AN IN-VIVO STUDY TO EVALUATE THE ANTI-ULCER ACTIVITY OF NĀRIKELĀ LAVANA IN PYLORIC LIGATION INDUCED ULCER IN WISTAR ALBINO RATS

AN IN-VIVO STUDY TO EVALUATE THE ANTI-ULCER ACTIVITY OF NĀRIKELĀ LAVANA IN PYLORIC LIGATION INDUCED ULCER IN WISTAR ALBINO RATS

Reshmi *, Abhaya Kumar Mishra, Sudhakar Bhat and Arun Mohanan

Department of Rasashastra and Bhaishajya Kalpana (Medicinal Chemistry and Pharmacy), Amrita School of Ayurveda, Amritapuri, Amrita Vishwa Vidyapeetham, Kollam, Kerala, India.

ABSTRACT: Lavana kalpanas are pharmaceutical preparation in which the ‘Lavana’ and the selected ‘auṣadhadravyā’ are igniting together in a closed sampuṭā to obtain the drug ash as medicinal product. Nārikelā lavana, one among the lavana kalpana mentioned in Bhaiṣajyaratnāvali 30^th chapter and in Rasa taraṇgiṇi14^th chapter. Nārikelā lavaṇa proved to have a high amount of electrolytes like calcium and potassium, which have antacid properties. Here the common ingredient in this Kalpana is lavana, which possesses madhura-lavanarasa & śītaviryā and having pittahara property which might be the reason for the extensive use of Lavaṇa Kalpanasin pittajanyavikaras. The higher alkalinity of Nārikelā lavaṇa may also play an important role in its mode of action. Materials and Methods: Nārikelā lavana was prepared as per the reference in Rasa taraṇgiṇi, and analytical parameters were tested. The experimental study was conducted in pyloric ligated Wistar albino rats. Then the data were analyzed statistically using One Way ANOVA followed by Dunnett’s multiple “t” test as a post hoc test. Results & Discussion: The analytical parameters like pH, LOD, Total ash, Acid insoluble ash, water-soluble ash etc. were tested, and the results were found to be within the permeable limits. The trial drug (Nārikelā lavana) shows significant results in total acidity, free acidity, and ulcer index by analyzing various parameters indicating the anti-ulcer activity. Conclusion: The obtained results clearly indicate Nārikelā lavana possesses anti-ulcer activity.

Keywords: Narikela lavana, Antiulcer study, In-vivo anti-ulcer study

INTRODUCTION: Peptic ulcer is the most predominant gastrointestinal disease; current therapy for Peptic ulcer is H2-receptor blockers, proton pump inhibitors, antacids, anticholinergics, and antibiotics.

Currently available treatments have limited efficacy and severe side effects. Hence, ulcer treatment is one of the challenging problems, and the researchers are looking forward to a drug with the minimal side effect, easily accessible & affordable.

Nārikelā lavaṇa, one among the Lavana kalpana mentioned in Bhaiṣajyaratnāvali 30^th chapter and in Rasa taraṇgiṇi 14^th chapter. Its main indication mentioned in both texts are śūla, especially in pariṇāmaśūla. By analyzing symptoms śūlac an be co-related to peptic ulcers.

Nārikelā lavana was proved to have a high amount of electrolytes like calcium and potassium, which has antacid properties. Here the common ingredient in this Kalpana is lavana, which possess madhura-lavanarasa & śītaviryā and having pittahara property which might be the reason for the extensive use of Lavaṇa kalpanas in Pittajanyavikaras. The higher alkalinity of Nārikelā lavaṇa may also play an important role in its mode of action.

Aims and Objectives: To analyze the pysico-chemical properties of Nārikelā lavaṇa and to experimentally evaluate the anti-ulcer activity of Nārikelā lavaṇa.

MATERIALS AND METHODS

Pharmaceutical Study: Pharmaceutically authentic and pure drugs were collected, and later Nārikelā lavaṇa was prepared according to the reference from Rasa taraṇgiṇi ¹. Nārikelā lavaṇa consists of only 2 ingredients- nārikelā and Saindhavā lavaṇa. A mature coconut devoid of outer fibrous part is taken & a hole is made into one of its eyes, and all the liquid inside is poured out. Saindhavā lavaṇa should be powdered well and poured into the coconut through the hole. The hole is now plugged with a mud cork. A layer of multanimitti smeared cloth was covered on the coconut and allowed it to dry, and the process was repeated 3 times. After proper drying of layers, it was subjected to Mahaputa ². The temperature was taken every 10 min using a thermocouple.

Analytical Study: Nārikelā lavaṇa was analyzed by doing the following tests: -Loss on Drying (LOD), Total ash, Acid insoluble ash, Water soluble ash, pH (5% solution) ^{3, 4}.

Experimental Study: Wistar strain albino rats weighing between 160 to 250g of either sex were used for the study. The Institutional Animal Ethical Committee approved all experimental protocols following the guideline formulated by CPCSEA and Approval No. SDMCRA/IAEC/AM-R-01. In pyloric ligation-induced ulcers, an experimental study was conducted in 3 groups, i.e., control, standard and trial groups with 8 Wistar albino rats, which were randomly selected. A day before dosing, the selected animals would be randomly divided into three groups comprising 4 male and 4 female Wistar albino rats.

The test drugs would be administered for 7 consecutive days. Ranitidine was administered for 4 consecutive days prior to pyloric ligation for the reference standard group. The control group, CMC solution and distilled water were also given for 7 consecutive days. Animals would be fasted for 36-40 hours by placing them in metabolic cages to prevent coprophagy but provided free access to water ad libitum. The dosing would be continued during this period. On the tenth day, one hour after dosing, pylorus was ligated by the method of Shay et al. (1945) ⁵.

Pre Operative: Rats were anesthetized with Inj. Ketamine 80mg/kg body weight (Intraperitoneal) Fig. 1 and Inj. Xyloxine 3mg/kg body weight (Intra Muscular) Fig. 2 for analgesic action. Shaving of the ventral part of the abdomen of rats was done ⁶ Fig. 3.

FIG. 1: FIG. 1: KETAMINE INJECTION (IP)      

FIG. 2: XYLOXINE INJECTION (IM)

FIG. 3: SHAVING OF VENTRAL PART

Operative Procedure: On the ventral part of the abdomen portion was opened in a layer by a small midline incision just below and lateral to the xiphoid process Fig. 4. Pyloric portion of the stomach was slightly lifted out, avoiding traction to the pylorus or damage to its blood supply. The pylorus was ligated with linen thread No.10 and stomach was replaced carefully Fig. 5. The incision was closed with interrupted sutures in layers & betadine ointment was applied over the sutures ⁷.

FIG. 4: ANESTHETIZED RAT ON DISSECTION TABLE          

FIG. 5: PYLORIC LIGATION

Post Operative Procedure: Each rat was kept in an individual metabolic cage. The animals were deprived of both food and water. Animals were euthanized under deep ether anesthesia at the end of 10 h after pyloric ligation ⁸. The abdominal cavity was reopened carefully, and the stomach was excised after tying the esophageal end to prevent loss of gastric contents during excision Fig. 6. Gastric contents were drained into tubes and centrifuged at 2000 rpm for 10 min Fig. 7. The gastric juice's volume and pH were noted and used for biochemical estimation. The stomach was opened along the greater curvature and washed under the running tap water. Then it was fixed on a wax board & observed the ulcers using a magnifying lens Fig. 8. After assessing the ulcer score, the glandular portion of the stomach was sent for histopathological assessment ^{9, 10}.

FIG. 6: PYLORIC LIGATED STOMACH

FIG. 7: COLLECTION OF GASTRIC JUICE             

FIG. 8: ULCER ASSESSMENT

RESULTS: The data obtained were analyzed using Graph pad in stat version 3.05 by student “t” test for comparison between two positive control groups and the rest of the data were analyzed by One Way ANOVA followed by Dunnett’s multiple “t” test as a post hoc test for determining the level of significance of the observed effects. Eight parameters were analyzed during the study.

pH of Gastric Juice: The data in the Table 1 shows there was an increase in gastric pH in the standard group when compared to the control group, the observed increase was found to be statistically very significant and there was an increase in gastric pH in test group when compared to the control group, the observed increase was found to be statistically non-significant.

TABLE 1: EFFECT OF NĀRIKELĀ LAVANA ON PH OF GASTRIC JUICE

Data: MEAN±SEM, **p<0.01.

Volume of Gastric Juice: The data in the Table 2 shows there was a decrease in gastric juice volume in the standard group when compared to the control group, the observed decrease was found to be statistically non-significant and there was an increase in gastric juice volume in the trial group when compared to the control group, the observed increase was found to be statistically non-significant.

TABLE 2 EFFECT OF NĀRIKELĀ LAVANA ON VOLUME OF GASTRIC JUICE

Data: MEAN±SEM.

Free Acidity: The data in the Table 3 shows there was a decrease in free acidity in the standard group when compared to the control group, the observed decrease was found to be statistically significant, and there was a decrease in free acidity in the test group when compared to the control group, the observed decrease was found to be statistically very significant.

TABLE 3 EFFECT OF NĀRIKELĀ LAVANA ON FREE ACIDITY

Data: MEAN±SEM, *P<0.05, **P<0.01

Total Acidity: The data in the Table 4 shows there was an increase in total acidity in the standard group when compared to the control group, the observed increase was found to be statistically non-significant and there was a decrease in total acidity in the test group when compared to the control group, the observed decrease was found to be statistically significant.

TABLE 4 EFFECT OF NĀRIKELĀ LAVANA ON TOTAL ACIDITY

Data: MEAN±SEM, *P<0.05.

Ulcer Index: The data in the Table 5 shows there was a decrease in ulcer index in the standard group, when compared to the control group, the observed decrease was found to be statistically very significant, and there was a decrease in ulcer index in the test group when compared to the control group, the observed decrease was found to be statistically very significant.

TABLE 5: EFFECT OF NĀRIKELĀ LAVANA ON ULCER INDEX

Data: MEAN±SEM, **P<0.01

Carbohydrate Estimation: The data in the Table 6 shows there was a decrease of total carbohydrates in standard group when compared to the control group, the observed decrease was found to be statistically very significant and there was decrease in total carbohydrate in test group when compared to the control group, the observed decrease was found to be statistically non-significant.

TABLE 6: EFFECT OF NĀRIKELĀ LAVANA ON CARBOHYDRATE ESTIMATION

Data: MEAN±SEM, **P<0.01

Protein Estimation: The data in the Table 7 shows there was a decrease in total protein in the standard group when compared to the control group, the observed decrease was found to be statistically non-significant, and there was decrease in total protein in test group when compared to the control group, the observed decrease was found to be statistically non-significant.

TABLE 7: EFFECT OF NĀRIKELĀ LAVANA ON PROTEIN ESTIMATION

Data: MEAN±SEM

Peptic Activity: The data in the Table 8 shows there was increase of peptic activity in the standard group when compared to the control group, the observed increase was found to be statistically non-significant and there was a decrease in peptic activity in test group when compared to the control group, the observed decrease was found to be statistically non-significant.

TABLE 8: EFFECT OF NĀRIKELĀ LAVANA ON PEPTIC ACTIVITY

Data: MEAN±SEM

Histopathological Examination of Stomach Tissue: Histopathological examination of stomach tissues was done and the following results were obtained.

Control Group: Stomach tissue sections of normal control group rats showed normal cytoarchitecture. Fig. 9 & 10. The data in Tables 9, 10, and 11 show the results of histopathological examination of stomach tissue of the control group, standard group, and trial group, respectively.

TABLE 9: SHOWING THE RESULT OF HISTOPATHOLOGY OF THE CONTROL GROUP

Standard Group

TABLE 10: SHOWING THE RESULTS OF HISTOPATHOLOGY OF STANDARD GROUP

Trial Group

TABLE 11: SHOWING THE RESULTS OF HISTOPATHOLOGY OF THE TRIAL GROUP

Photomicrograph of Histopathology of Normal Stomach Tissue:

FIG. 9: NORMAL STOMACH TISSUE    

FIG. 10: ABSENCE OF NECROSIS/EROSION

Photomicrograph of Histopathology of Control Group:

FIG. 11: ODEMA AND INFLAMMATION OF SUBMUCOSA     

FIG. 12:  MUCOSAL EROSION

Photomicrograph of Histopathology of Standard Group:

FIG. 13: ABSENCE OF ULCER              

FIG. 14: MUCOSAL EROSION ABSENT

Photomicrograph of Histopathology of Trial Group:

 FIG. 15: ULCERATED TISSUES NOT SEEN   

FIG. 16: ABSENCE OF MUCOSAL EROSION

DISCUSSION: The outcome of the experimental study has been provided in the form of a consolidated table as follows for easy comparison and discussion. Data in Table 12 shows the SD in total acidity, free acidity & ulcer index, indicating the anti-ulcer activity. A decrease in the total acidity implies reduced HCl along with organic acids. A decrease in the free acidity implies reduced HCl secretion. A decrease in ulcer index implies prevention of ulcer formation. By Analysing the ayurvedic pharmacological properties of the formulation, the main ingredients - Nārikelā & Saindhavā lavaṇa possess Madhural avanarasa, śītavīrya, laghu-snigdhaguna, madhuravipāka and vātapittahara properties. Madhura and lavanarasa of the formulation will pacify the vitiated vāta. Madhura vipāka and śītavīrya of the formulation may increase the mucosal secretion and thereby preventing the ulceration. Madura rasa is Sandhanakrit, which also might help in healing the ulcer. Pharmacological studies on the drug, Nārikelā revealed the presence of alkaloids, tannins, resins, and phenolic compounds such as terpenoids, steroids etc ¹¹.

Tannin and alkaloids were proved to be having anti-ulcer activity, which prevents ulceration ¹². The presence of phenolic compounds acts as anti-oxidant, that reduces the formation of free radicals and thus potentially protects the cell from oxidative damage ¹³. An analytical study on the formulation revealed higher alkalinity of 9.64, shows the significant effect on preventing the ulceration. Ash value was found to be 90.84%, indicating the richness of minerals present in the sample, which inturn raises its therapeutic efficiency. Higher water-soluble ash (87.4%) and lower acid insoluble ash (1.24%) in turn indicating its higher solubility and the absence of impurities, respectively.

Low LOD value (1.04%) ensures a longer shelf life of the sample. In an in-vivo study, a significant decrease in total acidity, free acidity and ulcer index were found compared to the pyloric ligated control group. Phytochemicals like tannin, and alkaloids were proved to have anti-ulcer activity by reducing the acid secretion might be the reason behind this significant decrease. The presence of phenolic compounds acts as anti-oxidant, thus repairing the damaged cells, and higher alkalinity of the formulation also prevents the formation of an ulcer. In the histopathology of the test drug group, the ulceration was found to be minimal when compared to control group. While considering the overall factors related to the mechanism of healing ulcer, the main three things to be considered are acid neutralization, reducing the secretion and mucosal protection.

Saindhavā lavaṇa in the formulation helps in neutralization and reducing the acid secretion. Nārikelā, in the formulation proved to have anti-oxidant might help in mucosal protection. By considering these factors, we can claim that our formulation Nārikelā lavana act in preventive way than curative in ulceration.

TABLE 12: SHOWING THE RESULTS OF VARIOUS PARAMETERS TESTED IN GASTRIC JUICE OF WISTAR ALBINO RATS

Where, NSD- Non significant decrease; SD- significant decrease; NSI- Non significant increase; SI-Significant increase

CONCLUSION: The analytical parameters like pH, LOD, Total ash, Acid insoluble ash, Water soluble ash etc were tested and the results was found to be within the permeable limits. The trial drug (Nārikelā lavana) shows significant results in total acidity, free acidity, and ulcer index by analyzing various parameters indicating the anti-ulcer activity. Thus, the obtained results clearly indicate Nārikelā lavana possesses anti-ulcer activity.

ACKNOWLEDGEMENT: I would like to express my sincere gratitude towards Dr. Ramesh N.V (HOD, Dept. of RSBK), Dr. Dhanya, Dr. Vineeth P. K (Assistant professor, Dept. of RSBK), Bri. Shylaja (Quality control laboratory), Dr. Aansu susan Varghese, Dr. Devipriya. S, Dr. Lekshmi C. S, Dr. Sangeetha Nandakumar N. K, Dr. Veena. G-PG scholars, Amrita School of Ayurveda, for their valuable suggestions and support.

CONFLICTS OF INTEREST: We declare no conflicts of interest

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    How to cite this article:

    Reshmi S, Mishra AK, Bhat S and Mohanan A: An in-vivo study to evaluate the antiulcer activity of Nārikelā lavana in pyloric ligation induced ulcer in Wistar albino rats. Int J Pharm Sci & Res 2022; 13(9): 3715-22. doi: 10.13040/IJPSR.0975-8232.13(9). 3715-22.

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