Methanol, ethanol, ethyl acetate, chloroform, toluene, n- hexane, hydrogen peroxide, sodium phosphate, acetic acid, potassium dichromate, glacial acetic acid, DPPH, ascorbic acid, ferric chloride, ferrozine, EDTA, sodium acetate, TPTZ, ferrous sulphate, etc. are purchased from Eswarr Scientific and Co., Karumandapam, Trichy- 620 001.

Healthy unripened Areca catechu L. nuts were collected from Kollam district of Kerala, India. It was dehusked and dried for three weeks. The dried seeds were powdered. The plant Areca catechu and Areca catechu L. nut were authenticated by JNTBGRI, Thiruvananthapuram, Pin 695 562, Kerala, India, and voucher specimens (Specimen Numbers TBGT/95955 & TBGT/95956) are deposited at the herbaria of the same research institute.

Soxhlet extraction is a very useful method for extraction purposes especially in plant materials (Abubakar et al 2020). Soxhletation is a process of extraction conducted in a device called soxhlet (Syintia et al. 2019). The powdered Areca catechu L. nut was subjected to extraction using the soxhlet apparatus (Anajwala et al. 2010). Repeat the same with various solvents based on polarity for about 16–24 hours (Kalaivani et al. 2019). The Soxhlet extractor is placed onto a flask containing the extraction solvents (Hirondart et al. 2020). The Soxhlet is then equipped with a condenser was used to get the extracts used in this study (Gopalasatheeskumar 2018). The powdered Areca catechu L. nut (25 g) was made as a roll with quality tissue paper and placed in the thimble (Maria et al. 2012). The solvent was heated to reflux (Abdul et al. 2012). The solvents used in the extraction are methanol, n- hexane, ethyl acetate, toluene, and chloroform. The process was continued up to when the solution became colorless in the thimble (Naczk et al.2006). The flask was removed from the Soxhlet extractor and poured into 250 ml beaker and evaporated to get the different solvent extracts of Areca catechu L. nut (Naveen et al. 2010).

DPPH assay method is very simple and used to find the overall antioxidant capacity of the sample (Moniruzzaman et al. 2012). For the DPPH reagent, the concentration used was 2.5 mg/ml. One ml of ethanol solution was added to the test tubes followed by the addition of Areca catechu L. nut extracts, 0.1 ml of 25–100 µg /ml (1 mg/ml stock). The DPPH reagent was added to each tube in a dark environment. Then, it was wrapped with aluminum foil and incubated on the shaker at room temperature for 15 minutes. The absorbance was recorded at 517 nm. Ascorbic acid was used as the standard at the same concentration (Athavale et al. 2012).

$\text{DPPH scavenging effect}(\%)=\left(1-\frac{Ac}{As}\right)·100$

Where A_c is the absorbance of the control which contains DPPH solution and A_S is the absorbance presence of different solvent extracts of Areca catechu L. nut (Gulcin et al. 2010). The percentage (%) inhibition of DPPH activity is used to find the change in absorbance between the sample and control. Ascorbic acid in ethanol was taken as a positive control and the assays were repeated for getting a triplicate of each sample concentration (Tuekaew et al. 2014).

The ability of plant extract to scavenge hydrogen peroxide is determined by using the reaction mixture containing 0.5ml of H₂O₂ (1ml of 30% of H₂O₂ was made up to 45 ml with distilled water), 1 ml of sodium phosphate buffer (pH 7.4), and 0.4 ml water. 0.1ml of the sample (25–100 µg/ml), was added to start the reaction. 2 ml dichromate acetic acid reagent (Dichromate acetic acid -5% potassium dichromate with glacial acetic acid in ratio 1:3) was added after 1 min to stop the reaction. The tubes were heated for 10 minutes, cool and the green color appeared was read at 240 nm using a spectrophotometer. Extracts (25–100 μg/ml) in distilled water is mixed to H₂O₂ and absorbance at 340 nm is noted after 10 min against a blank solution missed with phosphate buffer without hydrogen peroxide (Alam et al. 2013).

${\mathrm{H}}_2{\mathrm{O}}_2\text{scavenging effect}(\%)=\left(1-\frac{Ac}{As}\right)·100$

Where A_C is the absorbance of the control and As is the absorbance of different solvent extracts of Areca catechu L. nut (Gulcin et al. 2010).

The reaction mixture contains 1.0 ml of various concentrations of the herbal extract (25–100 µg /ml) and 0.05 ml of 2 mm FeCl₂. The reaction was carried out by the addition of 0.2 ml of 5 mm ferrozine. The reaction mixture was prepared and kept for 10 min and the absorbance of the reaction mixture was measured at 562 nm against a reagent blank. A smaller absorbance of the reaction mixture showed a higher ferrous ion chelating ability. All the reagents except the sample contained in the control. EDTA was used as a standard for comparison (Shahat et al. 2013).

$\text{Ferrous ion}\left({\mathrm{Fe}}^{2+}\right)\text{chelating effect}(\%)=\left(1-\frac{Ac}{As}\right)·100$

Where A_C is the absorbance of the control and A_S is the absorbance of different solvent extracts of Areca catechu L. nut (Gulcin et al. 2010).

By mixing 300 micromoles sodium acetate buffer (pH 3.6), 10.0 micromoles TPTZ (tripyridyltriazine) solution, and 20.0 micromoles FeCl₃.6H₂O solution in a ratio of 10:1:1 in volume for making FRAP reagent. Samples at different concentrations (25 – 100 μg/ml) were then added to 3 ml of FRAP reagent and the reaction mixture was incubated at 37 °C for 30 min. The increase in absorbance at 593 nm was measured and compared with known standard ascorbic acid (Amin et al. 2013). Freshly prepared FeSO₄ was used for calibration. The antioxidant property based on the ability to reduce Fe³⁺ irons of the sample was calculated from the linear calibration curve and expressed as mmol FeSO₄ equivalents per microgram of the sample. Using the linear regression (y = mx + c), half-maximal inhibitory concentration (IC₅₀) was calculated (Irshad et al.2012). It is also obtained from logistic regression model as

$Y=\frac{\text{Max}}{1+\left(\frac{x}{I{C}_{50}}\right)·\text{Hill coefficient}}$

All assays were repeated for getting a triplicate and statistical analysis was done by ANOVA. The data were interpreted as mean ± SD.