EVALUATION OF PLUMERIA ACUTIFOLIA POIR FLOWER EXTRACT FOR ANTIULCER ACTIVITY

M. Pharm Dissertation Protocol Submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru

BYMR. NAVEEN KUMAR G R

B. Pharm.

UNDER THE GUIDANCE OF

PROF. ITTAGI SHANMUKHA M. Pharm. (Ph.D).

P. G. DEPARTMENT OF PHARMACOLOGYS. C. S. COLLEGE OF PHARMACY,

HARAPANAHALLI-583131.2013-2014

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BENGALURU

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01 Name and Address of the Candidate

NAVEEN KUMAR G R S/O RUDRESH G MBEHIND ANJANEYA TEMPLE , SHAMANURDAVANAGERE-577004

S02 Name of the Institution

T. M. A. E. Society’sS. C. S. College of Pharmacy,Harapanahalli – 583 131 Davangere( dist) Karnataka

03 Course of the StudyBranch

M. Pharm. (Pharmacology)

04 Date of Admission to course 30-07-2013

05 Title of the Topic

EVALUATION OF PLUMERIA

ACUTIFOLIA POIR FLOWER EXTRACT

FOR ANTIULCER ACTIVITY

06

Brief resume of the intended work6.1. Need for the Study Enclosure – I

6.2. Review of the Literature Enclosure – II

6.3. Objective of the Study Enclosure – III

07

Materials and Methods7.1. Source of data Enclosure – IV

7.2. Methods of collection of data Enclosure – V7.3. Does the study require any Investigations on animals? If yes give details

Enclosure – VI

7.4. Has ethical clearance been obtained form your institution in case of 7.3.

Yes, Registration No: 157/PO/C/ 1999/ CPCSEA(Copies enclosed)

08 List of References (About 4 – 6) Enclosure – VII

09 Signature of the Candidate (NAVEEN KUMAR G R)

10 Remarks of the Guide

The present research work is original and not published in any of the journals. This work can be carried out in our Pharmacology laboratory.

11

Name and Designation of (in Block Letters)

11.1. Guide

11.2.Signature

11.3.Co-Guide (if any)

11.4.Signature

11.5. Head of the Department

11.6. Signature

Prof. ITTAGI SHANMUKHA M. PHARM.,(PhD)

P.G. Dept. of PharmacologyS.C.S. College of PharmacyHarapanahalli-583 131.Davangere. (Dist.) Karnataka

------------

Prof. A. VEERANA GOUDA M. Pharm.Head of the dept. of pharmacologyS.C.S. College of PharmacyHarapanahalli-583 131.Davangere. (Dist.) Karnataka

12

Remarks of the Principal

12.1. Signature

The present study is permitted to perform in the Pharmacology laboratory of our institution and the study protocol has been approved by IAEC.

Dr.R.Nagendra RaoPrincipal

ENCLOSURE-I06. Brief resume of Intended Work6.1 Need for the study.

It is a common practice to use allopathic drugs and medicines to treat many ailments. These

medicines possess severe toxicity on many systems and even damage many organs as well. More

over these drugs cannot be used in a patient who is suffering with many organ damages. Hence

researchers are looking at new remedial measures to treat various organ damages.

Numerous physiological and biochemical processes in the human body may produce

oxygen-derived free radicals and other reactive oxygen species (ROS) as byproducts. These are

involved in many neurodegenerative diseases/disorders and also in causing several organ damages.

Further, overproduction of such free radicals can cause oxidative damage to biomolecules (e.g.

lipids, proteins, DNA), eventually leading to many chronic diseases/disorders in humans1. Though

these free radicals are generated, human system can able to neutralize these generated free radicals.

But in many instances these free radicals may resistance to our immune system and cause severe

various organ damages including gastrointestinal tract, kidney, liver and brain. Since ancient

periods, plants are used as food as well as medicine and their usage has become common as they

lack severe adverse reactions. There are many plants which possess free radical scavenging

activity2-3 and are best exploited as medicines by many folklore practitioners to treat various organ

disorders.

Ulcer is defined as the erosion in the lining of the stomach or duodenum and is caused by

the disruptions of the gastric mucosal defense and repair systems. Ulcer in the stomach is called

gastric ulcer and in the duodenum is called duodenal ulcer, together peptic ulcer4.

Peptic ulcer is one of the most common, chronic gastrointestinal disorders in the modern

era. Now it has become a common global health problem affecting a large number of people

worldwide5. In an attempt to search a potent and safe medication for peptic ulcer, Hibiscus Rosa

Sinensis is being screened for its anti ulcer activity.

Treatment should aim at relieving the symptoms with healing and preventing from further

recurrences. Usually, an ulcer will take 3-6weeks to heal and it would not produce any scar. Mostly,

many people do not care about their stomach disorders. They temporarily manage their complaints

such as gas trouble, poor appetite and pain with antacid by purchasing in local medical shops. There

is a study which shows antacids will produce more sodium in blood which in turn gives risk to heart

attack or heart disorders.

When suffering is constant, some people give no importance to chest pain or heart burn.

Also, everyone should be aware that the same set of symptoms may occur in the case of ischaemic

heart disease-heart attack, so regular checkup of B.P and E.C.G is a must. Peptic ulcers will get

worse if they are not cared for by taking the right food and sticking to the timings and treated well.

In allopathic system of medicine, they depend mainly on antacids which give very fast relief.

Treating peptic ulcer is tough, since the digested treat cannot take rest for healing and is also

exposed to irritants which hinder the healing process. By using herbal plants, besides treating the

disease, it helps in improving body’s defense mechanism fight against various disease conditions,

general resistance, also to avoid recurrences.

Literature survey revealed that it is used traditionally in the treatment of allergies,

inflammation, leprosy and ascites. Used as carminative; diuretic, laxative6 and antimicrobial7 agent.

The juice of stem bark from this plant has been applied to carious teeth, ulcers and wounds, as

rubifacient and anti-rheumatic. The plumeria acutifolia Poir, was found to contains abundant

amount of phytoconstituents like quercitrin, rutin, lupeol-acetate, tannins, iridiods, alpha-amyrin,

beta-amyrin, alkaloids, plumieride, fulvoplumierin, Plumieric-acid, oxymethyldioxycinnamic-acid,

caoutchouc, cerotic-acid, Plumericine, Isoplumericine, beta-dihydroplumericic-acid. Since the plant

possess free radical scavenging phytoconstituents like poly phenols, tannins and flavonoids has

been reported to possess anti ulcer8-9, hence the study plant has been selected to screen for antiulcer

activity.

ENCLOSURE-II6.2 Review of Literature6,7,9:

The Plant plumeria acutifolia poir belongs to family Apocynaceae is reported to have

numerous applications in traditional medicinal system. The genus Plumeria is named in honor of

the seventeenth-century French Botanist Charles plumieria.The common name “Frangipani” came

from an Italian noble family. It is the native of Mexico and widely seen in temples and

Mohammedan burial grounds in India. Familiarly known as temple tree, jasmine tree, tree of love

and in kannada known as deva kanigale, go sampigae.

About the plant:Scientific classification9

Kingdom : PlantaeUnranked : AngiospermsUnranked : EudicotsUnranked : AsteridsOrder : GentianalesFamily : ApocynaceaeSubfamily : RauvolfioideaeTribe : PlumerieaeGenus : PlumeriaSpecies : Plumeria acutifolia

Description9:

A small deciduous tree with thick branches and copious milky juice, Leaves spirally

arranged, 15-30cm. long oblog-lanceolate or oblanceolate, with an intramarginalvein, acute at both

ends; petiole 3.8-6.3 cm long, stout, glabrous. Flowers are 5cm long. Across, white with a yellow

centre, externally tinged with pink, very fragrant in terminal panicles; peduncles about 12.5cm long.

Stamens inserted near the base of the corolla-tube, included.

Chemical Constituents10,11 :

The plumeria acutifolia Poir, was found to contains abundant amount of phytoconstituents

they are,

Bark: contains Iridiods, tannins, alkaloids, plumieride, and fulvoplumierin.

Latex: Plumieric-acid, lupeol-acetate, oxymethyldioxycinnamic-acid, lupeol,

Acetyl-lupeol, alpha-amyrin, beta-amyrin, caoutchouc, cerotic-acid

Root: Plumericine, isoplumericine, beta-dihydroplumericic-acid, beta hydroplumericine,

iridiods, tannins and alkaloids.

Flower: Quercetin, rutin, Quercetin-glycoside, phenyl-ethyl-alcohol, kaempferol,

Kaempferol -glycoside, l-(+)-bornesitol, linalol, citronellol, farnesol and geraniol

Medicinal Uses6, 7

The plant has been mentioned in ancient literature as anti-inflammatory, anti-allergic, diuretic,

carminative, laxative, used in the treatment of leprosy and ascites. It also possesses anti-microbial

activity & anti-ulcer activity. The stem bark of this plant has been employed against abscess,

gonorrhea and fevers. The juice of stem bark from this plant has been applied to carious teeth,

ulcers, wounds, rubifacient and anti-rheumatic.

ENCLOSURE -Ш

6.3 Objectives of the study:The objective of study is to evaluate the gastro protective effects of flower extract on

experimentally induced models in rats.

01. To prepare various extracts (petroleum ether extract, chloroform extract, alcoholic

Extract and aqueous extract) by successive extraction technique.

02. To identify the phytoconstituents present in the flower extract.

03. Quantification of phytochemicals.

04. To assess the antioxidant property.

05. To assess gastro-protective activity against experimentally induced gastric

ulceration in rats.

ENCLOSURE – IV7. Material & methods:

7.1 Source of data:

Whole work is aimed to generate data from the laboratory that is experiments on animals.

Albino rats will be used for this purpose.

Chemicals:

Nitroblue tetrazolium, nicotinamide adenine dinucleotide, phenazine methosulpahate,

phenyl hydrazine hydrochloride, aspirin, deoxyribose, trichloro acetic acid, ethanol, ketamine,

anesthetic ether, tetraethoxy propane, glutathione reductase, glutathione, topfer’s reagents,

phenolphthalein, sodium carbonate ,aspirin,catechol,tannic acid, sodium hydroxide, Folin ciocalteau

reagent and phosphomolibdic acid etc.

ENCLOSURE – V

7.2 Materials and methods

The whole study is divided into four phases to generate the data as follows.

Phase 01. Preparation of extract and Identification of phytoconstituents12, 13.

The flower extract will be prepared by successive soxhlation i.e. extracting dried powder

with the solvents of increasing order of polarity i.e. Pet. ether (60-80), chloroform (59.5-61.5),

70% ethanol (64.5-65.5) and water. Extracts will be concentrated under reduced pressure.

Phase 02. Quantitative determination of total phenol, flavonoid and tannin content by

Spectrophotometry:

Quantification of total phenolic content 14 :-

The total phenolic content of extract will be determined by taking aliquots of the extracts into 10ml glass

tube and the volume will be made up to 3ml with distilled water. Then 0.5ml of Folin ciocalteau reagent (1:1

with distilled water) and 2 ml sodium carbonate (20%) will be added subsequently in each test tube. A blue

color will be developed in each test tube because the phenols will undergo complex redox reaction with

phosphomolibdic acid in Folin ciocalteau reagent in alkaline medium. This results in a blue colored complex,

molybdenum blue. The test solutions will be warmed for 1min, cooled and the absorbance will be measured at

650nm using known concentration of catechol. The concentrations of phenols in the test samples will be

calculated from the calibration plot and expressed as mg catechol equivalent of phenol per gram of sample.

Quantification of total flavonoid content 14 :-

To determine the total flavonoidal content, the stock solutions of extract will be prepared with

ethanol to a suitable concentration for analysis. For determination of total flavonoidal content,

aliquots of each extract will be pipetted out in series of test tubes and the volume will be made up to

1ml with distilled water. Sodium nitrite (5%; 0.3ml) will be added to each test tube and incubated

for 5minutes at room temperature. Aluminium chloride solution (10%; 0.06ml) will be added and

incubated for 5minutes at room temperature. Sodium hydroxide (1M; 0.25ml) will be added and

total volume will be made up to 3ml with distilled water. Absorbance will be measured at 510nm

against a reagent blank using U.V. spectrometer and concentration of flavonoids in the test sample

will be determined and expressed as mg equivalent per gram of sample.

Quantification of tannins 15 : -

The tannins will be identified using FeCl3 and gelatin tests. For this purpose, 0.1g of flowers extract will be

transferred to a 100ml flask. 50ml of water will be added and boiled for 30min. After filtration with cotton

filter, the filtrate will be transferred to a 500ml volumetric flask and the volume will be made up to the mark

with distilled water. 0.5 ml aliquots will be transferred to the vials, 1ml 1% K 3Fe(CN)6 and 1 ml of 1% FeCl3

will be added and the volume will be made up to 10ml with distilled water. After 5 min the solution will be

measured calorimetrically at 720nm. The total content of tannins present in the plant extract will be obtained

from standard calibration curve which will be made by taking the tannic acid as standard.

Phase 03. Antioxidant property:

Super oxide anion scavenging activity.16

Hydroxyl radical scavenging activity.17

DPPH.17

Phase 04. Gastro protective activity models:-

1. Aspirin plus pylorus ligation induced gastric ulcer in rats18:

Wistar albino rats weighing 150-200 gm of either sex will be dividing into 6 groups; each group

consists of 6 animals as follow:

Group 1 : Negative control .

Group 2 : Positive control (Aspirin 200mg/kg, p.o+pylorus ligation)

Group 3 : Standard (sucralfate 100mg/kg p.o)

Group 4 : 70% ethanolic extract (lower dose)

Group 5 : 70% ethanolic extract (medium dose)

Group 6 : 70% ethanolic extract (higher dose)

Aspirin once daily for three days will be administering to all the animals except negative control

group. The animals are will be kept for fasting for 24 hours before pylorus ligation. On the fourth day, four

hours after the pyloric ligation the animal will be sacrifice and stomach will be dissecting out to collect the

contents for estimation of free acidity, total acidity and then open along the greater curvature, wash with

saline and examine for ulcer score and ulcer index. The stomach tissue homogenate will be prepared using

0.15M KCl for the estimation of Glutathione level, catalase, and super oxide dismutase.

2. Ethanol induced ulcer 19:

Albino rats of either sex weighing between 150 – 200 gm will be dividing into 6 groups

of 6 animals each.

Group 1 : Negative control Group 2 : Positive control (1 ml/200 gm of 95% alcohol p.o.) Group 3 : Standard (sucralfate 100 mg/kg p.o) Group 4 : 70% ethanolic extract (lower dose)Group 5 : 70% ethanolic extract (medium dose)Group 6 : 70% ethanolic extract (higher dose)

All groups of animals receiving the following treatments for 5 days Groups 1, 2 receive a

vehicle 10 ml/kg, group 3 standard drug ( Sucralfate) and groups 4,5,6 will be giving 70% ethnolic

extract of plumeria acutifolia as lower,medium,higher doses respectively. On the 5th day, 1h after

final dose of treatment, the gastric ulcers will be induced in rats by administering 95% ethanol for

all the groups except negative control group. After 1h animals will sacrifice and stomach will be

incised along the greater curvature and examine for ulcer score and ulcers index20. The stomach

tissue homogenate will be prepared using 0.15M KCl for the estimation of glutathione level,

catalase, and super oxide dismutase.

Phase 05. Histopathological studies:

Work plan details:

Total duration for the completion of proposed research work may be eight months

Inclusion criteria for the selection of animals: Adult albino rats weighing between 150-200 g

of either sex will be used. The animals will be kept under standard conditions of light and

dark cycle with food and water ad libitum. Animals will be acclimatized to laboratory

condition before the test.

Exclusion criteria: Any animal not conforming with above criteria are not selected for

the experiment.

Study sampling: Each model of organ toxicity requires six groups of six animals each.

Parameters of study:

Ulcer index, free acidity, total acidity, ph.

Tissue antioxidant: glutathione, catalase, Superoxide dismutase (SOD), Lipid peroxidation

and Protein estimation.

Statistical analysis: The results obtained from the above investigation will be subjected to

statistical analysis using one way ANOVA followed by Turkey- Kramer Multiple

Comparison test.

ENCLOSURE – VI

7.3 Does the study require any investigation or interventions to be conducted on patients or

other humans or animals? If so, please describe briefly.

Yes, study requires investigation on rats and mice. The effect of test extracts

will be studied on various parameters as stated above in objectives of the study.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Registration No: 157 / 1999/ CPCSEA.

The present study is applied for permission from Institutional Animal Ethics

Committee.

ENCLOSURE – VII

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