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PHARMACOLOGICAL STUDIES ON
Tricalysia sphaerocarpa (Dalzell ex Hook. F,) Gamble
By
G. ANANDHI
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
Submitted by
G. ANANDHI
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
DECLARATION
(G. ANANDHI)
Place: Puducherry
Date:
ACKNOWLEDGEMENT
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
Table 20: Chemical groups obtained from GC-MS Analysis of Methanol extract of
Tricalysia sphaerocarpa
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 31: Effect of formulation Test extract of Tricalysia sphaerocarpa on body weight
in Normal and Alloxan induced diabetic rats
INTRODUCTION
ancient aphorism. Nature is the supreme creation and man has completely been
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
countries has let to intensify efforts on the documentation of ethno medicinal data
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
1
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),
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
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
2
The most valuable of the drug and drug plant has been standardized as a result of
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
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
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
plants used by the indigenous people of world shows unknown compounds with
plants’ of immense food and medicinal value to the modern civilization. Seventy
3
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
medicine for various biological activities is a necessary first step in the isolation
from them, identification of crude drugs, extraction of the principle drugs, study
of their antimicrobial activities and their potential use as antioxidants are essential
pharmacological investigations.
1.1. Pharmacognosy:
structural, physical, chemical and sensory characters of crude drugs along with
their history, method of cultivation, collection and preparation for the market
and chemical testing. There are five methods of evaluation crude drugs namely
4
Morphlolgical or Organoleptic, Microscopical or Histological, Physical, Chemical
and Biological.
by which the drugs are identified. It also includes those of colour, odour, taste,
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
identification is done to identify the parts of the crude drug. The measurement of
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
1.2. Phytochemistry:
weight chemical compounds that are produced and accumulated by the plants.
5
saponins. Phytochemical analysis of plants, used in folklore has yielded a number
are important substances for the study of their traditional uses through the
act as a disease curing agents. About 3000 materials from 2764 plant species have
1992).
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
1.3. Pharmacology
cultures implementing them the role of natural products, herbal medicines, tribal
6
and traditional medicines is being increasingly appreciated during the recent years
for the prevention and cure of human ailments (Janardhanan et al., 2006)
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
risk. Most of the antioxidants in a typical diet are derived from plant sources and
activity, while others, such as the mono-phenols are weak antioxidants. The main
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
spices and aromatic herbs have been investigated for antioxidant activity
7
provide protection against oxidative degradation of foods by free radicals (Gulcin
et al., 2002).
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).
most prevalent human illness by the year 2020. Various antidepressants, ranging
1996, Stepaniuk et al., 2008) and also may pose a risk factor for development of
existing drug therapies and the remaining 70% fail to achieve complete remission
8
(Kulkarni et al., 2009). Herbal drug used in depression are Centella asiatica ,
food interactions. In this context, novel approaches are being tried to find more
(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
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
diabetes that are the major causes of morbidity and death (Kameswararao, 2003).
35%. Currently, there are over 150 million diabetic patients worldwide and this is
9
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
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).
they contain many bioactive substances with therapeutic potential. In recent years,
a large number of medicinal plants have been already tested for their antidiabetic
traditional acceptability and availability, low costs and lesser side effects.
10
the present work has been taken up to evaluate the medicinal potential of
11
CHAPTER 2
REVIEW OF LITERATURE
chronic diseases such as cancer, cardiovascular diseases and stroke. The positive
niruri, Jushi Singh (2011) on many endangered medicinal plants are some of the
examples to be mentioned.
12
This chapter encompasses the published literature on medicinal uses and
various studies on the genus Tricalysia a member of the family Rubiaceae which
family, madder family or bedstraw family. Members of the coffee family tend to
about 611 genera and more than 13,000 species are placed in Rubiaceae. This
fifth largest by number of genera. The group contains many commonly known
and the horticulturally valuable madder (Rubia), west indian jasmine (Ixora),
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,
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.
13
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
(George Usher, 1984). It is also found in central and south Maharashtra Sahyadris
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
to past disturbance (Mani and Parthasarathy, 2006). The wild coffee Tricalysia
and the site was dominated by 33% of the stand (Venkateswaran and
of anti-cancer plants in Ogun state, Nigeria, revealed that the bark of Tricalysia
14
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
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
al., 1996).
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.
15
determined. On the basis of the crystal structure of 1, the structures of the other
of spectroscopic evidence.
(1-4) having a cafestol-type carbon framework were isolated from the wood of
extract of the stem of Tricalysia dubia, together with 5 known rearranged ent-
16
Xu et al., (2010) isolated two new ent-kaurane glycosides namely
position from the roots of Tricalysia okelensis and their structures established by
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
sphaerocarpa through GC-MS analysis among which fatty acid was the major
major compound with peak area 35.77% and retention time 21.865 min, followed
17
1. Pharmacognosy
• Anatomical studies
• Histochemical localization
• Fluorescence analysis
2. Phytochemistry
3. Pharmacology
significance.
18
CHAPTER 3
The plant of Tricalysia sphaerocarpa was collected from the sacred grove
Mamunivar Centre for Post Graduate Studies, Lawspet, Puducherry, for future
reference.
Kingdom :- Plantae
Phylum Trachiophyta
Class Magnoliopsida
Order Gentianales
Family Rubiaceae
Genus Tricalysia
19
Synonyms:
Vernacular names:
Tamil : irrukulimaram
Kannadam : kaadukafibija.
Trunk /bark : trunk flutted, bark whitish, smooth, fissured when matured; balze
yellowish. The tree outer bark is often attacked by termites giving creamish
appearance.
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
20
secondary nerves 5-8 pairs, hairy domatia present at axils, tertiary nerves broadly
reticulate.
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
mammals and birds (Gamble 1921, Gamble 1993, Sasidharan 2004, Almeida
3.4. Ecology
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
al., 2009). The bark of Tricalysia macrophylla K. Schum is used for the
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
Tricalysia sphaerocarpa taste and smell like coffee and the infusion of leaves of
21
T.singularis (Korth.) K.Schum. is used as a beverage in Andala (Sumatra)
Tricalysia dubia
Tricalysia okelensis
The plant is collected from the wild growing in the natural environment of
(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
22
dried under shade in the laboratory for 3 to 4 days and the dried materials were
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
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
They were further washed thoroughly with distilled water, stained with safranin
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
23
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
For calculation the number of epidermal cells were counted in both the surfaces.
Stomatal number:
the leaf (Evans, 1996). For calculation the number of stomata at different region
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
Where,
S.I=S/E+SX100
Stomatal types:
24
Palisade Ratio:
each upper epidermal cell (Evans, 1996). The semi permanent mounts of cleared
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
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:
stem and root were taken using Olympic Nikon (Japan) Automatic Camera
25
(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
proteins. The colour and results are recorded . The tests were as follows:
Lignin:
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
Tannin:
Mucilage:
Starch:
Alkaloid:
26
Proteins:
covered and allowed to stand for 24 hours. Change to yellow colour indicates the
presence of proteins.
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
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:
the country concerned. The quality and purity required is achieved by standards
(numerical values) also given in the official work of reference. The powdered
27
Determination of Total Ash:
crucible which is previously ignited and weighed. The powdered drug is spread in
temperature not exceeding 450 C until free from carbon. The crucible is cooled
The total ash values were determined by the ash obtained form leaf and
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
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
28
Moisture content
The collected materials were chopped into small pieces separately, shade-
dried, and coarsely powdered using a pulverizor. The coarse powder were
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
Extractive values by (i) Batch process (Kokate, 1986) and (ii) Successive
water and filtered. PH of the filtrate was determined by using digital PH meter.
following the method of Harborne (1998) and Trease and Evans (1983).
The substance was mixed with little amount of dilute hydrochloric acid
29
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.
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
was added in boiling water bath for 10 minutes. Purple or bluish colour indicates
benzaldehyde) was added which turns into pink colour indicating the presence of
catechins.
junction of the two liquid layers and upper layer appears bluish green.
30
Test for Coumarins:
To the substance, a drop of sodium sulphate was added which turns into
presence of flavonoids.
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
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.
solution were added. Bluish green or red colour is the indication of the presence
of phenol.
31
Test for Phlobotannins:
boiled with 1 % aqueous HCl is the evidence for the presence of phlobotannins.
A little of the substance is shaken with water and copious lather formation
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
To 3ml test solution a few drops of dilute iodine solution was added. The
32
Test for Terpenoids (Salkowski test):
sulphuric acid were carefully added to form a layer. A reddish brown colour
chloride solution. Then one ml of extract was added to it. The formation of pink
isothermal temperature at 50ºC for 2 minutes, then increased to 200ºC at the rate
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
were identified.
33
Identification of Compounds:
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
(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
antioxidant molecules and free radicals, which results in the scavenging of free
(1958). An aliquot of 0.5 ml of sample solution in methanol was mixed with 2.5
and incubated for 37 minutes in the dark at room temperature. The absorbance
34
(BHT) was used as a positive control. DPPH free radical scavenging ability (%)
hydrogen peroxide.
SOD activity was assayed by determining the inhibition rate of nitro blue
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.
The method of Benzie and strain (1996) was adopted for the assay. The
35
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
Selection of extracts
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
36
Administration of the extracts
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
guidelines. Control groups were given only the vehicle (0.2% v/v Tween-80
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
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
at 25±1 °C. All the rats of either sex were divided in to four different groups of 6
37
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
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).
according to the methods described by Steru et al., (1985). Briefly, rat both
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
methanolic extract 100 and 200mg/kg and diazepam (3mg/kg) by placing mouse
38
on a wooden board. The number of head dips and time spent in each dip was
Statistical analysis
The immobility time in tail suspension test and forced swimming test was
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
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,
Experimental Design
39
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.
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
Alloxanization. Rats with blood glucose level above 250 mg/dl were considered
Experimental design
Group 1 - Normal control and received vehicle i.e. 1% Gum acacia Solution,
1ml/kg/BW
40
Group 6 - Received Glibenclamide 5mg/kg/BW, orally on 3rd day after
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,
Fasting Blood Glucose was estimated from the tail vein prior and 1, 3 and
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,
Diabetic Rats
After 15 days of treatment, overnight fasted rats were sacrificed and blood
was collected. The serum was separated and analyzed for lysosomal enzymes
41
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
(India) Limited (CONTOURTMTS Blood Glucose Meter with same Test Strips )
as follows:
Principle:
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
After the reaction time, the glucose concentration in the sample is displayed. No
calculation is required.
42
Principle
SGOT
α -Keto glutarate + L-asparate L -glutamate + Oxalacetate
pH 7.4
pyruvic acid. Rate of reaction is then determined by the estimation of pyruvic acid
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
43
Procedure
Tube No. 1 2 3 4 5
and measured the absorbance of all the five tubes against purified water on a
Test procedure
Serum 0.1 ml
44
Mixed well and allowed to stand at room temperature for 10 minutes. Estimated
Principle
SGPT
α -Keto glutarate + L-alanine L -glutamate + pyruvate
pH 7.4
corresponding hydrazone, which gives the brown color in alkaline medium and
Reagents
45
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
and measured the absorbance of all the five tubes against purified water on a
Test procedure
Mix well and allow to stand at room temperature for 10 minutes. Estimated with
46
Determination of Serum alkaline phosphatase (SALP)
Principle
in serum / plasma.
Alkaline Phosphatase
PNPP + H2O P- nitrophenol + Phosphate
The method has been recommended by the German Society of Clinical Chemistry
Reagents
Reagents 1: Substrate
Reagents 2: Buffer
buffer (Reagent 2). Mixed to dissolve by slow stirring to ensure uniform mixing.
47
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.
Calculation
We can also measure the change of optical density directly from Bio Chemical
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
standard.
48
Determination of Glutathione- Peroxidase activity:
at 340 nm. One unit of GPx is equal to the number of nano moles of NADPH
In animals, catalase was present in all major body organs, especially being
1963).
The catalase activity was assayed by the method of catalase catalyses the
2H2O2 2H2O + O2
Reagents
49
Phosphate buffer - 0.01M, pH 7.0: 173 mg of disodium hydrogen phosphate and
Hydrogen peroxide – 0.2M: 2.27 ml h hydrogen peroxide was made upto 100 ml
Procedure
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
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
50
CHAPTER 4
RESULTS
4.1. Pharmacognosy
4.1.1. Anatomy
peels of the leaf and stem, clearing of leaf, maceration of stem and transverse
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,
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
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
51
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 ±
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.
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
52
Table 1: Quantitative values of foliar epidermis of Tricalysia sphaerocarpa
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
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).
53
4.1.1.8. Transverse Section of Root:
The root in cross section is circular. The rhizodermis is peeled off. The 8-
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
circular, thick walled, narrow xylem fibres. The vessels include both wide and
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
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.
54
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
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
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
Root
Powdered root material was creamy white in day light, and pale yellow
chloroform, ethanol and n-butyl alcohol, creamy white in water. Under UV light,
55
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
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
stem powder was orange colour in acetone, yellow in benzene and water, pale
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
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,
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%
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.
56
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
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
4.2. Phytochemistry
insoluble ash and water soluble ash of leaf, stem, root and fruit of Tricalysia
Leaf
In leaf the moisture content, total ash, acid insoluble ash and water soluble
Stem
In stem the moisture content, total ash, acid insoluble ash and water
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
The results of extractive values are given in Table 8 and 9 and figure 1 and 2.
57
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
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
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
Various parts PH
Leaf 6.4
Stem 6.7
Root 6.8
Fruit 6.8
4.2.2.1. Batch Process
Leaf
Stem
aqueous extract (20%) ethanol (20%) acetone (18%), benzene (11%), n-butanol
Root
and diethylether(5%).
Fruit
(9%), chloroform (6%), benzene(5%) and the lowest value was found in
diethylether(3%).
58
4.2.2.2. Successive Process
Leaf
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
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%).
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
Table 11-14.
59
Leaf
extract, only the alkaloids were present and others were absent. In chloroform
were present and the other phytocompounds were absent. In ethylacetate extract,
(starch) and steroids were present and the others absent. Gums, oils and resins are
Stem
diethylether extract, only the alkaloids were present and other phytocompounds
60
Table 11: Phytochemical colour reactions of various extracts of stem of Tricalysia
sphaerocarpa
(starch), saponins, terpenoids are present and the others chemical compounds
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
protein, phenolic group and saponins in methanol, aqueous and the powder drug
(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
group, saponins, steroids and flavanoids in methanol and aqueous extract and the
61
Table 13: Phytochemical colour reactions of various extracts of root of Tricalysia
sphaerocarpa
protein, phenolic group, saponins were present and the others were absent. In
polysaccharide (starch) and glycosides were present and the others were absent.
polarity. Hence, the methanol extract of leaves, stem, root and fruit were
active fraction.
extract of leaf. Of which 9 belong to fatty acids (Oleic acid, Octadecanoic acid,
62
dihydro-, Bicyclo[3.1.1]heptane,2,6,6-trimethyl-, 2AH-Cyclobut[a]indene-2a-
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
acid,(z,z)- and 9,17-octadecadienal, (z)- with the peak area 11.54 % and retention
quantity with the peak area 0.51 % and retention time 22.8 minutes (Table 15 and
Figure 3).
63
Table 15: GC-MS Analysis of Methanol Extract of Leaf of Tricalysia sphaerocarpa
acid, methyl ester, Octadecanoic acid, methyl ester, Eicosanoic acid, methyl ester)
acid, n-Hexadecanoic acid). Of which one compound belongs to the class sugars
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
the peak area 0.90 % and retention time 12.21 minutes (Table 16, Figure 4).
64
Table 16: GC-MS Analysis of Methanol Extract of Stem of Tricalysia sphaerocarpa
1a,2,5,5,5a,6,9,10,10a, octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-
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
fluorine was found to be as in least quantity with the peak area 28 % and
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
65
Table 17: GC-MS Analysis of Methanol Extract of Root of Tricalysia sphaerocarpa
propanamine was found to be as in least quantity with the peak area 12.4 % and
antibiotic with the peak area 9.27 % and retention time 25.7 minutes (Table 18,
Figure 6).
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
66
Table 19: Combined Table for GC-MS Analysis of Methanol Extract of Tricalysia
sphaerocarpa
Table 20: Chemicals groups obtained from GC-MS Analysis of Methanol Extract of
Tricalysia sphaerocarpa
4.3. Pharmacology
7.76, 62.43 ± 6.74 at 50µg/ml and 51.59 ± 5.95 at 10µg/ml. In petroleum ether
42.65 ± 5.82 at 50µg/ml and 28.67 ± 5.76 at 10µg/ml. In water extract, the
± 5.85, 47.48 ± 4.92 and 35.53 ± 4.81 respectively. All the extracts showed dose
higher activity was observed in the chloroform extract and the results are
activity at 100µg/ml was 61.77 ± 5.98, 49.87 ± 4.76 at 50µg/ml and 32.02 ± 2.84
67
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
38.49 ± 2.97, 29.45 ± 2.46 at 50µg/ml and 10.78 ± 1.79 at 10µg/ml. All the
extract and the results are presented in the table 22 and figure 8.
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,
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
methanol extract as 7.95 ± 6.59 at 100µg/ml, 6.12 ± 5.39 at 50µg/ml and 4.78 ±
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
68
minimum value was observed in petroleum ether extract as 4.78 ± 6.79 at
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.
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).
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
time **140 seconds (P<0.01) to that of standard **135 seconds (P<0.01). These
69
Table 25 : Effect of methanolic extract of Tricalysia sphaerocarpa on Immobility time in
FST
Table 27: Effect of methanolic extract of T. sphaerocarpa. in Hole Board Test (HBT)
test. It is quite evident that none of the drug treated animals showed excellent
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
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
70
4.3.2.3. Anti - diabetic Activity
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
71
Table 28 : Effect of Test extract on blood glucose level in normal fasted rats
Table 29 : Effect of Test extract on blood glucose level in Alloxan-induced diabetic rats
(Single-dose short term study)
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
treatment, 50.3 ± 2.00 mg/dl after 6 hour of treatment. The maximum
However, the effects of other tested doses of Tricalysia sphaerocarpa were less
Diabetic Rats
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
72
± 2.80 mg/dl after the treatment of 1 hour, 299.6 ± 1.26 mg/dl after 3 hour of
daily once)
plasma glucose lowering effect. The present study indicates that alloxan induced
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
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 ±
73
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 Blood glucose level (mg/dl)
(dose mg/kg;
p.o)
Day 3 Day 6 Day 9 Day 12 Day 16
Normal 56.1±1.02 61.0±1.20 61.0±0.82 62.8±2.10 58.9±4.40
control
Diabetic 312.5 306.1±2.12a 290.8 ± 0.42a 279.2 ± 2.12a 278.2 ± 2.20a
control ±1.20 a (1.7) (6.8) (10.3) (11.96)
Test extract 291.2 280.5± 1.40c 260.2 ± 2.30c 230.0 ± 4.02c 192.1 ± 0.62c
(125) ± 2.26 b (4.4) (11.3) (27.1) (33.5)
Test extract 290.2 270.2± 4.20c 228.0 ± 0.48c 184.0 ± 2.70c 172.2 ± 1.50c
(250) ± 0.01 c (6.4) (20.7) (35.1) (39.9)
c c c
Test extract 280.0 252.2± 2.12 220.0 ± 2.12 182.4 ± 2.10 162.2 ± 2.10c
(500) ± 2.21 c (10.6) (22.6) (36.4) (35.7)
c c c
Glibenclamide 278.5 240.2± 2.12 210.6 ± 1.40 182.5 ± 0.86 164.5 ± 2.12c
(5) ± 2.30 c (1.5) (22.5) (35.8) (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
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).
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
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
74
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).
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
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
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).
75
Table 32 : Effect of formulation Test extract on biochemical parameters in Alloxan
induced diabetic rats.
levels, SGOT (90.36 ± 0.06 IU/L), SGPT (55.34 ± 0.45 IU/L), Alkaline
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
diabetic control group showed that normal architecture of pancreas with acini of
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
lobules. No fibrosis or inflammation was found. The test extract (500 mg/kg)
showed stroma into Mules like the standard Glibenclamide (Plate 9).
76
CHAPTER 5
DISCUSSION
Natural products have been the most successful source of drugs for ever.
one of the frontier areas in phytochemistry. Plants are probably the best cell
5.1. Pharmacognosy :
structural, physical, chemical and sensory characters of crude drugs along with
their history, method of cultivation, collection and preparation for the market
and chemical testing. There are five methods of evaluation of crude drugs namely
and Biological.
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
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
77
used as significant parameters in the angiosperm taxonomy as well as phylogeny.
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.
of the adaxial epidermis of leaf, the epidermal cell number, stomatiferous abaxial
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
tannineferous idioblasts. All these characters are typical to this plant which
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).
tannin and lignin. The present study reports the presence of starch, alkaloid and
78
protein in leaf, stem and root; tannin in leaf and stem. Lignin only in root.
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 also included, in which the type of vessels, tracheids, fibres etc. are
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
79
5.2. Phytochemistry :
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
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
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
plants, many of which have been used in established eastern medicine for
80
thousands of years (Gurudev Singh Raina, 2013). Flavonoides are also shown to
plant kingdom. They occur in all parts of the plants. Phenols are said to offer
associated with the health benefits derived from consuming high levels of fruits
and vegetables (Ka¨ hko¨ nen et al., 1999). Hence presence of phenolic
(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
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,
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).
revealed the presence of tannins, flavonoids, steroids and saponins (Aiyegoro and
81
Okoh, 2010). Phytochemical screening of the plants (Carica papaya, Magnifera
the presence of alkaloid, flavonoid, saponin, terpenoid, steroid and sterols in the
Methanolic extract of leaves, stem, root and fruit were subjected to GC-
MS analysis. This analysis were carried out to detect the possible compounds
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
aldehydes group, aromatic ketones group, aromatic ethers, phenolic group and to
healthy source of fat in the diet. Many fatty acids are known to have antibacterial
82
hexadecanoic acid, octadecanoic acid and oleic acids are among the fatty acids
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
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
of behenic acid yields behenyl alcohol. Pracaxi oil from the seeds of Pentaclethre
acid, and is used in hair conditioners. Nonadecanoic acid found in ox fats and
also been reported from the genus Streptomyces, along with its biological
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
83
Squalene, an isoprenoid compound structurally similar to beta-carotene, is
human tissues, with the greatest concentration in the skin, where it is one of the
protecting human skin surface from lipid peroxidation due to exposure to UV and
dose-dependent manner. Squalene may also act as a "sink" for highly lipophilic
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
2012), and also from the root of Bulbophyllum kaitense (Kalairasan et al., 2012).
84
n-hexadecanoic acid has anti inflammatory property. The rigorous use of
2012). n-hexadecanoic acid, oleic acid are the two major compounds present in
acid(Z,Z)- has been reported from the ethanolic extract of stem and root of the
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)-
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
Gaultheria fragrantissima (Padmavathy et al., 2014). The oil yielded from the
acid, octadecanoic acid, docosanoic acid, beter-sitosterol, eicosanoic acid and the
85
Stem
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,
hexadecanoic acid. n-hexadecanoic acid has also reported from the stem of
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,
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).
have also been recommended for their cholesterol lowering abilities, although
more study is needed to determine which compounds perform this function, and
86
how they work in the body. It has been already reported form the pseudobulb of
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
Root
five belongs to heterocyclics groups, one to aromatic ester groups, one to fatty
isolated from the ethanolic extract of the stems of Kadsura heteroclite (Wang et
and anxiety. Its also lower blood pressure in Hypertension patients, stimulate the
87
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
one to thiophosphates group, one to antibiotic and three to the other unclassified
vitro culture studies of DSG shows good stability in aqueous solution and retains
acid (Z,Z,Z-) are the common compounds seen both in stem and leaf. Most of the
leaf, stem, root and fruit of Tricalysia sphaerocarpa show antibacterial, antifungal
88
sedative, antiarthritic, antioxidant and anticancer properties and hence the plant
5.3. Pharmacology
Hydrogen peroxide and SOD Scavenging assay. The present study reveals, the
DPPH, FRAP and Hydrogen peroxide assay, but in SOD assay, the maximum
increases the activity) and ferrous ion chelating abilities from the methanolic
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
89
Methanolic extracts of Cassia fistula showed the highest amount of reducing
capacity (Irshad et al., 2012). Riaz et al., (2012) reported highest total antioxidant
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
The present findings obtained from FST, TST and HBT clearly reveal the
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
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
90
in the time spent immobile by mice in FST (Vikas Gupta et al., 2009). The
activity due to its reduction in the immobility period (Jamwal Neetu Singh et al.,
swimming time and decreased immobility time (Sangavai et al., 2013). Caffeine,
agents for depression treatment (Pravin Popatrao Kale et al., 2010). Celastrus
2014).
blood glucose levels up to 6 hr. except the lowest dose. The maximum
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
91
treatment. Oral administration of the extract showed a significant (P<0.01)
groups. The group treated with 500 mg/kg showed the decrease the lysosome
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
reported by previous workers are in conformitry into the present findings. The
(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
group (Masih et al., 2011). The aqueous and methanolic extract of Gongronema
(Akah et al., 2011). The methanolic and ethanolic extracts 200 mg/kg b.wt. of
and Alloxan induced diabetic rats (Ravinder Sangala et al., 2011). Aqueous and
92
activities in alloxan-induced hyperglycemic rats without significant change in
like bodyweight, and lipid profiles along with serum (Ahmed et al., 2005).
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
induced animal model (Arya et al., 2013). The methanol extract of Costus
and serum enzymes (SGOT, SGPT, ALP) in alloxan induced diabetic rats
93
Figure 1: Extractive values of various parts of Tricalysia sphaerocarpa by Batch
process.
35
30
25
20
15
10
0
Leaf Stem Root Fruit
Values in %
Acetone Benzene Chloroform Diethyl ether
Ethanol n-butyl alcohol Methanol Water
25
20
15
10
0
Leaf Stem Root Fruit
Values in %
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
S,S1-3,8-diazaundecamethylene bis[hydrogenthiosulfate]
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
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
Nonadecanoic acid
Figure 8: Anti oxidant activity -DPPH Scavenging assay:
100
90
80
70
60
50
40
30
20
10
0
10µg/ml 50µg/ml 100µg/ml
% of radicle scavenging
120
100
80
60
40
20
0
10µg/ml 50µg/ml 100µg/ml
% of radicle scavenging
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
10µg/ml 50µg/ml 100µg/ml
% of radicle scavenging
Figure 11: Anti oxidant activity - Superoxide dismutase (L-methionine and NBT
assay)
12
10
8
6
4
2
0
10µg/ml 50µg/ml 100µg/ml
% of radicle scavenging
important substances for the study of their traditional uses through the
can act as disease curing agents. The integrated research in drug discovery have
attracted and provided multidisciplinary research platforms. The present work has
sphaerocarpa.
chemicals through in vivo and in vitro studies i.e., the antioxidant, antidepressant
94
Pharmacognostical parameters like leaf constants, microscopy, physico-
chemical analysis, fluorescence analysis are a few of the basic protocol for
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.
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
alkaloid and protein in all the plant parts studied, tannin in leaf and stem, lignin
light and UV light. In root, it is mostly yellow in day light and pale yellow to dark
95
yellow in UV light . In fruit, it is mostly light brown in day light and creamy
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.
compounds from stem, 30 from leaf, 8 from root and 10 compounds from fruit)
acid and 9,12,15- octadecatrienoic acid (Z,Z,Z-) are the three common
followed by methanol, petroleum ether and water extracts. All the extracts
FRAP assay, the maximum value was observed in chloroform extract, followed
96
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
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
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.
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
inflammation was found. The various extracts (petroleum ether, chloroform and
methanol) of stem possess significant antioxidant activity and the methanolic stem
97
Thus the study concludes that the plant may be used as a potential drug for
phytochemical and pharmacological studies which are reported for the first time
from Tricalysia sphaerocarpa, may pave way for new drugs discovery.
98
Plate 1
Morphology of Tricalysia sphaerocarpa
-----X
--X -----Ph ----Sc
-----GT
--ICR
----CR
---DS
---GS
---SS
---HS
---MS ---BS
--MS
----GS
--SS
----DS
----VLT
----VI
----S
----Sc
----TI ----Ve
----Cu
----Ve
----SG
----Ve
----Ta
----P
--PP
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Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.
e - ISSN - 2249-7722
Print ISSN - 2249-7730
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.
~ 47 ~
Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.
Flowers minute, white, scented, Fruits greenish yellow, cuticle. 2 layers of palisade parenchyma are seen below
berry globose, the seeds flat, smooth, leaves elliptic or the upper epidermis. Stomata are seen only on the abaxial
lancaolate, obtusely acute, smooth, the main nerves about surface.
6-8 pairs, not prominent, nor the reticulation.
Stem: The stem in its outline is mostly dumble shaped.
Anatomical features:- Epidermis is unilayered with high cuticle. Cortex is
Leaf peelings: parenchymatous with 4-6 layered. Even the very younsg
Adaxial : Cells small in size, cell wall undulate, thin, no stem undergoes secondary thickening. The secondary
stomata. xylem and phloem are continous. A single layer of
scleroids are seen as an outer ring. The pith is very broad
Abaxial: Intercostal region: cells irregular, wall undulate, and formed of circular parenchymatous cells.
cell medium sized, stomata rubiaceous type, different in
size. Root: The root in its outline is circular and formed of 8-
Costal region: Cells small when compared to the 10 layers of parenchyma cells. The rhizodermis peeled
intercostal region, cell wall undulate, giant size stomata off. Secondary xylem and phloem are present. There is no
are seen, unicellular hairs present. Epidermal cell number, cortex. Rays are clearly seen. Xylem seen in the centre
stomatal number, stomatal index, palisade ratio, vein islet and the phloem towards the periphery.
number and veinlet termination number have been
calculated and presented in the Table 1. Phytochemical studies - The preliminary phytochemical
Stem peeling: Cells fairly large, cell wall thick, straight, studies in methanol, aqueous and powder drug revealed
basal cells very broad, tip cells tapering, stomata frequent, the marked presence of carbohydrate, glycosides,
rubiaceous type. alkaloids, tannin, flavanoids, moderate presence of
Venation pattern: Veins reticulate, showing lateral protein, phenol, terpenoids and saponin and absence of
branches; cells elongated with thin walls; vein-islet fairly triterpenoids, anthraquiones, catachins, coumarins (Table
large, each islet containing 3-4 termination points. Vein- 4).
islet number and veinlet termination number are presented Histochemical colour reactions - Presence of starch,
in Table 1. protein and tannin and absence of lignin and mucilage
Transverse section: Leaf: The midrib portion of the leaf (Table 3). The total ash, acid insoluble ash and water
is not much differentiated form the lamina. It is slightly soluble ash were 4 percent, 0.84 percent and 3.24 percent
thicker than the lamina. The xylem is omega shaped with respectively (Table 2). Fluorescence analysis shows
10-15 rays of xylem cells. Phloem is seen on the abaxial mostly dark green in day light whereas orange in UV light
surface. Both the epidermis is uniseriate, with thick (Table 5).
~ 48 ~
Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.
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.
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4. Suseela A and Pream S. Pharmacognostic studies on Lagascea mollis. J. Phytol. Res, 20(1), 2007, 95-102.
~ 49 ~
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
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.
65
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
the solvents was removed in vacuum and stored at 4ºC. spectrum obtained through GC –MS compounds present
The resulted extracts were subjected to preliminary in the plants sample were identified.
phytochemical screening and GC-MS analysis.
Histochemical color reactions were done by treating free Identification of Compounds
hand sections of stem with different reagents. The individual compounds were identified from
Phytochemical tests were done with dried powdered drugs methanol extract based on direct comparison of the
as well as different solvent extracts [1]. retention times and their mass spectra with the spectra of
known compounds stored in the spectral database, NIST
Gas Chromatography-Mass Spectrometry (GC-MS) (version year 2005).
analysis
GC-MS analysis was performed with GC Clarus RESULTS
500 Perkin Elmer equipment. Compounds were separated Phytochemical screening
on Elite-1 capillary column (100% The preliminary phytochemical study in
Dimethylpolysiloxane). Oven temperature was methanol, aqueous and powder drug shows the similar
programmed as follows: isothermal temperature at 50ºC results. It revealed the presence of carbohydrate,
for 2min, then increased to 200ºC at the rate of 10ºC/min, glycosides, alkaloids, protein, phenolic group, steroid,
then increased up to 280ºC at the rate of 5ºC/min held for saponins, flavanoids and terpenoids in methanol, aqueous
9 min. Ionization of the sample components was and the powder drug and absence of triterpenoids,
performed in the El mode (70 eV). The carrier gas was anthraquiones, catachins, coumarins and tannins. In
helium (1ml/min) and the sample injected was 2μl. The diethyl ether extract, only the carbohydrate is present and
detector was Mass detector turbo mass gold-Perkin Elmer. others are absent. In chloroform extract, alkaloids,
The total running time for GC was 36 min and software carbohydrates, flavonoids, glycosides, protein, phenolic
used was Turbomass 5.2. Using computer searches on a group, saponins, terpenoids are present. In ethyl acetate
NIST Ver.2.1 MS data library and comparing the extract, only carbohydrates, glycosides and steroids are
present(Table 1).
66
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
67
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
Fig 1. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the methanolic extract of stem of T.
sphaerocarpa
N-Hexadecanoic Acid
3-O-Methyl-D-Glucose
Ergot-5-En-3-Ol
Sitosterol
Stigmasterol
68
Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.
REFERENCES
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Nirali Prakashan. 1996, 140-141.
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Vol 5 | Issue 1 | 2014 | 53-56.
e - ISSN 2249-7544
Print ISSN 2229-7464
INTERNATIONAL JOURNAL
OF
PHYTOPHARMACY RESEARCH
www.phytopharmacyresearch.com
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%).
53
Vol 5 | Issue 1 | 2014 | 53-56.
analysis enumerated with molecular formula, retention found to be present as the major compound with peak area
time, molecular weight and peak area% (Table 1; Fig.1). 35.77% and retention time 21.865minutes followed by
GC-MS analysis of an methanolic extract of leaves of Octadecanoic acid with peak area 18.81% and retention
Tricalysia sphaerocapa showed 30 compounds. Of which time 20.093 minutes, followed by 9,12,15- octadecatrienic
9 compounds belongs to the group fatty acids, 4 belongs to acid with peak area 12.32% and retention time
aliphatic & aromatic bicyclics, 3 belongs to aromatic 15.01minutes. 2,2-Dimethylindene,2,3-dihydro-, 2-(2-
hydrocarbons, 2 belongs to aromatic nitriles, aliphatic Hydroxyphenyl)buta-1,3-diene, 1,2,3,4,8,9-hexahydro-
aldehydes, aromatic ketones and aromatic dicarboxylic 4,4,8-trimethyl-,(+)- was found to be as least quantity with
esters each and 1 compounds belongs to aromatic alcohols, the peak area 0.51% and retention time 22.882 minutes.
phenolics, terpenoids, barbiturates, pyrimidinedione, Some of the important structure of phytocomponents was
aromatic ethers each. Among this, Eicosanoic acid was given below (Fig. 2).
54
Vol 5 | Issue 1 | 2014 | 53-56.
Fig 1. Gas Chromatography - Mass Spectrometry (GC-MS) Chromatogram of the methanolic extract of leaves of T.
sphaerocarpa
55
Vol 5 | Issue 1 | 2014 | 53-56.
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