INTRODUCTION:

Diarrhoea is characterized by an increased frequency of bowel movements, watery stools and abdominal pain¹. It leads to cause malnutrition and deaths among children in the developing countries of the world today². According to the World Health Report, diarrhoea is the cause of 3.3% of all the deaths in the world. The worldwide distribution of diarrhoea accounts for more than 5-8 million deaths each year in children who were aged less than 5 years. The use of traditional medicine to combat the consequences of diarrhoea has been emphasized by the WHO in its diarrhoea control programme^3,4. Many synthetic chemicals are available for the treatment of diarrhoea, but they are not safe⁵.

It has been estimated that 50% of healthy individuals experience heartburns on a daily basis. The major cause of heart burn is peptic ulcer which occurs due to – stress, chronic use of non steroidal anti-inflammatory drugs, alcohol, cigarette smoking, genetic predisposition, and diet and Helicobacter pylori infection⁶. The current therapeutic approach to gastric ulceration is to achieve inhibition of gastric secretion, promotion of gastric protection, blockage of apoptosis, and epithelial cell proliferation for effective healing⁷.

In recent years, focus on plant research has increased worldwide and several studies had showed immense potential of medicinal plants⁸. Herbal medicines derived from plant extract, are increasingly being recognized in treating various clinical diseases, with relatively little knowledge of their modes of action⁹. Luffa aegyptiaca syn L.cylindrica (L.) fruit is commonly called sponge gourd, loofa, vegetable sponge, bath sponge or dish cloth gourd, is a member of cucurbitaceous family. The Plant is widely distributed throughout India. Its fruit is used in the traditional medicine as an anthelmintic, carminative, laxative, depurative, emollient, expectorant, and diuretic and lactagogue and are useful in fever, syphilis, tumours, bronchitis, splenopathy and leprosy¹⁰. It is used as a vegetable either prepared likes quash or eaten raw like cucumbers^{11, 12}. Its seeds have been used in the treatment of asthma, sinusitis and fever¹³.

The present study was aimed at evaluation of the antidiarreal and antiulcer activities of hydroalcoholic extract of Luffa aegyptiaca fruits in animal models.

MATERIALS AND METHODS:

Plant collection and authentication: The fruits Luffa aegyptiaca were collected from the surroundings of Nilgiris District, Tamilnadu. The plant was identified and authenticated by the field botanist Dr. S. Rajan, Survey of medicinal plants and collection unit (Central council for Research in homeopathy), Nilgiris District, Tamilnadu.

Extract Preparation:

The fruits were shade dried and made into a coarse powder which was passed through a 40-mesh sieve to get a uniform particle size and then used for extraction. A weighed quantity (500 g) of the powder was then subjected to continuous hot extraction in Soxhlet apparatus with hydroalcoholic solvent (ethanol 50% + water 50%) and the residual marc was collected. The extract was filtered through a cotton plug, followed by whatman filter paper no.1. The extract was evaporated under reduced pressure using a rotovac evaporator at a low temperature (40-60⁰C) until all the solvent had been removed to give an extract sample with a yield of 18% w/w, 16 % w/w and 13% w/w in relation to the dried starting material. Preliminary phytochemical analysis was carried out to identify the presence of phytoconstituents in the crude extract.

Phytochemical Screening:

The preliminary phytochemical investigation for fruit extracts of Luffa aegyptiaca was carried out in order to detect various phytoconstituents as per standard methods¹⁴.

Animals used:

Albino wistar rats of either sex weighing between 150 to 200 gm are used for the following activities. The animals were housed under standard conditions of temperature (25±2°C) and relative humidity (30-70%) with a 12:12 light-dark cycle. The animals were fed with standard pellet diet (VRK Nutrition, Pune) and water ad libitum. All animals experiment were carried out in accordance with the guidelines of CPCSEA (Reg No.1675/RO/c/12/CPSEA) and study was approved by the IAEC (Institutional Animal Ethical Commity) of Delve labs pvt ltd., with approval No.0010/13.

Anti-diarrheal activity:

Antidiarrheal activity was carried out by using Castor oil induced diarrhea method. In the present study, animals were divided into four groups of six rats each and fasted overnight before the test with free access to water. The animals were treated as follows:

Group I - Control + Castor oil (1ml)

Group II - Hydroalcoholic Extract of Luffa aegyptiaca fruits 250 mg/kg body weight + Castor oil (1ml)

Group III - Hydroalcoholic Extract of Luffa aegyptiaca fruits 500 mg/kg body weight + Castor oil (1ml)

Group IV - Standard drug (Diphenoxylate) 5 mg/kg body weight + Castor oil (1ml)

After 30 minutes of this treatment, each rat received 1 ml of castor oil orally. Each rat was then housed separately in perforated cage over a clean filter paper. . The numbers of wet feacal dropping were measured for six hours¹⁵.

Anti-ulcer activity:

Antiulcer activity was carried out by using Pylorus ligation method induced gastric ulcer. In this study animals were divided into 4 groups, each comprised 6 rats.

Group I - Control (normal saline)

Group II - Pyloric ligation

Group III - Pyloric ligation + Hydroalcoholic Extract of Luffa aegyptiaca fruits 500 mg/kg body weight

Group IV- Pyloric ligation + Standard drug (Famotidine 10 mg/kg)

Hydroalcoholic leaf Extract of Luffa aegyptiaca fruits (500 mg/kg) was administered for a period of 7 days. Group I were fed with saline solutions for 7 days, Group III were given extract 500mg for 7 days, Group IV were fed with standard famotidine for 7 days. Group II, III & IV were induced ulcers by Pyloric Ligation. On the 7th day normal saline, Famotidine and Hydroalcoholic extract of fruits were administered 1hr prior to pyloric ligation. After the pretreatment period of 1h animals were anaesthetized using pentobarbitone (35 mg/kg, i.p.), the abdomen was opened and pylorus ligation was done without causing any damage to its blood supply. The stomach was replaced carefully and the abdomen was closed in two layers with interrupted sutures. After 4 hours of pylorus ligation, stomachs were dissected out and cut open along the greater curvature and examined for ulcer index¹⁶. The gastric juice was titrated against 0.01N sodium hydroxide using Topfer’s reagent as indicator to find out the free acidity and total acidity¹⁷. Acidity was expressed as:

Acidity = Volume of NaOH x Normality x 100 mEq/1

0.1

The ulcer index was determined using the formula: Ulcer index =10/X

Where X = Total mucosal area / Total ulcerated area

STATISTICAL ANALYSIS:

All the biochemical results were expressed as mean + standard error of means (SEM). Data were analyzed by turkey’s range tests using sigma stat version- 3.5 software. A probability value of p < 0.05 was considered to be statistically significant.

RESULTS:

PHYTOCHEMICAL SCREENING:

Phytochemical Screening of Luffa aegyptiaca fruits shows the presence of Alkaloids, Saponins, Flavanoids, Diterpenes, Phenols, Tannins and Phytosterols as given in table 1.

Table No.1: Phytochemical Screening of Luffa aegyptiaca fruits Linn.

S.NO	Constituents	Results
1	Alkaloids	+
2	Saponins	+
3	Flavanoids	+
4	Diterpenes	+
5	Phenols	+
6	Tanins	+
7	Phytosterols	+
8	Proteins	-
9	Gums and Mucilage	-

+ = Presence - = Absence

ANTI DIARRHEAL ACTIVITY:

Diarrhea induced by Castor oil were significantly reduced by pretreatment with a dose of 250 mg/kg and 500 mg/kg Hydroalcoholic extract of Luffa aegyptiaca fruits , in a dose related manner when compared to control. The number of wet feaces at a dose of 5 mg/kg of diphenoxylate, 250 mg/kg and 500 mg/kg of the extract of Luffa aegyptiaca fruits were shown in table 2. The Percentage inhibition at a dose of 5 mg/kg of diphenoxylate, 250 mg/kg and 500 mg/kg of the extract of Luffa aegyptiaca fruits were 98.33, 59.25, 72.22 respectively. The percentage inhibition of hydroalcoholic extract (500mg/kg) was found to be closer to the reference standard drug diphenoxylate (5mg/kg) respectively.

Table 2: Effect of Hydroalcoholic extract of Luffa aegyptiaca fruits at different dose levels castor-oil induced diarrhea and their percent protection in rats

Groups	Treatment	No. of wet feaces	% Protection
Group I	Control+ Castor oil (1ml)	5.4 ± 0.24	-
Group II	Hydroalcoholic extract (250mg/kg)+ Castor oil (1ml)	3.20 ± 0.02	59.25
Group III	Hydroalcoholic extract (500mg/kg) + Castor oil (1ml)	1.50 ± 0.12	72.22
Group IV	Standard (Diphenoxylate)+ castor oil (1ml)	0.09 ± 0.36	98.33

Graph 1: Effect of Hydroalcoholic extract of Luffa aegyptiaca fruits at different dose levels castor-oil induced diarrhea (Number of wet feaces) in rats

Graph 2: Hydroalcoholic Extract of Luffa aegyptiaca fruits at different dose levels and their percent protection

The Ulcer Index of Hydroalcoholic Extract 500mg/kg were significantly reduced when compared to Pylorus Ligated rats. Ulcer Protective action at a dose of 500mg/kg of Hydroalcoholic Extract of Luffa aegyptiaca was found to be closer to the reference drug Famotidine (10mg/kg) respectively, ulcer index and percentage protection were given in table no.4.

Table No.4: The effect of Hydroalcoholic Extract of Luffa aegyptiaca on Ulcer Index and Percentage Protection

Treatment Groups	Ulcer index	% Protection
Group I	0.00 ± 0.00	0.00
Group II	7.54 ± 0.02	0.00
Group III	3.1 ± 0.01	82.6
Group IV	2.4 ± 0.02	95.8

Figure 1: Effect of Hydroalcoholic Extract of Luffa Aegyptiaca Fruits on Group I – Control

Figure 2: Effect of Hydroalcoholic Extract of Luffa Aegyptiaca Fruits on Group II– Pylorus Ligated

Figure 3: Hydroalcoholic Extract of Luffa Aegyptiaca fruits on Group Iii – Pylorus Ligated + Extract 500mg/Kg

Figure 4: Hydroalcoholic Extract of Luffa Aegyptiaca fruits on Group IV – Standard (Famotidine 12mg/Kg)

DISCUSSION:

Castor oil is an effective laxative. It decreases fluid absorption, increases secretion in the small intestines and colon and affects smooth muscle contractibility in the intestine. Castor oil produces diarrhea due to its active component recinoleic acid. Several mechanisms have been supposed to be involved in the antidiarrhoeal effect of castor oil¹⁸. These include inhibition of intestinal Na+, K+-ATpase activity to reduce normal fluid absorption. Activation of adenylate cyclase or mucosal cAMP-mediated active secretion, stimulation of prostaglandins formation, platelets-activating factor and most recently nitric oxide has been claimed to contribute to diarrheal of castor oil¹⁹. Despite the fact that numerous mechanisms have been proposed for the diarrheal effect of castor oil, it has not been possible to define its correct mechanism of action ²⁰.

Phytochemical screening revealed the presence of alkaloids, glycosides, sugars, terpenes and flavonoids. Earlier studies have shown that the anti-dysenteric and antidiarrhoeal properties of medicinal plants were due to the presence of tannins, alkaloids, saponins, flavonoids, sterols and/or triterpenes and reducing sugars^{21, 22}. Hence, tannins, sterols and/or tri terpenes may be responsible for the mechanism of action of the antidiarrhoeal activity of Hydroalcoholic Extract of Luffa aegyptiaca Fruits. This could be due to the fact that the extract increased the re-absorption of water by decreasing the intestinal motility, as was observed by the decrease in the intestinal transit in the charcoal meal test. The Present Study revealed that the hydroalcoholic extract of Luffa aegyptiaca fruits extract treated group showed less number of feacal droppings than the control group and the percentage protection of the hydroalcoholic extract of Luffa aegyptiaca extract treated group is better than the control. From the results it has been revealed that, pretreatment with 250 mg/kg and 500 mg/kg dose of Hydroalcoholic extract of Luffa aegyptiaca fruits had significantly produced antidiarrheal properties.

Peptic ulcer disease results from imbalance between aggressive factors and the maintenance of the mucosal integrity through endogenous defense mechanism⁶. Cyto-protective action by drugs has been considered to be due to the generation of prostaglandins or blockade of back diffusion of H+ ions ²³ will be the major mechanism which is responsible for antiulcer activity. It has also been reported that the presence of phytoconstituents tannins ²⁴, terpenoids ²⁵, sterols and flavonoids may be responsible for antiulcer activity ²⁶ which is in agreement with our findings. The hydroalcoholic extract of Luffa aegyptiaca fruits extract significantly reduced the gastric acid secretion. Recent reports and extensive literature review indicated that flavonoids and tannins showed cytoprotective action by increasing mucosal content of prostaglandins and mucous in gastric mucosa ²⁷.

CONCLUSION:

The results of phytochemical investigation of hydroalcoholic extract of Luffa aegyptiaca fruit shows the presence of alkaloids, saponins, flavanoids, diterpenes, phenols, tannins and phytosterols and biological screening results shows significant antidiarrhoeal and antiulcer effectiveness of the extract. Further research work can therefore be carried out in order to isolate bioactive molecules that are responsible for these biological activities for their development as novel antidiarrheal and antiulcer drugs.

REFERENCE:

1. Ezekwesili CN, Obiora KA, Ugwu OP. Evaluation of the anti-diarrhoeal property of the crude aqueous extract of Ocimum gratissimum L. (Labitae) in rats. Biokemistr. 16 (2): 2004; 122-31.

2. Victoria CG, Bryce J, Fontain O, Monsch R. Reducing deaths from diarrhea through oral rehydration therapy. Bulletin of World Health Organization. 78: 2000; 1246-55.

3. Inayathulla Shariff W R, Asit K, Sikarwar A, Mukesh S. Evaluation of the anti-diarrhoeal activity of the Crataeva nurrvala root bark in experimental animals. International Journal of Pharmacy and Pharmaceutical Sciences. 2: 2010; 158-61.

4. World Health Organization, World Health Report, WHO, Geneva. 2004; 120-25.

5. Hardman JG, Limberd LE. The Pharmacological Basis of Therapeutics. In: Goodman and Gillman’s (Eds), 10th edition, Macgraw Hill, New York. 1992; 914-31.

6. Amol N. Patil, Manjari G. Advani, S.N. Mali, Sudhir Pawar, Sanket B.Raut. “Evaluation of anti-ulcer effect of amlodipine in gastric ulcer models in rats” Indian journal of pharmacology. 44(3): 2012; 387-389.

7. Edwin S, Edwin J, Deb L, Goyal S, Gupta S. Anti-ulcer and Antioxidant activities of Psidium Guajava Linn. Indian Drugs. 44(5): 2007; 395-397.

8. Dahanurkar SA, Kulkarni RA, Rege RA. Pharmacology of medicinal plants and natural products. Indian J Pharmacol. 32: 2000; 81-118.

9. Begum N, Mayuren C, Balaji N, Chinnapa Reddy Y, Ara-vind Kumar K. Evaluation of Hepatoprotective activity of Aqueous extract of Curcuma Longa in carbon tetra chloride induced hepatotoxicity in Rats. Adv Pharmacol Toxicol. 9(3): 2008; 33-36.

10. Yoganandam GP, Ilango K, Kumar Sunil, Elumalai A. In-vitro antioxidant activity of L. cylindrica seeds oil. Journal of Global Pharma Technology. 2(3):2010; 93-97.

11. Yang Y, Ma X, Wu W, Guo P. Biological characters of the different varieties for Luffa cylindrica. Zhong Yao Cai. 22:1999; 165–167.

12. Oboh IO, Aluyor EO. Luffa cylindrica-an emerging cash crop. African J Agric Res. 4: 2009; 684–688.

13. Nagao T, Lanaka R, Iwase Y, Hanazone H, Okabe H. Studies on the constituents of Luffa acutangula Roxb. Clin Pharm Bull. 39: 1991; 599-606.

14. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 14 th ed. Nirali Prakashan; Pune. 2007. p. 297.

15. Venkateswara Rao Ch, Madhavan V, Sairam K, Vikas K. Antidiarrhoeal activity of the Cinnamomum tamala in experimental rats. J Nat Med. 62: 2008; 396-402.

16. H.Shay, S.A. Komarov, S.S. Fels, D. Meranze, M. Gruenstein, H. Siplet, Gastroenterology., 1945, 5^th edn. , 43–61.

17. S.K. Kulkarni, Handbook of Experimental pharmacology, Vallabh Prakashan, New Delhi, 1999, 3^rd edn., 148–150.

18. Izzo AA. Castor oil: An update on mechanism of action Phytotherapy research. 10: 1996; 109-111.

19. Mascolo N, Izzo AA, Ganginella TS and Capasso F.Relationship between nitric oxide and plateletactivating factor in castor oil induced mucosal injury in the rats’ duodenum. Naunyn Schmiedebergs Arch Pharmacology. 353:1996; 680-684.

20. Mascolo N, Izzo AA, Autore G, Barbato F and Capasso F. Nitric oxide and castor oil induced diarrhea. Journal of pharmacology and experimental therapeutics. 268: 1994; 291-295.

21. Galvez J, Zarzuelo A, Crespo ME, Utrilla MP, Jimenez J, Spiessens C, de Witte P. Anti-diarrhoeic activity of the Scleroarya birrea bark extract and its active tannin constituents in rats. Phytother Res. 5: 1991; 276-78.

22. Longanga Otshudi A, Vercruysse A, Foriers A. Contribution to the ethno botanical, phytochemical and pharmacological studies of traditionally used medicinal plants in the treatment of dysentery and diarrhea in the Lamella area, Democratic Republic of Congo (DRC). J. Ethanopharmacol. 71(3): 2000; 411-23.

23. De B, Maiti RN, Joshi VK, Agrawal VK Goel RK. Effect of some Sitavirya drugs on gastric secretion and ulceration. Ind J Expt Biol. 35:1997; 1084 - 1087.

24. Bodhankar SL, Jain BB, Ahire BP, Daude RB, Shitole PP. The effect of Rabeprazole and its isomers on aspirin and histamine-induced ulcers in rats. Indian J Pharmacol. 38(5): 2006; 357-358.

25. Patil VP, Viswanathswamy AHM, Thippeswamy AHM, Kamblekar YJ, Hallikeri CS, Hatapakki BC. Gastroprotective and Anti-ulcer properties of clozapine in pylorus ligated rats. Int J Pharmacol Biol Sci. 2(1): 2008; 121-126.

26. Mohammed A, Ravikumar J, Santosh HY, Nagashruthi MN. Anti-ulcer activity of Anisochilus carnosus leaf extract in pylorus ligated rats. Indian Drugs. 45(12): 2008; 979-981.

27. Dandagi PM, Patil MB, Mastiholimath VS, Gadad AP, Kul-karni A.R. Anti-ulcer activity of extracts of Calotropis Gi-gantea root extracts in pylorus ligated rat model. Int J Pharmacol Biol Sci. 2(1): 2008; 127-132.

Received on 04.08.2014 Modified on 18.08.2014

Res. J. Pharmacology & P’dynamics. 6(4): Oct. - Dec.2014; Page 184-189

Groups	Treatment	Gastric Volume (mL/100g)	Free acidity (mEq/100g)	Total acidity (mEq/100g)
Group I	Control	1.3 ± 0.43 ml	20.2 ± 0.45	45.1 ± 1.02
Group II	Pylorus Ligated	4.64 ± 0.11 ml	48.4 ± 1.56	98.4 ± 1.32
Group III	Ehanolic Extract (500mg/kg)	3.01 ± 0.2 ml	32.4 ± 0.56	66.4 ± 1.45
Group IV	Standard (famotidine 10mg/kg)	1.42 ± 0.4 ml	24.2 ± 1.32	47.6 ± 1.66