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Medicago sativa against gastric ulcer in rodents” synopsis for m. Pharm dissertation


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Evaluation of aqueous and alcoholic extracts of leaves of

Medicago sativa against gastric ulcer in rodents”

SYNOPSIS FOR

M. PHARM DISSERTATION


SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

SUBMITTED BY

REVATHI.GALI

I M. PHARM

DEPARTMENT OF PHARMACOLOGY

PES COLLEGE OF PHARMACY

BANGALORE– 560050

(2009-2011)


RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION




1.


NAME AND ADDRESS OF THE CANDIDATE.


REVATHI.GALI

LOCAL ADDRESS

P.E.S. COLLEGE OF PHARMACY

50 feet road, Hanumanthnagar,

B.S.K 1st stage, Bangalore – 560 050

PERMANENT ADDRESS

D/O.G.VenkateswaraRao,

H.NO:6-57/2A,

Hyderabad road,

Jaggayyapet-521175,

Krishna dist, Andhra Pradesh.




2.


NAME OF THE INSTITUTION.


P.E.S. COLLEGE OF PHARMACY

50 feet road, Hanumanthnagar,

B.S.K 1st stage, Bangalore – 560 050



3.


COURSE OF STUDY AND SUBJECT.


M.PHARM.

PHARMACOLOGY.



4.


DATE OF ADMISSION TO COURSE.


20th May2009


5.


TITLE OF THE TOPIC:
“Evaluation of aqueous and alcoholic extracts of leaves of

Medicago sativa against gastric ulcer in rodents





6. BRIEF RESUME OF THE INTENDED WORK
6.1 NEED FOR THE STUDY:
Gastric hyperacidity and gastro duodenal ulcer is a very common global problem today. The etiopathogenesis of peptic ulcer has changed from Schwartz dictum ‘No acid (gastric juice)-No ulcer’ to ‘No mucosal damage-No ulcers’.

Peptic ulcer occurs in that part of the gastrointestinal tract which is exposed to gastric acid and pepsin, i.e. the stomach and duodenum. The etiology of peptic ulcer is not clearly known, it results probably due to an imbalance between the aggressive (acid, pepsin and H.pylori) and the defensive (gastric mucus and bicarbonate secretion,prostaglandins,nitric oxide, innate resistant of the mucosal cells) factors.1

Hyper secretion of gastric acid is a pathological condition, which occurs due to uncontrolled secretion of hydrochloric acid from the parietal cells of the gastric mucosa through the proton pump H+ K+ ATPase. Even the normal rate of acid secretion may cause ulceration in the breached mucosa when some gastro protective factors are lost. Peptic ulcer disease is one of the most common gastrointestinal disorders, which causes a high rate of morbidity particularly for the population of non-industrialized countries.2

Several factors are implicated in the pathogenesis of gastric ulcer including: increased acid-pepsin secretion, impaired bicarbonate neutralization, impaired mucus secretion and precipitate lesions on the mucosal layer.3,4

Extracts of leaves and flowers of Medicago sativa are used to treats low cholesterol, diuretic, anti-inflammatory, anti fungal, treats anemia, bone and joint disorders, colon and digestive disorders, skin disorders and ulcers. It helps to reduce the effects of rheumatoid arthritis and other arthritic conditions. Medicago sativa tea can be useful in treating diabetes, stimulating appetite and for use as a general tonic The plant is a kidney tonic, prostatic tonic, reproductive tonic, musculoskeletal tonic, and glandular tonic. Medicago sativa has been one of the best herbal treatments for arthritis, gout and rheumatism. Now there is no scientific evidence for the evaluation of antiulcer activity of Medicago sativa. Hence present study is proposed to evaluate alcoholic and aqueous extracts of leaves of Medicago sativa for antiulcer activity.5

6.2 REVIEW OF THE LITERATURE:


PLANT PROFILE
Introduction

Medicago sativa is a species belonging to the family Fabaceae. Medicago sativa is commonly known as Lucerne or alfalfa, the plant species is native to central Asia (Armenia, Persia etc).There exists wild types in the Caucasus in the mountain regions of Afghanistan, Iran and adjacent regions.6

Synonym(s)

“Spanish clover”, “California clover”, “Chilean clover”, “Buffalo herb”, “Father of all foods” (Al-fal-fa), “Lucerne”, “ cultivated Lucerne”, “purple medic”, “purple medick”, “purple medicle”, “medicago”.Spanish: “alfalfa”. “medicago”. French: “Feuille de Luzern”6


Botanical description.6

Alternate trifoliate, with triangular –subulate stipules, dentate, the inferior third fused with the base of the petiole, up to 17mm long, the petiole is grooved,1cm to 6cm long. Three leaflets are present; the middle one is born from a petiolule longer than the lateral petiolules, 3mm to 6mm long. All the three leaflets are denticulate apical half or the apical third. The leaflets of the leaves are obviate or orbicular; the leaflets of the upper leaves are obanceolate to oblong, 1.5 to 3.5cm long by 0.5 to 2.5cm wide. 6


Ecology and distribution.6
History of cultivation
The plant species is native to central Asia (Armenia, Persia etc) 6


chemical constituents:7 -

Alkaloids (asparagine, trigonelline), amino acids, sugars, beta carotene, chlorophyll, cumarins, digestive enzymes, phytosterols, phytoestrogen, flavones, flavanoids, saponins, minerals( Ca, P, Mg, Ph, K, Fe, Zn, Cu, Al, Se, Si, Sa, Ni), vitamins(A, B1, B6, C, B12, D, E, K, niacin, folic acid).7


Uses:7-

Medicago sativa has many medicinal uses both where it is native and introduced. Extracts of bark, leaves and flowers have many uses such as anti-atherogenic, anti-bacterial against some gram-positive bacteria, anti-Cancer, anticholesterolemic, anti diabetic, anti-rheumatic, anti-spasmodic, appetizer, cardiotonic, diuretic, estrogenic, fever, lactagogue, nutritive, thyrotrophic-releasing hormone (TRH) activity, tonic.7
Traditional medicinal uses:8-

In traditional use Alfalfa Leaf to treat ulcers, arthritis pains and fluid retention8




Literature-

Extract of alfalfa seed (ethanolic 50 % v/v) prevents the development of plaque formation and hyperlipidaemia in cholesterol fed rabbits. It inhibits the elevation of serum total cholesterol, triglycerides, phospholipids, LDL-cholesterol and total cholesterol/phospholipid ratio, while HDL-cholesterol/total cholesterol ratio increases, which is associated with a reduced incidence of atherosclerosis.9


Alfalfa was reported to be hypocholesterolemic and antiatherosclerotic. Saponin glycosides were suggested to be responsible for this activity by neutralizing cholesterol in the stomach, enabling it to be excreted from the body. All alfalfa extracts possessed antiatherosclerotic activity as observed by the almost normalization of the aortic sections upon concomitant use of alfalfa extracts with cholesterol-enriched diet.10
Alfalfa (Medicago sativa) is a leguminous plant with high contents of phytoestrogen and saponin which are both useful in preventing cardiovascular disorders.11
Many medicinal plants have been used for the treatment of diabetes mellitus in Indian system of medicine and in other ancient systems of the world. Medicinal plants are significant source of biological compounds and many of the currently available biological compounds have been derived directly or indirectly from them. The major focus of this review is on anti-diabetic activity of various medicinal plants in which Medicago sativa is one. Incubation of glucose-responsive BRIN-BD11 cells with aqueous extract of Medicago sativa (Lucerene) showed dose dependent stimulation of insulin secretion at low glucose concentration. Administration of Lucerne in the diet (62.5g/kg) and drinking water (2.5 g/l) reduced the hyperglycaemia in streptozotocin diabetic mice.12
Fenugreek (Trigonella foenum graecum L.) seeds extract (FSE) and lucerne (Medicago sativa L.) extract (LDE) modulate post-challenge carbohydrate metabolism in type 2 diabetes animal model.

It seems that this effect mediated by enhancing insulin secretion is related only for LDE-treatment and observed only in n5-STZ rats.13




6.3 OBJECTIVE OF STUDY:-


  • Preparation of alcoholic and aqueous extracts of leaves of Medicago sativa.

  • Determination of LD50 of the extracts of Medicago sativa as per OECD guidelines.

  • Evaluation of antiulcer activity on the Medicago sativa extracts.


7. MATERIAL AND METHODS:-
SOURCE OF DATA:-
Whole work is planned to generate data from laboratory studies i.e. experiments are performed as described in references. Experimental studies in journals and in text books available with college and various institutions.

IISc library, Bangalore.

PESCP library, Bangalore

RGUHS digital library(Helinet), Bangalore


Web sites: www.sciencedirect.com

www.pubmed.com

www.google.com

www.ijp-online.com


7.1 PLAN OF WORK:

The whole study is divided in to following phases:


Phase I: Collection of plant material.
The leaves of the Medicago sativa plant will be collected from wild source and will be authentified. They will be subjected for alcoholic and aqueous extractions. The concentrated extract will be useful for our studies.
Phase II: Preparations of extracts.
Dried powder leaves will be successively extracted with alcohol (70%) and water in a soxhlet apparatus for 72 hours. The residue obtained after extraction is dried. The residue mass obtained is evaporated and reduce temperature to dryness. A % yield of all the two extracts will be determined.


Phase III: Determination of LD50 of the aqueous extract of Medicago sativa as per OECD guidelines.14

Female Swiss albino mice (18-20 g) are individually identified and allowed to acclimate to the laboratory condition for 7 days before the start of the study. Only one mouse receives single dose at a particular time. First animal receives a dose of 175 mg/kg and is observed for any toxicity signs, survival or death up to 48 hrs.. If the first animal died or appeared moribund, the second animal receives a lower dose (55mg/kg). The dose progression or reduction factor is 3.2 times of the previous dose. If no mortality is observed in the first animal then the second animal receives a higher dose (55 mg/kg). Dosing of the next animal is continued depending on the outcome of the previously dose for a fixed time interval (48 hours). The test is stopped when one of the stopping criteria is observed.

5 reversals occur in any 6 consecutive animals tested.


3 consecutive animals died at one dose level.
Survived animals are observed for long-term outcomes for a period of 14 days. The acute oral toxicity values are calculated using AOT 425 software (Environmental Protection Agency, USA) based on the short term (48 hours) and long term outcome (14 days).14
PHASE IV:

For determination of antiulcer activity the following experiments are to be performed.

All the models used in the pharmacological experiments will consist of the below 7 groups consisting of 6 animals in each group. Separate set of animals shall be used for individual test (or) models.

GROUP CLASSIFICATION:


TREATMENT

DRUG

DOSE

Group-1

Vehicle

-

Group-2

Control

-

Group-3

Omeprazole ( Standard )

10mg/kg

Group-4

Alcoholic extract of Medicago sativa

High dose

Group-5

Alcoholic extract of Medicago sativa

Low dose

Group -6

Aqueous extract of Medicago sativa

High dose

Group-7

Aqueous extract of Medicago sativa

Low dose

Evaluation of anti-ulcer activity will be done using following models:


ANTI-ULCER ACTIVITY


  1. Pylorus ligation induced ulcer model

  2. Indomethacin induced ulcer model

  3. Cold stress induced ulcer model


1.PYLORUS LIGATION INDUCED ULCER MODEL15,16

Forty two rats of either sex were randomly allotted to seven groups each consists of six animals. Group I served as the vehicle control; Group II served as the pylorus ligation control (4 h); Group III served as standard control; Group IV and V –alcoholic extract; and Group VI and VII-aqueous extract; were administered orally respectively by gavage using feeding needle. Albino rats of either sex weighing 150-200 g were used. After 18 hours of fasting, ulcer induction was undertaken. The rats were quickly and mildly anaesthetized with ether and the abdomen was cut open through a midline incision. The pylorus was secured and ligated with silk sutures, after which the wound was closed and the animal were allowed to recover from anesthesia. After ligation of the pylorus, drinking water was withheld and the gastric examinations were under-taken 19 hours after pylorus ligation. The animals were sacrificed with an overdose of ether and the stomachs were removed after clamping the esophagus. The gastric contents were collected

through the esophagus. The gastric juice was centrifuged and volume was recorded.15,16
Total acidity of gastric juice

An aliquot of 1ml of gastric juice was taken in to a 50 ml conical flask and two drops of phenolphthalein indicator was added and titrated with 0.01N NaOH until a permanent pink color was established. The volume of 0.01N NaOH consumed was noted. The total acidity was expressed as meq/l by the following formula: N × 0.01× 36.45 × 1000; which N is volume of NaOH consumed, 40.0 is molecular weight of NaOH. 0.01 is normality of NaOH and 1000 is the factor to be respected in liter.


Macroscopic evaluation of stomach

The stomachs were opened along the greater curvature, rinsed with saline to remove gastric contents and blood clots and examined by a ×5 magnifier lens to assess the formation of ulcer. The number of ulcers was counted. Ulcer scoring was undertaken.

The scores were: 0 = no ulcer, 1= superficial ulcer, 2= deep ulcer, 3= perforation. Ulcer area was assessed by using 3 M scaled surgical transpore tapes, which was fixed on a light and transparent sheet. Each cell on the tape was 1mm2 in area, so the number of cells was counted and the ulcer

Area was measured for each stomach. Ulcer index was measured by using following formula.

UI = UN + US + UP × 10-1

Which UI = ulcer index, UN = mean of ulcer number, US =mean of ulcer score, UP = ulcer probability (incidence %) for each group.15, 17.


2.INDOMETHACIN INDUCED ULCER MODEL17,18
Forty two rats of either sex were randomly allotted to seven groups each consists of six animals. Group I served as the vehicle control; Group II served as the indomethacin control; Group III served as standard control; Group IV and V –alcoholic extract; and Group VI and VII-aqueous extract; were administered orally respectively by gavage using feeding needle.

Albino rats of either sex weighing 150-200 g were used. The test drugs are administered orally in 0.1% Tween 80 solution 10 min prior to oral indomethacin in a dose of 20 mg/kg (4 mg/ml dissolved in 0.1%Tween 80solution). Six hours later, the rats are sacrificed in ether anesthesia and their stomachs removed.


Formol-saline (2% v/v) is then injected into the totally ligated stomachs for storage overnight. The next day, the stomachs are opened along the greater curvature, then washed in warm water, and

examined under a 3-fold magnifier.

The scores were: 0 = no ulcer, 1= superficial ulcer, 2= deep ulcer, 3= perforation. Ulcer area was assessed by using 3 M scaled surgical transpore tapes, which was fixed on a light and transparent sheet. Each cell on the tape was 1mm2 in area, so the number of cells was counted and the ulcer

area was measured for each stomach (15).Ulcer index was measured by using following

Formula.


UI = UN + US + UP × 10-1

Where UI = ulcer index, UN = mean of ulcer number, US =mean of ulcer score, UP = ulcer probability (incidence %) for each group.


3.COLD STRESS INDUCED ULCER MODEL17,18
Forty two rats of either sex were randomly allotted to seven groups each consists of six animals. Group I served as the vehicle control; Group II served as the cold stress control ; Group III served

as standard control; Group IV and V –alcoholic extract; and Group VI and VII-aqueous extract; were administered orally respectively by gavage using feeding needle.

Albino rats of either sex weighing 150-200 g were used. Rats were deprived of food, but not water, for about 18 h before the experiment. The experimental rats were immobilized by strapping the fore and hind limbs on a wooden plank and kept for 2 h, at temperature of 4-6 oC. Two hours later, the animals were sacrificed by cervical dislocation. Stomach were incised along the greater curvature and kept overnight in 10% formalin solution. Next day, the ulcers were examined under a magnifying lens.

The scores were: 0 = no ulcer, 1= superficial ulcer, 2= deep ulcer, 3= perforation. Ulcer area was assessed by using 3 M scaled surgical transpore tapes, which was fixed on a light and transparent sheet. Each cell on the tape was 1mm2 in area, so the number of cells was counted and the ulcer area was measured for each stomach Ulcer index was measured by using following formula.


UI = UN + US + UP × 10-1

Where UI = ulcer index, UN = mean of ulcer number, US =mean of ulcer score, UP = ulcer probability (incidence %) for each group.17,18



7.2 STATISTICAL ANALYSIS

Values will be expressed as mean ± SEM from 6 animals. Statistical difference in mean will be analyzed using one-way ANOVA (analysis of variance) following Turkey’s multiple comparison test p<0.05 will be considered statistically significant.



7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION TO BE CONDUCTED ON PATIENTS OR ANIMALS?

Yes, the entire experimental models require usage of laboratory animals.



7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN 7.3?

Yes ethical clearance has been obtained. (Copy enclosed).



8. References:

1. K D Tripathi. Essentials of MEDICAL PHARMACOLOGY. 5 ed; 2003.


2. Falk GW. Cecil essentials of medicine; 2001.

3. Kent-Lioyd K, Debas H. Physiology of the gastrointestinal tracts1994


4. Glavin GB, Szabo S. Experimental gastric mucosal injury. Laboratory models reveal mechanisms of pathogenesis and new therapeutic strategies. 1992;6: 825-31.
5. http://www.herbalremedies.com/alfalfa-information.html#2
6. http://herbalguides.com/guides/alfalfa
7. http://www.ann.com.au/herbs/Monographs/medicago.htm
8. http://www.viable-herbal.com/Singles/herbs/s188.htm
9. Dixit VP, Joshi S, Prabha Jain C. Prevention of aortic lesions and hyperlipidaemia by alfalfa seed extract in cholesterol fed rabbit. J Biosci. 1986; 10:251–6.
10. Khaleel AE, Mohamed Z G, Shohda A, Maraghy-EL., Hifnawy., Mohamed S, et al. Study of Hypocholesterolemic and Antiatherosclerotic Properties of Medicago sativa L. Cultivated in Egypt. Journal of Food and Drug Analysis. 2005; 13:212-8.
11. Asgary S, Moshtaghian J, Hosseini M, Siadat H. Effects of alfalfa on lipoproteins and fatty streak formation in Hypercholesterolemia rabbits. Pak J Pharm Sci. 2008; 21:460-4.
12. Dinesh Kumar B, Mitra A, Manjunatha M. In vitro and In vivo studies of Antidiabetic Indian medicinal plants. Journal of Herbal Medicine and Toxicology. 2009;3(2):9-14.
13. Winiarska H, Dworacka M, Borowska M, iewicz-Kozłowska TB, Gorecki P, Mścisz A, et al. The effects of plant extracts of Medicago sativa and Trigonella foenum-graecum on postprandial glucose levels in type 2 diabetic rats. herba polonica. 2007; 53.
14. 423 Acute Oral toxicity - Acute Toxic Class Method (Updated Guideline, adopted 20th December 2001)

15. Minaiyan M, Ghassemi D, Mohammadzadeh B. Anti-ulcer effect of Tripleurospermum disciforme (C.A. Mey) Shultz Bip on pylorus ligated (Shay) rats. Research in Pharmaceutical Sciences. 2006;1:15-21.


16. Shay H, Komarow S, Fels S, Meranze D, Gryenstein M, Siplet H. A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology. 1945; 5:43-61.
17. Vogel GH, Vogel WH, Scholkens BA, Sandow J, Muller G, Vogel WF. Pharmacological assays. In: Drug discovery and evaluation. . 2 ed; 2002.
18. Rao CV, Ojha SK, Radhakrishnan K, Govindarajan R, Rastogi S, Mehrotra S, et al. Antiulcer activity of Uteria salicifolia rhizome extract. . Journal of Ethno pharmacology 2004; 91:243-9.






SIGNATURE OF THE CANDIDATE




(REVATHI.GALI)







REMARKS OF THE GUIDE












NAME AND DESIGNATION OF THE

GUIDE
SIGNATURE




Mr. R. Srinath. .

Asst.Professor

Department of Pharmacology

P.E.S college of Pharmacy

Bengalooru-560050








CO-GUIDE

SIGNATURE




NOT APPLICABLE







HEAD OF THE DEPARTMENT
SIGNATURE




Mr. R. Srinath.

Asst.Professor & HOD

Department of Pharmacology

P.E.S college of Pharmacy

Bengalooru-560050.









REMARKS OF THE PRINCIPAL

FORWARDED FOR APPROVAL











SIGNATURE OF THE PRINCIPAL




Prof.Dr.S.Mohan

Principal & Director

PES College of Pharmacy

Bangalore-560050.







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