SCREENING OFTECTONA GRANDIS LINN.BARK EXTRACT FOR INFLAMMATORY BOWEL DISEASE IN RATS
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore– 560041
By
Miss.SHRUTI.B.DESAIB.Pharm
Under the Guidance of
Dr. Preeti KulkarniM.Pharm, Ph.D.
Department of Pharmacology
S.E.T’s COLLEGE OF PHARMACY
S. R. Nagar, Dharwad–580002
2013-2014
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA
ANNEXURE –II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1. / NAME OF THE CANDIDATE AND ADDRESS / SHRUTI.B.DESAIDEPT. OF PHARMACOLOGY,
SET’s COLLEGE OF PHARMACY,
S.R.NAGAR,
DHARWAD-580002
2. / NAME OF THE INSTITUTION / SET’s COLLEGE OF PHARMACY,
S.R.NAGAR,
DHARWAD-580002
3. / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOLOGY
4. / DATE OF ADMISSION TO THE COURSE / JULY- 2013
5. / TITLE OF THE TOPIC:
“SCREENING OF TECTONA GRANDIS LINN. STEM BARK EXTRACT FOR INFLAMMATORY BOWEL DISEASE IN RATS”
6.0
7.0
8.0 / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:
Inflammatory bowel disease (IBD) is a spectrum of chronic idiopathic inflammatory intestinal conditions. IBD causes significant gastrointestinal symptoms that include diarrhoea, abdominal pain, bleeding,anemia and weight loss. IBD conventionally is divided into two major subtypes: Ulcerative colitis and Crohn's disease. Ulcerative colitis is characterized by confluent mucosal inflammation of the colon starting at the anal verge and extending proximally for a variable extent (e.g.,proctitis, left-sided colitis, or pancolitis). Crohn's disease, by contrast, is characterized by transmural inflammation of any part of the gastrointestinal tract but most commonly the area adjacent to the ileocecal valve.1 Both diseases increase the risk of adenocarcinoma of the colon in the affected area.2Ulcerative colitis is most prevalent in North America, North Europe and Australia. The prevalence is roughly 10 times lower in southern and Eastern Europe, Africa, Asia and South America. Some reports have indicated that disease is now seen with increased frequency in parts of Asia such as India, Bangladesh and Japan.3
Specific goals of pharmacotherapy in IBD include controlling acute exacerbations of the disease, maintaining remission, and treating specific complications such as fistulas.1The main drugs used in the treatment of ulcerative colitis and crohn’s disease are the aminosalicylates and corticosteroids.4Although many types of treatment have been proposed and clinically proven, additional therapeutic approaches are needed because many patients do not satisfactorily respond to the currently available options or show significant side effects due to their prolonged use. Therefore there is need to develop safe and effective alternative therapeutic agents for treatment of IBD. Considering oxidative stress as one of the factor in IBD, antioxidants could be expected to provide relief.5Keeping this in view, an attempt was made to search for the plants containing antioxidants for the evaluation of effectiveness in improving IBD. In our literature search, Tectonagrandis Linn. Bark, which contains phytoconstituents such as flavanoids, alkaloids, tannins, anthraquionones and saponins6, has been already proved for its anti-oxidant activity. Therefore it is been hypothesized that the bark may be useful in treating IBD. However there are no scientific data regarding the utility of TectonagrandisLinn. Bark extract in the treatment of IBD. Thus has been selected for the present study.
6.2 Review of literature :
Inflammatory bowel disease including Ulcerative colitis and Crohn’s disease are among the most challenging human illness. Although the etiology of ulcerative colitis is largely unknown, the current literature suggests that multiple immune, genetic, and environmental factors influence both the initiation and progression of colitis.7 Subsequent activation of these cells cause a self augmenting cycle of cytokine production, cell recruitment and inflammation.8,9 This uncontrolled immune system activation results in a sustained massive production of cytokines such as tumor necrosis factor-α and interleukins (IL-1α and IL-8).10,11 In addition to cytokines, leukotrienes, thromboxane, platelet-activating factor, nitric oxide and reactive oxygen species are released from activated mucosal cells.12-14
The Plant Literature:
Tectonagrandis Linn. (TG) is commonly known as “ teak” belongs to Verbenaceae family.Its large deciduous tree 30-35 m. tall with light brown bark , leaves simple, opposite , broadly elliptical or acute or acuminate, with minute glandular dots , flowers are white in colour, small and have pleasant smell. The plant TectonagrandisLinn.is probably the most widely cultivated high value hardwood in the world and is native to India, Myanmar and south Asian countries. It is now one of the most important species of tropical plantation forestry. The whole plant is medicinally important and many reports claim to cure several diseases according to Indian traditional system of medicine. The survey reveals that the plant is used in treatment of urinary discharge, bronchitis, cold and headache, in scabies, used as laxative and sedative, as diuretic, anti- diabetic, analgesic and anti-inflammatory. The various constituents isolated are jugulone, which are been reported to anti-microbial activity, betulinaldehyde shows anti-tumor activity. Lapachol shows anti-ulcerogenic activity.
Taxonomical classification:
Kingdom : Plantae – plants
Subkingdom: Tracheobionta – Vascular plants
Divison : Mangnoliphyta – Flowering plants
Class: Magnoliopsida – Dicotyledons
Subclass: Astridae
Order: Lamiales
Family : Verbenaceae
Other names of the plant:
English: Indian Teak, Teak.
Hindi: Sagwan, Sagun.
Kannada: Sagwani
Sanskrit: Anila, Halmika.
Traditional uses of the plant:
Bark: Used as astringent, constipation, anthelmintic. It is used in bronchitis, hyperacidity, dysentry, verminosis, burning sensation, diabetes, difficult labour, and leprosy, skin diseases.
Leaves: Cooling, heamostatic, anti-inflammatory and vulnery. They are useful in treatment of piles, leucoderma and dysentry. Oil extracted from the wood is best for headache, biliousness, burning pains particularly over a region of liver.
Roots: In anuria and retention of urine.
Flowers: Oil extract from the flowers is useful in scabies, and promotes the hair growth.
Phytochemical constituents: Several classes of phytochemicals like alkaloids, glycosides, saponins, steroids, flavanoids, proteins and carbohydrates have been reported in T grandis.
Teak wood contains Napthaquinone (lapachol, deoxylapachol, 5-hydroxy lapachol), Napthaquinone derivatives : - dehydrolapachone, β- dehydrolapachone, tectol and dehydro-tectol,
Anthraquinonederivatives :Tectoquinone, 1-hydroxy-2-methylnapthaquinone and also Betulinic acid ,β-sitosterol and squalene15.
Lapachol Alphalapachone
Betullinic acid
β-sitosterol
6.3 Objective of study:
The objective of proposed study is to include the effect of TectonagrandisLinn.stem.Bark extract on experimental models of IBD by assessing the following parameters:
1To induce enterocolitis using Indomethacin.
2To induce ulcerative colitis using acetic acid.
3Dextran sodium sulfate induced colitis
4Evaluation:
- Macroscopic evaluation (Scoring of ulcers)
- Microscopic evaluation (Histopathology)
- Estimation of biochemical parameters :
- Myeloperoxidase(MPO)
- Lipid peroxidase
- Glutathione
- Measurement of physical parameters
- Change in body weight
- Change in colon weight
MATERIALS AND METHODS:
7.1 Source of Data :
The source of data are:
- Laboratory experiments on animals
- Literature survey
- Books and journals
- Internet
Materials:
- Collection, identification and authentification of the plant by a Botanist.
- Chemicals and Drugs: Indomethacin, Acetic acid and Psyllium Husk,
Drug Preparation :
The Ethanolic extract of TectonagrandisLinn. (TGEE) and aqueous extracts of dried powder of stem bark (TGAE) will be prepared by using Soxhlet and simple maceration methods, respectively. The alcoholic extract will be concentrated to dryness under reduced pressure and controlled temperature (400-500c) with a Rota vapor. The extract will be dried in order to produce a dark brown solid extract16.
Animals :
Albino wistar rats of either sex weighing 200-250g will be used. The animals will be maintained under controlled conditions of temperature (23 ± 2C) and 12-h light-dark cycles. The animals are randomized into standard and control experimental groups and housed separately in sanitized polypropylene cages containing sterile paddy husk as bedding. They will have free access to standard pellets as basal diet and water ad libitum.
Acute toxicity studies :
Acute toxicity studies will be carried out for TGEE( Ethanolic extract of TectonagrandisLinn.) and TGAE( Aqueous extract of TectonagrandisLinn.) using Acute Toxic class method as described in OECD guidelines no.423.Acute toxicity studies were carried out on wistar rats according to standard procedures.16
Methods :
1)INDOMETHACIN INDUCED ENTEROCOLITIS:
The study comprise of 5 different groups of albino wistar rats in each as follows:
Group I / Normal or untreated animals.
Group II / Control animals receive only indomethacin (7.5mg/kg) s.c.
Group III / Animals treated with indomethacin (7.5mg/kg) s.c+ lower dose. (TGEE)
Group IV / Animals treated with indomethacin (7.5mg/kg) subcutaneous + higher dose. (TGEE)
GROUP V / Animals, which will receive Prednisolone (2mg/kg p.o)andindomethacin (7.5mg/kg).
Animals pretreated with TectonagrandisLinn.bark extractfor 7 days will be administered Indomethacin (7.5 mg/kg, s.c.) on 8th and 9th day of treatment. Extract will be administered till 11th day. On the 11th day the animals will be sacrificed by cervical dislocation and dissected. Ileum and colon will be taken out to assess inflammation, based onphysical parameters, macroscopyand microscopic features. Quantification of inflammation would be done using biochemical assay (MPO, lipid peroxides,GSH).3,5
2) ACETIC ACID INDUCED ULCERATIVE COLITIS :
The study comprises five different groups of six albino wistar rats in each as follows :
Group I / Normal or untreated animals.
Group II / Control animals receive only 2ml of 4% (v/v) acetic acid.
Group III / Animals treated with 2ml of 4% (v/v) acetic acid + lower dose(TGEE)
Group IV / Animals treated with 2ml of 4% (v/v) acetic acid + higher dose(TGAE)
Group V / Animal treated group, which will receive Prednisolone
(2mg/kg p.o) and 2ml of 4% (v/v) acetic acid.
Animals will be treated withTectonagrandisLinn. Bark extract (500mg/kg) for 7days. On the 8th day, overnight fasted animals will be anaesthetized using pentobarbitone sodium and 2 ml of 4% acetic acid solution will be instilled into rectum.After 48h animals will be sacrificed by cervical dislocation and dissected to remove colon. Waste material will be removed from colon and it will be flushed with saline gently.Inflammation will be assessed based on physical parameters, macroscopyand microscopic features. Quantification of inflammation would be done using biochemical assay (MPO and lipid peroxides).3,5
3) DEXTRAN SODIUM SULFATE INDUCED COLITIS:
The study comprise of five different groups of six albino wistar rats in each as follows:
Group I / Normal or untreated animals.
Group II / Control animals receive only Dextran sodium sulfate (3%w/v in distilled water).
Group III / Animals treated with Dextran sodium sulfate (3%w/v in drinking water) + lower dose.(TGEE)
Group IV / Animals treated with Dextran sodium sulfate (3%w/v in drinking water) + higher dose.(TGEE)
GROUP V / Animal treated group, which will receive Prednisolone (2mg/kg p.o) and Dextran sodium sulfate (3%w/v in drinking water).
DSS (3%) given in distilled water will be administrated to the rats for a period of 7 days. The animals will have unlimited access to food. They will be under daily control, regarding their behavioral actions, change of body weight as well as diarrhea. The animals will be sacrificed by cervical dislocation and dissected to remove colon. Waste material will be removed from colon by flushing with saline gently. Inflammation will be assessed based on physical parameters, macroscopy and microscopic features. Quantification of inflammation would be done using biochemical assay (MPO, GSH& lipid peroxides)3,5.
1)
2)4)ANTI-OXIDANT ACTIVITY OF BARK EXTRACT OFTECTONA GRANDISLINN:
The bark extract of Tectonagrandis will be taken and subjected to anti-oxidant Screening by chemical methods at different concentration .The free radical scavenging property of extracts will be analysed by 1, 2-diphenyl 1-phenyl hydrazil.Anti-oxidants status of all scavenge those free radicals at different concentration will be analysed.17
Stastical analysis:
The data obtained will be expressed in MEAN +SEM and analysed by one way ANOVA followed by Turkey’s test.
7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS/ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.
Yes. The investigation involves adult Wistar ratsasan experimental animal
7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?
The copy of the ethical clearance certificateobtained from Institutional Animal Ethical Committee (IAEC) is attached.
REFERENCES :
- Sellin JH, Pasricha PJ. Pharmacotherapy of inflammatory bowel disease. In: Brunton LL, Lazo JS, Parker KL, editors.Goodman and Gilman’s The Pharmacological Basis of Therapeutics. 11th Ed. New York (NY): McGraw Hill; 2006. p. 1009-19.
- Dudzinski DM, Serhan. Pharmacology of eicosanoids. In: GolanDE, Tashjian AH, Armstrong EJ, Armstrong AW, editors. Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy. 2nd ed. Philadelphia (PA): Lippincott Williams and Wilkins; 2008. p. 758.
- Jagtap AG, Shirke SS, PhadkeAS. Effect of polyhedral formulation on experimental models of inflammatory bowel diseases. J Ethnopharmacol 2004; 90:195-204.
- Bennett PN, Brown MJ. Clinical Pharmacology. 9th Ed. New Delhi: Elsevier; 2006. p. 645-8.
- Hagar HH, Medany AE, Eter EE, Arafa M. Ameliorative effect of pyrrolidinedithiocarbamate on acetic acid induced colitis in rat. Eur J Pharmacol 2007; 554:69-77.
- Goswami.D.V, Patil.M.J, Modi A, Tiwari R.Pharmacognostic and Phytochemical investigation of stem bark of Tectonagrandis Linn. Ind J of Pharm and Bio sci2010:1(2):1
- Storber W, Ludviksson BR, Fuss IJ. The pathogenesis of mucosal inflammatory in murine models of inflammatory bowel disease and crohn’s disease. Ann Intern Med 1998; 128:848-56.
- Sartor RB. Pathogenesis and immune mechanism of chronic inflammatory bowel disease. Am J Gastroenterol 1997; 92:5-11
- Shanahan F. Inflammatory bowel disease: Immunodiagnostics, immunotheraputics, and ecotherapeutics. Gastroenterology 2001; 120:622-35.
- Inoue S, Masumoto T, Iida M. Characterization of cytokine expression in the rectal mucosa of ulcerative colitis: Correlation with disease activity. Am J Gastroenterology 1999; 94:2441-6.
- Ogata H, Hibi T. Cytokines and anti-cytokine therapies for inflammatory bowel disease. Curr Pharm Des 2003; 9:1107-13.
- Pololsky DK. Inflammatory bowel disease. N Engl J Med 1991; 325:1008-16.
- MacDonald TT, Monteleone G, Pender SI. Recent developments in the immunology of inflammatory bowel disease. Scand J Immunol 2000; 51:2-9.
- Woywodt , Lodt se. Scand J Immunol 2005200SI. Recent developments in the immunology of inflammatrory tivity. Am J Gasteroentero11111111 A, Ludwig D, Neustock P. Mucosal cytokine expression, cellular markers and adhesion molecules in inflammatory bowel disease. Eur J GastroenterolHepatol 1999; 11:267-76.
- Khera N, Bhargava S. Phytochemical and Pharmacological Evaluation of Tectonagrandis.Linn. Int J of Pharm and Pharm Sci 2013;5(3): 923-925
- Asif M. In vivo analgesic and anti-inflammatory effects of TectonagrandisLinn.stem bark extracts. Malaysian J of Pharm Sci 2011;9(1-11): 3
- Krishna MS, Nair JA, Anti-bacterial,Cytotoxic and anti-oxidant potentials from different extracts of leaves, barks and wood of Tectonagrandis . Int j Pharma Sci Drug Res.2010; 2 Suppl 2: 155-158.
9 / SIGNATURE OF THE CANDIDATE
10 /
REMARK OF THE GUIDE:
The above information and literature has been extensively investigated, verified and is found to be correct. The present study will be carried out under my supervision and guidance.11 / 11.1 NAME AND DESIGNATION
OF GUIDE
11.2 SIGNATURE / Dr. PREETI KULKARNI.M.Pharm., Ph.D.
PROFESSOR AND PRINCIPAL,
DEPT.OF PHARMACOLOGY,
S E T’sCOLLEGE OF PHARMACY,
S. R. NAGAR,
DHARWAD-580002
11.3 HEAD OF THE DEPARTMENT
11.4 SIGNATURE / Dr. A R. KULKARNI.M.Pharm., Ph.D.
PROFESSOR AND HEAD,
DEPT.OF PHARMACOLOGY,
S E T’sCOLLEGE OF PHARMACY,
S. R. NAGAR,
DHARWAD-580002
12 / 12.1 REMARKS OF THE PRINCIPAL / The above mentioned information is correct and I recommend the same for approval.
12.2 SIGNATURE / Dr. V H. KULKARNI.M.Pharm., Ph.D.
PRINCIPAL,
S E T’sCOLLEGE OF PHARMACY,
S. R. NAGAR,
DHARWAD-580002
1