Protective Effect of Hydrolea zeylanica Vahl. Leaf extract in Ethanol and Cold Restraint Stress Induced Ulcers in Rats

 

Mohammad Shamim Qureshi1*, A. Venkateshwar Reddy2, G.S. Kumar3

1R & D cell, Jawaharlal Nehru Technological University, Kukatpally Hyderabad, - 50035, Telangana, India

2Anwarul Uloom College of Pharmacy, New Mallepally, Hyderabad – 500001, Telangana, India

3School of Pharmacy, International Medical University, Bukit Jallil, Kuala Lumpur 57000. Malaysia

*Corresponding Author E-mail: sports78@gmail.com

 

ABSTRACT:

The present study was designed to investigate the Antiulcer activity of methanolic extract of the leaves of Hydrolea zeylanica (L) Vahl using two different models namely Ethanol and cold restraint stress induced ulcers models in rats. The extract was administered at a dose of 250 and 500 mg/kg orally 30 min prior to ulcer induction. Ranitidine (50 mg/kg) was used as a reference standard. The antiulcer activity was accessed by determining and comparing the ulcer index in the test group with that of the standard group. Gastric volume, total acid and free acid were estimated in rats. Hydrolea zeylanica leaf methanolic extract (HZLME) (500 mg/kg) showed maximum inhibition of gastric acid, free acid and total acid respectively. The ulcer index in the Hydrolea zeylanica leaf methanolic extract treated animals was found to be significantly less in all the models compared to standard drug treated group. However the antiulcer activity of Hydrolea zeylanica leaf methanolic extract was, found to be lesser than that of ranitidine. The results suggest that Hydrolea zeylanica possesses significant antiulcer property which could be due to cytoprotective action of the drug or strengthening of gastric and duodenal mucosa with an enhancement of mucosal defence mechanism.

 

KEYWORDS: Hydrolea zeylanica, Cold Restraint Stress, Total Acidity, Ulcer Index.

 

 


INTRODUCTION:

Peptic ulcer is a gastro intestinal disorder due to an imbalance between the disruptive factors like acid, pepsin, Helicobacter pylori and defensive factors like prostaglandins, bicarbonate secretion, gastric mucus, and innate resistance of the mucosal cell factors[1]. Generally peptic ulcer develops when disruptive factors prevail over the defensive factors[2]. The major factors that interfere  the equilibrium between disruptive factors and defensive factors are Helicobacter pylori, acid–pepsin hyper secretion, Non-steroidal Anti-inflammatory drugs (NSAIDs), sometimes idiopathic due to usage of tobacco, psychological stress, rapid gastric emptying and Zollinger-Ellisson syndrome where there is a high and uncontrollable production of acid which also leads to ulcer formation[3,4].

 

Synthetic drugs such as proton pump inhibitors, cytoprotectants, H2 receptors antagonist, demulcents, Anti cholinergics, antacids and prostaglandin analogues are used for the cure of ulceration but these drugs produce several adverse effects. So herbal medicines are considered as better alternatives for the treatment of peptic ulcer[5]. For example, proton pump inhibitors (Omeprazole, Lansoprazole) may cause constipation, nausea, abdominal pain, diarrhoea and H2 receptor antagonists (cimetidine) may cause gynaecomastia, loss of libido. Due to the occurrence of many side effects by use of synthetic drugs for many diseases, medicinal plants are regarded as the main source of new drugs as they have less or no adverse effects. As herbal medicines are considered as safe for the cure of ulcers with lesser adverse effects, economical, effective, relatively less toxic, extensive research is carried out in search for potent antiulcer agents of plant origin[6,7]. Hydrolea zeylanica (L) Vahl, family Hydrophyllaceae is found throughout India in moist and swampy places[8]. It is an annual herb with procumbent and branching stems up to 30 cm. long. The leaves are dark green, narrow and pointed at the tip and are arranged alternately on the swollen, spongy stems. The stems growing above water are firmer and sturdier[9]. It is also known as Koliary and used for Antiseptic[10], Anthelmintic[11] and Antidiabetic activity[12].

 

MATERIAL AND METHODS:

Plant Material

The leaves of Hydrolea zeylanica (L) belonging to the family Hydrophyllaceae were collected from local area of Chittoor district (India). The plant was identified and authenticated by Dr. K. Madhava Chetty, Plant taxonomist (IAAT:357), Department of botany, Sri Venkateswara University Tirupati. A.P. India. The plant bearing voucher No. 1012 (10/12/2014) was deposited at Department of botany Sri Venkateswara University Tirupati, A.P.

 

Chemicals

Mayers reagent, Dragendroffs reagent, ferric chloride, Ammonia solution, chloroform and distilled water were procured from the central drug store, Anwarul Uloom College of Pharmacy, New Mallepally, Hyderabad, India. The solvents (Analytical grade) used for extraction were obtained from SD fine chemicals. Standard drug ranitidine was procured from Mercury medicare, Hyderabad, India.

 

Preparation of extracts

The leaves of Hydrolea zeylanica (L) were shade dried and reduced to coarse powder in a mechanical grinder. The powdered material obtained was then subjected to successive extraction by Hot Percolation Method using petroleum ether, chloroform, and methanol solvents in a soxhelet extractor[13].  The different extracts obtained were evaporated using a rotary evaporator to obtained semisolid mass. The extracts thus obtained were subjected to phytochemical screening and the Hydrolea zeylanica leaf methanolic extract (HZLME) was used for further studies.

 

Experimental Animals

Swiss albino mice weighing 20-30gm were used to assess acute toxicity whereas adult male wistar rats weighing 150-200 gm were used to evaluate Anti-ulcer activity by Ethanol induced and Cold Restraint Stress induced ulcers models. The animals were maintained under standard laboratory conditions in polypropylene cages under 12 hr light/dark cycle, controlled temperature (24 ± 2°C), fed with commercial pellet diet (Nutrivet life science) and water ad-libitum in an animal house approved by Committee for the Purpose and Supervision on Experiments on Animals (Reg no. 1534/ PO/a/11/CPCSEA.). All the animals were acclimatized to the laboratory environment for 10 days before commencement of the experiments. The experimental protocol was approved by Institutional Animal Ethical Committee (IAEC) No.-IAEC/AUCOP/2016/01, Anwarul Uloom College of Pharmacy, New Mallepally, Hyderabad, Telangana, India.

 

Preliminary Phytochemical Screening

The qualitative chemical tests were carried out for the identification of the different phytoconstituents present in the HZLME using standard procedures. They are usually tested for the presence of alkaloids, flavonoids, phenols, tannins, glycosides, triterpenes, steroids and saponins[14,15].

 

Acute Toxicity Study

Acute oral toxicity study was performed as per OECD- 423 guidelines category IV (acute toxic class method,). Albino rats (n = 3) of either sex selected by random sampling technique were employed in this study. The animals were kept fasting for 4 h with free access to water only. HZLME was administered orally with maximum dose of 2000 mg /kg body weight by gastric intubation. The mortality was observed for three days. If mortality was observed in 2 out of 3 animals or 3 out of 3 animals then the dose administered was assigned as toxic dose. If mortality was observed in 1 animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed then the procedure was repeated for further higher dose such as 3000 mg/kg of body weight[16].

 

Evaluation of anti-ulcer activity

The anti-ulcer activity was determined by two experimental models namely ethanol-induced gastric ulcer and cold restraint stress-induced ulcer.

 

Ethanol - Induced Gastric Ulcer Model

The animals were divided into five groups of six rats each. Group I normal control(Distilled water), Group II (negative control) received absolute ethanol (1 ml/animal); Group III was treated with ranitidine (100 mg/kg); Groups IV and V were treated with HZLME 250 and 500 mg/kg, respectively[17]. The rats were fasted for 24 h and they received 1 ml of absolute ethanol orally. Standard and test drugs were administered orally 30 min before the administration of ethanol (1ml.). The animals were sacrificed after 1 h of ulcerogen administration, and their stomachs were excised and the gastric contents were aspirated. The gastric contents were subjected to centrifugation at 1000 rpm for 10 min for the determination of pH (digital pH meter) and ulcer index. The stomachs were washed with normal saline and kept in 10% formalin for histopathological          studies [18.19].

 

Ulcer index is determined as follows:

Ulcer index = 10/x, 

where “x” is total mucosal area/total ulcerated area.

 

Cold Restraint Stress-Induced Ulcers model

The ulcers were induced by subjecting the animals to cold restraint stress. The standard group received Ranitidine (50 mg/kgpo) and to the test groups HZLME 250 and 500 mg/kg was administered orally 30 minutes prior to subjecting the animals to cold stress. The animals were placed in a restraint cage and the cage was placed in cold water at a temperature of 2°C for 3 h. The animals were sacrificed after three hours, the gastric contents were collected and the gastric volume, pH, free acidity and total acidity were determined. The stomachs were excised, and the ulcer count was done[20, 21].

 

Collection and Measurement of Gastric juice

The stomachs were excised carefully by keeping the oesophageal end closed and opening along the greater curvature, luminal contents were removed. The gastric contents were collected and centrifuged at 1000 rpm for 10 min. The centrifuged samples were decanted and volume of gastric juice was noted.

 

Determination of pH of Gastric juice

1 ml of the supernatant liquid was diluted to 10 ml using distilled water. The pH of the solution was recorded by digital pH meter.

 

Estimation of Free and Total Acidities

One ml of gastric juice was pipetted into a 100 ml conical flask, 2 or 3 drops of Topfer’s reagent was added and titrated with 0.01 N Sodium hydroxide until all traces of red colour disappears and the colour of the solution turns to yellowish orange. The volume of alkali added was noted. This volume corresponds to free acidity. Then 2 or 3 drops of phenolphthalein solution was added and the titration was continued until a definite red tinge appears. Again the total volume of alkali added was noted. This volume corresponds to total acidity. The total Acidity was calculated by using the formula[22-28].

 

                 Vol. of NaOH × N × 100 mEq/L

Acidity   =-----------------------------------------------

                                                  0.1

 

Assessment of Ulcer Index

Mean ulcer score for each animal is expressed as Ulcer Index. The stomachs were washed in running water to detect ulcers in the glandular portion of the stomach. The numbers of ulcers per stomach were noted and severity scoring was done microscopically with the help of hand lens (10X) and scoring method was done as per  Kulkarni [29].

 

0 = Normal Stomach                                      

0.05 = Red Colouration

1 = Spot Ulcers  

1.5 = Haemorraghic Streaks

2 = Ulcers > 3 mm but < 5 mm

3 = Ulcers > 5 mm

 

Histopathological Evaluation

The stomach from all groups of animal were removed rapidly, opened along the greater curvature, and thoroughly rinsed with ice-cold saline. After recording the ulcers produced in the stomach, a longitudinal section of the gastric tissue was taken from the anterior part of the stomach and fixed in a 10% formalin solution. After 24 h of fixation followed by embedding in a paraffin block, it was cut into sections of 5 micron onto a glass slide and stained with hematoxylin-eosin for histological assessment of the gastric mucosa according to Bancroft et al[30].

 

Statistical Analysis

The values are expressed as Mean ± Standard Error of Mean. P< 0.01 was considered significant and is denoted as **. Data of gastric juice, pH and Ulcer score was analysed by One-way Analysis of Variance whereas free and total acidities were analyzed by Kruskal – Wallis test (non-parametric ANOVA) followed by Dunnett’s multiple comparison post-hoc test using GraphPad Instat version 3.10 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com

 

RESULTS

The Preliminary Phytochemical screening carried out on HZLME revealed the presence of phytoconstituents such as glycosides, flavonoids, sterols, tannins, alkaloids and phenolic acids (Table 1). The extract did not produce any toxic symptoms of mortality up to the dose level of 5000 mg/kg body weight in the treated animals, and hence it was considered safe for further pharmacological screening. Effects of methanol extract of Hydrolea zeylanica on ulcer index induced by ethanol in rats are shown in Table 2 and Figure 1.  Hydrolea zeylanica showed a dose dependent curative ratio compared to ulcer control groups. The extracts exhibited an inhibition percentage of 43.41 and 76.29 at doses of 250 and 500 mg/kg doses respectively; and, the standard drug, ranitidine, exhibited an inhibition percentage of 82.56. The effects of methanolic extract of Hydrolea zeylanica on acid parameters showed significant (P<0.001) effect at 250 mg/kg and 500 mg/kg doses compared to ulcer control animals. The volume of acid secretion, total and free acidity was decreased and pH of the gastric juice was increased compared to ulcer control group. (Table 2)In water immersion stress induced ulcers, the mean score value of ulcer inhibition was found to be significant (P<0.001) for 250 mg/kg and 500 mg/kg of the extract(HZLME). The percentage ulcer inhibition was found to be 50.21 and 70.98 for 250 mg/kg and 500 mg/kg respectively, than that of the standard which was found to be 81.38. (Table 3 and figure 2)

 

Histopathological findings

A.   Negative control

Stomachs of the negative control group showed aggregates of inflammatory cells, mucosal ulceration, degenerated epithelial cells and necrosis. Severe oedemas, inflammatory infiltration, congestion in vascular space were observed in submucosa. The results of histopathological studies are shown in figure 1 (A).

 

B.   Standard

Stomach mucosa of rats treated with the standard drug (ranitidine) was found to be intact, while few scattered lymphocytes were present indicating inflammation. Thick arrow in Figure 1(B).

 

C.   HZLME 250mg/kg.

HZLME 250mg/kg treated group revealed gastric ulceration and haemorrhage in mucosa. Submucosa showed moderate oedema and few scattered inflammatory cells. A result is shown in figure 1 (C).

 

D.   HZLME 500mg/kg.

Mucosa of rats treated with HZLME 500mg/kg was found to be normal. Submucosa showed mild oedema and scattered inflammatory infiltration. A result is shown in figure 1 (d).

 

Table 1. Preliminary phytochemical screening of leaf of Hydrolea zeylanica.

Phytochemical Screening

Results

 

Test for glycosides

+

Test for flavonoids

+

Test for sterols

+

Test for saponins

-

Test for terpenoids

-

Test for alkaloids

+

Test for tannins

+

Test for phenolic acids

+

+ Presence of the constituents. -  Absence of the constituents.


 

Table 2. Effect of HZLME on gastric volume (mL), pH, Free and total acidity (mEq/L) and ulcer index on Ethanol induced ulcers in rats.

Groups

Free Acidity

Total acidity

Volume of Gastric juice (ml.)

pH of Gastric acid

Ulcer Score and % Ulcer protection

Group-I Control (Distilled water)

47.30±2.73

73.48±3.33

1.10±0.10

1.89±0.12

00.00±0.00

-

Group-II -ve control (Ethanol 1ml/kg)

68.12±2.42

64±1.00

6.11±0.10

2.11±0.07

3.91±0.44

-

Group-III Standard

(Ranitidine 50mg/kg)

26.11±2.33**

36.67±0.33*

4.03±0.05***

3.73±0.04***

0.68±0.12***

82.56 %

Group-IV Test-I 250mg/kg

(HZLME)

39.22±1.77*

61.33±1.22

4.95±0.07**

3.21±0.04**

2.21±0.16***

43.41 %

Group-V Test-II 500mg/kg

(HZLME)

30.64±0.74*

48.83±0.87*

4.68±0.06**

3.48±0.09***

0.88±0.15***

76.29 %

Note: Data is expressed as Mean ± SEM; n = 6 per group. * indicates p< 0.05, ** indicates p< 0.01 and *** indicates p< 0.001.

 

 

Table 3. Effect of HZLME on gastric volume (mL), pH, Free and total acidity (mEq/L) and ulcer index on Cold Restraint Stress-Induced Ulcers in rats.

Groups

Free Acidity

Total acidity

Volume of Gastric juice (ml.)

pH of Gastric acid

Ulcer Score and % Ulcer

protection

Group-I Control (Distilled water)

50.20±2.33

67.44±3.23

2.10±1.11

1.90±0.11

00.00±0.00

-

Group-II -ve control (Ethanol 1ml/kg)

72.11±0.10

67.5±1.60

3.63±0.05

2.13±0.07

3.85±0.25

-

Group-III Standard

(Ranitidine 50mg/kg)

29.21±2.31**

44.17±1.01**

1.9±0.08***

3.68±0.06***

0.78±0.12***

81.38 %

Group-IV Test-I 250mg/kg

(HZLME)

41.32±1.67*

50.83±1.16*

2.81±0.06**

3.03±0.04**

1.91±0.04*

50.21 %

Group-V Test-II 500mg/kg

(HZLME)

31.34±0.34*

47.17±1.16**

2.01±0.07**

3.33±0.04**

1.11±0.21**

70.98 %

Note: Data is expressed as Mean ± SEM; n = 6 per group. * indicates p< 0.05, ** indicates p< 0.01 and *** indicates p< 0.001.

 

 

(A)   - control

(B) Std. (Ranitidine) 50 mg/kg

 

 

(C)  HZLME 250mg/kg

(D) HZLME 500mg/kg

Figure 1. Histopathology of stomach of Ethanol-induced ulcer models.

 

 

 

 

 

(D) - Control

(E) HZLME 250mg/kg

(F) HZLME 500mg/kg

Figure 2. Histopathology of the Stomach of Cold Restraint Stress-Induced Ulcers models.

 


DISCUSSION:

Ethanol induced gastric damage showed gross mucosal lesion, including long hemorrhage bands and petechial lesion. Animals pretreated with methanol extract of Hydrolea zeylanica and standard drug ranitidine showed very mild lesions and sometimes no lesion at all, when compared to ulcer control group. Ethanol produces severe gastric hemorrhagic lesions. Ethanol increases super oxide anion and hydroxyl radical production and lipid peroxidation in the gastric mucosa. These and other reactive metabolites react with most of the cell components, changing their structures and functions, or contributing to other mechanisms that finally promote enhanced oxidative damage. Ethanol-induced gastric mucosal injury is associated with extensive damage to mucosal capillaries and increased vascular permeability[31]. Water immersion stress is one of the best models of stress in rats to induce ulcer. This model provides both physiological stress as well as emotional stress to the animal. In case of water immersion induced stress in rats, the leaf extract showed significant ulcer inhibition in a dose dependant manner. The phytoconstituents like flavonoids, tannins, terpenoids, and saponin have been reported in several anti-ulcer literatures as possible gastro protective agents. Flavonoids, tannins and triterpenes are among the cytoprotective active materials for which anti ulcerogenic efficacy has been extensively confirmed[32]. It is suggested that these compounds will be able to stimulate mucus, bicarbonate and prostaglandin secretion, and counteract with the deteriorating effects of reactive oxidants in gastrointestinal lumen[33]. Tannins may prevent ulcer development due to their protein precipitating and vasoconstriction effects. Their astringent action can help precipitating micro proteins on the ulcer site, thereby forming an impervious layer over the lining that hinders gut secretions and protects the underlying mucosa from toxins and other irritants[34-37]. Alkaloids and terpenoids prevent ulcers induced by stress. Similarly, the methanol extract of Hydrolea zeylanica leaf, showed the presence of flavonoids, glycosides, tannins, and alkaloids. These phytoconstituents present in the extract could be the possible potential Antiulcer agents involved in the prevention of gastric lesion in rats. Further, studies are under process to isolate the possible phytoconstituents responsible for the anti-ulcer activity.

 

CONCLUSION:

The present study showed that methanolic extract of leaves of Hydrolea zeylanica have shown significant effect on the ulcer induced by two Models such as ethanol and cold restraint stress induced ulcers models. The maximum ulcer protection of HZLME has been shown in the Ethanol induced models and significant effect was found at 500 mg/kg dose levels.

 

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Received on 02.12.2016          Modified on 19.12.2016

Accepted on 26.12.2016        © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(1): 49-54.

DOI: 10.5958/0974-360X.2017.00012.9