“EVALUATION OF ANTIOSTEOPOROTIC ACTIVITY OF root extracts ofRubia cordifolia in rats”
SYNOPSIS FOR:
M. PHARM DISSERTATION
SUBMITTED TO:
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BENGALOORU, KARNATAKA.
SUBMITTED BY:
SHIVAKUMAR K S
I M. PHARM
DEPARTMENT OF PHARMACOLOGY
P.E.SCOLLEGE OF PHARMACY
BENGALOORU – 560050
(2009-2011)
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BENGALOORU, KARNATAKA.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR
DISSERTATION
1.0 / NAME AND ADDRESS OF THE CANDIDATE. / SHIVAKUMAR K Sp.e.s.college of pharmacyHANUMANTHANAGAR, 50 FT. ROAD, BENGALOORU-560 050.
PERMANENT ADDRESS
MADAGA HARANAHALLI (AT)
SURAGIHALLI (POST)
SHIKARIPUR (Tq)
SHIMOGA (Dt)
577427
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 OF ANTIOSTEOPOROTIC ACTIVITY OF root extracts ofRubia cordifolia in rats”
6.0 / 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, to disability and even to 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. In Ayurveda(Sandhaniya)Rubia cordifolia is used as a bone mender or used to heal fractured bones4. Yan-Bin W. et al. have reported the isolated Anthraquinones such as Physcion etc.from Morinda officinalis having Antiosteoporotic activity on osteoblasts and osteoclasts5. Since various Anthraquinones such as Physcion etc. also found in roots of Rubia cordifolia, we plan to explore the Antiosteporotic activity of root extracts of Rubia cordifolia using suitable animal model.
6. 2 objective of the study:
- To investigate anti-osteoporosis activity of aqueous and ethanolic root extracts of Rubia cordifolia in bilateral overiectomized female rats.
- To investigate anti-osteoporosis activity of aqueous and ethanolic root extracts of Rubia cordifolia in methyl prednisolone induced female rats.
Biochemical parameters measured in this study are serum calcium, alkaline phosphotase and tartrate resistant acid phosphatase by using suitable diagnostic kit.
measurement of bone length, weight (femur); bone volume, bone density (femur); ash content, scanning electron microscopy of femur, bone impact test, quantitative X-ray analysis are performed under collaboration with IISc.
6.3 PLANT PROFILE:
1.Rubia cordifolia:
Vernacular name: English: Indian madder
Kannada : Manjusta, Citravalli, Somalate, Siragatti
Sanskrit : Manjistha, Yojanavalli
Hindi : Mamjith, Majith
Description: Scabrous climber; stem four-gonous; leaves whorld, ovate-acuminate, five nerved from base, scabrid, base rounded-cordate, margin entire; flower small, greenish, in axillary and terminal cymes; calyx truncate, glabrous; corolla rotate to shortly companulate, lobes five, united in the middle into an inflated, short tube; stamens five, exserted; ovary depressed globuse with one ovule per cell, style two, stigmas capitates; drupe globose fleshy, purplish6.
Habitate: The plant is seen in the evergreen forests of peninsular India. Also reported from Greece, Africa and other Asiatic countries like China, Japan, Afghanisthan, Vietnam and malesia.
Part Used: Roots
Chemical Constituents: A new quinizarin-1,4-dihydroxy-6-methylanthraquinone-along with 1-hydroxy-2-methyl anthraquinone, nordamnacanthal and physcion isolated from roots;
7.0 / Four new quinones-1-hydroxy-2-methoxyanthroquinone, 1,4-dihydroxy-2-methyl-5-methoxyanthraquinone, 1,3-dimethoxy-2-carboxyanthraquinone and rubiadin-isolated from roots; 1,4-dihydroxy-2-methylanthraquinone and 1,5-dihydroxy-2-methylanthraquinone along with 3-prenyl-5-methoxy-1,4-naphthoquinone isolated from roots; Rubiconmaric acid, rubifolic acid, 1-hydroxy-2-methyl-9,10-anthraquione, 1,3-dihydroxy-2-ehoxymethyl-9,10-anthraquinone, Alizarin, mollugin, primeveroside, ruberythric acid, lucidin and cyclicheptapeptides isolated from roots7.
Uses: Anti-inflammatory, antidysentric, anthelmintic, emmenagogue, diuretic, galacto-purifier, alterant, ophthalmic, antiseptic. They are used in rheumatoid arthritis, neuralgia, cephalgia, diarrhea, skin diseases, leucoderma, ulcers, amenorrhoea, dysmenorrhoea, diabetes, fever, slow healing of broken bones, urethrorrhea, haemorrhoids, jaundice, hepatopathy, arthralgia and debility8.
6.4REVIEW OF THE LITERATURE ON TITLE PLANT:
Rubia cordifolia is also called as Manjishta, is an important medicinal plant used in the Ayurvedic medicinal system. Rubiadin, a major constituent isolated from Rubia cordifolia Linn. has a potent hepatoprotective action against carbon tetrachloride induced hepatic damage in rats9. Aqueous root extract of Rubia cordifolia showed Antihyperglycemic activity in streptozotocin-induced Diabetic rats10. One of the result showed that alizarin isolated from roots extract of Rubia cordifolia possessed significant modulatory role against the genotoxicity of mutagens11. In an invitro study, ethanolic and hexane extracts of roots of Rubia cordifoliashowed significant free radical scavenging activity12. Ethanolic root extract of Rubia cordifolia showed protective effect against cognitive deficits induced by beta amyloid peptide in mice13.
Some of the scientific reports on plant based preparations or agents having antiosteoporosis activity are given here under:
Ethanol extract from the root of Morinda officinalis showed antiosteoporotic activity in Ovariectomized Rats14.The ethanolic extracts of Cissus quadrangularis showed anti osteoporosis activity in ovariectomized rats15.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 rats16.A study proposed that aqueous black tea extract (camellia sinensis) may be assessed as a phytoestrogenic compound for prevention against estrogen deficiency-related osteoporotic damages17.The n-BuOH 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 ofosteoporosis18.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 deficiency19.
MATERIAL AND METHODS:
7.1 SOURCE OF DATA:
The plant material 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, may be obtained from standard journals and text books available within the college or from other pharmacy colleges or from libraries of National Institutes or through internets from industry.
7.2 METHODS OF COLLECTION DATA:
The whole study is divided into following phases:
Phase 1: Collection of plant material.
The roots of Rubia cordifolia will be procured from authenticated supplier from Bengalooru
and it will be authenticated by Taxonomist, Dept. of Botany, Bengalooru University,
Bengalooru.
Phase II: Preparations of extracts.
Dried powder leaf material will be successively extracted with alcohol (70%) and with water in a soxhlet apparatus for 72 hours. The residue obtained after extraction will be dried. The
residue mass obtained will be evaporated and reduce temperature to dryness. A percentage yield of the two extracts will be determined.
Phase III: Preliminary Phytochemical investigation.
Preliminary Phytochemical investigation will be done as described in Practical Pharmacognosy-techniques and experiments.20
Acute oral toxicity of plant extract(AOT).
Acute oral toxicity is carried out as per OECD 425 guidelines. Female Swiss albino mice (18-20 g) 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). The dose 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)21.
II. PHARMACOLOGICAL EVALUATION.
Animals: Spraque-Dawley Rats and Wister Rats having weight of 120-150 g will be selected for all the experiments. Animals will be kept in our animal house at an ambient temperature of
25C and 45-55% relative humidity with a 12/h dark: 12 hour light cycle. Animals will have free access to food and water ab-libitium.
1: Assessment of Anti-Osteoporosis activity of root extracts of Rubia cordiofolia in bilateral ovariectomized female rats.
Animal used :- Spraque-Dawley RatsSex :- Female
Weight :- 120-150g
Number of animal in each group:- 07
Procedure:
The evaluation of osteoporosis is followed accordingly. Initially testing is carried with the 3 months old female, virgin Spraque-Dawley rats. All the animals are allowed free access to distilled water throughout the experimental period. After 1 week, rats are randomized into 7 groups of 7 each. GROUP I rats are sham operated and GROUP II to V are bilaterally overiectomized under anesthesia. The animals are allowed a week to recover and then treated with aqueous and alcoholic extracts of Rubia cordifolia in various doses for twelve weeks. Body weights and serum analysis of all animals are recorded during the experimental. At the end of the study period Biochemical parameters viz.serum calcium, alkaline phosphotase, tartrate resistant acid phosphotase. Biomechanical and histopathological parameters vizmeasurement of bone length, width, weight, density, scanning electron microscopy studies, impact test, quantitative x-ray analysis and ash content are studied22.
Treatment protocol:
TREATMENT / DRUG / DOSE
Group 1 / Sham control / -
Group 2 / Ovariectomized Control receives vehicle only / -
Group 3 / Ovariectomized +standard drug Raloxifen. / 5.4mg/kg, p.o.
Group 4 / Ovariectomized + aqueous extract / low dose#
Group 5 / Ovariectomized + aqueous extract / high dose#
Group 6 / Ovariectomized + alcoholic extract / low dose#
Group 7 / Ovariectomized + alcoholic extract / high dose#
# Dose of extracts will be fixed after the outcome of acute oral toxicity test.
2: Assessment of Osteoporosis activity in Methyl Prednisolone induced female rats18
Animal used :- Wister Rats
Sex :- Female
Weight :- 120-150g
Number of animal in each group:- 07
Procedure:
Initially testing is carried with the 6 months old Wister rats. 47 animals are divided into 7 groups of 07 each. Group I will be considered as normal control and administered with normal saline s.c. GROUP II animals serves as negative control receives prednisolone s.c once in a week, at a dose of 7mg/kg for 8 weeks.GROUP III animals administered with prednisolone along with standard drug Alendronate. group IV, V, VI & VII animals administered with prednisolone along with low and high doses of aqueous and alcoholic extracts of Rubia cordifoliaby p.o. route for eight weeks. During the experiment Biochemical
parameters viz. serum calcium, phosphorus and serum alkaline phosphotase will be estimated. At the end of the experiment the femur bone from each group is collected for its histopathological examination23.
Treatment protocol:
TREATMENT / DRUG / DOSE
Group-1 / Control receives saline only / -
Group-2 / Methyl prednisolone / 7 mg/kg s.c.(once in a week for 8 weeks)
Group-3 / Methyl prednisolone + standard drug Alendronate / 4mg/kg s.c
Group-4 / Methyl prednisolone + aqueous extract / Low dose #
Group-5 / Methyl prednisolone + aqueous extract / High dose #
Group-6 / Methyl prednisolone + alcoholic extract / Low dose #
Group -7 / Methyl prednisolone + alcoholic extract / High dose #
# Dose of extracts will be fixed after the outcome of acute oral toxicity test.
8.0 / 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 investigation to be conducted on patients
Or other humans or animals?
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 of IAEC clearance has been attached]
REFERENCES:
1.Peak WA, Burckhardt P, Christiansen C. Consensus development conference: Diagnosis, prophylaxis and treatment of osteoporosis. Am J Med. 1993;94(6):646-50.
2.Melton LJ, Chrischilles EA, Cooper C, Lane AW, Riggs BL. Perspective: how many women have osteoporosis? . J Bone Miner Res. 1992;7:1005-10.
3.Chrischilles E, Shireman T, Wallace R. Costs and health effects of osteoporotic fractures. Bone. 1994;15(4):377-86.
4.Herbalayurveda [online].Aug 2006[cited 2009 Dec 13];Available from: URL:
5.Yan-Bin W, Cheng-Jian Z, Lu-Ping Q, Lian-Na S, Ting H, Lei J, et al. Antiosteoporotic Activity of Anthraquinones from Morinda officinalis on Osteoblasts and Osteoclasts. Molecules. 2009;14:573-83;.
6.Sivaranjan VV, Indira B. Ayurvedic drugs and their plant sources. 1 ed. New Delhi.: Mohan primalani for Oxford and IBH Publishing Co. Pvt. Ltd.; 2002..
7.Rastogi R, Mehrotra BN. Compendium of Indian medicinal plants. Lucknow: Central drug research institute; 1984.
8.Vaidhyaratnam PS. Indian medicinal plants a compedium of 500 species. Chennai: Orient Longman pvt.Ltd; 2004.
9.Guntupalli M, Mohana R, Chandana VR, Palpu P, Annie S. Hepatoprotective effects of rubiadin, a major constituent of Rubia cordifolia Linn. J Ethnopharmacol. 2006;103:484–90.
10.Baskar R, Bhakshu LM, Vijaya Bharathi G, Sreenivasa Reddy S, Karuna R, Kesava Reddy G, et al. Antihyperglycemic activity of aqueous root extract of Rubia cordifolia. in
streptozotocin-induced diabetic rats. Pharma Biol. 2006;44(6): 475-9.
11.Prabhjit K, Madhu C, Subodh K, Neeraj K, Bikram S, Satwinderjeet K. Modulatory role of alizarin from Rubia cordifolia L. against genotoxicity of mutagens. Food Chem Toxicol. 2009.
12.Prabhjit K, Bikram S, Subodh K, Satwinderjeet K. In vitro evaluation of free radical scavenging activity of Rubia cordifolia L. . J Chin Clin Med. 2008;3:278-84.
13.Chitra V, Pavan Kumar K. Neuroprotective studies of Rubia cordifolia Linn. on β-amyloid induced cognitive dysfunction in Mice. Int J Pharm Tech Res. 2009;1:1000-9.
14.Nan L, Lu-Ping Q, Ting, Han., Yan-Bin Wu, Zhang Q-Y, Hong Z. Inhibitory effects of Morinda officinalis extract on bone loss in ovariectomized rats. Molecules. 2009;14:2049-61.
15.Annie S, Saleemulla K, Malini S. Antiosteoporotic effect of ethanol extract of Cissus quadrangularis Linn. on ovariectomized rat. Journal of Ethnopharmacology. 2003;89(2-3):245-50.
16.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. Journal of Ethnopharmacology. 2007;109(1):165-9.
17.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 Pages. 2005;77(24):3049-57.
18.Cui-Cui H, Rong-Rong H, Yasuhiro T, Shigetoshi K, Li. J-X. Osteoprotective effect of extract from Achyranthes bidentata in ovariectomized rats
Journal of Ethnopharmacology. 2009.
19.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.
20.Khandelwal KR. Practical Pharmacognosy-techniques and experiments. Pune: Nirali Prakashan; 1996.
21. Acute oral toxicity studies [online].Dec 2001[cited 2009 Dec 09]; Available from:
URL:
22.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 Jul-Aug;69(7-8):461-8.
23.Naghavi M, Mesagarzadeh DD. On the attempt to establish a model on steroid induced osteoporosis in bones of rats. . Acta Med Iran. 1975;18(3-4):175-93.
9.0 / NAME OF THE CANDIDATE / SHIVAKUMAR K.S
10.0 / SIGNATURE OF THE CANDIDATE / (SHIVAKUMAR K.S)
11.1 / REMARKS OF THE GUIDE
11.2
11.3
11.4
11.5
12.1
12.2 /
NAME AND DESIGNATION OF THE
GUIDE
SIGNATURE
HEAD OF THE DEPARTMENT
SIGNATURE
REMARKS OF THE PRINCIPAL
SIGNATURE / MR.MUKUND HANDRAL
ASST. PROFESSOR,
DEPT. OF PHARMACOLOGY
P.E.S.COLLEGE OF PHARMACY
.
Mr.SRINATH.R
HOD& ASST PROFESSOR
DEPT. OF PHARMACOLOGY
P.E.S.COLLEGE OF PHARMACY
FORWARDED FOR APPROVAL
Prof. Dr. S.Mohan
PRINCIPAL AND DIRECTOR
P.E.S.COLLEGE OF PHARMACY
BENGALOORU -560 050