INTRODUCTION:

Inflammation is a complex biological response stimuli, pathogens, irritants characterized by redness, warmth, swelling and pain prolonged inflammations leads to the rheumatoid arthritis, atherosclerosis, heavy fever, ischemic heart diseases¹. Inflammation is a common manifestation of infectious disease like leprosy, tuberculosis, syphritis, asthma, inflammatory bowel syndrome nephritis vascularitis, celeic disease, auto-immune disease etc. Anti-inflammatory drugs like NSAIDS used to reduce the swelling and pain of inflammation. But these agents carry the risk of gastro-intestinal toxicity, cardiovascular and other toxicity for prolonged use for anti-inflammatory drugs having less severe side effects to use for chronic inflammatory disease as well ^2,3. Therefore, in recent time, more interest is shown in alternative and nature drugs for treatment of various diseases, but there is a lack of proper scientific evidence. Arthritis is an auto immune disorder characterized by pain swelling and stiffness. Its prevalence depends upon age. It occurs more frequently in women than in men. It is an inflammation of synovial joint due to immune mediated response. All antiinflammatory drugs are not antiarthritic because it does not suppress T-cell and B-cell mediated response Epidemiological studies overall show a female to male radio of about 3:1.These are many class of anti-arthritis drugs are available like NSAIDS, Monoclonal anti-bodies, uricosuric agents, gold compounds, anti-cytokinine immunosuppressant like Glucocorticoids, etc⁴. But this all class of drugs is responsible for symptomatic relief to evaluate the drug which actually prevent cause of arthritic there is requirement of evaluative model which produce arthritis in vial same that produce in humans. Animal models of arthritis are used to potential anti-arthritic drugs for clinical use. Therefore morphological similarities of human disease and capacity of the model to predict efficacy in humans are important criteria in humans are important criteria in model selection ^5,6. Arthritis involves the breakdown of cartilage and affects the weight beating joints such as Feet, Knees, Hips, Spine, Finger, Thumb joints. Controlling the production of auto antigen and inhibits denaturation of protein in rheumatic disease plays a major role in treating arthritis. Ficus carica (Common Figs) is the one of the important medicinal plant in Moraceae family, which is distributed in India, Pakistan, Afghanistan, Russia, Iran, Middle East, North Africa and Europe, Today the United States, Turkey, Greece and Spain are the primary producer of dried figs ⁷. Ficus carica root and leaves are used in the native system of medicine in different disorders such as gastrointestinal (colic, indigestion loss of appetite and diarrhea), respiratory (sore throats cough and bronchial problems), inflammatory and cardiovascular disorders and as antispasmodic ^8,9. Since there is no scientific evidence for antiinflammatory and antiarthritic activity of Ficus carica, therefore current research is aimed to evaluate the anti-inflammatory and antiarthritic activity of Ficus carica.

MATERIALS AND METHODS:

Preparation of plant material:

The fresh leaves of Ficus carica was collected from Periyakuppam Kambar Street, Thiruvallur District, Tamil Nadu, and India. Month of March 2017 and authenticated by Professor P. Jayaraman and the authentication number is PARC/2017/3512, The collected leaves were cleaned, washed with distilled water, dried under sunshade in dark room, and powdered by using mechanical mixer. After size reduction leaves were sieved under sieve No. 40 and sieve No. 60, stored in airtight container at room temperature.

Extraction of the plant material:

An aqueous extract of leaf of Figus carica (AELF) was prepared by heating the mixture of powder with water at 50-60º C till water reduces to 1/8th of its volume. This procedure involves simple decoction process to obtain the soluble materials being extracted from the crude raw plants, which was then cooled and filtered. The filtrate that obtained by decoction process was then concentrated. The concentrated aqueous extract of Figus carica (AELF) was stored at 2-5 °C until completion of study ^10,13.

Preliminary phytochemical analysis:

The obtained extract was subjected to phytochemical evaluation and identified the various plant constituents present in the test sample qualitatively ^11,12,14,15.

In vitro anti-arthritic activity:

Bovine serum denaturation method:

About 0.05ml of various concentrations (50, 100, 250µg/ml) of AELF and standard drug diclofenac sodium (50, 100, 250µg/ml) were taken respectively and 0.45ml (0.5%w/v BSA) mixed. The samples were incubated at 37⁰C for 20 minutes and the temperature was increased to keep the samples at 57⁰C for 3 minutes. After cooling, add 2.5 ml of phosphate buffer to the above solution. The absorbance was measured using UV –visible spectrophotometer at 255nm. The control represents 100% protein denaturation^16,17. The results were compared with diclofenac sodium. The percentage inhibition of protein denaturation can be calculated as

Percentage inhibition=100-[optical density of test solution -optical density of control] X 100.

Egg albumin denaturation method:

The reaction mixture (5ml) consisted of 0.2ml of egg albumin (from fresh hen’s egg) 2.8ml of phosphate buffered saline (pH 6.4) and 2ml of varying concentrations (100, 250, 500µg/ml) of AELF. A similar volume of double distilled water served as a control. Next the mixtures were incubated at 37±2⁰C in a BOD incubator for 15 min and then heated at 70⁰C for five min, after cooling their absorbance was measured at 600 nm by using the vehicle as a blank. Diclofenac sodium in the concentrations of 100, 250, 500 µg/ml was used as the reference drug and treated similarly for the determination of absorbance^18,19. The % inhibition of protein denaturation was calculated by using the following formula.

%Inhibition=100 [A1-A2/A1].

Where, A1=absorbance of control, A2=absorbance of the test sample. Each experiment was done in triplicate and the average was taken. The extract concentration for 50% inhibition (IC₅₀) was determined by the dose response curve.

RESULTS AND DISCUSSION:

The preliminary phytochemical studies revealed that the presence of Terpenoids, Flavonoids, Alkaloids, Tannins, phenols, Saponins, Steroids, sterols, Carbohydrates and Protein in AELF which is shown in Table 1.

Table 1: Preliminary phytochemical study of AELF

S. No.	Name of the secondary metabolite	Inference
1	Terpenoids	Present
2	Flavonoids	Present
3	Alkaloids	Present
4	Glycoside	Absent
5	Tannins and phenols	Present
6	Saponins	Present
7	Steroids and sterols	Present
8	Carbohydrates	Present
9	Protein	Present

In vitro antiarthritic activity of AELF by BSA method was shown in table 2 and figure 1. The results revealed that the percentage inhibition of denaturation of protein was increased with dose dependent manner. There is no significant difference between the standard and AELF which confirms the antiarthritic activity of AELF.

Table 2: In vitro antiarthritic activity of AELF by bsa method

CONCENTRATION (μg/ml)	% inhibition of denaturation of protein
CONCENTRATION (μg/ml)	plant extract	Standard
100	42.4	54.73
250	63.27	73.12
500	76.9	86.44

Figure 1: Percentage inhibition of denaturation of protein by BSA method

In vitro antiarthritic activity of AELF by egg albumin method was shown in table 3 and figure 2. The results revealed that the percentage inhibition of denaturation of protein was increased with dose dependent manner. There is no significant difference between the standard and AELF which confirms the antiarthritic activity of AELF.

Table 3: In vitro antiarthritic activity of AELF by egg albumin method

Concentration (μg/ml)	% Inhibition of denaturation of protein
Concentration (μg/ml)	Plant Extract	Standard
100	51.12	68.11
250	58.75	70.83
500	64.64	76.16

Figure 2: Percentage inhibition of denaturation of protein by egg albumin method

DISCUSSION:

Arthritis is one of the foremost health troubles worldwide, leading cause of disability in western and developing countries. Therapies developed along the principles of western medicine are often limited in efficacy, Carry the risk of adverse effects, and are often too costly, especially for the developing world. Therefore, treating arthritis with plant derived Compounds which are accessible and do not require laborious pharmaceutical synthesis seems highly attractive. Antiarthritic activity of aqueous extract of Ficus carica was studied significantly by using in vitro inhibition of protein denaturation method (BSA, egg albumin). Extract of Ficus carica at 3 different concentration provided significant protection against denaturation of proteins. The plant contains secondary metabolites such as Steroids, Triterpenoids, alkaloids and flavonoids. These constituents are responsible for this activity. Mechanism of denaturation probably involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding. Hence proper isolation of active principles might help in the findings of new lead compounds in the fields of antiarthritic drug research obtained data stated that Ficus carica extract could be used as potent antiarthritic agent.

CONCLUSION:

Antiarthritic activity of aqueous extract of Ficus carica was studied significantly by using in vitro inhibition of protein denaturation method (BSA, egg albumin). Extracts of at Ficus carica three different concentration provided significant protection against denaturation of proteins. The plant contains secondary metabolites such as Steroids, Triterpenoids, alkaloids and flavonoids. These constituents are responsible for this activity. Further study needed to isolation of active principles responsible for antiarthritic activity, in future it would be important to understand molecular mechanism of Ficus carica.

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Received on 29.01.2020 Modified on 18.02.2020

Asian J. Research Chem. 2020; 13(2):151-154.

DOI: 10.5958/0974-4150.2020.00030.9