antidiabetic and antioxidant potential of plumeria alba linn FLOWERS extract on streptozotocin induced diabetic rats
M.PHARM DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA.
BY
HAJI KHAJA MOINUDDIN
B.Pharm.
UNDER THE GUIDANCE OF
Dr.VEERENDRA SHASTRI
M.Pharm.Ph.D
PROFESSOR
DEPARTMENT OF PHARMACOLOGY
KARNATAKA COLLEGE OF PHARMACY
BIDAR-585403
2011-2012
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the Candidate and Address (In block letters) / HAJI KHAJA MOINUDDINS/O SYED AHMED ,
NEAR K.M.P SCHOOL JANWADA ROAD NAWADGERI BIDAR-585401
KARNATAKA.
2. / Name of the Institution / KARNATAKA College of Pharmacy, BIDAR – 585 403.
3. / Course of Study and Subject / M.PharmA. (PharmacOLOGY)
4. / Date of Admission to Course / 09-09-2011
5. / Title of the Topic / Antidiabetic and antioxidant pOTENTIAL of plumeria alba linn FLOWERS extract on streptozotocin induced diabetic rats
6. / BRIEF RESUME OF THE INTENDED WORK
6.1 Need for the study:
Since ancient times, plants have been an exemplary source of medicine. Research conducted in last few decades on the plant mentioned in ancient literature or used traditionally1. Ornamental plants are grown for decorative purposes in gardens and landscape design projects, as house plants, for cut flowers and specimen display. Ornamentals and flowers crops are not only grown for the display of aesthetic features , but also have some nutritive and medicinal properties. There has been renewed interest in utilising garden environments as therapeutic entities to enhance the process of healing that occurs in healthcare environments. By minimizing the stress response, therapeutic gardens can promote recovery from illness or preserve health. From centuries roses have been valued for their culinary, medicinal, cosmetic and aromatic properties2. The use of plant compounds for pharmaceutical purpose has gradually increased in India, about 80% of individuals from developed countries use traditional medicine, which involves compounds derived from medicinal plant. Therefore, such plants should be investigated to better understand their properties, safety and efficiency3. Herbs are widely exploited in the traditional medicine and their curative potentials are well documented4.
Diabetes is a chronic disorder in metabolism of carbohydrates, proteins, and fat due to absolute or relative deficiency of insulin secretion with/without varying degree of insulin resistance5,6. Also, it may be defined as a disease where the body either produces little
Insulin/ceases to produce insulin, or becomes progressively resistant to its action7. It has now become an epidemic with a worldwide incidence of 5% in the general population. The number of adults with diabetes in the world will rise from 135 million in 1995 to 300 million in the year 20258. The countries with the largest number of diabetic people in the year 2025 will be India, China and United States9. There are more than 30 million people with diabetes mellitus in India and the incidence is increasing10. Also, there are many patients in the community with undiagnosed diabetes. Decreased physical activity, increasing obesity, stress and changes in food consumption have been implicated in this increasing prevalence in the past two decades. Diabetes is being projected as the World’s main disabler and killer in the next 25 years11.
Patients with diabetes experience significant morbidity and mortality from microvascular (Retinopathy, neuropathy, nephropathy) and macrovascular complications
(Heart attack, stroke and peripheral vascular disease). The cost of treating diabetes and associated complications exceeds $ 100 billion per year. The complications are far less common and less severe in people who have well-controlled blood sugar levels. Acute complications include diabetic ketoacidosis, nonketotic hyperosmolar coma, and diabetic coma. In case of chronic complication, chronic elevation of blood glucose level leads to damage to blood vessels. In diabetes, the resultant problems are grouped under "microvascular disease” (due to damage to small blood vessels) and "macrovascular disease" (due to damage to the arteries)12.
Microvascular disease leads to retinopathy, neuropathy and nephropathy (nephropathy leads to anaemia)13,14. Macrovascular disease leads to cardiovascular disease, mainly by accelerating atherosclerosis. These disorders include: (1) Coronary artery disease, leading to- myocardial infarction (heart attack) or angina, (2) Stroke (mainly ischemic type), (3)
Peripheral vascular disease, which contributes to intermittent claudication (exertion-related foot pain) as well as diabetic foot12.
Reactive oxygen species (ROS), which include free radicals such as super oxide anion radicals (O2), hydroxyl radicals (OH) and non-free-radical species such as H2O2 and singlet oxygen (1O2) are various forms of activated oxygen15,16,17. The importance of free radicals and ROS has attracted increasing attention over the past decade. These molecules are exacerbating factors in cellular injury and in the aging process18. In living organism various reactive ROS can form in different ways. Normal aerobic respiration stimulates polymorphoneuclear leukocytes and macrophages, and peroxisomes appear to be the main endogenous sources of most the oxidants produced by cells. Exogenous sources of ROS include tobacco smoke, certain pollutants, organic solvents, and pesticides19, 20, 21. The free radicals are generated endogenously due to the metabolism of various xenobiotics, environment pollutants, etc. or oxidative stress. These free radicals interact covalently with membrane macro molecules and denature them. In addition, such covalent interaction may oxidize the lipids of the membrane (lipid peroxidation) 15, 16. This lipid peroxidation initiates cascade of biochemical reactions leading to cellular necrosis18. However, there are certain inbuilt antioxidant systems like tissue GSH, superoxide dismutase, catalase etc. to scavenge the free radicals and protect the organs. But several times the generation of free radicals are so high such that they may over power the inbuilt antioxidant systems and damage the cells21.
World health organization (WHO) has recommended the evaluation of traditional plant treatments for diabetes as they are effective, non-toxic, with less or no side effect and are considered to be excellent candidates for oral therapy22. Recently there are many medicinal plants possessing experimental and clinical antidiabetic activity that have been used in traditional systems of medicine23.
7. / The present work was under taken to explore the antidiabetic activity of the flowers of Plumeria alba (Apocynaceae). 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 glycosides24. The flowers of the plant Plumeria alba are reported to contain steroid, flavonoid and alkaloids1. Genus of Plumeria alba showed the presence of tannin, carbohydrates, glycoside, steroid and flavonoid1. The root bark of Plumeria alba had shown the presence of iridiods, tannins and alkaloids25.
Recent studies on diabetes claims that the flavonoids possess antidiabetic activity with antioxidant potential26,, however the literature reveals no scientific data on antidiabetic effect of Plumeria alba flowers. In view of this, the present study is taken up to investigate the possible antidiabetic with antioxidant potential of Plumeria alba flowers in diabetic rats.
6.2 Review of the literature of Plumeria alba Linn:
Botanical Classification27:-
Kingdom: Plantae
Order : Gentianales
Family : Apocynaceae
Subfamily : Rauvolfioideae
Tribe : Plumeriae
Genus: Plumeria
Species : P.alba
Vernacular Name28:
English : White frangipani,white frangipani
Kannada : Deva Champaka
Hindi : Golainchi / champa
Sanskrit : Kananakaravira
Telugu : Veyyivarahalu
Tamil : Peru, Perumallari, Perugalli
Plumeria alba (Apocynaceae) is native to Mexico, Central America and Caribbean, Brazil, Southern and southeastern Asia27.
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 fascicles29. 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 Colombia27.
Flowers: are twisted in the bud and consist of a calyx reduced to collar with 5 dark marks; a corolla tube expanding into 5 waxy ovate/elliptic lobes (c.4x3cm) which are white to pinkish at the top, and yellow at the base and an ovary below the tube like a small spot.
Plumeriaflowers are most fragrant at night in order to luresphinx mothsto pollinate them. The flowers have no nectar, and simply dupe their pollinators. The moths inadvertently pollinate them by transferring pollen from flower to flower in their fruitless search for nectar.
The literature survey reveals that the Plumeria alba possesses various bioactive compounds such as sterols, carbohydrates, tannins, triterpenoids and iridoid glycosides24. The aerial part of the plant Plumeria alba are reported to contain steroid, flavonoid and alkaloids1. Genus of Plumeria alba showed the presence of tannin, carbohydrates, glycoside, steroid and flavonoid1. The root bark of Plumeria alba had shown the presence of iridiods, tannins and alkaloids25. The plant is reported to contain amyrinacetate, mixture of amyrins, ß-sitosterolscopotein, iriddoids isoplumericin, plumeride, plumeride coumerate and plumeride coumerate glucoside30,31. The fresh leaves and bark contain pluieride, resinic acid, fulvoplumierin, a mixture of terpenoids, sterols and plumieride1. The bark of plumeria alba contains cytotoxic iridoids, fulvoplumierin, allamcin, allamandin, 2,5-dimethoxy-p-benzoquinone, plumericin and lignan liriodinndrin1.
Medicinal uses 32:
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 acure for itch.
10.Decoction of flower is used in diabetes in Mexico 1.
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 rats33.
2. The methanolic extract of Plumeria alba has shown to possess antitumour activity against dalton lymphoma ascites in mice34.
3. Essential oil of Plumeria alba flower has reported antimicrobial activity against gram positive and gram negative bacteria35.
Review of literature, till date, regarding Plumeria alba was carried out by chemical abstract, biological abstract, medicinal abstract and other national and international scientific journals. The aerial part of the plant Plumeria alba are reported to contain steroid, flavonoid and alkaloids1. The plant is reported to contain amyrinacetate, mixture of amyrins, ß-sitosterolscopotein, iriddoids isoplumericin, plumeride, plumeride coumerate and plumeride coumerate glucoside31,32. The fresh leaves and bark contain pluieride, resinic acid, fulvoplumierin, mixture of terpenoids, sterols and plumieride1. The bark of Plumeria alba contains cytotoxic iridoids, fulvoplumierin, allamcin, allamandin, 2,5-dimethoxy-p-benzoquinone, plumericin and lignan liriodinndrin1. 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 activity36etc. These active constituents and the above mention activities in turn appear to correlate with some other biological activities37. Our literature survey revealed that the different parts of Plumeria alba have been screened for various pharmacological activities but antidiabetogenic and antioxidant activities were not investigated in Plumeria alba Flowers so far. Therefore, the present study is planned to investigate the possible antidiabetic and antioxidant potential of Plumeria alba flowers on streptozotocin induced diabetic rats. Hence this study is essential and justifiable.
6.3 OBJECTIVES OF THE STUDY:
The literature review reveals that antidiabetic activity and antioxidant potential of Plumeria alba flowers have not been reported. In view of this, the present study is aimed to investigate the antidiabetic and antioxidant potential of the Plumeria alba flowers with the following objectives:
1. Identification and authentication of the plant material.
2. 214Extraction of Plumeria alba flowers with suitable solvents, such as petroleum ether, chloroform, ethanol and water.
3. To carryout preliminary phytochemical analysis of crude extracts for the detection of the type of phytoconstituents present.
4. To evaluate the effect of Plumeria alba flowers extract on antioxidant enzyme (in-vitro).
5. To study the toxicity for determination of LD50 of the extract in mice.
6. To evaluate the effect of Plumeria alba flowers extract for the hypoglycemic and antidiabetic activity in healthy and streptozotocin induced diabetes in albino rats respectively.
7.Materials & methods:
7.1 Source of data:
Whole work is aimed to generate data from the laboratory that is experiments on
animals. Albino rats and mice of either sex will be used for this purpose.
The scheme of proposed work is as follows:
a) Collection of flowers and preservation.
b) Shade drying, coarse powdering of flowers38,39.
c) To prepare extracts from suitable solvents38,39,40.
d) Preliminary phytochemical investigation of various extracts39,41,42.
e) Study of the toxicity for determination of LD50 of the extract in mice43.
f) To establish the pharmacological activities of extract after subjecting it to preliminary
qualitative phytochemical analysis.
7 7.2 Methods of collection of Data:
Plant Material: The Plumeria alba flowers are found throughout India. Collection of flower is ideal if collected in the winter season.
The whole study is divided into 4 phases.
Phase I:
1. Preparation of various solvent extracts 38,39,40 :
It is planned to dry the flowers 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 (60-80oC), chloroform (59.5-61.5oC), ethanol (64.5-65.5oC) and water. If further required the shade- dried powder is extracted directly with70% ethanol (hydro-alcoholic extract). 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.
The solution of various solvent extracts of Plumeria alba flowers is planned for the following investigation.
2. Preliminary phytochemical screening 39,41,42.