Determination of Total Phenolic Content and Antioxidant Activity of the Whole Plant Extracts of Argyreia imbricata (Roth) Sant. & Patel

Sebastin V*1, Gopalakrishnan G2, Sreejith M3, Anoob Kumar K I4 1Department of Pharmaceutical Chemistry, Malik Deenar College of Pharmacy, Seethangoli, Kasaragod, Kerala, India 2Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, Tamil Nadu, India 3Department of Pharmaceutical Chemistry, Nazareth College of Pharmacy, Thiruvalla, Pathanamthitta, Kerala, India 4Department of Pharmaceutical Chemistry, KVM College of Pharmacy, Cherthala, Alappuzha, Kerala, India

Plants of the genus Argyreia posses' ethno medicinal importance and various pharmacological activities are also reported. In this study, phenolic content, anti-oxidant potential of different extracts of Argyreia imbricata was evaluated. Extraction of powdered whole plant material was done with different solvents viz., petroleum ether, chloroform, ethyl acetate and methanol by soxhelation. The estimation of phenolics in the selected extracts of Argyreia imbricata was done by using a Folin-Ciocalteau reagent. The antioxidant potential of different extracts was evaluated by total anti-oxidant capacity, DPPH, hydroxyl and nitric oxide radical scavenging and reducing power assay methods. In the phenolics estimation, the methanol extract showed a signi icant presence of phenolic content followed by ethyl acetate extract. In case of anti-oxidant activity, the methanol and ethyl acetate extract showed a significant activity based on their concentration. The indings of this study clearly revealed the potential of methanol extract of A. imbricata in phenolic content and anti-oxidant activity. Also, the ethyl acetate extract showed signi icant results. Further studies on identi ication, isolation of the active constituents, toxicity evaluations may give useful results.

INTRODUCTION
The genus Argyreia belongs to the Convolvulaceae family is a larger one, consist of more than 200 species that have a wide distribution in Asian countries, including all over India (Traiperm et al., 2017;Sebastin et al., 2019). Various species of this genus have ethnomedicinal importance and diverse pharmacological activities of the Argyreia plants were described in various reports (Modi et al., 2010;Joseph et al., 2011;Jaiswal and Tailang, 2018). Argyreia imbricata, another one plant of this genus, is commonly found in south India at an altitude up to 300m mean sea level, which has not been previously explored scienti ically to our knowledge, was selected for our research.
Initially, extraction, preliminary phytochemical evalutaion and the spectral evaluation of active constituents in the extracts of the whole plant material of Argyreia imbricata was done (Sebastin et al., 2019). As a part of the investigation of different pharmacological activities, the anti-in lammatory activity (Sebastin et al., 2020) and the anti-diabetic activity (Sebastin et al., 2021) of the extracts were evaluated successfully. Now, it was focused on the evaluation of total phenolic content and anti-oxidant activity of the prepared extracts of the whole plant Argyreia imbricata.

Collection and extraction of plant material
The entire plant Argyreia imbricata was collected from the Mekkarai, a village located nearby the hillocks of the Western Ghats in the Tirunelveli District of Tamil Nadu, India. The collected material was dried and powdered after the proper identiication and authentication. The powdered material of the whole plant was extracted by soxhelation with the solvents in the rising order of polarity, such as petroleum ether, chloroform, ethyl acetate, and methanol (Sebastin et al., 2019). The dried extracts obtained were utilized for further experiments.

Total phenolic content estimation
In our preliminary phytochemical evaluation (Sebastin et al., 2019), only the ethyl acetate and methanol extract of the Argyreia imbricata showed the presence of phenolic compounds. Based on this inding, these two extracts were selected for the estimation of total phenolic content, which was done in accordance with the procedure of (Francis and Andrew, 2010;Senguttuvan et al., 2014;Baba and Malik, 2015) with slight modi ication.
Test extracts (0.5ml) of test extracts in different concentration were mixed with an equal volume of Folin-Ciocalteau reagent and 1ml of 20% sodium carbonate solution. The volume of the mixture was made in to 10ml by distilled water. Shaken well and kept at the dark condition for 45min., followed by the absorbance of the solution was measured spectrophotometrically (725nm). Blank was prepared. A calibration curve of gallic acid was prepared, and the linearity was obtained. The total phenolic content in the test extracts was expressed as milligram of gallic acid equivalent (mg GAE/g extract) by using the standards curve.

In vitro antioxidant activity
Antioxidant activity of all the extracts of A. imbricata was evaluated by total antioxidant capacity, DPPH, hydroxyl and nitric oxide radical scavenging and reducing power assay methods.

Total antioxidant capacity (TAC)
Evaluation of the total antioxidant capacity of the 0.1ml of test extracts (100µg/100µl) was mixed with the reagent (0.6M sulphuric acid, 4mM ammonium molybdate and 28mM sodium phosphate). The tubes containing the reaction mixture were covered with silver foil and kept in the water bath (95 • C) for 1.5h. Cooled to room temperature and the absorbance of reaction mixtures was measured spectrophotometrically at 695nm. Gallic acid was used as the standard. The total antioxidant capacity was calculated by the formula T = C V M T -Total antioxidant content (mg/g) of the extracts; C -Concentration of Gallic acid (mg/ml) obtained from the calibration graph; V -Volume of the extract taken (ml); M -the weight of the extract (g); TAC is expressed as milligram Gallic acid equivalents (mg GAE)/g of extract.

DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay
DPPH assay of the tests was carried out with reference of (Mol et al., 2016;Jemli et al., 2016). Reaction mixture contains 1ml of test extracts in different concentration (50, 100, 250, 500 and 1000µg/ml) and 1ml of methanolic solution of DPPH (0.1mM). It was kept in 30min. Incubation in dark at room temperature. Then, the absorbance of the reaction mixtures was measured at 517nm, using methanol as the blank, DPPH in methanol as the control and ascorbic acid as the standard control. The inhibition percentage of DPPH radical by the tests was identiied by The percentage of inhibition against concentration was plotted as a graph. The IC 50 values of the samples were assessed from the regression equation of the graph.

Hydroxyl radical scavenging assay
It was assessed in reference with (Batool et al., 2019). Deoxyribose (0.1ml; 2.8mM) prepared in potassium phosphate buffer (20mM; pH 7.4). To this EDTA (0.1mM), ferric chloride (200µl; 100mM), H 2 O 2 (100µl; 200mM) and 100µl of the test extracts in different concentration were added. After the addition of ascorbic acid (100µl; 300mM) to this mixture, it was kept in 37 • C for 1h. then trichloroacetic acid (1ml; 2.8%w/v) and 1%w/v thiobarbituric acid (1ml) prepared in 50mM NaOH were added, and the whole reaction mixture was kept in the water bath (15min) for boiling. Then the absorbance of cooled was measured spectrophotometrically (532nm). Normal and standard control were prepared. Quercetin was applied as the standard control. Suppression of deoxyribose degradation was calculated by Nitric oxide radical scavenging assay The nitric oxide radical scavenging potential of the tests was evaluated in reference with the procedure of (Francis and Andrew, 2010;Batool et al., 2019). The reaction mixture (5ml) was prepared by mixing sodium nitroprusside (5mM) in phosphate buffer (pH 7.3) and test extracts in different concentration. After the addition of 1ml of Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% naphthyl ethylene diamine dihydro chloride) to the reaction mixture, it was kept in 25 • C (in front of a 25W tungsten lamp) for 3h. The nitric oxide radical thus formed was interacted with oxygen to produce nitrite ion, which was measured spectrophotometrically (546nm). Normal and standard control were prepared. Ascorbic acid was employed as the standard control. The percentage inhibition nitric oxide radical formation was determined by

Reducing power assay
Evaluation of the antioxidant activity of test extracts by reducing power assay method was done in reference with the procedure of (Jemli et al., 2016;Moonmun et al., 2017). 500µl of the test extracts in different concentration (50, 100, 250, 500, 1000µg/ml) was mixed with 1.5ml of sodium phosphate buffer (0.2M; pH 6.6) and 1.5ml of 1% potassium ferricyanide. This mixture was incubated at 50ºC for 20min. Then, 5ml of 10% trichloroacetic acid was added to the incubated mixture and centrifuged at 3000rpm for 6min (4 • C) and the upper layer solution was collected. 1.5ml of the collected upper layer solution was mixed with an equal volume of distilled water and 300µl of 0.1% fresh ferric chloride solution and kept for 10min. Then, the absorbance of the reaction mixture was read at 700nm. Ascorbic acid was used as the standard for comparative evaluation.

RESULTS AND DISCUSSION
Results of total phenolic estimation are shown in Tables 1 and 2 and Figure 1. The results clearly indicated a signi icant presence of phenolic content in the methanol extract followed by the ethyl acetate extract (Table 2).
Regarding with antioxidant evaluation, the result of the total antioxidant capacity of the extracts tested by phosphomolybdate assay is shown in Table 3.
Generally, a higher absorbance value indicated the greater antioxidant capacity of test extracts. In this evaluation, the ethyl acetate and methanol extracts of A. imbricata showed a signi icant activity comparing with others, such as petroleum ether and chloroform extracts. The ethyl acetate extract showed the absorbance value of 1.788 and TAC of 87.63mgGAE/g followed by methanol extract with an absorbance value of 1.767 and TAC of 1.767mgGAE/g comparing with the absorbance value of 2.115 of the standard gallic acid.
In the case of evaluation of antioxidant activity by DPPH assay, the result is shown in Table 4. The results indicated a concentration based rise of percentage inhibitory activity of test extracts. The tested methanol extract showed a signi icant percentage inhibition score of 84.78, followed by the ethyl acetate extract showed 83.95 as the score of percentage inhibition. The IC 50 value of both the extracts was found as 108.85µg/ml. These results are signi icant one when comparing with the scores of the standard ascorbic acid.
In the case of hydroxyl radical scavenging assay, a concentration depended rise of percentage inhibition was found in the results. It was strongly revealed by the methanol extract in 1000µg/ml concentration with the score of percentage inhibition of 89.43±0.345 followed by the ethyl acetate extract in the same concentration with the percentage inhibition score of 76.34±0.078.
The score of methanol extract is a signi icant one comparing with the score of the standard, quercetin, which showed 91.13±0.174 percentage inhibition in the concentration of 100µg/ml (Table 5) The results of the nitric oxide radical scavenging       In this method also the methanol extract (1000µg/ml) showed a signi icant score next to that the same concentration ethyl acetate extract showed a signi icant score comparing with other tested extracts and the standard ( Table 6).
The result of an antioxidant evaluation of A. imbricata extracts by reducing power assay is shown in Table 7.
Generally, increased absorbance of the reaction mixture indicated increased reducing power. From the results, it was found that the methanol extract in 1000µg/ml concentration showed 2.625 as the absorbance score followed by the ethyl acetate extract in the same concentration showed 2.623 absorbance score, which clearly indicated the antioxidant potential of above said extracts comparing with the standard, ascorbic acid.

CONCLUSION
In the present study, the whole plant Argyreia imbricata was collected and after the identi ication and authentication, the collected material was dried, powdered and extracted by soxhelation with solvents of ascending order of polarity viz., petroleum ether, chloroform, ethyl acetate, and methanol. Selected extracts were subjected to the estimation of phenolic content and all the extracts prepared were evaluated for the anti-oxidant activity by different methods. In both the estimation of phenolic content and evaluation of antioxidant activity, the methanol extract of the whole plant A. imbricata showed signi icant results and next to that the ethyl acetate extract. Our future studies in the direction of identiication and isolation of the active constituents and toxicity evaluations of these extracts may give valuable results for further researches.

ACKNOWLEDGEMENT
Authors are thankful to Mr. Kumaran J., M. Pharm., (Pharmaceutical Biotechnology), for his help in the preparation of this manuscript.