Antidiabetic and Antiatherogenic Effects of Methanolic Extract of Seeds of Hippophae Rhamnoside

“Antidiabetic and Antiatherogenic effects of methanolic extract of seeds of Hippophae rhamnoside Linn.”

M. Pharm Dissertation Protocol Submitted to

RajivGandhiUniversity of Health Sciences, Karnataka

Bangalore – 560041

By

Mr.Chirag G Gediya,B.Pharm

Under the Guidance of

Dr. Kalyani Divakar,M.Pharm,Ph.D

H.O.D OF PHARMACOLOGY

Department of Pharmacology,

Acharya & B.M.ReddyCollege of Pharmacy,

Soldevanahalli, Chikkabanavara (Post)

Hesaraghatta Main Road,

Bangalore – 560090

2008-09

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1 / Name of the candidate and address / Mr. Chiragkumar G Gediya
S/O Ghanshyambhai M Gediya
#13/ Ishwarkrupa society,
B/H Trikamnagar-2,
L.H. Road, Varachha,
Surat-395010
Gujarat
2 / Name of the institution / Acharya & B.M. Reddy College Of Pharmacy
Soldevanahalli,
Hesaraghatta Road,
Chikkabanavara (Post),
Bangalore -560090
Phone No: 080-65650815
3 / Course of study and subject / M.Pharm
(Pharmacology)
4 / Date of admission / May 2008
5 / Title of the topic / “Antidiabetic and Antiatherogenic effects of methanolic extract of seeds of Hippophae rhamnosideLinn.”
6.
6.1
6.2
6.3 / Brief resume of intended work
Need for the study
Review of Literature
Aim and Objectives of the study / Enclosure I
Enclosure II
Enclosure III
7.
7.1
7.2
7.3
7.4 / Materials & Methods
Source of data
Methods of collection of data ( Including sampling ,procedure if any)
Does the study require investigation on animals?
If yes give details
Has ethical clearance been obtained from your institution in case of 7.3 / Enclosure IV
Enclosure V
Enclosure VI
Yes (Copy Enclosed)
8. / List of references (about I-VI) / Enclosure VII
9. / Signature of the candidate
10. / Remarks of the guide
11.
12. / Name & Designation of
Guide
Signature of Guide
Head of the Department
Signature of HOD / Dr. Kalyani DivakarM.Pharm, Ph.D
H.O.D, Department of Pharmacology,
Acharya & B.M.ReddyCollege of Pharmacy,
Dr. Kalyani DivakarM.Pharm,Ph.D
H.O.D, Department of Pharmacology
Acharya & B.M.Reddy College of Pharmacy.
13. / Remarks of the Principal
Signature / PRINCIPAL
Dr. Divakar Goli Pharm, Ph.D
Acharya & B. M. Reddy College of Pharmacy.

Enclosure - I

6. BRIEF RESUME OF INTENDED WORK

6.1 Need for the study :-

Diabetes mellitus is a chronic metabolic disorder affecting approximately 5% of the world’s population. It is characterized by dysregulation in carbohydrate, protein and fat metabolisms caused by the complete or relative insufficiency of insulin secretion and/or insulin action. According to WHOprojections, the diabetic population is likely to increase to 300 millions or more by the year 2025. Currently available therapies for diabetes include insulin and various oral antidiabetic agents such as sulfonylureas, bigunides, α-glucosidase inhibitors, glinides, which are used as monotherapy or in combination to achieve better glycaemic regulation. Many of these oral antidiabetic agents have a number of serious adverse effects. Thus, the management of diabetes without any side effects is still a challenge.1

Atherosclerosis leading to coronary artery disease (CAD) has assumed a pandemic proportion over the world and has become the most common cause of death in developed as well as developing nations. Atherosclerosis is primarily a lipid disorder affecting the arteries. Increased intracellular generation of reactive oxygen species has been proposed as a mechanism to tissue injury with a variety of pathological processes like ischaemia, inflammation, atherosclerosis and thrombosis. Lipids undergo peroxidative changes in the arterial wall, which eventually produce tissue injury. Increased free radicals can cause abnormal function of endothelial cells via reduced NO availability, and is believed to be an early event in atherogenesis. Hence, compounds that can scavenge the excess of free radicals formed, inhibit their production, or protect membranes from peroxidation are of wide therapeutic value.2

Atherosclerosis is one of the major vascular complicationof diabetes. Substantial evidence supports the central role of Ox-LDL in atherogenesis. Several factors are throught to promote lipoprotein oxidation in diabetes: enhancement of auto-oxidative glycosylation in the presence of high glucose concentration; increased production of free radicals and lipid peroxidation in the setting of hyperglycemia; and the decrease in antioxidant pathways.3

Medicinal properties of sea buckthorn has been ascribed to its important phytochemicals, like flavonoids, carotenoids, fatty acids, etc. It has been found that beta-carotene, flavonoids, fatty acids etc, are important medicinal phytochemicals. Sea buckthorn has been shown to have a potent antioxidant activity. Both the flavonoids and the oils from sea buckthorn have several potential applications. There are several areas of research that have been important points for their use: as an aid to patients undergoing cancer therapy; a long-term therapy for reduction of cardiovascular risk factors; treatment of gastrointestinal ulcers; internal and topical therapy for a variety of skin disorder and as a liver protective agent and a remedy for liver cirrhosis.4

The seed residue of Hippophae rhamnoside L. (FSH) and fruit residue of Hippophae rhamnoside L.(FFH) can decrease the levels of blood glucose and lipid in normal mice, and their effect on glycometabolism may be related to the control of glyconeogenesis.5

The antidiabetic and antiatherogenic effects of seed residue of Hippophae rhamnoside Linnhave not been investigated scientifically. Therefore, thepresent study is planned to find out the antidiabetic and antiatherogenic effects of methanolic extract of seeds of H. rhamnoside L.(MESH).

Enclosure-II

6.2 Review of Literature:-

Plant selected for the study:6

Name: Hippophae rhamnoside Linn.

Family: Elaeagnaceae

Description of plant:6

It is a medium sized deciduous tree or large shrub with 2.5-6 m in height. The main trunk has a thick and rough bark.

Young Branches- Smooth, grey, light ash coloured with needle shaped thorns.

Leaves - Grow either in alternate or in cluster.

- Each leaf is Elongated or elongate-spathulate with green upper surface and silvery tinged ashy green lower surface.

Flowers - Blooms when leaf is still in bud condition.

Fruits- Narrow-elliptic or Oval, yellow-orange berry with its external surface covered with silvery dust particles.

-Taste is sour.

The plant is not self-fertile. It flowers in April and the fruits are collected through August to October.

Distribution of plant:6

Sea buckthorn is a widely distributed in Asia such as Tibet, India, China, Rusia, Turky, Europe, North America, United States, Canada, Finland, Germany and some other European countries such as Britain, Italy, Spain.

Chemical Constituents:3

It contains vitamin C, mineral elements, monosaccharides sugar, organic acids, free amino acids, large amount of carotenoids and vitamin E, volatile compounds, and different flavonoids like quercetin, myricetin, isorhamnetin and kaempferol. It also contains fatty acids, triacylglycerol, glycerophopholipids, phytosterols, zeaxanthin esters, alpha-tocopherol and phenolic compounds. The oil content of the whole fresh berries, including seeds, varied from 2 to 11% and of the seeds from 4 to 17%.

Traditional Uses:6

Different parts of H. rhamnoides L. have been used for the treatment of diseases in traditional medicine in various countries in the world. It has long been used for relieving cough, aiding digestion, invigorating blood circulation and alleviating pain since ancient time. The extracts of H. rhamnoides L. Branches and leaves are also used in the treatment of diarrhea. Leaves are used in gastrointestinal and dermatologic disorders and have been applied as compress form in rheumatoid arthritis in the middle Asia. Flowers of H. rhamnoides L. are used as skin softener in Tajikistan.For its haemostatic and anti-inflammatory effects, fruits ofthe plant are added to prescriptions in pulmonary, gastrointestinal, cardiac, blood and metabolic disordersin Indian and Tibetian medicine.After reviewing the ancient literature Li and Guo point out that sea buckthorn is a mild drug with the characteristic effects such as lowering fever, diminishing inflammation,counteracting toxicity and abscesses, treating coughhand colds, keeping warm, easing respiration, clearing sputum, having mildly laxative effect, treating tumors, especially ofthe stomach and the esophagus, and treating different kindsof gynecological diseases in Tibetian medicine.Oil extracts obtained from fruits are used in liver diseases,inflammatory processes, absorption disorders in thegastrointestinal system, and are applied externally in hemorrhage. Juice, syrup, and oil of the fruits have been usedas pain killer, to heal wounds, in ulcer and other diseases ofthe stomach, cancer, and as a metabolism regulator in traditional medicine. The freshly pressed juice is used in the treatment of colds, febrile conditions, and exhaustion. Oil from fruits and seeds is used in the treatment of eczema, lupus erythematosus, chronic wounds that are difficult to heal, inflammatory diseases, erosion of the cervix uteri, in the treatment of burns and frozen parts of the body. Also, its berry oil is reported to treat skin disease and thrombosis.Oil extracts are used externally in dermatologic diseases such as eczema, psoriasis, lupuserythematosus, and chronicdermatoses. In ophthalmology, they are used in the treatment of keratitis, trachoma, injuries or burns of eye lid, conjunctivitis.

Therapeutic uses with scientific support :

1. The alcoholic extracts of leaf, fruit and berries of seabuckthorn (SBT) showed antioxidative effect against chromium (VI), sodium nitroprusside, and hypoxia induced oxidative stress in albino rats.7-9
2. The aqueous extract of seabuckthorn (Hippophae rhamnoides L.) seeds showed antioxidant and antimicrobial activities.10
3. Sea buckthorn berry oil (SBO)andtotal flavones of Hippophae Rhamnoides L.(TFH) inhibited platelet aggregation.11,12
4. The methanolic extract of “seabuckthorn” (Hippophae rhamnoides L.) berries showed in vitro anti-mutagenic activity.13
5. In vitro anti-tumor activity conducted on isorhamnetin flavonol aglycone isolated from Hippophae rhamnoides L. against human hepatocellular carcinoma cells(BEL-7402).14
6. The leaf and berries extract of Seabuckthorn (SBT) showed anti-inflammatory activity.15-17
7. Sea buckthorn seed and pulp oils showed both preventive and curative effects against experimental gastric ulcers in rats.18
8. Sea buckthorn extract appraised the effect on liver fibrosis by significantly decreasing the serum levels of laminin(LN), hyaluronic acid(HA), collagen types III and IV, total bile acid (TBA). So, sea buckthorn may be a hopeful drug for prevention and treatment of liver fibrosis.19
9. The total flavones extracted from seed residues of Hippophae rhamnosides L. showed antihypertensive effect on sucrose-fed rats.20
10. Effects of total flavones of Hippophae rhamnoids L.(TFH)performed in healthy human subjects, showed improvement of myocardial contractility, strengthened cardiac pump function, decreased peripheral vessel resistance and increased vessel elasticity.21
11. The seed oil, total flavones, procyanidins and leaf extract of seabuckthorn (Hippophae rhamnoides) againsthypoxia induced cerebral vascular injury, stress of patellar tendon in a rat model, acetic acid-induced lesions in the rat stomach and cutaneous excision-punch wound model respectively, promoted wound healingwhich might be due to increased antioxidant levels in the granulation tissue.22-25
12. Theflavonoid from seed residue of Hippophae rhamnoside L.(FSH) and fruit residue of Hippophae rhamnoside L.(FFH) decreased the levels of blood glucose and lipid in normal mice, and their effect on glycometabolism might be related to the control of glyconeogenesis.5
13. Seabuckthorn berries showed protection against radiation induced mitochondrial and genomic DNA from radiation-induced damage. The herbal preparation of Hippophae rhamnoidesshowed radioprotective effectagainst whole body lethal Irradiation in mice.26-28
14. Effect of dietary supplementation with sea buckthorn (Hippophae rhamnoides) seed and pulp oils was performed on fatty acid composition of skin glycerol phospholipids of patients with atopic dermatitis.29
15. The seabuckthorn seed oil offered a protection against sulfur dioxide induced injury to organs such as liver, lung, kidney and spleen.30

Enclosure – III

6.3 Aim and Objectives of the study:-

AIM:

The present study is intended to investigate the antidiabetic and antiatherogenic effects of methnolic extract of seeds of Hippophae rhamnoside Linn.(MESH)

OBJECTIVES:

1.To collect and prepare of the MESH.

2. To perform the Preliminary Phytochemical screening of MESH.

3. To perform the acute toxicity study of MESH.

4. To study the influence of MESHon glucosetolerance of normal rats.

5. To study the antidiabetic andantiatherogenic effects of MESHby estimating the following parameters.

• Serum Blood glucose

• SerumTotal cholesterol
• Serum HDL cholesterol
• Serum LDL cholesterol
• Serum Triglycerides
• Liver glycogen
• Liver Glucose-6-phosphatase
• Liver Hexokinase
• Serum Malondialdehyde

Enclosure – IV

7. Materials and Methods:-

7.1 Sources of data:-

The sources of data will be obtained from:-

1. Laboratory based studies.
2. National and international journals.
3. Internet.

Enclosure – V

7.2 Method of collection of data(including sampling procedure if any):-

Field and laboratory studies:

Field work:

The seeds of the Hippophae rhamnoside L.will be collected, and it will be authenticated.

Laboratory work:-

Animals

Male albino wistar Rats (150-250 g) will be selected for all the experiments. They will be housed in cages on a 12-h light:12-h dark cycle and will be allowed free access to laboratory diet.

Solvents used

Methanol.

Chemicals and reagents used

Alloxan, Gluburide, Glucose, 80% Ethanol, Perchloric acid, Anthrone reagent, Sucrose/EDTA buffer, Glucose-6-phosphate, Imidazole buffer, TCA:ascorbate buffer, Ammonium molybdate, Sodium citrate, Tris-magnesium chloride buffer, Glucose-6-phosphate dehydrogenase, Commercially available kits.

Data analysis

Statistical analysis of the data will be evaluated by analysis of variance(ANOVA) followed by dunnett’s test.

Methodology:-

(1)Preparation of the methanolic extract of seeds of hippophae rhamnosides L.(MESH):31

Dried seeds of Hippophae rhamnoides L. will be powdered and successively extracted in a Soxhlet extractor for 8 hours with methanol. The extract will be used to study the antidiabetic and antiatherogenic effects of seeds of Hippophae rhamnoside L.

(2)Phytochemical analysis:32

Phytochemical constituents will be detected by phytochemical tests.

(3)Pharmacological study design:-

1. Acute toxicity study of MESH:-

Acute toxicity study will be conducted according to OECD guidelines 425.

1. Influence of MESH on oral glucose tolerance:-33

The animals will be fasted overnight before commencing the experiment.

Overnight fasted Rats (18 h) will be divided into four groups of six rats each.

Group I: Glucose (2.5 g/kg, p.o.)

Group II: Glyburide (10 mg/kg, p.o.)

Group III: 1/3rd of LD50 of extract

Group IV :1/7th of LD50 of extract

All rats will be loaded with 2.5 g/kg, p.o., glucose solution after 30 minutes of drug administration.

Blood samples will be collected by the retro-orbital puncture (ROP) method just prior to drug administration and 30, 60, and 120 min after glucose loading.

Serum glucose level will be measured.

1. Influence of MESH on serum glucose in alloxan-induced diabetic rats:-33

Induction of experimental diabetes:1

Healthy adult Wistar male ratswill be injected with a single intraperitoneal injection of 120 mg/kg of alloxan. Alloxanic rats will be used when the elevated glucose level in plasma will be effective and permanent.

Overnight fasted diabetic rats will be divided into five groups of six rats each.

Group IV: Normal control group

Group V:Diabetic control group

Group VI:Diabetic group receiving Glyburide (10 mg/kg, p.o.)

Group VII:Diabetic group receiving 1/3rd of LD50 of MESH

Group VIII: Diabetic group receiving 1/7th of LD50 of MESH.

Study involves administration of vehicle, glyburide, or different doses of MESHfor a period of 28 days. Serum glucose levels will be estimated on days 7, 14, 21, and 28. Mean change in serum glucose will be calculated.

On completion of the treatment, blood sample will be collected and the following parameters for all the groups of animals will be measured:34

• Total cholesterol
• HDL cholesterol
• Triglycerides
• LDL cholesterol

For the determination of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol, the following Friedwald’s formula will be used.

VLDL cholesterol = triglyceride/5

LDL cholesterol = Total cholesterol - (VLDL + HDL cholesterol)

Animals will be sacrificed and liver will be dissected out and will be used for biochemical assays of the following parameters:34

• Glycogen level
• Glucose-6-phosphatase level
• Hexokinase level

Glycogen level:

Homogenize the collected liver separately in warm ethanol at a concentration of 100 mg/ml and then centrifuge at 10,000 rpm for 20 min.Collect the residue and dried in a boiling water bath. To each residue, add distilled water and perchloric acid. Carry out the extraction at low temperature for 20 min.Centrifugethe collected extract at 10,000 rpm for 15 min and transfer the supernant to a test tube and make up the volume up to 1 ml by addition of distilled water. To each tube, add anthrone reagent and incubate the whole at 95°C in a boiling water bath for 10 min. Measure the absorbance of the samples at 630 nm after cooling the tubes to room temperature. Express the amount of glycogen in tissue samples as microgrammes of glucose per milligramme of tissue.

Glucose-6-phosphatase level:

Homogenize the collected liver in ice-cold sucrose solution (250 mM). To sucrose/EDTA buffer, add 100 mM glucose-6-phosphate, imidazole buffer (100 mM, pH 6.5) and liver homogenate with thorough mixing. Incubate the tubes at 37°Cfor 15 min. Add TCA:ascorbate (10%:3%, w/v), and centrifuge the solution at 3000 rpm for 10 min. To the clear supernatant, add ammonium molybdate (1%, w/v) and sodium citrate (2%, w/v) and measurethe absorbance at 700 nm. Express the enzyme activity as units per grammeof wet tissue.

Hexokinase level :

Prepare the excised liver tissue homogenate in normal saline. To homogenate, add Tris–magnesium chloride buffer (200 mM Tris and 20 M MgCl2, pH 8.0), along with 0.67 M glucose, 16 mM ATP, 6.8 mM NAD and 300 U/ml glucose-6-phospate dehydrogenase solution. Mix the solution thoroughly, and measurethe absorbance at 340 nm after 5 min of incubation. Express the activity as units per gramme of wet tissue.

1. Influence of MESH on atherogenesis:-2

The rats will be divided into five groups VIII, IX, X, XI, XII of 6 rats each.The groups IX, X, XI and XII will receive atherogenic diet daily for a period of 30 days.

Group VIII: Normal

Group IX: Control (atherogenic diet alone)

Group X: Lovastatin (5 mg/kg)

Group XI :1/3rd of LD50 of extract

GroupXII:1/7th of LD50 of extract

At the end of the experimental period, all the animals will be fasted overnight and sacrificed. The following biochemical parameters will be estimated in serum:

• Total cholesterol
• HDL cholesterol
• LDL cholesterol
• Triglycerides
• Malondialdehyde

Malondialdehyde level:36

The assay mixture consists of serum 0.9% NaCl, 3% of sodium dodecylsulfate, thiobarbituric acid reagent and heat for 75 min at 95°C. Thereafter, add the mixture to cold 0.9% NaCl and cool in tap water and extract by adding n-butanol. After centrifugation at 3000 rpm for 15 min, the butanol phase will be assayed spectrophotometrically at 532 nm. Amounts of 0, 20, 40, 60 and 80 nmol of tetramethoxypropane serve as the external standard and will be assayed in the previously described fashion. Serum cholesterol will be expressed as nmol/ml.