Evaluation of Antiosteoporotic Activity of Aqueous And
“EVALUATION OF ANTIOSTEOPOROTIC ACTIVITY OF AQUEOUS AND
ALCOHOLIC EXTRACTS OF SEEDS OF SESAMUM INDICUMIN RATS”
SYNOPSIS FOR
M. PHARM DISSERTATION
SUBMITTED TO
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
BENGALOORU, KARNATAKA.
SUBMITTED BY
NITEEN HARI SHANBHAG
I M. PHARM
DEPARTMENT OF PHARMACOLOGY
P.E.SCOLLEGE OF PHARMACY
BENGALOORU – 560050
(2009-2011)
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
BENGALOORU, KARNATAKA.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR
DISSERTATION
1.0 / NAME AND ADDRESS OF THE CANDIDATE. / NITEEN HARI SHANBHAGLOCAL ADDRESS
p.e.s.college of pharmacyHANUMANTHANAGAR, 50 FT. ROAD, BENGALOORU-560 050.
PERMANENT ADDRESS
# 230-E, oppositeDivinekidSCHOOL,Ranade road, Tilakwadi, Belgaum-590 006.
2.0 / NAME OF THE INSTITUTION. / P.E.S.COLLEGE OF PHARMACY
Hanumanthanagar, 50 feet road, BENGALOORU – 560050.
3.0 / COURSE OF STUDY AND SUBJECT. / M.PHARM.
PHARMACOLOGY.
4.0 / DATE OF ADMISSION TO COURSE. / 20th MAY, 2009
5.0 / TITLE OF THE TOPIC:
“ Evaluation ofAntiosteoporotic activity of aqueous and alcoholic extracts of seeds of Sesamum indicum in rats”
6.0
7.0 / 6.1. NEED FOR THE STUDY:
Osteoporosis is the most frequent metabolic condition experienced by elderly individuals. It is a systemic skeletal disease characterized by low bone mass and micro architectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture1. Osteoporosis constitutes a major public health problem, leading to a decrease in the quality of life, disability and even death through its major symptomatic manifestation, the fracture particularly of the hip.
Using the WHO definition of osteoporosis, it has been estimated that 54% of all postmenopausal white woman have osteopenia, and another 30% have osteoporosis. About 70% of women older than 80 years have osteoporosis2.Clinical interest in osteoporosis related to fractures and associated disability. The lifetime risk of a hip, wrist, or clinically diagnosed vertebral fracture is about 40% for white woman and 13% for white men3.
In ancient system of medicine, a several number of herbal plants have been used for osteoporosis, bone calcification and fracture.Sesame seeds are very good source of calcium. Just a quarter-cup of sesame seeds supplies 35.1% of the daily value for calcium. Calcium helps to prevent osteoporosis. Very recently phytoestrogens have received enormous attention for their beneficial effects in osteoporosis4.Sesamin, a sesame lignan, was recently reported to be converted by intestinal microflora to enterolactone, a compound with estrogenic activity and also an enterometabolite of flaxseed lignans, which are known to be phytoestrogens5.
Since sesame seeds contain calcium as well as phytoestrogens which are beneficial in osteoporosis, it was thought worthwhile to investigate for its antiosteoporotic activity.
6.2objective of the study
- To investigate anti-osteoporosis activity of aqueous and ethanolic extractsof seeds of Sesamum indicum in bilateral ovariectomized female rats.
- To investigate anti-osteoporosis activity of aqueous and ethanolic extracts of seeds of Sesamum indicum in heparin induced osteoporosis in rats.
- Parameters measured in this study are serum calcium, alkaline phosphotase (ALP) and tartrate resistant acid phosphatase (TRAP) by using suitable diagnostic kit.
- measurement of bone length, width and weight (femur); ash content, scanning electron microscopy of femur, bone impact test, quantitative X-ray analysis will be performed undercollaboration with IISc.
Sesamum indicumLinn. (Pedaliaceae) is an annual shrub with white bell-shaped flowers with a hint of blue, red or yellow with branches or without branches. Sesame oil plays a prominent role inIndian Ayurvedic medicine. It is rubbed into the skin during abhyanga, a form of Indian massage which improves the flow of energy and helps to remove the impurities from the body4. Sesame seeds and oil possess good wound healing activity6. The ethanolic extract of sesame seeds showed good analgesic and antioxidant activity7. Sesaminol glycosides one of the most abundant lignan glycosides in sesame seed showed protective effect against cognitive deficits induced by beta amyloid peptide in mice8.
The flower extract of sesame possess antineoplastic effect. Flavanoids from Sesamum indicum have hypolipidaemic and hypoglycemic activities as well.If taken internally it prevents hair loss and graying, convalescence, chronic dry constipation, dental caries, osteoporosis, stiff joints, dry cough. It has marked ability to increase milk production in nursing mothers4.
Some of the scientific reports on plant based preparations or agents having antiosteoporosis activity are given here under:
The ethanolic extracts of Cissus quadrangularis showed anti osteoporosis activity in ovariectomized rats9.
The stem bark extracts of Erythrina variegate L. has been reported to suppress the high rate of bone turnover induced by estrogen deficiency, inhibit bone loss and improve the biomechanical properties of bone in ovariectomized rats10.
A study proposed that aqueous black tea extract (camellia sinensis) may be assessed as a phytoestrogenic compound for prevention against estrogen deficiency-related osteoporotic damages11.
The n-butanolic soluble fraction of the root of Achyranthes bidentata showed effective at preventing bone loss in ovariectomized rats and has a great potential as an alternative tool for the treatment ofosteoporosis12.
Another study showed that the waterextract of wood of Taxus yunnanensis may be useful for treatment of postmenopausalosteoporosis, especially for prevention of bone fracture induced by estrogen deficiency13.
PLANT PROFILE
- Sesamum indicum :
Kannada:Yellu.
English: Gingelly seed, Sesamum, Sesame14.
Description: Erect herbs. Leaves simple, variable, lobed or pedatisect. Flowers white or pink, in axillary racemes. Capsule oblong- quadrangular, deeply 4-grooved; seeds- black, brown or white in colour15.
Habitat: This small bush is indigenous to India and extensively cultivated in the warmer regions.
Part Used:Seeds and the fixed oil expressed from the seeds.
ChemicalConstituents:Seeds contain fixed oil 50 to 60%. Also contains proteins 22%, carbohydrates 18%, mucilage 4%, and woody fibre 4%. Oil contains 70% of liquid fat consisting of glycerides of oleic & linoleic acids and 12 to 14% of solid fats- stearin, palmitin & myristin; a crystalline substance sesamin and a phenol compound sesamol. A Flavanoid glucoside- pedallin is present in the leaves14.
Uses:Used in tooth disorders, wound healing, diarrhea, hemorrhoids, neurological deficit disorders, fracture healing, anti-allergic, veneral diseases, aphrodisiac, graying of hairs, baldness, and sinusitis. Seeds are lactagogue and emmenagogue. Leaves are demulcent16.
MATERIAL AND METHODS:
7.1 SOURCE OF DATA :Sesame seeds will be procured from authenticated suppliers. Whole experiment is planned to generate data from laboratories studies. Experiment will be performed as described in the standard bibliography, obtained from standard journals and text books available within the college or from other pharmacy colleges or from libraries of National Institutes or through internet.
Websites:
7.2 METHODS OF COLLECTION OF DATA:
The whole study is divided into following phases:
PHASEI.Collection of plant material.
The seeds of Sesamum indicum will be procured from authenticated supplier from Bengalooruand it will be authenticated by Taxonomist, Dept. of Botany, BangaloreUniversity, Bengalooru.
PHASE II. Preparation of extracts.
Seeds will be successively extracted with water and alcohol (70%) in a soxhlet apparatus for 72 hours. The residue obtained after extraction is dried. The residue mass obtained is evaporated to dryness. A % yield of all the two extracts will be determined.
PHASE III. Preliminary Phytochemical investigation.
Preliminary phytochemical investigation will be done as described by Practical Pharmacognosy- Techniques and Experiments17.
PHASE IV. Acute oral toxicity of plant extract18.
Acute oral toxicity is carried out as per OECD 425 guidelines. Female Swiss albino mice (18 to 25 grams) are individually identified and allowed to acclimate to the laboratory condition for 7 days before the start of the study. Only one mouse receives single dose at a particular time. First animal receives a dose of 175 mg/kg and is observed for any toxicity signs, survival or death up to 48 hrs. If the first animal died or appeared moribund, the second animal receives a lower dose (55mg/kg).Thedose progression or reduction factor is 3.2 times of the previous dose. If no mortality is observed in the first animal then the second animal receives a higher dose (55 mg/kg). Dosing of the next animal is continued depending on the outcome of the previously dose for a fixed time interval (48 hours). The test is stopped when one of the stopping criteria is observed.
5 reversals occur in any 6 consecutive animals tested.3 consecutive animals died at one dose level.
Survived animals are observed for long-term outcomes for a period of 14 days. The acute oral toxicity values are calculated using AOT 425 software (Environmental Protection Agency, USA) based on the short term (48 hours) and long term outcome (14 days).
PHASEV. PHARMACOLOGICAL EVALUATION.
Animals: Spraque-Dawley Rats and Wistar Rats having weight of 180-200g will be selected for all the experiments. Animals will be kept in our animal house at an ambient temperature of 23 ± 2C and 45-55% relative humidity with a 12 hour dark: 12 hour light cycle. Animals will have free access to food and water ad libitium.
1: Effect of seed extracts of Sesamum indicum in bilateral ovariectomizedfemale rats19.
Animal used : -Spraque-Dawley (SD) Rats.Sex :- Female
Weight :- 180-200g
Number of animal in each group:- 6
Procedure:
3 months old female, virgin Spraque-Dawley rats are taken. All the animals are allowed free access to distilled water throughout the experimental period. Experimental animals are randomized into 7 groups of 6 each. GROUP I will be sham operated and serves as basal control. GROUP II to VIIwill be bilaterally ovariectomized under anesthesia. GROUP II will receive vehicle and serves as ovariectomized control, GROUP III is treated with the standard drug Raloxifen (5.4 mg/kg). The animals will be allowed a week to recover and then GROUP IV to VII will betreated with seed extracts (aqueous & alcoholic) of Sesamum indicum in various doses for twelve weeks. Body weights and serum analysis (serum- calcium, ALP, TRAP) of all animals will be recorded during the experimental. At the end of the study period measurement of bone length, width, weight, scanning electron microscopy studies, impact test, quantitative x-ray analysis, ash content and histopathological parameters will be studied.
Treatment protocol:
Rescue experiment is performed in female rats (n=6). The animals are administered low and high doses of seed extracts (aqueous & alcoholic) ofSesamum indicum by p.o. route. The control group animals will receive vehicle.
Group 1 / Sham control
Group 2 / Ovariectomized Control
Group 3 / Ovariectomized + Treated with Standard (Raloxifen 5.4mg/kg)
Group 4 / Ovariectomized + Treated with low dose of aqueous extract
Group 5 / Ovariectomized + Treated with high dose of aqueous extract
Group 6 / Ovariectomized + Treated with low dose of alcoholic extract
Group 7 / Ovariectomized + Treated with high dose of alcoholic extract
# Dose of extracts will be fixed after the outcome of acute oral toxicity test.
2: Effect of seed extracts of Sesamum indicum on heparin induced osteoporosis in female rats20.
Animal used: - Wistar albino Rats.
Sex :- Female
Weight :- 180-200g
Number of animal in each group:- 6
Procedure:3 months old Wistar rats are taken and are allowed free access to distilled water throughout the experimental period. . Experimental animals are randomized into 6 groups of 6 each. The animals in GROUP 1 will receive a daily dose of water for injection (same volume as that of heparin sodium injection) and GROUP 2 to 6 will receive a daily dose of 1000 IU/Kgof unfractionated heparin subcutaneously. GROUP 2 will receive standard drug and GROUP 3 to 6 are treated with seed extracts (aqueous & alcoholic) of Sesamum indicum in various doses for on.Body weights and serum analysis (serum- calcium, ALP, TRAP) of all animals will be recorded during the experimental. At the end of the study period measurement of bone length, width, weight, scanning electron microscopy studies, impact test, quantitative x-ray analysis, ash content and histopathological parameters will be studied.
Treatment protocol:
Rescue experiment is performed in female rats (n=6). The animals are administered low and high doses of seed extracts (aqueous & ethanolic) of Sesamum indicum by p.o. route. The control group animals will receive vehicle.
Group 1 / Control, receiving vehicle only
Group 2 / Heparin + Treated with standard ( Alendronate)
Group 3 / Heparin + Treated with low dose of aqueous extract
Group 4 / Heparin + Treated with high dose of aqueous extract
Group 5 / Heparin + Treated with low dose of alcoholic extract
Group 6 / Heparin + Treated with high dose of alcoholic extract
# Dose of extracts will be fixed after the outcome of acute oral toxicity test.
Statistical analysis :
All the values will be expressedas mean±SEM. The data willbe analyzed by using one way ANOVA followed by suitable post-hoc test. Statistical significance will be set at p< 0.05.
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 experimental models require usage of laboratory animals.
7.4 Has Ethical Clearance been obtained from your institution in case of 7.3?
Yes, ethical clearance has been obtained.(copy enclosed)
8.0 / REFERENCES :
01.PeakWA, Burckhardt P, Christiansen C. Consensus development conference: Diagnosis, prophylaxis and treatment of osteoporosis. Am J Med. 1993;94(6):646-50.
02.Melton LJ, Chrischilles EA, Cooper C, Lane AW, Riggs BL. Perspective: how many women have osteoporosis? J Bone Miner Res 1992;7:1005-10.
03.Chrischilles E, Shireman T, Wallace R. Costs and health effects of osteoporotic fractures. Bone. 1994; 15(4):377-86.
04.Chakraborthy GS, Sharma G, Kaushik KN. Sesamum indicum: A review. J Herb Med Toxicol 2008;2(2):15-9.
05.Jose L, Pen a, Satu M, Heinonen., Anna M, Aura., et al. Dietary Sesamin Is Converted to Enterolactone in Humans. J. Nutr. 2005.
06.Kiran K, Asad M. Wound healing activity of Sesamum indicum L seed and oil in rats. Indian J Exp Biol. 2008 46(11):777-82.
07.Nahar L, Rokonuzzaman. Investigation of the analgesic and antioxidant activity from an ethanol extract of seeds of Sesamum indicum. Pak J Biol Sci. 2009;12(7):595-8.
08.Um MY, Ahn JY, Kim S, Kim MK, Ha TY. Sesaminol glucosides protect beta-amyloid peptide-induced cognitive deficits in mice. Biol Pharm Bull. 2009;32(9):16-20.
09.Annie S, Saleemulla K, Malini S. Antiosteoporotic effect of ethanol extract of Cissus quadrangularis Linn. on ovariectomized rat. J Ethnopharmacol. 2003;89(2-3):245-50.
10.Yan Z, Li. X-L, La PW, Chen. B, Chow. H-K, Wu. C-F, et al. Anti-osteoporotic effect of Erythrina variegata L. in ovariectomized rats. J Ethnopharmacol. 2007;109(1):165-9.
11.Das. AS, Das. D, Mukherjee. M, Mukherjee. S, Mitra. C. Phytoestrogenic effects of black tea extract (Camellia sinensis) in an oophorectomized rat (Rattus norvegicus) model of osteoporosis. Life Sciences. 2005;77(24):3049-57.
12.Cui-Cui H, Rong-Rong H, Yasuhiro T, Shigetoshi K, Li. J-X. Osteoprotective effect of extract from Achyranthes bidentata in ovariectomized rats J Ethnopharmacol. 2009.
13.Yin J, Tezuka Y, Subehan L, Shi M, Nobukawa, Nobukawa T, et al. In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis. Phytomedicine. 2006;13(1-2):37-42.
14.Singh MP, Himadri P. The medicinal herbs and their formulations; 2005.
15.Yoganarasimhan SN. Medicinal plants of India; 2001.
16.Rakesh Sharma, Rajesh A. Herbal Drugs, A twenty first century perspective; 2006.
17.Khandelwals K. Practical Pharmacognosy-techniques and experiments: Nirali Prakashan; 1996.
18. Acute oral toxicity studies [online].Dec 2001[cited 2009 Dec 09]; Available from:
URL:
19.Gnudi S, Giardino R, Mongiorgi R, Fini M, Zati A, Figus E, et al. Evaluation of an experimental model of osteoporosis induced in the female rat through ovariectomy. Boll Soc Ital Biol Sper. 1993;69(7-8):461-8.
20.Prabhakar reddy N. Evaluation of OST-6, a herbal formulation and a comparative study of its individual drugs for bone remineralization activity in osteoporosis. Bangalore: Rajiv Gandhi university of health sciences; 2001.
9.0 / NAME OF THE CANDIDATE / NITEEN HARI SHANBHAG
10.0 / SIGNATURE OF THE CANDIDATE
11.1 / REMARKS OF THE GUIDE / FORWARDED WITH A REQUEST
FOR APPROVAL
11.2
11.3
11.4
11.5
12.0
12.1 /
NAME AND DESIGNATION OF THE
GUIDE
SIGNATURE
HEAD OF THE DEPARTMENT
SIGNATURE
REMARKS OF THE PRINCIPAL
SIGNATURE /
Mr. SRINATH.R
HOD &ASST. PROFESSOR
DEPT. OF PHARMACOLOGY
P.E.S.COLLEGE OF PHARMACY
BENGALOORU-560050.
Mr. SRINATH.R
HOD &ASST. PROFESSOR
DEPT. OF PHARMACOLOGY
P.E.S.COLLEGE OF PHARMACY
BENGALOORU-560050.
FORWARDED FOR APPROVAL
Prof.Dr.S.MOHAN
PRINCIPAL AND DIRECTOR
P.E.S.COLLEGE OF PHARMACY
BENGALOORU -560050.