Antiulcer Activity of 50% Hydroethanolic Leaf Extract of Ruellia tuberosa l. and Dipteracanthus patulus (Jaca) On Pylorus Ligated  Gastric Ulcer Model

 

A Manikandan and D Victor Arokia Doss*

Department of Biochemistry, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India.

 

 

ABSTRACT

The antiulcer activity of 50% hydroethanolic leaf extract of Ruellia tuberose  L.  and Dipteracanthus patulus (Jaca) was evaluated in rats against pylorus ligated gastric ulcer model. The plant extract were administered orally at a dose of 500mg/kg and famotidine at the dose of 20 mg/kg (standard drug). Ulcer index was common parameter studied in the ulcer model, further the gastric pH, total and free acidity, gastric volume and antioxidant leaves such as SOD, CAT, GSH and the effect on LPO and protein were analysed. Both the extracts produced significant reduction in ulcer index, along with increase in the antioxidant enzyme and protein leaves as compared to control group and the lipid peroxidation was reduced in the treated and drug administered groups. Thus the plant extracts possess, significant antiulcer as well as antioxidant property.

 

KEYWORDS: Antiulcer, antioxidant, ulcer index,  Ruellia  tuberose .L,  Dipteracanthus patulus

 

INTRODUCTION

Peptic ulcer disease is one of the major health problem with multifactorial etiology. The gastric ulcer develops with increase in acid and the breakdown of mucosae defense (Konturek, 1985). Formation of ulcer is due to the loss of balance between gastro- protective and aggressive factors. The reduction in gastro-protective factors such as mucus, bicarbonate secretion and gastric mucosal blood flow and enhancement of aggressive factors, such as increase of acid/ pepsin secretion, H. pylori infection result in gastric ulceration (Miller, 1987; Ernst and gold, 2000). The initial step in the ulcer development is mucosal damage, which is due to the oxidative stress (OS) by reactive oxygen species (ROS), hypersecretion of hydrochloric acid through H+, K+ - ATPase action (Phull et al., 1995).

 

The ulceration can be controlled by stimulation of gastric mucin synthesis, increasing antioxidant levels in the stomach, scavenging of  ROS, inhibition of H+, K+-ATPase and Helicobacter pylori growth (Bandyopadhyay et al., 2002). The anti-secretory drugs such as omeprazole, lansoprazole, histamine, H2- receptor blockers – ranitidine, famotidine are being used to control acid secretion. These drugs produce potential adverse effect on human health (Wardum et al., 2005).

 

In traditional medicine of India, several plants have been employed to treat gastrointestinal disorders including gastric ulcers. Due to lack of side effects compared to synthetic drugs approximately 60% of the world’s population depends entirely on such natural medications (Sathyavathi et al., 1987). The percent study was undertaken to investigate the anti ulcer effect of the hydro-ethanolic extract of  Ruellia tuberosa. L (HERT) and Dipteracanthus patulus (Jaca) (HEDP) against pylorus ligated rats.

 

 


Table 1.  Effect of 50% hydroethanolic extracts of Ruellia tuberasa L. and Dipteracanthus patulus (Jaca) on total and free acidity, ulcer index, pH and volume  of gastric juice in pylorus ligated rats.

Groups

Total acidity

(m EN/L)

Free acidity

(m EN/L)

Ulcer index

pH

Volume of gastric juice (ml)

Ulcer control (Plyethelene glycol 5ml/kg bw)

96.7±7.51

76.6±5.3

35.3±2.02

1.5±0.80

1.81±0.08

Ulcer + Famotidine (20mg/kg bw)

28.9±1.52

16.1±0.83

10.5±0.87

4.0±0.97

1.00±0.06

Ulcer + 50% HERT (500mg/kg bw)

38.1±2.61a*

23.9±1.2a*

14.3±0.83a*

3.9±0.97a*

1.1±0.08a*

Ulcer + 50% HEDP (500mg/kg bw)

41.8±2.52b*

33.2±1.91b*

16.4±0.84b*

3.8±0.98b*

1.4±0.11b*

50% HERT and HEDP – Hydroethanolic extract of Ruellia tuberose L. and Dipteracanthus patulus (Jaca) respectively,

All values represent Means ± SD, n=6 in each group, p< 0.05 as compared to control (ulcer).

a – Group III Vs Group I ,  b – Group IV Vs Group I ,  * - significance

 

Table -2: Effect of 50% hydroethanolic extracts of Ruellia tuberosa L. and Dipteracanthus potulus (Jaca)  on SOD, CAT, GSH, protein and Lipid Peroxidation in pylrous ligated rats.

Groups

SOD (U/mg protein)

Catalase U/mg tissue)

Glutathione (n moles/mg)

Protein mg/g tissue

Lipid Peroxidation

(n moles / g tissue MDA)

Ulcer control

(Plyethelene glycol 5ml/kg bw)

16.63±0.84

13.27±0.82

115.1±0.36

1.48±0.09

1.28±0.10

Ulcer + Famotidine

(20mg/kg bw)

18.62±1.05

18.78±0.95

138.8±0.09

2.29±0.07

0.86±0.08

Ulcer + 50% HERT

(500mg/kg bw)

17.83±0.09a*

17.88±0.92a*

136.4±0.08a*

2.11±0.07a*

1.02±0.08a*

Ulcer + 50% HEDP

(500mg/kg bw)

17.0±0.06b*

16.50±0.85b*

126.9±0.08b*

2.02±0.08b*

1.20±0.07b*

 

 

 

 

 

 

 

 

 

 

 

50% HERT and HEDP – Hydroethanolic extract of Ruellia tuberosa L. and Dipeteracenthus patulus (Jaca) respectively, All values represent Means ± SD, n=6 in each group  p<0.05 as compared to control (ulcer). a – Group III Vs Group I, b – Group IV Vs Group I, * - significance

 

 


MATERIALS AND METHODS:

Collection of Plant Material:

Fresh leaves of Ruellia tuberose  L. and and Dipteracanthus patulus (Jaca) were collected from ABS (altogether Botanical species) medicinal plant garden, Karipatti, Salem, Tamil Nadu, India. The plant was identified by the  Botanical survey of India (BSI), Tamil Nadu Agriculture University (TNAU) (No: BSI / SC/ 5123/08-09 / Tech – 229and 182).

 

Extraction Procedure:

The fresh leaves collected were shade dried for five days and crushed into coarse powder. The powder thus obtained was cold macerated with 50% ethanol for 3 days. The suspension was filtered and residue was removed. Then it was evaporated to dryness in a rotary evaporator. Dark brown colored crystals were obtained. This residue was used for further studies.

 

Animals:

Male Wistar rats weighing 120-180g were fed with standard pellet diet (M/s Hindustan Lever foods, Bangalore, India) and were provided water ad libitum. Animals were housed under standard environmental conditions. All animal experiments were carried out at Periyar college of Pharmacy, Trichy, Tamilnadu, India (CPCSEA/265) and according to the guidelines of the Institutional Animals Ethics Committee. The experimental rats were divided into four groups with six animals each group. Group – I Ulcer control fed with 5ml/kg 2% w/v polyethylene glycol. Group – II Drug control administered with famotidine 20 mg/kg. Group – III (50% HERT) and Group IV (50% HEDP) were treated with plant extracts as a concentration of 500mg/kg.

 

Pylorous ligation – induced gastric ulceration:

Albino rats were fasted for 24 hours but water was allowed ad libitum prior to pylorus ligation. Under mild ether anesthesis, abdomen was opened by a small midline incision below the xiphoid process. The stomach was placed back carefully and the abdominal opening was closed by sutures. The drugs were administered orally 2 hours  pylorous ligation (Shay et al., 1945). The animals were sacrificed after 6 hours for observation of gastric lesions (Gupta et al., 1985). Samples of gastric juice were collected and centrifuged at 2500 g for 10 minutes to remove debris. Total acidity was assessed by titration against 0.01N NaOH to pH 7.0.

 

The freshly excised stomach samples were examined macroscopically for hemorrhagic lesions in the glandular mucosa. Immediately after the animals were sacrificed by decapitation, their stomach were dissected out, cut along the greater curvature and the mucosae were rinsed with cold normal saline to remove blood contaminant if any. Then the ulcer index was determined. (Shay et al., 1945). The gastric tissue samples were stored at -80°C until determination of Gultathione (GSH), LPO, Superoxide dismutase, catalase and protein activity.

 

Biochemical Estimations:

Tissue samples were homogenized in 10 volumes of ice cold 10% trichloro acetic acid (TCA) in an tissue homogenizer for 30 seconds. Homogenized tissue samples were centrifuged at 3000 rpm for 15 min at 40°C. Supernatant was used for the analysis.

 

The Supernatant obtained was used to determine the lipid peroxidation (Nichans and Samuelson, 1968). Glutathione (GSH) content was determined as described by Moron et al., 1979. The activity of superoxide dismutase (SOD) was assayed by measuring the reduction in the NBT in the presence of SOD (Kakkar et al., 1984) and catalase was assayed by the decomposition of H2O2 (Sinha, 1972). The protein content of the homogenate was determined by Lowry’s method (Lowry et al., 1951).

 

Statistical Analysis:

Results were expressed as means ± SD and were analysed for statistically significant difference using one-way analysis of variance (ANOVA). P value less than 0.05 was considered significant  use for statistical calculations.

RESULTS:

Pylorus ligation consistently caused lesions in the mucosa of the granular stomach, pretreatment of rats with 50% HERT and HEDP plant extracts reduced the gastric ulcerogenesis significantly and decreased the ulcer index values. Gastric acidity was significantly higher in the control group. Treatment with the plant extracts, caused a significant decrease in gastric acidity. Lipid peroxidation was found to be reduced in treated groups than the ulcer control. GSH, SOD, and catalase were found to be decreased in control, the drug and plant extract treated groups showed a significant increase in enzyme levels (Table 2). Protein levels improved in the treated groups compared to the control. The 50% hydroethanolic extract of Ruellia tuberosa  L. showed comparatively better result than 50% Hydroethanolic extract of Dipteracanthus patulus (Jaca).

 

DISCUSSION:

The etiology of peptic ulcer is unknown in most of the cases, it is generally accepted that it results from an imbalance between aggressive factors and the maintenance of mucosal integrity through the endogenous defense mechanisms (Piper and Stiel, 1986). To achieve the balance, different therapeutic agents are used to inhibit the gastric acid secretion and to boost the mucosal defense mechanisms by increasing mucus production, the surface epithelial cell stabilization and for interference with the prostaglandin synthesis (Dhuley, 1999).

 

Pylorus ligation induced ulcers are thought to be caused due to increased presence of acid and pepsin in the stomach. The essential criteria, which determine the status of mucosal defense barrier against the acid – pepsin is the quality and quantity of mucus secretion (Sanyal et al., 1983). Increased mucus production by the gastric mucosal cells can prevent gastric ulceration by several mechanisms.

 

The preliminary phytochemical studies revealed the presence of flavonoids, phenols in hydroethanolic extract of Ruellia tuberosa  L. and Dipteracanthus Patullus (Java); various flavonoids and phenolics have been reported for its anti-ulcerogenic action via antioxidant activity (Bhattacharya et al., 2007) with good levels of gastric protection (Alarcon et al., 1994, Parnar and Shikha Parnar, 1998). Thus the plant treated groups showed a considerable increase in the depleted levels of antioxidant enzymes. Such as SOD, CAT, GOD, GSH levels.

 

Measurement of the ulcer indices of the rats demonstrated that treatment with the plant extracts led to faster ulcer healing compared to control group. It decreased the ulcer progression and promoted healing of gastric lesions induced by pylorus ligation. It may be due to the presence of secondary metabolites in the plant extracts.

 

Mucosal damage can be easily produced by the generation of exogenous and endogenous active oxygen and free radicals (Naito et al., 1995). Increase in mucus production usually assists the healing process by protecting the ulcer crater against irritant stomach secretions (HCC and Pepsin), (Bemier and Florent, 1986). The antioxidant and free radical scavenging activity of these plant extracts were previously determined (data not shown), and it might be the factor  protecting the mucosal layer from the damage.

The mechanism of gastro-protective effect may be due to the reduction in the free radicals level and lipid peroxidation. Besides, presence of phyto-constituents in this plant like flavonoids, phenols, glycosides and terpenoids might be responsible for these actions. Future studies may be directed to identify the phytconstituents responsible for these pharmacological actions of Ruellia tuberosa L. and Dipteracanthus Patulus (Jaca).

 

REFERENCE:

1.       Alarcon, D.L., Martin, M.J and Motilva, V., J. Ethanopharma Col., 1994, 42, 161-170.

2.       Bandyopadhyay U, Biswas K, Chatterjee R, Bandyopadhyay D, Chattopadhyay I, Ganguly CK, Chakraborty T, Bhattacharya K, Banerjee RK. Gastroprotective effect of Neem (Azadirachta indica) bark extract: Possible involvement of H (+) – K(+) – ATPase inhibition and scavenging of hydroxyl radicals. Life sci 2002; 71: 2845-2865.

3.       Bemier, J.J and Florent, C.: les defences de J’ estomac, Recherche, 117, 614-621, 1986.

4.       Bhattacharya S.R., Chaudhuri, S. Chattopadhyay, and S.K. Bandyopadhyay, Healing properties of some Indian medicinal plants against indomethacin – induced gastric ulceration in rats, J. Cein. Biochem, Nutr, 41, 106-114, 2007.

5.       Dhuley J N, Protective effect of Rhina X, a herbal formation against physical and chemical factors induced gastric and duodenal ulcers in rats. Indian J Pharmacol 1999; 31: 128-32.

6.       Ernst PB, Gold BD. The disease spectrum of Hericobacter pylori: the immunopathogenesis of gastroduodenal ulcer and gastric cancer. Annu Rev Microbiol 2000; 54: 615-640.

7.       Gupta, M.B., Nath, R., Gupa, G.B and Bhargava, K.D., Cein. Experimental pharmacol, physiol, 1984, 12,61.

8.       Kakkar, P., Das. B and Viswanathan, P.N, 1984. A modified spectrophotometric assay of superoxide dismutase. Biophys, 21, 130-132.

9.       Konturek S.J. Gastric cytroprotection. S and J Gastroenterol 1985; 20: 543-53.

10.     Lowry .O.H., Rosebrough, N.J., Farr, A.L and Randall, R.J (1951) J. Biol Chem, 193-265.

11.     Miller TA. Mechanisms of stress – related mucosal damage, AMJ Med. 1987; 83:8-14.

12.     Moron, M.S., Depierre, J.N and Manner vik, V.C 1979, levels of glutathione, glutathione reductose and glutathione-s-transferase activity in rat lung and liver, Biochem. Biophy Acta., 582, 67-68.

13.     Naito, y., Yoshikawa. T., Matsuyama, K.Yagi, N., Arai, M., Nakamura, Y., Nishimura, S., Yoshida, N., and Kondo, M.: Effects of oxygen radical scavengers on the quality of gastric ulcer hearing in rats, J. Cein, Gstroenterol., 21, S82-86, 1995.

14.     Nichans, W.G and Samuelson N, B. 1968, formation of malonaldihyde from phospholipids arachidonate during microsomal lipid peroxidation, Eur.J. Biochem, 6, 126-130.

15.     Parmar, N.S and Shikha Parmar. Indian J. Physiol, Pharmacol., 1998, 48, 343-351.

16.     Phull PS, Green CJ, JAcyna MR. A radical view of the stomach: the role of oxygen – derived free radicals and anti-oxidants in gastro duodenal disease. Eur J Gasthroenterol Hepatol 1995; 7:265-275.

17.     Piper DW, Stiel DD. Pathogenesis of chronic peptic ulcer, current thinking and clinical implications. Med Prog 1986; 2:7-10.

18.     Sanyal AK, Mitra PK, Goel RK. A modified method to estimate dissolved mucosubetanees in gastric juice. Ind J. Exp. Biol 1983: 21:78-80.

19.     Sathyavathi GV, Gupta AK, Tandon N. Medicinal plants of India. New Delhi, India, Indian Council of Medical Research (ICMR) 1987:2.

20.     Shay, H. Kamorow, S.A., Fele, S.S., Meranz., D., Gruensteinh and Siplet, H., gastroenterology, 1945, vol.5, p.43.

21.     Sinha A.K., 1972. Colorimetric Assay of Catalase. Anal. Biochem, 47, 389-392.

22.     Waldum H7, Gasto Fsson B, Foss mark R, Quigstad G. Antiulcer drugs and gastric cancer. Dig Dis SCT 2005; 50 Suppl 1: S39-S44.

 

 

Received on 06.09.2009

Accepted on 11.10.2009     

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 1(3): Nov. – Dec. 2009, 198-200