In-Vitro Evaluation Of Anti-Inflammatory Activity Of Methanolic Extract Of Bauhinia Racemosa Lam. With Diclofenac

Abstract

Commercially available synthetic anti-inflammatory medications have their own restrictions due to adverse side effects, there has always been a need for anti-inflammatory phytotherapeutic substances. Therefore, novel, highly effective anti-inflammatory medicines derived from natural sources are being investigated. Bauhinia racemosa Lam. (Fabaceae) is found throughout India. It has been found to have a range of therapeutic properties. In the present study, the potential anti-inflammatory activity of Methanolic extract of Bauhinia racemosa Lam. were evaluated and compared with standard drugs. Extraction of Bauhinia racemosa Lam. leaves was done as per the standard method. Different concentrations of the extracts were used for anti-inflammatory activity by inhibition of egg albumin denaturation. The plant have shown to have anti-inflammatory activity in a dose-dependent manner than standard drug diclofenac. Further studies on the identification of the active principles present in the leaf extract need to be done.

Keywords

Introduction

Bauhinia racemosa Lam. belongs to the Caesalpiniaceae Family. It occurs in China, Ceylon, and India. Numerous traditional therapeutic applications for plant parts have been documented, including the use of flowers as a diuretic. Dried leaves, flowers, and buds are used to treat dysentery. Root bark is applied to liver irritation (1,2). Seeds are an aphrodisiac and tonic. Chemical bio constituents such as ?–sitosterol and ?–amyrin were separated from the stem bark, (3,4) besides these bio compounds, at least two flavanols (kaempferol and quercetin) and two coumarins (scopoletin and scopoline) were separated from the leaves of the plant (5). Stilbene (resveratrol) was extracted from B. racemosa heart wood (6) The fresh flower buds of the plant showed antiulcer activity (7).

Inflammation is the body's protective response to tissue injury, microbial infection, allergies, or chemical irritants. It is a crucial immunological response that starts the healing process for injured tissue and works to eliminate dangerous stimuli. The symptoms of inflammation include heat, redness, discomfort, and swelling (8). Protein denaturation is defined as a process in which a protein is denatures as a result of external forces such as heat, strong acid or strong base, an organic solvent, or a concentrated inorganic salt. This means the proteins' secondary and tertiary structures get disoriented (9,10). Enzymes lose activity because substrates can no longer bind to the active site. Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed treatment around the world due to their proven effectiveness in minimizing pain and inflammation. NSAIDs have been linked to the prevention of protein denaturation, which functions as an antigen and triggers autoimmune conditions (11). These medications have a number of side effects, the most serious of which is stomach irritation, which can lead to the development of gastric ulcers (12). Medicinal plants are well recognized as an important source of novel chemical compounds with potential therapeutic effects. In recent years, the use of herbal medicine and natural products has expanded because of minimal cost and lesser side effects (13). Many natural plant compounds have been utilized for ages in Ayurvedic medicine to inhibit inflammatory pathways with limited side effects (14). The aim of the study was to evaluate the anti-inflammatory activity of Bauhinia racemosa Lam. using the albumin denaturation method as an indirect measure against inflammation.

MATERIALS AND METHODS

Collection and Authentication of Plant Materials

The plant material for the research work was collected from Rani Baug, Mumbai. The leaves were collected during May and June 2023. All the unwanted particles were removed and cleaned to make it free from any dust. The authentication of the plant material (Accession no. PD-431) was done at Blatter Herbarium, Mumbai.

Drying, processing, and storage of plant powder

The collected plant material was dried in the oven at 40° C for seven days. The dried plant material was powdered and passed through a series of sieves to obtain different particle-sized materials. The smallest particle-sized material (0.2 mm) was used for carrying out instrumental analysis and anti-microbial activities whereas the remaining material was used to carry out other activities.

Preparation of Methanolic Extract

About 3 g of dried sample powder was weighed and the extraction process was carried out by using 300?ml of methanol in Soxhlet apparatus for 8 hours. The extract was used for further analysis.

Assessment of In-Vivo anti-inflammatory activity by protein denaturation

Inhibition of albumin denaturation assay (15)

The reaction mixture (5?ml) contained 200??l of hen's egg albumin (fresh), 2.8?ml of phosphate buffer (pH?6.4), and 2?ml of various concentrations (100-1000??g/ml) of the standard drug (diclofenac sodium 200??g/ml), and 2?ml of distilled water was used instead of extract or diclofenac to prepare the control. The reaction mixtures were incubated for 15?min at room temperature in a BOD incubator, and then the reaction mixtures were heated at 70°C for 5?min. After cooling, the absorbance of reaction mixtures was measured at 660?nm using a UV-visible spectrophotometer using the buffer as the blank. The inhibition percentage of egg albumin denaturation was calculated using the following equation.

 

 

RESULTS AND DISCUSSION

The effect of different concentrations of Bauhinia racemosa Lam. of methanolic extract on inhibition of albumin denaturation is illustrated in Fig. 1 and Table 1. Protein denaturation is a well-known cause of inflammation. As a part of the investigation on anti-inflammatory activity, ability of different concentrations of Bauhinia racemosa Lam. methanolic extracts showed differential inhibitory activity (Table 1). Leaf extract showed inhibitory activity of 37,40,50,54 and 56% respectively, which could be comparable with the commercially available synthetic anti-inflammatory drug Diclofenac (Figure 1).

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Table 1: Effect of B. racemosa Lam. methanolic extract on protein denaturation

Protein denaturation is the most common cause of long-term inflammation. Inhibiting such forms of denaturation may therefore have a beneficial therapeutic impact on inflammation. Furthermore, stabilizing the regenerative molecules may result in better therapeutic outcomes. Mizushima and Kobayashi (1968) (16) have shown that phytochemicals inhibit protein denaturation and they have anti-inflammatory activity. Sakat et al., (2010) (17) have also slightly modified the technique to screen the anti-inflammatory activity in vitro. Recently, methanolic extract of Enicostemma axillare has been studied successfully (18). The relation between in vitro protein denaturation and in vivo anti-inflammatory action has been seen in Mikania scandens (19). Hence, this study utilized these methods to evaluate in vitro anti-inflammatory activity.

In the current study, it was seen that as the concentration of extract increased, there was a steady rise in anti-inflammatory activity. In relevance to dose-dependent protein protective activity, the change in concentration of extract showed a proportional increase in activity. Although the percentage inhibition values in the extract at high concentrations approached more than 50%, this study solely used egg albumin to evaluate the extract.  In a clinical scenario, there are many more proteins, that are involved in inflammation and repair. While an indication is provided for potential protein protective activity, in vivo testing is essential to prove the clinical utility of the extracted phytochemicals. Further studies on the identification of the active principles present in the leaf extract are warranted to ascertain its possibilities. In the future, oral bio-availability and mechanism of action should be elaborated.

CONCLUSION

As a part of the investigation on anti-inflammatory activity, the ability of different concentrations of B. racemosa Lam. methanolic leaf extracts showed differential inhibitory activity. The inhibition rate of egg albumin denaturation for methanol extraction increased gradually with an increase in concentration. Significantly higher inhibition was observed at the concentration of 1000 ?g/ml than the reference drugs. Our present study concludes that  B. racemosa Lam. has anti-inflammatory properties which could be due to the presence of active constituents in the plant extract. Hence, B. racemosa Lam. may serve as one of the anti-inflammatory herbal drug source.

CONFLICT OF INTEREST

There is no conflict between the authors.

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