PHYTOCHEMICAL SCREENING AND EVALUATION OF ANTI-BACTERIAL AND

ANTHELMINTIC ACTIVITIES OF BARK OF PLANT CASSIA FISTULA

ABSTRACT

Plants are of great value in the field of treatment and cure of diseases. Interestingly, it is
estimated that more than 25% of the modern medicines are directly or indirectly derived from
plants. Cassia (Caesalpiniaceae) is one of the largest genera with about 2500 species and has
drawn the attention worldwide. It has been reported that it possesses several pharmacological
actions like anti-bacterial, anthelmintic, hepatoprotective, anti-inflammatory, antitussive,
antifungal, anti-oxidant and wounds healing properties. This study deals with phytochemical
screening and evaluation of anti-bacterial and anthelmintic activities of the bark extracts of
Cassia fistula. Microscopic and macroscopic studies, loss on drying, extraction with different
solvents (methanol, acetone, distilled water) had been carried out. The anti-bacterial assay was
done against two gram +ve and two gram –ve bacteria by agar cup diffusion method. The
anthelmintic activity was done on Pheretima posthuma(earthworm) by recording the paralyzing
and death time under different concentrations. The loss on drying was calculated to be 17%.
Maximum extraction yield was given by methanol. The major chemical constituents of the bark
is reported to be anthraquinones and dihydroxyanthraquinones. Along with these glycosides,
tannins, saponins, flavonoids, terpenoides were also present. The test for alkaloids and steroids
was negative. Methanolic extract showed the maximum activity, be it anti-bacterial or
anthelmintic. The extract was effective only against gram +ve bacteria. Among Staphylococcus
aureus and Streptococci faecalis, the former was more susceptible. The activity against gram –ve
bacteria was nil. In case of anthelmintic activity, the concentration of 50 mg/ml was effective
and near to the paralyzing and death time as recorded for the 10 mg/ml concentration of the
standard drug. However, aqueous extract was more effective than the acetone extract.
 CHAPTER 1
INTRODUCTION

1 Introduction

1.1 Background

From the beginning of human civilization people are indebted to nature in many ways 1. Among
the various gifts of nature, plants are the essential ones. Plant species are used for medicinal
purposes as well as human diet and also as preservatives 2. Medicinal plants are of great value in
the field of treatment and cure of diseases3.
Over the last few years, researchers have aimed at identifying and validating plant-derived
substances for the treatment of various diseases. Interestingly it is estimated that more than 25%
of the modern medicines are directly or indirectly derived from plants. It is worth mentioning
that Nepalese medicinal plants are considered as a vast source of several pharmacologically
principles and compounds that are commonly used as home remedies against multiple ailments 4.
Since early 1990s, the use of forest products for medicine has been emerging as a vital income
generating resource for the development of various social groups, hence, there is an increased
attention for their long-term sustainability 5.
Nepal is regarded as one of the main source of medicinal plants since Vedic age and Nepal
possesses 2.2% of the total global flora. Among them 245 flora are endemic. The flora of Nepali
Himalaya contains 10,167 of which over 7,000 are flowering plants and over 1600 species are
medicinal and aromatic herbs 6, 7.
The use of medicinal herbs in Nepal dates back to at least 500 AD, medicinal and aromatic
values which are being used traditionally as medicine while some are still to be explored
scientifically for their medicinal value. Traditional medicine in Nepal has strong cultural and
religious background. It exists in different ways such as ethnic and tribal group, ritual or
ceremonial practices, spiritual practices, diet or self-healing. Indigenous and local communities
have been using traditional knowledge under local laws, customs and traditions. Traditional
medicine practitioners in Nepal are accepted as traditional healers: their indigenous knowledge
and technology have been transferring from generations. They are the major contributors of
health services in Nepal. People have belief on their knowledge and faith on their formulations.
It is most reliable, easy to access, affordable, and comparatively more effective system if
formulations are followed sincerely 6, 8.

The World Health Organization estimated that 80% of the populations of developing countries
rely on traditional medicines, mostly derived from plant sources, for their primary health care
needs. Modern pharmacopoeia still contains at least 25% drugs derived from plants and many
other synthetic analogues built on plants derived prototype compounds 9. At present there are
many chemical substances derived from plants that are considered as important drugs. Several of
the drugs used today are simple synthetic modifications or copies of the naturally obtained
substances 10.
Traditional medicine is based on various systems including Ayurveda, Siddha, Unani and
Homoeopathy. The evaluation of these drugs is primarily based on phytochemical,
pharmacological and allied approaches including various instrumental techniques such
chromatography, microscopy and others. With the emerging worldwide interest in adopting and
studying traditional systems and exploiting their potential based on different health care systems,
the evaluation of the rich heritage of traditional medicine is essential 11.
Cassia (Caesalpiniaceae) is one of the largest genera with about 2500 species widely distributed
throughout the tropical and subtropical regions of the world. This genus is prized for its
agricultural, economical, and medicinal virtues. Hence Cassia has drawn the attention
worldwide. One such important medicinal plant species which belongs to this genus is Cassia
fistula 12.
Cassia fistula Linn. (Cassia) family Caesalpiniaceae (Fabaceae) commonly known as Amulthus
and in English popularly called “Indian Laburnum” has been extensively used in Ayurvedic
system of medicine for various ailments. It is deciduous and mixed-monsoon forests throughout
greater parts of India, ascending to 1300 m in outer Himalaya. It is widely used in traditional
medicinal system and has been reported to possess hepatoprotective, anti-inflammatory,
antitussive, antifungal, anti-bacterial, anthelmintic, anti-oxidant and wounds healing properties
11
.
1.2 Plant profile

Scientific classification 13
Kingdom: Planate
Subkingdom: Tracheobinota
Super Division: Spermatophyta
Division: Mangoliophyta
Class: Magnoliopsida
Sub Class: Rosidae
Order: Fabales
Family: Fabaceae
Genus: Cassia
Species: fistula

Vernacular names 13
Nepali Rajbrikshya
Bengali Bundaralati, Sonalu, Soondali, Sondal
English Indian Laburnum, Purging Fistula, Cassia, Golden Shower.
Hindi Sonhali, Amultus
Sanskrit Nripadruma
Arab Khayarsambhar
Oriya Sunaari

Urdu Amaltaas

1.3 Plant description

1.3.1 Morphology

It is a deciduous tree with greenish grey bark, compound leaves, leaf lets are each 5-12 cm long
pairs. A semi-wild tree known for its beautiful bunches of yellow flowers and also used in
traditional medicine for several indications. A fruit is cylindrical pod and seeds many in black,
sweet pulp separated by transverse partitions. The long pods which are green, when unripe, turn
11
black on ripening after flowers shed . Pulp is dark brown in colour, sticky, sweet and
mucilaginous, odour characteristic, and somewhat disagreeable 14. Drug occurs in flat or curved
thick pieces; outer surface smooth to rough with warty patches; greenish grey to red; inner
surface rough, reddish with parallel striations; fracture, laminate; odour, sweet and characteristic;
taste, astringent 15.
A tree 6-9 m high; trunk straight; bark smooth and pale grey when young, rough and dark brown
when old; branches spreading, slender. Leaves 23-40 cm long; main rhachis pubescent; stipules
minute, linear-oblong, obtuse, pubescent. Leaflets 4-8 pairs, ovate or ovate-oblong, acute, 5-12.5
by 3.8-9.5cm, bright green and glabrous above, paler and silvery-pubescent beneath when young,
the midrib densely pubescent on the underside, base cuneate; main nerves numerous, close,
conspicuous beneath; petiolules 6-10 mm long, pubescent or glabrous. Flowers in lax racemes
30-50 cm. long; pedicels 3.8-5.7 cm. long, slender, pubescent and glabrous. Calyx 1 cm long
divided to the base, pubescent; segments oblong, obtuse. Corolla 3.8 cm across, yellow; stamens
all antheriferous. The pods are pendulous, cylindric, nearly straight, smooth, shining, brown-
black, indehiscent, with numerous (40-100) horizontal seeds immersed in a dark coloured
sweetish pulp. Seeds broadly ovate, 8mm. long, slightly less in breadth, and 5mm thick 16. The
fruit pods are 40-70 cm long and 20-27mm in diameter, straight or slightly curved, smooth but
finely striated transversely, the striations appearing as fine fissures. The rounded distal ends bear
a small point marking the position of the style. The dorsal suture appears as a single vascular
strand and the ventral suture as two closely applied strands. Internally the pod is divided by thin,
buff coloured, transverse dissepiments at intervals of about 0.5cm. Each compartment contains
one seed which is flat, oval, reddish brown with a well-marked raphe. The seed contains a
whitish endosperm in which the yellowish embryo is embedded 17.

1.3.2 Geographical Distribution

The trees of Cassia Fistula are found in deciduous and mixed monsoon forests throughout greater
parts of India, Nepal, China ascending to 1300 m in outer Himalaya and inner hills. In eastern
region, it occurs as a scattered tree throughout the forest areas 11. The plant is cultivated as an
ornamental throughout India 18.

1.3.3 Chemical constituents 25

Table 1 Chemical Constituents of Cassia Fistula

Secondary metabolites Plant parts/organs

Oxyanthraquinone,dihydroxyanthraquinone Bark
 (-) epiafzelechin, (-) epiafzelechin-3-Oglucoside,(-) Leaves
epicatechin, procyanidinB2, biflavonoids, triflavonoids,
rhein,rheinglucoside, sennoside A,sennoside B,
chrysophanol, physcion

Kaempferol, leucopelargonidintetramer (with free glycol Flower

unit), rhein, fistulin, alkaloids, triterpenes

Rhein, volatile oil, waxy and resinousDerivatives Fruit pulp

Fistulic acid, 3-formyl-1-hydroxy-8-methoxyanthaquinone, Pods

3B-hydroxy-17-norpimar-8(9)-en-15-one

Chrysophanol Seed

Rhamnetin-3-O-gentiobioside Roots
1.3.4 Traditional Uses

11, 14, 16, 19, 20, 21, 22

The root is prescribed as a tonic, astringent, febrifuge and strong purgative . The
leaves extract reduced mutagenecity in E. coli 15. Extract of the root bark with alcohol can be
used for backwart fever. The leaves are laxative and used externally as emollient, a poultice is
11, 15, 19, 21
used for chilblains, in insect bites, swelling, rheumatism and facial paralysis . Leaves
possess anti periodic and laxative properties, the leaves are used in jaundice, piles, rheumatism
ulcers and also externally skin eruptions, ring worms, eczema. The leaves and bark mixed with
16, 21
oil are applied to pustules, insect bites . The roots are used in chest pain, joint pain, and
migraine and blood dysentery. The extract of the root lowered the blood sugar level up to 30 %
23
.
11, 16
Leaves and flowers are both purgative like the pulp . Ashes from burnt pods mixed with little
11
salt are used with honey taking 3- 4 times to relieve cough . Root is useful in fever, heart
diseases, retained excretions and biliousness 19. Fruits are used as catharatic and in snake bite.
20, 22
Juice of leaves is used in skin diseases . Flowers and pods are used as purgative, febrifugal,
biliousness and astringent. The ethanolic 50% extract of pods show antifertility activity in female
albino rats. The heated pods are applied to swellings on the neck due to cold. The fruits are
16, 21
reported to be used for asthma . Pulp is given in disorders of liver. The drug is used as
analgesic as an antipyretic, it is a remedy for malaria and fever. It is also applied in blood
poisoning, anthrax and antidysentric, leprosy and antidiabetic, for the removal of abdominal
obstruction 21. The extract of the flower inhibits the ovarian function and stimulate the uterine
function in albino rats. Fruits are used in the treatment of diabetes 15, antipyretic, abortifacient,
demulcent, lessens inflammation and heat of the body; useful in chest complaints, throat
troubles, liver complaints, diseases of eye and gripping 16. Juice of leaves is useful as dressing for
ringworm, relieving irritation and relief of dropsical swelling. The pulp of the fruit around the
seeds is a mild purgative 11, 16, 19, 21, 24. It is also used in biliousness and in diabetes. Externally, it is
11, 16, 19, 20, 21, 22
useful for evacuation in flatulent colic, as dressing for gouty or rheumatic joints .
The pith is particularly useful if there is swelling in stomach, liver or intestine. The seeds are
11, 16, 19, 20, 21, 22
emetic, used in constipation and have cathartic properties . The seeds are slightly
16, 21
sweet and possess laxative, carminative, cooling, improves the appetite , and antipyretic
activity. They are useful in jaundice, biliousness, skin disease and in swollen throat. A seed dried
produce marked hypoglycaemic activity 21. Seed powder is used in amoebiasis 18, 21
. The fruit
pulp is used for constipation, colic, chlorosis and urinary disorders 18. The bark possess tonic and
antidysentric properties, it is also used for skin complaints, the powder or decoction of the bark is
administered in leprosy, jaundice, syphilis and heart diseases. The aqueous extract of the root
bark exhibits anti-inflammatory activity. The root is used in cardiac disorders biliousness,
21, 24
rheumatic condition, haemorrhages, wounds, ulcers and boils and various skin diseases . The
stem bark is used against amenorrhoea, chest pain and swellings 15.

1.3.5 Anti-bacterials and Anthelmintics

Anti-bacterials are a type of drug used in the treatment and prevention of bacterial infections.
They may either kill or inhibit the growth of bacteria. A limited number of antibiotics also
possess antiprotozoal activity. Antibiotics are not effective against viruses such as the common
cold or influenza, and their inappropriate use allows the emergence of resistant organisms. Drugs
which inhibit viruses are termed antiviral drugs or antivirals rather than antibiotics. Sometimes
the term antibiotic (which means "opposing life") is used to refer to any substance used against
microbes, synonymous with antimicrobial. Some sources distinguish between antibacterial and
antibiotic; antibacterials are used in soaps and disinfectants, while antibiotics are used as
medicine.
Anthelmintics are a group of antiparasitic drugs that expel parasitic worms (helminths) and other
internal parasites from the body by either stunning or killing them and without causing
significant damage to the host. They may also be called vermifuges (those that stun) or
vermicides (those that kill). Anthelmintics are used to treat people who are infected by
helminths, a condition called helminthiasis. These drugs are also used to treat infected animals.
Pills containing anthelmintics are used in mass deworming campaigns of school-aged children in
many developing countries. For example, the treatment of choice for soil-transmitted helminths
is mebendazole and albendazole and praziquantel for schistosomiasis.
 CHAPTER 2
OBJECTIVES
2 General objective

The general objective of this study is to perform phytochemical screening which comprises of
the preliminary tests for the identification of various secondary metabolites like alkaloids,
glycoside, tannins, flavonoids, etc. Along with the identification, some biological evaluations are
also to be conducted.

2.1 Specific objectives

 To check the solubility of the grinded dry bark of Cassia fistula.

 To use different solvent system for extraction by soxhelation.
 To compare the yield obtained from different solvent extract.
 To determine the phytoconsituents.
 To study the antibacterial activity of different solvent extract of Cassia fistula.
 To study the anthelmintic activity of different solvent extract of Cassia fistula.
 CHAPTER 3
Literature Review

3 Literature review

Vimalraj, Kumar et al. 2009 conducted a study on anti-bacterial activity of the stem bark
extract of Cassia fistula and found that the alcohol extract showed greater inhibition against
S. aureus compared to aqueous extract which was tested to bacterial isolates include S.
aureus MTCC 443, Bacillus subtilis MTCC 441, E.coli MTCC443, and few field isolates.25
M. Irshad, Man Singh, et. al., conducted an assessment of anthelmintic activity of Cassia
fistula and found that the concentration of 100 mg/ml of extracts caused more significant
paralysis as well as death of worms as compared to reference drug Piperazine citrate at dose
of 10 mg/ml. The correlation coefficient between paralysis and death time of Pheretima
posthuma by seeds and pulp were 0.9986 and 0.9976 respectively. It was concluded that
Cassia fistula can be used as anthelmintic.26
Ahmed and Baig conducted a study on the anti-microbial efficiency of different solvent
extracts of Cassia fistula and found that the ethanolic and aqueous extracts of C. fistula could
be used as a potential anti-microbial source for various infections.27
Ilavarasan et. al., conducted a study and found Cassia fistula to have variety of
phytochemicals constituents. It is reported that, the presence of flavonoids in Cassia fistula
may be responsible for the anti-oxidant and anti-inflammatory effect possessed by the plant.28
Kulkarni, Govindappa et. al., conducted a study on evaluation of antioxidant activity of
different parts of Cassia fistula and found that the methanolic and ethanolic extract of
different parts of the plant were in order: steam bark> leaves> flower> pulp since they
yielded highest concentration of total phenolics 69.4% in steam bark and lowest in pulp
2.12%.29
M. Kadhim, I. Hameed et. al., conducted a study on in vitro antibacterial, antifungal and
phytochemical analysis of methanolic extract of fruit of Cassia fistula. Methanolic extract of
bioactive compounds of Cassia fistula was assayed for in vitro antibacterial activity against
eleven pathogenic bacteria by using the diffusion method in agar. The zone of inhibition
were compared with different standard antibiotics. The diameters of inhibition zones ranged
from 1.00±0.05 to 6.02±0.23 mm for all treatments.30
S. M. Seyyednejad, H. Motamedi et al., conducted a study on the anti-bacterial activity of
organic extracts of Cassia fistula. The extract of C. fistula was effective against B. cereus, S.
aureus, E. coli, and K. pneumonia. The most susceptible microorganisms to ethanolic and
methanolic extracts were E. coli and K. pneumonia respectively. The MIC and MBC of
ethanolic extracts against S. aureus, E. coli, S. epidermidis, and K. pneumoniae were also
determined.31
K. Gobianand, P. Vivekanandan et. al., conducted a study on anti-inflammatory and
antipyretic activities of Cassia fistula (Golden shower) in Wistar albino rats. They tested the
ethanolic extracts at various doses (50, 100, 250, 500 and 750 mg/kg by weight) for its anti-
inflammatory effect and the results were compared with standard drugs (diclofenac and
indomethacin). The results indicated that the ethanolic extracts significantly inhibited both
the carrageenan-induced hind paw oedema and cotton-pellet granuloma in a dose dependent
manner.32
T. Bhakta et al. (1998) reported that the methanol extract of leaves of C. fistula (collected
from India in 1995) was investigated for its effect on a cough model induced by sulfur
dioxide gas in mice. The extract exhibited significant, dose-dependent antitussive activity
compared with the control. The antitussive activity was comparable with that of codeine
phosphate, a prototypes antitussive agent. C. fistula extract (400 and 600 mg/kg, p.o.)
inhibited coughing by 44.44 and 51.85%, respectively, with respect to the control group 33.
U. K. Mazumder et al (1998) showed that the methanol extract of seeds of C. fistula was
tested for different pharmacological actions in mice. The extract significantly potentiated the
sedative actions of sodium pentobarbitone, diazepam, meprobamate and chlorpromazine. It
also potentiated analgesia induced by morphine and pethidine in a dose-dependent manner.
The extract also influenced behaviour in mice 34.
Kumar et al. (1998) studied that the methanol extract of fruits of C. fistula inhibited the 5-
lipoxygenase catalysed formation of leukotriene B4 in bovine polymorphonuclear leukocytes
(IC50 value of 38 micro g/ml). Lipid peroxidation in bovine brain phospholipid liposomes
induced with 2,2'-azo-bis-(2-amidinopropane) dihydrochloride (AAPH) was inhibited (IC50
of 40 micro g/ml). A linear correlation was obtained between the effects of the extract in the
2 assays suggesting a redox-based mechanism for the inhibition of the 5-lipoxygenase
enzyme 35.
M. J. Mukhopadhyay et al., anthraquinone glycosides of Cassia fistula were investigated
for their ability to induce a clastogenic effect on the bone marrow cells of Swiss albino mice.
The endpoints screened were chromosomal aberrations and frequency of aberrant cells. Oral
exposure to doses of these anthraquinones and their equivalent amount in leaf and pod
extracts did not induce significant numbers of chromosomal aberrations or aberrant cells. The
results indicate that anthraquinone sennoside B and rhein are weakly genotoxic. Pure
sennoside B and rhein were weakly clastogenic. Crude extracts of C. fistula (leaves and pods)
each containing sennoside B and rhein were also weak clastogens. The CA/cell and % DC
were lower than those induced by an equivalent amount of pure sennoside B. Therefore,
these phytolaxatives do not behave as potent clastogens and pods or leaves of C. fistula can
be used as an alternative source of sennosides 36.
T. Bhakta et al. (2001) examined the methanol extract of buds of C. fistula for its antipyretic
action on normal body temperature and yeast-induced pyrexia (fever) in rats. The extract
showed significant activity in both the models at doses of 200 and 400 mg/kg. At a dose level
of 200 mg/kg, the extract caused significant lowering of normal body temperature up to 3 h.
At 400 mg/kg dose, it caused significant lowering of body temperature up to 6 h after
administration. In the model of yeast-provoked elevation of body temperature, the extract
showed dose dependent lowering of body temperature up to 4 h at both the dosage levels.
The results obtained are comparable to those for paracetamol, a standard antipyretic agent 37.
P. Siddhuraju et al. (2002) investigated the antioxidant properties of 90% ethanol extracts
of leaves, and 90% methanol extracts of stem bark, pulp and flowers from Cassia fistula. The
antioxidant activity power was in the decreasing order of stem bark, leaves, flowers and pulp
and was well correlated with the total polyphenolic content of the extracts. The reason for
low antioxidant activity in the flower and pulp fractions could be the presence of some
prooxidants, such as chrysophanol and reducing sugars which dominate the antioxidant
compounds present in the extracts. Thus, the stem bark had more antioxidant activity in terms
of reducing power, inhibition of peroxidation, O2 - and DPPH radical scavenging ability 38.
M. A. Akanmu et al. (2004) discussed the in-vitro effect of Cassia fistula infusion on
isolated guinea-pig ileum. The acute and sub-chronic toxicity of the infusion of C. fistula and
Cassia acutifolia sp. Del. Pod-(Senokot tablet) as the reference drug were also determined.
The results obtained for C. fistula infusion when compared with senokot tablet showed that
the infusion of Cassia fistula pods possessed very low levels of toxicity, having the LD 50 of
6600 mg/kg and also without any pathological effects on the organs examined
microscopically. It is therefore concluded from the study that C. fistula pod infusion could be
safely utilized as laxative drugs and as a substitute for the official Senna 39.
T. Bhakta et al. (1999) evaluated that the extract of leaves of C. fistula was tested for
antiinflammatory effects, and compared with those of phenylbutazone, using carrageenan,
histamine and dextran-induced paw oedema assays in rats. Potent antiinflammatory activity
against all phlogistic agents was noted 40.
T. Bhakta et al. (1998) reported that the methanolic extract of C. fistula leaves was
examined for its wound healing property in the form of an ointment in two types of wound
models in rats; excision wound model and incision wound model. The ointment of the leaf
extract of two different concentrations (5 and 10% w/w ointment of leaves extract in simple
ointment base) responded significantly in both models of wounds tested. The results were
also comparable to that of the standard drug, nitrofurazone, in terms of wound contraction
ability, epithelization period, tensile strength and regeneration of tissue at wound area 41.
Pradeep Kannampalli, et al. (2007) evaluated that the hepatoprotective and antioxidant
effect of Cassia fistula leaf extract on liver injury induced by diethyl nitrosamine (DEN) was
investigated. Wistar rats weighing 200±10 g was administered a single dose of DEN (200
mg/kg b.w., i.p.) and left for 30 days. For hepatoprotective studies, ethanolic leaf extract
(ELE) of C. fistula Linn. (500 mg/kg b.w., p.o.) was administered daily for 30 days. AST,
ALT, ALP, LDH and bilirubin were estimated in serum and liver tissue. Lipid peroxidation
(LPO), SOD and CAT were also estimated in liver tissue as markers of oxidative stress. DEN
induced hepatotoxicity in all the treated animals were evident by elevated serum ALT, AST,
ALP and bilirubin levels and a simultaneous fall in their levels in the liver tissue after 30
days. Induction of oxidative stress in the liver was evidenced by increased LPO and fall in
the activities of SOD and CAT. ELE administration for 30 days prevented the DEN induced
hepatic injury and oxidative stress. In conclusion, it was observed that ELE of C. fistula
protects the liver against DEN induced hepatic injury in rats 43.
Padma Singh et al. (2006) tested the leaf extract of Cassia fistula for antifungal activity
against Candida albicans. Extracts of the leaves of Cassia fistula were prepared in acetone,
diethyl ether and methanol. The antifungal activity was performed by paper disc diffusion
assay. The methanol extract showed highest activity i.e., upto 21 mm which was comparable
with the standard antifungal antibiotic, clotrimazole 44.
 CHAPTER 4
Materials and Methods

4 Materials and Methods

4.1 Materials
4.1.1 Plant collection and authentication

The bark of plant Cassia fistula was collected locally from the forest of Chinde danda, Dharan-
24, Sunsari, Nepal during the month of July to August and authenticated by Department of
Botany, Post Graduate Campus, Biratnagar.

4.1.2 Bacteria and Earth-worm availability

The pathogenic bacterial species (S. aureus, S. faecalis, E. coli, Salmonella typhi) were collected
from the Department of Microbiology, BPKIHS Dharan, Sunsari, Nepal. The adult earthworms
Pheritima posthuma were made available from the Laboratory of Microbiology of Sunsari
Technical College, Dharan, Sunsari and washed with normal saline to remove all faecal matter
and were used for anthelminthic property.

4.1.3 Chemical requirements

Table 2 Chemicals and reagents required

S. N. Chemical and Reagents Company / Manufacturer

1. Sulphuric acid Qualigens

2. Ferric Chloride Loba Chemie fine

3. Sodium hydroxide Ranbaxy fine chemicals

4. Acetic anhydride E. Merck

5. Chloroform E. Merck

6. Acetic acid (glacial) Qualigens

7. Mayer’s reagent Loba Chemie fine

 8. Benzene Nike chemicals

9. Ammonia Nike chemicals

10. DMSO Qualigens

11. Methanol Nike chemicals

12. Acetone Qualigens

13. Hexane Qualigens

14. Ethanol Manoj

15. Glycerin Jyoti labs

16. Saffranin S.D. fine chem

17. Nutrient Broth Hi-media

18. Mueller-Hinton agar Hi-media

19. Albendazole Am-tech

20. Chloramphenicol disc Ranbaxy

4.1.4 Equipment’s requirements

Table 3 Equipments and apparatus required

S. N. Chemical and Reagents Company

1. Digital Incubator Digilab

2. Laminar air flow + U.V. Sonar

3. Heating Mantle Accura

4. Hot air oven Digilab

5. Microscope Jaskins

6. Weighing Balance Gupta & Sons

7. Water bath Sonar

 8. Glasswares: Petri plates, test tubes, beakers, -
volumetric flasks, pipette, soxhlet apparatus

9. Others: forceps, inoculating loop, borer, spatula, -

grinder, sieve

4.2 Methods
4.2.1 Pharmacognostic analysis
4.2.1.1 Microscopic Study

Fresh stem bark material was collected and preserved in glycerin and alcohol mixture (1:1) for
softening. Sections of fresh barks were cut in transverse and longitudinal planes, stained with
crystal violet and basic fuchsin and mounted on microscope for microscopical characters. A
small amount of powder boiled in water, stained in safranin and mounted in glycerin and
observed for microscopical characters

4.2.1.2 Macroscopical study

Macroscopical study was carried out with naked eye which gave details concerning the plant
aspect, general appearance, colour and odour.

4.2.2 Loss on drying/ Moisture content

About 10gm of the powdered crude plant was accurately weighted in a tarred dish and dried in
an oven at 100-105o c for 15 min. It was then cooled in a desiccator and again weighted. The loss
on drying was calculated with reference to the amount of dried powder taken. This process was
repeated for three times for more accurate results.
LOD (%) = (initial sample weight – sample weight after drying) x 100 % / initial
sample weight
4.2.3 Extraction

The stem barks of C. fistula were collected locally from the forest of Chinde danda, Dharan-24,
Sunsari, Nepal during the month of July to august and authenticated by Department of Botany,
PG Campus, Biratnagar.
The plant materials were cut into small pieces and were shade dried at room temperature and
they were dried in a hot air oven. Dried sample was crushed into powder by electric
blender(electric grinder) and the coarse powder was passed through sieve no. 40 to collect the
fine powder which was subjected to extraction by using suitable solvents in the soxhlet apparatus
followed by evaporation of solvent and drying the extract and storing at 4o C.`

4.2.3.1 Extraction of Plant

Table 4 Solvents used for extraction

S.N. Plant parts Quantity Solvent Amount

1. Powder of stem bark 25 gm Methanol 160 ml

2. Powder of stem bark 25 gm Acetone 160 ml

3. Powder of stem bark 25 gm Distilled water 160 ml

4.2.3.2 Extraction Procedure

25 gm of dried powder of stem bark was extracted with 160 ml of each solvent: methanol,
acetone and distilled water. Extracts were concentrated by evaporation. The concentrated
products were weighted and yield percentage was calculated. The extracts were stored for further
study.

4.2.4 Preliminary phytochemical Analysis

The Casssia fistula L. plant bark was collected and dried under shade condition, grind to powder
using an electric blender and dissolved separately in 100 ml of solvent. This solution was kept
under room temperature for seven days to allow the extraction of compounds from the stem bark.
The solution of each sample was stirred after every 24 hours using sterile glass rods. After seven
days, the solution was filtered through what man No-1filter paper. The solvent was evaporated
and sticky substance obtained that was stored in there refrigerator and suspended in 10%
(DMSO) Di-methyl Sulfoxide prior to use. Chemical tests were carried out with both the plants
extracts and on the powder specimens using standard procedure to identify the constitutions as
described by Harborne, the specific procedure involved for the evaluations of a particular group
of chemical is mention below.

4.2.4.1 Tannins

1 ml of water and 1-2 drops of ferric chloride solution were added in 0.5ml of extracted solution.
Blue color was observed for tannis and green black for methanolic tannin.

4.2.4.2 Saponins (Foam test)

Small amount of extract was shaken well with little quantity of water. If foam produced persist
for ten minutes if the presence of saponins.

4.2.4.3 Flavonoids (Alkaline Reagent test)

Extractions were treated with few drops of sodium hydroxide solution. Formation of intense
yellow color, which because colors on addition of acid, Indicates the presence of flavonoids.

4.2.4.4 Steroids

2 ml of acetic anhydride was added to 0.5g extract of each sample with 2 ml H 2SO4. The color
changed from violet or blue or Green in some samples indicating the presence of steroids.

4.2.4.5 Terpenoides (salkowski test)

5 ml of each extract was mixed in 2 ml of chloroform, and concentrated H2SO4 (3ml) was
carefully added to form a layer. A reddish brown coloration of the interface was formed to show
the presence of terpenoids.

4.2.4.6 Cardiac glycosides (Keller-Killani test)

5 ml of each extract was treated with 2 ml of glacial acetic containing drop of ferric chloride
solution. This was underplayed with 1 ml of concentrated sulphuric acid. A brown ring of inter
face indicates a deoxysugar characteristics of carotenoides. A violet ring may appear below ring
while in the acetic layer, a greenish ring may from just gradually throughout thin layer.

4.2.4.7 Alkaloids

Alkaloids are basic nitrogenous compounds with definite physiological and

pharmacological activity. Alkaloids solution produces while yellowish precipitated a few drops
of Mayer’s reagents are added.

4.2.4.8 Anthraquinones

Born trigger's test will be used for detecting the presence of anthroquinone. In this case 0.5g of
planet extract was shaken with benzene layer separated and half of its own volume of 10%
ammonia solution added. A pink, red, or violet coloration in the ammonia phase indicated the
presence of anthraquinone.

4.2.5 Anti-bacterial assay

4.2.5.1 Extract Preparation

The plant materials was cut into small pieces and was shade dried at room temperature and they
were dried in a hot air oven. Dried sample was crushed into powder by electric blender(electric
grinder) and the course powder was passed through sieve no. 40 to collect the fine powder which
was subjected to extraction by using suitable solvents in the soxhlet apparatus. Then the extract
was passed through Whatman No.1 filter paper and the filtrate was concentrated by evaporation
in order to reduce the volume. The paste like extract was stored in labeled screw capped bottles
and kept in refrigerator at 4°C.

4.2.5.2 Microorganisms

Bacterial strains consisted of E.coli, Staphylococcus aureus, Streptococcus faecalis and

Salmonella typhii.

4.2.5.3 Evaluation of Antibacterial activity

The antibacterial activity of solvent extracts of various plant parts were evaluated through agar
cup diffusion method.
4.2.5.4 Antibacterial agent

Chloramphenicol was used as the standard drug.

4.2.5.5 Plant Extracts

The antibacterial screening was performed in the stem bark extract of plant using methanol,
acetone and distilled water. The concentrations of stem bark solvent extract of plant were
prepared by dissolving in suitable solvent i.e. sterile distilled water and DMSO whose
concentration were as follows 800 mcg/ml, 400 mcg/ml, 200 mcg/ml, 100 mcg/ml, 50 mcg/ml,
25 mcg/ml.

4.2.5.6 Dilution Technique

a. Preparation of stock solution: 16 mg of each plant extract was weighted in the
weighing balance then dissolve in 10 ml distilled water in a separate clean and
dried bottles and make the concentration 1600 mcg/ml and kept in UV chamber
for sterilization(24 hr).
b. Preparation of dilution from stock solution
A. 800 mcg/ml solution: from the micropipette 1 ml of stock + 1 ml SDW
B. 400 mcg/ml solution: 1 ml of solution A + 1 ml SDW
C. 200 mcg/ml solution: 1 ml of solution B + 1 ml SDW
D. 100 mcg/ml solution: 1 ml from C + 1 ml SDW
E. 50 mcg/ml solution: 1 ml from D + 1 ml SDW
F. 25 mcg/ml solution: 1 ml from D + 1 ml SDW

4.2.5.7 Procedure for antibacterial assay

Antibacterial test of plant extracts were carried out by agar cup diffusion method.

4.2.5.8 Procedure of agar cup diffusion method

In this technique, petridishes of Muller Hinton agar were prepared by pouring melted agar media
previously inoculated with selected microorganism. After the solidification of agar, cup was
made with the help of borer (6mm) and cups filled with solution of suitable concentration of
sample and standard respectively and was inoculated at 37 0 c for 24 hours. The antimicrobial
agent diffuses through agar around its cups and produces a characteristics zone of inhibition of
microorganism sensitive to the sample, the diameter of which can be measured.
Requirements

1. Muller Hinton Agar

2. Sterile distilled water

3. Membrane filter

4. Measuring cylinder

5. Conical flasks

6. Sterile cork borer (diameter 6 mm)

7. Micropipette (1 ml)

8. Petri plate containing nutrient agar

9. Sterile cotton swab

10. Inoculating loop

11. Forceps, ruler

4.2.6 Anthelminthic activity

Observation was made for the time taken to paralysis and death of individual worms, Pheretima
posthuma. Time for paralysis was noted when no movement of any sort can be observed except
when the worms were shaken vigorously. Death was concluded when the worms lost their
motility followed with fading away of their body color.

Requirements:

1. Earthworm
 2. Petri dish

3. Distilled water

4. Normal saline

5. Timer

6. Test tubes

7. Pipette and micropipette

8. Standard drug (Albendazole)

4.2.6.1 Procedure

Indian adult earthworms (Pheretima posthuma) were collected from moist soil and washed with
normal saline to remove all fecal matter were used for the anthelmintic study. The earthworms of
3-5 cm in length and 0.1-0.2 cm in width were used for all the experimental protocol. The
different concentration of plant extracts were prepared in distilled water. The concentrations
made were 10 mg/ml, 20 mg/ml, 25 mg/ml and 50 mg/ml for each solvent extracts. The standard
drug was prepared in distilled water at a dose level of 10 mg/ml. The earthworm which served as
normal control received D/W only. Then the earthworm received the standard drug i.e.
albendazole at a dose level of 10 mg/ml and remaining earthworm received different
concentration of 10 mg/ml, 20 mg/ml, 25 mg/ml and 50 mg/ml of each solvent extracts.
Observation was made for the time taken to cause paralysis and death of individual worms.
Paralyzing and death time were concluded when the worms lost their motility followed with
fading away of their body colours.

4.2.6.2 Preparation of solution of standard drugs

300 mg of albendazole was dissolved in 30 ml of 6% DMSO in distilled water to make

concentration of 10 mg/ml.
Preparation of each plant extracts: in 30 ml solution
For 10 mg/ml (300 mg in 30 ml distilled water)
For 20 mg/ml (600 mg in 30 ml distilled water)
For 25 mg/ml (750 mg in 30 ml distilled water)
For 50 mg/ml (1500 mg in 30 ml distilled water)
All the earth worms were placed in petri dish containing 30 ml of all the solutions. Time of
paralysis and death of the worm was noted19.
 CHAPTER 5
RESULTS

5 Results

5.1 Pharmacognostical study

5.1.1 Microscopical Study
Table 5 Microscopic characteristics of Cassia fistula

Transverse section Outer most cork consisting of 17-21 layers of rectangular to

tangentially elongated cells arranged in vertical rows. Presence of
calcium crystals.

Phelloderm 35-45 layered, often interspersed with groups of stone cells, lignified
with ramified pits, presence of calcium oxalate crystals.

Secondary phloem abundant, consisting of phloem parenchyma, sieve tubes, phloem

fibers, medullary rays and crystalliferous fibers.

Medullary rays uni-or biseriate, rarely triseriate, cells radially long, rectangular

Powder microscopy pieces of cork cells with dense contents; many stone cells isolated or in
groups; cortical parenchyma cells with starch grains, crystals of various
shapes and sizes

5.1.2 Macroscopical studies

Table 6 Macroscopic characteristics of Cassia fistula

The stem bark is 3-6 mm thick with lenticular horizontal markings

General appearance and brownish dots over surface. Fracture is fibrous and striations are
vertical.

Surface structure Smooth and slender when young and rough when old

Color Pale grey when young and dark brown when old.

Odour Disagreeable

Taste Bitter
5.2 Loss on drying

Initial weight (weight of petri plate + plant powder before drying) = 84.6 gm + 10 gm = 94.6 gm
Final Weight (weight of petri plate + Plant powder after drying in oven) = 92.9 gm
Weight of sample after drying = 92.9 gm – 84.6 gm = 8.3 gm
Therefore,
LOD (%) = (initial sample weight – sample weight after drying) x 100 % / initial sample weight
= (10 gm – 8.3 gm) x 100 % / 10 gm
= 17 %

5.3 Extractive` Values

The yield percentage for each extract was calculated and it was found to be the highest in case of
methanol extract. Table no. 7 shows the respective yield for each extracts.

5.4 Phytochemical screening

Phytochemical screening of the plant showed the presence of different constituents in different
solvent extracts. The phytochemical analysis showed that the various groups that were found to
be present in the different extracts are listed in table no. 8

CHAPTER 6
DISCUSSION

6 Discussion

In this study the plant part taken is the stem bark of plant Cassia fistula. It was grinded and
extracted using solvents: methanol, acetone and distilled water. The extract yield is in order:
methanol>acetone>distilled water.
Cassia fistula contains a number of chemical constituents which can be evidenced during
phytochemical screening among them some of chemical constituents shows anti- bacterial as
well as anthelmintic activities.
Phytochemical analysis of the crude extracts (Phytochemical screening, LOD) revealed presence
of flavonoids as one of the chemical constituent. Studies by Nwokonkwo, 2009, reported that
polyphenolic compounds show anthelmintic activity. The anti-bacterial activity is shown by the
presence of anthraquinones and dihydroxyanthraquinones; both of them are glycosides.
This study reveals that methanolic, acetone and aqueous extracts of bark consists of tannins,
saponins, flavonoids, terpenoids, anthraquinone glycosides. As we focus on the anti-bacterial
activity, it reveals that maximum activity is shown by the methanolic extract against gram +ve
bacteria, Staphylococcus aureus. The study by T. Ranjith Vimalraj et. al., has also demonstrated
that the methanolic extract of the bark is more efficient than aqueous extract against S. aureus.
Seyyed Mansour Seyyednejad et. al. demonstrated that the extracts of flower is effective against
B. cereus, S. aureus, S. epidermidis, E. coli and K. pneumoniae. The most susceptible
microorganisms to ethanolic and methanolic extracts were E. coli and K. pneumoniae
respectively. Also, B. cereus and S. aureus showed least sensitivity to ethanolic and methanolic
extracts respectively. This demonstrates that the actual anti-bacterial constituent from the extract
can be used as a narrow spectrum antibiotic for Staphylococcus aureus.
The anthelmintic activity of C. fistula may be due to phenolic compound, tannins that can bind to
free proteins in the gastrointestinal tract (GIT) of host animal or glycoprotein on the cuticle of
the parasite and may cause death( Athnasiadou and Bate-Smith et. al.). As a vermicidal,
albendazole causes degenerative alterations in the intestinal cells of the worm by binding to the
colchicine-sensitive site of tubulin, thus inhibiting its polymerization or assembly into
microtubules. The loss of the cytoplasmic microtubules leads to impaired uptake of glucose by
the larval and adult stages of the susceptible parasites, and depletes their glycogen stores.
Degenerative changes occur in the endoplasmic reticulum, the mitochondria of the germinal
layer, and the subsequent release of lysosomes results in a decreased production of ATP. Due to
diminished ATP production, the parasite is immobilized (paralysis) and eventually dies. In this
study, the shortest time of paralysis and death was found at dose 50 mg/ml which was near to the
paralyzing and death time as recorded for the 10 mg/ml concentration of the standard drug,
albendazole. This type of study by M. Irshad et. al., reported shortest time of paralysis and death
at dose of 100 mg/ml using fruit pulp and seed extracts which was near to the paralyzing and
death time as recorded for the 10 mg/ml concentration of the standard piperazine citrate.
 CHAPTER 7
CONCLUSION

7 Conclusion

This work comprised of plant description, phytochemical constitution, anti-bacterial and

anthelmintic activities of stem bark of Cassia fistula Linn. (Fabaceae). Loss on drying was found
to be 17%, which gives a hint that the powder is slightly hygroscopic in nature. The
phytochemicals comprised by the extracts were tannins, saponin, flavonoids, terpenoids and
anthraquinone glycosides. No alkaloids, steroids, and cardiac glycosides were detected. The
extract yield was maximum for methanol as compared to acetone and distilled water. The anti-
bacterial activity was evaluated based on the inhibition zone using agar cup diffusion method.
The methanolic extract had a better and maximum activity against Gram +ve S. aureus. Acetone
and aqueous extracts had lesser susceptibility and had no activities against Gram –ve bacteria.
The anthelmintic activity was also more efficient by methanolic extract than acetone and
aqueous extracts. However, aqueous extract was more effective than the acetone extract. This
plant has many other essential features which needs to be explored. As very less study on this
plant has been done, it has a virgin area of research. I recommend further studies on different
parts of this plant and the need for isolation of the active chemical constituents along with
moulding it into formulations for use.