1. INTRODUCTION:

The free radicals are highly reactive chemical species produced in the body and have the potential to damage cells, DNA and other biomolecules, resulting in diseases such as cancer, ulcer, cardiovascular and neurodegenerative ailments¹. Protection against free radicals can be enhanced by ample intake of dietary antioxidants. Antioxidants may be of great benefit in improving the quality of life by preventing or postponing the onset of degenerative diseases.

Ulcers are open sores on the skin or mucus membrane characterized by a superficial loss of tissue. Mouth ulcer, esophagus ulcer, peptic ulcer and genital ulcer are the different types of ulcer¹. Alcohol intake, smoking, excessive stress, usage of non-steroidal anti-inflammatory drugs and H. pylori bacterial infection are major causes of peptic ulcer characterized by inflammation, mucosal bleeding and abdominal pain in patients².

Antiulcer medications with proven efficacy in the treatment of acute ulcers include H₂-receptor antagonists, H⁺/K⁺ATPase (proton pump) inhibitors, antacids, sucralfate and prostaglandin analogues³, but clinical evaluation of these drugs has shown side effects, incidence of relapses and drug interactions. Hence, herbal medicines are generally useful in such chronic cases, wherein drugs are required to be used for long periods.

Herbal drugs obtained from plants are believed to be much safer, this has been proved in the treatment of various ailments. Nearly 80% of the world population use traditional medicine, mainly medicinal plants, to cure illness. The plant Ziziphus rugosa Lam. of Rhamnaceae family is a large straggling scandent armed shrub, large elliptic subcordate leaves, paniculate flowers and wood is reddish, moderately hard. The fruits are edible and rich in nutrients⁶. The plant finds medicinal values in the traditional system of medicine. The phytochemical study reported the presence of flavonoids, alkaloids, terpenoids, carbohydrares etc⁷. Ethnobotanical investigations reported that plant bark is used in the treatment of ulcer, astringent and as antidiarrhoeal. The plant bark is also proved as antidiabetic, antifungal, analgesic and anti-inflammatory drug^(8-11). Despite of the traditional use, its antiulcer property has not been studied clinically. Therefore, the present work was carried out to study the efficacy of ethanolic extract of bark of Ziziphus rugosa Lam. on gastric ulcer induced by indomethacin and ethanol in animal models.

2. MATERIAL AND METHODS:

2.1 Collection and extraction of plant materials:

The bark of Ziziphus rugosa Lam collected from ALN Rao Memorial Ayurvedic medical college campus, Koppa, Chikkamagalore Dist, Karnataka. India. It was authenticated by Botanist Dr. Prakash Kumar Jha of ALN Rao Memorial Ayurvedic medical college. The bark was shade dried, coarsely powdered and extracted with ethanol using soxhlet apparatus (60–80°C). Then the extract was filtered, concentrated to dryness and stored for further use.

2.2 ANTIOXIDANT ACTIVITY^{12, 23, 24}

2.2.1 DPPH radical scavenging activity assay:

The DPPH assay is a simple, rapid and widely used method to evaluate antioxidant activity. The free radical scavenging activity of extract was evaluated. Serial dilutions of plant extract were prepared, 1 mL of test samples were mixed with 1 mL of 0.2 mM DPPH in methanol solution. After shaking vigorously, the mixture was incubated in dark at room temperature for 30 min, and the absorbance (Abs) was measured using a UV-visible spectrophotometer (Shimadzu) at 517 nm. DPPH methanol solution was used as a control. Percentage radical scavenging activity was calculated by using the following formula^12,26.

2.2.2 Free radical scavenging ability by the use of a stable ABTS radical cation assay:

The ABTS assay can be used to determine the antioxidant capacity. In the presence of hydrogen-donating antioxidants, the ABTS gets reduced. The antioxidant activity is proportional to the decrease in absorbance. ABTS solution was prepared by mixing 7 mM of ABTS and 2.45mM of potassium persulphate in water, which was incubated for 12–16 hours in dark at room temperature. Before use, the ABTS solution was diluted to get an absorbance of 0.7±0.02 at 734nm by using a UV-visible spectrophotometer (Shimadzu). Samples were prepared in methanol with dilutions of different concentration. A sample (10μL) was placed in a test tube and mixed thoroughly with 2.99mL ABTS radical working solution. Absorbance of the resulting clear mixture was recorded at 734 nm. Percentage radical scavenging activity was calculated by using the following formula¹².

2.3 Experimental animals:

Experimental animals, Wistar albino rats weighing 150-250gms were procured from animal house of Sree Siddaganga College of Pharmacy, Tumkur, Karnataka, India. They were maintained in polypropylene cages under standard environmental conditions (14 h dark/10 h light cycles; 25±2°C temperature; 35–60% humidity with air ventilation), fed with standard pellet diet and fresh water ad libitum. The study protocol was approved from the Sree Siddaganga College of Pharmacy Institutional Animal Ethics Committee (IAEC) under the reference no. SSCPT/IAEC.Clear/202/2019-20.

2.4. Acute toxicity study:

Acute toxicity study for ethanolic extract of of Ziziphus rugosa (ZREE) Lam. bark was conducted as per OECD guidelines 423¹³ using Wistar Albino rats. Each animal was administered with ethanolic solution of the extract by oral route. The animals were observed for any changes continuously for the first 2 hours and upto 24 hours for mortality²⁵.

2.5. Antiulcer activity:

In all the experimental models, Wistar Albino rats of either sex weighing 150- 250gm were selected and divided into five groups of six animals each. Animals were fasted for 24 hour before the study, but had free access to water. Group I treated as Normal control, received only distilled water, group II and III treated as treatment groups, received ethanolic extract of of Ziziphus rugosa (ZREE) Lam. bark extract of 100 and 200mg/kg, (p.o.) for 7 days (once a day) respectively. Group IV received ranitidine 50mg/kg (p.o.) and group V as inducer control received ethanol 80% (1ml/animal p.o) or Indomethacin (50mg/kg).^14-15

2.5.1 Ethanol induced model^16-20

In this method, Wistar albino rats were fasted in individual cages for 24 hours. ZREE or reference drug or control was given orally 30 min prior to administration of 80% ethanol (1 ml/animal). After 1 hour the rats were euthanized with excess of anesthetic ether and stomach was cut open along the greater curvature, cleaned with normal saline and the inner surface was examined using magnifier. Gastric volume, pH, Total acidity, Ulcer index and % ulcer protection were measured.¹⁷

2.5.2 Indomethacin induced ulcer model^17-20

Wistar Albino rats were fasted in individual cages for 24 hours. ZREE or reference drug or control was given orally 30 min prior to administration of Indomethacin (50mg/kg). After 1 h the rats were euthanized with excess of anesthetic ether and stomach were cut open along the greater curvature, cleaned with normal saline and the inner surface was examined using magnifier. Gastric volume, pH, Total acidity, Ulcer index and % ulcer protection were measured.¹⁷

2.5.2,1 Determination of gastric volume:

After sacrificing the rat, the stomach portion was removed. The gastric contents were transferred into the centrifuge tube, centrifuged and filtered. The supernatant liquid was then transferred to a measuring cylinder and the volume was measured.

2.5.2,2 Determination of pH of gastric content:

One ml of the gastric juice was collected, diluted with 1 ml water and the pH was directly measured by using Digital pH meter

2.5.2,3 Determination of free acidity and total acidity:

The gastric contents were centrifuged and filtered. One ml of the gastric juice was collected and the solution was titrated against 0.1N sodium hydroxide using 2 to 3 drops of topfer’s reagent as indicator, to the end point when the solution turned to yellowish orange colour. This indicated the volume of NaOH required for neutralizing the free hydrochloric acid present in the gastric juice. Then 2 to 3 drops of phenolphthalein solution was added and titration was continued until a definite red colour appeared. The difference between the two readings indicated the volume of NaOH required to neutralize combined acid present in the gastric juice. The sum of the two titrations was the total acid present in the gastric juice. Acidity was calculated by using formula^{14, 27,28}.

2.5.2,4 Determination of ulcer index

The stomach was opened along the greater curvature. The following scores/ratings as described by Takagi and Okabe²⁰ was used to evaluate the ulcer index as well as the severity of gastric lesions.

0 = no lesion, 1 = mucosal oedema and petechiae, 2 = one to five small lesions (1-2mm),

3 = more than five small lesions or one intermediate lesion (3-4 mm),

The ulcer index and the percentage protection are given by the following equations.

3. STATISTICAL ANALYSIS:

All values were reported as mean ± standard deviation. The statistical significance of differences between groups was assessed using one-way analysis of Variance (ANOVA) followed by Dunnett's test.

4. RESULTS AND DISCUSSION:

The ethanolic extract of Ziziphus rugosa bark was analyzed for antioxidant and antiulcer activities in two models.

4.1 Antioxidant activity:

The antioxidant capacity of the ethanolic extract of Ziziphus rugosa bark was evaluated for DPPH and ABTS reducing assays. ZREE has exhibited potential radical scavenging activity in both assays as shown in fig1. Where 125µg/ml concentration of ZREE has more scavenging effect of 55.84% than other concentration in DPPH assay. In ABTS assay 1000µg/ml concentration of ZREE has more scavenging effect of 81.69% than other. In DPPH assay it confirms that the scavenging effect is not dose dependent as high concentration has less scavenging effect.

Fig. 1 DPPH and ABTS antioxidant potential values of ethanolic extract of Ziziphus rugosa Lam. bark

4.2 In-vivo Ethanol induced Ulcers:

Oral administration of ethanol causes linear hemorrhagic lesions, extensive sub mucosal edema, mucosal friability, inflammatory cell infiltration, and epithelial cell loss in the stomach 21-22. Animals pretreated with ZREE (100 and 200mg/kg) and ranitidine (50mg/kg) has significantly protected gastric lesions induced by ethanol, in comparison to the inducer control group (Table 2,3 and Figure 2, 3 and 4).

4.3 In-vivo Indomethacin induced Gastric Ulcer:

Gastric ulcer induction by NSAIDs occurs through the inhibition of cyclooxygenases and thus prostaglandin synthesis 21-22. Prostaglandins are responsible for regulating mucosal cell renewal. Thus, the suppression of prostaglandin synthesis by NSAIDs causes increased susceptibility to gastric mucosal lesions. The administration of indomethacin (50mg/kg), a nonsteroidal anti-inflammatory drug (NSAID), caused injury to the gastric mucosa in inducer control group. Animals pretreated with ZREE (100 and 200mg/kg) and ranitidine (50mg/kg) has significantly protected gastric lesions induced by indomethacin, (Table 2,3 and Figure 2,3 and 4).

Table 2: Effect of Ethanolic extract of Ziziphus rugosa (ZREE) and ranitidine (50mg/kg/p.o.) on ulcer index and percentage protection on rats

Parameters		Normal	ZREE Treated 100mg/kg	ZREE Treated 200/kg	Std Drug Ranitidine	Inducer control
Ulcer Index	Ethanol induced	0.32	3.32	2.33	2	6
Ulcer Index	Indomethacin induced	0.32	2.48	1.84	1.16	6
Percentage protection	Ethanol induced	100%	58.5%	70.88%	75%	00
Percentage protection	Indomethacin induced	100%	56.66%	68.88%	81%	00

Results represents mean ± S.E.M., n=6. Statistical analysis was done by one-way ANOVA followed by Dunnett's test, ** p < 0.01; *** p < 0.001 compared to normal

Table 3: Effect of Ethanolic extract of Ziziphus rugosa (ZREE) and ranitidine (50mg/kg/p.o.) on gastric volume, pH and total acidity on rats.

Parameters		Normal	ZREE Treated 100mg/kg	ZREE Treated 200mg/kg	Std Drug Ranitidine	Inducer control
Gastric volume	Ethanol induced	2.10±0.043	3.13±0.043	2.77±0.049	2.45±0.076**	3.13±0.033
Gastric volume	Indomethacin induced	2.00±0.052	2.50±0.058	2.217±0.048**	2.150±0.022**	3.117±0.04
pH	Ethanol induced	4.55±0.043	3.88±0.048**	4.15±0.034***	4.25±0.022***	3.38±0.083
pH	Indomethacin induced	4.317±0.083	3.683±0.065	4.07±0.031**	4.100±0.026**	3.317±0.091
Total acidity	Ethanol induced	76.67±2.11	105±2.24	90±2.58***	85±2.24***	126.67±3.33
Total acidity	Indomethacin induced	81.67±3.07	108.33±3.07**	96.67±2.11	88.33±1.67***	130.00±3.65

Results represents mean ± S.E.M., n=6. Statistical analysis was done by one-way ANOVA followed by Dunnett's test, ** p < 0.01; *** p < 0.001 compared to normal.

Fig. 2 Effect of ethanolic extract of Ziziphus rugosa Lam.bark on Ulcer

5. CONCLUSION:

Our study showed that ethanolic extract of Ziziphus rugosa Lam. bark has potent antioxidant activity and ulcer protective activity. The antiulcer activity may be mediated by its antioxidant activity. Further work is necessary to elucidate the actual mechanism involved in the anti-ulcer activity.

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Received on 26.01.2020 Modified on 12.03.2020

Research J. Pharm. and Tech. 2020; 13(10):4720-4724.

DOI: 10.5958/0974-360X.2020.00831.8