INTRODUCTION:

Plant derived substance have recently become of great interest owing to their versatile applications. Nearly 80% of world’s population relies on traditional medicine for primary health care most of which involve the use of plant extracts.¹

Macrotyloma uniflorum,the whole seeds of horse gram are also known as Kulthi bean, Madras gram. In India it is prescribed for the treatment of jaundice and water retention. In Kerala, Tamil Nadu, Maharashtra, South Canara, this herb is traditionally use for special dishes.

In Indo-Pak subcontinent, traditionally the seeds of Macrotyloma uniflorum are use as medicine for asthma, colic, diarrhoea, dysuria, kidney stones, leucorrhoea, obesity and splenomegaly.^2,3

Peptic ulcer can also be stated as stomach ulcer, is a break or pore formation in the stomach or duodenum lining. Various etiological factors like Helicobacter pylori⁴infection, non-steroidal anti-inflammatory drugs like NSAIDs, tobacco smoking, alcohol, stress are responsible for ulcer production^,5

Now a day’s ulceration constitutes a major problem in a vast developing country like India. According to the latest WHO data published in May, 2014, 0.96% or 85,487 deaths are occurring by Peptic ulcer disease. In case of ulceration, India get 26^th rank in the world for death rate, which is 9.12 per 100,000 population.⁶

Herbs stand always a good role in ulcer healing and increasing its popularity by time and age due to its fewer side effects and most cost effective nature. Few herbs are already established in the market for treating ulcer and its pain management like banana, cabbage and its juice, cayenne pepper, Indian gooseberry⁷, wild carrot⁸, curcumin⁹etc.

Macrotyloma uniflorum is used in colic from the ancient time in the folk medicine but there is no complete scientific data available. The gastro protective effect of phenolic acid present in the Macrotyloma uniflorum is proven by indomethacin induced and absolute alcohol induced anti ulcer model.¹⁰Hence here is an attempt to validate the ulcer protective activity of Macrotyloma uniflorum by using two different models such as pyloric ligation and stress induced anti-ulcer model scientifically.

MATERIALS AND METHODS:

Experimental animals:

Albino wistar rat weighing 150-200g either sex is used for the study in different models. The animals were purchased from authorised animal breeder 1 week prior to the study, so that animal could adapt to the new environment. Animals were well maintained under standard hygienic conditions, at a temperature (22±2ºC), 65% relative humidity (45-64%), and 12 hr light and dark cycle in NSCBIP animal house. Animals were housed in groups of 4 in polypropylene cages (32×24×16) cm along with commercial food pallets and tap water ad libitum. On every alternative day cages were cleaned. Paddy husk purchased from the local market, sterilised and used as bedding material, which was changed every day. The cages were maintained clean and all experiments were conducted between 10am to 6pm.

Extracts:

At first seeds were purchased from the market, and then these seeds were washed with fresh water. After washing these seeds were shed dried. Seeds were powdered by using mixture grinder. The coarse powder obtained from the seeds was subjected to soxhlet extraction for consecutive 48 hrs by using 70% hydro-alcoholic mixture.

Chemical and drugs:

Formalin, ethanol, sodium hydroxide (NaOH), hydrochloric acid (HCl), was obtained from LOBA CHEMIE PVT. LTD.Ranitidine (Rantac (R) 150mg by JB chemicals and pharmaceutical ltd.), and famotidine (Famocid 20mg by Sun pharmaceutical industries ltd.) were purchased from chemist. Di-ethyl ether was purchased from MERCK Specialities Pvt. Ltd, Mumbai.

Statistical analysis:

The data are expressed as Mean±SEM for each treatment group. The significance among the groups was assessed using one way analysis of variance (ANOVA) followed by Dunnet’s comparison test between the data of control and treated groups. p>0.05 were considered significant.

Preliminary Phytochemical Investigations of the Extracts:

Ethanol extracts of Macrotyloma uniflorum was evaluated for phytochemical investigation to check the presence of various phytoconstituents like alkaloid, carbohydrates, saponin, flavonoids, glycosides, tannins and proteins.^11,12,13

Experimental model:

i) Antiulcer activity by pyloric ligation:-

Albino wister Rats were divided into three groups, each group consisting of six animals. Animals were fasted for 24 h. One group received normal saline 2ml/kg (control), the second group received ranitidine 20mg/kg by oral route (standard) and the third group received alcoholic extract of M. uniflorum (150mg/kg) by oral route, 30 min prior to pyloric ligation. After 4h the animals were sacrificed and the gastric content was collected. The total volume of gastric content was measured. The gastric contents were centrifuged for 10 min at 1000rpm. The supernatant liquid was separated out and 1ml of this was diluted to 10ml with distilled water. The solution was titrated against 0.01N NaOH using phenolphthalein as indicator, to the endpoint when the solution turned to orange colour. Titration was further continued obtain pink colour. The volume of NaOH required was noted and was taken as corresponding to the total acidity^14,15,16. Acidity was expressed as:

Acidity = (Volume of NaOH × normality × 100)/0.1 m Eq/L¹⁴

ii) Stress induces antiulcer activity by water immersion model:

It has been shown that exposure of rats to resistant stress significantly increases gastric acid secretion for a few hours when the restraint animals are subjected to additional water immersion. The development of gastric acid secretion may be enhanced by exposure to water immersion.

The procedure for inducing ulcers with the water-immersion-stress-induced ulcer model, include animals being fasted for a period of 24 hours before the experiment. The animals were divided into three groups each containing 6 no of animals. One group received normal saline 2ml/kg (control), the second group received famotidine 20mg/kg by oral route (standard) and the third group received alcoholic extract of M. Uniflorum (150mg/kg) by oral route, after 30 min of the treatment the animals were placed individually in a restricted cage and immersing them vertically in water tank (20ºc-23ºc) for 4 hours. After 4 hours the animals were taken out from the water immersion cage and sacrificed. The stomach of each animal was isolated, mounted and the number of ulcer formation was counted for evaluation.^17,18

RESULT:

Extract Preparation:

The seeds of Macrotyloma uniflorum were subjected to soxhlet extraction by using 70% ethanol for consecutive 48 hours and 10% yield were obtained.

Preliminary phytochemical investigation:

The extracts obtained from the seeds of Macrotyloma uniflorum were subjected for preliminary investigation and followed observation are found.

Sl. No.	Test names	Results
1	Test for Alkaloid	Positive
2	Test for Carbohydrates	Positive
3	Test for Steroids,	Positive
4	Test for Saponins	Positive
5	Test for Tannins	Positive
6	Test for Protein and Amino acid	Positive
7	Cardiac glycoside	Positive

DISCUSSION:

Macrotyloma uniflorum, the whole seeds of horse gram are also known as Kulthi bean, Madras gram and used in various ailments in folklore medicinal system, is reported as anti-alopecic, anti-cirrhotic, anti-neuropathic, cholesterolytic, lipotropic and a sweetener. It has also been widely use in treatment of kidney stone, inflamed joints, fever, musculoskeletal disorders, sinus wounds, localize abdominal tumours, different ulcers and as a hepato- protective.

Though Macrotyloma uniflorum is traditionally used as a gastro protective and ulcer healing, there was always a need to reveal the anti ulcer potential of the same scientifically. Macrotyloma uniflorum has already shown potent antioxidant activity and a potent antioxidant is obvious to show the healing capability¹⁹ as per as general pharmacology concern.

Panda V. et. al studied the gastro-protective effects of the phenolic acids of Macrotyloma uniflorum (horse gram) on experimental gastric ulcer models in rats. This seeds are rich in phenolic acids, p-coumaric acid being the most abundant phenolic acid. The study established the antiulcer and antioxidant activity of the hydroalcoholic extract of the seeds of M. uniflorum and p-coumaric acid against indomethacin and absolute ethanol induced ulcers in rats.¹⁰

From the above study it is also proven that Macrotyloma uniflorum has significant antiulcer activity, but unfortunately the study has been done only on two selective models as alcohol induced antiulcer model and indomethacin induced antiulcer model but other few important models were not incorporated to evaluate the antiulcer properties.

In the present study there was an attempt to find out the antiulcer properties of Macrotyloma uniflorumin two different models like pyloric ligation model and stress induced antiulcer model, so here it may also validate the antiulcer activity of Macrotyloma uniflorumin stress related condition.

In the pyloric ligation model the untreated control has shown 4.3 mEq/L of acidity whereas the ranitidine treated standard group shown 2m Eq/L and M. uniflorum has shown 2.8 mEq/L acidity respectively.

In the stress induced antiulcer model the activity was more prominent, in the untreated control there was 26 no of sores were present whereas the standard group shown only 1 number of ulcer sore and in the Macrotyloma uniflorum treated group there was 5 number of ulcer sore present.

From the above findings it is established that Macrotyloma uniflorumis having significant antiulcer activity while compare with the untreated control group, this finding not only exactly correlate the previous antiulcer activity of Macrotyloma uniflorum done by other researchers, but also establish its potency against stress related ulcer condition.

ACKNOWLEDGMENTS:

It gives me great pleasure to acknowledge, with deep appreciation, to all those who have extended their cooperation throughout the period of my project work. I take this opportunity to express my deep gratitude to the principal, management and dept. of pharmacology, NSCBIP for guiding and helping me throughout this research.

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Received on 08.03.2019 Modified on 14.05.2019

Research J. Pharm. and Tech 2020; 13(2):853-856.

DOI: 10.5958/0974-360X.2020.00161.4