Protective effect of ethanolic extract of Terminalia bellirica against transient global ischemia induced brain damage in normal and STZ-induced diabetic rats
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore – 560 041
By
Miss P.M.SWETHA B.Pharm
Under the Guidance of
Mr. MANJUNATHA .P.M M. Pharm, (Ph.D)
Asst. Professor
Department of Pharmacology,
Acharya & B.M.Reddy College of Pharmacy,
Soladevanahalli, Chikkabanavara (Post),
Hesaraghatta Main Road, Bangalore – 560 090.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1. / Name of the candidate &Address. / P.M.Swetha,
D-no:,10-44/18
Ponnakalava road
Sivaji nagar,
Yogimallavaram,
Thiruchanur,
Andhra Pradesh.
2. / Name of the Institution. / Acharya & B.M. Reddy College Of Pharmacy.
Soladevanahalli, Hesaraghatta Road,
Chikkabanavara Post,
Bangalore-560090.
Phone No: 080 2839 6011.
Fax No: 080 2839 3541.
3. / Course of the study
& subject. / M. Pharm (Pharmacology)
4. / Date of admission. / 17th June 2011
5. / Title of the Topic / Protective effect of ethanolic extract of Terminalia Bellirica against transient global ischemia induced brain damage in normal and STZ-induced diabetic rats
6.
6.1
6.2
6.3 / Brief resume of intended work
Need of the work
Review of Literature
Aim and Objective of the study / Enclosure I
Enclosure II
Enclosure III
7.
7.1
7.2
7.3
7.4 / Materials & Methods
Source of data
Methods of collection of data
( Including sampling ,procedure if any)
Does the study require investigation on animals?
If yes give details
Has ethical clearance been obtained from your institution in case of 7.3 / Enclosure IV
Enclosure V
Enclosure VI
Applied for animal ethical clearance
8. / List of references / Enclosure VII
9. / Signature of the candidate
10. / Remarks of the guide
11.
11.1
11.2
11.3
11.4 / Name & Designation of
Guide
Signature of Guide
Head of the Department
Signature of HOD / Mr. MANJUNATHA.P.M. M.pharm,( Ph.D)
Assistant Professor.
Department of Pharmacology,
Acharya & B.M.Reddy College of Pharmacy,
Soladevanahalli, Chikkabanavara (Post),
Hesaraghatta Main Road, Bangalore– 560 090.
Dr.Kalyani Divakar M.Pharm, Ph.D
Professor & HOD
Department of Pharmacology,
Acharya & B.M.Reddy College of Pharmacy,
Soladevanahalli, Chikkabanavara (Post),
Hesaraghatta Main Road, Bangalore – 560 090.
12.
12.1
12.2 / Remarks of the Principal
Principal
Signature of the principal / Dr. DIVAKAR GOLI M.Pharm, PhD
Acharya & B.M.Reddy College of Pharmacy,
Soladevanahalli, Chikkabanavara (Post),
Hesaraghatta Main Road, Bangalore – 560 090.
Enclosure - I
6. BRIEF RESUME OF INTENDED WORK
6.1 NEED OF THE WORK:
Ischemic stroke accounts for 88% of all strokes and is associated with a high incidence of morbidity and mortality. 1 Stroke has been ranked third most common cause of death worldwide and cerebrovascular diseases are considered second most frequent causes of deaths in the year 2020. 2 Hence, a major challenge that confronts medical researchers is the development of therapeutic agents, which prevent neuronal degeneration induced by ischemia, hypoxia and other traumatic insults to the CNS. Natural products probably represent an ideal source to develop safe and effective agents for the management of stroke and deserve scientific probe. Growing evidence supports the role of oxidative stress as one of the primary factors in brain injury mediated by cerebral ischemia and stroke. 3 The brain is particularly vulnerable to oxidative stress because of its high rate of oxidative metabolic activity, intense production of reactive oxygen species (ROS) metabolites. ROS namely superoxide and hydroxyl free radicals, together with hydrogen peroxide have been proposed to cause neurotoxic effect and initiate a free radical-mediated chain reaction causing additional damage to diverse areas in the brain.4 Therefore, oxidative injury could be one possible cellular cascade affecting all organs and tissues during ischemia. Many antioxidants are reported to reduce ROS-mediated reactions and rescue neurons from ischemia/reperfusion induced neuronal loss in animal models of cerebral ischemia.5, 6
Herbal medicines possess the natural antioxidents which are capable of preventing the oxidative damage in diabetes with high oxidative stress. The major advantage of promoting the use of an herbal drug over synthetic drug is that they have less toxicity in comparison to other contemporary drugs available in the market. In the present study we will evaluate ‘Ethanolic extract of Terminalia bellirica’ against transient global ischemia induced brain damage in normal and STZ-induced brain damage in rats. T.bellirica is one of the oldest medicinal herbs of India, is an ingredient in Indian Ayurvedic formulation ‘TRIPHALA’ used for treatment of digestive and respiratory disorders. “Terminalia bellirica” is belonging to family: Combretaceae. It is locally called by different names in different languages: English- Belliric myrobalan, Sanskrit- Vibhitaki Terminalia bellirica synonyms.7
Enclosure – II
6.2 REVIEW OF LITERATURE:
SCIENTIFIC PUBLICATIONS:
· The analgesic and anti-pyretic activities of ethanolic and aqueous extracts of T.bellirica fruits has been investigated in acetic acid induced writhing, Eddy’s hot plate method and brewer’s yeast induced fever models in mice and rats.8
· Effect of continuous administration of dried 75% methanolic extract of T.bellirica suspended in water was studied in alloxan induced hyperglycaemia and anti-oxidant defence mechanism in rats.9
· Effect of benzene and ethanol extract of T.bellirica barks has been studied on activities of accessory reproductive ducts in male rats.10
· Protective effect of T.bellirica fruit extract and its active constituent, gallic acid has been reported against carbon tetrachloride intoxification which causes liver and kidney damage.11
· The free radical scavenging activity and anti-oxidant potential of acetone extract/fractions of T.bellirica fruit was investigated.12
· Alcoholic and water extracts of T.bellirica showed significant anti-salmonella activity.13
· Growth inhibitory effects of T.bellirica extracts were studied against human hepatocellular carcinoma and lung cancer cells.14
· Water, acetone and chloroform extracts of T.bellirica were examined for their antimutagenic potency using the emes salmonella/microsome assay.15
· The fruits of Terminalia bellirica are used for the medicinal purpose. It is used both, internally as well as externally. The seed oil or the paste of its fruit is applied externally on the swollen and painful parts. The paste of its fruits is applied on the eyelids in conjunctivitis Bibhitaka (Terminalia belerica).16 The Terminalia belerica fruits extract has demonstrated significant anti-tumorous, anti-cancerous and selective toxicity against various types of cancer cells.7
Enclosure – III
6.3 AIM AND OBJECTIVE OF THE STUDY
AIM:
In the present study, different doses of ethanolic extract of Terminalia bellirica to be tried to assess their neuroprotective effect in transient global ischemia induced brain damage in normal and STZ-induced diabetic rats.
OBJECTIVE OF THE STUDY:
To evaluate the neuroprotective potentials of ethanolic extract of Terminalia bellirica in transient global ischemia induced brain damage in normal and STZ-induced diabetic rats by estimating the following parameters.
I. Behavioral studies
- Locomotor activity21
- Beam walking test22
- Hanging wire test23
II. Biochemical estimations:
- Measurement of total protein24
- Measurement of lipid peroxidation25
- Measurement of total thiols26
- Measurement of Glutathione27
- Measurement of brain infract size28
III. Histopathological studies29
Enclosure – IV
7. Materials and Methods:
7.1 SOURCE OF DATA:
The source of data will be obtained from,
Ø Laboratory based studies.
Ø Literature survey, CD ROM.
Ø National & International Journals.
Ø Text books.
Ø Internet.
The sources of data are from experiments on animals which involves the following:
To Evaluate of neuroprotective activity of ethanolic extract of Terminalia bellirica on experimental animals (Albino Wister rats).
Enclosure – V
7.2 METHOD OF COLLECTION OF DATA
Plant materials
The dried powder of Terminalia bellirica fruits will be procured from commercial vendors. Authentified and herbarium preparation done earlier.17
Preparation of ethanolic extract of T.bellirica:
Dried powder of T.bellirica fruits was extracted with ethanol (90%) at a temperature of 77-790C for 48h by using soxhlet extractor. After completion of extraction, remaining solvent was removed by evaporation till the solid mass was obtained. The dried extracts will be stored in airtight container and finally the extracts thus obtained are subjected to phytochemical analysis17.
Dose selection of ethanolic extract of terminalia bellirica:
Dose of T.bellirica was selected from acute toxicity studies performed by Trivedi V P, et al.18 According to results of the reference study, maximum tolerable dose of T.bellirica fruit extract was found to be 1000mg/kg i.p. in mice. Hence, in present study we have selected 1/7th and 1/3rd (150mg/kg and 350mg/kg) dose of T.bellirica as a low dose and high dose respectively.
Acute toxicity studies
Earlier acute oral toxicity studies were carried out for ethanolic extract of T.bellirica using acute toxic class method, according to OECD guidelines No. 423. Based on toxicity data two dose of 150 and 350 mg kg-1 will be selected (OECD, 1996).17
Drugs:
Ethanolic extract of T.bellirica will be suspended in distilled water using Tween 80 (1% v/v). Two doses of ETA (150 and 350 mg kg-1) will be chosen and administered orally17.
Chemicals:
1,1,3,3-tetraethoxyproprane, NADH, nitro blue tetrazolium(NBT), phenazine methosulphate will be procured from Sigma- Aldrich chemicals Ltd, St. Louis, USA. 5, 5’-Dithiobis (2-nitrobenzoic acid) (DTNB), reduced glutathione will be obtained from Himedia Laboratories, Mumbai. All the other chemicals to be procured from Merck laboratories, nice chemicals, Loba chemie, and Sd. fine chemicals will be of analytical grade.
Animals:
100 healthy Wister albino Rats (200-260g) of either sex will be procured from registered suppliers. The animals will be housed in standard environmental condition & provided with food & water ad libitum. All experiments will be conducted in accordance with the guidelines of CPCSEA, Government of India.
ENCLOSURE – VI
7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly.
To evaluate neuroprotective activity of ethanolic extract of Terminalia bellirica on experimental animals (Albino Wister rats).
Experimental protocol
Animals will be divided into five groups of 10 rats each. To obtain maximum data from small number of animals, each group will be subjected to Locomotor activity, Beam Walking test and Hanging wire test before focal cerebral ischemia and after 24 h reperfusion; finally subjected to examine cerebral infarct volume, biochemical parameters and histopathological studies.
The first group will sham operated [rats were subjected to surgical procedure, but did not suffer middle cerebral artery occlusion (MCAO), except for exposure of right internal carotid artery (ICA) and right external carotid artery (ECA)]. The second group received 350 mg/kg, dose of ETA and subjected to sham operation. The third group served as control group (vehicle treated) i.e., rats were orally administered vehicle [simple syrup I.P. + Tween 80 (1%, v/v), 10 ml/kg] for 7 days (pre-treatment) before subjecting to 30 min MCAO followed by reperfusion for 24 h. Fourth and fifth group of rats, orally received 150 mg/kg and 350 mg/kg, dose of ETA respectively. Vehicle or drugs were fed once daily for 7 consecutive days prior to the experiment.
a) Induction of transient cerebral ischemia:
Transient cerebral ischemia was produced by following the method of Iwasaki, et al.19 Rats will be anesthetized by giving Thiopentone sodium (40 mg/kg) i.p. surgical technique and placed in dorsal recumbence. A longitudinal incision of 1.5cm in length was made in the midline of the ventral cervical skin. The right common carotid artery, ICA and ECA were exposed and carefully isolated. A nylon monofilament (40mm in length and 0.24mm in diameter), its tip rounded by flame-heating, was inserted from the lumen of the ECA to that of the right ICA to occlude the origin of the right middle cerebral artery (MCA). The right MCA was occluded for 30 min, and there after brain was allowed to be reperfused with blood by withdrawing the of the nylon thread. 24 h after reperfusion, rats were decapitated. Temperature was maintained at 37±0.5ºC throughout the surgical operation.
b) Induction of Diabetes:
Diabetes will be induced with a single dose of 45mg/kg i.p injection of Streptozotocin (STZ) in 10mM citrate buffer (pH 4.5) after an overnight fast. Three days after the STZ injection, rats with blood glucose levels greater than 250 mg/dl will be considered to be diabetic.20
Diabetic rats will be divided into five groups of 10 rats each. To obtain maximum data from small number of animals, each group will be subjected to Locomotor activity, Beam Walking test and Hanging wire test before focal cerebral ischemia and after 24 h reperfusion; finally subjected to examine cerebral infarct volume, biochemical parameters and histopathological studies.
The first group will be sham operated [diabetic rats will be subjected to surgical procedure, but did not suffer middle cerebral artery occlusion (MCAO), except for exposure of right internal carotid artery (ICA) and right external carotid artery (ECA)]. The second group of diabetic rats will receive 350 mg/kg, dose of ETA and subjected to sham operation. The third group of diabetic rats will serve as control group (vehicle treated) i.e., rats will be orally administered with vehicle [simple syrup I.P. + Tween 80 (1%, v/v), 10 ml/kg] for 7 days (pre-treatment) before subjecting to 30 min MCAO followed by reperfusion for 24 h. Fourth and fifth group of diabetic rats, orally receive 150 mg/kg and 350 mg/kg, dose of ETA respectively. Vehicle or drugs were fed once daily for 7 consecutive days prior to the experiment and further subjected to transient cerebral ischemia.
I. Behavioral Procedures:
1) The locomotor activity will be recorded by using actophotometer (Inco pvt.ltd. ambala, India) before locomotor task; animals will be placed individually in the activity meter for 2 min for habituation. Thereafter, locomotor activity will be recorded using actophotometer for a period of 5 min.21
2) Beam walking test is used to evaluate gross vestibule motor function. The apparatus consisted of a rod 120 cm in length and with a diameter of 2.3 cm. A wooden box (20 cm X 20 cm X 10 cm) will be set at one end of the rod as a nest for motivating the animal to cross the beam. The apparatus will be suspended 50 cm above a cushion, which protected the animals against fall injury. Rats will be trained twice daily for 2 days before BCCA occlusion and assessed for motor coordination after 24 h of reperfusion. The time taken to traverse the beam will be recorded. The cut-off time will be taken as 120 s.22
3) Hanging wire test is used to measure forelimb grip strength of the rats. In this test, animals will be suspended by the forelimbs on a wire (45 cm long and 0.3 cm diameter) stretched between two posts 40cm above a foam pillow. The time(s) until the animal fell will be recorded. The cut-off time will be taken as 90 s.23