Anti-inflammatory activity of acacia catechu-bark aqueous solution in aspirin induced gastric ulcer in rodents

INTRODUCTION

In this modern era of science, focus has been shifted towards the search of medicine from the natural sources assuming that they will have good safety profile and tolerability. Nowadays, people prefer using medicines made from the plant resources because they consider them safer than the commercial pharmaceutical drugs. Antioxidants are the drugs which decrease the oxidative stress thus reducing the oxidative damage to the body. Plants are a rich source of antioxidants.^1,2

Acacia catechu is widely available throughout the Indian subcontinent. Its main constituents are catechins, epicatechins, epigallocatechin, gallate, rocatechin, phologlucin and protocatechuic acid. Catechin has high content of antioxidants and a high antioxidant and anti-inflammatory potential. These are used to treat many inflammatory conditions like boils, ulcer, skin lesions, pharyngitis, leucorrhea, erysipelas, spongy gums, diarrhea and hypertrophy of gums.^2-6

The uses of Acacia catechu can be found in the history from the date betel leaves were used. In 15^th century, it was exported to Europe and in 17^th century it was named as Acacia catechu. During the 19^th century it was commercially used in France. It’s of two types, dark and pale catechu, which were used commercially.⁴

Antioxidant potential was studied by Priyadarsini et al. They studied its antioxidant potential as inhibition of lipid peroxidation by radiation in the rat liver. This antioxidant potential is not unique to the acacia catechu and many other plants have been studied for this. Acacia catechu is one of them.^4-12 Studies have shown inverse relationship between disease and food intake rich in antioxidants. Though use of commercially prepared antioxidants has resulted in liver damage and even cancer, efforts are being made to look for antioxidants from natural sources like plants.^14,15

The aim of this study was to look for anti-inflammatory potential of Acacia catechu in albino rats. We assessed this by measuring inflammatory infiltrate in the gastric ulcers induced by aspirin in rodents.

METHODS

This research was carried out at Postgraduate Medical Institute, Lahore after taking ethical permission from ethical committee. 48 male and female albino rats, weighing 150-250 gm were purchased from the University of Veterinary and Animal Sciences Lahore. All the animals were examined thoroughly and weighed before the commencement of experiment. The rats were housed in experimental research laboratory of Postgraduate Medical Institute, Lahore, under controlled conditions of temperature 22±0.5°C, humidity (50±10%). Aspirin in powder form was taken from BDH (British drug houses) Limited, Poole England. Bark of Acacia catechu was obtained from Botany Department of Government College University, Lahore.

Rats were randomly divided using balloting method into four groups, comprising of twelve animals each. The animals in each group were numbered, using colours on their tails and these identification points were checked each time before intervention. Group A served as a control and was further subdivided into three groups A₁, A₂ and A₃ which were given 4 ml of distilled water orally for 3, 7 and 14 days. Group B was further subdivided into three groups B₁, B₂ and B₃ which were given aspirin at a dose of 100 mg/kg body weight, dissolved in 4 ml of distilled water orally for 3, 7 and 14 days, respectively.¹⁶

Group C was further subdivided into three groups C₁, C₂ and C₃ which were given 100 mg/kg body weight of aspirin along with 250 mg/kg body weight of Acacia catechu, dissolved in 4 ml of distilled water orally for 3, 7 and 14 days.

Group D was further subdivided into three groups D₁, D₂ and D₃ which were given 100 mg/kg body weight of aspirin along with 500 mg/kg body weight of Acacia catechu, dissolved in 4 ml of distilled water orally for 3, 7 and 14 days.¹⁷

Animals of group A₁, B₁, C₁ and D₁ were sacrificed on day three, animals of group A₂, B₂, C₂ and D₂ were sacrificed on day 7, similarly animals of A₃, B₃, C₃ and D₃ were sacrificed on day 14.

Statistical analysis

The data was added and analysed using SPSS 20 (Statistical Package for Social Sciences). Mean±SD was calculated for inflammatory cells, in the mucosa and submucosa of stomachs. One way ANOVA was applied to compare means of inflammatory cells among the control and experimental groups. Post hoc Tukey test was used for multiple comparisons.

P value of ≤0.05 was considered as statistically significant.

RESULTS

Inflammatory cells were counted in lamina propria and submucosa of the stomach in the ulcer base.

On day 3, few inflammatory cells were seen in subgroup A₁. Highest number was seen in subgroup B₁. Subgroup C₁ had milder inflammation as compared to subgroup D₁.

On day 7 very few inflammatory cells were observed in control subgroup A₂. Marked increase in inflammatory cells was seen in subgroup B₂. An increased inflammatory response in subgroup B₂ indicated a continued process of tissue injury. Inflammatory cells in subgroup C₂ and subgroup D₂ were less as compared to subgroup B₂.

On day 14, very little number of inflammatory cells were observed in control subgroup A₃. Marked increase was seen in subgroup B₃. Inflammatory cells in subgroup C₃ and subgroup D₃ showed little increase as compared to control group. Acacia catechu inhibited inflammatory cell infiltration hence inhibiting inflammation. A dose of 500 mg/kg however showed better results than a dose of 250 mg/kg.

Mean number of inflammatory cells in control and experimental groups is shown in Table 1 and Figure 1.

After applying post hoc Tukey test, it was seen that number of inflammatory cells was significantly less in subgroup A₁ as compared to subgroup B₁ (p=0.000) as well as subgroup C₁ (p=0.003) and subgroup D₁ (p<0.05). Statistically significant difference between inflammatory cell count in subgroup B₁ with subgroup C₁ and subgroup D₁ was noted (p=0.000). Difference between subgroup D₁ with subgroup A₁ (p=0.666) and subgroup C₁ (p=0.333) was also significant. (Figure 1, Table 2). Inflammatory cell count in B₂ was strikingly higher than subgroup A₂, subgroup C₂ and subgroup D₂ (p=0.000).

Statistically significant increase in inflammatory cells was also seen in subgroup C₂ as compared to subgroup A₂ and subgroup D₂ (0.000). The difference between inflammatory cell count in A subgroup 2 and subgroup D₂ was not statistically significant (p=0.067) (Table 2).

Statistically significant difference in subgroup B₃ as compared to subgroups A₃, C₃ and D₃ (p=0.000) was seen. Difference between inflammatory cells in subgroup A₃ and subgroup D₃ was (p=0.774) which was not statistically significant. The difference between inflammatory cell count in subgroups A₃, C₃ and D₃ was also statistically significant (p=0.000) (Table 2).

*p value ≤0.05 is statistically significant.

*p value ≤0.05 is statistically significant.

DISCUSSION

Significant increase in number of inflammatory cells in subgroups B₁, C₁ and D₁ as compared to subgroup A₁ was noticed. However, number of inflammatory cells in subgroups C₁ and D₁ was significantly less than subgroup B₁. In normal wound healing, platelets release various growth factors as well as proinflammatory cytokines like IL-1 that attract neutrophils to the wound site.¹⁸ Neutrophils infiltrate the site of injury within an hour. Cellular debris is phagocytosed and bacteria are killed by free radicals generated by neutrophils.¹⁹ Macrophages migrate to the site of injury at about 48 to 96 hours after tissue injury.²⁰ They phagocytose poly morphonuclear leucocytes and become predominant cells in the wound by Day 2 after injury.²¹ However abundant neutrophils along with macrophages were present in subgroup B₁ denoting ongoing inflammatory process. In a comparative study, effect of aspirin on pyloric ligated rat models of gastric ulcers was evaluated. Results showed ulcer crater containing distorted gastric glands, inflammatory exudates and cellular debris.²²

Flavocoxid obtained from Acacia catechu decreases pro inflammatory cytokines as TNF-α, hence inhibiting ROS formation resulting in reduced leukocyte infiltration.²³

Day 7

Significant increase in number of inflammatory cells in subgroup B₂ as compared to control group A₂ was noticed. Functional neutrophils have life-spans of around 2 days at the site of injury. After completing their task, they undergo apoptosis and get phagocytosed by the macrophages.¹⁹ Macrophages are stimulated by hypoxia and induce angiogenesis in the granulation tissue.²⁴ As the inflammation resolves numbers of neutrophils and macrophages are reduced.²⁵ Number of inflammatory cells in subgroup B₂ on day 7 was significantly more as compared to subgroup B₁ on day 3. In a study, role of neutrophils in the pathogenesis of indomethacin induced ulceration was studied on gastric antrum of rat. Results revealed that there was a time dependent increase in the extent and severity of ulceration and neutrophil infiltration into the gastric antrum after treatment with indomethacin.²⁶ Flavocoxid containing catechin from Acacia catechu attenuates inflammation and neutrophil invasion in a carrageenan-induced paw edema model.²⁷ Number of inflammatory cells in subgroup B₂ was significantly greater than subgroup C1.Similarly subgroup D₁ showed significantly decrease in inflammatory cells as compared to subgroup C₁.

Day 14

Significant increase in number of inflammatory cells in subgroup B₃ as compared to control group A₃ was noticed. Inflammation lasts as long as there is debris in the wound. In an in vitro study on dermal equivalent model, it was found that that the presence of macrophages actually delays wound contraction. Hence the withdrawal of macrophages from the site of wound may be essential for subsequent healing.²⁸ Presence of macrophages in wound indicates ongoing leucocyte infiltration. Inflammation and neutrophil infiltration are vital in causing gastric damage induced by NSAIDs.²⁶ The inflammation results in increased TNF-α production, which enhances neutrophil-derived superoxide generation.²⁶ This stimulates IL-1 production resulting in neutrophil accumulation.²⁹ Inhibition of inflammation by Acacia catechu can be attributed to quercetin. In an experimental study it was found that quercetin had an inhibitory effect on the secretion of inflammatory factors TNF-α, IL-1β and IL-6 of cardiac fibroblasts hence inhibiting their inflammatory secretions.³⁰ Arjmandi et al, in 2014 conducted a 1-week clinical trial on osteoarthritic patients to examine the effectiveness of UP446 from Acacia catechu and Scutellaria baicalensis extracts (500 mg/day) to detect selected biomarkers of inflammation in comparison to naproxen (440 mg/day). Serum interleukins IL-1β, IL-6 and TNF-α were inhibited indicating a decrease in inflammation hence improving the symptoms of knee osteoarthritis.³¹ Another component of Acacia catechu is tannin which is present abundantly in its bark. Anti-inflammatory effect of Syzygium cumini bark which is rich in tannins, was investigated in animal models of formaldehyde induced paw edema and cotton pellet granuloma in rats. The study proved that the extract has a potent anti-inflammatory action without any adverse effect on gastric mucosa.³²

This was a short duration study, so its prolonged efficacy as antiulcer and anti-inflammatory effect is unclear.

CONCLUSION

In conclusion, the current study provides initial data on the inflammatory and antiulcer activity of Acacia catechu bark and justifies its uses. The main chemical constituents of Acacia catechu such as flavonoids, alkaloids and tannins have been shown to possess multiple medicinal properties. It is posited in this in this study that protective effects of Acacia catechu are due to its antioxidant properties.

Recommendations

Further research with extracts prepared with ethyl acetate, ethanol, and methanol on gastric mucosa should be evaluated. Future researches should focus on biochemical assays to show that there was reduced oxidative stress in Acacia catechu treated animals. Although Acacia catechu has been used for years, in traditional medicine there is still a need for well-controlled animals and human studies to evaluate its safety and efficacy.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the Institutional Ethics Committee

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