Fresh leaves of MKL were obtained from the market. Specimens were collected in the month of September, 2016. Authentication and verification of the plants were done by proper authorities.

  The leaves were thoroughly washed and shade dried, grinded into fine powders and stored in air tight containers. The soxhlet apparatus was used for extraction. The resultant extracts were filtered using Whatman filter paper no. 1, concentrated by evaporation and collected in petri dishes. The final yield of MKL leaf extracts were 66.3 grams (26.5%), stored in a refrigerator at 4 °C in labelled air tight containers.

  This was carried out as per OECD guidelines. The rats were randomly selected, marked for identification, and kept in their cages for seven days prior to dosing. Animals were fasted prior to dosing (food was withheld for 3-4 hours). The dose was calculated according to the fasted body weight. Post fasting, the first animal was dosed at 175 mg/kg body weight with aqueous extract of study drug by gavage. Food was withheld for a further 1-2 hours. The animal was observed for mortality for 48 hours. Then a second animal was dosed at 550 mg/kg body weight by the same process. Again after 48 hours a third animal was dosed at 2000 mg/kg and observed for the next 48 hours for mortality. All the above animals were observed for a period of 14 days and were found to be alive at 2000 mg/kg. Two doses of the study drug were selected i.e. 200 mg/kg, and 400 mg/kg.

The study had 2 experimental models.

• Ulcer induction by pyloric ligation [7]
• Ulcer induction by cold and restraint stress [8]

  For each model, 5 groups of 6 animals each were selected. Group I (Normal control) served as a common for both models. Thus, for both models, a total of 54 animals were taken, detailed as follows [9-11].

Table 1: Duration and route of drug administration: All study drugs were administered orally for 7 days, using an oral feeding tube for rats.

  Under proper aseptic and antiseptic conditions, ulcer induction was done by pylorus ligation as per the standard procedure [7].

   Under proper aseptic and antiseptic conditions, the animals were starved and on 7th day, ulcers were induced by cold while putting the animal in restraint [8].

  Following variables were measured in both models in the study:

• Ulcer index
• Percentage of ulcer protection
• Additionally following variables were studied in pyloric ligation model,
• Gastric content volume
• Free acidity
• Total acidity
• pH

   The ulcers were evaluated quantitatively using ulcer index and scoring number [13]. Percentage ulcer protection was also calculated. Volume, pH, free and total acidities of gastric content was determined. Titration was done with 0.01 N NaOH, till total acidity was achieved.

   Stomach tissues were fixed in 10% formalin for 24 hrs, then embedded in paraffin. Small sections were made (3-5 µm) and stained with H&E dye and examined under light microscopy.

• Analysis done using the Graph pad prism 5.01 software.
• Between the groups, the data was compared using One way ANOVA test followed by post hoc Tukey’s test.
• The level of significance for each comparison in the analysis was calculated at 0.05.

   The ulcers were evaluated quantitatively using ulcer index and scoring number [13]. Percentage ulcer protection was also calculated. Volume, pH, free and total acidities of gastric content was determined. Titration was done with 0.01 N NaOH, till total acidity was achieved.

   The ulcers were evaluated quantitatively using ulcer index and scoring number [13]. Percentage ulcer protection was also calculated. Volume, pH, free and total acidities of gastric content was determined. Titration was done with 0.01 N NaOH, till total acidity was achieved.

Results

Pyloric ligation induced ulcer model

• Ulcer index: On comparison using one-way ANOVA, the mean ulcer index between the groups was significantly different (P < 0.001) (Table 2). On post hoc analysis, it was higher in all pyloric ligated groups than the normal control group. In all treatment groups, the mean ulcer index was significantly lower than the disease control group (P < 0.001). On comparison with ranitidine treated group, it was significantly higher in aqueous extract Murraya koenigii 200 mg/kg (p = 0.002) and 400 mg/kg (p = 0.041). The mean ulcer index in the groups treated with both doses of MKL were comparable (p = 0.92) as shown in table 3.
• Percentage of ulcer protection: Compared to the disease control group, the ulcer protection in ranitidine group was 64.12%; in groups treated MKL in doses of 200 and 400 mg/kg, it was 45.2% and 50.07%, respectively (Table 4)
• Volume of gastric juice: On comparison using one-way ANOVA, the mean volume of gastric juice between the groups was significantly different (P < 0.001) (Table 2). On post hoc analysis, volume was significantly higher (P < 0.001) in induced groups than the normal control group. It was significantly lower in the ranitidine (P < 0.001), Murraya koenigii 400 mg/kg (p = 0.042) treated groups than the disease control group. Compared to ranitidine treated group, the mean volume in MKL 200 mg/kg (P < 0.001) and 400 mg/kg (p = 0.001) was significantly higher (Table 3).
• pH of gastric juice: On comparison using one-way ANOVA, the mean pH of gastric juice between the groups was significantly different (P < 0.001) (Table 2). On post hoc analysis, the mean pH was significantly lower in the disease control (P < 0.001) and AEMK200 (P < 0.001) groups than normal control. The mean pH was significantly higher in the all treatment groups than the disease control group (P < 0.001). On comparison with ranitidine treated group, the mean pH of gastric juice in MKL 200 mg/kg (P < 0.001) treated groups was significantly lower. The mean pH in the group treated with MKL 400 mg/kg (P < 0.001) was significantly higher than the 200 mg/kg treated group (Table 3).
• Free acidity of gastric contents: On comparison using one-way ANOVA, it was observed that the mean free acidity of gastric juice between the groups was significantly different (P < 0.001)(Table 2). On post hoc analysis, it was observed that the mean free acidity of gastric juice was significantly higher (P < 0.001) in all induced groups than the normal control group. The mean free acidity of gastric juice was significantly lower in the all treatment groups than the disease control group (P < 0.001). On comparison with ranitidine treated group, the mean free acidity of gastric juice in aqueous extract MKL 200 mg/kg (P < 0.001) and 400 mg/kg (P < 0.001) treated groups was significantly higher. The mean free acidity of gastric juice in the group treated with aqueous extract MKL 400 mg/kg (P < 0.001) was significantly lower than the 200 mg/kg treated group (Table 3).
• Total acidity of gastric contents: On comparison using one-way ANOVA, it was observed that the mean total acidity of gastric juice between the groups was significantly different (P < 0.001) (Table 2). On post hoc analysis, it was observed that the mean total acidity of gastric juice was significantly higher (P < 0.001) in all induced groups than the normal control group. The mean total acidity of gastric juice was lower in the all treatment groups than the disease control group (P < 0.001). On comparison with ranitidine treated group, the mean total acidity of gastric juice in aqueous extract MKL 200 mg/kg(P < 0.001) and 400 mg/kg (P < 0.001) treated groups was significantly higher. The mean total acidity of gastric juice in the group treated with aqueous extract MKL 400 mg/kg (P < 0.001) was significantly lower than the 200 mg/kg treated group (Table 3).

Table 2: Pyloric ligation model.
Effect on ulcer index, volume of gastric juice, Ph of gastric juice, free acidity of gastric juice and total acidity.

Table 3: Post hoc analysis using Tukey’s test.

Table 4: Percentage of ulcer protection.

• Ulcer index: On comparison using one-way ANOVA, it was observed that the mean ulcer index between the groups was significantly different (P < 0.001) (Table 5). On post hoc analysis, it was observed that in all induced groups, the mean ulcer index was significantly higher (P < 0.001) than the normal control group. The mean ulcer index in all treatment groups, the mean ulcer index was significantly lower than the disease control group (P < 0.001). On comparison with ranitidine treated group, the mean ulcer index in aqueous extract MKL 200 mg/kg (P < 0.001) and 400 mg/kg (p = 0.013) treated groups was significantly higher. The mean ulcer index in the groups treated with both doses of MKL were comparable (p = 0.4) (Table 6).
• Percentage of ulcer protection: Compared to the disease control group, the ulcer protection in ranitidine group was 67.9%; in groups treated with aqueous extract of MKL in doses of 200 and 400 mg/kg, it was 47.49% and 54.82%, respectively (Table 4).

Table 5: Cold restraint model.

Table 6: Post hoc analysis using Tukey’s test.

Figure 1: Histopathological images of gastric mucosa after Hematoxylin-eosin staining under light microscope (pyloric ligation ulcer model).

Figure 2: Histopathological images of gastric mucosa after Hematoxylin-eosin staining under light microscope (Cold restraint stress induced ulcer model).

  Docked structure of ligand-1 with HIV-1 capsid-A protein is presented at figure 1. It is observed that as our ligand is small it easily goes to the binding site. But it is too small to tightly fit to the binding cavity. As a result, binding energy is very small, only -4.9 kcal/mol. There is only one hydrogen bonding formation is observed which is presented in figure 2. This hydrogen bonding is made by a glutamate residue which is 155th residue. All other interactions are Van der Waals interaction. Though its binding energy is small, its binding frequency is very high, as high as 30.0%. It is because its size is very small and its charge density is very high. It has three interaction zones which is shown in HB plot presented in figure 3. Estimated inhibition constant for this ligand is 250 μ mole.

  Docked structure of ligand-2 is presented in figure 4 and interaction plot is presented in figure 5. Binding affinity of ligand-2 is -5.13 kcal/mol and binding frequency is 50.0% which is very high. Thus, ligand-2 is an improved inhibitor. It is observed that ligand-2 is attached to one side of the cavity. Thus, its binding frequency is very high. Inhibition constant of this ligand is 174.9 μ mole.

Figure 3: HB plot of ligand-1 with p24 HIV protein.

Figure 4: Docked structure of ligand-2 with p24 HIV protein.

Discussion

  The present study demonstrated the antiulcer activity of aqueous extract of leaves of MKL in animal models of pyloric ligation and cold restraint induced gastric ulcers. MKL leaf extract has demonstrated antiulcer activity in some animal studies. These plants are easily available throughout the country in abundance, thus, giving us the opportunity to develop a cheap and easily available alternative for management of peptic ulcers. Thus, these plants were chosen and their antiulcer efficacy was evaluated in comparison to that with ranitidine.

  In the present study, induction of ulcer by pyloric ligation or cold restraint stress was seen in the disease control groups, which was evident from the increase in ulcer index in comparison to the normal control group. Other variables also differed significantly.

  With treatment by aqueous extract of MKL, the ulcer index was lower compared to the disease control group, but higher compared to ranitidine. Dose dependent effect was not observed. The ulcer protection was 50% at 400 mg/kg dose and 45.2% at 200 mg/kg dose. The gastric juice volume was lower at 400mg/kg dose compared to disease control group. Compared to ranitidine, at both doses the gastric volume was higher. The gastric pH was increased compared to disease control group. In comparison to ranitidine, only 400 mg/kg dose gave comparable results. pH was higher compared to 200 mg/kg dose, representing a dose dependent effect. The free and total acidity of gastric contents was reduced than disease control group, however, the effect was not comparable to the ranitidine group, and also a dose dependent effect was observed with better response at higher dose. These results revalidate the findings of some other previous studies [13-16].

  The ulcer index in the aqueous extract of MKL at both doses was lower than the disease control group, but was higher than ranitidine group, and similar at both doses. The ulcer protection provided was lower than that by ranitidine treatment at both doses.

  From the findings of the study, it was evident that the aqueous extracts of leaves of MKL have antiulcer activity. This activity measured in terms of ulcer index and ulcer protection was evident compared to the disease control group. Ulcer protection was found to be more at higher doses compared to the lower doses. The pH, volume, free and total acidity represent secretory activity in the stomach. The effect on these variables demonstrated anti-secretory activity of the aqueous extract of MKL. Antiulcer activity of aqueous extract of Murraya koenigii leaves in the dose of 200 mg/kg and 400 mg/kg has been demonstrated in few studies. Patidar., et al. [17] and Sharma., et al. [18] reported antiulcer activity in terms of substantial ulcer percentage protection and changes in the volume, pH, free and total acidity of gastric juice as observed in number of ulcer models induced by various methods. Thus, the present study findings are in agreement with the previous studies evaluating the antiulcer activity of aqueous extract of leaves of MKL. A compound isolated from the leaves, 1,3,5-trihydroxy-7-methylanthracene-9,10-dione, has been found to be responsible for the antiulcer activity. However, the leaves contain numerous other phytoconstituents which have not yet been evaluated for their antiulcer potential.

Conclusion

  The present study adds to evidence of antiulcer efficacy of aqueous extracts of leaves of Murraya koenigii. Though not as good as ranitidine, it is a promising agent for antiulcer compounds. Research should be encouraged to identify and isolate the active phyto-constituents from the aqueous extracts of the leaves of these plants. The available evidence of antiulcer activity provides satisfactory evidence for further advanced studies with more pure and refined products for testing its efficacy in human. Development of compounds as antiulcer agents may have an additional advantage of being cheap and easily available.

Conflict of Interest

Nothing to declare.