PHYTOCHEMICAL ANALYSIS AND INVITRO ANTIOXIDANT

ACTIVITY OF CASSIA OCCIDENTALIS

BY

KHALID SHEHU

UG16/SCBC/1110

A PROJECT SUBMITTED TO THE DEPARTMENT OF

BIOCHEMISTRY, FACULTY OF SCIENCES, GOMBE STATE
UNIVERSITY, GOMBE

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR

THE AWARD OF B. SC (HONS) DEGREE IN BIOCHEMISTRY

AUGUST, 2021.

i
 DECLARATION
I hereby declared that this PROJECT is a product of my research efforts; undertaken under the

supervision of Prof. (Mrs) H. Hamza and has not been presented elsewhere for the award of a degree or

certificate. All sources have been fully acknowledged.

_____________________________ __________________________

Khalid Shehu Date

(UG16/SCBC/1110)

ii
 CERTIFICATION
The project has been examined and approved for the award of degree of B.Sc (Hons) Biochemistry
for its contribution to knowledge and literary presentation

___________________________ ___________________________
Prof. (Mrs) H. Hamza Date
(Project supervisor)

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 APPROVAL PAGE
This is to certify that the research work for this project and its subsequent preparation by Khalid

Shehu (UG16/SCBC/1110) were carried out under my own supervision.

____________________________ ________________
(External Examiner) Date

___________________________ ________________
Prof. (Mrs) H. Hamza Date
(Project Supervisor)

____________________________ ________________
Dr. Fatima Umar Maigari Date
(Head of Department)

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 ACKNOWLEDGEMENT

All Praises be to Allah (S.W.T) the most beneficial the most merciful for giving me the

opportunity, gift of life, wisdom, strength and resources needed for the completion ofthisproject

research work.

Special and sincere gratitude goes to my beloved family especially my prayer warriors(my

parents) for their supports and prayers from the beginning till the end of my program as

undergraduate.And I also extended my special gratitude and appreciation to my beloved sister

(HajiyaAishatuAlbasu) for her prayers and financial support at the same time throughout my

studies, Thank you for always being there.

A Faithfull gratitude also goes to my workaholic project supervisor, Prof. (Mrs) H. Hamza. Thank

you for the professional guidance through my first research project with discipline and advices

throughout the entire processes.

To the entire Biochemistry department staff, the HOD Dr. Fatima Umar, my lecturers especially Dr.

AisamiAbubakar, Dr. Lazarus,Mr. Jahlil James and MallamAlhassanSiddan, the laboratory staff

especially Mr. Musa, Mr. Sadisu for giving me a listening ears through the entire process and

guiding me step by step on how to and how not to do in my work.

To my fellow projects mates and friends, especially Wasinda Julius janet, Ahmed Yahaya, Ali A 3.

Zainab and Muhammad Mahmud. Thank you for being supportive.

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 DEDICATION
This project work is dedicated to my beloved sister, who encouraged and motivated me to study. It is
also dedicated to my parents, who taught me that even the largest task can be accomplished if it is
done one step at a time.

And above all, to Allah (S.W.T.) the almighty, giver and sustainer of life.

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DECLARATION.......................................................................................................................................ii
CERTIFICATION....................................................................................................................................iii
APPROVAL PAGE....................................................................................................................................iv
ACKNOWLEDGEMENT.........................................................................................................................v
DEDICATION..........................................................................................................................................vi
LIST OF TABLES....................................................................................................................................ix
LIST OF FIGURES...................................................................................................................................x
ABSTRACT...............................................................................................................................................xi
CHAPTER ONE........................................................................................................................................1
BACKGROUND OF THE STUDY..........................................................................................................1
STATEMENT OF THE RESEARCH PROBLEM................................................................................2
AIM AND OBJECTIVES.........................................................................................................................2
SIGNIFIACANCE OF THE STUDY.......................................................................................................3
JUSTIFICATION OF THE STUDY........................................................................................................3
SCOPE AND LIMITATION....................................................................................................................3
CHAPTER TWO.......................................................................................................................................4
LITERATURE REVIEW.........................................................................................................................4
PLANT DESCRIPTION...........................................................................................................................4
TAXONOMIAL CLASSIFICATION......................................................................................................4
BOTANICAL NAME................................................................................................................................4
LOCAL NAME..........................................................................................................................................5
PYTHOCHEMICAL CONSTITUENT...................................................................................................5
PHARMACOLOGICAL ACTIVITIES..................................................................................................6
CHAPTER THREE.................................................................................................................................13
MATERIALS AND METHODS............................................................................................................13
APPARATUS AND EQUIPMENTS......................................................................................................13
REAGENT USED....................................................................................................................................13
COLLECTION AND IDENTIFICATION OF PLANT MATERIAL................................................13
METHODS..............................................................................................................................................14

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METHOD OF EXTRACTION...............................................................................................................14
PHYTOCHEMICAL SCREENING......................................................................................................14
QUANTITATIVE ANALYSIS...............................................................................................................15
ANTIOXIDANT ACTIVITY METHODS............................................................................................16
CHAPTER FOUR...................................................................................................................................18
RESULT AND DISCUSSION................................................................................................................18
PHYTOCHEMICAL CONSTITUENTS (QUALITATIVE ANALYSIS)..........................................18
Phytochemical screening (quantitative analysis)...................................................................................19
CHAPTER FIVE.....................................................................................................................................29
Summary..................................................................................................................................................29
Conclusion................................................................................................................................................29
Recommendation.....................................................................................................................................29
Reference..................................................................................................................................................30
Appendix..................................................................................................................................................32

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LIST OF TABLES
Table 1:Table of the Qualitative determination of cassia occidentalis leaves extract.

Table 2:Table of Quantitative determination of cassia occidentalis leaves extract.

Table 3: Total antioxidant capacity showing the % inhibition and IC50

Table 4: Hydrogen peroxide scavenging assay showing the % inhibition and IC50

Table 5: Ferric reducing antioxidant activity showing the % inhibition and IC50

Table 6: statistical analysis

Table 7: mean of the absorbance

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LIST OF FIGURES
Figure 1: Histogram showing the antioxidant activity of cassia occidentalis

Figure 2: Concentration-dependent inhibition of cassia occidentalis leaves extract andAscorbic

acid (standard) using total antioxidant capacity

Figure 3: Concentration-dependent inhibition of cassia occidentalis leaves extract and

Ascorbic acid (standard) using hydrogen peroxide scavenging activity.

Figure 4: Concentration-dependent inhibition of cassia occidentalis leaves extract and Ascorbic

acid (standard) using reducing power activity

Figure 5: concentration dependent absorbance of cassia occidentalis leaves extract and ascorbic
acid using total antioxidant capacity

Figure 6: concentration dependent absorbance of cassia occidentalis leaves extract and ascorbic
acid using hydrogen peroxide scavenging activity

Figure 7: concentration dependent absorbance of cassia occidentalis leaves extract and ascorbic
acid using ferric reducing antioxidant activity.

Figure 8: concentration dependent absorbance of ascorbic acid standard

Table 9: Total Antioxidant Capacity of cassia occidentalisLeaves expressed as μg AAE/g in

different concentration.

Table 10: hydrogen peroxide scavenging activity of cassia occidentalisLeaves expressed as μg

AAE/g in different concentration.

Table 11: Ferric reducing antioxidant power activity of cassia occidentalisLeaves expressed as
μg AAE/g in different concentration

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 ABSTRACT
Plants play a vital role in drug development,due to the promising biologicalactivitiesagainst
diseases.Cassia occidentalisLinn which belong to the family Leguminaceae.Phytochemical
components and the antioxidant activity ofCassia occidentalisEthanolic extract have assessed.
The results have shown that ethanolic extract of Cassia occidentalis leavesshowed marked
antioxidant activity due to the presence of bioactive compounds which is alkaloids (1.4%),
flavonoids (11.2%), saponins (18%), tannins (38.3µg/ml), sugar and quinone.Anthraquinone,
coumarin, phenol and glycoside sugar were not detected.The ethanolic extract of the leaves of
Cassia occidentaliswere subjected to phytochemical analysis by standard qualitative and
quantitative analysis and the invitro antioxidant activity was evaluated by determination of total
antioxidant capacity, hydrogen peroxide H 2O2 radical scavenging activity and Ferric reducing
anti-oxidant potential (FRAP). The analyses revealed that the ethanolic extract of cassia
occidentaliswas able to efficiently scavenge the free radicals in a dose dependent manner. The
statistical analysis of cassia occidentalis showed that there is no significance differences between
total antioxidant capacity and hydrogen peroxide compared to the ascorbic acid standard due to
their mean differences 0.0238 and 0.1478 respectively. Whereas ferric reducing antioxidant
capacity shows slightly significance difference compared to the standard because of their mean
difference 0.2186.

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 CHAPTER ONE

BACKGROUND OF THE STUDY

Natural sources like plants play a vital role in drugdevelopment. This is due to the promising

biologicalactivity of plant materials in treating infectious diseases successfully without any side

effects. This attracted the scientist worldwideand started exploring the plant kingdom. One

among such herb is Cassia occidentalisLinn. which belong to the family Leguminaceae. The

potential activity of these herbs in prevention and treatment of diseases depend on their bioactive

compounds or phytochemicals. Plant-derived bioactive compounds have been found to stimulate

osteoblast differentiation and bone formation.These bioactive compounds include flavonoids,

coumarins, ligninspolyphenols, terpenoids,carotenoids, alkaloids andanthraquinoe(emodin) (Je

Taeet al., 2004).Plant extracts containing the phytochemical anthraquinone (emodin) have

gained momentum in being used incosmetic, dye, food, and pharmaceutical industries due to

their wide therapeutic and pharmacological properties (Alveset al., 2004).The leaf is recognized

as anti-neuralgic, purgative (intreatment of diarrhea and dysentery) and vermifuge. Oxidative

stress is caused due to the release of free radicals like superoxide anions, hydrogen peroxide, and

hydroxyl, nitric oxide and peroxy nitrite. These free radicals plays an important role in the

pathogenesis of neurodegenerativedisorders, atherosclerosis, diabetes, inflammation,aging

(Burns, et al.,2001).Antioxidants are compounds which act as radical scavengers when added to

the food products prevent the radical chain reaction of oxidation, delay or inhibit the oxidation

process and increase the shelf life by retarding the processes of lipid per oxidation. This article

takesinterest in proving the antioxidant activity of Cassiaoccidentalisalong with the detailed

analysis of the phytochemicals present in this herb (Young, 2001).

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Plants are important source of drugs; especially in traditional medicine. It is a common practice

in Nigeria and other parts of the world to use plant in the form of crude extracts, decoction,

infusion or tincture to treat common infection and chronic conditions. According to WHO, over

70% of the world population rely on medicinal plants for primary health care, and there are

reports from various researchers on natural substances of plant origin which are biologically

active, with desirable antimicrobial and antioxidant properties. Despite tremendous progress in

human medicines, infectious diseases caused by bacteria, fungi, viruses and parasites are still a

major threat to public health the impact is particularly high in developing countries due to

relative unavailability of medicines and the emergence of widespread drug resistance.

STATEMENT OF THE RESEARCH PROBLEM

In recent times, there has been increasing trends of diseases that are associated with free-radical

generation in the body; subsequently damaging the cellular biomolecules and finally leading to

disease conditions. The usage of the synthetic antioxidant may have adverse side effects. Hence

there is a need for natural antioxidants which could be considered as safer without any side

effects.

AIM AND OBJECTIVES

Aim of the study

 The aim of this research is to investigate the phytochemical components and to assess the

antioxidant activity ofCassia occidentalisEthanolic Extract.

Objectives of the study

 To carry out qualitative phytochemical analysis

 To assess free radical scavenging activity of Cassia occidentalisEthanolic Extract.

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SIGNIFIACANCE OF THE STUDY
There is an increasing interest in natural antioxidants which prevent oxidative damage. Natural

antioxidants increase the antioxidant capacity of plasma and reduce the risk of disease. Free

radicals can be scavenged by the natural antioxidant which are safe with no any side effects.

Therefore it is necessary to explore their antioxidant antioxidant potential of medicinal plant

found within the community.

JUSTIFICATION OF THE STUDY

This research work ought to find the vital bioactive phytochemical components present in the

plant extract with antioxidants activities in order to explore the medicinal benefits of the plant

against many diseases, thus replace the use of synthetic antioxidants which have side effects. In

ethnomedicine, cassia occidentalis has been used in the treatment of many diseases such as

cancer, diabetics etc. Therefore this study can also provide scientific prove for the

ethnomedicinal benefits of the plant.

SCOPE AND LIMITATION

This research work is only limited to phytochemical screening and in vitro antioxidant activity of

cassia occidentalis.

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 CHAPTER TWO

LITERATURE REVIEW

PLANT DESCRIPTION
Cassia occidentalisLinn, usually grows in the southern part of india which is known as Kasmard

in Sanskrit, Kasondi in Hindi and coffe Senna in English. The plant belongs to

Caesalpiniaceaefamily.The common name is Ponnavarai in Tamil. The roots, leaves and seeds

are the parts of the plant used. It is an erect herb, commonly found by road sides, ditches and

waste dumping sites. Cassiaoccidentalis has been widely used as traditional medicine in African

country suchsuch Nigeria, Cameroon and Ghana. The entire parts of the plant have medicinal

values (Mohammed M, et al., 2012).

TAXONOMIAL CLASSIFICATION
Kingdom - plantae

Subkingdom - Tracheobionta

Phylum -Tracheophyta

Class - magnoliopsida

Subclass - Rosidae

Order -Fabales

Family - Leguminos

Genus - Cassia

Specie - Occidentalis

BOTANICAL NAME
Cassia occidentalis

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LOCAL NAME
English - Senna occidentalis

Hausa- majamfari

PYTHOCHEMICAL CONSTITUENT
Phytochemical screening of the plant showed the presence of carbohydrates, saponins, sterols,

flavonoids, resins, alkaloids, terpenes, anthraquinones, glycoside and balsam. Presence of these

metabolites strongly concluded the great potential of the plant as a source of phytomedicines. As

the flavonoids and resins are present, it might be responsible for its anti-inflammatory properties.

Chinese folkloric medicine contains flavonoids which has anti-inflammatory effect on both acute

and chronic inflammation. Alkaloids are known for decreasing blood pressure, balancing the

nervous system in case of mental illness and antimalarial properties. Tannins help in wound

healing and anti-parasitic. Presence of terpenes suggests possessing anti-tumor and anti-viral

properties. Eudesmanesesquiterpenes have been reported to contain antibacterial properties.

Saponins are believed to have antioxidant,anti-cancer, anti-inflammatory, and anti-viral

properties. The anthraquinones, emodin and chrysophanone have been reported to possess

wound healing properties. Other compounds reported in literature include, 1,8-dihydroxyl-2-

methyl anthraquinone, 1,4,5-trihydroxy-3-methyl-7-methoxy anthraquinone, cassiaoccidentalin

A, B and C, which are C-glycosides, achrosine, anthrones, apigenin, aurantiobtusin, campesterol,

cassiollin, chrysoobtusin, chrysophanicacid, chrysarobin, chrysoeriol, essentialoils, funiculosin,

galactopyranosyl, helminthosporin, islandicin, kaempferol, lignoceric acid, linoleic acid,

linolenic acid, mannitol, mannopyranosyl, matteucinol, obtusifolin, obtusin, oleic acid, physcion,

quercetin, rhamnosides, rhein, rubrofusarin, sitosterols, and xanthorin. Pharmacognistic analysis

of the plant showed 10%moisture thuslesssensitive for microbial attack and 7.4% total ash value

5
indicates the low amount of inorganic substance. It contained 5.3% of acid insoluble ash value

suggested that the soluble inorganic component is small. The alcohol and water extractive values

were 7.7% and 15.1% respectively showed that water is a better solvent of bulk extraction than

alcohol(SaganuwanAS2006).

PHARMACOLOGICAL ACTIVITIES
Antimicrobial activity

A study was carried on Cassia occidentalisantimicrobial properties. Test was conducted with

four different extracts such as methanol, aqueous, benzene, petroleum ether and chloroform

extract. Among which methanol extract showed positive against P.aeruginosa, K. pneumoniae,

P. mirabilis, E. coli, S. aureus and S.epidermidis; aqueous extract was effective against P.

vulgaris, K.pneumoniaeand P. aeruginosa; benzene and petroleum ether extracts was active

against P. mirabilis and E. coli; chloroform extract was found to be very inactive against all

tested strains. Another study reported maximum activity against Salmonella typhiand minimum

with Shigella spp. This study concluded that antibacterialactivity of Cassia occidentalisleaves of

ethanol and water extractwere increase with higher concentration (Mohammed M, et al., 2012).A

report with Cassia occidentalisflower extractshowedmaximum inhibition against Klebsiella

pneumonia and no activity against Staphylococcus aureus, Streptococcus pneumoniae,

andPseudomonas aeruginosa. Thus the flower extract of Cassiaoccidentaliscan be used to

treatKlebsiellaassociated ailment such as pneumonia, bronchitis and other diseases known to

cause by K.pneumonia (Daniyanet al., 2011). A report states that the E. coli was sensitive to

methanol, hexane, chloroform and aqueous extract of leaves ofC.occidentalis(Saganuwan 2006).

Similarly, Jain and his coworkers observed that the metabolite rich fraction of (anthraquinones)

leaves, pods, flowers and callus were effective against E.coli.Yet other study showed that the

6
petroleum ether and ethanolic extract of leaves ofC.occidentaliswas active against E. coli. With

Chloroform and aqueous extract the inhibition was not observed againstE.coli. Based on these

experiments we can clearly say that changes in the activities of plant extracts might be due to

spatial and temporal variations.P.aeruginosashowing multidrug resistance is highly challenging

to treat by conventional antibiotics (Jainet al., 1998). A study tested the efficiency of leaf extract

of C. occidentalisagainst the growth of P.aeruginosaand found that the microbial growth was

highly inhibited. And the crude extracts was effective on some microbes such

asStaphylococcusaureus, Escherichia coli, Bacillus subtilisandCandida albicanswhich was a

common causative agent of urinary tract infection and diarrhea diseases (Mohammed et al.,

2012). As this plant has potential antimicrobial activity but invivo studies with the extract should

becarriedout to confirm that the zone of inhibition is not only by the sensitivity of the microbes

also the concentration is highly essential when using for treatment (Basri 2005).

Antioxidantactivity/Hepatoprotectiveactivity

The aqueous–ethanolic extract of leaves of C. occidentaliswas tested

for hepatoprotective activity on liver damage in rat which was induced by paracetamol and ethyl

alcohol by monitoring serum transaminase, alkaline phosphatase, serum cholesterol, serum total

lipids and histopathological alterations. They found that the leafextract had shown significant

hepatoprotective activity ( Jafriet al., 1999). Some other observations had found that the seed

extracts of C. occidentalis limits the DNA degradation caused by iron (II)-driven Fenton

reaction. It is notable that inhibition of DNA damage may be due to their capability of strong

ferrous ion chelation. Further, they proposed that the scavenging activity towards free radicals

might be the reason. C. occidentalisis an ingredient in Himoliv, a polyherbalayurvedic

formulation. It is also proved that it prevents the carbon tetra chloride induced hepatotoxicity in

7
rats (Bhattacharyya D, et al., 2003). Based on the observation they suggested that Himoliv

increases the protective enzymes superoxide dismutase (SOD) and catalase in liver homogenate

of rats (Kolhapure SA 2004). It is also present in other polyherbal formulation Liv.52 tablet and

syrup extensively used for Hepatitis A (HA). For the preparation of this syrup, other plants

included Capparisspinosa, Cichoriumintybus, Solanum nigrum, Terminaliaarjuna,

Achilleamillefoliumand Tamarixgallicaetc along with C.occidentalisare present. A study with 50

clinical samples over 30 years with 4490 patients was performed to identify the efficacy with

short and long term safety of Liv.52 in Hepatitis A (Tonaet al., 2001). This study concluded that

Liv.52 tablets and syrups are potential and safer for hepatitis A.

Larvicidal Activity

The larvicidal and pupicidal potential of Cassia Occidentaliswas analyzed in a study against the

larvae of Anopheles Stephensi. The ethanol extract of Cassia Occidentaliswere found to be more

effective against larva and pupa respectively. The smoke toxicitystudy was also conducted and

identified that it was more effective against the Anopheles stephensi(AbiramiDhandapani 2011).

Smoke exposed gravid females oviposited fewer eggs when compared to those that were not

exposed. Yet another study reveal that seed oil creates increase in mortality of C. maculatuseggs.

Based on numerous trials with pure compounds suggested that fatty acids (linoleic, oleic and

stearic) are responsible for C. occidentalistoxicity (Lienardet al., 1993). The oviposition of C.

maculatuswas not reduced by C. occidentalisseed oil at 10 ml/kg seed.

8
Antimalarial activity

C. occidentalisplant extract was proved to have effective antimalarial activity. A study with

ethanolic, dichloromethane and lyophilized aqueous extracts of C. occidentalis root bark was

tested for antimalarial activity against PlasmodiumbergheiANKA. They tested its toxicity by

treating the orally and found that there was no toxic effect or mortality in mice with a single

dose, of 500 mg/kg of body weight, or same dose given twice weekly for 4 weeks. The extracts

produced significant chemo suppressions of parasitemia with 200 mg/kg dose when administered

orally. C.occidentaliswas found to be potential with 60% chemo suppression. They also found

that the ethanolic extract is more active than the lyophilized aqueous extract. C. occidentalisleaf

extract with ethanol and chloroform was found to possess better antimalarial activity. When

tested with 6 μg/ml concentration more than 60% inhibition was observed against the parasite

(Tonaet al., 2001).

Immunosuppression

To determine the Immunosuppression, cyclophosphamide (CP) was administered

intraperitoneally in a single dose of 50 mg/kg b.w. Body weight, relative organ weight, lymphoid

organ cellularity,hemagglutination titer (HT); plaque forming cell (PFC) assay and quantitative

hemolysis of SRBC (QHS) were analyzed in animals. It has suppressive effects on lymphoid

organ weight and cellularityand other parameters of humoral immunity. The CP-exposed animals

were administered with plant extract and showed better humoral responses. The plaque forming

cells were found to be morein CP-treated animals after C. occidentalisadministration. In

QHSassay, also C. occidentalisshowed protection in CP-treated animals. They also found out

that the bone marrow cell counts were much higher in plant extract treated animal which were

reduced in CPtreated animals. They suggest that modulating the hepatic drug metabolizing

9
enzymes might be the mechanism for hematotoxic and immunotoxic responses of

cyclophosphamide (Bilal Bin-Hafeezet al., 2001).

Anti-inflammatory activity

Cassia occidentalisleaf powder was tested for anti-inflammatory activity and

Cardiospermumhalicacabumaerial parts with ethanol extract was assayed in male albino rats

using carrageenan-induced rat paw edema. At 2000 mg/kg dose the C. occidentaliswas found to

be active at maximum level and 500 mg/kg was found to be the minimal active dose for C.

halicacabum. The efficiency was tested in cotton pellet granuloma assay and observed that the

transudative, exudative and proliferative components of chronic inflammation were suppressed

by these drugs. Lipid peroxide content and γ-glutamyltranspeptidase and phospholipase A2

activity in the exudate of cotton pellet granuloma was lowered with the usage of these drugs. In

normalized cotton pellet granulomatous rats, increased alkaline phosphatase

activitywithdecreased A/G ratio of plasma were found after the treatment. C. occidentalispowder

and C.halicacabumextract were able tostabilize the human erythrocyte membrane against

hypotonicity-induced lysis. It is likely that these drugs may exert their anti-inflammatory activity

by inhibition of phospholipase A2, resulting in the reduced avail ability of arachidonic acid, a

precursor of prostaglandin biosynthesis, and/or by stabilization of the lysosomal membrane

system (Sadiqueet al., 1987).

Toxicity Studies

Acute toxicity test was conducted in a report with Cassia occidentalis and found that this plant

did not show any hazardous symptoms or death. With the sub acute treatment, the Cassia

occidentalisdoesn’t change body weight gain, consumption of foodandwater and the profiles of

10
hematological and biochemical. Also, no changes were seen in macroscopical and

microscopicalaspect of organs in the animals. Thus they conclude that acute administration of

Cassia occidentalisis not toxic. Histopathological analysis showed no cell death,necrosis or

inflammation of the liver and kidney. The leaves of this plant are thus found to be safe with no

adverse effect on the liver and kidney function sat the doses administered (Tanimu 2012).

Another study had investigated the effect sofCassiaoccidentalisoral administration during

pregnancy in female Wistar rats. They found that there was no statistically significant changes

between control and test groups with respect to fetuses, placentae and ovaries weights; number

of implantation and resorption sites; number of corpora lutea in the ovaries and pre- and post-

implantation loss rates (Aragaoet al., 2009).

Antianxiety and Antidepressant activity

Around 5% of world’s population was affected by anxiety and depression a widespread

psychiatric disorder. Previously, plants and formulations were used to treat anxiety and

depression over decades. A recent report has studied the antianxiety and antidepressant activity

of ethanolic and aqueous extracts o fCassiaoccidentalisleaves in rodents. Exposing the rats to

unfamiliar aversion in different methods like elevated plus maze model and actophotometer

antianxiety activity was tested. Less aversion fear elicits antianxiety activity. Antidepressant

activity was analyzed by despair swim test and tail suspension test. Reduced immobility time

elicits antidepressant activity. They conclude that ethanolic and aqueous extracts of Cassia

occidentalisleaves possess antianxietyand antidepressant activity. Ethanolic extract of Cassia

occidentalis leaves showing more significant activity over the aqueous extracts (Saba Shafeen, et

al., 2012).

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Analgesic and antipyretic activity

Cassia occidentalisLinn was screened for analgesic and antipyretic activity. Ethanol and water

extracts of Cassia occidentalisleaves were screened in mice which was induced by acetic acid

and tested for hot plate and tail immersion assay, and also in yeast induced pyrexia method in

rats. They found that the ethanol and waterextracts of Cassia occidentalispossess antinociceptive

and antipyretic properties. Highest inhibition dose was found to be as

300 mg/kg. The report clearly mentioned that both the ethanolic and water extracts of Cassia

occidentalisshowed significant effect on pyrexia induced by yeast (Karpakavalliet al., 2011).

Antidiabetic activity

The aqueous extract of C. occidentaliswas tested for antidiabetic activity and the study proved

that there was a significant reduction in fasting blood glucose levels in the normal and

alloxaninduced diabetic rats. They also tested for other extracts include petroleum ether and

chloroform extracts and concluded that activity from day 14 and activity from 7 days

respectively. Specific variations were seen in serum lipid profiles (cholesterol and triglyceride),

serum protein, and changes in body weight by aqueous extract treated-diabetic animals, when

compared with the diabetic controland normal animals. Histopathological studies have also

revealed that pancreas of the animals showed regeneration by extract which were necrosed

earlier (Laxmiet al., 2010).

12
 CHAPTER THREE

MATERIALS AND METHODS

APPARATUS AND EQUIPMENTS

i. Beaker, Conical flask, Stirrer, Whatman No. 1 Filter paper, Funnel, Spatula, Cuvette,
Measuring cylinder, Dropper, Syringe, Test tubes and Micropipette. (Pyrex glasses
company, VMR, USA)
ii. Orbital shaker (Bibby scientific LTD UK)
iii. Weighing balance (Adam laboratory, model No: AE438181850)
iv. Hot plate (Veestar model-ECF2, china)
v. Incubator (Genlab thermal engineers)
vi. Spectrophotometer (Model No: C.E. 7400)
vii. Rotary evaporator (Stuart RE300DB)

REAGENT USED
Magnesium acetate (JHD china), Distilled water (FTH Gombe-Dialysis center), Sodium
hydroxide (Hopkins and Hycians LTD, England), Magnesium turnings (East Anglia chemicals,
England), Ferric Chloride (Qualikems fine chemicals PV LTD china), Concentrated Sulphuric
acid (Sigma Aldrich, UK), Lead acetate (Lab. Tech. chemicals, Nigeria), Anthrone
(Lobachemiepvt, LTD India), Folin-Denis reagent (Lobachemiepvt, LTD India), Sodium
carbonate (Park scientific, LTD, UK), Ethanol (Sigma Aldrich, UK), Follin – Ciocalteau reagent
(Lobachemiepvt, LTD India), Acetic acid (Hydrite chemical co. USA), Ammonium hydroxide
(Griffin and George England), Diethyl ether (Lobachemiepvt, LTD India), N – Butanol
(Guaghuasci-tech LTD China), Methanol (Honeywell Riedel de aen, Germany), Sulfuric acid
(Sigma Aldrich, UK), Sodium phosphate, Ammonium molybdate (Kermel LTD), Phosphate
buffer (GH Tech. China), Potassium ferricyanide (DAMAO, China), Trichloroacetic acid (GH
Tech. China), Hydrogen peroxide (Sigma Aldrich, UK)

COLLECTION AND IDENTIFICATION OF PLANT MATERIAL

Cassia occidenlis leaves was collected in Gombe, Akko LGA, area of Zagaina, Gombe State.
The plant was identified and authenticated by a taxonomist at the Department of Botany
Sciences, Gombe State University.

13
METHODS
Preparation of cassia occidentalis leaves Extract

The Cassia occidentalis leaves of were collected fresh from its plant, shade dried for about 10
days. The dried leaves were pulverized into fine powder using mortar and pestle and sieved. The
fine powder was placed in a dry bottle and tied up in a black polythene to prevent light
penetration.

METHOD OF EXTRACTION
Fifty gms of the Cassia occidentalis leaves powder was weighed and placed in a 1000ml conical
flask. 50% ethanol was added. The mixture was stirred, mounted on an orbital shaker for an hour
and allowed to stand for 24 hours. After 24 hours the mixture was mounted again on the orbital
shaker and shaken for an hour. The mixture was filtered using a filter paper. The filtrate was
evaporated using the rotary evaporator and the crude extract was obtained and placed into an
empty beaker.

PHYTOCHEMICAL SCREENING
Cassia occidentalis leave extract was subjected to test for the presence of alkaloid,
anthraquinone, coumarin, flavonoid, phenol, quinone, saponin, tannin, sugar and glycoside.

Qualitative Analysis

Test for Alkaloid (Wagner’s Test): A few drops of Wagner’s reagent were added to few ml of
plant extract along the sides of test tube. A reddish- Brown precipitate confirms the test as
positive.

Test for Anthraquinone (Borntragor’s Test): Few drops of magnesium acetate solution were
added to 1ml of the extract; the formation of pink color showed the presence of anthraquinone.
(Okeniyiet al., 2012) (Falodunet al., 2006)

Test for Coumarin: To1.5ml of the extract in a watch glass few drops of alcoholic sodium
hydroxide was added, the appearance of yellow color indicated the presence of coumarin
(Okeniyiet al., 2012).

14
Test for Flavonoid (Shindo’s Test): To 1.3ml of the extract 0.5g of magnesium turnings was
added the mixture was boiled for 5 minutes; the appearance of orange to red color indicated the
presence of flavonoid (Okeniyiet al., 2012).

Test for Phenol: A few drops of ferric chloride solution were added to 2ml of the extract in a
watch glass; the appearance of bluish green color indicated the presence of phenol (Okeniyiet al.,
2012) (Falodunet al., 2006)

Test for Quinone: One ml of the extract was mixed with concentrated sulphuric acid. The
appearance of the color formation signified that quinone was present (Okeniyiet al., 2012)
(Falodunet al., 2006)

Test for Saponin (Frothing Test): To 2.5ml of the extract a few drops of distilled water was
added and the mixture was shaken vigorously, a cupious lather formation was noticed which
indicated the presence of saponin, and the absence of the cupious lather meant the absence of
saponin (Okeniyiet al., 2012) (Falodunet al., 2006)

Test for Tannin (Wohler’s Test): A few drops of basic lead acetate solution were added to
1.6ml of the extract; the appearance of a white precipitate indicated the presence of tannin in
some of the plant extract. (Falodunet al., 2006)

Test for Sugar: To 2.5 ml of the extract in 150ml beaker, and a small quantity of anthrone and a
few drops of concentrated sulphuric acid were added, a green coloration of mixture indicates the
presence of sugar (Okeniyiet al., 2012)

Test for Glycoside: To 2.5ml of the extract with few drops of anthrone in a watch glass, one
drop of concentrated sulphuric acid was added and made into a paste, and heated gently over a
water bath; a dark green coloration indicates the presence of glycoside. (Falodunet al., 2006)

QUANTITATIVE ANALYSIS
Determination of Tannin

To 0.5g of sample was weighed and placed into a 250ml conical flask. 75ml of distilled water
was added. The mixture was boiled for 30 minutes and centrifuged at 2000rpm. The supernatant
was collected in a 100ml volumetric flask and the volume was made up to 100ml. One ml of the

15
sample extract was transferred into 100ml volumetric flask containing 75ml of distilled water.
0.5ml of Folin-Denis reagent and 1ml of sodium carbonate was added. The absorbance was read
at 700nm after 30 minutes. Standard tannic acid graph was prepared using 0- 100µg tannic acid.

Determination of Flavonoid

To 50cm3 40% aqueous ethanol in 250 ml beaker to 150g of sample was added. The mixture was
covered and allowed to stand for 24hours at room temperature. The supernatant was discarded
and the residue was re-extracted (three times) with the same volume of ethanol. Filter paper was
used to filter mixture and left to evaporate to dryness.

Determination of Alkaloid

Five gms of the sample was weighed into a 250ml beaker and 200ml of 20% acetic acid in
ethanol was added and covered to stand for 4 hours. This was filtered and the extract was
concentrated using a water bath to one quarter of the original volume. Concentrated ammonium
hydroxide was added drop wise to the extract until the preparation was complete. The whole
solution was allowed to settle and the precipitate was collected by filtration and weighed
(Okeniyiet al., 2012) (Falodunet al., 2006)

Determination of Saponin

Five gms of the powder extract was taken and heated at 55 ℃ in 100ml of 20% ethanol for 4
hours and filtered and the residue treated with 100ml of 20% ethanol and combined the filtrate
which was concentrated to 40ml by heating in a water bath at 90 ℃ . The filtrate treated with
20ml diethyl ether shaken vigorously in a separating funnel. Discarded the ether layer and the
aqueous layer was mixed with 60ml n-butanol and the solution was evaporated to dryness
followed by heating the residue to constant weight in an oven. The saponin content was
calculated as percentage of the crude drug taken.

ANTIOXIDANT ACTIVITY METHODS

Total Antioxidant Capacity (Phosphomolybdic Acid Method)

The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent
formation of a green phosphate/Mo (V) complex at acid pH. To 0.3 ml extract (25µg/ml,

16
50µg/ml, and 100µg/ml) was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28
mM sodium phosphate and 4 mM ammonium molybdate) was added. In case of blank 0.3 mL of
methanol was used in place of extracts. The tubes containing the reaction solution were capped
and incubated in a boiling water bath at 95°C for 90 min. After cooling to room temperature, the
absorbance of the solution was measured at 695 nm using a spectrophotometer. The antioxidant
capacity of each sample was expressed as ascorbic acid (A.A) equivalent.

Ferric Reducing antioxidant Power (FRAP) Method

To 1 mL of sample 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium

ferricyanide (1%) were added and mixed. The reaction mixture was incubated at 50 0C for 20
minutes. Then 2.5 mL of trichloroacetic acid (10%) was added and centrifuged for 10 minutes.
An aliquot 2.5 mL was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl 3 (0.1%). The
absorbance of all solutions was measured at 700 nm and expressed as mg of ascorbic acid
equivalent per g of extract (mg AAE/g extact).

Hydrogen peroxide (H2O2) scavenging assay

A solution of hydrogen peroxide (40mM) is prepared in a phosphate buffer (50mM pH 7.4). The
concentration of hydrogen peroxide was determined by absorption at 230nm using
spectrophotometer. The extract of different concentration (20-60µg/ml) in distilled water is
added to hydrogen peroxide and absorbance at 230nm is determined after 10 min against a blank
solution containing phosphate buffer without hydrogen peroxide. The percentage of hydrogen
peroxide scavenging is calculated as follows:

% scavenged (H2O2) = [(Ai – At)/Ai] × 100

Where Ai is the absorbance of control and At is the absorbance of test.

17
 CHAPTER FOUR

RESULTS AND DISCUSSION

PHYTOCHEMICAL CONSTITUENTS (QUALITATIVE ANALYSIS)

Table 1:Qualitative phytochemical constituentsof Cassia occidentalis leaves extract.

Phytochemicals Test used Inference

Alkaloid Wagner’s Test ++

Anthraquinone Borntragor’s Test --
Coumarin --
Flavonoid Shindo’s Test ++
Phenol --
Quinone +++
Saponin Frothing Test ++
Tannin Wohler’s Test +
Sugar +++
Glycoside --

Key: + Present

+ + Moderately present

+ + + Adequately present

-- Absent

The potential activity of these herbs in prevention and treatment of diseases depend on their
bioactive compounds or phytochemicals. Plant-derived bioactive compounds have been found to
stimulate osteoblast differentiation and bone formation. These bioactive compounds include
flavonoids, alkaloids, quinone, saponin, tannin and sugar are present as shown in table 1
(Srividyaet al., 2017)

18
PHYTOCHEMICAL SCREENING (QUANTITATIVE ANALYSIS)

Table 2:Quantitative phytochemical constituentsof Cassia occidentalis leaves extract.

Phytochemical Amount

Tannin (µg/ml) 38.3

Flavonoid (%) 11.2

Alkaloid (%) 1.4

Saponin (%) 18

19
The phytochemicals found in the leaves extracts were further subjected to quantitative analysis to
estimate the phyto compounds present. The results shows that total saponin has highest % of
18% followed by flavanoid 11.2% while alkaloid has 1.4% were all determined by weight
percentage of Cassia occidentalis leaves extract (Table 2) only tannin is calculated in µg/ml
concentration as 38.3µg/ml.

Phytochemicals have various pharmacological activities against ailments such as rheumatism,

diarrhea, dysentery etc. Plant-derived phytochemicals such as tannins, phenolic acids,
flavonoids, anthocyanins and proanthocyanins, lignans, stilbenes, coumarins, quinones,
xanthones, catechins, emodins etc., could delay or prevent the onset of degenerative diseases
because of their redox properties, which allow them to act as hydrogen donors, reducing agents,
hydroxyl radicals (-OH.) or superoxide radical (O2) scavengers. (Srividyaet al., 2017) Ethanolic
extract of Cassia occidentaliswhich was quantitatively assessed for phytochemical analysis
revealed the presence of Alkaloids, flavonoid, tannin and saponin. (Srividyaet al., 2017)

These bioactive compounds may be the probable reason for the free radical scavenging activity
of the extract. (Srividyaet al., 2017)

20
IN VITRO ANTIOXIDANT

Table 3:Total antioxidant capacity the % inhibition and IC50

Concentration Ascorbic acid Cassia occidentalis

(µg/ml) % inhibition % inhibition

20 12 0.4

40 38 8.4

60 44 18.8

80 51 20.20

100 77 33.6

IC50 55.53 52.45

21
Table 4:Hydrogen peroxide scavenging assay the % inhibition and IC50

Concentration Ascorbic acid Cassia occidentalis

(µg/ml) % inhibition % inhibition

20 12 3.6

40 38 22.9

60 44 42.00

80 51 41.00

100 77 30.00

IC50 55.53 80.12

22
Table 5:Ferric reducing antioxidant activity the % inhibition and IC50

Concentration Ascorbic acid Cassia occidentalis

(µg/ml) % inhibition % inhibition

20 12 40.00

40 38 41.95

60 44 60.00

80 51 53.54

100 77 63.90

IC50 55.53 67.43

23
Table 3 to 5 and Figure 2 to 4 shows the hydrogen peroxide, total antioxidant capacity and ferric
reducing antioxidant power scavenging activity of Cassia occidentalisleaf extract. In these
analyses standard ascorbic acid was used as a positive control. The antioxidant property was
expressed as inhibition concentration, IC50. The concentration of extract was used to calculate
the inhibition concentration IC50 in μg/ml.
Cassia occidentaliswas able to effectively scavenge the free radicals in different concentrations
in a dose dependent manner in all the assays.

The total antioxidant capacity and the standard ascorbic acid were calculated at 50% inhibition
(IC50) as 52.45% and 55.53% respectively (Table 3). The hydrogen peroxide scavenging assay
and the standard ascorbic acid were calculated also at 50% inhibition (IC50) as 80.12% and
55.53% respectively (Table 4). Also ferric reducing antioxidant activity together with the
standard were calculated at 50% inhibition (IC50) as 67.43% and 55.53% respectively.

24
Table 6:statistical analysis

Dunnett's multiple Mean 95.00% Below Summa Adjust A-?

comparisons test Diff. CI of diff. threshol ry ed P
d? Value
AscA vs. TAC 0.023 -0.1724 to No Ns 0.9786 B TAC
8 0.2200
AscA vs. FRAP 0.218 0.02245 to Yes * 0.0278 C FRAP
6 0.4148
AscA vs. HPA - -0.3440 to No Ns 0.1625 D HPA
0.147 0.04835
8

The statistical analysis of cassia occidentalis showed that there is no significance differences
between total antioxidant capacity and hydrogen peroxide compared to the ascorbic acid
standard. Ferric reducing antioxidant capacity shows slightly significance difference compared to
the standard. (Table 3)

25
 Comparison of Antioxidant Activity of
Cassia (Senna) Occidentalis Leaves and Ascorbic Acid
0.8

0.6

0.4

0.2

0.0

-0.2
AscA TAC FRAP HPA

Figure 1:Histogram showing the antioxidant activity of Cassia occidentalisby total antioxidant
capacity, hydrogen peroxide and ferric reducing power methods using ascorbic acid as standard.

Total antioxidant power

100
% inhibition

50

0
0 20 40 60 80 100 120
concentration in µg/ml

% ih asc %ihi Ext

Figure 2:Concentration-dependent inhibition of cassia occidentalisleaves extract and

Ascorbic acid (standard) using total antioxidant capacity

Hydrogen peroxide
100
% inhibition

50

0
0 20 40 60 80 100 120
Concentration in µg/ml

% inh std %inh ext

Figure 3:Concentration-dependent inhibition of cassia occidentalisleaves extract and

Ascorbic acid (standard) using hydrogen peroxide scavenging activity.

26
 Ferric reducing antioxidant power
100
% inhibition

50

0
0 20 40 60 80 100 120
concentration in µg/ml

%inh asc %inh ext

Figure 4:Concentration-dependent inhibition of cassia occidentalisleaves extract and Ascorbic

acid (standard) using reducing power activity

Total Antioxidant Capacity of Cassia (Senna) Oc-

cidentalis Leaves and Ascorbic Acid
0.800
0.600
Absorbance

Ascorbic Acid
0.400
Cassia (Senna) Occiden-
0.200 talis
0.000
10 30 50 70 90 0
11
Concentration (µg/ml)

Figure 5: concentration dependent absorbance of cassia occidentalis leaves extract and ascorbic
acid using total antioxidant capacity

Hydrogen Peroxide Activity of Cassia (Senna)

Occidentalis Leaves and Ascorbic Acid
1.000
Absorbance

Ascorbic Acid
0.500
Cassia (Senna) Oc-
cidentalis
0.000
10 30 50 70 90 0
11
Concentration (µg/ml)

27
Figure 6: concentration dependent absorbance of cassia occidentalis leaves extract and ascorbic
acid using hydrogen peroxide scavenging activity

FRAP activity of Cassia (Senna) Occidentalis

Leaves and Ascorbic Acid
0.800
0.600
Absorbance

Ascorbic Acid
0.400 Cassia (Senna) Occiden-
0.200 talis
0.000
10 30 50 70 90 0
11

Concentration (µg/ml)

Figure 7: concentration dependent absorbance of cassia occidentalis leaves extract and ascorbic
acid using ferric reducing antioxidant activity.

28
 CHAPTER FIVE

SUMMARY
Cassiaoccidentalis has been widely used as traditional medicine in African country such such
Nigeria, Cameroon and Ghana. The entire parts of the plant have medicinal values. The roots,
leaves and seeds are part of the plant used. It is an erect herb, commonly found by road side,
ditches and waste dumping sites.The potential activity of these herbs in prevention and treatment
of diseases depend on their bioactive compounds or phytochemicals. Plant-derived bioactive
compounds have been found to stimulate osteoblast differentiation and bone formation. These
bioactive compounds include flavonoids, alkaloids, quinone, saponin, tannin and sugar.

CONCLUSION
Cassia occidentalis has numerous potential to consider as useful medicinal plant against various
diseases. Based on the phyto compounds present. The results shows that total saponin has highest
% of 18% followed by flavanoid 11.2% while alkaloid has 1.4% were all determined by weight
percentage of Cassia occidentalis leaves extract, only tannin is calculated in µg/ml concentration
as 38.3µg/ml.

RECOMMENDATION
 Further studies has to be carried out to use the phytochemicals in pharmaceutical industry
as a substitute for medicine.

29
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LarvicidalAnd Smoke Repellent Activities Of Cassia Occidentalis L. (Caesalpiniaceae)
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LaxmiVerma, Anirudh Khatri, Basant Kaushik, Umesh K Patil, Rajesh S Pawar. (2010)
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31
 APPENDIX
Table 7:mean of the absorbances

Concentration AscA TAC FRAP HPA

(µg/ml)
20 0.187 0.332 0.168 0.563
40 0.391 0.399 0.216 0.479
60 0.443 0.406 0.186 0.487
80 0.488 0.458 0.270 0.632
100 0.703 0.498 0.279 0.790

Ascorbic Acid Standard Graph

0.800
0.600 f(x) = 0.00564833333333333 x + 0.103633333333333
Absorbance

R² = 0.924522811449627
0.400
0.200
0.000
10 20 30 40 50 60 70 80 90 100 110
Conecntrations (µg/ml)

Figure 8:concentration dependent absorbance of ascorbic acid standard.

Total Antioxidant Capacity of Cassia (Senna) Oc-

cidentalis Leaves expressed as μg AAE/g
7042.86
6328.57
5275.00 5400.00
4078.57

20 40 60 80 100

32
Table 9:Total Antioxidant Capacity of cassia occidentalisLeaves expressed as μg AAE/g in
different concentration

Hydrogen Peroxide Activity of different extracts of

Cassia (Senna) Occidentalis Leaves expressed as μg
AAE/g
12257.143
9435.714
8197.619
6697.619 6852.381

20 40 60 80 100

Table 10:hydrogen peroxide scavenging activity of cassia occidentalisLeaves expressed as μg

AAE/g in different concentration.

FRAP of Cassia (Senna) Occidentalis Leaves

expressed as μg AAE/g
2971.429 3132.143

2007.143
1471.429
1150.000

20 40 60 80 100

Table 11: Ferric reducing antioxidant power activity of cassia occidentalisLeaves expressed as
μg AAE/g in different concentration.

33