Invitro Ef icacy of antioxidant activity in ethanolic and aqueous leaf extracts of Andrographis paniculata Nees and Rhinacanthus nasutus Kurz

Akilandeswari G1, Bupesh G2,3, Vijaya Anand A*4, Saradhadevi K M1, Mayur Mausoom Phukan3, Meenakumari K2 1Department of Biochemistry, Bharathiar University, Coimbatore, Tamil Nadu, India 2Research and Development Wing, Cancer and Virology Laboratory, Sree Balaji Medical College and Hospital, Bharath University (BIHER), Chennai, Tamil Nadu, India 3Department of Forest Science, Central University of Nagaland, Lumami, India 4Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India


INTRODUCTION
All living organisms involves many biological processes which produce harmful intermediates known as reactive oxygen species (ROS) or free radicals (Sen et al., 2010;Santhi et al., 2019). Radiation, bacteria, virus, toxins, smoking and alcohol are also reported to produce ROS. Over production of ROS implicated as a potential contributor to the pathogenesis and complications of some disease like diabetes, cancer, atherosclerosis, arthritis and ageing process (Khalaf et al., 2008). Oxidative stress is an imbalance between oxidants and antioxidants that cause damage in the biomolecules like nucleic acid, protein, DNA and RNA (Droge, 2002).
Antioxidant acts as a defense mechanism that protect against deleterious effect of oxidative reaction produced by ROS (Jayachitra and Krithiga, 2012). The antioxidant nutrient may be a major importance in disease prevention (Chanda and Dave, 2009). These days there has been a surge of interest for the helpful possibilities of therapeutic plants as cancer prevention agents is decreasing such free radical incited tissue injury (Pourmorad et al., 2006). Examinations of phenolic mixes in medicinal plants have picked up signi icance because of their high cancer prevention agent action. It has extraordinary incentive in forestalling the beginning or movement of numerous human ailments (Chang et al., 2007).
Medicinal plants which were studied in the recent investigation is Andrographis paniculata Nees and Rhinacanthus nasutus Kurz, belongs to the family Acanthaceae. Plenty of A. paniculata were distributed in India and China. It is an annual plant with 1-3 ft high. The A. paniculata leaves are used as antibacterial, antiviral, hepatoprotective, and hypoglycemic agent. The leaves of the plants which contains lavinods, terpinods, tannins etc (Hidalgo et al., 2013;Suja et al., 2017). R. nasutus are widely distributed in South China and India and this has been used for the treatment of diabetes, hepatitis, cancer, and hypertension etc (Siripong et al., 2006). The leaves of the plant has a rich source of triterpinoids, lavinoids, and steroids etc (Kupradinun et al., 2009). The present study was to evaluate the antioxidant activity of aqueous and ethanolic extract of A. paniculata and R. nasutus by in vitro model.

Plant material and extraction
The fresh leaves of A. paniculata and R. nasutus were collected in and around Trichy and Coimbatore. The plants were identi ied and are authenticated with the specimen at St. Joseph's College Herbarium, "THE RAPINAT",Department of Botany, Trichy, Tamil Nadu. The plant parts were dried and then those dried plant parts are exposed to electrical blender to deliver a coarse powder. The plant was then extracted by utilizing water and ethanol. 5 g of plant powder was macerated with 100 ml of water and ethanol independently in a closed lask for 24 h. The substance was shaken as often as possible during the initial 6 h and permitted to stand for 18 h. At that point the iltrate was dried in a tarred latbottomed dish at 105 • C until consistent weight was acquired. The concentrates were preserved in the fridge for further examinations.

DPPH radical scavenging assay
The free radical scavenging capacity of the ethanolic concentrate of A. paniculata and R. nasutus was determined utilizing the DPPH strategy followed by Vijayakumar et al. (2015). DPPH (200 µM) solution was set up in 95% methanol. From the stock plant extract solution of different concentrations (25, 50, 75, 100 µg/ml) were taken in four test tubes. 1 ml of newly prepared DPPH solution was incubated with test drug and after 15 min, the absorbance was taken as 517 nm utilizing a spectrophotometer. Standard ascorbic acid was utilized as reference.

Control − T est
Control × 100 ABTS + radical scavenging assay ABTS+ assay was done by the technique for Gülçin et al. (2009). Changing the concentration (25, 50, 75, 100 µg/ml) of plant extracts and standard ascorbic acid solutions were taken into series of test tubes. 2 ml of ABTS solutions was included and the volume was made up to 1 ml with ethanol. To the control 2 ml of ABTS solution and 1 ml with ethanol was included. The solutions were perused promptly at 734 nm.

Calculation
ABTS + Scavenging activity (%) = Ferric reducing power assay FRAP assay was utilized by the technique of (Vijayalakshmi and Ruckmani, 2016). 1 ml of differing concentrations (25, 50, 75, 100 mg/ml) of plant extract was blended in with 3.5 ml phosphate buffer and 3.5 ml of potassium ferricyanide. The blend was incubated at 75 • C for 25 min. Aliquots of 3.5 ml of trichloroacetic acid were added to the blend, which was then centrifuged at 3000 rpm for 15 min. The upper layer of the solution (3.5 ml) was blended in with equivalent volume of distilled water, to this 1 ml of newly prepared ferric chloride solution was included and the absorbance was estimated at 700 nm. The expanded absorbance of the reaction blend demonstrates increase in reducing power.

A T est A Blank × 100
Superoxide radical scavenging activity    The PMS-NADH system (phenazine methosulfate, nictotinamides adenine dinucleotide system) of (Fontana et al., 2001). It was utilized for the age of superoxide anion. 0.2-1 ml of ethanol and plant extract was blended in with 0.5 ml NBT and 0.5 ml NADH was blended. The reaction blend incubated at 50 • C for 5 min. After 5 min absorbance of the blend was estimated at 560 nm against the blank. The level of hindrance was dictated by looking at the consequences of control and test.

RESULTS AND DISCUSSION
The stable DPPH radical model is a broadly utilized, generally quick and exact strategy for the assessment of free radical scavenging activity. DPPH is a steady free extreme it turns into a stable diamagnetic particle when it acknowledges an electron or hydrogen radical. Antioxidant on association with DPPH both exchange electron or hydrogen atom into to DPPH and hence killing its free radical character and convert it to 1,1-diphenyl-2picryl hydrazine and the level of staining shows the scavenging action of the drug (Vasanth and Bupesh, 2019). The reduced limit of DPPH radical is controlled by the abatement in its absorbance at 517 nm prompted by cancer prevention agents (Saran et al., 2019). The decrease in absorbance of DPPH radical achieved by malignant growth prevention agents because of the reaction between cancer prevention agent particles and radical progression which realizes the looking of the radical by hydrogen donation. It is visually noticeable as an adjustment in shading from purple to yellow. Henceforth, DPPH is commonly used as a substance to evaluate the antioxidant activity (Braca et al., 2001;Usmani, 2013). Table 1 shows the inhibition action of ethanolic extracts of A. paniculata and R. nasutus shows most elevated hindrance of 66.44% in 100 µg/ml focuses and least inhibition of 36.86% in 25 µg/ml and the IC 50 esteems is 60 µg/ml and the ethanolic concentrate of R. nusutus shows 42.58% most noteworthy and 36.05% lower inhibition in 100 µg/ml and 25 µg/ml individually and the outcomes were contrasted and standard ascorbic acid which utilized as a source of perspective and the IC 50 value is 76 µg/ml. In this examine, ABTS is changed over to its radical cation by the expansion of potassium persulfate. This ABTS radical cation is blue in shading and absorbs light at 734 nm. The ABTS radical cation is responsive towards most antioxidants, including thiols, phenolics, and ascorbic acid. During this response, the blue ABTS radical cation is converted to its colorless, neutral form (Ragavendra et al., 2013). The FRAP test used to measure the decreasing capability of a cell reinforcement responding with a ferric tripyridyltriazine (Fe3+-TPTZ) complex and delivering a colored ferrous tripyridyltriazine (Fe2+-TPTZ). The free extreme chain breaking happens through giving a hydrogen molecule. At low pH of about 3.6, decrease of Fe3+-TPTZ complex with blue shaded Fe2+-TPTZ happens, which has an absorbance at 593 nm. The outcomes acquired are highly reproducible and related directly to the molar convergence of the cancer prevention agents present. This is according to the results point by point by Benzie et al. (1999); Jeong et al. (2004). Superoxide dismutase (SOD) is a signi icant protein in a antioxidant resistance framework (Curtis et al., 1972). SOD converts over the superoxide anion into hydrogen peroxide and in this way decreases the harmful impact. The level of restraint of superoxide by SOD may decrease the cell harms. The current investigation demonstrates that the expanding grouping of the concentrate has a most extreme inhibitory action of SOD.