1
ANTIBACTERIAL, ANTHELMENTIC AND
PHYTOCHEMICAL INVESTIGATION ON THE POD
EXTRACTS OF Cassia fistula Linn.
Dissertation submitted to the University of Kerala
in partial fulfilment of the requirements for the
Degree of Master of Science in Botany
Candidate code : 09144011
Exam Code : 64506304
Subject Code : BO201
University of Kerala
August 2011
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Dr. Oommen P. Saj Department of Botany
Associate Professor of Botany University College
Thiruvananthapuram
CERTIFICATE
This is to certify that this project report entitled ANTIBACTERIAL,
ANTHELMINTIC AND PHYTOCHEMICAL INVESTIGATIONS ON THE
POD EXTRACT OF Cassia fistula Linn. is an authentic record of bonafide
research work carried out by Miss. Sumi S. Under my supervision and
guidance for partial fulfilment of the requirements of Degree of Master of
Science in Botany of the University of Kerala and further that no part of it has
been presented earlier for any degree or diploma in any of the Universities.
Dr. Oommen P. Saj
Trivandrum
September, 2011
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DECLARATION
I, Sumi S. hereby declare that this dissertation entitled
ANTIBACTERIAL, ANTHELMINTIC AND PHYTOCHEMICAL
INVESTIGATIONS ON THE POD EXTRACT OF Cassia fistula Linn. is
an authentic record of the original research work carried out by me under the
supervision and guidance of Dr. Oommen P. Saj, Associate Professor of
Botany, University College, Thiruvananthapuram in partial fulfilment of the
requirements for Degree of Master of Science in Botany of the University of
Kerala and further that no part it has been presented earlier for any degree or
diploma in any of the Universities.
Tvpm Sumi S.
August 2011 MSc Botany
University college
Thiruvananthapuram
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ACKNOWLEDGEMENT
In the first place I would like to thank our God almighty and to my
parents who have shown keen interest through my carrier. My endeavour stands
incomplete without dedicating my gratitude to a few people who have contributed a lot
towards the successful completion of my project work.
This work is carried out at Department of Botany, University
college, Thiruvananthapuram under the supervision of Dr. Oommen P. Saj, Assosiate
Professor of Botany University College, Thiruvananthapuram. I express my indebtedness
and gratitude towards him for his liberal mind, compassion, constant encouragement and
continual support throughout the course of the investigation.
I express my sincere thanks to Dr. Premlal, Principal, University
College ,Thiruvananthapuram and Sri. V.A. Saseendhra Babu , Head ,Department of
Botany, University College ,Thiruvananthapuram for providing necessary facilities to
carry out the present work.
I express my gratitude to all my teachers of Department of
Botany, University College, Thiruvananthapuram. I also thank the laboratory staff of
Botany, University College, Thiruvananthapuram for proper assistance during the course
of this work.
I also express my thanks to Miss. Vinni K.V, Miss. Ayanaravi, and
other M .Phil and PhD students for the encouragement and co-operation rendered during
this project work.
I am thankful to all my classmates and friends for their valuable
support and help during the course of work.
Once again I thank all who gave support to complete the project
successfully
Sumi S.
Thiruvananthapuram ,
August 2011
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CONTENTS
1. INTRODUCTION
2. REVIEW OF LITERATURE
3. MATERIALS AND METHODS
3.1. Taxonomic Position
3.2. Morphology
3.3. Collection of Material
3.4. Preparation of Extract
3.5. Phytochemical Screening of plant
3.5.1 Test for Reducing Sugar
3.5.2 Test for Flavonoid
3.5.3 Test for Steriods and Terpenoids
3.5.4 Test for Tannin
3.5.5 Test for Coumarin
3.5.6 Test for Saponin
3.5.7 Test for Anthraquinone
3.5.8 Test for Glycosides
3.5.9 Test for Alkaloids
3.5.10 Test for Phlobotannin
3.5.11Test for Iridoids
3.6 Quantitative test for plant tissue
3.6.1 Determination of Moisture
3.6.2 Total Carbohydrate
3.6.3 Estimation of Protein
3.6.4 Total Phenol Estimation
3.6.5 Estimation of Tannin
3.6.6 Test for Chlorophyll
3.6.7 Test for Aminoacid
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3.7 Chromatographic Studies
3.7.1 Thin Layer Chromagraphy
3.7.2 Preparation of chromoplate
3.7.3 Application of Samples on chromoplate
3.8 Anthelmintic Studies
3.9 Antibacterial Study
3.9.1 Sterilization of glasswares
3.9.2 Preparation of disc for bacterial study
by disc diffusion method
3.9.3 Preparation of Nutrient agar media for
Bacterial growth
3.9.4 Preparation of peptone water for
subculturing the bacteria
3.9.5 Inoculation
4. RESULTS
4.1 Phytochemical analysis of pod extract
4.2 Phytochemical analysis by TLC
4.3 Anthelmintic activity
4.4 Antibacterial study
5. DISCUSSION
6. SUMMARY AND CONCLUSION
7. REFERENCES
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Abstract
Cassia fistula Linn. (Leguminosae) is a tree distributed throughout
South India. It is known as Golden shower tree. Only a few works were done
previously on Cassia fistula pod. Present study is concentrated on
antimicrobial, anthelmintic and phytochemical aspects of the Cassia fistula
pod. For this the fruit extracts were used. The methanolic extract of Cassia
fistula seed was investigated for potential antimicrobial activity against
medicinally important bacteria. The extract from the pods of Cassia fistula
Linn. were investigated for their anthelmintic activity. Four concentrations
(1%, 2%, 5%, crude, albendazole ) of extract were studied, which involve
determination of time of paralysis and death of worms. As the plant possess
several secondary metabolic compounds, it provides protection against insect
attack and invasion of pathogen. These properties can be investigated by a
thorough analysis. So that the economic potentialities can be exploited
commercially.
Key words:
Cassia fistula Linn. Pod extract, Anthelmintic activity, Antimicrobial activity
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1. INTRODUCTION
Plants are the gift of nature.Without plants life is not possible on earth,
as they provide all the necessary amenities for the life. Research in medicinal
plants is yielding as entitled as and will go on yielding much information which
is likely to be used in medicine. Medicinal plants and herbs continue to be the
source of proven medicines and new revolutionary drugs. Plants identified as
“Rasayans” in Indian Ayurvedic system of medicine have various
pharmacological properties, an entire section of Materia Medica is devoted to
drugs entitled as „Rasayanas‟(Chopra et al,1996).
Based on current research on medicinal plants, it is proved that they will
play important roles in human health. The use of various herbal remedies and
preparations are described throughout human history representing the origin of
modern medicines, mainly as self prescribed products. This type of usage of
herbal drugs is typically based on simple matching of a particular herbs to
particular disease or symptoms (Lee, 2003).
Two drastic diseases that widely affect human population are cancer and
parasitic worm infections. World Health Organization (WHO) estimated that
staggering two billion people harbour parasitic worm infections. The parasitic
worms also infect livestocks and crops, affecting food production with a
resultant economic impact. Despite this prevalence of parasitic infections,
research on anthelmintic drug is poor as per WHO, only few drugs are
frequently used in the treatment of these parasitic infections. Cancer is another
serious disease which widely spread among populations (Aswarmanoj et al.
2008; Pagaria et al. 2009; Martin, 1997). Over three quarter of the world
population relies mainly on plants and plant extract for health care. More
than30% of the entire plant species, at one time or other was used for medicinal
purposes. It is estimated that world market for plant derived drugs may account
for about Rs 2,oo,ooo crores. Presently, Indian contribution is less than
Rs.2000 crores. (Chimmad et al.)
The growing awareness of the harmful side effects of chemotherapy has
made people to explore the time tested remedies from traditional alternative
medicine. India being a tropical country blessed with vast natural resources
and ancient knowledge for its judicious utilization (Basu, 2002). It perhaps the
largest producer of medicinal herbs and is rightly called the Botanical Garden
of the world. Tribal people are the true conservationist of forests and they have
their own indigenous ways of protecting and managing the plant resources. The
9
dynamic flora of India is matched by some 400 different ethanic groups of
varying levels of accualisation.
The plant in our fields and forests are amazing chemical factories.
carbohydrates, fats, and proteins are the plant products we know best. The
plants manufacture these from the carbon dioxide of the air plus the water and
the minerals of the soil, and store them away in roots, stems, leaves, and seeds.
But some plants make other highly complicated chemicals that are extremely
valuable to us as medicines (Millient, 1977).
The forest in India is the principal respository of large number of
medicinal and aromatic plants, which are largely collected as raw materials for
manufacture of drugs and perfumery products. About 8,000 herbal remedies
have been codified in Ayurveda. The Rigveda (5000BC) has recorded 67
medicinal plants, Yajurveda (1,400-1000 BC) 81 species , Charak Samhita
(700 BC) and Sushrut Samhita (200 BC) had described properties and uses of
1100 and 1270 species respectively, in compounding of drugs and these are
still used in the classical formulation in the Ayurvedic system of medicine.
Unfortunately, much of the ancient knowledge and many valuable plants are
being lost at an alarming rate. With the rapid depletion of forests, impairing the
availability of raw drugs, Ayurveda, like other systems of herbal medicines
has reached a very critical phase.
Plant cells produce a vast amount of secondary products. Many of these
are highly toxic and are often stored in specific vesicles or in the vacuole.
Several studies indicate that this kind of storage functions on one hand as a
detoxification of the plant itself and generates on the otherhand a reservoir of,
for example , nitrogen rich molecule. Some secondary plant products can be
reversibly degraded and are fed into the basic metabolism while others cannot.
Although secondary plant products are very common, this does not mean that
every plant can produce every product. Some compounds are restricted to
single species, others to related groups.
Most of the plant resources, especially in the tropical regions, are rich in
plant secondary metabolites, and the lack of information on the appropriate
methods for their determination has been the main bottle neck in better
understanding the enzymes and biochemical pathways in their synthesis, the
genes responsible for controlling major biochemical processes and the
physiological significance of plant secondary metabolites and the exploiting the
beneficial effects of these phytochemicals. Secondary metabolites produced
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are defensive in function so they may produce negative effects on other
organisms. This can be exploited and used against many pathogenic
organisms. Antimicrobial substances are present in almost all plant species and
several studies have been conducted which revealed the presence of
compounds with antimicrobial properties in various plant parts
(Aswal et al. 1984).
Most of the plants possess various pharmacological properties such as
antiaging, anticancer, antistress, antibacterial etc. Cassia fistula Linn is one of
such plant which is reported as tonic and used in various disease conditions.
Leaves are the most complex organs of plant, where much of plants chemical
activity, including photosynthesis is carried out. As a result leaves get
accumulated with various primary and secondary metabolites (Martin, 1997).
Cassia fistula Linn is cultivated throughout tropical countries of the
world, from West Indies to India, from South Africa to Egypt(Wart, 2000) .The
plant was even referred to as „purging cassia‟ in Europe in the thirteenth
century (Thomas, 1988). Cassia fistula has been used extensively in the
folklore medicine for the treatment of a variety of diseases. The pulp of ripe
fruits has a mild, pleasant purgative action and is also used as an anti-fungal
drug (Kasuko and Nagayo, 1951).
Butanol extract of the residue of Cassia fistula from a 70% alcohol
fraction has been shown to have anti- viral effect, while hot water extracts have
proved useful in the treatment of uterine, menstrual disorders and fever (Kaji
and Khorara, 1965; Sen and Sukla,1968). Other ethanomedical uses of
Cassia fistula plant include its anti-diarrhoeal and anti-dysentery
(Manandhar, 1989) and in the treatment of diabetes and skin diseases(Chopra
et al. 1992). Recently its hepato –protective activity has been evaluated ( Batna
et al. 2001).
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2. REVIEW OF LITERATURE
Traditional medicine can be a very important source of
active plant constituents. The smoking of Datura in case of asthma, use of
Nuxvomic, in paralysis and the use of croton as a purgative can be definitely
traced to have originated from ancient India. In some countries like India
pharmaceutical companies are already marketing preparation of tablets, capsule
etc; made directly from the appropriate plant extract for the treatment of
specific disease like Hepatitis. The expansion of those markets, while being a
commendable goal, accelerate the depletion of the resources.
Inhibitory substance present in the plant exudates of plants
directly affect the microorganism or encourage certain groups to dominate the
environment and functions as antagonists as pathogen. Plant materials contain
specific bioactive ingredients, used in Ayurvedic, Sidha and Unani school of
medicine. Various extract have been shown to be beneficial in the control of
Hepatitis and in some cases specific antiviral, antibacterial, antimalarial and
immune modulating factors have been implicated, which are very potential
antiviral, antibacterial, antimalarial ingradient. Almost all plant species produce
antimicrobial substances. Several studies have been conducted which reveal the
presence of compounds with antimicrobial activities in various plant parts
(Aswal et al. 1984).
Plants have well defined mechanism by synthesising
defence substance. A number of other biologically active compounds like
ephedrine, hordenin, mescaline cucohygrine, rieinune, piperine, coline,
colchicines, etc. are extracted from other plants (Chan et al. 1990). Alkaloids
having the antimicrobial activity now used for preparing medicine to various
diseases in humans and animals.
Due to increasing development of drug resistance in
human pathogen as well as the appearance of undesirable effect of some
antimicrobial, there is a need to search for new agents. Many reports have
shown that some Cassia species contain antimicrobial substances particularly
Cassia fistula Linn. (Fuzelliier et al. 1982, Crockett et al. 1992, Agarkar and
Somchit et al. 2003). Many Cassia species are grown as ornamental plants
throughout Thailand (Gritsanapan and Nualkaew, 2001).
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Cassia fistula Linn. Cassia alata Linn. and Cassia tora
Linn. (Leguminosae) are recommended for primary health care in Thailand to
treat ringworm and skin diseases (Farnsworth and Bunyaprapatsara, 1992).
Among the three species of Cassia, Cassia alata Linn. (Leguminosae) has most
activity (Souwalak, 2004).
The use of herbal medicine predates the introduction of
antibiotics and predates social, economic, and religious barriers. Cassia fistula
Linn. is locally used in Nigeria in the treatment of several infection which
included ringworm, parasitic skin diseases, (Dalziel, 1956; Palanichami et al.
1990). The leaves are reported to be useful in treating convulsion, gonorrhoea,
heart failure, abdominal pains, Oedema and also used as purgative
(Ogunti et al. 1993).
A study in Malaysia (Ibrahim et al. 1995) reported that
ethanolic extract of Cassia fistula Linn. showed high activity against
dermatophytic fungi, Trichophyton mentagrophytes var. Intergigitale,
Trichophyton mentagrophytes var. Mentographytes, Trichophyton rubrum and
Microsporium gypseum (MIC:125 mg/ml) and Microsporium canis (MIC:25
mg/ml). Several studies (Akinsinde et al. 1995; Akinyemi et al. 2000) have
been conducted to provide scientific basis for the efficiency of plants used in
herbal medicine.
Cassia fistula Linn has been reported to contain
anthraquinone the principal laxative constituent of many plants used as
purgative (Ogunti et al. 1993). The dichloromethane fraction of the flower
extract was found to be the most effective (Khan et al. 2001).
The essential oil obtained by hydrodistillation of leaves of
Cassis fistula Linn was analysed using capillary gas chromatography. Forty
four compounds representing 5.2% of the oil were identified, among them
linalool (23.01%) borneol (86.6%) and pentadecanal (9.3%) were major
constituents. The antioxidant activity of the oil was found to be low compared
to be that of butylated hydroxytoluene (Agnaniet et al. 2006).
The extract of Cassia alata Linn plant contain many
compounds such as anthraquinone, Chrysoeriol-7-0-(2”-0-beta-D-
mannopyranosyl)-beta-D-allopyranoside, rhammetin-3-0-(2”-0-beta-D-
mannopyranosyl), Physcione, Kaempferol, Kaempferol-3-gentiobioside, Aloe
emodin, Chrysophanol, Deoxycoelutain, Sennoside A, Sennoside B,
Sennoside C, Sennoside D, rhein, rhein methyl ester diacetate, beta-sitosterol,
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isochrysophanol, 4,5- dihydroxy-2-hydroxyantron, 4,5-dihydroxy-1-
hydroxyantron, emodin, galactomannan, chrysophanic acid,
anthraquinones-glycosides etc (Zhari, 1999; Moriyama et al. 2003).
From the chloroform extracts of seed kernels of
Caesalpinia crista Linn shows two methyl migrated cassane-type
furanoditerpenes, caesalpinnis and two normal cassane type furnoditerpenes
were isolated together with eight known cassane type diterpenes (Kalauni et al.
2006).
The search for biologically active compounds has been
vigorous in recent years due to the growing cases of microbial resistance to the
time honoured antibiotics (Dimayuga et al. 1991). Many of the active
compounds have been shown to have antimicrobial properties (Crowan, 1999).
Phospholipase C gamma inhibitory activities of prenylated flavanoids isolated
from Erythrina senegalensis (Leguminosae) were reported (Fernandes, 1996).
Glycrrhiza glabra (Leguminosae) was tested for its
antiulcer and antioxidant activity in rats (Batna, and Balaraman, 2006).
Pepticare possesses antiulcer activity, a herbomineral formulations of the
Ayurveda medicine consisting of herbal drugs. The nutrient composition was
similar to other conventional legumes. Anti nutritional factors such as tannins
(10.71 mg/g), free phenols (2.27 mg/g) phytates (2.16 mg/g) and trypsin
inhibitors could be substantially eliminated by soaking pressure, cooking
(Ghasolia and Jain, 2005).
The studies had revealed that different solvent extracts of
the Cassia fistula Linn leaf showed different bioactivities but all did cause an
immediate decrease in motor activity, hyperanemia, micturition, diaorrhea etc.
The hexane extract exhibited an analgesic and anti-inflammatory activity,
chloroform extract gave anti-mutagenic effect while ethyl acetate extract
showed anti inflammatory activity (Khan et al. 2001).
Analgesic activity of Cassia alata Linn leaf extract was
tested against mice, maximum analgesic activity of extract was apparent 120
minute after injection. The compound namely kaempferol-3-sophoroside
influence the analgesic activity (Palanichamy and Nagarajans, 1990).
The aqueous extract of Bauhinia forficate was able to
neutralize the serine protease involved in cloating activity (Oliveira et al.,
2005).
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Antimutagenic activity is also present in the extract of
Cassia alata Linn leaf. Antimutagenic activity guided fractionation of an
extract prepared from the thorns of Gleditsia sinensis ( Leguminosae) involve
the isolation of one triterpenoid and four steroid ( Lim et al. 2005).
A combination of the ethanolic extracts of Cassia fistula
leaf Ocimum sanctum have shown anticryptococus activity (Somachit et al.
2003). A preliminary finding in animal studies suggested that Cassia fistula
Linn. had a positive fertility effect on the reproductive functions of female
mice (Azmahani et al. 2001).
Cassia fistula Linn. has been used extensively in the
folklore medicine for the treatment of a variety of diseases. The pulp of the ripe
fruits has a mild, pleasant, purgative action and is also used as an antifungal
drug (Kasuko and Nagayo, 1951). Butanol extract of the residue of the Cassia
fistula Linn. from a 70% alcohol fraction has been shown to have antiviral
effect, while hot water extract has proved useful in the treatment of uterine,
menstrual disorder and fever (Kaji and Khorara 1965 ; Sen and Shukla 1968).
Other ethanomedical uses of Cassia fistula Linn. plant include its
antidiarrhoeal and antidysentery (Manandhar 1989) and in the treatment of
diabetics and skin diseases (Chopra et al. 1992).
The aqueous extract of the pods of Cassia fistula Linn
cultivated in lle-lfe Nigeria were investigated for pharmacological and
toxicological properties. The in vitro effect of Cassia fistula Linn. infusion on
isolated guinea pig ileum was examined. The acute and subchronic toxicity of
the infusion of Cassia fistula Linn and Cassia accutifolia Del. pod as the
reference drug (senokot tablet) were also determined (Bakta et al. 2001)
The seed of Cassia fistula Linn is used as anthelmintic.
The ethanolic extract of Caesalpinia bonducella seed kernel has been subjected
for its antipyretic and antinociceptic in a adult albino rats or mice. Caesalpinia
bonducella seed kernel possess potential antipyretic and antinociceptic
activities and thus validates its use in the treatment of pain and pyretic
disorders (Archana et al. 2006).
Hyperglycaemic effect was also reported in Cassia fistula
Linn pod extract oral effectiveness of Cassia fistula Linn mesocarp on
stereptozotocin induced hyperglycemia in rats has been studied and result
compared with glybenclinide (Palanychami et al. 1988) reduce the blood sugar
level. Aqueous and ethanolic extracts of Ceasalpinia bonducella showed potent
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hypoglycaemic activity in chronic type 2 diabetic model (Chakrabarthi et al.
2005). So tribal people of Andaman and Nichobar island used this as a remedy
for Diabetics mellitus.
Anti inflammatory effect of flavonoids isolated from
Caesalpinia pulcherrima Linn. Anti inflammatory activities for the first time of
flavonoids namely 5,7-dimethoxy flavonone (1), 5-7-dimethoxy flavonone (2),
isobonducellin (3), 2-hydroxy-2,3,4, 6-tetramethoxy- chalcone (4) and
bonducellin (5). All of them isolated from this plant was studied in
Lipopolysaccharide and interferon. These compounds significantly and dose-
dependently inhibited inflammatory mediators (Rao et al., 2006). Anti-
inflammatory activities of heat treated Cassia fistula leaf extract and kaemferol
3-0-genitiobioside (K3G) isolated from Cassia fistula as an abundant flavonoid
glycoside were studied by comparing their activities of sun dried Cassia fistula
leaf extract. Observed strong inhibitory effects on concavalin-A induced
histamine release from rat peritoneal exudates cells both in the extract of heat
treated and sun dried Cassia fistula leaf. Furthermore, the heat treated leaf
extract exhibited stronger inhibitory effects than the effects of the sun dried
leaf extract at low concentration in studies of concanavalin A induced
histamine release, 5-lipoxygenase inhibition and also inhibition of cyclo
oxygenase, where as kaemferol 3-0,gentiobioside showed weak inhibitory
effect of concanavalin A induced histamine release 5 liproxygenase
( Moriama et al ., 2003).
The antidiarrhoeal potential of the ethanolic extract of
stem bark of Butea monosperma has been evaluated using several experimental
model. The extract inhibited castor oil induced diarrhoea and also reduced
gastro intestinal motility after charcoal meal administration. Use of this herbal
remedy as a non specific treatment for diarrhoea in folk medicine
(Gunakkurnu et al. 2005).
The methanol extract of stem bark of Bauhinia recemosa
show anti-inflammatory, analgesic and antipyretic effect (Gupta et al., 2005).
The ethanol extract Indigofera tinctoria shows chemopreventive effect of
N-nitrosodiethyl amine induced phenobarbitol promoted liver tumours in rats
(Joshi, 1996).
The methanol extract of Cassia fistula seeds were
investigated for potential antimicrobial activity against medicinally important
bacterial, yeast ad fungal strains. The antimicrobial activity was determined in
16
methanol extract using disc diffusion technique and broth dilution method. The
extract was effective on tested microorganism and the minimum inhibitory
concentration values were found in the range of 1.563 - 50.00 mg/ml. Apart
from that the methanolic extract Cassia fistula seeds was further tested for in
vivo brine shrimp lethality test. The test exhibited no significance toxicity
against Artemia salina. The Cassia fistula seeds extract with high LC50 value
signified that this plant is not toxic to human ( Bhattachatargee, 1998).
17
3. MATERIALS AND METHODS
3.1 TAXONOMIC POSITION
According to Bentham and Hooker’s system of classification (1866), the
systematic position of Cassia fistula Linn is as follows
Class - Dicotyledonae
Sub class - Polypetalae
Series - Calyciflorae
Order - Rosales
Family - Leguminosae
Sub family - Caesalpiniaceae
Genus- - Cassia
Species - fistula Linn.
3.2 Morphology.
It is a deciduous, medium sized tree with a gray, smooth, exfoliating bark. 4-8
pairs of leaflets are seen, distinctly stalked, oblong or ovate, with a silvery
pubescence. The flowers are bright yellow, in axillary, pendulous, raceme; the
pods are cylindrical, smooth, hard, dark brown or black; the seeds light brown,
hard, shiny, biconcave and are embedded in sweetish pulp.
3.3. Collection of material
Cassia fistula Linn. pods were collected from the local areas of Kollam
and also collected from Palayam. Collected materials were cleaned, weighed,
and dried in shade. The dried material was again weighed and powdered and
stored for further works.
3.4. Preparation of extract
The phytochemicals present in the plant material was extracted by the
distillation method using soxhlet apparatus. The solvent, methanol was used for
the separation of chemical component. About 100 gm of plant tissue were
weighed and shade dried for 10 days. The dried materials were powdered and
50gm of powder sample was packed in a thimble and kept in soxhlet apparatus.
The whole apparatus was kept over a heating mantle and was heated
continuously for 8 hours at boiling point of each solvent. The extract was
18
concentrated to dryness and the residues were transferred to a preweighed
sample bottle and were stored in a desiccator for further studies
( Harborne, 1973).
3.5. Phytochemical screening of plant extracts
Different biochemical parameters like reducing sugar,
Flavonoid, Terpenoid, Tannin, Coumarin, Saponin, Anthraquinone, glycosides,
phlobotannin, iridoids, alkaloids etc. are tested.
3. 5. 1. Test for reducing sugars (Fehling’s test)
The aqueous methanol extract was added to boiling Fehling
solution in a test tube, a brick red colour indicates the presence of reducing
sugars.
3. 5. 2. Test for flavonoids (Shinoda test)
Take 1ml of the extract and add a few magnesium turnings,
followed by the addition of con. HCl drop by drop. Development of pink
colour indicates the presence of flavonoids.
3. 5. 3. Steroids and Terpenoids (Libermann-Burchard Test)
Take 2 ml of extract, dry and dissolve in chloroform. Add a
few drops of acetic anhydride and conc.H2SO4 and keep undisturbed for few
minutes. Formation of green colour indicates the presence of steroids, while
pink colour indicates the presence of terpenoids.
3. 5. 4. Test for Tannins
To 1ml of the extract, add 2 drops of 5% FeCl3. Presence of
dirty green precipitate indicates the presence of tannin.
3. 5. 5. Coumarin
1 ml of the extract was dissolved in methanol followed by
the addition of a few drop of alcoholic NaOH . Concentrated HCl was added
through the sides of the test tubes which shows the appearance and
disappearance of yellow colour, indicates the presence of coumarin.
3. 5. 6. Test for saponin
5 ml of the extract was shaken with 5ml of distilled water
and was heated to the boiling point. Froathing indicates the presence of
saponin.
19
3. 5. 7. Test for Anthraquinones
To 0.5 gm of powdered material add 10 ml of 1% HCl and
boiled for 5 minutes. Filter the sample and allowed to cool. Partition the cool
filtrate against equal volume of chloroform. Carefully transfer the chloroform
layer into clean test tubes. Shake with equal volume of 10% ammonia solution
and allow the layer to separate. Presence of delicate rose pink colour indicates
the presence of combined anthraquinones.
3. 5. 8. Glycosides (Killer-Killiani Test)
To 0.5 gm of extract diluted to 5ml with distilled water and
add 2ml of glacial acetic acid and containing one drop of ferric chloride
solution .This was underplayed with 1ml of conc. H2SO4. Brown ring at the
interface the presence of glycosides.
3.5.9 Test for Alkaloids.
5 ml of extract evaporated to dryness. Residue heated on a
boiling water bath with 2% HCl. Then filtered, treated Mayers reagent. Yellow
precipitate indicating the presence of alkaloid.
3.5.10 Test for phlobotannin.
Take 1 ml extract and boiled with 5 ml of 1% HCl red
precipitate showed the presence of phlobotannin.
3.5.11 Test for Iridoids.
Take extract, add 1ml reagent like Asperulin, Accubin and Monotropin. The
mixture was heated over a flame. Development of a light blue colour indicated
the presence of iridoids.
3.6. Quantitative test for plant tissue
3. 6. 1. Determination of moisture
5 gm of material was taken in a pre-weighed petridish. The
petridish was placed without lid into an oven at 1100C for three hours. The
petridish was taken out and closed immediately with a lid. The dish was cooled
in a desiccator and weighed. The amount of moisture of the material was
calculated from the difference in weight (Sadasivam, 1987).
3.6.2 Total carbohydrate
Weighed amount of fresh tissue was homogenized with
distilled water. The homogenate was filtered using a two layered cheese cloth.
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The filtrate was then centrifuged at 10,000gm for 15min. The supernatant was
collected and the volume was made up to 25ml using distilled water. An
aliquot of sample was pipetted out and 4ml Anthrone reagent added. It was
then kept in a boiling water bath for 10 min.The tubes were cooled and the
absorbance was measured at 530nm. The amount of total carbohydrate present
was determined using the standard graph of glucose ( Roe et al.1965).
3. 6. 3. Estimation of protein
Total protein present in the plant was estimated by Lowrey‟s method .
1 gm powdered plant material was homogenized in 5ml of 0.1 M PO4 buffer.
The homogenate was filtered through double layered cheese cloth and
centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected and
the volume was made up to 1.5ml by PO4 buffer. After that 1.5ml of Bradford
reagent was added and kept it for 5 minutes. The absorbance was recorded
spectrophotometrically by using appropriate blank at 595nm.The protein
content was calculated from the standard graph of BSA or Bovine Serum
Albumin (Lowrey, 1959).
3. 6. 4. Total Phenol Estimation
Estimation of total phenols was performed by the method of Mayr et al.,
(1995).Weighed amount of fresh tissue was chopped and put in boiling
methanol (80%) for 10 minutes and refluxed. The refluxed matter was
homogenized. The homogenate was filtered and centrifuged at 10,000 rpm for
10 min. the supernatant was collected and the volume was made up to 20 ml
using 80% methanol. 0.5ml Folin –Ciocalteau reagent followed by 2ml 20%
sodium carbonate was added to an aliquot of sample and mixed thoroughly.
The reaction of phenol with phosphomolybdic acid in Folin –Ciocalteau
reagent in alkaline medium produced a blue coloured complex. The tubes were
kept in a boiling water bath for one minute, cooled and centrifuged, supernatant
was taken and the absorbance was measured at 650 nm. The amount of total
phenols present was determined using a standard graph of catechol
(Malick, 1980).
3. 6. 5. Estimation of tannin
Tannin present in the sample was estimated by
Folphosphotungsto molybdic acid in alkaline solution to produce highly
coloured blue solution, the intensity of which is proportional to the amount of
tannin. The intensity is measured at 700nm. 2gm of powered tissue was taken
21
in a 250ml conical flask and 75ml distilled water was also added. It was then
heated gently for 30 minutes and centrifuged at 2000rpm for 20 minutes. The
supernatant was collected and made upto 100ml with distilled water. An
aliquot of 1ml extract was made up to 75ml with distilled water. 5ml Folin-
Denis reagent and 10 ml sodium carbonate was also added. Then the solution
was diluted to 100ml with distilled water. The absorbance was recorded
spectrophotometrically at 700nm after 30 minutes against suitable blanks. Total
amount of tannic acid was calculated from the standard graph of tannic acid
(Sadasivam, 1987).
3.6.6 Test for Chorophyll
Take 1gm tis