Evaluation of Antidiabetic Potential of Leaf and Flower Extract of Jasminum grandiflorum on Alloxan Induced Diabetic Rats.

M. PHARM DISSERTATION PROTOCOLSUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

BYSANTOSH KUMAR B.Pharm.

UNDER THE GUIDANCE OF

DR. SHIVAKUMAR SWAMY M.Pharm, Ph.D.,

HOD & PRINCIPALMALLIGE COLLEGE OF PHARMACY, BANGALORE

MALLIGE COLLEGE OF PHARMACY#71 SILVEPURA, BANGALORE 90

Rajiv Gandhi University of Health Sciences,

1

Karnataka, Bangalore.Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01Name and Address of the Candidate

SANTOSH KUMARS/O:- SRI DWARIKA PRASAD SAHUQuarter no:- 2226, Sector- 8/C, E Road.Bokaro steel city,(Jharkhand) pincode-827009

02 Name of the Institution Mallige College Of Pharmacy#71 Silvepura,Chikkabanavara PostBangalore 90

03 Course of the Study Branch M.Pharm, (Pharmacology)

04 Date of Admission to course 23-11-2010

05 Title of the TopicEvaluation of Antidiabetic Potential of Leaf and Flower Extract of Jasminum grandiflorum on Alloxan Induced Diabetic Rats.

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 anyInvestigations on animals?If yes give details

Enclosure – VI

7.4. Has ethical clearance beenobtained form your institutionin case of 7.3.

Yes

08 List of References Enclosure – VII

2

09 Signature of the Candidate(SANTOSH KUMAR)

10 Remarks of the Guide

The present research work is original and not published in any of the journals with best of my knowledge upon extensive literature review. This work will be carried out in the Pharmacology laboratory by Mr. Santosh Kumar under my supervision.

11

Name and Designation of (in Block Letters)11.1. Guide

11.2.Signature

11.3.Co-Guide

11.4.Signature

11.5. Head of the Department

11.6.Signature

Dr. SHIVAKUMAR SWAMY M. Pharm., Ph. D.,HOD & PRINCIPALMallige College Of Pharmacy,Bangalore, Karnataka.

Mr. Prashanth Baganal M.Pharm.,

Dr. SHIVAKUMAR SWAMY M. Pharm., Ph. D.,Principal & HODMallige College Of Pharmacy,Bangalore, 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. Shivakumar Swamy)

3

Enclosure: I

06. Brief resume of the intended work:

Introduction: Diabetes mellitus is an endocrine disorder that is characterised by hyperglycaemia 1

resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of

diabetes is associated with long-term damage, dysfunction, and failure of different organs, especially

the eyes, kidneys, nerves, heart, and blood vessels. Long-term complications of diabetes include

retinopathy with potential loss of vision; nephropathy leading to renal failure; peripheral neuropathy

with risk of foot ulcers, amputations, and Charcot joints; and autonomic neuropathy causing

gastrointestinal, genitourinary, and cardiovascular symptoms and sexual dysfunction. Patients with

diabetes have an increased incidence of atherosclerotic cardiovascular, peripheral arterial and

cerebrovascular disease. Hypertension and abnormalities of lipoprotein metabolism are often found in

people with diabetes.2

Reports from the International Diabetes Federation (IDF) indicate that the prevalence of

diabetes mellitus has reached epidemic levels globally. Estimates for 2010 indicate that 285 million

adults have diabetes in the seven regions of the IDF. These numbers represent an increase of 39 million

from 2007 and an expected continued increase to 439 million in 2030. It is believed that by 2025, more

than 75% of the world population with diabetes will reside in developing countries and the countries

with the largest populations of adults with diabetes will include: India, China and the United States.

India has 50.8 millions of people with diabetes in 2010 and expected to increase by 87 million by 2030.

The highest number of deaths attributable to diabetes is expected to occur in countries with large

populations-1,008,000 deaths in India3.

The present pharmaceutical drugs are either too expensive or have undesirable side effects.

Treatment with sulphonylureas and biguanides are also associated with side effects4.

Compared with synthetic drugs, drugs derived from the plants are considered to be less

toxic with fewer side effects.

However, for a number of reasons, complementary medicine has grown in popularity in

recent years. Dietary measures and traditional plant therapies as prescribed by Ayurvedic and other

indigenous systems of medicine are used commonly in India. Many indigenous Indian medicinal plants

have been found to be useful to successfully manage diabetes and some of them have been tested and

their active ingredients isolated. The World Health Organisation (WHO) has also recommended the

evaluation of the plants effectiveness and conditions where we lack safe modern drugs5.

4

6.1 Need For The Study:

On careful observation of the chemical constituents of Jasminum grandiflorum, it is

observed that it contains many chemical constituents which are already proved for their anti-diabetic

activity. The extensive literature survey of J. grandiflorum reveals that its anti diabetic property is not

scientifically tested and established.

In the present scenario the health providers looks at natural sources to maintain health

conditions rather than synthetic drugs. It is also established beyond doubt that, the drugs derived from

plants sources have less toxic and hence proved beneficial for ailments which require lifelong

treatment.

In the recent past years many medicinal plants are screened for their hypoglycemic property

and quite a few of them are already successful in entering the market.

Considering all the above points we felt, it is worth to investigate the hypoglycemic

property of Jasminum grandiflorum.

5

Enclosure: II

6.2 Review of literature

Jasminum grandiflorum, also known variously as the Spanish jasmine, Royal

jasmine, Catalonian jasmine, among others (chameli in Hindi) is a species of jasmine native to

South Asia. Family:- Oleaceae.

Classical name6 : mallige, Jati, Sauanasyayani, Sumama, Chetika, Hridyagandha, Malati,

Rajaputrika.

DISTRIBUTION7:

Jasminum grandiflorum L. (Family: Oleaceae) exhibit awide ecological range and found

extensively all over India. The plant is cultivated in well drained loamy soil and also on a variety of

soils such as black ,lateritic and clay loam with good drainage system as the plant is highly

susceptible to water logging The harvesting of the flower is done in the month of May to December

(in South India) and July to November (in North India)

Botanical Description7 :

It is a climbing shrub, the leaves are opposite, with 3 to 7 lance-shaped, Entire ovate to

somewhat elliptic in shape with acuminate mucronate apex, petiole almost lacking, imparipinnately

compound, with three paired foliates ending with a single leaf at the tip. The leaflets are elongate-

lanceolate, acute, 7 to 11 terminal leaflet somewhat large than laterals, narrowing at the base, ovate-

lanceolate, acute or acuminate, laterals ovate, terminal one larger than laterals and often partially

united with surfaces with a ciliate margin. Flowers are terminal and axillary cymes, calyx lobes long

and linear, more than half as long as the corolla tubes. The fruit is a black berry, elliptic, globose

berries when ripe.

CHEMICAL COMPOSITION7, 8:

Leaves:- The leaves are found to contain many chemicals, main constituents are 2”-

epifraxamoside, demethyl-2”-epifraxamoside, jasminanhydride, oleacein, 2-(3,4-dihydroxy phenyl)-

ethanol, isoquercitrin, ursolic acid , resin, salicylic acid, jasminine, indoleoxygenase, 3,4-dihydroxy

benzoic acid, 2-hydroxy-30, 40-dihydroxyacetophenone, oleanolic acid, B- Sitosterol, catechin, and

gallic acid.

6

Flowers: The flowers are known for their fragrance. These contain- Cis-3-hexenol, 2-vinyl

pyridine, indole, myrcene, linalool, geranyl linalool, α-terpineol, geraniol, linalyl acetate, nerolidol,

phytol, isophytol, farnesol, eugenol, benzyl alcohol,p-cresol, methyl benzoate, benzyl cyanide,

benzyl acetate, methyl dihydrojasmonate, methylanthranilate, jasmone, methyl- N-methyl

anthranilate, vanillin, cis-3-hexenyl benzoate, benzylbenzoate, methyl palmitate, methyl linoleate,

jasgranoside, jaspolyoside, 8-epi-kingiside, 10-hydroxy- oleuropein, 10-hydroxy ligstroside,

oleoside-7,11-dimethyl ester , 3-O-α-L-rhamnopyranosyl,(1→2)-β-D-xylopyranosyl-hederagenin-28-

O-β-galactopyranosyl(1→6)-β-D-galactopyranosylester, hederagenin-3-O-β-D-

glucopyranosyl(1→3)-α-L-arabinopyranoside,2-α,3β,23-trihydroxyolean-12-en-28-oic –O-β -D-

glucopyranosyl ester, hederagenin-3-O-β-D-xylopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-

arabinopyranoside,2α,3β,23-trihydroxyolean-12-en-28-oic–O-α\-L-rhamnopyranosyl(1→4)β-D-

glucopyranosyl(1→6)-β-D-glucopyranosyl ester, hederagenin-3-O-α-L-rhamnopyranosyl (1→2)-α-L

arabinopyranoside, kaempferol-3-O,α-L-rhamnopyranosyl(1→3)-[α-L-rhamnopyranosyl(1→6)β-D-

galactopyranoside, kaempferol-3-O-rutinoside, 7-ketologanin, oleoside-11-methylester, 7-glucosyl-

11- methyl oleoside, ligstroside and  oleuropein.

Jasmine oil: - Oil obtained from its flowers mainly contains Methyl jasmonate , benzyl

benzoate, linalool, linalyl acetate, benzyl alcohol,indole, jasmone, methyl anthranilate, P-cresol,

geraniol, racemic-(5-pent-2-enyl)-5,1-pentanolide,benzyl benzoate, nerol, 1-α-terpineol, d and dl-

linalool, γ-jasmolactone, farnesol, nerolidol and eugenol.

Jasminum grandiflorum (Family: Oleaceae) commonly called as Spanish jasmine, Royal

jasmine, Catalonian jasmine, found and cultivated in many South Asian. The different parts of plant

are used traditionally in ayurveda for the treatment of various ailments as fallows, The whole plant is

used as bitter, astringent, acrid, thermogenic, aphrodisiac, antiseptic, anodyne,

depurative,emmenagogue, emollient, diuretic, anthelmintic, deobstruant, dentrifrice, suppurative and

tonic7.

The roots are useful in cephalalgia, vitiated condition of vata, paralysis, facial

paralysis,mental debility, chronic constipation, flatulence, strangury, sterility,

dysmenorrhoea,amenorrhoea, ringworm, leprosy, skin diseases and giddiness9.

The leaves are useful in odontalgia, fixing loose teeth, ulcerative stomatitis, leprosy,

skindiseases, ottorhoea, otalgia, strangury, dysmenorrhoea, ulcers, wound and corns9, are chewed in

7

aphthae, stomatitis, toothache, ulcer in the mouth and leaf-juice or oilobtained from it is dropped in to

the ear7. A decoction of the leaf was also usedas a gargle.The oil cooked with juice of jati leaves was

prescribed in purulent discharge from the ear. Fresh juice of the leaves is a valuable application for

sort corns between the toes, for ulceration in the mouth,throat and gums, the leaves fried in ghee are

recommended to be applied7.

Flowers are useful in stomatopathy, cephalopathy, odontopathy, ophthalmopathy, leprosy,

skin diseases, pruritis, strangury, dysmenorrhoea, ulcers, as refrigerant, ophthalmic and

vitiatedconditions of pitta9. applied as a plaster to the loins, genitals and pubes as an aphrodisiac. The

plant isused in scorpion-string7. Charaka used the sprouts or dried flowers, inprescriptions, externally

in coryza, nasal hemorrhage and dermatosis. Sushruta used Malati as aningredient of a medicated

clarified butter for external application on infected wounds, for cleansing and sterilizing the interior

of ulcer, as an ingredient of hair oil for baldness andalopecia and as an ingredient of an eye-salve for

loss of vision.

The phytochemical investigation and pharmacological screening of the different parts of

plant has been done and several reports suggest that the ethanolic extract of plant screened for

Antitioxident10, Antiulcer11, Anticarcinogenic12, Wound healing13, Antiviral14, Spasmolytic activity15,

Anti-inflammatory16, Antimicrobial17, cytoprotactive18, Chemoprentative and Lipid peroxidative

activity19, Breast cancer19, Anthelmintic activity20 , Angiotention convertatin enzyme inhibitor

activity and evaluated21.

In the recent past many hypoglycaemic agents are introduced, still the diabetes and the

related complications continue to be a major medical problem not only in developed countries but

also in developing countries. Many Indian medicinal plants are reported to be useful in diabetes22,23.

Most of the plants having characteristic odour possess essential oil and bioflavonoids as

their main chemical constituents along with other components and some of them have been proved

for their antidiabetic activity.

8

For eg.

Table 1. List of some plants with their main chemical composition which have been proved as

antidiabetic.

Sl.no

Name and family

Parts used

Main constituent

Individual components Reference for composition

Reference for anti diabetic

1 Coriander

Coriandrum sativum L.

umbelliferae

seeds Essential oil Geranyl acetate, linalool,

 nerol, neral, linalool,

cis-dihydrocarvone and

 geranyl acetate,

α and β-pinenes,

dipentene (limonene),

p-cymene, α- and γ -terpinenes,

n-decanal, geraniol and

l-borneol.

24 33

2 CURRY LEAF Murraya koenigii (L.) (family: Rutaceae

Leaves Essential oil

flavonoids

α-pinene, sabinene, b-pinene, aterpinene, β-phellandrene, γ-terpinene and terpinen-4-ol .

7,4-1-diOMe Vitexin, 4-1-OMe Kaempferol, Vanillic acid, Syringic acid,

p-coumaric acid.

24,25 33

9

3 Bay leaf

Laurus nobilis

L.

(Lauracea)

Leaves Essential oil

Flavonoids

1-8- cineole, linalool,

α-ter-pinyl acetate and methyl eugenol

:flavones (apigenin and luteolin),

flavonols (kaempferol, myricetin, and quercitin.

sesquiterpene lactones , alkaloids,

glycosylated flavonoids, and monoterpene and germacrane alcohols

24 34

4 Tamarindus indica L.

Caesalpimiaceae

Leaves Volatile oil

Flavonoids

tannins

The major constituents of the leaf oil of tam-arind are linalool, anthranilate, benzyl ben-zoate and limonene. α-Pinene,β-pinene,nerol, etc.

taxifolin, apigenin, eriodictyol, luteolin, and naringenin

proanthocyanidin in form of catechin, epicatechin , procyanindin

26,27 33

5 Cinnamon

Lauraceae

Barks and Leaves

Volatile oil

flavonoids

benzaldehyde,linalool, αterpineol, geraniol, cinnamaldehyde, eugenol,cinnamyl alcohol,and coumarin. hydroxybenzaldehyde, cinnamic acid, cinnamyl acetate, and 3-phenylpropionaldehyde,

quercetin, kaempferol 

24,28 33

10

6 Azadirachta indica

(Meliaceae)

Leaves Essential oil

Flavonoids

Flavonol glycocides:-

Tannins:-

Linalool , Caryophyllene ,

α-Cubeben , α-Copaen ,

Allo-aromadendren

quercetin and Isorhamnetin. kaempferol, myricetin and quercetin. Nimbaflavanon

quercetin-3-galactoside, kaempferol-3-glucoside and myricetin 3’-L-arabinose.

gallic acid, (+) gallocatechin, (-) epicatechin, (+) ,catechin

29,30 33

7 Ocimum sanctum

Labiatae

Leaves

Essential oil

Phenol, alkaloid, tannin,

ascorbic acid

a-pinene, camphene , ß-pinene, myrcene, limonene , cis-ocimene , p-cymene,cis-3-hexenol, fenchyl acetate, camphor, linalool , fenchyl alcohol , methyl chavicol, -terpineol , citronellol , geraniol, methyl cinnamate and eugenol.

31 31

8 Allium sativum

Linn.

Lehsun Liliaceae

Roots Volatile oil

Allin, Allicin.

Sulphur containing compounds.

terpenes include citral, geraniol, limonene, alpha-and beta-phellandrene, ajoene and vinyldithiines

32 33

11

9 Adrak Zingiber

officinale

Zingiberaceae

Rhizome Sesquiterpene

Oxygenated monoterpenes

α-zingiberene, β-phellandrene,camphene; β-bisabolene,

geranial, geraniol ,nerol, 1,8-cineol,

α-terpineol, borneol, linalool, methyl nonyl ketone

24 33

10  Black pepper

Piper nigrum

Piperaceae

Seed Alkaloid,

volatile oil

(monoterpene hydrocarbon)

 

(oxygenated monoterpens)

oleoresin

Piperin

 camphene, δ3-carene,p-cymene, limonene, myrcene,

cis-ocimene, α-phellandrene, β-phellandrene and α- and β-pinenes, sabinene,α- and γ -terpinenes, terpinolene andα-thujene,

linalool, α-terpeneol, 1,1,4,trimethylcyclohepta-2,4-dien-6-ol, phellandral, pip-eritone, citronellal, nerol, geraniol, isopino-camphone, methyl citronellate, methylgeranate,

α-terpenyl acetate, terpenoleneepoxide and translimonene epoxide

24 35

12

Some of the aromatic flowers or herbs also have essential oil as their main chemical

components and their extract has been screened to have anti diabetic efficacy. Such as rosemary,

eucalyptus, rose,and champak etc.

Table II. List of some aromatic herbs (or) flowers with their main chemical composition which

have been proved as antidiabetic.

Sl.

no

Name and family Parts

used

Main

constituent

Individual components Reference

for

composition

Reference

for anti

diabetic

1. ROSEMARY

Rosmarinus

officinalis Labiatae

Herbs Essential oil -pinene, camphene, ß-

pinene, sabinene trace,

myrcene, -phellandrene, -

terpinene, limonene, 1, 8-

cineole, -terpinene , p-

cymene, terpinolene ,

camphor, copaene,

linalool, terpinen-4-ol,

caryophyllene,

-terpineol, thymol and

carvacrol

36 37

2. EUCALYPTUS

Eucalyptus spp.

Myrtaceae

Herbs Essential oil -pinene, ß-pinene,

myrcene,

limonene, 1-8 cineole, p-

cymene, -terpinene ,

terpinolene, citronellal,

linalool, iso-pulegol,

citronellol,

citronellyl acetate and

caryophyllene

36 33

13

3. ROSE

Rosa damascena

Rosaceae

Flowers Essential oil citronellol, paraffins,

geraniol, nerol,

ß-phenyl ethanol,

eugenol methyl ester,

linalool, and farnesol

36 38

4. CHAMPAK,

Michelia

champaca

Magnoliaceae

Flowers Essential oil cineole, iso-eugenol,

phenyl ethyl alcohol,

benzaldehyde, methyl

anthranilate, benzyl

alcohol, p-cresol and its

methyl ether.

36 39

As litreture reveals and on careful observation of Table 1, 2 and the chemical constituents

of Jasminum grandiflorum, it is observed that it contains many chemical constituents same as some

medicinal plants which are already proved for their anti-diabetic activity. The extensive literature

survey of J. grandiflorum reveals that its anti diabetic property is not scientifically tested and

established .

Considering all the above points we felt, it is worth to investigate the hypoglycemic

property of Leaf and Flower extract of Jasminum grandiflorum.

14

Enclosure: III

6.3 Objectives of the study

The present research work is an attempt to establish the possible antidiabetic efficacy using

alcoholic extract of leaves and flowers of Jasminum grandiflorum in rats with the following

objectives.

Collection and identification of plant.

Alcoholic extraction of leaves and flowers of Jasminum grandiflorum.

Qualitative estimation of phytochemical constituents.

Pharmacological Screening for antidiabetic activity.

o Effect of J.grandiflorum extract on Blood Glucose Level in normal albino rats.

o Evaluation of Antidiabetic activity in Alloxan induced experimental model.

o Evaluation of effect of extract on biochemical parameters and histopathological changes.

15

Enclosure: IV

7. Materials and methods:-

7.1 Source of data:-

The work is aimed to generate data from experiments to be conducted at pharmacology laboratory

of our institution. Albino rats and mice will be used for this purpose.

The experiments, which involves the following steps:

Collection and authentication of leaves and flowers of Jasminum grandiflorum.

alcoholic extraction of leaves and flowers of Jasminum grandiflorum

Qualitative estimation of phytochemical constituents.

Screening for antidiabetic activity.

16

Enclosure: V

7.2 Method of collection of data:

1) Collection and identification of plant material:

For this study, Leaves and flowers of Jasminum grandiflorum will be collected from the

surrounding gardens of Bangalore, Karnataka. The sample will be identified and authenticated by

the botanist.

2) Extraction of J. grandiflorum leaves and flowers:

Fresh leaves and flowers will be cleaned and shade dried at room temperature and will be

powdered mechanically. The powdered materials will be extracted with 70% alcohol by Soxhlet’s

extraction method40. The extracts will be concentrated using rotary flash evaporator and percentage

yield of the same will be recorded. Finally the extract will be used for qualitative phytochemical

analysis and to evaluate Antidiabetic activity.

3) Qualititative Phytochemical Analysis:

The crude extracts thus obtained will be subjected to preliminary phytochemical test following

the standard procedures described in the literature.

4) Screening of Antidiabetic activity

A. Effect of Jasminum grandiflorum leaf and flowers extract on blood glucose level on normal albino rats41.

Male albino strain rats weighing (160–200 g), aged 8-14 weeks older are to be equally divided

into four groups of six rats each. Animals belonging to Group I-normal control group will be

administered only vehicle and Group II-standard group will be administered reference drug

Glibenclamide (0.25 mg/kg, p.o.), while Group III- will be administered with Leaf extract of

J.grandiflorum (250 mg/kg, p.o.) and Group IV will be administered Flower extract of

J.grandiflorum (250 mg/kg, p.o.) respectively. Thereafter, blood samples will be collected from tail

vein prior to dosing and then at 30, 60, 90 and 120 min. and blood glucose level will be determined

by Glucose-Oxidase method42. And a data of % reduction of blood glucose will be recorded as

below.

17

Group I - normal control -only vehicle

Group II - standard group - Glybenclamide (0.25mg/kg, p.o.)

Group III - Leaf extract of J.grandiflorum (250 mg/kg, p.o.)

Group IV - Flower extract of J.grandiflorum (250 mg/kg, p.o.)

Data to be recorded:-

Effect of extract on Normoglycemia:

Group Treatment % Reduction in blood glucose level

0 min 30 min 60 min 90 min 120 min

I

II

III

IV

In the next part of the experiment, the normoglycemic studies will be carried out in the same

groups of animals which are used earlier. The Albino Rats will be administered extract of

J.grandiflorum (250 mg/kg, p.o.) daily for 28 days in group III and IV. Group I acts as control and

Group II animals are administrated Glybenclamide (0.25mg/kg, p.o.) and blood samples will be

collected from tail vein prior to dosing (day 0) and then at regular intervals of day 7, 14, 21 and 28

respectively and to be subjected to fasting blood glucose level. . The fasting blood glucose level

will be analyzed using Glucose-Oxidase- Method and a data of % reduction of blood glucose is to

be recorded.

Group I - normal control -only vehicle.

Group II - standard group - Glybenclamide (0.25mg/kg, p.o.)

Group III - Leaf extract of J.grandiflorum (250 mg/kg, p.o.)

Group IV -Flower extract of J.grandiflorum (250 mg/kg, p.o.)

18

Data to be recorded:-

Effect of extract on Normoglycemia

B.

Evaluation of Antidiabetic activity in Alloxan induced experimental model.

Chemical induction of diabetes by Alloxan in experimental animals (Rats) and its biochemical

conformation41, 43:-

Male albino strain rats weighing (160–200 g), will be used for this experiment and are to be

fed on commercial feeds and to be given water ad libitum .The animals are to be fasted from feeds

for 12 hours before the commencement of the experiment,but will be allowed water ad libitum.

Animals will be induced diabetes by a single dose subcutaneous injection of freshly prepared

Alloxan monohydrate (120 mg/kg) dissolved in normal saline (0.9% w/v NaCl in distilled water)41.

The rats will be treated with 20% glucose solution intraperitoneally after 6 hours.They are to be

kept for the next 24 hours on 5% glucose solution bottles in their cages to prevent hypoglycemia43.

Blood glucose level will be measured by using Glucose-Oxidase- Method and diabetes will be

confirmed after 72 hr of alloxanisation. Rats which will show marked hyperglycemia (FBG >250

mg/dl) after 72 h of injection will be selected for further studies41.

Effect of extract on blood glucose of alloxan induced diabetic rats.

Diabetic Albino Rats which will be selected will be divided into five groups (n=6) as

follows:

Group-I- Normal control rats (nonalloxanized) normal saline only;

Group-II- Diabetic control rats (Untreated, alloxanized);

Group-III - Diabetic rats- Glibenclamide (0.25 mg/kg; p.o.) as standard

reference drug.

Group IV - Diabetic rats (250mg/kg body weight) crude leaf extract and

Group V - Diabetic rats (250mg/kg body weight) crude flower extract and

respectively.

19

Group Treatment % Reduction in blood glucose level

1st day 7thday 14thday 21stday 28thday

I

II

III

IV

The treatment will be continued for a period of 28 days respectively following oral

administration by gastric intubation, using a force-feeding needle to the experimental animals.

Plasma glucose will be estimated on withdrawing blood samples from tail vein prior to dosing (day

0) and then at regular intervals of day 7, 14, 21and 28 respectively all groups of animals and a data

is to be recorded. The body weight, food and fluid intake of all groups of animals will be monitored

on a daily basis for 28 days at regular time. Fixed amount of rat chow and fluid will be given to

each rat and replenished the next day. At the end of 28th day, all the rats will be euthanized by

pentobarbitone sodium (60 mg/kg) and sacrificed by cervical dislocation.

Blood sample will be withdrawn from abdominal aorta into fresh centrifuge tubes and

centrifuged at 2,500 rpm for 15 min to obtain serum and plasma. Serum samples will be stored at -

20°C until utilized for further biochemical estimation parameters41.and the organs like Brain,

Spleen, Pancreas, Heart, Liver and Kidney are to be collected for Organ Weight Analysis and

histopathological investigation.

Data to be recorded:-

Effect of extracts on diabetic rats:

Group Treatment % Reduction in blood glucose level

0days 7thday 14thday 21stday 28thday

I

II

III

IV

V

20

C. Evaluation of effect of extract on biochemical parameters and histopathological changes.

1. Blood Analysis(Biochemical Parameters)

The parameters like cholesterol, HDL-cholesterol, triglycerides, LDL-C and VLDL will be

determined in serum using Auto analyzer. Liver profile parameters like total protein, albumin,

alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT),

and bilirubin are to be determined in serum using Auto analyzer. Blood urea nitrogen, Creatinine

and uric acid will be determined using auto analyzer. Atherogenic index has to be calculated by

using the following formula44. Anti-atherogenic index is expressed as percentage. Higher the index

the greater the anti-atherogenic potential and vice-versa.

Total cholesterol − HDL-C

2. Atherogenic index = X 100

HDL-C

3. Organ weight Analysis

At the end of the 28th day Brain, Spleen, Pancreas, Heart, Liver and Kidney will be carefully

dissected out and organs of all the animals will be examined macroscopically. The positions, shape,

sizes and colors of the internal organs will be visually observed for signs of gross lesions.

4. Histopathological investigation:-

Tissue Processing:

Pancreas, Heart, Liver and kidney tissues will be placed in 10% formalin (diluted to 10%

with 20 mM phosphate buffer pH 7.4) for 1 hr to rectify shrinkage due to high concentration of

formalin. The tissues will be dehydrated by ascending grades of isopropyl alcohol by immersing in

80% isopropanol overnight and 100% isopropyl alcohol for 1 hour. The dehydrated tissues will be

cleared in two changes of xylene, 1 hour each. The wax impregnated tissues will be embedded in

paraffin blocks using the same grade wax. The paraffin blocks are to be mounted and cut with

rotary microtome at 3 micron thickness. The sections will be floated on a tissue floatation bath at

40 °C and taken on glass slides and smeared with equal parts of egg albumin and glycerol. The

sections are then will be melted in an incubator at 60 °C and after 5 min the sections will be

allowed to cool.

21

Tissue Staining

The sections will be deparaffinised by immersing in xylene for 10 min in horizontal staining

jar. The deparaffinised sections will be washed in 100% isopropyl alcohol and will be stained in

Ehrlich’s hematoxylin for 8 min in horizontal staining jar. After staining in hematoxylin, the

sections will be washed in tap water and dipped in acid alcohol to remove excess stain (8.3% HCl

in 70% alcohol). The sections will be then placed in running tap water for 10 min for blueing (slow

alkalization). The sections will be counter stained in 1% aqueous eosin (1 gm in 100 ml tap water)

for 1 min and the excess stain is to be washed in tap water and the sections will be allowed to dry.

Complete dehydration of stained sections will be ensured by placing the sections in the incubator at

60°C for 5 min. When the sections will be cooled, they will be mounted in DPX mount having the

optical index of glass (the sections will be wetted in xylene and inverted on to the mount and to be

placed on the cover slip). The architecture is to be observed at low power objective under

microscope. The cell injury and over aspects are to be observed under high power dry objective45.

Light microscopic examination of the sections will be then carried out and micrographs will be

produced using Vanox-T Olympus photographing microscope. The histopathological examinations

will be reviewed by the pathologist.

6) Statistical Analysis:

The data obtained from the study will be subjected for statistical analysis using One-way ANOVA

followed by Turkey Kramer Multiple Comparison Test to assess the statistical significance of the

results.

7) Work plan details:

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

1. Collection of plant materials including authentication - One month.

2. Duration of experimentation on animals including

Preparation of crude extracts - Five months.

3. Literature collection - Two months.

4. Dissertation writing and communication of research

papers. - Two months.

22

Enclosure-VI

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

humans and animals? If so please describe briefly.

The proposed study requires the investigation on albino rats of either sex (Wistar Strain) weighing

150 - 200 gm for the Antidiabetic activity. Whereas albino mice of Swiss Strain will be utilized for

the acute toxicity study.

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

The present study protocol is approved from Institutional Animal Ethics Committee.

23

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