6.0 / BRIEF RESUME OF THE INTENDED WORK :
6.1  NEED FOR THE STUDY:
A Gastric ulcer is an erosion of the mucosa of the stomach. Because the normal stomach lining is adapted to resist the corrosive action of gastric juice, ulcer formation is the result of over secretion of HCl or under secretion of mucus[1]. Gastric ulcer diseases is believed to be due to an imbalance between aggressive and protective factors. The gastric mucosa is continuously exposed to potentially injurious agents such as acid, pepsin, bile acids, food ingredients, bacterial products (Helicobacter pylori) and drugs.[2]
Antiulcer drug is targeted at either counteracting aggressive factors (acid, pepsin, active oxidants, platelet aggravating factor (PAF), leukotrienes, endothelins, bile or exogenous factors including NSAIDs) or stimulating the mucosal defenses (mucus, bicarbonate, normal blood flow, prostaglandins (PG), nitric oxide). The aim of treating peptic ulcer disease are to relieve pain, heal the ulcer and prevent ulcer recurrence. Currently, efforts are on research of a suitable treatment from natural product sources. A large number of species and herbs have been evaluated by various researchers for their anti-ulcer effects to achieve a favourable outcome. [3],
Caesalpinia pulcherrima, an indigenous plant, widely grown in India, is known to be effective in the treatment of gastro-intestinal disorders. Extract of Caesalpinia pulcherrima is used as anti-ulcer agent and was found to almost protection against gastric ulcers. Research has demonstrated anti-inflammatory, antioxidant, analgesic, anticonvulsant, antiviral, antibacterial and antacid properties of caesalpinia pulcherrima.[4]
Ranitidine,a nonimidazole H2 blocker,inhibit acid production by reversibly competing with histamine for binding to H2 receptors on the basolateral membrane of parietal cells. The H2-receptor antagonists predominantly inhibit basal acid secretion, which accounts for their efficacy in suppressing nocturnal acid secretion. Because the most important determinant of duodenal ulcer healing is the level of nocturnal acidity, evening dosing of H2-receptor antagonists is adequate therapy in most instances.[5]
6.2 REVIEW OF LITERATURE:
6.2.1 NAME OF THE PLANT: CAESALPINIA PULCHERRIMA
Caesalpinia pulcherrima (Linn.) Swartz belonging to Caesalpiniaceae family.
Other Name: Peacock’s pride(English), Guletura(hindi), Kenjige(kannada), Sidhakya(Sanskrit), Mayurkonrai(Tamil)
Part used : Leaves, Bark, Root
Description : An erect, smooth shrub or small tree, 1.5 to 5 meters high. The branches are armed with a few scattered spines. Leaves are bipinnate, 4-8 pairs, 6 to 12 cm long. Leaflets are stalkless, 7 to 11 pairs, elliptic, and 1 ro 2 cm long. Flowers are red and yellow, borne on terminal racemes, about 4 cm in diameter. [4].
Chemical constituents : Isolated five flavonoids - 5,7-dimethoxyflavanone, 5,7-dimethoxy-3,4'-methylenedioxyflavanone, isobonducellin, 2'-hydroxy-2,3,4'-6'-tetramethoxychalcone and bonducellin, all with anti-inflammatory activities . Isolated from the stems, a cassane-type diterpene ester, pulcherralpin.[4]
Uses: C. pulcherrima is mainly used in the treatment of gastric ulcer, antibacterial. Decoction of roots used for fevers. Decoction of leaves used as mouth wash and gargle for mouth ulcers. Decoction of flowers used for erysipelas and inflammation of the eyes. Fruit is astringent and used for diarrhea and dysentery. In Ayurvedic medicine, used for fever, jaundice, colic, flatulence, malignant tumors. In the Amazon, leaf juice used for fevers; the flower juice for sores. In Jamaica, plant is used as a purgative[4]. Caesalpinia pulcherrima has a wide spectrum of biological and pharmacological actions including analgesic and anti inflammatory[6], anticonvulsant[7].
6.3 OBJECTIVES OF THE STUDY:
1. Extraction of Caesalpinia pulcherrima stem bark.
2. To study primary phytochemical constituent of Caesalpinia pulcherrima stem bark.
3. To evaluate antiulcer activity of Caesalpinia pulcherrima using vivo and vitro model.
·  Experimental model :
(1)  Pylorus Ligation
(2)  Water Immersion Stress Induced Ulcer
(3)  Indomethacin Induced Ulcer
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 are obtained from college library and electronic media.
7.2 COLLECTION OF PLANT MATERIAL:
DRUGS AND CHEMICALS:
All other chemicals and reagents will be of pure analytical grade and obtained from local suppliers.
PLANT MATERIAL AND EXTRACTINON OF CAESALPINIA PULCHERRIMA STEM BARK :
The bark of C.pulcherrima will be collected from Mangalore or local supplier. It was authenticated by Dr. U. Srinivasa, Department of Pharmacognosy and phytochemistry, Srinivas College Of Pharmacy, Mangalore, Karnataka, India.
Shade dried, coarsely powdered stem bark of Caesalpinia pulcherrima will extracted with petroleum ether (60-80◦) , choloroform, ethanol (95%) in soxhlet apparatus. The marc will be dried and shaken with warm distilled water and boiled. Each of the extractives obtained will subject evaporate to dryness under vaccum. The extractives will stored in a refrigerator for further use.[8]
ANIMALS:
Wistar albino rats (180 to 200 g) of either sex procured from Indian Institute of Sciences will be used for this study. They will be maintained under standard conditions (temperature 22 ± 2°C, relative humidity 60±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 humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the “National Academy of Sciences” and published 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 EXPERIMETAL MODELS:
7.3.1 PYLORUS LIGATION
Experimental Design:
The rats will be divided into 5 groups of 6 each. The ulcer will be induced from group II to group V by oral administration of aspirin (200 mg/kg) for 3 days and pylorus will be ligated on the fourth day following 36 hour fasting. The group I will be served as normal control. The different groups will be assigned as described below.[9],[10]
Group I : Vehicle control (0.1% Tween 80), p.o(per os-through mouth)
Group II : Toxic control, Aspirin (200 mg/kg), p.o
Group III : Aspirin (200 mg/kg) + Ranitidine (32 mg/kg), p.o
Group IV : Aspirin (200 mg/kg) + Hydroalcohoic extract of Caesalpinia pulcherrima
stem bark (75mg/kg) p.o
Group V : Aspirin (200 mg/kg) + Ranitidine (32 mg/kg)+ Hydroalcohoic extract of
Caesalpinia pulcherrima stem bark (75mg/kg) p.o
Four hours after the pyloric ligation, the animals will be sacrificed by decapitation. The stomach will be removed, opened along with greater curvature, fixed on a cork plate and the number and severity of ulcers is registered with a stereo-microscope using the following scores and the ulcer index is determined.
• 0 = no ulcer
• 1 = superficial ulcers
• 2 = deep ulcers
• 3 = perforation.
The gastric content will be titrated against 0.1 N NaOH to find out the free acidity and total acidity.
EVALUATION:
An ulcer index UI will be calculated:
UI = UN + US+ UP × 10-1
• UN= average of number of ulcers per animal
• US = average of severity score
• UP = percentage of animals with ulcer
7.3.2 WATER IMMERSION STRESS INDUCED ULCER
Experimental Design:
Stress ulcers will be induced by forced swimming in the glass cylinder (height 45 cm, diameter 25 cm) containing water to the height of 35 cm maintained at 25°C for 3hour. Rats will be fasted for 24h prior to the experiment and divided into 5 groups of 6 each. The different groups will be assigned as described below.
Group I : Vehicle control (0.1% Tween 80), p.o
Group II : Toxic control, Swim Stress
Group III : Swim Stress + Ranitidine (32 mg/kg), p.o
Group IV : Swim Stress + Hydroalcohoic extract of Caesalpinia pulcherrima stem
bark (75 mg/kg) p.o,
Group V : Swim Stress + Ranitidine (32 mg/kg) + Hydroalcohoic extract of
Caesalpinia pulcherrima stem bark (75 mg/kg) p.o
After the drug treatment animals will be allowed to swim in water for 3 hour. The stomach of each animal will be removed, opened along with greater curveture, washed with warmed water and the extent of gastric damage assessed.[11]
EVALUATION:
Gastric Index:
The mean count of measured lesions of each animal for each group will be calculated. Inhibition of the lesion production will be expressed as percentage value.
7.3.3 INDOMETHACIN INDUCED ULCER
Experimental Design:
Wistar rats weighing between 180 to 220 gm will be randomly divided into 5 groups of 6 each. The ulcer will be induced from group II to group V by oral administration of indomethacin (20 mg/kg). The group I will be served as normal control. The different groups will be assigned as described below.
Group I : Vehicle control (0.1% Tween 80) p.o
Group II : Toxic control, Indomethacin (20 mg/kg) p.o
Group III : Indomethacin (20 mg/kg) + Ranitidine (32 mg/kg) p.o
Group IV: Indomethacin (20 mg/kg) + Hydroalcoholic extract of Caesalpinia
pulcherrima stem bark (75mg/kg) p.o,
Group V : Indomethacin (20 mg/kg) + Ranitidine (32 mg/kg) + Hydroalcohoic
extract of Caesalpinia pulcherrima stem bark (75 mg/kg) p.o,
All the drug solutions will be prepared using 0.1% Tween 80 as emulsifying agent and given 0.2 ml/200 g 10 minute prior to oral indomethacin administration. After 6 hours, rats will be sacrificed and 2% v/v formal-saline injected into totally ligated stomach for overnight storage. The next day, stomach will be opened along with greater curveture, washed with warmed water, and examined under a 3-fold magnifier. The lengths of the longest diameters of the lesions are measured and summated to give a total lesion score (in mm) for each animal, the mean count for each group will be calculated.[12],[13]
EVALUATION:
Gastric Index:
The mean count of measured lesions of each animal for each group will be calculated. Inhibition of the lesion production will be expressed as percentage value.
7.4 STATISTICAL ANALYSIS:
The mean value + SD will be calculated for each parameter. For determination significant intergroup differences, each parameter will be analyzed separately and one way analysis of variance (ANOVA) will be carried out.
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 Wistar strain albino rats and Sprague-Dawley rats.
7.6 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION?
Yes. Ethical clearance has been obtained. (Copy enclosed)
8.0 / LIST OF REFERENCES:
1.  V. C. Scanlon, T. Sanders. Essential of Anatomy and Physiology, 5th Edn., F. A. Davis company, Philadelphia, 2007, 396.
2.  Toma W, Hiruma-Lima CA, Guerrero RO, Souza Brito AR. Preliminary studies of Mammea Americana L (Guttiferae) bark/latex extract point to an effective antiulcer effect on gastric ulcer models in mice. Phytomedicine 2005;12:345-50.
3.  Al-Yahya MA, Rafatullah S, Mossa JS, Ageel AM, Al-Said MS, Tariq M. Gastric antisecretory, antiulcer and cytoprotective properties of ethanol extract of Alpinia galanga willd in rats. Phytotherapy Research 1990;4:112-4.
4.  Kirtikar, K.R. and Basu, B.D. Eds., In; Indian medicinal plants, 2nd Edn., vol-2, Periodical Experts Book agency, New Delhi, 1991, p.p. 848.
5.  Goodman and Gilman. Manual of Pharmacology and Theraputics,11th Ed. (2006) ,The McGraw Hill company’s,624.
6.  Patel SS, Verma NK, Chatterjee C, Gauthanman K., Screening of Caesalpinia pulcherrima Linn flowers of analgesic and anti inflammatory activity IJARNP vol-3(3), p.p-1-5, sep-oct-2010.
7.  Dinesh, J.singh, Anupama B., Sunil K., Anticonvulsant effect of ethanol extract of Caesalpinia pulcherrima leaves, Brazilian journal of pharmacognosy, p.p-1410 october 2009.
8.  Srinivasa U., Neelakanta, Shabraya A.R, and Rao V.J., Analgesic activity of Clerodendrum Phlomidis Stem bark. Indian drugs vol-47, no-22 ,Feb 2010,57.
9.  Shay H, Komarov SA, Fels SE, Meraze D, Gruenstein M, Siplet H. A simple method for the uniform production of gastric ulceration. Gastroenterology 1945;5:43-61.
10.  Kulkarni SK, Handbook of Experimental Pharmacology, Edn 3, Vol I. Vallabh Prakashan, New Delhi 1999;148-150.
11.  . Shenoy AM, Shastry CS, Sridevi, Gopkumar P. Anti-ulcer activity of Naravelia zeylanica leaves extract. Journal of Pharmacy Research 2009;2(7):1218-1220.
12.  Djahanguiri B. The production of acute gastric ulceration by indomethacin in the rat. Scand J Gastroenterol 1969;4:265–267.
13.  Kitajima T, Yamaguchi T, Tani K, Kubota Y, Okuhira M, Inoue K, Yamda H. Role of endothelin and platelet-activating factor in indomethacin-induced gastric mucosal injury in rats. Digestion 1993;54:156–159.