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

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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

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

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

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

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

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

• 
  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.
  

IISc library, Bangalore.

PESCP library, Bangalore

RGUHS digital library(Helinet), Bangalore

The whole study is divided in to following phases:

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.

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:

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

1. 
   Pylorus ligation induced ulcer model
   
2. 
   Indomethacin induced ulcer model
   
3. 
   Cold stress induced ulcer model
   

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.

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.}

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.

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

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.

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

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.

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

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

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

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

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.

Asst.Professor

Department of Pharmacology

P.E.S college of Pharmacy

Bengalooru-560050

Asst.Professor & HOD

Department of Pharmacology

P.E.S college of Pharmacy

Bengalooru-560050.

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Principal & Director

PES College of Pharmacy

Bangalore-560050.