Evaluation of Antimutagenic,Antifungal and Anthelmintic Activity of Tylophora Indica Leaves

Evaluation of Antimutagenic,Antifungal and Anthelmintic Activity of Tylophora Indica Leaves

EVALUATION OF ANTIMUTAGENIC,ANTIFUNGAL AND ANTHELMINTIC ACTIVITY OF TYLOPHORA INDICA LEAVES

SYNOPSIS FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

BY

YASHAVANTH KUMAR N.L

I M.PHARM

DEPARTMENT OF PHARMACOLOGY

VISVESWARAPURA INSTITUTE OF PHARMACEUTICAL SCIENCES

BANGALORE-560070

(2013-2014)

RAJIV GANDHI UNIVERSITY OF HEA LTH SCIENCES

KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.0 / NAME OF THE CANDIDATE
AND ADDRESS(IN BLOCK
LETTERS) / YASHAVANTH KUMAR N.L S/O LAKSHMIKANTHAIAH N.V
NIDUVALALU VILLAGE (POST)
TUMKUR DISTRICT
PIN:572120
2.0 /

NAME OF THE INSTITUTION

/

VISVESWARAPURA INSTITUTE OF

PHARMACEUTICAL SCIENCES

BANGALORE-560070

3.0 /

COURSE OF STUDY AND SUBJECT

/

MASTER OF PHARMACY IN

PHARMACOLOGY

4.0 /

DATE OF THE ADMISSION

/ 26-07-2012
5.0 /

TITLE OF THE TOPIC:

EVALUATION OF ANTIMUTAGENIC,ANTIFUNGAL AND ANTHELMINTIC
ACTIVITY OF TYLOPHORA INDICA LEAVES
6.0 / BRIEF RESUME OF THE INTENDED WORK
6.1NEED FOR THE STUDY
The human body is continuously and unavoidably exposed to diverse chemical carcinogens namely polycyclicaromatic hydrocarbons, heterocyclic amines, and aromatic c amines, which in order to express their genotoxicity and carcinogenicity, must be metabolised to reactive intermediates that are capable of interacting covalently with DNA. Damage to DNA is likely to be a major cause of cancer and other diseases. Many naturally occurring compounds with anti-oxidant activity are known to protect cellular components from oxidative damage and prevent chronic degenerative diseasessuch as atherosclerosis and heart diseases, which are the leading causes of death in the human population. There has been growing interest in identification and use of herbal plants to prevent mutagenesis and carcinogenesis due to their relative nontoxic effects.1Nature has bestowed us with medicinal plants. There is a need to explore them for use as antimutagenic and anticarcinogenic food or drug additives.2Tylophora indica is one such plant scientifically proven for the treatment of bronchial asthma, cancer,rheumatism. It also has immunomodulatory, antioxidant, antiasthmatic, smooth muscle relaxant, antihistaminic, hypotensive, activities. Since, the constituents of Tylophora indica have anticancer,3antioxidant activities,4 it may be evaluated for anti-mutagenic activity.
Fungus infections are the diseases caused by growth of fungi in or on the body.5Tinea infections are among the most common dermatologic conditions throughout the world. Tinea corporis and tinea cruris, are superficial dermatophyte infections, commonly known as ‘‘ring- worm’’.6Tylophora indica has shown antifungal activity against fungi like A.niger ,A.fumigatus and T.viridae.7 It is used in traditional medicine for psoriasis and seborrheic dermatitis8 and hence can be suspected to treat ring worm infections which is a common fungal infection.
Helminthiasis is a macroparasiticdiseaseof humans and animals in which a part of the body is infected withparasitic wormssuch aspinworm,roundworm, ortapeworm. Worms often live in thegastrointestinal tract, but may also burrow into theliver,lymphatic system, or otherorgans.9Helminthiasis ishighly prevalent particularly in third world countries (Dhar et al., 1982) due to poor management practices. However, increasing problems of development of resistancein helminths due to synthetic anthelmintic drugs (Geert & Dorny, 1995; Coles, 1997) have led to the proposal of screening medicinal plants for their anthelmintic activity.10Tylophora indica is reported to have anti amoebic activity11 hence it may possess anthelmentic activity.
Hence the present study is to evaluate aqueous (AQLT)and alcoholic (ALLT) extracts of leaves of Tylophora indica for anti-mutagenic, antifungal, anthelmentic activity.
6.2REVIEW OF LITERATURE
Tylophora indica(Burm f.) Merill. (Family: Asclepidaceae) commonly known as Antmul is a twining perennial plant distributed throughout southern and eastern part of India in plains, forests, and hilly places. The plant is found growing normally in Uttar Pradesh, Bengal, Assam, Orissa, Himalayas and sub Himalayas in India. It is a branching climber or shrub that grows up to 1.5 meters, leaves are obvate-oblong to elliptic-oblong, 3-10cm long and 1.5-7cm wide. The plant has been traditionally used for the treatment of bronchial asthma, jaundice and inflammation. Its antitumor,immunomodulatory, antioxidant, antiasthmatic, smooth muscle relaxant, antihistaminic, hypotensive, antirehumatic activities are scientifically proven. In Ayurveda, the plant has been used in treatment of asthma, dermatitis and rheumatism. Although the leaf and root of this plant are widely used for treating jaundice in Northern Karnataka, there is a paucity of scientific evidence regarding its usage in liver disorder . The other reported activities include cytotoxic effect, immunomodulatory activity, anticancer activity and antiamoebic activity,3 antidiarrhoeal activity,12 antiulcer activity,13hepatoprotective activity,14 Endophytic fungi activity,15 diaphoretic, osteoarthritis pain, whooping cough16, anticataleptic activity aganist Haloperidol Induced Catalepsy,17 anthyperilipidemic activity.18It also seems to be a good remedy in traditional medicine as antipsoriasis, seborrheic dermatitis, anaphylactic, leucopenia ,and the plant was investigated by well-diffusion method against bacterial pathogens associated with HIV.8
The active constituents of Tylophora indica are phenanthroindolizidine alkaloids liketylophorine, tylophorinine, tylophorinidine and septidine. Recently some rare alkaloids namely tyloindicines A, B, C, D, E, F, G, H, I, and J, desmethyl- tylophorine, desmethyl tylophorinine, isotylocrebrine, anhydroustylophorinine, anhydrous- dehydrotylophorinine, γ- fagarine, skimmianine, 14- hydroxyisotylocrebrine, 4,6 desmethylisodroxy- o Methyltylophorinindine have been reported. The non-alkaloidal compounds isolated from Tylophoraindica are kaempferol, quercetin, α- and β-amyrins, tetratriacontanol,octaosanyloctacosanoate, sigmasterol, β-sitosetrol, tyloindane, cetyl-alcohol, wax, resin, coutchone, pigments, tannins, glucose, calcium salts, potassium chloride, quercetin and kaempferol. The new alkaloids include tyloindicines A-E, (+)-14- hydroxyisotylocrebrine and 4-6- desmethylisotylocrebrine. Tylophorine, 6- desmethyltylophorine, tylophorindine and 5-hydroxy-o-methyltylophorindine were the known alkaloids.3
Crude and pure extracts of Tylophora indica showed more antifeedant activity than stem and root against Spodoptera litura, a polyphagous pest on wide ranging crops..Similarly the crude extracts of leaf has exhibited higher antibacterial activity than root and shoot against Basillus subtilis, Staphylococcus aureus, Mycrococcus luteus and P.aergenosa. All the crude and pure compounds showed antifungal activity against Aspergillus niger, Aspergillus fumigatus and Trichoderma viridae.7
Methanolic leaves extract of Tylophora indica showed anti diabetic activity against alloxoneinduced diabetic in rats.18Root or leaf powder of Tylophora indica is used in, intermittent fever, as expectorant and administered in respiratory infections, bronchitis. Dried leaves are emetic, and expectorant.16The aqueous and alcoholic extracts of Tylophora indica leaves possess good diuretic activity.19The ethanolic extract of tylophora indica on carrageenan induced hind paw oedema and cotton pellet granuloma has shown to possess anti-inflammatory activity.20 Alcoholic and aqueous extracts of leaves of Tylophora indica are usedfor their antidiarrheal activity in rodents in three experimentally induced diarrhoeal models i.e. castor oil induced diarrhoea and PG-E2 induced enteropooling in rats and gastrointestinal motility test in mice.12 The hydroalcoholic leaf extract of Tylophora indica showed wound healing activity against experimentally induced wounds in rats in excision wound, incision wound, burn wound and dead space wound models.21The antioxidant activity of crude extract of Tylophora indica (leaves and stems)was determined by using DPPH free radical scavenging in which methanol and ethyl acetate extract showed highest antioxidant activity. Tylophorinidine hydrochloride showed promising antioxidant activity.4
Chemical carcinogens namely polycyclicaromatic hydrocarbons, heterocyclic amines, and aromatic amines,interact covalently with DNA . Damage to DNA is likely to be a major cause of cancer and other diseases.1Since the constituents of Tylophora indicahave anticancer3 and antioxidant activities4 it may be evaluated for anti-mutagenic activity.
Tinea infections are among the most common dermatologic conditions throughout the world. .Tinea corporis and tinea cruris are superficial dermatophyte infections, commonly known as ‘‘ring- worm’’.6 Tylophora indica has shown antifungal activity against fungi like A.niger ,A.fumigatus and T.viridae.7 It is used in traditional medicine for psoriasis and seborrheic dermatidis8 and hence can be suspected to treat ring worm infections which is a common fungal infection caused by genera Trichophyton, Microsporum and Epidermophyton.22.
Helminthiasis is a macroparasiticdiseaseof humans and animals in which a part of the body is infected withparasitic wormssuch aspinworm,roundworm, ortapeworm.9Tylophora indica is reported to have anti amoebic activity8 hence it may possess anthelmintic activity.
6.3 OBJECTIVE OF THE STUDY
1.To evaluate anti-mutagenic activity of Tylophora indica leaf extracts (aqueous and ethanolic) by using
a. Bone marrow micronucleus test in mice.
b. Chromosomal Abberation test in mice.
c. AMES test using Histidine requiring strains of Salmonella typhimurium TA98, TA100 and TA102.
2. To evaluate antifungal activityof Tylophora indica leaf extract against dermatophytes of genera Trichophyton, Microsporum and Epidermophyton.
3. To evaluate anthelmintic activityof Tylophora indica leaf extracts using Earth worm model.
MATERIAL AND METHODS
7.1 SOURCE OF DATA :
The data will be generated by performing experiments on animals. The standard information is collected from various journals, standard text books available in library of Visveswarapura Institute of Pharmaceutical Sciences, Indian Institute of Science, RGUHS-digital library and from various standard websites.
Web sites:






METHOD OF COLLECTION OF DATA:
The data will be generated by performing the experiments using animal models like mice.
7.2 METHODOLOGY :
The animals will be procured and kept in a cages at a standard laboratory temperature and humidity. The animals will be provided with standard feed and water ad libitum. Mice will be allowed to acclimatize to the laboratory conditions for a week performing the experiment on them
Dose selection :
A)Tylophora indica (Aqueous and Ethanolic extract) :Dose(200 and 400 mg/kg. b.w p.o.) were selected based on earlier studies.12
B) Cyclophosphamide :Dose (100 mg/kg b.w i.p.) were selected based on earlier studies2
C) Colchicine : Dose (4 mg/kg b.w i.p.)were selected based on earlier studies.2
. 1. To evaluate anti-mutagenic activityofTylophora indica leaf extracts (aqueous and ethanolic) by using
A. BONE MARROW MICRO NUCLEUS TEST26,27
ANIMALS REQUIRED
a) Species : Swiss albino mice
b) Weight : 25-30g
c) Gender : either sex
d) Number : 60 mice
Animals would be housed in plastic cages with paddy husk bedding. Animals would be provided with food and water ad libitum.
Grouping and treatment :
Group 01:(n=6) vehicle treated animals would receive vehicle orally for 7 consecutive days
Group 02 :(n=6) clastogenic challenge control group, animals would be injected with single dose of cyclophosphamide (i.p 100mg/kg ).
Group 03 :(n=6) would receive aqueous leaf extract of Tylophora indica (200mg/kg) orally for 7 consecutive days
Group 04 :(n=6) would receive aqueous leaf extract of Tylophora indica (400mg/kg) orally for 7 consecutive days
Group 05 :(n=6) would recieve ethanolic leaf extract of Tylophora indica (200mg/kg) orally for 7 consecutive days
Group 06 :(n=6) would receive ethanolic leaf extract of Tylophora indica (400mg/kg) orally for 7 consecutive days
Group 07 :(n=6) would be treated with aqueous leaf extract of Tylophora indica (200mg/kg) orally for 7 consecutive days, followed by cyclophosphamide (i.p.100mg/kg ) as challenge on 7th day.
Group 08 :(n=6) would be treated with aqueous leaf extract of Tylophora indica (400mg/kg) orally for 7 consecutive days, followed by cyclophosphamide (i.p.100mg/kg ) as challenge on 7th day.
Group 09 :(n=6) would be treated with ethanolic leaf extract of Tylophora indica (200mg/kg) orally for 7 consecutive days, followed by cyclophosphamide (i.p.100mg/kg ) as challenge on 7th day .
Group 10 :(n=6) would be treated with ethanolic leaf extract of Tylophora indica (400mg/kg) orally for 7 consecutive days, followed by cyclophosphamide (i.p.100mg/kg ) as challenge on 7th day .
Group 3 to 6 is to evaluate if Tylophora indicahas any mutagenic potential.
Animals would be sacrificed by cervical dislocation on 8th day. Femur and tibia would be isolated for extraction of bone marrow. Bone marrow cells would be processed for the micronucleus test.
B. CHROMOSOMAL ABERRATION TEST26,28
ANIMALS REQUIRED
a) Species : Swiss albino mice
b) Weight : 25-30g
c) Gender : either sex
d) Number : 60 mice
Animals would be housed in polypropylene cages with paddy husk bedding. Animals would be provided with food and water ad libitum.
Grouping and Treatment:
Group 01:(n=6) vehicle treated animals would receive vehicle orally for 7 consecutive days.
Group 02 :(n=6) clastogenic challenge control group, animals would be injected with cyclophosphamide (i.p 100mg/kg ) and colchicine.
Group 03 :(n=6) would receive aqueous leaf extract of Tylophora indica (200mg/kg) orally for 7 consecutive days.
Group 04 :(n=6) would receive aqueous leaf extract of Tylophora indica (400mg/kg) orally for 7 consecutive days.
Group 05 :(n=6) would receive ethanolic leaf extract of Tylophora indica (200mg/kg) orally for 7 consecutive days.
Group 06 :(n=6) would receive ethanolic leaf extract of Tylophora indica (400mg/kg) orally for 7 consecutive days.
Group 07 :(n=6) would receive aqueous leaf extract of Tylophora indica (200mg/kg) orally for 7 consecutive days, followed by cyclophosphamide (i.p.100mg/kg ) and colchicine as challenge on 7th day.
Group 08 :(n=6) would receive aqueous leaf extract of Tylophora indica (400mg/kg) orally for 7 consecutive days, followed by cyclophosphamide (i.p.100mg/kg ) and colchicine as challenge on 7th day.
Group 09 :(n=6) would receive ethanolic leaf extract of Tylophora indica (200mg/kg) orally for 7 consecutive days, followed by cyclophosphamide (i.p.100mg/kg ) and colchicine as challenge on 7th day.
Group 10 :(n=6) would receive ethanolic leaf extract of Tylophora indica (400mg/kg) orally for 7 consecutive days, followed by cyclophosphamide (i.p.100mg/kg ) and colchicine as challenge on 7th day.
Group 3 to 6 is to evaluate if Tylophora indicahas any mutagenic potential.
Animals would be sacrificed by cervical dislocation on 8th day.90 minutes prior to death each animal would be injected with 0.04% colchicine in a dose of 4 mg/kg i.p for mitotic arrest. Femur and tibia would be isolated for extraction of bone marrow. Bone marrow cells would be processed for the chromosomal aberration test.
AMES TEST
Antimutagenicity of Tylophora indica leafextract against direct acting mutagens would be determined according to the methods of Maron and Ames (1983). Histidine requiring strains of Salmonella typhimurium TA98, TA100 and TA102 will be used. For this 2ml of top agar containing 0.2 ml of 0.5mM histidine–biotin will be mixed with mutagens (NaN3, MNNG, or NPD), at different concentrations . Different concentrations of tylophora indica extract and 0.1 ml freshly grown typhimurium culture (1×109 cells/ml approximately) would be poured onto minimal agar plates and would be incubated at 37°C for 48 h. After the incubation, the revertant colonies would be counted using a colony counter.1
2. To evaluate antifungal activity of Tylophora indica leaf extract against dermatophytes of genera Trichophyton, Microsporum and Epidermophyton.
Antifungal activity of the crude extract would be tested using agar diffusion method described by Cheesbrough (2006). An antifungal drug (Ketoconazole) would be used as standard drug. The fungal isolates would be allowed to grow on a Sabouraud dextrose agar (SDA) (Oxoid) at 25°C until they sporulated. The fungal spores would be harvested after sporulation by pouring a mixture of sterile glycerol and distilled water to the surface of the plate and later scraping the spores with a sterile glass rod. The harvested fungal spores and bacterial isolates would be standardized to an OD600nm of 0.1 before use. One hundred microliter of the standardized fungal spore suspension would be evenly spread on the SDA (Oxoid) using a glass spreader. Wells would then bored into the agar media using a sterile 6 mm cork borer and the wells would be filled with the solution of the extract (10, 5, 2.5, 1.25, 0.625 and 0.325 mg/ ml), taking care not to allow spillage of the solution to the surface of the agar medium. The plates would be allowed to stand on the laboratory bench for 1 h to allow for proper diffusion of the extract into the media. Two hundred milligram (200 g) of the Ketoconazole drug would be dissolved in 100 mL of distilled water according to the manufacturers' instructions. 1 mg of the solution would be dispensed into the wells using sterile pipettes. Plates would be incubated at 25°C for 96 h and later observed for zones of inhibition. The effect of the extract on fungal isolates would compared with Ketoconazole at a concentration of 1 mg/ml.22
Minimum fungicidal concentration (MFC)
This would be carried out to assess the crude extract for fungicidal or fungistatic effect. It would be carried out as described by Cheesbrough (2006).22
3. To evaluate anthelmintic activityofTylophora indica leaf extracts(aqueous and ethanolic) using Earth worm model
a) Species : pherithema posthuma (Earth worms)
b) Size : 3-5 cm in length;0.1-0.2 cm in
width23
c) Number : 36
The anthelmintic activity would be carried as per the method of ajaiyeoba etal24 with minor modification .The assay is performed on pheretima posthuma due to its anatomical and physiological resemblance with intestinal roundworm parasites of human being.
Groupings :
Group 01 : Earth worms (n=6) would be placed in vehicle (control group)
Group 02 : Earth worms (n=6) would be placed in 10mg/ml Piperazine citrate(std)23
Group 03: Earth worms (n=6) would be placed in 50mg/ml of aqueous leaf extract of Tylophora indica
Group 04 : Earth worms (n=6) would be placed in 100mg/ml of aqueous leaf extract of Tylophora indica
Group 05 : Earth worms (n=6) would be placed in 50mg/ml of ethanolic leaf extract of Tylophora indica
Group 06 : Earth worms (n=6) would be placed in 100mg/ml of ethanolic leaf extract of Tylophora indica
The earth worms of respective groups would be placed into 10ml of the respective extract and the time taken for paralysis and death would be recorded. Maximum cut off time to observe or death would be 120mins.25 Paralysis is said to occur when the worms do not revive even in normal saline. Death is concluded when the worms lose their motility followed with fading away of their body colour.23
7.2.4 STATISTICAL ANALYSIS
The results will be expressed as mean ± standard error of mean. Statistical analysis will be done using one way ANOVA, followed by post hock analysis done by Dunnett’s test.
7.3 Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:
YES,Study requires investigation on animals. The effects of the drug will be studied on various parameters using mice as experimental animal model.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes, Ethical clearance certificate will be attached along with the hard copy.
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