Rajiv Gandhi University of Health Sciences s169
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE.
M. PHARM SYNOPSIS
YEAR OF ADMISSION- January 15, 2013
“EFFECT OF BLUMEA LACERA EXTRACT ON EXPERIMENTALLY INDUCED INFLAMMATORY BOWEL DISEASE IN RATS”
BY
Mr. SANTOSH BASNET
M. PHARM, PART-I
UNDER THE GUIDANCE OF
Mr. VIJAYA KUMAR S M. Pharm.,
LECTURER
DEPARTMENT OF PHARMACOLOGY
SREE SIDDAGANGA COLLEGE OF PHARMACY, B. H. ROAD, TUMKUR,
KARNATAKA - 572 102.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE.
PROFORMA FOR THE REGISTRATION OF SUBJECT FOR
DISSERTATION
1. NAME OF THE CANDIDATE : Mr. SANTOSH BASNET.
& ADDRESS I M. Pharm.
Department of Pharmacology
Sree Siddaganga College of Pharmacy
B. H. Road, Tumkur - 572 102.
2. NAME OF THE GUIDE : Mr. VIJAYA KUMAR S M. Pharm.,
Lecturer
Department of Pharmacology
Sree Siddaganga College of Pharmacy,
B. H. Road, Tumkur - 572 102.
3. NAME OF THE INSTITUTE : Sree Siddaganga College of Pharmacy
B. H. Road,
Tumkur,
Karnataka-572 102.
4. COURSE OF STUDY : Master of Pharmacy (Pharmacology)
5. DATE OF ADMISSION : January 15, 2013
6. TITLE OF THE SYNOPSIS :
“EFFECT OF BLUMEA LACERA EXTRACT ON EXPERIMENTALLY INDUCED INFLAMMATORY
BOWEL DISEASE IN RATS”
7.GENERAL DISCUSSION :
7.1 NEED FOR THE STUDY:
Inflammatory bowel disease (IBD) is a term mainly used to describe two diseases, Ulcerative colitis and Crohn’s disease. Both Ulcerative colitis and Crohn’s disease are chronic diseases that involve inflammation of the gastrointestinal tract. Ulcerative colitis only affects the colon (large intestine) and rectum, while Crohn’s disease can affect the entire digestive system; from the mouth to the anus.1 Inflammatory bowel disease are considered autoimmune diseases, in which the body’s own immune system attacks elements of the digestive system.2
The peak age of onset for IBD is 15 to 30 years old, although it may occur at any age. About 10% of cases occur in individuals younger than 18 years. Ulcerative colitis is slightly more common in males, whereas Crohn’s disease is marginally more frequent in women.3 The data that does exist suggest that the worldwide incidence rate of ulcerative colitis varies greatly between 0.5–24.5/100,000 persons, while that of Crohn’s disease varies between 0.1–16/100,000 persons worldwide, with the prevalence rate of IBD reaching up to 396/100,000 persons.4
The intestinal inflammation is histologically characterized by infiltration of polymorphonuclear leukocytes, monocytes and macrophages. They are activated by various inflammatory mediators including prostaglandins, leukotriene’s, platelet-activating factor and cytokines to synthesize and liberate reactive oxygen metabolites.5,6 The balance between the production of free radical and antioxidant defence in the body has important health implications: if there are too many free radicals or too few antioxidant for protection, a condition of oxidative stress develops, which may cause chronic and pre-treatment damage.7 A favourable correlation between the activity of free radicals in the intestine and the clinical disease activity has been exhibited indicating the significance of these substances in the inflammatory process.8
Most of the current therapies of IBD involves treatment with glucocorticoids, 5-aminosalicyclicacid (5-ASA) and immunosuppressive drugs, but these drugs are not safe as they may produces serious complications and toxic effects such as nephrotoxicity, pulmonary toxicity and male infertility.9,10 Therefore there is need to develop safe and effective alternative therapeutic agents for treatment of IBD. Considering oxidative stress and inflammatory mediators are the factors in IBD, hence, antioxidants11 and anti-inflammatory12 agents could be expected to provide relief from IBD.
Blumea lacera (Asteraceae) is a genus of flowering plants and has been reported to possess antioxidant property13 and used traditionally as anti inflammatory agent14.
No scientific data regarding the activity of Blumea lacera plant extract on IBD is available. Hence, the present study is designed to evaluate the effect of Blumea lacera plant extract on experimental models of IBD in rats.
7.2 REVIEW OF LIERATURE:
Blumea lacera (Family: Asteraceae) is an annual or biennial herb, erect 20-100 cm tall with a strong odour of turpentine. Stems are simple or more often branched, terete, ash colored, densely with silky hairs or glandular trichomes, pubscent; branches sometime ascending. Leaves sessile or petiolate, elliptic to oblong, lanate abaxially, tomentose adaxially, base attenuate, margin doubly serrate or sometimes slightly variously lyrately lobed, apex obtuse; veins 5-8 pairs. Many flowers arranged in axillary cymes or terminal panicle. Pappus is white. Fruit is an achene, oblong and not ribbed.14,15
Plant profile16
Kingdom : Plantae
Sub kingdom : Viridaeplantae
Infrakingdom : Streptophyta
Division : Tracheophyta.
Subdivision : Spermatophytina
Infradivision : Angeiospermae
Class : Magnoliopsida.
Superorder : Asternae
Order : Asterales.
Family : Asteraceae.
Genus : Blumea
Species : Lacera.
Vernacular names:
Kukundara, Tamrachuda, Sukshmapatra, Gaandhaari gida, Janglimuli, Kakronda.17
Distribution:
The plant occurs throughout the plains of India from the north-west ascending to 2,000 ft. in the Himalayas. It is a common roadside weed in Ceylon and Malaya. It is distributed to the Malay Islands, Australia, China, Nepal and Tropical Africa.18
Chemical Constituents:
It shows the presence of Camphor, cineol 66, d-fenchone 10 and citral; Diester of coniferyl alcohol, acetylenic compounds, a thiophene derivative; campesterol, hentriacontane, hentriacontanol, alpha-amyrin and its acetate, lupeol and its acetate and β-sitosterol.14
Claimed Therapeutic Uses of Blumea lacera:
· Sores and Wound healing19
· Treating Bronchitis20
· Anthelmintic21,22
· Astringent21,22
· Diuretics21,22
· Healing cuts23
· Anti inflammatory24
· Anti pyretic25
· Anti fungal26
· Anti microbial27
· Anti dysentric28
· Anti diarrhoea29
7.3 Objective of the study
The objective of the study is to investigate the effect of ethanolic extract of Blumea lacera in following experimentally induced IBD in rats.
A) Indomethacin induced Entero colitis (Crohn’s disease) in rats by assessing the following parameters;
1. Scoring of ulcers (macroscopic)
2. Estimating enzyme markers such as
· Myeloperoxidase
3. Estimation of antioxidant parameters
· Lipid peroxidase (LPO)
· Glutathione (GSH)
4. Estimation of serum parameters
· Lactate dehydrogenase (LDH)
5. Histopathology (microscopic)
B) Acetic acid induced Entero colitis (Ulcerative colitis) in rats by assessing the following parameters;
1. Clinical scoring
2. Scoring of ulcers (macroscopic)
3. Colon weight/length index
4. Colonic contractility studies
5. Estimating enzyme markers such as
· Myeloperoxidase (MPO)
6. Estimation of antioxidant parameters
· Lipid peroxidase (LPO)
· Glutathione (GSH)
7. Estimation of serum parameters
· Lactate dehydrogenase (LDH)
8. Histopathology (microscopic)
8.0 MATERIALS AND METHODS:
Chemicals:
Sulphasalazine, Indomethacin, Acetic acid, EDTA, Thiobarbituric acid, Lactate dehydrogenase Kit.
Instruments:
Hot air oven, homogenizer, UV spectrophotometer, Auto analyzer, Cooling centrifuge.
Experimental animals:
Albino Wistar male rats weighing 200 - 250 g will be used. The animals will be maintained under controlled conditions of temperature (23 ± 2° C) and 12 h light-dark cycles. The animals are randomized into experimental and control groups and housed each
in to sanitized polypropylene cages containing sterile paddy husk as bedding. They will have free access to standard pellets as basal diet and water ad libitum.
Selection of the dose:
Based on previous published article, 100 mg/kg and 200 mg/kg dose of Ethanolic extract of Blumea lacera (EEBL) will be selected and administered via. Oral route.25
8.1 METHODS:
Collection of plant material:
Plant will be collected from Gujarat state of India and authenticated by Botanist.
Preparation of plant extract:
The collected plant will be washed and will be air dried under shade. After drying, the plant material will be coarse powdered and subjected to hot extraction in soxhlet apparatus using 70% ethanol and residues of the solvent will be removed under reduced pressure. The yield will be dried in vacuum desiccators and stored for further use.
8.1.1 Indomethacin Induced Entero colitis: 30
The male Wistar albino rats (200 - 250 g) will be selected and randomized into following groups. Each group consists of six animals.
Indomethacin Induced Entero colitisGroups / Treatment
I / Normal untreated
II / EEBL (200 mg/kg, p.o)
III / Indomethacin (7.5 mg/kg, s.c)
IV / Indomethacin + EEBL (100 mg/kg, p.o)
V / Indomethacin + EEBL (200 mg/kg, p.o)
VI / Indomethacin + Sulphasalazine (100 mg/kg/day, p.o)
Animals will be pre-treated with EEBL for 7 days. The Indomethacin (7.5 mg/kg) will be given s.c on 8th and 9th day of treatment. Drug treatment will be continued till 11th day. On the 12th day after treatment, the animals will be sacrificed by cervical dislocation and dissected. Ileum and colon will be taken out to assess inflammation, based on macroscopic and microscopic features. Quantification of inflammation will be done by assessing the parameters like Myeloperoxidase (MPO), Lipid peroxidase (LPO) and Glutathione (GSH).
8.1.2 Acetic Acid Induced Ulcerative Colitis: 31
The male Wistar albino rats (200 - 250 g) will be selected and randomized into following group. Each group consists of six animals.
Acetic Acid Induced Ulcerative ColitisGroups / Treatment
I / Normal untreated.
II / Acetic acid (2 ml of 3%, intra rectally)
III / Acetic acid + EEBL (100 mg/kg, p.o)
IV / Acetic acid + EEBL (200 mg/kg, p.o)
V / Acetic acid + Sulphasalazine (100 mg/kg/day, p.o)
Animals will be pre-treated with EEBL for 7 days and fasted overnight. On the 8th day animal will be anaesthetized using ether and 2 ml of 3% acetic acid in 0.9 % saline was instilled into rectum. After 48 h 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 the macroscopic and microscopic features. Quantification of inflammation will be done by assessing the parameters like Myeloperoxidase (MPO), Lipid peroxidase (LPO) and Glutathione (GSH).
Macroscopic scoring for rat ileum: 32
Gross macroscopic findings will be ranked using the following criteria:
Score / Points / Particulars0 / No change in both serosa and mucosa
1 / Hyperemia and/or petechial bleeding
2 / Single mucosal erosion or ulceration
3 / Single mucosal erosion or ulcer with hyperemic, adhesive or hemorrhagic lesions in the serosa and mesentery’s
4 / Multiple erosions or ulcerations (less than 10 cm)
5 / Multiple erosions or ulcerations (more than 10 cm).
Clinical Activity Score:
Colitis will be quantified with a clinical score accessing weight loss, stool consistency and bleeding.
· Weight loss
Score/Points / Weight Loss0 / No Weight Loss
1 / 1 to 5%
2 / 5 to 10%
3 / 10 to 20%
4 / > 20%
· Stool consistency
Score/Points / Stool Particulars0 / Well-formed pellets
2 / Pasty and semi-formed stools that did not stick to the anus
4 / Liquid stools that did not stick to the anus
· Bleeding
Score/Points / Particulars0 / No gross bleeding
4 / Gross bleeding
Statistical analysis:
The analysis of data will be done by using one way ANOVA, followed by Tukey’s multiple comparison test by using Graphpad prism 5.0 software (Graphpad, San Diego, CA)
8.2. DOES the study require any investigation or interventions to be conducted on patients or other humans / animals? If so please described briefly.
Yes, the above study requires investigation to be done on the male Wistar rats, for evaluation of inflammatory bowel disease.
8.3. HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 8.2
The copy of the ethical clearance certificate obtained from Institutional Animal Ethical Committee (IAEC) is attached.
9.0 REFERENCES:
1. Daniel K. Podolsky, M.D. Inflammatory bowel disease. N Engl J med 1991; 325, 928-937.
2. Fiocchi C. Inflammatory bowel disease: Etiology and Pathogenesis. Gastroenterol 1998; 115, 182-205.
3. Loftus EV. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence and environmental influences. Gastroenterol 2004; 126, 1504-1508.
4. Lakatos PL. Recent trends in the epidemiology of inflammatory bowel diseass: up or down? World J Gastroentrol 2006; 12, 6102-6108.
5. Simmonds NJ, Rampton DS. Inflammatory bowel disease—a radical view.Gut 1993; 34, 865–868.
6. Lih-Brody L, Powell SR, Collier KP, Reddy GM, Cerchia R, Kahn E, et al. Increased oxidative stress and decreased antioxidant defenses in mucosa of inflammatory bowel disease.Dig Dis Sci 1996; 41, 2078–2086.
7. Abdollahi M, Ranjbar A, Shadnia S, Nikfar S, Rezaiee A. Pesticides and oxidative stress: a review.Med Sci Monit2004; 10, 144–147.
8. Nielsen OH, Ahnfelt-Ronne I. Involvement of oxygen-derived free radicals in the pathogenesis of chronic inflammatory bowel disease.Klin Wochenschr 1991; 69, 995–1000.
9. Di Paolo MC, Paoluzi OA, Pica R, Iacopini F, Crispino P, Rivera M, Spera G, Paoluzi P. Sulphasalazine and 5-aminosalicylic acid in long-term treatment of ulcerative colitis: report on tolerance and side-effects. Dig Liver Dis 2001; 33, 563-569.
10. Jankauskiene A, Druskis V, Laurinavicius A. Cyclosporine nephro toxicity: associated allograft dysfunction at low trough concentration. Clin Nephrol 2001; 56, 27-29.
11. Hanan HH, Azza EIM. Ameliorative effect of pyrrolidine dithiocarbamate on acetic acid induced colitis in rat. Europ J Pharmacol 2007; 554, 69-77.
12. Sushil KJ, Justin LR, and Jennifer LC. High glucose and ketosis (acetoacetate) increases, and Chromium niacinate decreases, il-6, il-8, and mcp-1 Secretion and oxidative stress in u937 monocytes .Anti-oxidants and Redox signalling: 2007; 9, 10.
13. Halder, Luke D. Evaluation of antibacterial, antioxidant and toxicological activity of crude extracts of Adiantum capillus-veneris, Blumea lacera, Cassia alata, and Cissus quadrangularis from Faridpur, Bangladesh. Available from:
URL:http://hdl.handle.net/10361/1497[ cited: 2013 Aug 25]
14. Database of Medicinal and Aromatic plants in Rajasthan. Available from: URL :http://bioinfo.bisr.res.in/project/domap/plant_details.php?plantid=0010&bname=Blumea%20lacera [cited: 2013 Aug 26]
15. Chinese plant names. Available from:
URL:http://www.efloras.org/florataxon.aspx?flora_id=3&taxon_id=200023555 [cited: 2013 Aug 26]
16. ITIS report: Taxonomy and Nomenclature. Available from:
URL:http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=505924 [ cited: 2013 Aug 27]
17. Medicinal plant with antimicrobial activity. Available from:
URL: http://parisaramahiti.kar.nic.in/Medicinal_plants_new/med%20plants/p20.html