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www.wjpr.net Vol 8, Issue 9, 2019.
Mahanthesh et al. World Journal of Pharmaceutical Research
400
BIOLOGICAL ACTIVITIES OF CASSIA OCCIDENTALIS LINN: A
SYSTEMATIC REVIEW
Mahanthesh M. C.*1, Manjappa A. S.
2, Sherikar A. S.
3, Disouza J. I.
4 and Shinde M. V.
5
Department of Pharmacognosy1, Department of Pharmaceutics
2,4, Department of
Pharmaceutical Chemistry3,5
Tatyasaheb Kore College of Pharmacy, Warananagar, 416 113, Maharashtra, India.
ABSTRACT
Cassia occidentalis L. (Kasamardah), Negro coffee, Family
leguminose, is an erect, perennial plant and have traditional practice, as
well as wide Phytochemicals and having diverse biological activities,
known to possess antiallergic, antibacterial, antidote for poison, blood
purifier, antifungal, antidiabetic, anti-inflammatory, antimutagenic,
psoriasis, melanoblast cell line leprosy and hepatoprotective activity.
Chemicals including achrosin, aloeemodin, cassia occidentanol I,
cassia occidentanol II, emodin, anthraquinones, anthrones, apigenin,
aurantiobtusin, campesterol, cassiollin, chryso-obtusin, chrysophanic
acid, chrysarobin, chrysophanol, chrysoeriol. The presented review
summarizes the botany, ethnopharmacologyquery, phytochemistry,
biological activity and toxicity of the Cassia occidentalis plant. It is accomplished that,
research on Cassia occidentalis suggests an enormous biological potential of this plant in
terms of research during the last few years, it is strongly believed that detailed information as
presented in this review on the phytochemical and various biological properties of the
extracts might provide detailed evidence for the use of this plant in different medicines.
KEYWORDS: Pharmacological activity, Phytochemicals, toxicity study and
ethanomedicinal uses.
1. INTRODUCTION
The worldwide current trend towards the utilization of natural plant remedies has created an
enormous need for information about the properties and uses of medicinal plants
World Journal of Pharmaceutical Research SJIF Impact Factor 8.074
Volume 8, Issue 9, 400-417. Review Article ISSN 2277– 7105
Article Received on
07 June 2019,
Revised on 27 June 2019,
Accepted on 17 July 2019,
DOI: 10.20959/wjpr20199-15430
*Corresponding Author
Dr. Mahanthesh M. C.
Department of
Pharmacognosy, Tatyasaheb
Kore College of Pharmacy,
Warananagar, 416 113,
Maharashtra, India.
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(www.organicfacts.net) and even through, the centuries, people have used the complex
properties found in plants to treat medical ailments (www.studiampress.in).
Human beings are not sufficiently aware that natural products drug discovery is important for
new generations as a tool for their health care. We know that for the major lethal diseases,
there are no truly effective drug treatments. In addition, drug resistance to existing
chemotherapeutic regimens for fungal and bacterial infections, AIDS, cancer, and malaria is
increasing. Because of the challenges for health care in the future, this is the call for decision-
makers, governments, international agencies, and pharmaceutical companies to commit to the
sustainable development of natural products as medicinal agents, particularly in developing
countries (http.sustainnabledevelpoment.un, org).
Modern pharmacological studies revealed that the Cassia occidentalis has several biological
activities such as blood purifier; expectorant, liver disease was studied (Jeri et al., 1999). And
in Unani system of medicine melanoblast cell line was reported(Sumathy et al., 2011),
traditional medicine anti-allergic, anti-inflammatory and anti-oxidant(Sreejith et al.,2010), the
relaxant(Ajagbonna et al., 2010) , anti-nociceptive activity(Sini et al., 2010) and skin diseases
like psoriasis; leprosy(Sathya et al., 2012) in antifungal, anti-diabetic was showed (Prabh et
al., 2011), hepato-protective(Uzzi and Grillo 2013). The hepatomyoencephalopathy in
children (Vashishtha et al., 2007) and hypolipidemic, antioxidant and antiatherosclerogenic
was reported (Fidèle et al., 2017).
In this study, traditional plants in ancient times as well as its current usage in different
population of the world. Although the progressive research work and literature report is on
the way for Cassia occidentalis, till date no review article with accurate information
regarding reported ethanobotanical, ethanobiological and phytochemical studies as well as
folklore use is available. In order to accomplish the mentioned goal, current review aim to
assess different aspects ie ethanobotanical, ethanopharmacological and phytochemical of
Cassia occidentalis, with regard to its folklore use along with current research work carried
out on the plant so for.
2. MATERIALS AND METHODS
The databases used for literature ie Google Scholar, Scopus, Web of Science, Science Direct,
Springer Link, Sci Finder and PubMed. The terminologies used in the review articles consists
of Keywords such as Ethnomedicinal, Ethnobotanical, Ethopharmacological, Phytochemical,
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Pharmacological, Antimalarial, Antidipresent, Antibacterial, Antifungal, Antioxidant,
Cytotoxicity, Neuroprotective, Glycosides, Flavonoids, and Alkaloids. The review reports
every aspect of plant including folk uses, surveys of ethanobotanical, ethno medicinal and
ethno pharmacological uses.
The literature searched is categorized under headings with detail explanation under individual
section as well as respective tables for summarization of data as follows.
3. Literature review
3.1. Plant description
The plant Cassia occidentalis a common weed, a diffuse offensively odorous undersharb,
subglabrous braches, leaflets 3-5 pairs flowers yellow, fruits cylindrical or compressed,
transversly septate glabrous pods containing 20-30 seeds (Arya Vaidya Sala 1994). The
whole plant as well as leaves flower, fruit are shown in [Fig1 respectively]. The Taxonomy
shown in [Table1] and plant brief description of Cassia occidentalis is as below.
Fig-01: Cassia occidentalis whole plant representing different parts of the plant.
Table 01: Taxonomy of Cassia occidentalis.
Cassia occidentali
Kingdom Plantae
Subkingdom Tracheobionta
Division Tracheophyta
Class Magnoliopsida
SubClass Rosidae
Order Fabales
Family Leguminose
Genus Cassia
Species Occidentalis
Botanical Name Cassia occidentalis
Synonym Senna occidentalis
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3.2. Plant
The plant is an eract tropical annual herb with leathery compound leaves growing up to 6 feet
tall. The seed pads are dark brown and curve slightly upword; the seeds are olive brown and
flattened on both ends. The seeds, which are in long pods, can be roasted and made into
coffee like drink.
3.3. Leaves
Leaves are alternate, even pinnately compound, each one with 4–6 pairs of nearly sessile,
opposite leaflets, with a fetid smell when crushed, each leaflet 4–6 cm long, 1.5–2.5 cm wide,
ovate or oblong, lanceolate with a pointed tip and fine white hairs on the margin. The rachis
has a large, ovoid, shining, dark purple gland at the base. Stipules are 5–10 mm long, often
leaving an oblique scar. Inflorescence is a compound of axillary and terminal racemes.
3.4. Flower
The flower is perfect, 2 cm long with 5 yellowish green sepals with distinct red veins and 5
yellow petals.
3.5. Fruits
The fruit is a dry, dehiscent, transversely partitioned, faintly recurved, laterally compressed,
sickle shaped legume (pod), 7–12 cm long, 8–10 mm wide, with rounded tip and containing
25–50 seeds. Seeds are oval shaped, 3.5–4.5 mm wide, flattened; pale to dark brown, slightly
shiny, smooth and with a round pointed tip (Roy et al., 1997; Long and Lakela 1976).
3.2. Geographical distribution
This species is widely scattered from Himalayas to the Westbengal, South India, Burma and
Ceylon(www.bioinfo.bisr.res.in) and found throughout India, up to an altitude of 1.500 mt
(Nadkarni 2005). Coffee senna grows throughout the tropics and subtropics including United
States from Texas to Iowa eastward, Africa, Asia and Australia (Khare 2007; Liogier 1998).
Growing abundantly on wastelands immediately after the rains.
3.2.1. Vernacular names
The Cassia occidentali is know with different common names and these synonym are vary
from one area, languages or district to the other as Chumhettet in Thailand (Stevens et al.,
2001). Barkichakor, Chilmile, Panwar and Tulo tapre in Nepals (Peter Hanelt et al., 1996).
Bricho, Brusca, Frijolillo, Guanina, Fihaari, Dirjinni in Spain and Somalia. Moshabela moha,
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Tsinyembane, Umnwanda nyoka in South Africa (Kee and Walter 1999). Inglatiang, Msalafu,
Mnubobundo, Mnika Uvunda, Mwingajini, Mrumbuzi, Mrambazi, Mbukomaviin Kenya
respectively (Anals Fac Farm Porto 1965).
Negro coffee in english, Kasaumdi in hindi, Ponnavirm in Malayalam, Kasamardah in
sanskrit and Kasinda in telugu(Arya Vaidya Sala 1994).
3.2.2. Ethano medicinal uses (http://www.rain-tree.com/fedegosa)
Ethano medicinal uses of different countries was shown in table 02
Table-02: Ethano medicinal uses of Cassia occidentalis.
Regions Ethano Medicinal Use
India
Abscesses, bites (scorpion), constipation, diabetes, edema, fever, inflammation,
itch, liver diseases, liver support, rheumatism, ringworm, scabies, skin diseases,
snakebite, wounds
Africa
Abscesses, bile complaints, birth control, bronchitis, bruises, cataracts,
childbirth, constipation, dysentery, edema, erysipelas, eye infections, fainting,
fever, gonorrhea, guinea worms, headache, hematuria, hemorrhages (pregnancy),
hernia, increasing perspiration, inflammation, itch, jaundice, kidney infections,
leprosy, malaria, pain (kidney), menstrual disorders, rheumatism, ringworms,
scabies, skin diseases, skin parasites, sore throat, stomach ulcers, stomachache,
swelling, syphilis, tetanus, worms, water retention, wounds
Brazil
Anemia, constipation, edema, fatigue, fever, gonorrhea, liver disorders, malaria,
menstrual disorders, skin problems, tuberculosis, urinary disorders, water
retention, weakness
Central
America
Abortions, antifungal, athlete's foot, birth control, constipation, diarrhea, fungal
infections, headache, menstrual disorders, menstrual pain, pain, respiratory
infections, ringworm, spasms, uterine pain, urinary tract infections, urinary
insufficiency, worms
Mexico
Chills, digestive sluggishness, dyspepsia, earache, eczema, edema, fatigue, fever,
headache, inflammation (skin), laxative, leprosy, nausea, pain, rash, rheumatism,
ringworms, skin problems, sores, stomachache, swelling, tumors, ulcers,
venereal disease, water retention, worms, yellow fever
Panama Colic, inflammation, spasms, stomach problems, worms, and as an antiseptic
Haiti
Acne, asthma, burns, colic, constipation, dropsy, eye infections, gonorrhea,
headache, malaria, rheumatism, skin rashes and infections, and to increase
perspiration
Amazonia For abdominal pain, birth control, bile insufficiency and malaria
Peru for asthma, bronchitis, fever and urinary insufficiency
3.2.3. Phytochemistry
The phytochemicals isolation from Cassia occidentalis yielded different com-pounds, most
of which are reported for the first time and belongs to the class of glycosides. The main plant
chemicals in Cassia occidentalis include: achrosin, aloe-emodin, emodin was reported
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(Anton et al., 1968) and similarly in another study, anthraquinones, anthrones, apigenin,
aurantiobtusin, campesterol, cassiollin, chryso-obtusin, chrysophanic acid, chrysarobin,
chrysophanol [Table-3] (Kudav and Kulkarni 1974). Ferol, lignoceric acid, linoleic acid,
linolenic acid, mannitol, mannopyranosyl, matteucinol, obtusifolin, obtusin, oleic acid,
physcion, quercetin, rhamnosides, rhein, rubrofusarin, sitosterols, tannins, and xanthorine was
showed (Chukwujekwu et al., 2016). In another study of phytochemicals of Cassia
occidentalis reveals that the nature and amount of phytochemicals vary according to climate.
For example stems, leaves and the root bark of the plant from Ivory Coast, Africa contain
small amount of saponins, no alkaloids, sterols, triterpenes, quinines, tannins and flavonoids.
However, a large amount of alkaloids were found in the stem, leaves and fruits from Ethiopia
(Smolenski et al., 1975). [Table 2].
Ararabinol isolated from the whole plant of Cassia occidentalis.ie bisanthraquinone has two
methoxyls instead of two methyls present in ararabinol (Sastry et al., 2011). and α L
arbinopyranoside, β D xylopyranoside and also β D galactopyranoside it was reported (Yadav
and Satnami 2011). Two pigments like 7-methyl physcin, 7-methyltorosachrysone[Table -3]
(Takahashi et al., 1976). In further investigation, new flavonoid compound cassioccidentalins
A, B and C(Kitanaka and Takido1992), and Isolation of cassiollin from Cassia occidentalis
(Ginde et al., 1970) [Table-3].
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Table 03: Reported Phytochemicals of Cassia occidentalis.
(A) Rhein (B) Aloe emodin
(C) Chrysophenol (D) Emodin
(E) Physcin (F) Occidentol I
(G) Occidentol II (H) 4, 4 5, 5 tetrahydroxy 2, 2, methoxy 9, 9
bianthraquinone
(I) α-L arabino pyranoside (J) β-D xylopyranoside
O
O
HHOOC
OH OH
O
O
HHOH2C
OH OHO
O
HH3C
OH OH
O
O
OHH3C
OH OH
O
O
HH3C
OH OH
OH OH O
CH3
OH
CH3
H
OHOHO
H
CH3
CH3
H
OH OH O
CH3
OH
CH3
H
OHOHO
H
CH3
CH3
CH3
OH O OH
OCH3
OCH3
OHOOH
CH3CH3
O
OOH
OH
O
OOH
OH
OH
OH
OH
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(K) β-D galactopyranoside
(L) Cassiaoccidentalis A, B and C
(A-R1& R
2=H, R
3=Rha. B-R
1=H, R
2=OH,
R3=Rha and C-R
1=CH3, R
2=OH, R
3=Rha)
(M) 7-methyl physcin (N) 7-methyltorosachrysone
(O) Cassiollin
4. Pharmacological activities
4.1 Antimicrobial activity
Cassia occidentalis leaf extracts obtained in different solvents showed high antimicrobial
activity on E. coli at concentration between 900–1000 mg. However, E. coli was found to be
most susceptible to a hexane extract at concentration ranges between 500–1000 mg but there
was no antimicrobial activity exhibited against other tested microorganisms (Pseudomonas
multocida, Salmonella typhi, Salmonella typhimurium, Salmonella pyogenes, Salmonella
pneumoniae) was reported (Saganuwan et al 2006) and in another study, examined leaf, seed
and pod for their antifungal acivity of fungi candida albicans, Aspergillus calvatus and
Aspergillus niger, different plants parts in terms of Minimal inhibitory concentration(MIC)
ranged between 200-1000 µg/ml, the extracts performed as good as or better than standard
drugs Nystatin and Greseofulvine with exception of activity of leaf extract against aspergilla
was studied(Vipul and Anjan 2011).
O
OOH
OH
OH
OH
CH3
O
O
O
OR3
CH3
OH
OH
OH O
R2
OR1
H
OH CH3 OH
O
CH3
CH3CH3
OH CH3 OH
H
OHCH3
CH3
CH3
CH2
OH
CH3
COOMe
OH
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Few observations have demonstrated that, different organic and aqueous extracts of leaves of
Cassia occidentalis L were screened for their antimicrobial activity against seven human
pathogenic bacterial and two fungal strains by disk diffusion assay ie P. aeruginosa, K.
pneumoniae, P. mirabilis, E. coli, S. aureus, S. epidermidis and fungus C. albicans, among
these extracts, methanol and aqueous extracts showed significant antimicrobial activity
against most of the tested microbes. The most susceptible microorganism was P. aeruginosa
(18mm zone of inhibition in aqueous extract) followed by P. mirabilis (15 mm zone of
inhibition in methanol extract) and Candida albicans (8 mm zone of inhibition in methanol
extract (Vedpriya et al., 2010).
The antibacterial activities of Cassia occidentalis were screened against Escherichia coli,
Klebsiella pneumoniae and Candida albicans, Staphylococcus aureus, Pseudimonas
aeruginosa and Salmonella typhi using agar well diffusion method at various concentrations
(80-360 mg/ml).
The minimum inhibitory concentration (MIC) was determined using serial dilution method,
while minimum bacterialcidal concentration (MBC) by plating various dilution of extract.
The significant antibacterial activities with MIC in the range of 160 mg/ml- 280 mg/ml, MBC
in range of 160 mg/ml – 320 mg/ml, The extract show the sensitive organisms were S. aureus
and P. aeruginosa while K. pneumonia show some degree of resistances compared to other
organisms tested was reported (Ndarubu et al., 2016).
4.2 Central Nervous system depressant
The leaf ethanol extract of Senna occidentalis possess CNS depressant effect was showed
(Ukwubile et al., 2017).
4.3 Anti-inflammatory activity
In another study, the methanolic extract of the leaves of Cassia italica was investigated for its
anti inflammatory activity in animal models. The extract at different doses used showed good
anti inflammatory activity which has been done significantly, by the formation of oedema
induced by carrageenan and formalin(Sermakkani and Thangapandian 2013) and the leaves
of Cassia occidentalis Linn obtained by cold extraction of mixture of equal proportions of
petroleum ether, ethyl acetate and methanol was chosen for pharmacological screening. In rat
paw edema model induced by carrageenan, the extract at the 400 mg/kg dose level showed
36.68% (p<0.001) inhibition of edema volume at the end of 4h. In the acetic acid-induced
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writhing test, the extract at the 200 and 400 mg/kg dose level showed 39.9% and 52.4%
inhibition of writhing, respectively (Kanakam et al., 2013).
4.4 Anticonvulsant activity The chloroform extract of whole plant of Cassia occidentalis
Linn has remarkable anticonvulsant activity against MES and PTZ induced convulsion was
studied (Mahanthesh and Jalalpure 2016).
4.5 Anti diabetics’ activity
In further investigation, Aqueous extract of Cassia occidentalis produced a significant
reduction in fasting blood glucose levels in the normal and alloxan-induced diabetic rats
(Laxmi Verma et al., 2010) and the leaf extract at different doses and times following in
normal and diabetic mice significantly reduced the blood glucose level to normal in diabetic
mice was studied(Onakpa et al., 2012),
animal group treated with 400mg/kgbwt of
Cassia.occidentalis root extract and this did not differ significantly from that of metformin-
treated rats. The present study shows that the extract has hypoglycaemic potential and is able
to correct the dyslipidemia associated with hyperglycaemia. This finding scientifically proves
its use in traditional medicine for the treatment of diabetes was reported (Garba et al., 2015)
and further more methanol fraction of plant leaves (COLMF) was tested against
streptozotocin-induced diabetic rats. Histopathological examination showed that COLMF
extract protected the pancreatic tissue from STZ-induced damage (Emmanuel et al., 2010).
4.6. Nephroprotective activity
The nephroprotective activity of the 70% hydroalcoholic extract of Cassia occidentalis Linn
was tested against gentamicin induced nephrotoxicity in rats. The degree of protection was
determined by estimating urinary creatinine, urinary glucose, urinary sodium, urinary
potassium, blood urea, serum creatinine levels and body weight of the animals. The In-vivo
antioxidant activity was determined by estimating the tissue levels of GSH, SOD, catalase
and lipid peroxidation. The results suggested that HACO possess nephroprotective activity
against gentamicin induced kidney damage was studied (Gowrisri et al., 2012).
4.7. Antitrypanosomal activity
Similarly in another study, in vitro and in vivo antitrypanosomal effects of the ethanol extract
of Senna occidentalis leaf were investigated. The crude extract exhibited an in vitro activity
against Trypanosomabrucei brucei as it completely eliminated parasites’ motility within 10
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minutes post incubation with 6.66 mg/ml of effective extract concentration was
showed(Ibrahim et al., 2010).
4.8. Myostimulant effect
It was observed that leaves of Cassia occidentalis shows myostimulant effect and is
characterized by the increase of the rhythm and the amplitude of isolated intestine muscle
(Mea Arsene et al., 2017).
4.9. Cytotoxicity activity
In a follow up study, in vitro cytotoxicity and antibacterial properties of plant via alcoholic,
hydro-alcoholic, and aqueous extracts against eight human cancer cell lines from six different
tissues and four bacterial strains. It was observed that aqueous extract had more potential than
hydro-alcoholic and alcoholic extracts against HCT-15, SW-620, PC-3, MCF-7, SiHa, and
OVCAR-5 human cancer cell lines at 100, 30, and 10 μg was studied (Madhulika and Ajit
2010) and in another study the hydroalcoholic extract of seeds on clonidine induced mast cell
degradation has been studied and is compared with the effect of oral administration of
Sodium cromoglycate as standard on rats. A clonidine resulted in mast cell degradation.
Extract of the seeds significantly increased the percentage protection against mast cell
degradation and is capable with protected in mast cell degradation is observed (Patel et al.,
2017).
4.10. Antipyretic and antioxidant activity
Furthermore observation based on antipyretic and antioxidant activity of Methanolic seed
extract of plant by different in-vitro models and evaluated as per standard procedure. Finally,
antipyretic (Brewer’s Yeast Pyrexia model) and antioxidant activity (DPPH and Hydrogen
Pyrode Method) was evaluated by adopting different methods(Vijay et al., 2017) and also
observed that, antioxidant potency of sequential organic and aqueous leaf extracts of Cassia
occidentalis was investigated, employing various model in vitro systems, such as nitric oxide
scavenging (NOS) activity, carotene–linoleic acid model system, hydroxyl radical scavenging
(HRS) activity, reducing power, metal chelating activity (MCA) and superoxide radical
scavenging (SRS) activity(Vedpriya et al., 2011).
The antioxidant activity of Cassia occidentalis was assessed by monitoring the levels of lipid
peroxides, antioxidant enzymes like glutathione peroxidase, glutathione reductase,
glutathione-Stransferase, superoxide dismutase and catalase, and non-enzymic antioxidants
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like reduced glutathione, vitamin-C, vitamin-E, cereloplasmin and uric acid in the liver
tissues the ethanolic extract significantly prevented was reported(Ravi kumar and Abbulu
2011) and in another study, the diuretic and antioxidant properties of aqueous extract of plant
leaves was studied (Fid`ele et al., 2015).
4.11. Toxicological studies
In few observations have demonstrated that, effects of oral sub-acute administration of plant
during pregnancy in female Wistar rats was studied (Yadav et al., 2010) and in similarly
another study a pre-clinical safety evaluation of hydroalcoholic extract of stem and leaf of the
plant, male and female wistar rats, acute or subacute administration of extract is not toxic in
both male and female wistar rats and suggesting a safety use by humans(Mirtes et al., 2011).
4.12. Other activity
It was observed that hypolipidemic and anti-atherosclerotic activity of the plant(Ntchapda et
al., 2017) and in similarly another study, mineral and proximate composition of plant leaf
was studied (Aja et al., 2017). It was reported anti-nociceptive activity(Sini et al., 2010).
In a
follow up study the wound healing property of methanolic crude extract of Cassia
occidentalis leaves and pure compound chyrophenol is isolated, from it was evaluate in
excision, incision and dead space wound models and it was found to possess significant
wound healing property(Saba Shafeen et al., 2012) and acute toxicity and diuretic activity of
ethanolic extract of extract (200 & 400mg/kg b.w, p.o) was evaluated on wistar strain albino
rats the activity was compared with furosemide was studied(Mittal Arun et al., 2011).
In another study the methanolic extract of Cassia occidentalis roots and its antitussive
activity in rats by sulpherdioxide induced caugh refelex method and it showed significant
antitussive activity which was compared with that of codeine phosphate was reported(Mittal
Arun et al., 2011).
5. CONCLUSION
The scientific research on Cassia occidentalis suggests an enormous biological potential of
this plant in terms of research during the last few years, it is strongly believed that detailed
information as presented in this review on the phytochemical and various biological
properties of the extracts might provide detailed evidence for the use of this plant in different
medicines. The phytochemical variations and efficacy of the medicinal values of plant is
dependent on ecological locations and seasons.
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Mahanthesh et al. World Journal of Pharmaceutical Research
412
Roasted seeds of this plant are very commonly used by local people of Haryana as a coffee
substitute and in curing several diseases. In Africa Cassia occidentalis used as folk medicine
for childbirth.
Furthermore, there is a demand to standardize the toxic properties, proper identification,
processing and removal of harmful properties and utilized to prepare superior medicine.
The crude extracts, fractions, sub-fractions as well as isolated compounds from Cassia
occidentalis along with phytochemical, biological and toxicological studies confirm the role
of these plants in its folkloric uses.
ACKNOWLEDGEMENT
Authors are thankful to Management, Shree Warana Vibhag Shikshan Mandal, and Principal,
Tatyasaheb Kore College of Pharmacy, Warananagar for their continuous support in carrying
out this project.
Conflict of Interest: Authors have no conflict of interest.
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