toxicity of Tephrosia calophylla by using Wister albino rats”
Synopsis for registration of M.Pharmacy dissertation.
Submitted to
RajivGandhiUniversity of Health Sciences, Bangalore– 560 041
Karnataka
In partial fulfillment
of the requirement for the Degree of
MASTER OF PHARMACY
IN
PHARMACOLOGY
By
P.Pradeep kumar.
I M.PHARM
Under the Guidance of
S. Vimal kumar
Assistant Professor
Department Of Pharmacology
DAYANANDA SAGARCOLLEGE OF PHARMACY
2010
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE - 560 041, KARNATAKA
ANNEXURE - II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the Candidateand Address / P.PRADEEP KUMAR.
S/o P. Chandra babu,
9-57,New pet bus stand,
Chandragiri.A.P.
2. / Name of the Institution / DayanandaSagarCollege Of Pharmacy
ShavigeMalleshwara Hills
Kumaraswamy layout,
Bangalore – 560 078
3. / Course of Study and
Subject / Master of Pharmacy in Pharmacology
4. / Date of Admission / 18th June 2009
5.Title of the Topic:“Anti-oxidant, Anti-ulcer activity and evaluation of sub-acute toxicity of Tephrosia calophylla by using Wister albino rats.”
6.0
7.0 / Brief resume of the intended work:
6.1-Need of the study:
A) Anti-Ulcer Activity:
Peptic ulcer disease (PUD) is a disorder of gastro intestinal tract which requires a well-targeted therapeutic strategy [1]. The main pathophysiologic condition is an imbalance distribution between offensive (acid, pepsin and pylori) and defensive factors like (musin, prostaglandin, bicarbonate, nitric oxide and growth factors) [1,2].Nearly 15,000 deaths occur in each year due to this disease [1]. PUD like gastric ulcer and duodenal ulcer s affects on a large number of world population and is induced by several factors like stress, smoking, nutritional deficiency and ingestion of non-steroidal anti-inflammatory drugs [2].
Mainly there are 2 treatments for treating ulcer:
1) By reducing the production of gastric acid.
2) With re-enforcing gastric mucosal protection.
The drugs used to treat ulcer like proton pump inhibitors, histamine receptor blockers, drugs affecting the mucosal barrier and prostaglandin analogs [1, 2].
Mostly the Indian medicinal plants (Allophylus serratus, Desmodium gangeticum, Ocimum sanctum, Convorulus pluricaluis, Bidens pilosa, Emblica officinalis) and its phytochemical constituents have been an extraordinary source of therapeutic agents for treating various disorders including PUD and shows encouraging for new findings.The development of tolerance and side effect on clinical treatment makes its arguable[1].This causes the development of new anti-ulcer drugs whichincludes herbal drugs[1, 2].
Tephrosia calophylla BEDD is a perennial under-shrub. It is rich in flavonoids and Isoflavonoids. Many experimental studies in flavonoid rich plants of genus –Tephrosia have demonstrated anti-oxidant, anti-ulcer, hepatoprotective and anti-hyperlipidemic effects. Hence in this study I have choosen Tephrosia calophylla plant for anti-ulcer activity.
B) Anti-Oxidant Activity:
Anti-oxidant plays an important role in food as a health protecting factor. Primary natural sources of anti-oxidants are whole grains, fruits and vegetables. Some anti-oxidants like vitamin C, vitamin E, carotenes, phenolic acids, and phytoestrogens having the capability to reduce free radicals level. Compounds like gallates acts as a strong anti-oxidant activity and monophenols acts as a weak anti-oxidant[3].
Free radicals are formed in the body at the time of drug metabolism, exposure to ionizing radiation, UV light, pollution etc[4].These are present in the form of Reactive oxygen species (ROS) and Reactive nitrogen species (RNS) like superoxide ions(O2)and hydroxyl radicals(OH) and in the form of no free radical species such as hydrogen peroxide(H2O2)[5].These oxygen species leads to many diseases like malaria, immune deficiency syndrome, heart diseases, stroke, arteriosclerosis, diabetes and cancer[5].
Recently the pharmacologists shows more interest on the development of new drugs based on herbal formulations and their extracts which are popularly used in our ancient traditional ayurvedic system of medicine. Due to the cost effectiveness and side effects most of the people prefer the herbal drugs. I selected the Tephrosia calophylla plant which is rich in flavonoids and Isoflavonoids.
Recently flavonoids have considerable interest because of their potential beneficial on human health and they have anti-viral, anti-allergic, anti-platelet, anti-inflammatory and anti-oxidant activities. Hence in this study I have chosen Tephrosia calophylla plant for anti-oxidant activity.
C) Sub-Acute Toxicity:
This study was performed to elucidate the possible sub-acute toxic effects of Chloroform and Methanolic extract of Tephrosia calophylla and some hematological parameters in rats.
.
Plant description:
Tephrosia PERS. (Leguminosae, Papilionoideae) is a large tropical and sub-tropical genus estimated to contain 300 species. Tephrosia calophylla BEDD.is a perennial under-shrub found widely in Talakona forest of Andhra Pradesh, South India. The genus Tephrosia is known to elaborate a rich variety of flavonoids and isoflavonoids. Phytochemical investigation of the whole plant of this hitherto university gated species has led to the isolation of a new coumestan, tephcalostan together with two known flavonoids,7-O-methylglabranin and kaempferol-3-O-beta-D-glucopyranoside.
6.2 – Review of the literature:
Through survey of the literature of the published works- both books and periodicals and internet, yielding the following information
· Ansarullah et al. (2009)[6] reported the Hyperlipidemic potential of a polyherbal preparation on Triton WR 1339 (Tyloxapol) induced hyperlipidemia.
· SeruGanapaty et al. (2009)[7] reported the chemical constituents of Tephrosia calophylla BEDD and isolated a new cytotoxic benzil and coumetan derivatives.
· Pennaka Hari kishore et al.(2003)[8] A new coumestan from Tephrosia calophylla.
· Amit khatri et al. (2009)[9]reported Hepatoprotective activity from aerial parts of Tephrosia purpurea L. and stem bark of Tecomella undulate.
· Bouramanaissay et al. (2005)[10] reported Flavonoids from Tephrosia deflexa and T.albifoliolis
· A.S. Damre et al. (2003)[11] Studies on the immunomodulatary activity of flavonoidal fraction of Tephrosia purpurea.
· A.M. Arriaga et al.(2009)[12]Anti-oxidant and larvicidal activities of Tephrosia egregia against Aedesa egypti.
· P.Pavana et al. (2007)[13] reported Anti-hyperglycemic and anti-hyperlipidemic effects of Tephrosia purpurea leaf extract in Streptozotocin induced in diabetic rats.
· S.S. Deshpande et al.(2008) [14] reported Pharmacological activity of Tephrosia purpurea in rats.
· K Soni, et al. (2006)[15] reported Anti-oxidant activity of fraction of Tephrosia purpurea(Linn).
· K Kavitha et al. (2006)[16] reported Anti-carcinogenic and anti-lipidperoxidative effects of Tephrosia purpurea(Linn.) Pers. in 7, 12-dimethylbenz (a) anthracene (DMBA) induced hamster buccal pouch carcinoma.
· Cesar C. Andrei. et al. (2000)[17] have isolated and reported C-prenyl flavonoids from roots of Tephrosia tunicate.
· Annalakshmi Chinniah et al. (2009)[18] evaluated the potential of Tephrosia purpurea as anti-Helicobacter pylori agent.
· Palanisamy Prabhakar et al. (1996)[19] evaluated a new flavone from Tephrosia hookeriana.
· P.Dharmanl, et al. (2006)[1] reported exploring Indian medicinal plants for anti-ulcer activity.
· V.V.Asha, et al. (2008)[2] reported gastroprotective effect of Dodonaea viscosa on various experimental ulcermodels.
· K.Indira Priyadarshini et al. (2005)[4] evaluated the anti-oxidant activity of different plant extracts and herbal formulations.
· Rumitshah et al. (2010)[3] reported in-vitro anti-oxidant activity of roots of Tephrosia purpurea(Linn).
6.3 – Objective of the Study:
The objective of the present investigation is to evaluate the Anti-oxidant, Anti-ulcer activity and sub-acute toxicity of Tephrosia calophylla.
Plan of work:
1. Collection and Authentication of plant material.
2. Extraction.
3. Phytochemical Evaluation.
4. Anti-ulceractivity.
a) Ethanol induced gastric ulcer
b) Indomethacin induced gastric ulcer
c) Pylorus ligation method
5. Anti-oxidant activity.
a) In-vivo
b) In-vitro
6. Sub-acute toxicity.
Materials and Methods:
7.1– Source of Data:
Ø Whole work is planned to generate data from laboratory studies i.e. experiments are performed as described in reference journals and in text books available with college and various institutions.
Data collected from:
Ø Biological abstract
Ø Chemical abstract
Ø K R Kirtikar & B D Basu
Ø Materia Medica
Ø Medicinal plants of India
Ø Wealth of India
Ø Indian journal of pharmacology
Ø International journal of pharmacology
Ø Journal of Ethnopharmacology
Ø International journal of green pharmacy
Websites:
Ø www.pubmed.com
Ø www.googlescholar.com
Ø www.academicjournals.org
Ø www.sciencedirect.com
7.2– Method of collection of data:
Plant materials –Tephrosia calophylla will be collected from the perennial under shrub found widely in Talakona forest of Andhra Pradesh, India[20]. The plant material will be identified taxonomically and authenticated by Gandhi Krushi Vignana Kendra Bangalore (GKVK). A voucher specimen of the collected sample will be deposited in the departmental herbarium for future reference.
The data and related literatures will be collected from various scientific national and international journal i.e. Indian Journal of Pharmacology, Planta Medica, etc.
7.3 -Does the study require any investigations or interventions to be conducted on Patients or other humans or animals? If so, Please describe briefly.
Yes, the study requires investigation on albino rats.
7.4 – Has ethical clearance been obtained from your Institution in case of 7.3?
Yes, Approved by CPCSEA
7.5 Method:
Preparation of extract:
The fresh leaves of Tephrosia calophylla were dried under shade and powder in a mixture grinder. The powder of plant material used for the extraction. Powder material was extracted according to standard maceration procedure by using the solvents like pet. ether, chloroform and methanol separately. Then each filtrate was concentrated separately on water bath to a thick paste and dried under vacuum [5].
Experimental design:
Evaluation of Antiulcer, Antioxidant activity and sub-acute toxicity will be done using following method:
Animals are divided into 5 groups containing 6 animals in each group for each model. After the acute toxicity studies, the dose will be fixed. The animals are grouped as follows
A)ANTI- ULCER ACTIVITY:
Group 1 : Normal control (Treated with 1% CMC)
Group 2 : Reference control (Ulcer control)
Group 3 : Treatment control 1 (Treated with Chloroform extract)
Group 4 : Treatment control 2 (Treated with Methanol extract)
Group 5 : Standard control
Drugs are administered by oral route. After final day of drug treatment ulcer development is examined under microscope and ulcer index was calculated.
a)Ethanol induced gastric ulcer[2]:
Animals were randomly divided into groups each of 6 rats. Gastric lesions were induced with ethanol at a dose of 8ml/kg administered per oral (p.o). Chloroform extract, Methanolic extract of Tephrosia calophylla, standard drug and saline were administered orally 30min prior to induction of gastric lesions. The animals were anaesthetized 6 h with ether and stomachs were incised along the greater curvature and the length of each ulcer was measured. The glandular portion of the stomach was immersed in sodium carbonate buffer (pH 10) for biochemical analysis.
b)Indomethacin induced gastric ulcer[2]:
Animals were divided into groups each of 6 rats. Gastric lesionswere induced with indomethacin (40 mg/kg) administered to rats after fasting for 24 h. chloroform extract and methanolic extract, standard drug and saline were administered orally 30min prior to induction of gastric lesions. The animals were sacrificed 4 h after treatment with the ulcerogenic agent to assess the ulcer size and antiulcer activity.
c)Pyloric ligation[2]:
Animals were divided into groups each of six rats that were fasted 24 h prior to receiving an oral dose of the saline, Chloroform extract and Methanolic extract of Tephrosia calophylla and standard drug. Pyloric ligation was done by ligating the pyloric end of the stomach of rats 1 h after drug administration. Animals were allowed to recover and stabilized in individual cage and were deprived of water during post-operative period. After 4 h of surgery, rats were sacrificed and gastric juice was collected and subjected to analysis. Gastriccontents were analysed for total acidity by titrating against 0.01N NaOH using phenolphthalein as indicator. The pH of gastric juice was measured by using pH paper strips of varying ranges. The colour of the pH paper after the procedure was matched with standard scale and the pH was recorded for different groups of animals.
Parameters evaluated:
· Measurement of ulcer index[2]
· Gastric glutathione assay[2]
· Estimation of alkaline phosphatase (ALP)[2]
B)ANTIOXIDANT ACTIVITY:
The anti-oxidant activity was evaluated with tissue homogenate (liver and stomach) from the animals using in anti-ulcer activity.
1)In-vivo:
a) Assay of malondialdehyde (MDA)[21]
b) Assay of superoxide dismutase (SOD )activity[21]
c) Assay of catalase activity[21]
d) Assay of total tissue sulfhydryl (thiol) group(reduced glutathione level)[21]
Drugs are administered by oral route. After final day of drug treatment blood is removed from all the animals and parameters like Total leukocyte count, lipid peroxidation, Super oxide dismutase, catalase, Reduced glutathione are estimated.
2)In-vitro:
a) Thiobarbituric acid (TBA) method[5] .
b) Ferric thiocyanate (FTC) method[5] .
c) Determination of DPPH radical scavenging activity[3] .
d) Superoxide free radical scavenging activity[3] .
e) Determination of nitric oxide radical scavenging activity[3].
C) SUB ACUTE TOXICITY: [22]
Four groups of animals were used, each of which contained 4 rats. Group 1 including control animals to which isotonic saline solution (ISS, 10 mL kg−1) will be injected intraperitoneally. Groups 2-4 were treated with extract (chloroform, methanolic) will be administrated intraperitoneally in different doses (equivalent to 10, 20 and 30 percentages of LD50 value), respectively with once dose in a volume of 10 mL kg−1 daily for 2 weeks.
Body weight of the animals was recorded at the beginning and after 7 and 14 days of experiment. Haematological and biochemical/serum profile parameters were estimated at the end of experiment. Animals of the different groups were sacrificed
by cervical dislocation 6 h after the last injection and liver and kidneys were removed. Tissue specimens were fixed in 10% buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin-eosin.
Expected outcome: From these observations, a conclusion may be drawn on the potential of herbs on experimental Anti-oxidant,Anti-ulcer and sub-acute toxicity in animal models which can be extrapolated to human beings.
Statistical analysis –The results were expressed as Mean ± SEM. The data was evaluated using one way ANOVA followed by Newman- keuls multiple range test & differences below p<0.05 are considered as significant.
8.0 / List of References:
1. P. Dharmanl, G.Palits. Exploring Indian medicinal plants for antiulcer activity; Indian Journal of Pharmacology, April(2006), 38(2):95-9.
2. M.Arun, V.V.Asha. Gastroprotective effect of Dodonaea viscosa on various experimental ulcer models; Journal of Ethnopharmacology, (2008):460-465.
3. Rumit shah, Heenakathad Rajrlseth, Naveenseth. Invitro Antioxidant activity of roots of Tephrosia purpurea linn; International Journal of pharmacy and pharmaceutical sciences, (2010), 2 (3): 30-33.