EVALUATION OF ANTI-OSTEOPOROTIC ACTIVITY OF SPINACIA OLERACEA
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE
BY
KASUNDRA GUNJANLAL SHANTILAL
Under The Guidance of
Mr ASHOKA SHENOY .M.PHARM
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
2011 – 2013
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
ANNEXURE-II
BANGALORE, KARNATAKA.
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.0 / NAME AND ADDRESS OF THE CANDIDATE / LOCAL ADDRESSSTEMS AND LEAVES, 1ST FLOOR, SALVADOOR VILLA, CHILAMBI DWARA, LADY HILL, MANGALORE -576006
PERMANANT ADDRESS
S/O KASUNDRA SHANTILAL P, MAMTA NIVAS, MAHEJAN PETH, BALAGI MANDIR ROAD, MUKHED, DIST NANDED, MAHARASTRA – 431715
2.0 / NAME OF THE INSTITUTION / SRINIVAS COLLEGE OF PHARMACY
VALACHIL,MANGALORE.-574143
3.0 / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOLOGY.
4.0 / DATE OF ADMISSION TO COURSE / 31-10-2011
5.0 / TITLE OF THE TOPIC / EVALUATION OF THE ANTI-OSTEOPOROTIC ACTIVITY OF SPINACIA OLERACEA
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, disability and even death through its major symptomatic manifestation, the fracture particularly of the hip. In 2010, there were 2.7 million hip fractures alone worldwide; with changes in population demographics, this figure is expected to rise to six million by 2050. More than 40% of woman will experience a fracture by the time they reach at the age of 70 years2.
The current drugs for treatment of osteoporosis like hormone replacement therapy (HRT), Calcitonin and Bisphosphonate are associated with major side effects. Renewed attention to alternative medicines and natural therapies has stimulated research interest in herbal medicines. Hence there is need for more efficacious agents that minimize bone loss and stimulate bone deposition with lesser side effects.
REVIEW OF LITERATURE:
6.2.1 Spinacia oleracea (spinach) is an edibleflowering plantin the family ofAmaranthaceae. It is native to central and South-westernAsia and commonly called as ‘Palak’. It is anannual plant , which grows to a height of up to 30cm. Theleavesare alternate, simple, and ovate to triangular-based, very variable in size from about 2–30cm long and 1–15cm broad, with larger leaves at the base of the plant and small leaves higher on the flowering stem. Different parts of this plant have been reported to posses anti-inflammatory, anti-oxidant and CNS depressive activity. The leaves used as cooling, emollient, anti-pyretic, hypoglycaemic, diuretic, laxative, digestible, anthelminthic, urinary concretions, sore throats, pain in the joints, flatulence throat. Spinach very rich in flavonoids such as querecetin, myricetin, kempeferol, apigenin, phenolic compounds such as para-coumaric acid, ferulic acid, ortho-coumaric acid. It contains high concentration of vitamin A, E, C, K, folic acid, oxalic acid and various minerals such as magnesium, calcium, phosphorous, iron, zinc, copper and potash.
The extracts of spinacea oleracea posses free radical scavenging activity.4 The Glycolipids
fractions from Spinach showed anti tumor activity by inhibition of DNA Polymerase.5 Leaves of Spinach showed anti diabetic activity in Alloxan-induced diabetic rats.6 The leaf extracts of spinach showed anti inflammatory potential.7 The aqueous extract of Spinach also have anti ulcer activity8. In vitro studies on leaves showed anthelminthic activity.9
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 Rats.10 Taxus yunnanensis may be useful for treatment of postmenopausal osteoporosis, especially for prevention of bone fracture induced by estrogen deficiency11. A study proposed that aqueous black tea extract (camellia sinensis) may be assessed as a phytoestrogenic compound for prevention against estrogen deficiency-related osteoporotic damages12 . In vivo experiment determined the antiosteoporotic activity of water extract of Dioscorea spongiosain extract, in which it inhibited the decrease in cancellous bone mineral content, cancellous bone mineral density, and cortical bone mineral content of the proximal tibia in ovariectomized rats.13
6.3 OBJECTIVES OF THE STUDY
1) To investigate anti-osteoporosis activity of in bilateral ovariectomized female rats.
2) To investigate anti-osteoporosis activity of in glucocorticoid induced female rats.
The Anti-osteoporotic activity will be evaluated using following parameters :
Biochemical parameters
i. Serum calcium
ii. Alkaline phosphatase (ALP)
iii. Tartarate resistant acid phosphatase
Biomechanical evaluation
i. bone length,
ii. bone weight (femur)
iii. bone volume, bone density (femur)
iv. ash content
v. 4th lumbar vertebra compression
Histomorphometrical study of the bone
7.0 / MATERIALS AND METHODS:
7.1 SOURCE OF DATA:
Experiment will be performed as described in the standard bibliography, literatures and text books. The reputed journals and publications are obtained from college library and through web search.
7.2 COLLECTION OF MATERIAL:
7.2.1 COLLECTION OF PLANT MATERIAL & EXTRACTION:
The Spinacia oleracea leaves will be collected from Mangalore district and will be authenticated by a Taxonomist. The powdered leaves will be extracted by hot percolation method (Soxhlet apparatus) using methanol solvent system. The extract will be filtered through a cotton plug followed by Whattman filter paper No.1 and then concentrated by using a rotary evaporator at low temperature. The extract will be preserved in airtight containers and kept at 4-5°C until further use.
7.2.2 DRUGS AND CHEMICALS:
Raloxifane, Alendronate, Prednisolone, Ketamine will be procured from Cipla Pharmaceuticals, Mumbai. Diagnostic kits to estimate serum calcium, tartarate resistant acid phosphatase and ALP will be procured from Sigma Diagnostic Laboratories, Surat.
7.2.3 ANIMALS
Female Wistar rats (150-180 g) will be procured from Indian Institute of Sciences. They will be maintained under standard conditions (temperature 22 ± 2oC, relative humidity 50±5% and 12 h light/dark cycle).The animals will be housed in sanitized polypropylene cages containing sterile paddy husk as bedding. They will have free access to standard pellet diet and water ad libitum. The Institutional Animal Ethics Committee approved the experimental protocol. All the animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the “National Academy of Sciences” andpublished by the “National Institute of Health”. All the procedures will be performed in accordance with Institutional Animal ethics committee constituted as per the direction of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), under ministry of animal welfare division, Government of India, New Delhi, India.
7.3 EXPERIMENTAL METHODS :
7.3.1 OVERIECTOMIZED OSTEOPOROSIS IN FEMALE RATS 14
EXPERIMENTAL DESIGN :
The female Wistar rats of will be divided randomly into five groups of six animals each. All animals except group I will be ovariectomized to induce osteoporosis. Treatment will be done for 90 days commencing from 22nd post ovariectomized day. All the treatment will be done using oral administration. The different groups will be assigned as below.
Group I : Sham control
Group II : Ovariectomized Osteoporotic Control
Group III : Ovariectomized + Standard Reference drug (Raloxifane 5.4mg/kg body weight)
Group IV : Ovariectomized + Spinacia oleracea extract (300 mg/kg)
Group V : Ovariectomized + Spinacia oleracea extract(600 mg/kg)
EVALUATION:
Biochemical Parameters:
The blood will be withdrawn from retro orbital sinus and serum will be separated for
estimation of biochemical parameters namely Serum calcium, Alkaline phosphatase (ALP), and Tartarate resistant acid phosphatase.
Biomechanical parameters:
Femur bones will be separated from adhering tissues for measurement of biomechanical parameters like bone length, bone weight, bone volume and bone density.
Ash content:
The ash content of bones will be measured by drying it in hot air oven for 24 hrs and heating inside muffles furnace for 6 hrs.
Compression IV lumbar vertebra:
The fourth lumbar vertebra will be located and then isolated. The fresh vertebra will be placed on the Monsanto hardness tester and compressed until it fractured. Histomorphometrical study of the bone: The separated bones will be fixed in 10% neutral formalin for further histomorphometrical study
7.3.3 METHYL PREDNISOLONE INDUCED OSTEOPOROSIS IN RATS15
EXPERIMENTAL DESIGN :
The animals will be randomly divided into five groups of six animals each. All animals
except group I will be induced osteoporosis by subcutaneous administration of Methyl Prednisolone every week throughout the experiment. All the treatment will be done using oral administration. Treatment will be done for 60 days. The different groups will be assigned as below.
Group I : Control receives saline only
Group II : Methyl Prednisolone (7 mg/kg sub cutaneous, once in a week for 8 weeks)
Group III : Methyl Prednisolone + standard drug Alendronate (4mg/kg sub cutaneous)
Group IV : Methyl Prednisolone + Spinacia oleracea extract (300 mg/kg)
Group V : Methyl Prednisolone + Spinacia oleracea extract (600 mg/kg)
EVALUATION:
Biochemical Parameters:
The blood will be withdrawn from retro orbital sinus and serum will be separated for estimation of biochemical parameters namely Serum calcium, Alkaline phosphatase (ALP), and Tartarate resistant acid phosphatase.
Biomechanical parameters:
Femur bones will be separated from adhering tissues for measurement of biomechanical parameters like bone length, bone weight, bone volume and bone density.
Ash content:
The ash content of bones will be measured by drying it in hot air oven for 24hrs and heating inside muffles furnace for 6 hrs.
Compression IV lumbar vertebra:
The fourth lumbar vertebra will be located and then isolated. The fresh vertebra will be placed on the Monsanto hardness tester and compressed until it fractured. Histomorphometrical study of the bone: The separated bones will be fixed in 10% neutral formalin for further Histomorphometrical study
7.4 STATISTICAL ANALYSIS:
The data will be expressed as Mean value + SEM and will be analyzed by the one-way ANOVA.
7.5 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.
Yes. Study requires investigation on Female Wistar rats.
7.6 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION?
Yes. Ethical clearance has been obtained. (Copy enclosed)
LIST OF 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. Riggs B L, Melton L J III. Invouluational osteoporosis. N. Engl. J. Med 1986; 1(1): 314-8.
3. Gaikwad Priyanka Subhash, Shete Rajkumar Virbhadrappa, Otari Kishor Vasant. Spinacia oleracea Linn: A pharmacognostic and pharmacological overview. IJRAP 2010; 1(1):78 -84.
4. Vasthi kennedi evanjelene, Evaluation of free radical scavenging activity and biological properties of spinacia oleracea. Int. J of eng. Sci. tech 2011; 3(1):25-30.
5. Naoki Maeda, Yasuo Kokai, Seiji Ohtani, Hiroeki S ahara, Takahiko Hada, Chisato Ishimaru et al, Anti-tumor effects of the glycolipids fraction from spinach which inhibited DNA polymerase activity. Nutrition and Cancer 2007; 57(2):216-23.
6. V. Gomathi, B. Jayakar, R. Kothai, G. Ramakrishnan, Antidiabetic activity of leaves of Spinacia oleracea Linn in Alloxan-induced diabetic rats. J. Chem. Pharm. Res. 2010; 2(4):266-74.
7. Anil Nagar, Alok kumar Shukla, Papaya Bigonia, Anti-infalmmatory activity potential of Spinaceae oleraceae leaf extract. J. nat pharm 2011; 2(1):80-7.
8. Kore Kakasaheb J, Shete Rajkumar V, Patel Apsari J, Kulkarni Jitendra B, Antiulcer activity of aqueous extract of spinacia oleracia in rats. IJRPC.2011; 2(3):654-61.
9. Shuchi Dave, Amrita Bhaiji, Uttam S.Baghel, S.K.Yadav, Vijay K.Sharma, In-vitro Anthelminthic Activity of Leaves of Spinacia oleracea. IJTPR.2010; 1(1):21-23.
10. 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(20):49-61.
11. Yin J, Tezuka Y, Subehan L, Shi M, Nobukawa, Nobukawa T, In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis.
Phytomedicine 2006; 13(1-2):37-42.
12. 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.
13. Jun YIN, Yasuhiro Tezuka, Kyoji Kouda, Quan le Tran, Tatsuro Miyahara, Yingjie c Hen.et.al. Antiosteoporotic Activity of the Water Extract of Dioscorea spongiosa. Biol Pharm Bull 2004 ; 27(4):583-6.
14. Bhagath Kumar Potu, Muddanna S Rao, Anti-osteoporotic Activity of the Petroleum Ether Extract of Cissus quadrangularis Linn in Ovariectomized Wistar Rats. Chang Gung Med J 2010; 33(3):252-57.
15. Iraj Ragerdi Kashani, Fatemeh Moradi, Prevention of methylprednisolone acetate-induced osteoporosis with calcium administration in rat model. Acta Medica Iranica 2009; 47(4):115-8.
9. / SIGNATURE OF THE CANDIDATE
10. / REMARKS OF THE GUIDE / RECOMMENDED AND FORWARDED
11. / NAME AND DESIGNATION
11.1 GUIDE / MR.ASHOKA SHENOY ,
ASSISTANT PROFESSOR,
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
11.2 SIGNATURE
11.3 CO-GUIDE (IF ANY) / N.A
11.4 SIGNATURE / ------
11.5 HEAD OF THE
DEPARTMENT / MR MOSES SAMUEL RAJAN
ASSOCIATE PROFESSOR
DEPARTMENT OF PHARMACOLOGY,
SRINIVAS COLLEGE OF PHARMACY,
MANGALORE – 574143
11.6 SIGNATURE
12. / 12.1 REMARKS OF THE CHAIRMAN AND THE PRINCIPAL / RECOMMENDED AND FORWARDED
12.2 SIGNATURE