Octadecanoic acid C18H32O2 284 20.093 18.81 11 Nonadecanoic acid C19H38O2 298 20.979 1.10 12 n-Hexadecanoic acid C16H32O2 256 18.176 10.41 13 Tetradecanoic acid C14H28O2 228 18.176 10.41 14 9,12,15-Octadecatrienic acid,
(Z,Z,Z)- C18H30O2 278 15.01 12.32
15 9,12-Octadecadienoic acid (Z,Z)-
19.977 11.54
16 Eicosanoic acid C20H40O2 312 21.865 35.77 17 Docosanoic acid C22H44O2 340 23.477 0.77 Aliphatic aldehydes 18 9,17-Octadecadienal, (Z)- C18H32O 19.977 11.54 19 1H-Benzimidazole, 5,6-
dimethyl- C9H10N2 146 22.882 0.51
Aromatic hydrocarbons 20 2-(2-Hydroxyphenyl)buta-
1,3-diene C10H10 130 22.882 0.51
21 Phenanthro[3,2-b]furan-7,11-dione,1,2,3,4,8,9-hexahydro-
C19H20O3 296 23.274 1.62
4,4,8-trimethyl-, (+)- 22 (2-Methoxyphenyl)carbamic
acid, naphthalene-2-yl ester C17H15O3N 281 23.129 1.38
Aromatic ketones 23 Chrysene-1,7(2H,8H)-dione,
3,4,9,10-tetrahydro-2,8-dimethyl-
C20H20O2 292 23.129 1.38
24 tert-Butyl(5-isoproply-2-methylphenoxy)dimethylsilane
C16H28OSi 264 23.129 1.38
Aromatic dicarboxylic esters 25 Bis(2-ethylhexyl) phthalate C24H38O4 390 23.216 1.42 26 Phthalic acid, di(2-
propylpentyl ester) C24H38O4 390 23.216 1.42
Barbiturates 27 cyclobarbital C12H16N2O3 236 23.477 0.77 Terpenoids 28 Squalene C30H50 410 25.322 10.87 Phenolics 29 4,6-Bis(1,1-dimethylethyl)-
4´-methyl-1-1´-biphenyl-2-ol C19H28O 272 23.274 1.62
Aromatic alcohols 30 Dibenzo[a,c]phenazin-10-ol C20H12N2O 296 23.274 1.62
64
4.2.5.2. GC-MS Analysis of Methanolic Extract of Stem
Totally 17 chemical compounds were identified from the methanolic
extract of stem. Of which one belongs to aliphatic hydrocarbons groups
(2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-), four
to steroid groups (Androstane-3,16-diol,(3β,5α,16α)-, Ergost-5-en-3-ol,(3β)-,
Stigmasterol, Τ-Sitosterol), five to fatty acid esters (Hexadecanoic acid, methyl
ester, 9,12-Octadecadienoic acid, methyl ester, (E,E)-, 6,9,12-Octadecatrienoic
acid, methyl ester, Octadecanoic acid, methyl ester, Eicosanoic acid, methyl ester)
three to fatty acid group (9,12,15-Octadecatrienic acid, (Z,Z,Z)-, Octadecanoic
acid, n-Hexadecanoic acid). Of which one compound belongs to the class sugars
(3-O-Methyl-d-glucose), one to the class tocopherols (t-Tocopherol), one to
aromatic nitrile (4-Hydroxy-3-methyl-beta-phenylcinnamonitrile). Among this,
octadecanoic acid was found to be present as major constituent with the peak area
29.88% and retention time 15.33 minutes, followed by n-hexadecanoic acid with
the peak area 15.10 % and retention time 12.80 minutes, and followed by 9,12,15-
octadecatrienoic acid,(z,z,z)- with the peak area 12.32 % and retention time 15.01
minutes. Hexadecanoic acid, methylester was found to be as least quantity with
the peak area 0.90 % and retention time 12.21 minutes (Table 16, Figure 4).
4.2.5.3. GC-MS Analysis of Methanolic Extract of Root
Totally 8 compounds were identified from the methanolic extract of root.
Of which five belongs to heterocyclics groups (3-chloro-2,4-dimethyl -12- thia-
1,5,6a,11, tetraaza-indeno[2,1-a]fluorine, oxitriptan, dl-5-hydroxytryptophan, 2-
methyl-5-p-dimethylaminophenyl oxadiazol, Benzo(b)thiophene,4-methyl), one
Table 16: GC-MS Analysis of Methanol Extract of Stem of Tricalysia sphaerocarpa
No. Name of the compound Molecular formula
Molecular weight
RT Peak area %
Sugars 1 3-O-Methyl-d-glucose C7H14O6 194 11.06 6.18 Aromatic acids & esters 2 1,2-Benzenedicarboxylic
acid, bis(2-methylproplyl) ester
C16H22O4 278 11.59 1.66
Steroids 3 Androstane-3,16-
diol,(3β,5α,16α)- C19H32O2 292 21.65 1.01
4 Ergost-5-en-3-ol,(3β)- C28H48O 400 29.71 4.40 5 Stigmasterol C29H48O 412 30.17 1.45 6 Τ-Sitosterol C29H50O 414 31.31 9.81 Fatty acid esters 7 Hexadecanoic acid, methyl
ester C17H34O2 270 12.21 0.90
8 9,12-Octadecadienoic acid, methyl ester, (E,E)-
C19H34O2 294 14.23 1.89
9 6,9,12-Octadecatrienoic acid, methyl ester
C19H32O2 292 14.31 1.03
10 Octadecanoic acid, methyl ester
C19H38O2 298 14.64 1.57
11 Eicosanoic acid, methyl ester
C21H42O2 326 17.34 1.42
Fatty acid 12 9,12,15-Octadecatrienic
acid, (Z,Z,Z)- C18H30O2 278 15.01 12.32
13 Octadecanoic acid C18H36O2 284 15.33 29.88 14 n-Hexadecanoic acid C16H32O2 256 12.80 15.10 Aromatic nitrile 15 4-Hydroxy-3-methyl-beta-
phenylcinnamonitrile C16H13NO 235 22.07 1.94
Aliphatic hydrocarbons 16 2,6,10,14,18,22-
Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-
C30H50 410 24.02 5.99
Tocopherols 17 Τ-Tocopherol C28H48O2 416 28.26 3.45
65
to aromatic carboxylic ester groups (Benzoic acid, 4-(3-hydroxy-3-methyl-1-
butynyl)-methyl ester), one to fatty esters (Hexadecanoic acid,
1a,2,5,5,5a,6,9,10,10a, octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-
dioxo-1H-2,8a-methano cyclopenta (a) cyclopropa (e) cyclodecen-5-yl ester) and
one to triterpenoid group (9,19-cyclolanostane-6,7-dione,3-acetoxy-). Among
this, benzo(b)thiophene, 4-methyl was found to be present as major constituent
with the peak area 100% and retention time 18.58 minutes, followed by 2-methyl-
5-p-dimethylaminophenyl oxadiazol with the same peak area and retention time
17.8 minutes, and followed by oxitriptan with peak area 100% and retention time
17.13 minutes. 3-chloro-2,4-dimethyl-12-thia-1,5,6a,11-tetraaza-indeno(2,1-a)
fluorine was found to be as in least quantity with the peak area 28 % and
retention time 7.94 minutes respectively (Table 17, Figure 5).
4.2.5.4. GC-MS Analysis of Methanolic Extract of Fruit
Totally 10 compounds were identified from the methanolic extract of fruit.
Of which three belongs to heterocyclics group (N-[4-(4-chlorophenyl)isothiazol-
5yl]-1-methylpiperidin-2-imine, N-[2-(1-piperazyl)ethyl]-N-[2-
thiophosphatoethyl]-1,3-propanamine, 5,8,15,18,23-pentaoxa-1,12-diazabicyclo
(10,8,5)-pentacosane), one to aliphatic aldehyde groups (4-octadecenal), one to
thiosulphate group (S,S1-3,8-diazaundecamethylene bis[hydrogenthiosulfate]),
one to thiophosphates group (2-[3-cyclohexylaminopropylamino]ethyl
thiophosphate), one to antibiotic (Deoxyspergualin) and three to others i.e.
unclassified (4,13,20-tri-O-methylphorbol 12-acetate, EPPS, 2-myristynoyl
pantetheine). Among this, S,S1-3,8-Diazaundecamethylene bis[hydrogen
Table 17: GC-MS Analysis of Methanol Extract of Root of Tricalysia sphaerocarpa
No. Name of the compound Molecular
formula Molecular weight
RT Peak area %
Heterocyclics 1 3-chloro-2,4-dimethyl-
12-thia-1,5,6a,11,tetraaza-indeno[2,1-a]fluorine
C16H10N4SCl 325.5 7.94 28
2 dl-5-hydroxytryptophan C11H12N2O3 220.2 16.6 100 3 oxitriptan C11H12N2O3 220.2 17.13 100 4 2-methyl-5-p-
dimethylaminophenyl oxadiazol
C11H13N3O 203.2 17.8 100
5 Benzo(b)thiophene,4-methyl
C9H8S 148.2 18.58 100
Aromatic carboxylic ester 6 Benzoic acid,4-(3-
hydroxy-3-methyl-1-butynyl)-methyl ester
C13H14O3 218.3 12.53 61.7
Fatty esters 7 Hexadecanoic
acid,1a,2,5,5,5a,6,9,10,10a,octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-dioxo-1H-2,8a-methanocyclopenta(a)cyclopropa(e)cyclodecen-5-yl ester
C36H52O 580 22.98 10.4
Triterpenoids 8 9,19-cyclolanostane-6,7-
dione,3-acetoxy- C32H50O4 498.7 11.86 14.9
Table 18: GC-MS Analysis of Methanol Extract of Fruit of T. sphaerocarpa
No. Name of the compound Molecular formula
Molecular weight
RT Peak area %
Antibiotics 1 Deoxyspergualin C17H37N7O3 512.96 9.27 25.7 Aliphatic aldehydes 2 4-octadecenal C18H34O 266.5 14.43 18.8 Heterocyclics 3 N-[4-(4-chlorophenyl)
isothiazol-5yl]-1-methylpiperidin-2-imine
C15H16ClN3S 305.8 15.02 16
4 N-[2-(1-piperazyl)ethyl]-N-[2-thiophosphatoethyl] -1,3-propanamine
C11H27N4O3PS 326.4 12.08 12.4
5 5,8,15,18,23-pentaoxa-1,12-diazabicyclo (10,8,5)-pentacosane
C18H36N2O5 370 19.15 61.2
Thiophosphates 6 2-[3-cyclohexylamino
propylamino]ethyl thiophosphate
C11H25N2SO4 296.4 18.9 47.9
Thiosulphates 7 S,S1-3,8-
diazaundecamethylene bis[hydrogenthiosulfate]
C9H22N2O6S4 382.5 17.2 83.7
Others 8 4,13,20-tri-O-
methylphorbol 12-acetate C24H35O7 435 25.97 12.5
9 EPPS C9H20N2SO4 252 20.63 15.9 10 2-myristynoyl
pantetheine C25H44N2O5S 484.7 10.88 15.4
66
thiosulfate] was found to be present as major constituent with the peak area 83.7%
and retention time 17.2 minutes, followed by 5,8,15,18,23-pentaoxa-1,12-
diazabicyclo(10,8,5)-pentacosane with the peak area 61.2% and retention time
19.15 minutes. N-[2-[1-piperazyl]ethyl]-N-[2-thiophosphatoethyl]-1,3-
propanamine was found to be as in least quantity with the peak area 12.4 % and
retention time 12.08 minutes respectively. Deoxyspergualin is found to be an
antibiotic with the peak area 9.27 % and retention time 25.7 minutes (Table 18,
Figure 6).
4.2.5.5. Comparative Analysis of Compounds Identified by GC-MS Analysis
Totally 65 compounds were identified from various parts of Tricalysia
sphaerocarpa through GC-MS analysis. Among this, 30 compounds were isolated
from leaf, 17 from stem, 10 from fruit and 8 from root. Octadecanoic acid, n-
hexadecanoic acid and 9,12,15- octadecatrienoic acid (Z,Z,Z-) are the common
compounds seen both in stem and leaf (Table 19). Totally 26 different groups of
compounds are seen. Among this 12 compounds belongs to fatty acid group, 8
compounds belongs to heterocyclics, 6 compounds belongs to fatty acid esters, 4
belongs to steroids, 4 belongs to aliphatic & aromatic bicyclics, 3 belongs to
aromatic hydrocarbons, 3 belongs to aromatic nitriles, 3 belongs to unclassified, 2
belongs to aliphatic aldehydes, 2 belongs to aromatic ketones, 2 belongs to
aromatic dicarboxylic esters, and one compound each belongs to triterpenoid,
antibiotic thiosulphates, thiophosphates, alphatic hydrocarbons, aromatic esters,
tocopherol, aromatic acid & esters, sugars, aromatic alcohols, phenolics,
terpenoids, barbiturates, pyrimidinedione and aromatic ethers. Fatty acid group
Table 19: Combined Table for GC-MS Analysis of Methanol Extract of Tricalysia sphaerocarpa
No. Name of the compound Leaf Stem root fruit activity 1 Bicyclo[3.1.1]heptane,
2,6,6-trimethyl-, [1R-1α,2β,5α]-
+ - - - No activity found
2 2,2-Dimethylindene,2,3-dihydro-
+ - - - No activity found
3 Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-
+ - - - No activity found
4 2AH-Cyclobut[a]indene-2a-carboxylic acid, 1,2,7,7a-tetrahydro, methyl ester
+ - - - No activity found
5 -Ethylbenzonitrile + - - - No activity found 6 4-Hydroxy-3-methyl-
beta-phenylcinnamonitrile
- + - - No activity found
7 2,5-Dimethylbenzonitrile + - - - No activity found 8 2,3,5,6-
Tetrafluoroanisole + - - - Raw material for
bioenergy, biomedicines 9 2,4(1H,3H)-
Pyrimidinedione, 5(trifluoromethyl)-
+ - - - Antiviral
10 Oleic acid + - - - Antibacterial, Antifungal 11 Octadecanoic acid + + - - Antibacterial, Antifungal 12 9,12,15-Octadecatrienic
acid, (Z,Z,Z)- + + - - No activity found
13 9,12-Octadecadienoic acid (Z,Z)-
+ - - - Anti-inflammatory, hypocholesterolemic, cancer preventive, insectifuge, antiarthritic, hepatoprotective, antiandrogenic, nematicide, antihistaminic, antieczemic
14 9,17-Octadecadienal, (Z)-
+ - - - antimicrobial
15 9,12-Octadecadienoic acid, methyl ester, (E,E)-
- + - - No activity found
16 6,9,12-Octadecatrienoic acid, methyl ester
- + - - No activity found
17 4-octadecenal - - - + No activity found 18 Octadecanoic acid,
methyl ester - + - - No activity found
19 Nonadecanoic acid + - - - Cytotoxic activities
20 Eicosanoic acid + - - - No activity found 21 Eicosanoic acid, methyl
ester - + - - No activity found
22 n-Hexadecanoic acid + + - - Antioxidant, Hypocholesterolemic, Nematicide, Pesticide, Lubricant, Antiandrogenic, anti inflammatory, Flavor, Hemolytic, 5-Alpha reductase inhibitor
23 Hexadecanoic acid, methyl ester
- + - - No activity found
24 Hexadecanoic acid,1a,2,5,5,5a,6,9,10,10a,octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-dioxo-1H-2,8a-methanocyclopenta(a)cyclopropa(e)cyclodecen-5-yl ester
- - + - No activity found
25 Tetradecanoic acid + - - - Antioxidant, Cancer preventive, Cosmetic, Nematicide, Lubricant, Hypercholesterolemic
26 Docosanoic acid + - - - Antimicrobial, lubricant, hair conditioners
27 1H-Benzimidazole, 5,6-dimethyl-
+ - - - No activity found
28 2-(2-Hydroxyphenyl)buta-1,3-diene
+ - - - No activity found
29 Phenanthro[3,2-b]furan-7,11-dione,1,2,3,4,8,9-hexahydro-4,4,8-trimethyl-, (+)-
+ - - - No activity found
30 (2-Methoxyphenyl)carbamic acid, naphthalene-2-yl ester
+ - - - No activity found
31 Chrysene-1,7(2H,8H)-dione, 3,4,9,10-tetrahydro-2,8-dimethyl-
+ - - - No activity found
32 tert-Butyl(5-isoproply-2-methylphenoxy)dimethylsilane
+ - - - No activity found
33 Bis(2-ethylhexyl) phthalate
+ - - - No activity found
34 Phthalic acid, di(2-propylpentyl ester)
+ - - - Antibacterial, Oral toxicity during pregnancy and sucking in the Long-Evans Rat
35 cyclobarbital + - - - Anesthetic, anticonvulsant, sedative, hypnotic, veterinary euthanasia agent
36 Squalene + - - - Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immunostimulant, Chemo preventive, Lipoxygenaseinhibitor, Pesticide
37 4,6-Bis(1,1-dimethylethyl)-4´-methyl-1-1´-biphenyl-2-ol
+ - - - No activity found
38 Dibenzo[a,c]phenazin-10-ol
+ - - - No activity found
39 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-
- + - - No activity found
40 3-O-Methyl-d-glucose - + - - No activity found 41 1,2-Benzenedicarboxylic
acid, bis(2-methylproplyl) ester
- + - - Used in preparation of perfumes and cosmetics, plasticized vinyl seats on furniture, cars, and clothing including jackets, raincoats, and boots and used in textiles, as dyestuffs, cosmetics, and glass making
42 Androstane-3,16-diol,(3β,5α,16α)-
- + - - No activity found
43 Ergost-5-en-3-ol,(3β)- - + - - No activity found 44 Stigmasterol - + - - Antimicrobial, Diuretic,
Antiinflammatory, Antiasthma, Antiarthritic, Anticancer
45 τ-Sitosterol - + - - Antibacterical activity
46 τ-Tocopherol - + - - Vitamin E, cosmetics 47 Deoxyspergualin - - - + Antitumour,
immunosuppressive activity
48 2-myristynoyl pantetheine
- - - + No activity found
49 N-[2-(1-piperazyl)ethyl]-N-[2-thiophosphatoethyl]-1,3-propanamine
- - - + No activity found
50 N-[4-(4-chlorophenyl)isothiazol-5yl]-1-methylpiperidin-2-imine
- - - + antipsychotic activity
51 5,8,15,18,23-pentaoxa-1,12-diazabicyclo(10,8,5)-pentacosane
- - - + No activity found
52 2-[3-cyclohexylaminopropylamino]ethyl thiophosphate
- - - + No activity found
53 EPPS - - - + No activity found 54 S,S1-3,8-
diazaundecamethylene bis[hydrogenthiosulfate]
- - - + No activity found
55 4,13,20-tri-O-methylphorbol 12-acetate
- - - + No activity found
56 3-chloro-2,4-dimethyl-12-thia-1,5,6a,11,tetraaza-indeno[2,1-a]fluorine
- - + - No activity found
57 dl-5-hydroxytryptophan - - + - insomnia, depression, anxiety, lower blood pressure in Hypertension patients, anti inflammatory, stimulate the production of antibodies
58 oxitriptan - - + - No activity found 59 2-methyl-5-p-
dimethylaminophenyl oxadiazol
- - + - antifungal
60 Benzo(b)thiophene,4-methyl
- - + - No activity found
61 Benzoic acid,4-(3-hydroxy-3-methyl-1-butynyl)-methyl ester
- - + - No activity found
62 9,19-cyclolanostane-6,7-dione,3-acetoxy-
- - + - immunosuppressive effect
*SOURCE: earlier reported Note : + = present; - = absent.
Table 20: Chemicals groups obtained from GC-MS Analysis of Methanol Extract of Tricalysia sphaerocarpa
Sl.No
Group No. of compounds Total of compounds leaf stem root fruit
1 Aliphatic & aromatic bicyclics
4 - - - 4
2 Aromatic nitriles 2 1 - - 3 3 Aromatic ethers 1 - - - 1 4 Pyrimidinedione 1 - - - 1 5 Fatty acids 9 3 - - 12 6 Aliphatic aldehydes 2 - - 1 3 7 Aromatic hydrocarbons 3 - - 3 8 Aromatic ketones 2 - - 2 9 Aromatic dicarboxylic
esters 2 - - - 2
10 barbiturates 1 - - - 1 11 terpenoids 1 - - - 1 12 Phenolics 1 - - - 1 13 Aromatic alcohols 1 - - - 1 14 sugars - 1 - - 1 15 Aromatic acids & esters - 1 - 1 16 steroids - 4 - - 4 17 Fatty acid esters - 5 1 - 6 18 Aliphatic hydrocarbons - 1 - - 1 19 tocopherols - 1 - - 1 20 heterocyclics - - 5 3 8 21 Aromatic esters - - 1 - 1 22 Thiophosphates - - - 1 1 23 Thiosulphates - - - 1 1 24 antibiotic - - - 1 1 25 triterpenoids - - 1 - 1 26 others 3 3 Total 30 17 8 10 65
67
plays a major role with 12 different chemical constituents with therapeutic
significance (Table 20).
4.3. Pharmacology
4.3.1. In-vitro Antioxidant Activity
4.3.1.1. DPPH Scavenging Activity
In stem, maximum activity was observed in chloroform extract. The
percentage of scavenging activities at 100µg/ml, 50µg/ml and at 10µg/ml were
observed to be 87.73 ± 8.37, 67.22 ± 7.23 and 58.68 ± 6.47 respectively. In
methanol extract, the percentage of scavenging activity at 100µg/ml was 71.62 ±
7.76, 62.43 ± 6.74 at 50µg/ml and 51.59 ± 5.95 at 10µg/ml. In petroleum ether
extract, the percentage of scavenging activity at 100µg/ml was 66.63 ± 7.23,
42.65 ± 5.82 at 50µg/ml and 28.67 ± 5.76 at 10µg/ml. In water extract, the
percentage of scavenging activity at 100µg/ml, 50µg/ml and 10µg/ml were 51.49
± 5.85, 47.48 ± 4.92 and 35.53 ± 4.81 respectively. All the extracts showed dose
concentration dependent activity in all the tested concentration. The significantly
higher activity was observed in the chloroform extract and the results are
presented in the table 21 and figure 7.
4.3.1.2. Iron Chelating activity (FRAP Assay)
In stem, maximum value of Iron Chelating activity was observed in
chloroform extract as 66.46 ± 6.21 at 100µg/ml, 41.24 ± 3.98 at 50µg/ml and
25.52 ± 1.95 at 10µg/ml. In methanol extract, the percentage of scavenging
activity at 100µg/ml was 61.77 ± 5.98, 49.87 ± 4.76 at 50µg/ml and 32.02 ± 2.84
at 10µg/ml. In water extract, the percentage of scavenging activity at 100µg/ml
Table 21: Antioxidant activity of various extracts using DPPH assay: Solvent % of radicle scavenging
10µg/ml 50µg/ml 100µg/ml Petroleum ether 28.674 ± 5.76 42.650 ± 5.82 66.638± 7.23 Chloroform 58.688±6.47 67.224± 7.23 87.728± 8.37 Methanol 51.595±5.95 62.439± 6.74 71.624± 7.76 Water 35.553±4.61 47.485± 4.92 51.496± 5.85 BHT 47.393± 5.89 69.784± 7.76 89.720± 8.80 All the values are expressed as mean ± SEM(n=6) Table 22: Antioxidant activity of various extracts using Iron chelating activity Solvent % of radicle scavenging
10µg/ml 50µg/ml 100µg/ml Petroleum ether 10.789± 1.79 29.456± 2.46 38.493± 2.79 Chloroform 25.523± 1.95 41.240± 3.98 66.458±6.21 Methanol 32.029± 2.84 49.879± 4.76 61.776± 5.98 Water 29.555± 3.65 40.788± 3.62 55.466± 5.72 EDTA 59.437± 5.77 70.125± 6.75 95.459± 8.39 All the values are expressed as mean ± SEM(n=6) Table 23: Antioxidant activity of various extracts using Hydrogen peroxide assay Solvent % of radicle scavenging
10µg/ml 50µg/ml 100µg/ml Petroleum ether 0.240±1.65 0.578±3.54 0.724±7.58 Chloroform 0.442±2.79 0.712±5.71 0.993±8.32 Methanol 0.378±2.43 0.562±3.45 0.728±7.61 Water 0.153±1.30 0.310±2.73 0.478±3.26 Ascorbic acid 0.994±1.18 1.120±8.41 1.440±8.72 All the values are expressed as mean ± SEM(n=6) Table 24: Antioxidant activity of various extracts using Superoxide dismutase assay Solvent % of radicle scavenging
10µg/ml 50µg/ml 100µg/ml Petroleum ether 1.988±3.75 2.590±4.21 4.778±6.79 Chloroform 2.322±3.98 5.330±4.65 6.901±6.37 Methanol 4.784±4.06 6.120±5.39 7.953±6.59 Water 2.850±3.22 4.875±6.32 5.445±5.25 Ascorbic acid 5.510±4.81 8.010±7.51 9.847±8.73 All the values are expressed as mean ± SEM(n=6)
68
was 55.46 ± 5.72, 40.78 ± 3.62 at 50µg/ml and 29.55 ± 3.65 at 10µg/ml. In
petroleum ether extract, the percentage of scavenging activity at 100µg/ml was
38.49 ± 2.97, 29.45 ± 2.46 at 50µg/ml and 10.78 ± 1.79 at 10µg/ml. All the
extracts showed dose concentration dependent activity in all the tested
concentration. The significantly higher activity was observed in the chloroform
extract and the results are presented in the table 22 and figure 8.
4.3.1.3. Hydrogen peroxide Assay
The results of hydrogen peroxide assay were presented in the table 23 and
figure 9. In stem, all the extracts showed dose concentration dependent activity in
all the tested concentrations. Chloroform extract showed the maximum value as
0.99 ± 8.32 at 100µg/ml, 0.71 ± 5.91 at 50µg/ml and 0.44 ± 2.79 at 10µg/ml,
moderate value was observed in methanol extract as 0.73 ± 7.61 at 100µg/ml,
0.56 ± 3.45 at 50µg/ml and 0.37 ± 2.43 at 10µg/ml followed by petroleum ether
extract as 0.72 ± 7.58 at 100µg/ml, 0.57 ± 3.54 at 50µg/ml and 0.24 ± 1.65 at
10µg/ml and the minimum value was observed in water extract as 0.47 ± 3.26 at
100µg/ml, 0.31 ± 2.73 at 50µg/ml and 0.15 ± 1.30 at 10µg/ml respectively. The
chloroform extract showed the high performance activity.
4.3.1.4. Superoxide dismutase (L- methionine and NBT Assay)
In stem, the maximum Superoxide dismutase activity was observed in
methanol extract as 7.95 ± 6.59 at 100µg/ml, 6.12 ± 5.39 at 50µg/ml and 4.78 ±
4.06 at 10µg/ml followed by the chloroform extract as 6.90 ± 6.37 at 100µg/ml,
5.33 ± 4.65 at 50µg/ml and 2.32 ± 3.98 at 10µg/ml, water extract showed 5.45 ±
5.95 at 100µg/ml, 4.87 ± 5.01 at 50µg/ml and 2.85 ± 3.22 at 10µg/ml and the
69
minimum value was observed in petroleum ether extract as 4.78 ± 6.79 at
100µg/ml, 2.59 ± 4.21 at 50µg/ml and 1.98 ± 3.96 at 10µg/ml respectively. It
shows that the methanolic extract to be the most potential scavenger. The results
of Superoxide dismutase assay were presented in the table 24 and figure 10.
4.3.2. In-vivo Pharmacological studies
4.3.2.1. Acute toxicity
No mortality was observed in the animals treated with 2000 mg/kg
methanol extract of stem. There were no signs of any toxicity.
4.3.3. Anti-depressant activity
The behavioral despair model was performed in order to investigate the
ability of this herbal drug in the elevation of suppressed mood, which is quite
common in today’s scenario. The results obtained from FST (Forced swimming
test), TST (Tail suspension test) and HBT (Hole board test) clearly reveled the
fact that this drug is potentially quite useful in cases of depression (Table 25-27).
The present findings suggested that methanolic extract when administered at an
acute oral dose of 200 mg/kg of body weight (P<0.01) reduced the immobility
time by 135 seconds as compared to the immobility time of control i.e. 190
seconds the time shown by animals treated with extract was found to be 170
seconds when it was compared with control and standard. The decrease in the
immobility time was quite close to that of standard. The time of mobility was
increased by methanolic extract at a dose of 200 mg/ml, shown the immobility
time **140 seconds (P<0.01) to that of standard **135 seconds (P<0.01). These
Table 25 : Effect of methanolic extract of Tricalysia sphaerocarpa on Immobility time in FST
Group no. Drug treatment Dose mg/kg Immobility period
I Contol (Distilled water) 5ml/kg 190 sec
II Methanolic extract 100 mg/kg 170 sec.
III Methanolic extract 200 mg/kg 135 sec.
IV Standard drug 30 mg/kg 140 sec.
Results are expressed as mean ±S.E.M [n=6]. Test group compared with control group p<0.01. test group compared with standard group p<0.01.
Table 26 : Effect of methanolic extract of T. sphaerocarpa in Immobility time in TST
Group no. Drug treatment Dose Immobility period I Control (Distilled water) 5ml/kg 195 sec.
II Methanolic extract 100 mg/kg 170 sec.
III Methanolic extract 200 mg/kg 165 sec.
IV Standard drug 30 mg/kg 135 sec.
Results are expressed as mean ±S.E.M [n=6]. Test group compared with control group p<0.01. test group compared with standard group p<0.01. Table 27: Effect of methanolic extract of T. sphaerocarpa. in Hole Board Test (HBT) Group Dose(i.p;mg/kg) No. of head dippings
per 3 min No. of line crossings per 3min
Control(distilled water)
5ml/kg 22 ± 1.43 18 ± 0.9
Standard(Diazepam) 4mg/kg 8 ± 0.71 10 ± 1.24
Test group-1 100mg/kg 18 ± 1.04 15 ± 0.55
Test group-2 200mg/kg 13 ± 1.01 14 ± 0.55
Results are expressed as mean ±S.E.M [n=6]. Test group compared with control group p<0.01. test group compared with standard group p<0.01.
70
results show that after standard i.e. Imipramine HCl (30 mg/kg), the methanolic
extract is most potent amongst all the treated groups.
Findings on tail suspension test were quite comparable to the previous FS
test. It is quite evident that none of the drug treated animals showed excellent
results compared to the standard. The immobility of Imipramine HCL (P<0.01) 30
mg/ kg was found to be 135 seconds. In this test the time of animals treated with
methanolic extract 100 mg/kg was found to be 170 seconds (P<0.01) when it was
compared to the control group of animals which was 195 seconds. The
immobility time of methanolic extract when given an acute dose of 200 mg/kg
each of body weight significantly reduced the time of immobility by 135 seconds
(P<0.01). The present findings on HBT suggested that methanolic extract when
administered at an acute oral dose of 200 mg/kg of body weight (P<0.01) reduced
the number of head dippings by 13 per 3 min and the number of line crossing per
3 minutes is 14. Adminstration of an acute oral dose of 100 mg/kg of body weight
(P<0.01) reduced the number of head dipping by 18 per 3 min and the number of
line crossing per 3 min is 15 as compared to control number of head dipping by
23 per 3 minutes and the number of line crossing per 3 min is 18 and as standard,
number of head dipping by 8 per 3 minutes and the number of line crossing per 3
minutes is 10. The results clearly revel the fact that standard treated animals
showed better response as compared to the plant extract treated groups but even
though methanolic extract 200 mg/kg treated group showed better response as
compared to standard drug treated group of animals (Plate 8).
71
4.3.2.3. Anti - diabetic Activity
4.3.2.3.1. Effect of Methanolic Stem Extract on Blood Glucose Level in
Normal Fasted Rats
All doses of methanolic stem extract of Tricalysia sphaerocarpa in normal
fasted rats, significantly (P<0.05) reduced the blood glucose levels up to 4 hour
except the lowest dose. The impairment of blood glucose levels of Tricalysia
sphaerocarpa was marked and dose dependent at each time point. The normal
control group showed 60.3 ± 1.70 mg/dl at 0 hour, 62.5 ± 2.12 mg/dl one hour
after the treatment, 61.0 ± 1.24 mg/dl 2 hours after the treatment, 60.2 ± 1.14
mg/dl after 4 hour of treatment, 58.2 ± 2.62 mg/dl after 6 hour of treatment. The
group treated with 125 mg/kg showed 62.0 ± 4.80 mg/dl after the treatment of 0
hour, 62.0 ± 1.90 mg/dl after the treatment of 1 hour, 61.0 ± 5.17 mg/dl after 2
hour of treatment, 59.0 ± 1.80 mg/dl after 4 hour of treatment, 60.3 ± 2.70 mg/dl
after 6 hour of treatment. The group treated with 250 mg/kg showed 64.1 ± 2.72
mg/dl after the treatment of 0 hour, 65.0 ± 1.22 mg/dl after the treatment of 1
hour, 63.5 ± 2.62 mg/dl after 2 hour of treatment, 60.3 ± 2.14 mg/dl after 4 hour
of treatment, 62.0 ± 1.40 mg/dl after 6 hour of treatment. The group treated with
500 mg/kg showed 70.0 ± 2.12 mg/dl after the treatment of 0 hour, 68.0 ± 2.01
mg/dl after the treatment of 1 hour, 64.1 ± 1.70 mg/dl after 2 hour of treatment,
59.0 ± 6.20 mg/dl after 4 hour of treatment, 63.0 ± 2.12 mg/dl after 6 hour of
treatment. The group treated with glibenclamide 5 mg/kg showed 61.1 ± 3.18
mg/dl at 0 hour the treatment, 57.0 ± 1.20 mg/dl after the treatment of 1 hour,
51.3 ± 2.24 mg/dl after 2 hour of treatment, 46.3 ± 2.34 mg/dl after 4 hour of
Table 28 : Effect of Test extract on blood glucose level in normal fasted rats Group Treatment (dose mg/kg, po)
Blood Glucose level (mg/dl) 0 hr 1 hr 2 hr 4 hr 6 hr
Normal control 60.3 ± 1.70 62.5 ± 2.12 61.0 ±1.24 60.2 ±1.14 58.2 ±2.62 Test extract (125) 62.0 ± 4.80 62.0 ±1.90
(1.5) 61.0 ±5.17 (4.5)
59.0 ±1.80 (10.6)
60.3 ±2.70a
(8.6)
Test extract (250) 64.1 ± 2.72 65.0 ±1.22 (3.1)
63.5 ±2.62 a
(5.3) 60.3 ±2.14 b
(10.3) 62.0 ±1.40b
(5.1)
Test extract (500) 70.0 ± 2.12 68.0 ±2.01a
(5.5) 64.1 ±1.70 b
(10.9) 59.0 ±6.20 b
(18.0) 63.0 ±2.12b
(12.5)
Glibenclamide (5) 61.1 ± 3.18 57.0 ± 1.20 (6.7)
51.3 ±2.24 b
(16.0) 46.3 ±2.34 b
(24.2) 50.3 ±2.00 b
(17.6) Values are mean ± SEM from 6 animals in each group. Figure in parenthesis indicates % fall in BGL as compared to 0 hr. Table 29 : Effect of Test extract on blood glucose level in Alloxan-induced diabetic rats (Single-dose short term study)
Group & Treatment
(dose mg/kg, po)
Blood Glucose Level (mg/dl)
0 hr 1 hr 3 hr 6 hr
Normal control 60.8±1.60 61.6±2.02 62.5±1.64 60.1±1.20
Diabetic control 295.8±1.96 a 306.1±2.80a
(-3.7) 299.6±1.26a
(-1.5) 300.3±1.82a
(-1.7) Test extract (125) 309.5±2.07 270.0±1.80 b
(11.9) 212.0±1.60 b
(31.4) 202.0±2.21 b
(32.1) Test extract (250) 302.3±1.00 241.0±1.12 b
(21.1) 200.3±1.06 b
(32.8) 199.0±2.10 b
(34.2) Test extract (500) 300.0±1.46 210.1±2.00 b
(29.7) 132.5±2.20 b
(54.8) 150.1±2.31 b
(49.3) Glibenclamide (5) 280.0±2.20 198.0±1.20 b
(29.4) 100.5±1.87 b
(60.1) 120.8±2.60 b
(54.1) Values are mean ± SEM from 6 animals in each group. Figure in parenthesis indicates % fall in BGL as compared to 0 hr. P value: <0.01; compared to a normal group, b diabetic group
72
treatment, 50.3 ± 2.00 mg/dl after 6 hour of treatment. The maximum
hypoglycemic activity was induced by 500 mg/kg dose at 4 hour by 18%.
However, the effects of other tested doses of Tricalysia sphaerocarpa were less
than the reference drug (Table 28).
4.3.2.3.2. Effect of Test Extract on Blood Glucose Level in Alloxan-induced
Diabetic Rats
4.3.2.3.2.1. Effect of single dose administration of Test extract on blood
glucose level in Alloxan-induced diabetic rats (Short Term Study):
All doses of methanolic stem extract of T. sphaerocarpa in alloxan-
induced diabetic rats, significantly (P<0.01) reduced the blood glucose levels up
to 6 hour except the lowest dose. The normal control group showed 60.8 ± 1.60
mg/dl at 0 hour, 61.6 ± 2.02 mg/dl after the treatment of 1 hour, 62.5 ± 1.64 mg/dl
after 3 hour of treatment, 60.1 ± 1.20 mg/dl after 6 hour of treatment. The group
treated with 125 mg/kg showed 270.0 ± 1.80 mg/dl after the treatment of 1 hour,
212.0 ± 1.60 mg/dl after 3 hour of treatment, 202.0 ± 1.60 mg/dl after 6 hour of
treatment. The group treated with 250 mg/kg showed 241.0 ± 1.12 mg/dl after the
treatment of 1 hour, 200.3 ± 1.06 mg/dl after 3 hour of treatment, 199.0 ± 2.10
mg/dl after 6 hour of treatment. The group treated with 500 mg/kg showed 210.1
± 2.00 mg/dl after the treatment of 1 hour, 132.5 ± 2.20 mg/dl after 3 hour of
treatment, 150.1 ± 2.31 mg/dl after 6 hour of treatment. The group treated with
glibenclamide 5 mg/kg showed 198.0 ± 1.20 mg/dl after the treatment of 1 hour,
100.5 ± 1.87 mg/dl after 3 hour of treatment, 120.8 ± 2.60 mg/dl after 6 hour of
treatment. The diabetic control group showed 295.8 ± 1.96 mg/dl at 0 hour, 306.0
73
± 2.80 mg/dl after the treatment of 1 hour, 299.6 ± 1.26 mg/dl after 3 hour of
treatment, 300.3 ± 1.82 mg/dl after 6 hour of treatment (Table 29).
4.3.2.3.2.2. Effect of multidose administration of Test extract on blood
glucose level in Alloxan-induced diabetic rats (long term study of 15 days
daily once)
The methanol extract of T. sphaerocarpa showed significant (P<0.01)
plasma glucose lowering effect. The present study indicates that alloxan induced
tissue injury is reversed by continuous administration of T. sphaerocarpa extract
with subsequent decrease in blood sugar. Alloxan treated diabetic rats showed
significant increase in the level of fasting plasma glucose levels when compared
to normal rats. Alloxan generate free radicals in the body, leads to tissue damage
including pancreas and would be responsible for increased blood sugar seen in the
animals. Oral administration of T. sphaerocarpa methanolic extract of 500 mg/kg
showed significant (P<0.01) plasma glucose lowering effect in 12 and 16 days of
treatment. The normal control group showed 56.1 ± 1.02 mg/dl on 3rd day, 61.0 ±
1.20 mg/dl on 6th day, 61.0 ± 0.82 mg/dl on day 9, 62.8 ± 2.10 mg/dl on day 12
and 58.9 ± 4.40 mg/dl on 16th day of treatment. The group treated with 125mg/kg
showed 291.2 ± 2.26 mg/dl on 3rd day, 280.5 ± 1.40 mg/dl on 6th day, 260.2 ±
2.30 mg/dl on day 9, 230.0 ± 4.02 mg/dl on day 12 and 192.1 ± 0.62 mg/dl on
16th day of treatment. The group treated with 250mg/kg showed 290.2 ± 0.01
mg/dl on 3rd day, 270.2 ± 4.20 mg/dl on 6th day, 228.0 ± 0.48 mg/dl on day 9,
184.0 ± 2.70 mg/dl on day 12 and 172.2 ± 1.50 mg/dl on 16th day of treatment.
The group treated with 500mg/kg showed 280.0 ± 2.21 mg/dl on 3rd day, 252.2 ±
Table 30 : Effect of multidose administration of Test extract on blood glucose level in Alloxan-induced diabetic rats (long term study of 15 days daily once)
Group & treatment
(dose mg/kg; p.o)
Blood glucose level (mg/dl)
Day 3
Day 6
Day 9
Day 12
Day 16
Normal control
56.1±1.02 61.0±1.20 61.0±0.82 62.8±2.10 58.9±4.40
Diabetic control
312.5 ±1.20 a
306.1±2.12a
(1.7) 290.8 ± 0.42a
(6.8) 279.2 ± 2.12a
(10.3) 278.2 ± 2.20a
(11.96) Test extract (125)
291.2 ± 2.26 b
280.5± 1.40c
(4.4) 260.2 ± 2.30c
(11.3) 230.0 ± 4.02c
(27.1) 192.1 ± 0.62c
(33.5)
Test extract (250)
290.2 ± 0.01 c
270.2± 4.20c
(6.4) 228.0 ± 0.48c
(20.7) 184.0 ± 2.70c
(35.1) 172.2 ± 1.50c
(39.9) Test extract (500)
280.0 ± 2.21 c
252.2± 2.12c
(10.6) 220.0 ± 2.12c
(22.6) 182.4 ± 2.10c
(36.4) 162.2 ± 2.10c
(35.7) Glibenclamide(5)
278.5 ± 2.30 c
240.2± 2.12c
(1.5) 210.6 ± 1.40c
(22.5) 182.5 ± 0.86c
(35.8) 164.5 ± 2.12c
(42.9) Values are mean ± SEM from 6 animals in each group. Figure in parenthesis indicates % fall in BGL as compared to Day 3. p values: <0.01, as compared to a normal group; c
diabetic control group b<0.05 compared to diabetic group.
Table 31 : Effect of formulation Test extract on body weight in Normal and Alloxan induced diabetic rats
Group & treatment (dose,
mg/kg; p.o)
Initial Body weight (g)
Final Body weight (g)
% increased/ decreased of body weight
Normal control 80.22±2.70 102.00±8.22 + 18.30
Diabetic control 99.60±10.20 60.12±2.90 - 41.92
Test extract (125) 82.60±12.34 50.82±8.92 - 34.80
Test extract (250) 84.22±10.22 63.44±12.12 - 25.44
Test extract (500) 88.12±8.44 74.10±4.22 - 19.00
Glibenclamide (5) 90.00±11.02 76.22±8.52 - 15.98
values are mean ± SEM from 6 animals in each group Where + indicates % increase of body weight.
- Indicates % decrease of body weight.
74
2.12 mg/dl on 6th day, 220.0 ± 2.12 mg/dl on day 9, 182.4 ± 2.10 mg/dl on day 12
and 162.2 ± 2.10 mg/dl on 16th day of treatment. The group treated with
glibenclamide 5mg/kg showed 278.5 ± 2.30 mg/dl on 3rd day, 240.2 ± 2.12 mg/dl
on 6th day, 210.6 ± 1.40 mg/dl on day 9, 182.5 ± 0.86 mg/dl on day 12 and 164.5
± 2.12 mg/dl on 16th day of treatment. The diabetic control group showed 312.5 ±
1.20 mg/dl on 3rd day, 306.1 ± 2.12 mg/dl on 6th day, 290.8 ± 2.12 mg/dl on day
9, 279.2 ± 2.12 mg/dl on day 12 and 278.2 ± 2.20 mg/dl on 16th day of treatment
(Table 30).
4.3.3.3. Effect of Formulation Test Extract on Body Weight in Normal and
Alloxan Induced Diabetic Rats
In the anti-diabetic activity, the effects methanolic extract of T.
sphaerocarpa on body weight was measured and were compared with normal and
diabetic control groups. Oral administration of the extract at the dose of 500
mg/kg showed a significant (P<0.01) decrease in body weight. In normal control
groups, the initial body weight was 80.22 ± 2.70 g, the final body weight was
102.00 ± 8.22 g and the percentage of body weight was increased by 18.30 In
diabetic control groups, the initial body weight was 99.60 ± 10.20 g, the final
body weight was 60.12 ± 2.90 g and the percentage of body weight was decreased
by -41.92. The groups treated with 125 mg/kg shows, the initial body weight was
82.60 ± 12.34 g, the final body weight was 50.82 ± 8.92 g and the percentage of
body weight was decreased by -34.80. In the groups treated with 250 mg/kg
shows, the initial body weight was 84.22 ± 10.22 g, the final body weight was
63.44 ± 12.12 g and the percentage of body weight was decreased by -25.44. The
75
groups treated with 500 mg/kg shows, the initial body weight was 88.12 ± 8.44 g,
the final body weight was 74.10 ± 4.22 g and the percentage of body weight was
decreased by -19.00. The groups treated with glibenclamide 5 mg/kg shows, the
initial body weight was 90.00 ± 11.02 g, the final body weight was 76.22 ± 8.52 g
and the percentage of body weight was decreased by -15.98 (Table 31).
4.3.3.4. Effect of Formulation Test Extract on Bio-Chemical Parameters in
Alloxan Induced Diabetic Rats.
In normal control groups, the lysosome enzyme levels, SGOT (72.12 ±
0.12 IU/L), SGPT (41.08 ± 0.36 IU/L), Alkaline phosphatise (132.90 ± 0.25
IU/L), and also in % of lipid peroxidation (60.22 ± 1.02). It also reduced the
enzymatic antioxidants like CAT (3.16 ± 0.14 U/mg), GPx (2.56 ± 0.10 U/mg). In
diabetic control groups, the lysosome enzyme levels, SGOT (138.20 ± 0.18 IU/L),
SGPT (98.16 ± 0.44 IU/L), Alkaline phosphatise (250.23 ± 0.22 IU/L), and also
in % of lipid peroxidation (102.0 ± 1.59). It also reduced the enzymatic
antioxidants like CAT (1.88 ± 0.169 U/mg), GPx (1.83 ± 0.175 U/mg). The group
treated with 125 mg/kg showed the lysosome enzyme levels, SGOT (115.8 ± 0.16
IU/L), SGPT (75.16 ± 0.58 IU/L), Alkaline phosphatise (216.30 ± 0.42 IU/L), and
also in % of lipid peroxidation (80.56 ± 0.04). It also reduced the enzymatic
antioxidants like CAT (2.20 ± 0.817 U/mg), GPx (2.10 ± 0.09 U/mg). The group
treated with 250 mg/kg showed the lysosome enzyme levels, SGOT (105.23 ±
0.06 IU/L), SGPT (64.66 ± 0.60 IU/L), Alkaline phosphatise (194.20 ± 0.14
IU/L), and also in % of lipid peroxidation (74.36 ± 0.15). It also reduced the
enzymatic antioxidants like CAT (2.46 ± 0.069 U/mg), GPx (2.26 ± 0.04 U/mg).
Table 32 : Effect of formulation Test extract on biochemical parameters in Alloxan induced diabetic rats.
Group & treatment(dose
mg/kg, po)
SGOT (IU/L)
SGPT (IU/L)
Alkaline phosphatise (IU/L)
% Lipid Peroxi-dation
CAT (U/mg)
GPX (U/mg)
Normal control 72.12 ± 0.12
41.08 ± 0.36
132.90 ± 0.25
60.22 ± 1.02
3.16 ± 0.14
2.56 ± 0.10
Diabetic control
138.20 ± 0.18 a
98.1 6± 0.44 a
250.23 ± 0.22 a
102.0 ± 1.59
1.88 ± 0.169 a
1.83 ± 0.175 a
Test extract 125
115.8 ± 0.16b
75.16 ± 0.58 b
216.30 ± 0.42 b
80.56 ± 0.04 b
2.20 ± 0.817 b
2.10 ± 0.09 b
Test extract 250
105.23 ± 0.06 b
64.66 ± 0.60 b
194.20 ± 0.14 b
74.36 ± 0.15 b
2.46 ± 0.069 b
2.26 ± 0.04 b
Test extract 500
90.36 ± 0.06 b
55.34 ± 0.45 b
169.20 ± 0.17 b
65.32 ± 0.58 b
2.76 ± 0.31 b
2.68 ± 0.17 b
Glibenclamide (5)
83.33 ± 0.18 b
48.30 ± 0.24 b
155.30 ± 0.40 b
60.93 ± 0.36 b
2.80 ± 0.23 b
2.50 ± 0.15 b
Values were expressed as Mean ± SEM of 6 rats in each group. p value: <0.01; compared to a normal group b diabetic
76
The group treated with 500mg/kg showed the decrease the lysosome enzyme
levels, SGOT (90.36 ± 0.06 IU/L), SGPT (55.34 ± 0.45 IU/L), Alkaline
phosphatise (169.20 ± 0.17 IU/L), and also in % of lipid peroxidation (65.32 ±
0.58). It also reduced the enzymatic antioxidants like CAT (2.76 ± 0.31 U/mg),
GPx (2.68 ± 0.17 U/mg). All the concentrations tested have dose dependent
activity in the experimental animals (Table 32).
4.3.3.5. Histopathological Studies
Microscopically examined pancreas section of normal rat group and
diabetic control group showed that normal architecture of pancreas with acini of
serous epithelial cells along with nest of endocrine cells separated by
fibrocollaoenous, stroma into lobules. No fibrosis or inflammation was found.
Pancreas section of rat treated with Test extract (125 mg/kg and 250 mg/kg)
showed that normal architecture of pancreas with acini of serous epithelial cells
along with nest of endocrine cells separated by fibrocollagenous, stroma into
lobules. No fibrosis or inflammation was found. The test extract (500 mg/kg)
showed stroma into Mules like the standard Glibenclamide (Plate 9).
77
CHAPTER 5
DISCUSSION
Natural products have been the most successful source of drugs for ever.
Researches on drug discovery especially in phytodrug investigation have become
one of the frontier areas in phytochemistry. Plants are probably the best cell
factories that can be exploited to produce a wide range of chemical compounds,
especially the low molecular weight secondary metabolites (Hadacek, 2002).
5.1. Pharmacognosy :
Pharmacognosy is defined as the scientific and systematic study of
structural, physical, chemical and sensory characters of crude drugs along with
their history, method of cultivation, collection and preparation for the market
(Evans, 1996). Identification of drugs can be done by morphological, histological
and chemical testing. There are five methods of evaluation of crude drugs namely
Morphlolgical or Organoleptic, Microscopical or Histological, Physical, Chemical
and Biological.
Micromorphology of vegetative and reproductive plant organs is the
object of research to resolve the taxonomic problems of critical species and
genera. Of the several traits on leaf surface, the stomata are perhaps the most
significant from the point of view of systematic and phylogeny. Stomata that are
highly characteristic of the epidermis occur in widely divergent parts of the plants
including common foliage leaves. Stomatal size is an ecologically important
attribute. The type, size, distribution and frequency of stomata have been
recognized to be specific to the taxa below the family and these characters were
78
used as significant parameters in the angiosperm taxonomy as well as phylogeny.
The importance of epidermal cell characters is now well established in taxonomic
considerations of angiosperms (Parveen et al., 2000). Microscopical evaluation of
the plant drugs helps to identify the organized drugs by their known histological
characters and used to confirm the structural details of the drugs from plant origin.
The anatomical features of T. sphaerocarpa such as the astomatic nature
of the adaxial epidermis of leaf, the epidermal cell number, stomatiferous abaxial
epidermis, rubiaceous type of stomata in various sizes, orientation and frequency,
palisade spongy ratious, vein islet number, veinlet termination number and
unicellular conical trichomes of the leaf traits are the characteristic to the plant.
The macerated elements showed characteristic vessel elements with tails on one
side or on both sides. Anatomy of the leaf, stem and root reveled unique features
such as hemispherical epidermal cells on the stem and midrib region of leaf with
arches of thick cuticle, discrete vessels in the secondary xylem, distribution of
tannineferous idioblasts. All these characters are typical to this plant which
would be very useful in correct botanical identification of crude samples.
Histochemical localization methods provides the authentic data on the
availability of chemical compounds by simple and quick methods. Histochemical
analysis is highly essential that will aid the pharmacognosist to locate chemical
substances and its properties in terms of cells, tissues and parts (Johansen, 1940).
Histochemical localization is performed for starch, alkaloid, protein,
tannin and lignin. The present study reports the presence of starch, alkaloid and
79
protein in leaf, stem and root; tannin in leaf and stem. Lignin only in root.
Mucilage is totally absent in all the plant part studied.
Fluorescence analysis showed that, the leaf powder is mostly green to
dark green in daylight whereas it is orange in UV light. In stem, it is mostly
yellow in both day light and UV light. In root, it is mostly yellow in day light and
pale yellow to dark yellow in UV light. In fruit, it is mostly light brown in day
light and creamy white to brown in UV light. When physical and chemical
methods are insufficient, as often happens with the powdered drugs, there are
methods such as fluorescence studies and microchemical tests to identify the
powdered drugs and their adulterants. In addition to this preliminary
phytochemical, histochemical tests using free hand sections of fresh parts,
microtome sectioning are used in identifying the adultered ones. Maceration
methods also included, in which the type of vessels, tracheids, fibres etc. are
considered in determining the genuiness of the drug (Kulkarni and Surekha,
1981). In Tricalysia sphaerocarpa, the libriform type of fibres, with thick
lignified walls and fairly very narrow lumen are seen.
A glimpse at the literature reveals that there is very little information on
the anatomical features of Rubiaceae members. Virtually Tricalysia sphaerocarpa
the present test plant has remained unexplored. Therefore, the present study marks
the first comprehensive report on the anatomical features of leaves, stem and root
of the test plant. Along with the physico-chemical studies, it would pave the way
for its botanical identification and to distinguish from adulterants and
substitutions during herbal drug quality control.
80
5.2. Phytochemistry :
Physicochemical properties are important parameters in detecting
adulteration on improper handling of the drug. In the evaluation of crude drug,
ash value and extractive values are important parameters. The estimation of ash
value is useful for detecting low-grade products, exhausted drugs and the drug
with excess of sandy matter. The determination of extractive values with array of
solvent gives information about extractable polar and non polar as well as total
extractable plant constituents (Rajan et al., 2013). Physicochemical evaluation of
crude drug involves the determination of the identity, purity and quality. Purity
depends upon the absence of foreign matter, whether organic or inorganic. While
quality refers essentially to the concentration of the active constituents in the drug
that makes it valuable to medicine. The present study reveals that the moisture
content, total ash, acid insoluble ash and water soluble ash are high in stem when
compared to leaf, root and fruit.
5.2.1. Phytochemical Screening
The preliminary phytochemical colour reactions reveals the marked
presence of alkaloids, cardiac glycosides, flavonoids, glycosides, reducing sugar
and non-reducing polysaccharide(starch) in methanolic extract, water extract and
powder as such in leaf, stem, root and fruit. Proteins, phenolic group, steroids, and
saponins are very low or sparingly observed in leaf, stem, root and fruit.
Terpenoids are present in leaf, stem and root, but it is absent in fruit. Flavonoids
encompass a huge array of biologically active compounds that are omnipresent in
plants, many of which have been used in established eastern medicine for
81
thousands of years (Gurudev Singh Raina, 2013). Flavonoides are also shown to
inhibit microbes which are resistant to antibiotics (Linuma et al., 1994).
Phenols the aromatic compounds with hydroxyl group are widespread in
plant kingdom. They occur in all parts of the plants. Phenols are said to offer
resistance to diseases and pests in plants. Phenolic compounds could be a major
determinant of antioxidant potentials of food plants and could therefore be a
natural source of antioxidants and therefore phenolic compounds have been
associated with the health benefits derived from consuming high levels of fruits
and vegetables (Ka¨ hko¨ nen et al., 1999). Hence presence of phenolic
compounds in Tricalysia sphaerocarpa plays an important role in antioxidation.
Saponins are a special class of glycosides which have soapy characteristics
(Fluck, 1973), and also an active antifungal agents (Sadipo et al., 1991). Tannins
are water – soluble polyphenols that are present in many plant foods and
precipitate proteins. Tannins have been reported to prevent the development of
microorganisms (antimicrobial agents) by precipitating microbial protein and
making nutritional proteins unavailable for them (Sadipo et al., 1991). The growth
of many fungi, yeasts, bacteria and viruses is inhibited by tannins (Chung et al.,
1998). Tannins are reported to have various physiological effects like anti–irritant,
antisecretolytic, antiphlogistic, antimicrobial and antiparasitic effects. Tannins are
known to be useful in the treatment of inflamed or ulcerated tissues and they have
remarkable activity in cancer prevention (Ruch et al., 1989; Motar et al., 1985).
The phytochemical analysis conducted on Helichrysum longifolium extract
revealed the presence of tannins, flavonoids, steroids and saponins (Aiyegoro and
82
Okoh, 2010). Phytochemical screening of the plants (Carica papaya, Magnifera
indica, Psidium guajava, Vernonia amygdalina) revealed the presence of
flavonoids, terpenoids, saponins, tannins and reducing sugars (Ayoola et al.,
2008). The studies of Shakeri et al., (2012) on phytochemical screening revealed
the presence of alkaloid, flavonoid, saponin, terpenoid, steroid and sterols in the
extracts of aerial parts of Anabasis aphylla.
5.2.2. GC-MS Analysis
Methanolic extract of leaves, stem, root and fruit were subjected to GC-
MS analysis. This analysis were carried out to detect the possible compounds
present in the active fraction. Phytochemicals were extracted best in methanol
(Bhaigyabati et al., 2011).
Leaf
In the present study, from the methanol extract of leaf, totally 30 chemical
compounds were identified of which 9 belong to fatty acids, four to aliphatic and
aromatic bicyclics, two each to aromatic hydrocarbons groups, aromatic nitrile
groups, aromatic dicarboxylic esters groups. Of which one compound belonged to
each of the class terpenoids, barbiturates, aromatic alcohols group, aliphatic
aldehydes group, aromatic ketones group, aromatic ethers, phenolic group and to
pyrimidinedione group. Among this, eicosanoic acid is found to be present as
major constituent, followed by octadecanoic acid. Oleic acid is an unsaturated
fatty acid present in several plants and being unsaturated is considered as a
healthy source of fat in the diet. Many fatty acids are known to have antibacterial
and antifungal properties (Russel, 1991). Dodecanoic acid, tetradecanoic acid,
83
hexadecanoic acid, octadecanoic acid and oleic acids are among the fatty acids
known to have potential antibacterial and antifungal activity (McGraw et al.,
2002; Seidel and Taylor, 2004). Oleic acid has been found to be fungistatic
against a wide spectrum of moulds and yeasts. For example, it was observed to
cause a delay of 6-8 hour in the germination of fungal spores, and was also found
to be effective at concentrations as low as 0.7% v/v (Sheba et al., 1999). It has
also been disclosed that these fatty acids have potential antibacterial and
antifungal principle for clinical application (Altieri et al., 2008). Docosanoic acid
also called as Behenic acid is a normal carboxylic acid, which is a saturated fatty
acid. Commercially, behenic acid is often used to give hair conditioners and
moisturizers due to their smoothing properties. It is also used in lubricating oils,
as solvent evaporation retarder in paint removers. As Amide an anti-foam is used
in the manufacturing of detergents, floor polishes and dripless candles. Reduction
of behenic acid yields behenyl alcohol. Pracaxi oil from the seeds of Pentaclethre
macroloba is a natural product with one of the highest concentrations of behenic
acid, and is used in hair conditioners. Nonadecanoic acid found in ox fats and
vegetables oils is used by certain insects as a phermone. Nonadecanoic acid has
also been reported from the genus Streptomyces, along with its biological
functions as anti-tumor agent and inhibition of IL-12 production (Yoo et al.,
2002). Nonadecanoic acid has already been isolated from several sources,
including a fungus (Juzlova et al., 1996), marine sponge (Mishra et al., 1996), and
plant (Hogg and Gillan, 1984; Fukunaga et al., 1989), and exhibits inhibitory
effects on fibrinolysis and plasmin activity (Kawashiri et al., 1986).
84
Squalene, an isoprenoid compound structurally similar to beta-carotene, is
an intermediate metabolite in the synthesis of cholesterol. In humans, about 60
percent of dietary squalene is absorbed. It is transported in serum generally in
association with very low density lipoproteins and is distributed ubiquitously in
human tissues, with the greatest concentration in the skin, where it is one of the
major components of skin surface lipids. Squalene is not very susceptible to
peroxidation and appears to function in the skin as a quencher of singlet oxygen,
protecting human skin surface from lipid peroxidation due to exposure to UV and
other sources of ionizing radiation. Supplementation of squalene to mice has
resulted in marked increases in cellular and non-specific immune functions in a
dose-dependent manner. Squalene may also act as a "sink" for highly lipophilic
xenobiotics. Since it is a nonpolar substance, it has a higher affinity for un-ionized
drugs. In animals, supplementation of the diet with squalene can reduce
cholesterol and triglyceride levels. In humans, squalene might be a useful addition
to potentiate the effects of some cholesterol-lowering drugs. The primary
therapeutic use of squalene currently is as an adjunctive therapy in a variety of
cancers. Although epidemiological, experimental and animal evidence suggests
anti-cancer properties, to date no human trials have been conducted to verify the
role this nutrient might have in cancer therapy regimens. Phthalic acid, di(2-
propyl pentyl)ester and squalene was found in the wood extractives of Melaleuca
leucadendrda (Xu et al., 2013). Squalene has already been reported from the
acetone extract of the glandular hair of fruit of Mallotus philippensis (Velanganni,
2012), and also from the root of Bulbophyllum kaitense (Kalairasan et al., 2012).
85
n-hexadecanoic acid has anti inflammatory property. The rigorous use of
medicated oils rich in n-hexadecanoic acid for the treatment of rheumatic
symptoms in the traditional medical system of India, Ayurveda (Aparna et al.,
2012). n-hexadecanoic acid, oleic acid are the two major compounds present in
the oil of Chasmanthera dependens (Modupe Ogunlesi et al., 2010). 9,12,15-
octadecatrenoic acid, methyl ester, (Z,Z,Z-), oleic acid, 9,12-octadecadienoic
acid(Z,Z)- has been reported from the ethanolic extract of stem and root of the
plant Mallotus philippensis (Velanganni et al., 2011). 2,3,5,6,tetrafluoroanisole
was already reported from Dinochloa puberula by Py-GC/MS and it used as raw
material for bioenergy and rare biomedicines (Qiang et al., 2008). 2,4,(1H,3H)-
Pyrimidinedione is used as an antiviral agent, and it reduces cellular cytotoxicity
and inhibits HIV type 1 and HIV type 2 (Buckheit et al., 2007). Phthalic acid,
di(2-propyl pentyl)ester and oleic acid was identified from the chloroform extract
of marine Kocuria sp. SRS88 by GC/MS and the chloroform extract showed
antibacterial activity (Ranganathan Sahadevan et al., 2014). Ethyl benzonitrile
was found to be the major constituents of the methanolic extract of leaf of
Gaultheria fragrantissima (Padmavathy et al., 2014). The oil yielded from the
ethanolic extract of the seeds of Brachystegia eurycoma showed n-hexadecanoic
acid, octadecanoic acid, docosanoic acid, beter-sitosterol, eicosanoic acid and the
oil showed antibacterial activity (Okenwa Uchenna Igwe et al., 2013).
Cyclobarbital is used as an anesthetic, anticonvulsant, sedative, hypnotic,
veterinary euthanasia agent.
86
Stem
From the methanol extract of stem, totally 17 chemical compounds were
identified of which one belongs to aliphatic hydrocarbons groups, four to steroid
groups, five to fatty acid esters. Of which one compound belongs to the class
sugars, one to the class tocopherols, one to aromatic nitrile. Among this,
octadecanoic acid was found to be present as major constituent, followed by n-
hexadecanoic acid. n-hexadecanoic acid has also reported from the stem of
Bulbophyllum kaitense (Kalairasan and Ahmed John, 2011). n-hexadecanoic
acid, octadecanoic acid, 9,12,15- octadecatrenoic acid, methyl ester, (Z,Z,Z-),
oleic acid, 9,12-octadecadienoic acid(Z,Z)- has been reported the ethanolic extract
of stem and root of the plant Mallotus philippensis (Velanganni and Kadamban,
2011). Steroid are abundant in nature, many derivatives of steroid have
physiological activity (Vollhardt et al., 1994). Steroids are used in medicine in the
treatment of cancer, arthritis or allergies and in birth control (Okwu et al., 2010).
Stigmasterol isolated from plants were reported to be involved in the synthesis of
many hormones like progesterone, androgens, estrogens and corticoids with
several pharmacological prospects such as antiosteoarthritic,
antihypercholestrolemic, antitumor, hypoglycaemic, antimutagenic, antioxidant,
anti-inflammatory and CNS effects. Stigmasterol does seem to be play a role in
reducing inflammation, which may because it is a precursor to chemical
compounds which can limit inflammatory processes. Sterols like stigmasterol
have also been recommended for their cholesterol lowering abilities, although
more study is needed to determine which compounds perform this function, and
87
how they work in the body. It has been already reported form the pseudobulb of
Bulbophyllum kaitense (Kalairasan and Ahmed John, 2011). Sitosterol is already
recorded from the ethanolic extract of leaf of Mallotus philippensis (Velanganni
et al., 2011). τ-Tocopherol is the analog of tocopherol (vitamin E). In cosmetics
and personal care products, tocopherol and other ingredients made from
tocopherol, including tocopherol esters are used in the formulation of lipstick, eye
shadow, blushers, face powders and foundations, moisturizers, skin care products,
bath soaps and detergents, hair conditioners, and many other products. Similar
results were reported from the pseudobulb of Bulbophyllum kaitense (Kalairasan
and Ahmed John, 2011).
Root
Totally 8 compounds were identified from the methanol extract. Of which
five belongs to heterocyclics groups, one to aromatic ester groups, one to fatty
esters and one to triterpenoid group. Among this, benzo(b)thiophene, 4-methyl
was found to be present as major constituent, followed by 2-methyl-5-p-
dimethylaminophenyl oxadiazol. Oxadiazol is a compound showing strong
antifungal activity (Zhang et al., 2013). 9,19-Cyclolanostane derivates was also
isolated from the roots of Actaea pachypoda. Cyclolanostane Triterpene
diglycosides isolated from the aerial parts of Cimicifuga foetida shows
immunosuppressive effect (Pan et al., 2009). Cyclolanostane Triterpene was also
isolated from the ethanolic extract of the stems of Kadsura heteroclite (Wang et
al., 2006). Tryptophan is a compound which is useful for insomnia, depression
and anxiety. Its also lower blood pressure in Hypertension patients, stimulate the
88
production of antibodies, reduce inflammation. Tryptophan supplementation may
inhibit the development of full-blown AIDS in persons infected with HIV virus
(www.biogenesis-antiaging.com).
Fruit
Totally 10 compounds were identified of which three belong to
heterocyclic group, one to aliphatic aldehyde groups, one to thiosulphate group,
one to thiophosphates group, one to antibiotic and three to the other unclassified
groups. Among this, S,S-3,8-Diazaundecamethylene bis[hydrogen thiosulfate]
was found to be present as major constituent, followed by 5,8,15,18,23-pentaoxa-
1,12-diazabicyclo(10,8,5)-pentacosane. Deoxyspergualin (DOS), a substance
composed of a guanidinic and a spergmidine moiety, was originally described as
an antitumor agent(Takeuchiii et al., 1981). Deoxyspergualin (DSG) has been
found to have an antitumour and immunosuppressive activity. It also acts as an
antimalarial agent (Ramya et al., 2007). Methyldeoxyspergualin (MeDSG) in in
vitro culture studies of DSG shows good stability in aqueous solution and retains
strong immunosuppressive activity (Odaka et al., 1998). Methylpiperidin isolated
from plants shows antipsychotic therapeutic potential (Fuchigami et al., 2012).
Octadecanoic acid, n- hexadecanoic acid and 9,12,15- octadecatrienoic
acid (Z,Z,Z-) are the common compounds seen both in stem and leaf. Most of the
compounds obtained through GC-MS analysis from the methanolic extract of
leaf, stem, root and fruit of Tricalysia sphaerocarpa show antibacterial, antifungal
and antiviral properties. Some of them have antitumour, anti-inflammatory,
antiasthma, antiarthritic, diuretic, antipsychotic, anesthetic, anticonvulsant,
89
sedative, antiarthritic, antioxidant and anticancer properties and hence the plant
Tricalysia sphaerocarpa have high medicinal value.
5.3. Pharmacology
5.3.1. Antioxidant Activity
The antioxidant activity of various extacts (petroleum ether, chloroform,
methanol, water) of Tricalysia sphaerocarpa was determined by DPPH, FRAP,
Hydrogen peroxide and SOD Scavenging assay. The present study reveals, the
maximum activity in chloroform extract, followed by the methanolic extract using
DPPH, FRAP and Hydrogen peroxide assay, but in SOD assay, the maximum
activity was observed in methanolic extract. Using DPPH radical scavenging
method, methanolic extract of some medicinal plants like Camellia sinensis,
Eugenia caryophyllus, Piper cubeba, Zingiber officinale, Trigonella foenum-
graecum and Elettaria cardamonum was found to have significant antioxidant
activity (Khalae et al., 2008). Al-fartosy (2011) reported strong antioxidant
activity as well as strong reducing power (increase in the extract concentration
increases the activity) and ferrous ion chelating abilities from the methanolic
extract of Inula graveolensa. Higher antioxidant potential of the Samanea saman
extracts (petroleum ether, ethyl acetate, chloroform, aqueous and HCl extracts)
was observed in both DPPH scavenging assay and reducing power assay
(Arulpriya et al., 2010). Crataegus. monogyna flowers, leaves and fruits had H2O2
radical scavenging, total antioxidant activity (Keser et al., 2012). Antioxidant
potential of various extracts of Cassia fistula was determined by the DPPH,
FRAP, Fe3+ reducing power, and hydrogen peroxide scavenging assay.
90
Methanolic extracts of Cassia fistula showed the highest amount of reducing
capacity (Irshad et al., 2012). Riaz et al., (2012) reported highest total antioxidant
activity from the chloroform fraction of Dodonaea viscose. The methanolic
extract of Leucas plukenetti Whole plant plays an important role in the
modulation of oxidative stress (Subhangkar Nandy et al., 2012). The methanolic
leaf extract of Moringa peregrine exhibited the scavenging acativity on DPPH
assay (Dehshahri et al., 2012). The ethyl acetate fraction of Tagetes erecta
ethanol extract was found to be the most effective in DPPH assay (Miglena
Valyova et al., 2012).
5.3.2. Anti - Depressant Activity
The present findings obtained from FST, TST and HBT clearly reveal the
methanolic extract of Tricalysia sphaerocarpa elevate the suppressed mood in
animal models. The decrease in the immobility time was quite close to that of the
standard i.e. Imipramine. It clearly reveals that the animals treated with methanol
extract 200 mg/kg showed better response than those treated with standard drug.
Saroj Kothari et al., (2010) reported the methanolic extract of Aegle marmelos
leaf showed significant antidepressant and anxiolytic activities. All doses of the
aqueous extract of Melissa officinalis, produced a significant reduction in
immobility along with an increase in climbing behavior which is similar to those
which have been observed with imipramine (Emamghoreishi and Talebianpour,
2009). The methanol extract at the dose of 100mg/kg of the leaves of Citrus
paradise var. foster markedly increased the average time spent in the open arms in
EPM and methanol extract at the dose of 400mg/kg showed a significant decrease
91
in the time spent immobile by mice in FST (Vikas Gupta et al., 2009). The
methanolic extract of Foeniculum vulgare possesses significant antidepressant
activity due to its reduction in the immobility period (Jamwal Neetu Singh et al.,
2013). The ethanolic extract of Caryophyllus aromaticus proposed antidepressant-
like effect of higher dose concentration (200mg/kg) and significantly increased
swimming time and decreased immobility time (Sangavai et al., 2013). Caffeine,
as a psychomotor stimulant, suggest a possible positive effect on dopaminergic
activity of caffeine augmentation (10 mg/kg or lower dose) with antidepressant
agents for depression treatment (Pravin Popatrao Kale et al., 2010). Celastrus
paniculatus seed oil showed significant antidepressant-like activity (Valeca et al.,
2014).
5.3.3. Anti - Diabetic Activity
The present study revealed that all dosess of Tricalysia sphaerocarpa
methanolic stem extract in normal fasted rats, significantly(P<0.05) reduced the
blood glucose levels up to 6 hr. except the lowest dose. The maximum
hypoglycemic activity was induced by 500 mg/kg dose at 4 hr. by 18%. In
alloxan-induced diabetic rats, it significantly(P<0.01) reduced the blood glucose
levels up to 3 hour except the lowest dose. The maximum hypoglycemic activity
was induced by 500 mg/kg dose at 3 hour. The present study indicates that
alloxan induced tissue injury is reversed by continuous administration of T.
sphaerocarpa extract with subsequent decrease in blood sugar. Oral
administration of T. sphaerocarpa methanolic extract of 500 mg/kg showed
significant (P<0.01) plasma glucose lowering effect in 12 and 16 days of
92
treatment. Oral administration of the extract showed a significant (P<0.01)
decrease in body weight in all the tested concentrations, compared to control
groups. The group treated with 500 mg/kg showed the decrease the lysosome
enzyme levels, SGOT, SGPT, Alkaline phosphatise, and also in % of lipid
peroxidation. It also reduced the enzymatic antioxidants like CAT, GPx,
compared to diabetic controls. Microscopically examined pancreas section of
normal rat group, diabetic control group, Test extract (125, 250 and 500 mg/kg)
and the standard showed that normal architecture of pancreas with acini of serous
epithelial cells along with nest of endocrine cells separated by fibrocollaoenous,
stroma into lobules. No fibrosis or inflammation was found. Similar results
reported by previous workers are in conformitry into the present findings. The
antidiabetic potential of the methanolic extract of Operculina turpethum stem and
root was evaluated in the Streptozotocin- induced type 2 diabetic models
(Pulipaka et al., 2012). The antidiabetic effects of the methanol and acetone
extract of Acalypha indica Linn. was evaluated in normal and Alloxan induced
diabetic rats. Decreased blood glucose level of the test animals shows that the
extract exhibit significant antidiabetic activity when compared to diabetic control
group (Masih et al., 2011). The aqueous and methanolic extract of Gongronema
latifolium leaves showed antidiabetic activity in alloxan induced diabetic rats
(Akah et al., 2011). The methanolic and ethanolic extracts 200 mg/kg b.wt. of
seeds of Annona squamosa as significant hypoglycemic activity in both normal
and Alloxan induced diabetic rats (Ravinder Sangala et al., 2011). Aqueous and
cold extracts of Terminalia catappa exhibited significant anti-hyperglycemic
93
activities in alloxan-induced hyperglycemic rats without significant change in
body weight. They also improved conditions of DM as indicated by parameters
like bodyweight, and lipid profiles along with serum (Ahmed et al., 2005).
Manikandan et al., (2013) reported the methanolic extract of Psidium guajava
leaves, showed its in vitro anti-diabetic activity. The aqueous and methanolic
extracts of aerial parts, viz. leaves, stem and seeds of the plant, Cassia
occidentalis possessed anti-hyperglycemic/ anti-diabetic activity against alloxan
induced animal model (Arya et al., 2013). The methanol extract of Costus
pictus(120mg/kg.p.o.) showed significant (p<0.001) reductions of blood glucose
and serum enzymes (SGOT, SGPT, ALP) in alloxan induced diabetic rats
(Nandhakumar Jothivel et al., 2007).
Thus the present study will be useful in the following ways.
In proper botanical identification of the crude drug of the plant studied
through pharmacognostical investigation
To identify the chemicals responsible for the medicinal properties of the
plant through various phytochemical studies
Pharmacological investigations on antioxidant activity, antidepressant
activity and antidiabetic activity of Tricalysia sphaerocarpa will provide
the first scientific report in medicinal science
It will also provide clues for new drug discovery.
Figure 1: Extractive values of various parts of Tricalysia sphaerocarpa by Batch process.
Figure 2: Extractive values of various parts of Tricalysia sphaerocarpa by Successive process
0
5
10
15
20
25
Leaf Stem Root Fruit
Values in %
Chloroform Diethyl ether Ethyl acetate Methanol
0
5
10
15
20
25
30
35
Leaf Stem Root Fruit
Values in %Acetone Benzene Chloroform Diethyl etherEthanol n-butyl alcohol Methanol Water
Figure 3: GC-MS chromatogram of Methanolic Leaf Extract of Tricalysia sphaerocarpa
Figure 4: GC-MS chromatogram of Methanolic Stem Extract of Tricalysia sphaerocarpa
Figure 5: GC-MS chromatogram of Methanolic Root Extract of Tricalysia sphaerocarpa
Figure 6: GC-MS chromatogram of Methanolic Fruit Extract of Tricalysia sphaerocarpa
Figure7(a): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa
2,5-Dimethylbenzonitrile 2-Ethylbenzonitrile 2,3,5,6-Tetrafluoro-4-Methylanisole
n-Hexadecanoic acid Tetradecanoic acid
Eicosanoic acid Oleic acid 9,12-Octadecadienoic acid(Z,Z-)
Docosanoic acid Bis(2-Ethylhexyl)Phthalate 3-O-Methyl-D-Glucose
Figure7(b): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa
Eicosanoic acid,methyl ester Octadecanoic acid
Ergost-5-en-3-ol Stigmasterol Cyclobarbital
1,2-benzenedicarboxylic acid bis(2-methylpropyl) ester
9,12,15-octadecatrienoic acid, (zzz)- 9,12-Octadecadienoic acid, methyl ester,(E,E)-
Figure7(c): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa
Hexadecanoic acid, methyl ester octadecanoic acid, methyl ester
9Z-octadeca-9,17-dienal 1H-Benzimidazole, 5,6-dimethyl-
1H-Indene-2-ethanol,2,3-dihydro- Oxitriptan
Benzoic acid,4-(3-hydroxy-3-methyl-1-butynyl)- methyl ester
Figure7(d): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa
2-[3-cyclohexylaminopropylamino]ethyl thiophosphate EPPS
S,S1-3,8-diazaundecamethylene bis[hydrogenthiosulfate]
2-methyl-5-p-dimethylaminophenyl oxadiazol Benzo(b)thiophene,4-methyl
Hexadecanoic acid,1a,2,5,5,5a,6,9,10,10a,octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-dioxo-1H-2,8a-methanocyclopenta(a)cyclopropa(e)cyclodecen-5-yl ester
Figure 7(e): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa
4,13,20-tri-O-methylphorbol 12-acetate 2-myristynoyl pantetheine
5,8,15,18,23-pentaoxa-1,12-diazabicyclo dl-5-hydroxytryptophan (10,8,5)-pentacosane
3-chloro-2,4-dimethyl-12-thia-1,5,6a,11,tetraaza- indeno[2,1-a]fluorine
4-octadecenal
Figure7(f): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa
N-[4-(4-chlorophenyl)isothiazol-5yl] Deoxyspergualin -1-methylpiperidin-2-imine
Squalene
Phthalic acid, di(2-propylpentyl) ester 9,17-Octadecadienal, (Z)-
Nonadecanoic acid
Figure 8: Anti oxidant activity -DPPH Scavenging assay:
0102030405060708090
100
10µg/ml 50µg/ml 100µg/ml
% of radicle scavenging
Petroleum ether Chloroform Methanol Water BHT
Figure 9: Anti oxidant activity - Iron chelating activity (FRAP)
0
20
40
60
80
100
120
10µg/ml 50µg/ml 100µg/ml
% of radicle scavenging
Petroleum ether Chloroform Methanol Water EDTA
Figure 10: Anti oxidant activity - Hydrogen peroxide assay
00.20.40.60.8
11.21.41.6
10µg/ml 50µg/ml 100µg/ml
% of radicle scavenging
Petroleum ether Chloroform Methanol Water Ascorbic acid
Figure 11: Anti oxidant activity - Superoxide dismutase (L-methionine and NBT assay)
02468
1012
10µg/ml 50µg/ml 100µg/ml
% of radicle scavenging
Petroleum ether Chloroform Methanol Water Ascorbic acid
94
CHAPTER 6
SUMMARY AND CONCLUSIONS
Chemical investigations of wild medicinal plants used by the indigenous
people of world show unknown compounds with promising biological activities.
Phytochemical analysis of plants, used in folklore has yielded a number of
compounds with various pharmacological activities. Hence medicinal plants are
important substances for the study of their traditional uses through the
verification of pharmacological effects and can be natural composite sources that
can act as disease curing agents. The integrated research in drug discovery have
attracted and provided multidisciplinary research platforms. The present work has
been concentrated to bring out the medicinal potential of Tricalysia
sphaerocarpa.
The important conclusion derived from this study is summarized in three
aspects viz, 1. Pharmacognostical studies for easy identification of plant species,
2. Phytochemical analysis to identify the chemicals responsible for the medicinal
properties of the plant, 3. To understand the Pharmacological properties of the
chemicals through in vivo and in vitro studies i.e., the antioxidant, antidepressant
activity and antidiabetic activity.
Tricalysia sphaerocarpa belongs to the coffee family (Rubiaceae), which
is commonly called as wild coffee, locally called as irrukulimaram in tamil, vella
by Srilankans and kadukafibija in kanada. The roasted seeds of Tricalysia
sphaerocarpa are used as a coffee substitute. Along with Tinospora giloy,
Argemone satyanashi, Tricalysia sphaerocarpa is traditionally used for sleep.
95
Pharmacognostical parameters like leaf constants, microscopy, physico-
chemical analysis, fluorescence analysis are a few of the basic protocol for
standardization of crude drugs. Hence, in the present work the pharmacognostical
standardization has been performed.
Microscopical analysis of cleared leaf showed the characteristic
polyhedral vein islets and vein terminations. The examination of the macerated
materials showed the characterstic fibres, vessel elements and broken parenchyma
cells. The epidermal studies revealed that the number of epidermal cells/mm2
,type of trichome, occurrence of stomata on the lower surface only, the number of
stomata /mm2 , type of stomata, the stomatal index are characteristic to this plant.
The anatomical studies revealed the presence of hemispherical shaped
epidermal cells with very thick arches of cuticle in leaf and stem that extends to
the radial walls also. Presence of tannineferous idioblast, discrete vessel elements
with simple perforation and tails at one or both ends are the salient features
present in Tricalysia sphaerocarpa. All the characters are typical to this plant
which would be very useful in correct botanical identity of crude samples.
The histochemical localization tests revealed the presence of starch,
alkaloid and protein in all the plant parts studied, tannin in leaf and stem, lignin
only in root and the absence of mucilage in all the parts.
In fluorescence analysis, leaf powder is mostly green to dark green in
daylight whereas it is orange in UV light. In stem, it is mostly yellow in both day
light and UV light. In root, it is mostly yellow in day light and pale yellow to dark
96
yellow in UV light . In fruit, it is mostly light brown in day light and creamy
white to brown in UV light.
Of the physico-chemical parameters studied the moisture content (20%),
total ash (5.16%), acid insoluble ash (1.06%) and water soluble ash (3.90%) were
high in stem when compared to all other parts of the plant. In both the batch
process and successive process the highest extractive values were recorded in
methanol extract of leaf, stem, root and fruit, when compared to other solvents.
The preliminary phytochemical screening of leaves, stem, root and fruit
revealed the presence of phytoconstituents like alkaloids, carbohydrates,
flavonoids, glycosides, proteins, phenolic group and saponins in methanolic
extract, water extract and powder as such.
By GC-MS analysis, totally 65 chemical compounds (17 chemical
compounds from stem, 30 from leaf, 8 from root and 10 compounds from fruit)
were identified from the methanolic extract. Octadecanoic acid, n- hexadecanoic
acid and 9,12,15- octadecatrienoic acid (Z,Z,Z-) are the three common
compounds seen both in stem and leaf.
Antioxidant activity was significantly higher in the chloroform extract,
followed by the methanolic extract of stem. By DPPH scavenging method, the
percentage of scavenging activity was found to be more in chloroform extract,
followed by methanol, petroleum ether and water extracts. All the extracts
showed dose concentration dependent activity in all the tested concentration. By
FRAP assay, the maximum value was observed in chloroform extract, followed
by methanol, water and petroleum ether extracts. By Hydrogen peroxide assay,
97
Chloroform extract showed the maximum value, followed by methanol,
petroleum ether and water extracts. By superoxide dismutase assay, the maximum
value was observed in methanol extract, followed by chloroform, water and the
minimum value was observed in petroleum ether extract. This activity may be due
to the presence of secondary metabolities such as flavonoids, tannins, and
steroids, all of which are known antioxidants.
In acute toxicity, no mortality was observed in the animals treated with the
dose of 2000 mg/kg methanol extract of stem. There were no signs of any
toxicity. The studies on anti-depressant activity clearly revealed that the animals
treated with methanol extract at 200 mg/kg had better response than those treated
with standard drug (Imipramine). In anti-diabetic activity, the methanolic extract
of stem, significantly reduced the blood glucose level in both normal fasted rats
and alloxan induced diabetic rats. It also reduces the body weight, and decrease
the enzymatic levels like SGOT, SGPT, Alkaline phosphatise, CAT and GPX. It
also reduced the lipid peroxidation level in all the tested concentrations.
Microscopically examined pancreas section of normal rat group, diabetic control
group, test extract (125, 250 and 500 mg/kg) and the standard showed the normal
architecture of pancreas with acini of serous epithelial cells along with nest of
endocrine cells separated by fibrocollaoenous, stroma into lobules. No fibrosis or
inflammation was found. The various extracts (petroleum ether, chloroform and
methanol) of stem possess significant antioxidant activity and the methanolic stem
extract possesses significant anti-depressant and anti-diabetic activities.
98
Thus the study concludes that the plant may be used as a potential drug for
antioxidation, depression and diabetes. The results of pharmaconostical,
phytochemical and pharmacological studies which are reported for the first time
from Tricalysia sphaerocarpa, may pave way for new drugs discovery.
Plate 1
Morphology of Tricalysia sphaerocarpa
Habit Basal portion
Trunk A twig with fruits
T.S and L.S of fruit showing flat seeds
Plate 2
Leaves of Tricalysia sphaerocarpa
Dorsal surface Ventral surface
T.S. of Lamina
Midrib Leaf blade
Cu: Cuticle; Ep: Epidermis; X:Xylem; Ph:Phloem; GT:Ground tissue; Pc: Palisade cells; Sc:Spongy cells
Leaf epidermal peel of Tricalysia sphaerocarpa
Adaxial Epidermis without Stomata Abaxial Epidermis with Stomata
ICR: Inter Costal Region; CR: Costal Region
--------EEpp
----------GGTT
----XX ----------PPhh
--------CCuu --------CCuu --------PPcc
--------CCRR
--------SScc
----IICCRR
----------XX
Plate 3
Size and Orientation of Stomata in Tricalysia sphaerocarpa
Various sizes of Stomata
Stomatal opening Stomata enlarged
Degenerated Stomata Unicellular Trichome GS: Giant Stomata; MS: Medium sized Stomata; SS: Small Stomata; BS: Blind Stomata; DS: Degenerated Stomata; HS: Half Stomata.
----MMSS
------BBSS
--------GGSS
----SSSS
------GGSS
------MMSS
------SSSS ------HHSS
--------DDSS
------DDSS
Plate 4
Vein islet and Veinlet termination of Tricalysia sphaerocarpa
Stem epidermal peel of Tricalysia sphaerocarpa
VI: Vein islet; VLT: Veinlet termination; S:Stomata.
--------VVLLTT
--------SS
--------VVII
Plate 5
T.S. of Stem of Tricalysia sphaerocarpa
Portion enlarged Outer portion
Epidermis enlarged Xylem showing vessels
Pith portion enlarged Sc: Sclerenchyma cells; Ve: Vessel element; TI: Tannineferous Idioblast.
--------SScc
--------VVee
--------VVee
--------TTII
--------CCuu
Plate 6
T.S. of Root of Tricalysia sphaerocarpa
Entire view portion enlarged
Xylem showing vessels SG: Starch grains; Ve:Vessel.
--------SSGG
--------VVee
Plate 7
Macerated elements of stem of Tricalysia sphaerocarpa
Vessel elements Single tailed vessel member Single fibre
Vessel member with pits Double tailed vessel member
Parenchyma cells Trachea P: Pits; PP: Perforation plate; Ta: Tail.
--------PP
----PPPP
--------TTaa
Plate 8
Effect of Methanolic Stem extract of Tricalysia sphaerocarpa
Anti - Depressant Activity
Forced swimming test (FST)
Tail suspension test (TST)
Hole Board Test (HBT)
Plate 9
Effect of Methanolic Stem extract of Tricalysia sphaerocarpa
Anti –Diabetic Activity
Histopathological Studies
Normal Control Diabetic Control Test extract (125)
Test extract (250) Test extract (500) Glibenclamide (5mg)
99
CHAPTER 7
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hope. WHO, Geneva. Xiao CH, Lu YR. 1987. Chinese medicinal chemistry. Shanghai Science
Technology Press. Shangai. 1-7. Xu K, Li K, Yun H, Zhong T and Cao X. 2013. A comparative study on the
inhibitory ability of various wood-based composites against harmful biological species. BioResources. 8(4): 5749-5760.
Xu W, Jacob MR, Agarwal AK, Clark AM, Liang ZS and Li XC. 2010. ent-
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Yoo JC, Han JM, Nam SK, Ko OH, Choi CH, Kee KH, Sohng JK, Jo JS, and
Seong CN. 2002. Characterization and Cytotoxic activities of Nonadecanoic acid produced by Streptomyces scabiei subsp. Chosunensis MO137 (KCTC 9927). The Journal of Microbiology. 40(4): 331-334.
Zhang MZ, Mulholland N, Beattie D, Irwin D, Gu YC, Chen Q, Yang GF and
Clough J. 2013. Synthesis and antifungal activity of 3-(1,3,4-Oxadiazol-5-yl)-indoles and 30(1,3,4-Oxadiazol-5-yl)methyl indoles. Eur. J. Med. Chem. 63: 22-32.
Zheng W, Wang SY. 2001. Antioxidant activity and phenolic compounds in
selected herbs. J Agric Food Chem . 49(11): 5165-5170. Zwenger S and Basu C. 2008. Plant terpenoids: applications and future potentials.
Biotechnol. Mol. Biol. Rev. 3(1): 001-007.
Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.
~ 47 ~
e - ISSN - 2249-7722
Print ISSN - 2249-7730
International Journal of Phytotherapy
www.phytotherapyjournal.com
PHARMACOGNOSTICAL AND PRELIMINARY
PHYTOCHEMICAL STUDIES ON TRICALYSIA SPHAEROCARPA
(DALZELL) GAMBLE
G. Anandhi* and A. Pragasam
Department of Plant Science, Kanchi Mamunivar centre for post graduate studies, Lawspet, Puducherry-605008.
INTRODUCTION
Human beings come on this earth as guests of
plants is a monumental ancient aphorism. Since time
immemorial, nature is own supreme creation, man has
completely been dependent on plants and as citizen
developed, he has learnt to emplit natural resources and to
make use of every bit of it. Infact from the start of life to
the last breath, almost every aspect of human life is
deeply associated with plants. Primitive man tried to cure
diseases from plants growing abundantly around him. His
experience through trial and taught him a lot about the
medicinal properties of different plants.
Survey on medicinal plants used by ethnic
people of north western Tanzania revealed the use of
Tricalysia sphaerocarpa (Dalzell)Gamble for various
ailments. Tricalysia pallensa root decoction is drunk
against malaria. Tricalysia coricea sbsp. Nyassaea root
decoction mixed with leaf juice is drunk, and the body
bathed with a root decoction. Tricalysia coriacea is also
used for skin diseases, epixstasis and malaria/ yellow
fever (jaundice). To the best of our knowledge, nobody
has investigated form the angle of anatomy,
histochemistry, phytochemistry, the present study was
taken up.
MATERIALS AND METHODS
Anatomical works were carried out in leaf, stem
and root by preparing the peelings and transverse
sections. Mature leaves were cleared by using 5% NaOH
and chloral hydrate solution, washed in water, stained and
mounted in 50% glycerine. Maceration was carried out
with stem and root materials following Jeffrey’s method
[1]. Histochemical color reactions were done by treating
free hand sections with different reagents. Phytochemical
tests were done with dried powdered drugs as well as
different solvent extracts [2].
RESULTS
Morphological features-A small tree with smooth leaves
and very small flowers. Leaves dark green,
Corresponding Author:-G. Anandhi Email: [email protected]
ABSTRACT
The present work has been taken up to study the crude drug of Tricalysia sphaerocarpa (Dalzell)Gamble of
the family Rubiaceae. The morphological characters of the plant; the anatomical characters of the leaf, stem, and root,
microscopic observations of the crude drug; qualitative analysis of primary and secondary metabolites such as
carbohydrates, alkaloids, tannins etc., of the powder as well as different solvent extracts of leaf and ash values were
studied. Qualitative phytochemical observations revealed the presence of many primary and secondary metabolites.
The values calculates/ data collected could be used for the identification and standardization of the powdered drug of
this taxon.
Key words: Tricalysia sphaerocarpa, Pharmacognosy, Phytochemistry.
Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.
~ 48 ~
Flowers minute, white, scented, Fruits greenish yellow,
berry globose, the seeds flat, smooth, leaves elliptic or
lancaolate, obtusely acute, smooth, the main nerves about
6-8 pairs, not prominent, nor the reticulation.
Anatomical features:-
Leaf peelings:
Adaxial : Cells small in size, cell wall undulate, thin, no
stomata.
Abaxial: Intercostal region: cells irregular, wall undulate,
cell medium sized, stomata rubiaceous type, different in
size.
Costal region: Cells small when compared to the
intercostal region, cell wall undulate, giant size stomata
are seen, unicellular hairs present. Epidermal cell number,
stomatal number, stomatal index, palisade ratio, vein islet
number and veinlet termination number have been
calculated and presented in the Table 1.
Stem peeling: Cells fairly large, cell wall thick, straight,
basal cells very broad, tip cells tapering, stomata frequent,
rubiaceous type.
Venation pattern: Veins reticulate, showing lateral
branches; cells elongated with thin walls; vein-islet fairly
large, each islet containing 3-4 termination points. Vein-
islet number and veinlet termination number are presented
in Table 1.
Transverse section: Leaf: The midrib portion of the leaf
is not much differentiated form the lamina. It is slightly
thicker than the lamina. The xylem is omega shaped with
10-15 rays of xylem cells. Phloem is seen on the abaxial
surface. Both the epidermis is uniseriate, with thick
cuticle. 2 layers of palisade parenchyma are seen below
the upper epidermis. Stomata are seen only on the abaxial
surface.
Stem: The stem in its outline is mostly dumble shaped.
Epidermis is unilayered with high cuticle. Cortex is
parenchymatous with 4-6 layered. Even the very younsg
stem undergoes secondary thickening. The secondary
xylem and phloem are continous. A single layer of
scleroids are seen as an outer ring. The pith is very broad
and formed of circular parenchymatous cells.
Root: The root in its outline is circular and formed of 8-
10 layers of parenchyma cells. The rhizodermis peeled
off. Secondary xylem and phloem are present. There is no
cortex. Rays are clearly seen. Xylem seen in the centre
and the phloem towards the periphery.
Phytochemical studies - The preliminary phytochemical
studies in methanol, aqueous and powder drug revealed
the marked presence of carbohydrate, glycosides,
alkaloids, tannin, flavanoids, moderate presence of
protein, phenol, terpenoids and saponin and absence of
triterpenoids, anthraquiones, catachins, coumarins (Table
4).
Histochemical colour reactions - Presence of starch,
protein and tannin and absence of lignin and mucilage
(Table 3). The total ash, acid insoluble ash and water
soluble ash were 4 percent, 0.84 percent and 3.24 percent
respectively (Table 2). Fluorescence analysis shows
mostly dark green in day light whereas orange in UV light
(Table 5).
Table 1. Quantitative values of foliar epidermis
Quantitative Values Abaxial Epidermis Adaxial Epidermis
Epidermal cell/mm² 1122.8/sq.mm 1414/sq.mm
Stomata/mm² 336/sq.mm -
Stomatal index 26.9 -
Palisade ratio 6.2 -
Vein islet number 93.8 -
Veinlet termination number 57.4 -
Table 2. Analytical ash values of leaf of T. sphaerocarpa
Parameter Results (%)
Total ash 4
Acid insoluble ash 0.84
Water soluble ash 3.24
Table 3. Histochemical colour reactions of leaf of T. sphaerocarpa
Test for Status of the substance
Starch +
Proteins +
Tannin +
Lignin -
Mucilage -
(+) Indicates presence; (-) Indicates absence.
Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.
~ 49 ~
Table 4. Phytochemical colour reactions of leaf of T. sphaerocarpa.
Phytochemicals Chloroform
extract
Diethylether
extract
Ethylacetate
extract
Methanol
extract
Aqueous
extract
Powder as
such
Alkaloids ++ ++ - ++ ++ ++
Anthraquinones - - - - - -
Carbohydrates ++ - ++ ++ ++ ++
Catechins - - - - - -
Coumarins - - - - - -
Flavonoids ++ - - ++ ++ ++
Gums, oils and resins - - - - - -
Glycosides ++ - ++ ++ ++ ++
Proteins + - - ++ ++ +
Phenolic group + - - + + +
Saponins + - - + + +
Tannins + - - + + +
Terpenoids + - - + + +
Triterpenoids - - - - - -
(++) Marked presence; (+) Moderate presence, (-) Absence
Table 5. Fluorescence analysis of leaf of T. sphaerocarpa.
Chemicals Leaf
Day light UVlight
Powder as such Green Yellow
Solvent
Acetone Dark green Orange
Benzene Dark green Orange
Chloroform Dark green Orange
Ethanol Green Orange
n-butyl alcohol Yellowish green Orange
Water Green Dark brown green
Reagents
10% FerricChloride Reddish brown Black
50% Sulphuric Acid Reddish brown Dark brown
1 NHCl Green Green
5% Ammonia Green Green
1% Thionyl Chloride Green Dark green
DISCUSSION AND CONCLUSION
The qualitative microscopic characters are useful
in the identification of the crude drug sample [3]. This
features are believed to be constant for a given species
[4]. Hence, the need for evolving criteria for standard
samples of crude drugs has become very important in
pharmacognosy. Methanolic extract, aqueous extract and
crude drug powder shows the similar results in the
phytochemical analysis. The plant T. sphaerocarpa was
subjected to pharmacognosticalstudies to identify the
plant materials and to differentiate them from the spurious
crude drugs. In light of the above, a combination of
characters such as epidermal cell number, stomatal
number, stomatal index, palisade ratio, venation pattern,
vein-islet numbers, vein-let termination number are found
to be very significant micro-morphological characters in
the identification of crude drug of Tricalysia
sphaerocarpa could be successfully used for the
identification of the powdered drug of this taxon.
REFERENCES
1. Johensen DA. Plant Microtechnique. Mc Graw Hill Book Co. inc, New York, 1940.
2. Khandelwal KR, Pawar AP, Kokate CK, Gokhale SB. Practical pharmacognosy techniques and experiments, IIIed .
Nirali Prakashan. 1996, 140-141.
3. Trease GE and Evans WC. Pharmacognosy, XIII ed. WB Saunders Ltd UK. 1996, 516-547.
4. Suseela A and Pream S. Pharmacognostic studies on Lagascea mollis. J. Phytol. Res, 20(1), 2007, 95-102.
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
65
PHYTOCHEMICAL SCREENING OF THE METHANOLIC
EXTRACT OF STEM OF TRICALYSIA SPHAEROCARPA (DALZELL
EX HOOK. F,) GAMBLE
G. Anandhi and Dr. A. Pragasam
Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Lawspet, Puducherry, India.
INTRODUCTION
Primitive man tried to cure diseases from plants
growing abundantly around him. His experience through
trial and taught him a lot about the medicinal properties of
different plants. The active secondary metabolites possess
various medicinal applications as drugs or as model
compounds for drug synthesis. Phytochemical analysis of
plants, used in folklore has yielded a number of
compounds with various pharmacological activities.
Hence medicinal plants are important substances for the
study of their traditional uses through the verification of
pharmacological effects and can be natural composite
sources that act a disease curing agents. Review of
literature revealed no work as been done form the angle of
histochemical analysis, preliminary phytochemical
screening and identification of phytocomponents by Gas
Chromatography-Mass Spectrometry (GC-MS) analysis
on this plant. Hence it was decided to do so.
MATERIALS AND METHODS
Collection of Plant material
The plant of Tricalysia sphaerocapa was
collected from the sacred grove of Thirumanikuzhi, of
Cuddalore district, Tamil Nadu. The collected plant
material was botanically identified. The species
conformation was engaged at French Institute Herbarium
(HIFP), Puducherry. The herbarium specimen was
prepared and deposited at the Department of Botany,
Kanchi Mamunivar Centre for Post Graduate Studies,
Lawspet, Puducherry, for future reference.
Preparation of the Extracts
The collected materials (stem) were chopped into
small pieces separately, shade-dried, and coarsely
powdered using a pulverizer. The coarse powder was
subjected to successive extraction with chloroform,
diethyl ether, ethyl acetate and methanol by Soxhlet
method. The extracts were collected and distilled off on a
water bath at atmospheric pressure and the last trace of
Corresponding Author:-G. Anandhi Email: [email protected]
International Journal of Pharmacotherapy
www.ijopjournal.com
ISSN 2249 - 7765
Print ISSN 2249 - 7773
ABSTRACT
Tricalysia is a genus of the plant family Rubiaceae. Approximately 50 species distributed in subtropical and
tropical regions in Asia and Africa. Some of these used as folk fore medicine as sedative, emetic, malaria/yellow fever,
skin diseases and also for urine disorders. Tricalysia sphaerocarpa is commonly known as wild coffee and its synonym
was Discospermum sphaerocarpum Dalzell ex Hook. F. Powdered materials were subjected to successive extraction with
chloroform, diethyl ether, ethyl acetate and methanol by soxhlet method for preliminary phytochemical screening and
methanol extract is used for GC-MS analysis to investigate the chemical components present in it. Totally 17 chemical
compounds were identified, among which 5 belongs to fatty acid esters groups, 4 belongs to steroid groups. Of which
octadecanoic acid (29.88%), n-hexadecanoic acid (15.10 %), 9, 12, 15-octadecatrienoic acid,(z,z,z)-(12.32 %) were the
major constituent identified.
Key words : GC-MS analysis, Tricalysia sphaerocarpa, Discospermum sphaerocarpum, Methanol extract.
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
66
the solvents was removed in vacuum and stored at 4ºC.
The resulted extracts were subjected to preliminary
phytochemical screening and GC-MS analysis.
Histochemical color reactions were done by treating free
hand sections of stem with different reagents.
Phytochemical tests were done with dried powdered drugs
as well as different solvent extracts [1].
Gas Chromatography-Mass Spectrometry (GC-MS)
analysis
GC-MS analysis was performed with GC Clarus
500 Perkin Elmer equipment. Compounds were separated
on Elite-1 capillary column (100%
Dimethylpolysiloxane). Oven temperature was
programmed as follows: isothermal temperature at 50ºC
for 2min, then increased to 200ºC at the rate of 10ºC/min,
then increased up to 280ºC at the rate of 5ºC/min held for
9 min. Ionization of the sample components was
performed in the El mode (70 eV). The carrier gas was
helium (1ml/min) and the sample injected was 2μl. The
detector was Mass detector turbo mass gold-Perkin Elmer.
The total running time for GC was 36 min and software
used was Turbomass 5.2. Using computer searches on a
NIST Ver.2.1 MS data library and comparing the
spectrum obtained through GC –MS compounds present
in the plants sample were identified.
Identification of Compounds
The individual compounds were identified from
methanol extract based on direct comparison of the
retention times and their mass spectra with the spectra of
known compounds stored in the spectral database, NIST
(version year 2005).
RESULTS
Phytochemical screening
The preliminary phytochemical study in
methanol, aqueous and powder drug shows the similar
results. It revealed the presence of carbohydrate,
glycosides, alkaloids, protein, phenolic group, steroid,
saponins, flavanoids and terpenoids in methanol, aqueous
and the powder drug and absence of triterpenoids,
anthraquiones, catachins, coumarins and tannins. In
diethyl ether extract, only the carbohydrate is present and
others are absent. In chloroform extract, alkaloids,
carbohydrates, flavonoids, glycosides, protein, phenolic
group, saponins, terpenoids are present. In ethyl acetate
extract, only carbohydrates, glycosides and steroids are
present(Table 1).
Table 1. Phytochemical colour reactions of stem of T. sphaerocarpa.
Phytochemicals Chloroform
extract
Diethyl ether
extract
Ethyl acetate
extract
Methanol
extract
Aqueous
extract
Powder as
such
Alkaloids ++ ++ - ++ ++ ++
Anthraquinones - - - - - -
Carbohydrates ++ - ++ ++ ++ ++
Catechins - - - - - -
Coumarins - - - - - -
Flavonoids ++ - - ++ ++ ++
Gums, oils and resins - - - - - -
Glycosides ++ - ++ ++ ++ ++
Proteins + - - ++ ++ +
Phenolic group + - - + + +
Saponins + - - + ++ ++
Steroids - - ++ ++ + +
Tannins - - - - - -
Terpenoids + - - + + +
Triterpenoids - - - - - -
Table 2. Histochemical colour reactions of stem of T. sphaerocarpa.
Test for Chemicals/reagents used Status of the substance
Starch Iodine solution +
Proteins Aqueous picric acid solution +
Tannin Dilute ferric chloride -
Lignin 1% potassium permanganate, 2% HCl, dil. Ammonia -
Mucilage Methylene blue reagent -
(‘+’’+’) presence, ‘-‘ absence)
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
67
Table 3. Analytical ash values of stem of T. sphaerocarpa.
Parameter Results %
Total ash 5.16
Acid insoluble ash 1.06
Water soluble ash 3.90
Table 4. Fluorescence analysis of stem of T. sphaerocarpa.
Chemicals Stem
Day light UVlight
Powder as such Creamy white Pale yellow
Solvent
Acetone Yellow orange
Benzene Pale yellow Creamy white
Chloroform Dark yellow Pale yellow
Ethanol Yellow Dark yellow
n-butyl alcohol yellow Creamy white
Water Creamy white Pale yellow
Reagents
10% Ferric Chloride Orange Black
50% Sulphuric Acid Reddish brown Black
1 NHCl Creamy white Yellow
5% Ammonia Pale yellow Yellow
1% Thionyl Chloride Light yellow Brown
Table 5. GC-MS Analysis of methanol extract of stem of T. sphaerocarpa
No. Name of the compound Molecular
formula
Molecular
weight
RT Peak
area %
Sugars
1 3-O-Methyl-d-glucose C7H14O6 194 11.06 6.18
Aromatic acids & esters
2 1,2-Benzenedicarboxylic acid, bis(2-methylproplyl) ester C16H22O4 278 11.59 1.66
Steroids
3 Androstane-3,16-diol,(3β,5α,16α)- C19H32O2 292 21.65 1.01
4 Ergost-5-en-3-ol,(3β)- C28H48O 400 29.71 4.40
5 Stigmasterol C29H48O 412 30.17 1.45
6 τ-Sitosterol C29H50O 414 31.31 9.81
Fatty acid esters
7 Hexadecanoic acid, methyl ester C17H34O2 270 12.21 0.90
8 9,12-Octadecadienoic acid, methyl ester, (E,E)- C19H34O2 294 14.23 1.89
9 6,9,12-Octadecatrienoic acid, methyl ester C19H32O2 292 14.31 1.03
10 Octadecanoic acid, methyl ester C19H38O2 298 14.64 1.57
11 Eicosanoic acid, methyl ester C21H42O2 326 17.34 1.42
Fatty acids
12 9,12,15-Octadecatrienic acid, (Z,Z,Z)- C18H30O2 278 15.01 12.32
13 Octadecanoic acid C18H36O2 284 15.33 29.88
14 n-Hexadecanoic acid C16H32O2 256 12.80 15.10
Aromatic nitrile
15 4-Hydroxy-3-methyl-beta-phenylcinnamonitrile C16H13NO 235 22.07 1.94
Aliphatic hydrocarbons
16 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-
hexamethyl-, (all-E)-
C30H50 410 24.02 5.99
Tocopherols
17 τ-Tocopherol C28H48O2 416 28.26 3.45
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
68
Fig 1. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the methanolic extract of stem of T.
sphaerocarpa
Fig 2. Structure of some phytocomponents
N-Hexadecanoic Acid
3-O-Methyl-D-Glucose
Eicosanoic Acid, Methyl Ester
Ergot-5-En-3-Ol
Stigmasterol
Sitosterol
Histochemical colour reactions
It reveals the presence of starch, protein and
absence of tannin, lignin and mucilage (Table 2). The
total ash, acid insoluble ash and water soluble ash were
5.16 percent, 1.06 percent and 3.90 percent respectively
(Table 3). Fluorescence analysis shows mostly yellow to
white in day light whereas black in UV light (Table 4).
Gas Chromatography-Mass Spectrometry (GC-MS)
analysis
Totally 17 chemical compounds were identified
of which 1 belongs to aliphatic hydrocarbons groups, 4
belongs to steroid groups, 5 belongs to fatty acid esters.Of
which1 compound belonged to the class sugars,
1compound belongs to the class tocopherol, 1 belongs to
aromatic nitrile.Among this, octadecanoic acidwas found
to be present as major constituent with the peak area
29.88% and retention time 15.33 minutes, followed by n-
hexadecanoic acid with the peak area 15.10 % and
retention time 12.80 minutes, and followed by 9,12,15-
octadecatrienoic acid,(z,z,z)- with the peak area 12.32 %
and retention time 15.01 minutes. Hexadecanoic acid,
methylester was found to be as least quantity with the
peakarea 0.90 % and retention time 12.21 minutes
respectively (Table 5; Fig.1). Some of the important
structures of phytocomponents were given below (Fig.2).
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
69
DISCUSSION
Ash values, fluorescence analysis and
histochemical studies were used for the standardization of
drug. Stigmasterol isolated from plants were reported to
be involved in the synthesis of many hormones like
progesterone, androgens, estrogens and corticoids [2] with
several pharmacological prospects such as
antiosteoarthritic, antihypercholestrolemic, antitumor,
hypoglycaemic, antimutagenic, antioxidant, anti-
inflammatory and CNS effects [3-6]. τ-Tocopherol is the
analog of tocopherol (vitamin E). Dodecanoic acid,
tetradecanoic acid, hexadecanoic acid and octadecanoic
acid are among the fatty acids known to have potential
antibacterial and antifungal activity [7-8]. Moreover, the
presence of various bioactive compounds confirms the
application of T. sphaerocarpa for various ailments by
traditional practitioners. Similarly, the same studies were
previously reported by the plants like Alseodaphne
semecarpifolia, Stylosanthes fruticosa, Cassia auriculata,
Wrightia tinctoria, Vernonia cinerea, Hugonia mystax [9-
15]. However, isolation of individual phytochemical
constituents may proceed to find a novel drug. In addition
to this, the results of the GCMS profile can be used as
phytochemical tool for the identification of the bioactive
components.
CONCLUSION
From the present study, it was concluded that the
plant T. sphaerocarpa are highly valuable in medicinal
usage for the treatment of various human ailments along
with the chemical constituents present in it. The
compounds needs further research on toxicological
aspects to develop safe drug.
ACKNOWLEDGEMENT
Authors thanks Mr. S. Kumaravel, Manager,
Quality Control, Food Testing Laboratory, Indian Institute
of Crop Processing Technology (IICPT), Thanjavur for
providing facilities to carry out the work.
REFERENCES
1. Khandelwal KR, Pawar AP, Kokate CK and Gokhale SB. Practical Pharmacognosy- techniques and experiments, III edn.
Nirali Prakashan. 1996, 140-141.
2. Kaur S, Singh HP, Batish DR, Kohli RK. J. Med. Plants Res., 5(19), 2011, 4788-4793.
3. Marquis VO, Adanlawo TA, Olaniyi AA. Planta Med., 31(4), 1977, 367-74.
4. Prestwich GD, Eng WS, Roe RM, Hammock BD. Arch. Biochem. Biophys., 228, 1984, 639.
5. Svoboda JA, Rees HH, Thompson MJ, Hoggard N. Steroids, 53(3-5), 1989, 329-43.
6. Chowdhury R, Rashid RB, Sohrab MH, Hasan CM. Pharmazie, 58(4), 2003, 272-273.
7. McGraw LJ, Jager AK, Van Staden J, Isolation of antibacterial fatty acids from Schotia brachypetala. Fitoterapia 73,
2002, 431-433.
8. Seidel V, Taylor PW, In vitro activity of extracts and constituents of Pelagonium against rapidly growing mycobacteria.
Int. J. Antimicrob. Agen., 23,2004,613-619.
9. Gook-Che J, Myoung-Soon P, Do-Young Y, Chul-Ho S, Hong-Sig S, Soo Jong U. Exp. Mol. Med., 37(2), 2005, 111-
120.
10. Charles A, Leo Stanly A, Joseph M, Alex Ramani V. Asian J. Plant Sci. Res., 1(4), 2011, 25-32.
11. Paul John Peter M, Yesu Raj J, Prabhu Sicis VP, Joy V, Saravanan J, Sakthivel S. Asian J. Plant Sci.Res., 2(3), 2012,
243-253.
12. Yesu Raj J, Paul John Peter M and Joy M. Asian J. Plant Sci. Res., 2(2), 2012, 187-192.
13. Jayamathi T, Komalavalli N, Pandiyarajan V. Asian J. Plant Sci. Res., 2(6), 2012 688-691.
14. Abirami P, Rajendran A. European J. Exp. Biol., 2(1), 2012, 9-12.
15. Vimalavady A, Kadavul K.Euro. J. Exp. Bio., 3(1), 2013, 73-80.
Vol 5 | Issue 1 | 2014 | 53-56.
53
e - ISSN 2249-7544
Print ISSN 2229-7464
CHARACTERIZATION OF THE METHANOLIC EXTRACT OF
LEAVES OF TRICALYSIA SPHAEROCARPA (DALZELL EX HOOK.F.)
GAMBLE BY GC-MS
Anandhi G1*, Pragasam A
1, Prakash Yoganandam G
2
1Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Puducherry, India.
2College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, Puducherry, India.
ABSTRACT
Tricalysia sphaerocarpa is commonly known as wild coffee and its basianym was Discospermum sphaerocarpum
Dalzell ex Hook. F. Gas Chromatography-Mass Spectrometry is an important technique used for metabolic profiling in plants
and also used for the qualitative and quantitative estimation of organic compounds. Totally 30 chemical compounds were
identified from the methanolic extract of the leaves of Tricalysia sphaerocarpa, among which fatty acid is the major group
consists of 9 compounds. Eicosanoic acid was found to be present as the major compound with peak area 35.77% and retention
time 21.865minutes, followed by octadecanoic acid (18.81%).
Keywords: GC-MS analysis, Tricalysia sphaerocarpa, Discospermum sphaerocarpum, Methanol extract.
INTRODUCTION
The genus Tricalysia, (Rubiaceae) comprises 50
species distributed in subtropical and tropical regions in
Asia and Africa, of which 2 species has been reported form
India [1]. Some of these used as folk fore medicine as
sedative, emetic, malaria, yellow fever, skin diseases and
also for urine disorders. Medicinal plants are important
substances for the study of their traditional uses through
the verification of pharmacological effects and can be
natural composite sources that act a disease curing agents.
So phytochemical investigation on the extract for their
main phytocompounds is very vital. Hence in the present
study, the methanolic extract of leaves of Tricalysia
sphaerocarpa were screened for Gas Chromatography-
Mass Spectrometry.
MATERIALS AND METHODS
Collection of Plant material
The plant of Tricalysia sphaerocapa was
collected from the sacred grove of Thirumanikuzhi, of
Cuddalore district, Tamil Nadu. The collected plant
materials were botanically identified. The species
confirmation was engaged at French Institute Herbarium
(HIFP), Puducherry. The herbarium specimen was
prepared and deposited at the Department of Botany,
Kanchi Mamunivar Centre for Post Graduate Studies,
Lawspet, Puducherry, for future reference.
Gas Chromatography-Mass Spectrometry (GC-MS)
analysis
GC-MS analysis was performed with GC Clarus
500 Perkin Elmer equipment. Compounds were separated
on Elite-1 capillary column (100% Dimethylpolysiloxane).
Oven temperature was programmed as follows: isothermal
temperature at 50ºC for 2min, then increased to 200ºC at
the rate of 10ºC/min, then increased up to 280ºC at the rate
of 5ºC/min held for 9 min. Ionization of the sample
components was performed in the El mode (70 eV). The
carrier gas was helium (1ml/min) and the sample injected
was 2μl. The detector was Mass detector turbo mass gold-
Perkin Elmer. The total running time for GC was 36 min
and software used was Turbomass 5.2. Using computer
searches on a NIST Ver.2.1 MS data library and
comparing the spectrum obtained through GC –MS
compounds present in the plants sample were identified [2-
3].
Identification of Compounds
The individual compounds were identified from
methanolic extracts based on direct comparison of the
retention times and their mass spectra with the spectra of
known compounds stored in the spectral database, NIST
(Version year 2005).
RESULTS
The compounds were identified by GC-MS
Corresponding Author: Anandhi G Email:- [email protected]
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Vol 5 | Issue 1 | 2014 | 53-56.
54
analysis enumerated with molecular formula, retention
time, molecular weight and peak area% (Table 1; Fig.1).
GC-MS analysis of an methanolic extract of leaves of
Tricalysia sphaerocapa showed 30 compounds. Of which
9 compounds belongs to the group fatty acids, 4 belongs to
aliphatic & aromatic bicyclics, 3 belongs to aromatic
hydrocarbons, 2 belongs to aromatic nitriles, aliphatic
aldehydes, aromatic ketones and aromatic dicarboxylic
esters each and 1 compounds belongs to aromatic alcohols,
phenolics, terpenoids, barbiturates, pyrimidinedione,
aromatic ethers each. Among this, Eicosanoic acid was
found to be present as the major compound with peak area
35.77% and retention time 21.865minutes followed by
Octadecanoic acid with peak area 18.81% and retention
time 20.093 minutes, followed by 9,12,15- octadecatrienic
acid with peak area 12.32% and retention time
15.01minutes. 2,2-Dimethylindene,2,3-dihydro-, 2-(2-
Hydroxyphenyl)buta-1,3-diene, 1,2,3,4,8,9-hexahydro-
4,4,8-trimethyl-,(+)- was found to be as least quantity with
the peak area 0.51% and retention time 22.882 minutes.
Some of the important structure of phytocomponents was
given below (Fig. 2).
Table 1. GC-MS Analysis of methanolic extract of leaf of T. sphaerocarpa
No. Name of the compound Molecular formula Molecular
weight RT Peak area %
Aliphatic & Aromatic bicyclics
1 Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-, [1R-
1α,2β,5α]- C10H18 138 16.942 0.91
2 2,2-Dimethylindene,2,3-dihydro- C11H14 146 22.882 0.51
3 Bicyclo[3.1.1]heptane, 2,6,6-trimethyl- C10H18 138 16.942 0.91
4 2AH-Cyclobut[a]indene-2a-carboxylic acid,
1,2,7,7a-tetrahydro, methyl ester C13H14O2 202 13.456 2.89
Aromatic nitriles
5 -Ethylbenzonitrile C9H9N 131 13.456 2.89
6 2,5-Dimethylbenzonitrile C9H9N 131 13.456 2.89
Aromatic ethers
7 2,3,5,6-Tetrafluoroanisole C7H4F4O 180 13.863 1.05
Pyrimidinedione
8 2,4(1H,3H)-Pyrimidinedione, 5(trifluoromethyl)- C5H3F3N2O2 180 13.863 1.05
Fatty acids
9 Oleic acid C18H34O2 282 20.979 1.10
10 Octadecanoic acid C18H32O2 284 20.093 18.81
11 Nonadecanoic acid C19H38O2 298 20.979 1.10
12 n-Hexadecanoic acid C16H32O2 256 18.176 10.41
13 Tetradecanoic acid C14H28O2 228 18.176 10.41
14 9,12,15-Octadecatrienic acid, (Z,Z,Z)- C18H30O2 278 15.01 12.32
15 9,12-Octadecadienoic acid (Z,Z)- 19.977 11.54
16 Eicosanoic acid C20H40O2 312 21.865 35.77
17 Docosanoic acid C22H44O2 340 23.477 0.77
Aliphatic aldehydes
18 9,17-Octadecadienal, (Z)- C18H32O 19.977 11.54
19 1H-Benzimidazole, 5,6-dimethyl- C9H10N2 146 22.882 0.51
Aromatic hydrocarbons
20 2-(2-Hydroxyphenyl)buta-1,3-diene 22.882 0.51
21 Phenanthro[3,2-b]furan-7,11-dione,1,2,3,4,8,9-
hexahydro-4,4,8-trimethyl-, (+)- C19H20O3 296 23.274 1.62
22 (2-Methoxyphenyl)carbamic acid, naphthalene-2-
yl ester C17H15O3N 281 23.129 1.38
Aromatic ketones
23 Chrysene-1,7(2H,8H)-dione, 3,4,9,10-tetrahydro-
2,8-dimethyl- C20H20O2 292 23.129 1.38
24 tert-Butyl(5-isoproply-2-
methylphenoxy)dimethylsilane C16H28OSi 264 23.129 1.38
Aromatic dicarboxylic esters
25 Bis(2-ethylhexyl) phthalate C24H38O4 390 23.216 1.42
26 Phthalic acid, di(2-propylpentyl ester) C24H38O4 390 23.216 1.42
Barbiturates
27 cyclobarbital C12H16N2O3 236 23.477 0.77
Terpenoids
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28 Squalene C30H50 410 25.322 10.87
Phenolics
29 4,6-Bis(1,1-dimethylethyl)-4´-methyl-1-1´-
biphenyl-2-ol C19H28O 272 23.274 1.62
Aromatic alcohols
30 Dibenzo[a,c]phenazin-10-ol C20H12N2O 296 23.274 1.62
Fig 1. Gas Chromatography - Mass Spectrometry (GC-MS) Chromatogram of the methanolic extract of leaves of T.
sphaerocarpa
Fig 2. Structure of Some Important Phytocompounds
Squalene Docosanoic acid Eicosanoic acid
Oleic acid n-Hexadecanoic acid 2,5-Dimethylbenzonitrile
2-Ethylbenzonitrile Cyclobarbital 2,3,5,6-Tetrafluoro-4-
methylanisole
Octadecanoic acid 1H-Benzimidazole,5,6-dimethyl- (9Z)-octadeca-9,17-dienal
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DISCUSSION
n-Hexadecanoic acid is also known as Palmitic
acid. n-Hexadecanoic acid, Octadecanoic acid, 9,12-
octadecadienoic acid (Z,Z)-, 9,12,15-octadecatrienoic acid ,
methyl ester (Z,Z,Z)- was already reported in the leaf of
Mallotus philippensis [4]. 9,12,15-octadecatrienoic acid ,
n-Hexadecanoic acid, Octadecanoic acid was also reported
in the methanolic extract of stem of Tricalysia
sphaerocarpa [5]. Secondary metabolites in plant products
are responsible for several biological activities in man and
animals [6]. The active components usually interfere with
growth and metabolism of microorganisms in a negative
manner [7]. Phenolic compounds and steroidal compounds
which are more effective in higher concentrations
inhibiting the growth of all fungi[8]. The fatty acids being
effective in the treatment of asthma, rheumatoid arthritis,
inflammatory bowel diseases [9]. Esters are functionally
used in the design of “Prodrugs” [10], terpenes are anti-
allergic [11] and antimicrobial agents [12]. Squalene is the
Triterpene compound showed activity against
Antibacterial, Antioxidant, Antitumor, cancer preventive,
Immunostimulant, Chemo preventive, Lipoxygenase
inhibitor, pesticide [13].
CONCLUSION
From the present study, it was concluded that the
plant T. sphaerocarpa are highly valuable in medicinal
usage for the treatment of various human ailments along
with the chemical constituents present in it. The
compounds needs further research on toxicological aspects
to develop a safe drug.
ACKNOWLEDGEMENT
Authors are thankful to Mr. P. Gopal, Technical
Manager, Sargam Laboratory Pvt. Ltd. Guindy, Chennai
for providing facilities to carry out the work.
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