In-vitro Antioxidant and Anti-inflammatory Potential of Ficus infectoria Fruits

Background: Antioxidants are chemical substances, either synthetic or natural, that can avert or slow down a range of cellular damage. Objectives: The purpose of this study was to verify the antioxidant and anti-inflammatory properties of alcoholic and aqueous extracts of Ficus infectoria fruits. Materials and Methods: DPPH free radical quenching test, reducing power technique, and hydrogen peroxide scavenging tests were used to assess antioxidant efficacy. In vitro anti-inflammatory experiments were also investigated for membrane stabilization potential and inhibition of proteins from denaturation. Results: Fruits included alkaloids, steroids, phenolics, flavonoids, and amino acids, according to phytochemical study. The total phenolic and flavonoid content of the methanolic and aqueous preparations were determined to be 576.16 ± 129.10 and 416.12 ± 112.01 mg/g Gallic Acid Equivalents (GAE) and 423.17 ± 56.48 and 253.35 ± 24.07 mg/g Rutin Equivalents (RE) respectively. In comparison to the benchmarks Rutin and ascorbic acid, both preparations showed significant DPPH and H 2 O 2 quenching efficacy. As compared to the standard Indomethacin, both preparations have strong membrane stabilising and protein denaturation inhibitory potencies Conclusion: The current findings point to Ficus infectoria ’s antioxidant and anti-inflammatory properties. However, the active ingredients relevant for the activity must be identified and purified, and the exact mechanism must be determined.

and free radicals, resulting in the production of adhesion molecules and inflammatory mediators.Furthermore at the site of inflammation, free radicals involve other cellular components leads to loss of function and even death. [8]icus infectoria, often known as Philkan/white Figure, is a member of the Moraceae family.It is an important medicinal plant that is widely spread throughout the world's tropical and subtropical regions.It is a massive deciduous, fast-growing, densely foliaceous tree with a beautifully shaped crown that may reach a height of approximately 20 metres. [9]The bark of the plant has traditionally been used to treat ulcers and leucorrhoea, as well as to get rid of round worms.The leaves are also used to treat a number of skin conditions.The pharmacologically active elements found from the leaves of Ficus infectoria include -amyrin, -amyrin, lupeol, stigmasterol, and compesterol.Infectorin, bergapten, scutellarein, bergaptol, scutellarein glucoside and sorbifolin, have also been isolated from the complete plant. [10,11]The anti-oxidant and anti-inflammatory effects of aqueous and alcoholic preparations of Ficus infectoria fruits, as well as phytochemical screening, were investigated in this work.

Plant material collection and extract preparation
Mature fruits from Ficus infectoria were harvested in October 2019 from the Guru Nanak Dev University (GNDU) campus in Amritsar, Punjab.Dr. Jatinder Kaur, Head of Department, Botanical and Environmental Sciences GNDU, validated the plant, and the specimen was preserved in the GNDU herbarium under the voucher number 1793-GNDU.After drying in the shade, the fruits were roughly pulverized.500 g of powdered drug was treated to a 12-hr hot extraction with methanol using the Soxhlet device.Whatman filtration paper no. 2 was used to filter the extract.Under vacuum, the extract was concentrated.The aqueous extract was made using a simple maceration process.

Procurement of chemicals
All the chemicals utilized were procured from reputable vendors such as Rankem Gurugram, Haryana 122002, Central Drug House (P) Ltd -CDH Ansari Rd, Daryaganj, New Delhi, Delhi 110002.

Determinations of DPPH free radical quenching
With slight modifications, the technique described by Braca et al. was used to determine the DPPH free radical quenching capability of alcoholic and aqueous extracts of Ficus infectoria fruits.Simply, a freshly made solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was placed in test tubes, followed by various dilutions of fruit extracts (25 g/mL to 125 g/mL).For 30 min, the mixture was left at room temperature.A control sample was generated using a similar process but without the test sample.Using Ascorbic acid as a reference, the absorbance of the incubated solution was measured spectrophotometrically at 517 nm.Methanol was utilised as a blank solution throughout the operation. [12]

Reducing power estimation
The reductive efficacy of said preparations was estimated using Oyaizu's method with minimal modifications.Simply, the test blend was made by combining various dilutions of Ficus infectoria fruit extracts (100-500 g) with 0.2 M phosphate buffer (pH 6.6) 2.5 mL, followed by 1 percent potassium ferricyanide 2.5 mL [K 3 Fe(16)N 6 ], and incubating for 20 min at 500C before adding 10 percent trichloroacetic acid 2.5 mL.After centrifuging the whole mix at 3000 rpm for 10 min, the top layer of solution (2.5 mL) was sorted off and dissolved in distilled water (2.5 mL), followed by 0.1 percent FeCl 3 0.5 mL.A similar method was used to make standard ascorbic acid.At 700 nm, the absorbance of the solution was determined spectrophotometrically against a blank solution.The enhanced absorbance of said blend is exactly proportional to the reducing power. [13,14] 2 O 2 Scavenging Potency: The H 2 O 2 scavenging potency of F. infectoria fruit extracts was computed utilizing Ruch et al. method with slight changes.In a 7.4 pH phosphate buffer, a 40 mM H 2 O 2 solution was freshly produced.Various dilutions of the fruit extract prepared in distilled water ranging from 250-1250 µg/mL were combined into a 0.6 mL H 2 O 2 solution.The absorbance of the blend was determined spectrophotometerically at 230 nm after 10 min, using a blank consisting of phosphate buffer devoid of H 2 O 2 .The % H 2 O 2 scavenged by the test and standard solution was calculated using the following formula: The absorbance of the control solution is Ao, whereas the absorbance of the test and standard solutions is A1. [15,16]tal phenolics: The phenolic content in aqueous and alcoholic preparations of Ficus infectoria fruits was determined using the Folin-Ciocalteu technique.The extract was dissolved in distilled water to make a stock concentration of 1 mg/mL.In a test tube, 0.5 mL of extract was combined with 2.5 mL of 10% Folin-Ciocalteu's reagent made in distilled water, then 2.5 mL of 7.5 percent NaHCO 3 .The complete reagents, except the test solution, were mixed to make the blank solution.The absorbance was measured spectrophotometrically at 765 nm after incubating the reaction mixture at 45°C for 45 min.The entire analysis was carried out in triplicate using Gallic acid as a standard, and the findings were represented as mg GAE / gram of dry weight. [17]otal flavonoids: The flavonoid content of F. infectoria fruit extract was determined using Quettier's method.The residue was dissolved in alcohol at a conc. of 1 mg/mL for the estimate.I mL of 2% AlCl 3 was added to the methanolic solution.At 370°C, the reaction mixture was incubated for 1 hr.Each test analysis was carried out three times.At 415 nm, the absorbance was measured spectophometrically.The calibration curve was created using Rutin as a reference.The results were expressed in milligrams of rutin equivalents per milligram of dry extract. [18]ti-inflammatory potency The human red blood cell (HRBC) membrane stabilization method: 10 ml of blood were drawn in the heparinized vials from the healthy human subjects who did not have the prior 2 weeks history of the administration of anti-inflammatory drugs.Following that, the blood was centrifuged for ten minutes at 3000 rpm.The packed cells were suspended in an equivalent amount of saline solution after the supernatant was removed.The tubes were centrifuged and washed once more to get the clear supernatant.Dissolving human red blood cells (HRBC) in normal saline yielded a 10% suspension.After then, the suspension was held at 40°C without being disturbed.The test sample was made by combining 0.5 mL of HRBC suspension with different concentrations of fruit extracts in 1 mL of Phosphate buffer (pH 7.4), then adding 2 mL of hypotonic or hypo saline solution.Taking the different concentrations of Indomethacin as a standard, the same suspension was prepared.After incubating all the samples for 30 min at 37°C, the reaction mixtures were centrifuged at 3000 rpm.The supernatant liquid was separated and the content of hemoglobin was determined spectrophotometrically at the wavelength of 560 nm.The % membrane-stabilizing potency was estimated as follows. [19,20]× optical density of drug % Stabilizing activity 100 optical density of control respectively.At the very same amount, standard ascorbic acid had a reducing power of 1.529.Table 3.

H 2 O 2 Scavenging Potency
The ability of alcoholic and aqueous preparations of F. infectoria to scavenge H 2 O 2 was assessed as a percentage inhibition of scavenging.At a concentration of 1000 µg/mL, the alcoholic and aqueous preparations inhibited 78.41 percent and 61.25 percent of the enzymes, respectively, comparing to 98.72 percent for normal ascorbic acid.Figure 3 and Table 4.
Total phenolic and flavonoid content: Figure 4 depicts the total phenolic and flavonoid levels of alcoholic and aqueous F. infectoria rhizome preparations.Total phenolics in alcoholic and water preparations (mg/g) in Gallic acid equivalent were 576.16 and 416.21 mg/g, respectively, while total flavonoids in alcoholic and aqueous extracts (mg/g) in Rutin Equivalents were 423.17 and 253.35 mg/g (RE) Table 5.

Membrane stabilization
At a dosage of 125 µg/mL, the percentage membrane stabilising efficacy of methanolic and aqueous preparations of F. infectoria was 82.09 and 65.47 percent, respectively, while indomethacin exhibited 96.76 percent membrane stabilising potency.Table 6 and Figure 5.

Protein denaturation
Methanolic and aqueous preparations of F. infectoria inhibited protein denaturation by 67.07 and 47.17 percent, respectively, at a conc. of 500 g/mL, while indomethacin inhibited 92.36 percent at the same concentration.Tables 7 and Figure 6.

CONCLUSION
In comparison to benchmarks like ascorbic acid, rutin, and Indomethacin, the findings show that both alcoholic and aqueous preparations of Ficus infectoria fruits have high antioxidant and anti-Inhibition of protein denaturation: Protein denaturation assay was performed using slightly modified procedure prescribed by Sakat et al. 0.2 mL of egg albumin was taken from fresh hen's egg and dissolved in 2.8 ml of phosphate buffer saline of pH 6.4.To this solution, different concentrations of the fruit extract were added and the final volume was adjusted to 5 mL with double distilled water.After incubating the test samples at 37.2°C for 15 min, all of the reaction mixtures were heated for 20 min at 51°C before chilling.Against a blank, the turbidity intensity was calculated to be 660 nm.The extract's % protein denaturation inhibition was then calculated as: [21] − = × Abs control Abs sample Percentage inhibition 100 Abs control

RESULTS AND DISCUSSIONS
Phytochemical analysis of fruits revealed the presence of alkaloids, steroids, phenolics, flavonoids and amino acids Table 1.

DPPH quenching potency
At a concentration of 250 µg/ mL, the capacity of alcoholic and aqueous preparations of F. infectoria to scavenge DPPH free radicals was determined as inhibitory activity, which was 90.17 percent and 82.12 percent, respectively, while as rutin had an inhibitory activity of 97.89 percent.Figure 1 and Table 2.

Reducing power
In comparison to ascorbic acid, both the preparations of F. infectoria showed good reducing power.Figure 2. At 500 µg/mL, the alcoholic and aqueous preparations had reducing powers of 1.054 and 0.978,

Figure 1 :
Figure 1: DPPH quenching potency of aqueous and alcoholic extract of Ficus infectoria.Figure 2: Reducing power potency of aqueous and alcoholic extract of F. infectoria.

Figure 2 :
Figure 1: DPPH quenching potency of aqueous and alcoholic extract of Ficus infectoria.Figure 2: Reducing power potency of aqueous and alcoholic extract of F. infectoria.

Figure 3 :
Figure 3: H 2 O 2 scavenging potency of alcoholic and aqueous extract of F. Infectoria.

Figure 4 :
Figure 4: Total Phenolic and flavonoid content of Ficus infectoria fruits.

Figure 5 :
Figure 5: In vitro membrane stabilization potency of methanolic and aqueous extracts of F. infectoria.

Table 3 : Reducing power potency of Ficus infectoria.
inflammatory activity.However, in comparison to aqueous preparation, alcoholic preparation demonstrated statistically prominent potency.To back up the current findings, more research is being done to isolate, identify, and describe the active principle(s).