Evaluation of Gastroprotective and Anti-Diarrhoeal Properties of Methanolic Extract of Plumeria alba Leaves
M.PHARM DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA
BY
ANSARI MEHBOOB ALI
B.Pharm.
UNDER THE GUIDANCE OF
MOHAMMED SAIFUDDIN KHALID
M.Pharm.
ASST. PROFESSOR
DEPARTMENT OF PHARMACOLOGY
LUQMAN COLLEGE OF PHARMACY
GULBARGA-585102
2013-2014
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the Candidate and Address (In block letters) / ANSARI MEHBOOB ALIAZAD NAGAR ,AKBAR LALA COMPOUND,NEAR GLASS FACTORY,SUBEDAR ENGLISH HIGH SCHOOL,GHATKOPER ,MUMBAI (WEST),40008
2. / Name of the Institution / Luqman College of Pharmacy, gULBARGA – 585 102.
3. / Course of Study and Subject / M.Pharm (PharmacOLOGY)
4. / Date of Admission to Course / 05/07/2013
5. / Title of the Topic / Evaluation of Gastroprotective and Anti-Diarrhoeal Properties of Methanolic Extract of Plumeria alba Leaves.
6. / BRIEF RESUME OF THE INTENDED WORK
6.1 Need for the study:
Gastric ulcer, the most common disorder of GIT has multifunctional causes in its pathophysiology1. Ulcer is a local defect or excavation of the surface of an organ or tissue that is produced by the sloughing (shedding) of inflammatory necrotic tissue2. It occurs only when an inflammatory necrosis exists on or near the surface3. The sites for ulcerations are the stomach, duodenum and intestinal ulcers4.
In the acute stage there is infiltration by polymorphs with vasodilatation while long- standing ulcers develop infiltration by lymphocytes plasma cells and macrophages with associated fibroblastic proliferation and scarring5.The pathogenesis and in the treatment of acid- peptic conditions have occurred culminating in the discovery of helicobacter pylori and proton pump inhibitors. Drug treatment of peptic ulcer is targeted at either counteracting these aggressive factors or stimulating the mucosal defense6. Proton pump inhibitors have become the drugs of choice to promote healing from erosive esophagitis to peptic ulcer disease because of acid production7.
Diarrhoea is a major public health problem in developing countries and is said to be endemic in many regions of Asia and is the leading cause of high degree of morbidity and mortality which contributes to the death of 3.3 to 6 million children annually8. Multiple drug resistance among Enteropathogens in various geographic regions presents a major threat in the control of diarrhoea8. Therefore indigenous medicinal plant as an alternative to antibiotics are said to play a significant role here. This particular aspect of using medicinal plants as a remedy or home cure for diarrhoea is applied in our study8.
Diarrhoea is associated with an increased frequency of bowel movements with the production of soft or watery stools. It may be defined as the passage of more than 300ml of liquid faeces in 24 hours. This results in fluid and electrolyte loss that may lead ultimately to death, particularly in young children. Pain, urgency, perianal discomfort and incontinence often accompany it. Low-volume, painful, bloody diarrhoea is known as dysentery. Diarrhoea may be due to a specific disease of the intestines or secondary to a disease outside the intestines. For instance, bacillary dysentery direct affects the gut, while diabetes mellitus causes neuropathic diarrhoeal episodes. Diarrhoea can be divided into acute or chronic forms. Infectious diarrhoea is often acute; diabetic diarrhoea is chronic. Whether acute or chronic, diarrhoea has the same pathophysiologic cause that helps in the identification of specific treatments.
Several herbs and shrubs are useful as medicines as reported by many scientists9. Many such herbs, shrubs and plants are known to protect the organs from the environmental, chemical and occupational challenges. Mimosa pudia is one such green leaf shrubs10. This is abundantly grown and used as medicinal plant11.The plant is said to possess principle constituents like alkaloid12, Mimosine12, tannin12, adrenaline12, ascorbic acid12, fiber12, β-carotene12 etc. The present constituents like tannin13, ascorbic acid13, fiber13 and β-carotene13 have said to posses Anti-ulcer14,15,16 property.
It has been reported that the pharmacological significance was noted due to the presence of various bioactive compounds in the Plumeria alba such as sterols, carbohydrates, tannins, triterpenoids and iridoid glycosides17. The flowers of the plant Plumeria alba are reported to contain steroid, flavonoids and alkaloids18. Genus of Plumeria alba showed the presence of tannin, carbohydrates, glycoside, steroid and flavonoid18. Flavonoids are said to possess anti-diarrhoeal property19.The root bark of Plumeria alba had shown the presence of iridiods, tannins and alkaloids20. The constituents like tannin13, have said to posses anti-ulcer14,15,16 ,property. Plumeria alba has shown a better antioxidant property in comparative bioactivity studies21. Therefore, there is a possibility that the antioxidants may have a protective role in ulcer healing. However, the literature reveals no scientific data on antiulcer and antidiarrhoeal effect of Plumeria alba leaves.
Keeping this in view, the present study is taken up to evaluate the possible antiulcer & antidiarrhoeal properties of Plumeria alba leaves in experimental animals. Hence, it is thought that the present study is highly justifiable and more needful.
6.2 Review of the literature of Plumeria alba Linn:
Botanical Classification22:-
Kingdom: Plantae
Order : Gentianales
Family : Apocynaceae
Subfamily : Rauvolfioideae
Tribe : Plumeriae
Genus : Plumeria
Species : P.alba
Vernacular Name23:
English : White frangipani,white frangipani
Kannada : Deva Champaka
Hindi : Golainchi
Madrasi: Simaiyarali
Sanskrit : Kananakaravira
Telugu : Veyyivarahalu
Tamil : Peru, Perumallari, Perugalli
Plumeria alba (Apocynaceae) is native to Maxico, Central America and Caribbean, Brazil, Southern and southeastern Asia22.
Plumeria alba is a small lacticiferous tree or shrub grows 4.5m high, occasionally grown in the gardens. The plant is mainly grown for its ornamental and fragrant flowers. Leaves lanceolate to oblanceolate, flowers white, fragrant, in corymbose fascicles24. Each of the separate species of Plumeria bears differently shaped alternate leaves with distinct form and growth habits. The leaves of P. alba are quite narrow and corrugated, whereas leaves of P. pudica have an elongated shape and glossy, dark-green color. P.pudica is one of the everblooming types with non-deciduous evergreen leaves. Another species that retains leaves and flowers in winter is P.obtusa though its common name is "Singapore," it is originally from Colombia22.
The literature survey reveals that the Plumeria alba possesses various bioactive compounds such as sterols, carbohydrates, tannins, triterpenoids and iridoid glycosides17. The aerial part of the plant Plumeria alba are reported to contain steroid, flavonoid and alkaloids18. Genus of Plumeria alba showed the presence of tannin, carbohydrates, glycoside, steroid and flavonoid18. The root bark of Plumeria alba had shown the presence of iridiods, tannins and alkaloids20. The plant is reported to contain amyrinacetate, mixture of amyrins, ß-sitosterolscopotein, iriddoids isoplumericin, plumeride, plumeride coumerate and plumeride coumerate glucoside25,26. The fresh leaves and bark contain pluieride, resinic acid, fulvoplumierin, a mixture of terpenoids, sterols and plumieride18. The bark of plumeria alba contains cytotoxic iridoids, fulvoplumierin, allamcin, allamandin, 2,5-dimethoxy-p-benzoquinone, plumericin and lignan liriodinndrin18.
Medicinal uses 27:
1. Used as plaster over hard tumours.
2. Used as a cure for gonorrhoea.
3. Used to dispel indolent swelling.
4. Used as rubefacient in rheumatism.
5. Used as strong purgative.
6. Used in treatment of diarrhoea.
7. Used as ague.
8. Used as purgative.
9. Used as a cure for itch.
10. Used as violent cathartic.
Reports from modern literature of the plant Plumeria alba L.:
1. The methanolic extract of Plumeria alba and aqueous extract of Alove vera has been reported for hepatoprotective activity against CCL4 induced hepatotoxicity on male wister rats28.
2. The methanolic extract of Plumeria alba has shown to possess antitumour activity against dalton lymphoma ascites in mice29.
3. Essential oil of Plumeria alba flower has reported antimicrobial activity against gram positive and gram negative bacteria30.
Review of literature, till date, regarding Plumeria alba was carried out by chemical abstract, biological abstract, medicinal and aromatic plant abstract, and google, Wikipedia, Materia medica, Indian medicinal plant, Indian herbal drugs and other national and international scientific journals. The aerial part of the plant Plumeria alba are reported to contain steroid, flavonoid and alkaloids18. The plant is reported to contain amyrinacetate, mixture of amyrins, ß-sitosterolscopotein, iriddoids isoplumericin, plumeride, plumeride coumerate and plumeride coumerate glucoside26,27. The fresh leaves and bark contain pluieride, resinic acid, fulvoplumierin, mixture of terpenoids, sterols and plumieride18. The bark of Plumeria alba contains cytotoxic iridoids, fulvoplumierin, allamcin, allamandin, 2,5-dimethoxy-p-benzoquinone, plumericin and lignan liriodinndrin18. The flavonoids are polyphenolic compounds and reported to exhibit various pharmacological activities such as CNS activity, cardiotonic activity, lipid lowering activity, antioxidant activity, hepatoprotective activity, hypoglycemic activity31etc. These active constituents and the above mention activities in turn appear to correlate with some other biological activities32. Our literature survey revealed that the different parts of Plumeria alba have been screened for various pharmacological activities but antiulcer and antidiarrhoeal activities were not investigated in Plumeria alba leaves so far. Therefore, the present study is planned to evaluate the potential Gastroprotective and Anti-diarrhoeal property of methanolic extract of Plumeria alba leaves in different experimental models of ulcer and diarrhoea. Hence this study is essential and justifiable.
6.3 OBJECTIVES OF THE STUDY:
The objective of the proposed study is to evaluate the Gastroprotective and Anti-diarrhoeal properties at different doses of methanolic extract of Plumeria alba var.alba leaves in rats.
Specific Objectives:
1. Collection and extraction of Plumeria alba (Apocynaceae) leaves.
2. Authentication and characterization of the plant material.
3. To prepare leaves extract of Plumeria alba with methanol.
4. To carryout preliminary phytochemical analysis for the detection of the type of phytoconstituents present in methanolic extract.
5. To determine the dose range of methanolic extract of Plumeria alba leaves by conducting acute toxicity studies as per OECD guidelines.
6. To evaluate the anti-ulcer effect of methanolic extract of Plumeria alba leaves in albino rats by following models:
i. Pylorus ligation induced ulcer model.
ii. Aspirin induced ulcer model.
7. The evaluate the anti-diarrheal effect of methanolic extract of Plumeria alba leaves in albino rats by following models:
i. Castor-oil induced diarrhoea.
ii. Prostaglandin-E2 induced enteropooling model.
7. / Materials and methods:
7.1 Source of data:
Data will be obtained from CD-Rom, Internet facilities, Literatures, related articles, books from libraries of Luqman College of Pharmacy, Gulbarga, Gulbarga University, Gulbarga etc., and other Research Publications and Journals.
Web sites: www. sciencedirect.com
www. pubmed.com
www. google.com
www. ijp-online.com
www. freemedicaljournals.com
www.elsevier.com
7.2 Methods of collection of Data:
The data collected will be based on animal experimentation as per the parameters studied under each animal model, which are mentioned under the objectives of the study.
The Plumeria alba leaves are found throughout India. The experiment will be conducted using different animal models and data will be generated from such experimental studies as mentioned under the objective of the study. Chemicals and reagents will be procured from standard companies. The data collected will be based on animals experimentation as per the parameter studied under each animal model. The doses of extract will be selected on the basis of our preliminary toxicity studies as per OECD guidelines. The control animal receives only the vehicle (2% w/v gum acacia) in the same volume and through same route of administration.
Methodology:
1.Preparation of various solvent extracts 33,34,35:
It is planned to dry the leaves under shade at room temperature and pulverized. Then the powder obtained is subject to successive Soxhlet extraction with the solvents with increasing order of polarity i.e. petroleum ether, chloroform, methanol and water. The extract is allowed to concentrate under reduced pressure (bath temperature 5oC) and store in air tight container in refrigerator below 10oC. All these extracts are used for biological investigations and in vivo studies, after subjecting it to preliminary qualitative phytochemical analysis.
2.Preliminary phytochemical screening 35,36,37:
It is planned to carry out the preliminary phytochemical investigation of different extracts of Plumeria alba leaves for detection of various phytochemical by standard method described in standard books.
Experimental animals:
In-bred healthy Wister rats weighing 150-250g and Swiss albino mice of either sex weighing 20 to 30g will be included for the study. Rats and mice will be housed in polypropylene cages (six per cage) with stainless steel grill top, bedded with paddy husk. Rats and mice will be maintained under controlled temperature at 25oC ± 2oC with 12 hr light/ dark cycle in a well-ventilated animal house. All Rats and Mice will have a free access to food (pellet chow) and water ad libitum. Institutional Animal Ethics Committee approval for the experimental protocol has been obtained (copy enclosed); animals will be maintained under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).
Determination of Acute toxicity studies (LD50)38.
It is further planned to study the acute toxicity of methanolic extract of Plumeria alba leaves in albino mice of either sex (20 to 30g). Fixed dose method (OECD guideline number 420) of CPCSEA will be adopted for toxicity studies to obtain dose range of methanolic extract of Plumeria alba leaves.
Work Protocol:
Evaluation of Anti-Ulcer activity by following methods:
Method 1: Pylorus ligation induced ulcer model 39,40,41.
Grouping:
Albino Wistar rats of either sex weighing between 150-250g are selected and shall be divided into 4 groups containing 6 rats each.
Group I - Control (2% w/v gum acacia) 1x 6 = 6 Rats.
Group II - Lansoprazole 8 mg/kg, in 2% w/v gum acacia, p.o) 1x 6 = 6 Rats.
Group III - Methanolic extract of Plumeria alba leaves (p.o.) dose 1 1x 6 = 6 Rats