In vitro Evaluation of Anti-Inflammatory Activity of Symplocos racemosa Using Protein Denaturation Assay

Introduction: Recently there is considerable awareness and interest in the field of herbal medicine due to its natural origin and lesser side effects compared to Allopathy. Selected herbal plants like Symplocos racemosa, commonly known as lodhra, are found mainly in plains and lower hills of Bengal. The word ‘Lodhra’ means ‘Propitious’. Symplocos racemosa is an important Indian traditional drug used in many Ayurvedic and herbal formulations for treatment of liver as well as uterine disorders and leucorrhea. Ethnobotanical Literature indicates use of Symplocos racemosa Original Research Article Janani et al.; JPRI, 33(47B): 715-720, 2021; Article no.JPRI.74406 716 in treatment of eye disease, skin disease, ear disorders, liver and bowel complaints, tumours,uterine disorders, spongy and bleeding gums, asthma, fever, snakebite, gonorrhoea and arthritis. Aim: To analyse the anti-inflammatory activity of Symplocos racemosa using protein denaturation assay. Materials and Methods: 2 g of Lodhra bark powder is mixed with 100 ml distilled water & boiled for 20 min at 50°C. The extract is filtered using whatman filter paper & concentrated to 10 ml.1 ml each of Bovine serum albumin is added to various fixations of plant extract (10μL 50 μL) and the anti inflammatory activity was evaluated by analysing the percentage inhibition. Results: From this study, it is evident that Lodhra has significant anti-inflammatory activity. At 50μl concentration, the plant extract shower higher antiinflammatory activity of 76%. Conclusion: Symplocos racemosa extract has proved to exhibit effective antiinflammatory activity. Further studies have to be carried to analyse the other properties of this herb, which can be incorporated successfully in the pharmaceutical industry.


INTRODUCTION
Ayurveda is the most ancient medical system practised in India [1]. Recently there is considerable awareness and interest in the field of herbal medicine due to its natural origin and lesser side effects compared to Allopathy [2]. Selected herbal plants like Symplocos racemosa, commonly known as lodhra, are found mainly in plains and lower hills of Bengal [3] . Symplocos racemosa grow up to 6-8.5m tall, found in the plains and lower hills throughout north and East India, ascending in the Himalayas up to an elevation of 1400 m, Bengal, Assam and Chota Nagpur [4]. Symplocos is a genus of ericales, containing about 250 species native to Asia, Australia and the Americas. About 68 species are found in India, of which only a few are of economical importance. The word 'Lodhra' means 'Propitious' [5]. The Lodhra leaves appear simple, alternate, spiral; petiole up to 1.5cm long, plano convex in cross-section, glabrous;lamina 6.5-12.5 3-4.3cm, oblateolate to narrow elliptic, narrow apex, canaliculate midrib, 6-12 pairs of secondary nerves [6]. The Lodhra bark is greyish in colour, lenticellata and blaze creamy.
Symplocos racemosa is an important Indian traditional drug used in many Ayurvedic and herbal formulations for treatment of liver as well as uterine disorders and leucorrhea [7]. Symplocos racemosa belongs to the unite Eric family of symplocaceae, known as Lodhra in Sanskrit; it is a small evergreen tree, found throughout the tropics and subtropical countries [8]. Ethnobotanical Literature indicates use of Symplocos racemosa in treatment of eye disease, skin disease, ear disorders, liver and bowel complaints, tumours,uterine disorders, spongy and bleeding gums, asthma, fever, snakebite, gonorrhoea and arthritis [9]. Majority of the phytopharmacological reports on stem bark of the plant include anticancer,hepatoprotective, anti-oxidant, antiandrogenic, anti-diabetic, wound healing and anti-inflammatory effects [10].
Phytochemicals studies indicated the presence of many phenolic glycosides like symploca-side, triterpenoids like betulinic acid, acetyl oleanolic acid and oleanolic acid and flavonoids like Quertin,anthrasinins like 3-mono glucofuranoside of 7-O methyl leucopelargonidin glycosides, symposides, tannins like allergic acid, alkaloids like loturine, loturidine and colloturine [11][12][13]. Inflammation is the body's first response to infection or injury and is critical for both innate and adaptive immunity [14,15]. It can be considered as part of the complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells, or irritants [16]. Natural components and phytoconstituents are able to interfere with the inflammatory mechanisms by preventing prolonged inflammation which promotes health [17]. Symplocos racemosa has a wide spectrum of uses like anti-androgenic, anti -inflammatory, antiulcer, hepatoprotective, wound healing activities. Our team has extensive knowledge and research experience that has translate into high quality publications [18][19][20][21][22].
This study particularly aims at analysing the anti-inflammatory properties of Symplocos racemosa.

Preparation of Plant Extract
Powdered Symplocos racemosa is purchased commercially from herbal health center, in Chennai. 2 grams of the herbal powder is dissolved in 100 ml of distilled water. Then, the mixture was boiled for 20 minutes at 50 °C in a heating mantle. Afterwards, The extract is filtered finely using a whatman filter paper and allowed to stand undisturbed for about 20 minutes. 10 ml of the filtered plant extract is obtained by placing it in a hot water bath for up to 10 minutes and used for performing an anti-inflammatory assay.

Evaluation of Anti Inflammatory
Activity Using Albumin Denaturation Assay

Statistical Analysis
By means of SPSS software version 22, the association between the concentration and percentage of inhibition of protein denaturation for the Symplocos racemosa plane extract as well as diclofenac was assessed using paired t tests. The p value was found.

RESULTS
In order to determine the anti-inflammatory activity of the Symplocos racemosa plant extract, we have taken different concentrations (10 μl, 20 μl, 30 μl, 40 μl and 50 μl) and compared it with the standard anti-inflammatory drug (Diclofenac). The percentage of inhibition increases gradually, as we raise the concentration of the plant extract.
The results depicted the biosynthesised Symplocos racemosa has a percentage of inhibition of protein denaturation upto 76%, which is close to the standard drug used (86%) (Fig. 1). At the wavelength of 660nm, absorbance values were calculated using a UV spectrophotometer ( Table 1). The results were statistically analysed by SPSS software through chi square tests. P value is 0.220(p>0.05), which is statistically insignificant. Thus, there exists not a noteworthy statistical difference between the standard drug and the plant extract, this proved the good antiinflammatory activity of Symplocos racemosa.

DISCUSSION
From the advent of mankind, several medicinal plants have been exploited for various research purposes to investigate their properties which can be applied in industrial and medical fields. A lot of artificial drugs like NSAIDs which are used against inflammation are effective, but have many side-effects like gastrointestinal and renal damage. Symplocos racemosa has been used in standardisation of Ayurvedic formulations and can pave the way for global acceptance of traditional medicinal systems [23].

Fig. 1. The bar graph represents the comparison between the Symplocos racemosa and the standard anti-inflammatory agent. The X axis represents the percentage of inhibition of protein denaturation and the Y axis represents the concentration of the Symplocos racemosa plant extract in microlitres and the standard anti-inflammatory agent (Diclofenac). P value=0.220 (>0.05), which is statistically insignificant
A study has been reported that the methanolic extract of leaves of Symplocos racemosa in the concentration of 1000 milligram/ml exhibited 67 % protection against inflammation [24]. Another study revealed that the bark of Symplocos racemosa showed significant anti-inflammatory activity at 300 and 500 mg/kg doses. L. Kaviya et al., has proved that at 50 µl concentration, 81% maximum anti-inflammatory activity was shown by Lodhra bark and the efficiency of standard drugs to nearly 90% . Suppression of inflammation is by spatially and temporarily controlled production of mediators. Chronic inflammation is associated in case of cardiovascular diseases, atherosclerosis, Type two diabetes, Rheumatoid arthritis and several types of cancers. Inflammatory biomarkers involve cytokines, acute phase reactants… The elevators in inflammation involve interleukins IL-6, C-reactive proteins, lipoprotein associated phospholipases. Infectious, Non-infectious agents, damaged cells, toxic compounds, pathogens trigger inflammatory signalling pathways. Commonly, Nuclear factor kappa-light chain enhancer of activated B cells (NF-KB), Mitogen-activated protein kinase (MAPK), Janus kinase(JAK) signal transducer and activator of transcription (STAT) pathways are associated. Huong et al., in his study had revealed that the genus symplocaceae inhibits the NO production and the expression of iNOS and COX-2 proteins, thus acting against inflammation [25]. To lessen the side-effects and toxicity, Biologically prepared herbs act as an excellent alternative for commercially available synthetic drugs. Medicinal plants have a wide range of phytochemicals like secondary metabolites which are potent and safe to use. Understanding the role and mechanisms of low-grade inflammation, both as a contributor to those chronic diseases as well as to the overall health status is essential to provide clues for the development of innovative, more efficient, therapeutic and preventive strategies. The interplay between inflammation and metabolic imbalances will have its vast impact on important homoeostatic mechanisms like the Intrinsic circadian rhythm, Autophagy and cell senescence . The limitations of this study is that it made use of only one herb and analysed its anti-inflammatory activity. Moreover, No trials were performed against microbes to test its efficacy. In future, these drawbacks could be satisfied by using more herbs and analysing the other therapeutic properties of Symplocos racemosa.

CONCLUSION
In this study, Symplocos racemosa (Lodhra bark) extract has been prepared using a simple extraction method. This extract has been proved to exhibit significant anti-inflammatory activity with increasing concentration. It can act as a better alternative when compared to commercial steroidal and non-steroidal anti-inflammatory drugs prevailing in the society. Further studies have to be carried out to analyse other properties of Symplocos racemosa that can be incorporated successfully in the medical and pharmaceutical industry.

CONSENT
It is not applicable.

ETHICAL APPROVAL
For conducting the study, Ethical clearance was obtained from the institutional review board.

ACKNOWLEDGEMENT
 Saveetha Dental College and Hospitals  K K traders, Mayiladuthurai, Tamilnadu.