Indian Journal of Animal Research

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Anti-inflammatory Activity of Azima tetracantha Root Bark Extract in Rats

R. Nagulsamy1, L. Kalaiselvi1,*, S.P. Preetha1, R.P. Aravindh Babu1, S. Ramesh1
1Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Chennai-600 007, Tamil Nadu, India.
Inflammation is associated with many pathological conditions such as arthritis, inflammatory bowel syndrome, cancer, etc. The side effects associated with the long term use of current anti-inflammatory drugs necessitates the need for the development of new drug with fewer side effects. The present study was aimed at evaluating anti-oxidant and anti-inflammatory activity of Azima tetracantha root bark, a plant widely used by tribal people and in Siddha medicine for the treatment of arthritis and rheumatism. Aqueous and methanolic extracts of Azima tetracantha root bark were prepared. Total phenol, flavoid, alkaloid, terpenoid and saponin content were analyzed. The anti-oxidant activity was determined by DPPH assay and total antioxidant capacity. The anti-inflammatory activity was evaluated by carrageenan induced paw edema model in rats. The total phenol and flavonoid contents were higher in methanolic extract compared to aqueous extract. The extracts were found to have anti-oxidant activity and dose-dependent anti-inflammatory activity in both male and female rats with highest average inhibition at 400 mg/kg. The results confirm the traditional use of this plant against inflammatory disorders and could be source for the development of novel drugs.
Inflammation plays a very important role in tissue healing but uncontrolled chronic inflammation is associated with many disease conditions such as arthritis, asthma, atherosclerosis, cancer, diabetes, Alzheimer’s disease, Parkinsons disease, etc (Borquaye et al., 2020). Chronic inflammatory diseases continues to be one of the major health problem and it is usually treated using non-steroidal anti-inflammatory drugs (NSAIDs), steroids and immunosuppressants (Hussain et al., 2020). NSAIDs are most commonly used to control inflammation and pain in those conditions but their long term treatment can cause serious adverse effects such as gastric ulceration and bleeding, liver damage, kidney damage, stroke, cardiovascular complications and so on. Hence, there is an absolute need for alternate drugs with maximal efficacy and minimal side effect, which necessitates the pursuit of novel drugs from alternate systems of medicine.
Azima tetracantha is widely used in the alternative systems of medicine for the treatment various disease conditions including rheumatoid arthritis, cough, cold, fever, body pain, bronchitis, asthma, dropsy, etc. (Kekuda and Raghavendra, 2017). The roots of the plant are used in the herbal formulation called Pilavaikkalimbu, which is used topically in the treatment of tumors. The root of this plant is one of the components of Parangichakkai Choornam, polyherbal Siddha formulation which is indicated in the treatment of various diseases of pitha and kabha origin (Kumarasamy and Kumarswamy, 2014). There are studies that have corroborated pharmacological actions of leaf extract of this plant. However, there is little information about phytochemical composition and pharmacological actions of root extracts. Since the root bark is used by traditional healers and in Siddha medicine, this study was attempted to elucidate phytochemical composition and to validate anti-oxidant and anti-inflammatory potential of aqueous and methanolic extract of root bark of A. tetracantha.
The present study was conducted at Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Chennai from March 2022 to May 2022.
Azima tetracantha whole plant was collected and authenticated by Botanist at Department of Medicinal Botony, Government Siddha Medical College, Arumbakkam, Chennai. Aqeuous and methanolic extracts of root bark were prepared by maceration technique, lyophilized and stored at 4°C. The total yield of aqueous and methanolic extracts was 12% and 4%, respectively.
The qualitative phytochemical screening tests were carried out as per the procedure described by Deyab et al., (2016). The total phenol content in the extracts was determined by Folin- Ciocalteau method using gallic acid as a standard (Singh and Maurya, 2009). The total flavonoid content of the extracts was determined by aluminium chloride method (Kamtekar et al., 2014) using Catechin as a standard. The alkaloid content of the extract was determined by titrimetric method as described by Debnath et al., (2015).  The terpenoid and saponin contents of the extract were determined as per the method described by Malik et al., (2017) and Ezeonu and Ejikeme (2016). respectively
The free radical scavenging activity of plant extracts were evaluated using DPPH (2, 2-diphenyl-1-picryl hydrazyl) (Zahin et al., 2009). The total antioxidant capacity of the extract was measured by phosphomolybdenum method as described by Wan et al., (2011) and expressed as ascorbic acid equivalent (AAE) per g of extract.
The anti-inflammatory activity was evaluated by carrageenan induced paw edema model in Wistar rats. The rats were maintained under standard conditions of relative humidity and temperature and provided with rodent pellet feed and water ad libitum. The protocol was approved by the Institutional Animal Ethical Committee, Madras Veterinary College (12/SA/IAEC/2022). The animals were acclimatized and randomly divided into 6 groups as given in the experimental design below (Table 1).

Table 1: Experimental design.

Carageenan was injected into sub-plantar region of hind paw to induce acute inflammation. Paw edema was measured before and at various time intervals after carrageenan injection. The anti-inflammatory activity was calculated as percentage inhibition of oedema in comparison to the carageenan control using the formula,
Vc = Oedema volume of carrageenan control group.
Vt = Oedema volume of treatment group.
The results were expressed as mean±standard error of the mean (SEM) and statistically analysed by one-way ANOVA followed by Duncan’s Posthoc analysis. Values were considered significantly different at P<0.05.
The alternative systems of medicine have gained attention in the recent years. With the increased use of herbal drugs, validation of efficacy of the herbal plants by scientific studies becomes essential. A. tetracantha root is widely used in the siddha medicine and also by tribal people and traditional healers in the treatment of various disease conditions including rheumatoid arthritis. In the present study, the preliminary phytochemical analysis of A. tetracantha root bark extracts revealed the presence of various phytochemicals which includes terpenoid, phenol, alkaloid, flavonoid, saponin, tannin, quinine and carbohydrates (Table 2).

Table 2: Phytochemical screening of Azima tetracantha root bark extract.

The total phenolic and total flavanoid content was found to be higher in methanolic extract compared to aqueous extract (Table 3). Our findings is consistent with several reports of higher total phenol content in  methanol extract than the other solvents which could be due to more effective extraction of polyphenolic chemicals in methanol (Jung et al., 2004; Choi et al., 2007). The terpenoid content in the methanolic extract was higher than aqueous extract whereas aqueous extract had higher saponin content. Phytochemical data on A. tetracantha root bark are very limited and to the best of our knowledge, this is the first report of the phytoconstituent in aqueous root extracts of A. tetracantha.

Table 3: Quantitative phytochemical analysis of Azima tetracantha root bark extract.

In DPPH assay, methanolic extract showed higher antioxidant activity with an IC50 of 0.226 mg/mL compared to aqueous extract (IC50-1.19 mg/mL) (Fig 1). The total antioxidant capacity of aqueous and methanolic extracts of A. tetracantha root were 117.81±13.91 and 126.35±126.35 mg AAE per g extract.

Fig 1: DPPH free radical Scavenging activity.

Phytochemicals such as phenols and flavonoids contributes to the antioxidant activity of the plant extracts (Ravipati et al., 2012). In the present study, we found higher amount of total phenol and flavonoids in methanolic extract compared to aqueous extracts and this was reflected by higher antioxidant activity in methanolic extract compared to aqueous extract.
The anti-inflammatory effect of the extracts was evaluated in carrageenan-induced paw edema model.  In the present study, carrageenan control rats showed increased paw edema which increases in time with maximum edema at 3-4 h after carrageenan injection. Both the extracts of A. tetracantha caused reduction in paw edema compared to carrageenan control which was significant at all time points in both male and female rats (Fig 2 and 3). The % anti-inflammatory activity are given in Table 4 and Table 5. Both the extracts showed dose-dependent inhibition of paw edema from 1 h and it continued up to 12 h in both male and female rats with maximum activity at 400 mg/kg. In male rats, inhibition of inflammation in rats treated with high dose of extracts was higher and did not differ significantly from the diclofenac standard.

Table 4: Per cent inhibition of Inflammation in Male rats.


Table 5: Per cent inhibition of inflammation in female rats.


Fig 2: Changes in Paw edema volume in male rats.


Fig 3: Changes in paw edema volume in female rats.

The carragenan induced paw edema is a well established animal model to evaluate anti-inflammatory property of the herbal products. The development of carrageenan induced paw edema occurs in two phases. The first phase is observed within 1-2 h of induction and is mediated by histamine, serotonin and bradykinin. The second phase (3-6 h) is mediated by prostaglandins, nitric oxide and various cytokines including IL-6, IL-1β, TNF-α (Karim et al., 2019). In present study, we observed inhibition of edema formation by the extracts of A. tetracantha in both first phase and second phase and maximum inhibition was noticed with methanolic extract at 400 mg/kg. 
The results of the study demonstrated antioxidant and anti-inflammatory activity of A. tetracantha and provides scientific validation and justification for the traditional use of this plant in inflammatory conditions. The activity could be due to phytochemicals such as to phenols, flavonoids and terpenoids present in the extract. Further studies are warranted to isolate the bioactive compound and to elucidate the mechanism of action.
This study was funded by Tamil Nadu Veterinary and Animal Sciences University, Chennai.
The authors declare that they have no conflict of interest.

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