On valorization of solvent extracts of Terminalia arjuna (arjuna) upon DNA scission and free radical scavenging improves coupling responses and cognitive functions under in vitro conditions

Chronic diseases have been treated using the phytochemical concepts of ethnomedicinal plant-derived herbal products. Terminalia arjuna, a significant ethnomedicinal plant, was revisited and reconnoitred for antioxidant, free radical scavenging, and DNA nicking inhibiting activity under H2O2 conditions using 21 solvent extracts. Ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid, and nitrous oxide scavenging (%) were found to have a strong positive association and interaction (PCA 1 explains 84.54% variation) with ethanol bark (Etoh-AB) (Meoh-AF). TPC (144.67–1794 µg/mL GAE) and TFC (2.5–34 µM Fe(II)/g were highest in Etoh-AB. In a pattern of combined solvent extracts, Etoh-AB had the highest antioxidant capacity, accompanied by Etoh-AL ≥ Meoh-AB ≥ Dw-AF. With R2 = 0.94, the DNA nicking inhibition behaviour parameters relative front, relative quantity, band (%), and lane (%) formed a positive significant (p < 0.01) connection. For the first time, we show that Etoh-AB nicks supercoiled, circular plasmid DNA in a way that is comparable to normal antioxidants. Normal antioxidants with the ability to prevent DNA nicking include Butylated hydroxy anisole < Butylated hydroxy toluene < ascorbic acid < and Gallic acid. Gallic acid (m/z 170.0208 g/mol) and Ellagic acid (m/z 302.0063 g/mol were present in high concentrations in solvent extracts. 0.48 mg was found to be the effective concentration for inhibiting relative DNA nicking. The current study is the first of its kind to show that steroid concentrations are higher in bark fractions of acetone, ethanol, and methanol. Furthermore, T. arjuna solvent extracts provide a wealth of information on phytochemical profiling, antioxidant ability, and DNA nicking inhibition, which may be useful for exploring the natural way and further research to develop a remedy against geriatric chronic disease. Despite the fact that ethanol is very close to methanol in terms of solvent toxicity, the current study identified it as the preferred solvent. Thus, the current research revisits previous studies and explores the potentiality of non-polar and polar aprotic and polar protic solvent systems, which lend credence to bioactive compounds that may be useful in isolating and formulating safe and cost effective herbal medicament for livestocks and aquaculture, and drugs for deoxygenerative human diseases, and can also be investigated further to instil environmental frugality.

DPPH inhibition activity. The DPPH inhibition (%) of bark showed maximum activity, Etoh-AB (36.44-73.78%) followed by Meoh-AB (24.66-72.44%), Acet-AB (14.44-65.11%) and Dw-AB (18.88-45.55%). For fruit extracts, maximum activity showed by Etoh-AF (22.35-72.84%) followed by Dw-AF (47.55-66.88%), Acet-AF Table 1. Qualitative phytochemical analysis of solvent extracts of T. arjuna +++, high concentration; ++, moderate concentration; +, low concentration; ND, not detected, table showed that Etoh-AB showed a higher profile for tannins and phenolic, alkaloids and flavonoids. Similarly, high polar solvent extracts also showed better profiling as compared to apolar and aprotic solvent extracts.  www.nature.com/scientificreports/ DNA nicking inhibition activity of the four standards tested, followed by ascorbic acid > BHT > BHA. According to the gel, Etoh-AB, Meoh-AB, and Acet-AB have the highest activity, followed by Etoh-AF, Acet-AF, Chlo-AL, Etac-AL, and others, all of which have relatively similar effects on plasmid DNA. Quantitatively, the protective effects can be seen in the relative front value (RF), relative quantity (RQ), band (%), and lane (%) values. The RF values vary insignificantly (p > 0.05) when compared to the standard controls, with the highest value reported for Etoh-AB, followed by (0.66 ± 0.13), Meoh-AB (0.66 ± 0.11), Acet-AB (0.62 ± 0.14), and Dw-AB (0.61 ± 0.14), respectively. A similar pattern was observed for fruit and leaf solvent extracts. The pattern for RQ value was the same, with maximum values for Etoh-AB (1.20 ± 0.29 ng), Meoh-AB (1.16 ± 0.30 ng), Acet-AB (0.97 ± 0.34 ng) and Dw-AB (0.84 ± 0.37 ng), respectively, and almost the same trend was observed for fruit except for the Dw and Acetone fractions, but for leaf the trend changes depending on operation. For RQ, there was a substantial difference (p < 0.05) between Etoh-AB and Dw-AB. One-way ANOVA revealed 11 and 12 solvent extract groups for band (%) and lane (%), respectively, indicating major differences between solvent extracts ( Over all association patterns, significance, networking and ordination scaleogram among solvent extracts based on antioxidant potential. The antioxidant ability of the solvent extracts is used to network. Dw-AL in a centric role, separating extracts of high polar and non-polar or polar aprotic solvents. Solvent extracts with high antioxidant potential are distributed on the far left hand side, while less active solvent extracts are distributed on the far right hand side (Fig. 2). www.nature.com/scientificreports/ Solvent extract networking at 50% edge cutoffs reveals more than 50% similarity between connecting edges of nodes. Node diameter is proportional to the number of edges connected to it, and edge thickness is proportional to similarity. Starting from the extreme right, the antioxidant activities of Hex-AL, Etac-AF, Chlo-AB, Chlo-AL, Chlo-AF, Acet-AL, and Hex-AL are more than 50% identical. On the other hand, Dw-AL has 19 connecting edges that bind the aforementioned group to high polar solvent extracts such as Etac-AB, Acet-AF, Etac-AL, Acet-AB, Etoh-AL, Meoh-AL, Dw-AF, Dw-AB, Etoh-AB, Meoh-AB. The linking edges of Chlo-AB and Hex-AF are 7, 7, 7, 8 and 8, respectively. Other solvent extracts with common similarity patterns include Acet-AF, Dw-AF, Meoh-AB, Etoh-AB, and Etoh-AF (Fig. 3).
The correlation degree that DPPH has a highly important (P0.01) positive correlation with other antioxidants is as follows in ascending order: ABTS > NO scav. > TPC > FLV > FRAP, which can be expressed in (%) correlation as 99.1, 99 > 83 > 77.4 > 69.3, respectively. Similarly, ABTS and NO scav. (%) showed a correlation in the same way that DPPH did. FRAP, on the other hand, displayed a highly important (p = 0.01) positive correlation with all antioxidants, with the highest correlation with DPPH (0.693). TPC and FLV displayed a highly significant (p < 0.01) positive association with each other, in comparison to FRAP and scavenging antioxidants such as DPPH, ABTS, and NO (Table 3).   (Table 3).
Etoh-AB has the highest numerical value of 23.7, followed by Meoh-AB and Acet-AB. Dw counter parts had the lowest values. According to the PCA scatter bi-plot, PCA components 1 and 2 explained 84.54 and 12.52% of the difference, respectively (Fig. 5). The antioxidant potential of solvent extracts Etoh-AB >, Meoh-AB > Acet-AB > Meoh-AL is strongly associated with DPPH, ABTS, NO scavenging activity, and FRAP potential in ascending order. TPC and FLV, on the other hand, are highly intracorrelated with the operation of Dw-AF >, Etoh-AL > Meoh-AF > Etoh-AF > Acet-AF. On the other side of the axis, solvent extracts such as Chlo-AL and Chlo-AF are strongly correlated with each other but have no correlation with regular antioxidants. Similarly, the association between Dw-AL, Acet-AL, Hex-AL, Chlo-AB, Hex-AB, Hex-AF, and Etac-AF and standard antioxidants is weak.
Phytochemical analysis using Liquid chromatography coupled to electrospray-Orbitrapmass spectrometry. The data obtained from anQ-Exactive plus-Orbitrap hybrid mass spectrometer showed elution profile of different metabolites as their retention time Fig. 6a-r.
For thorough study, the samples were divided into groups, such as bark, leaf, and fruit, and individual samples were examined within each group. Gallic acid (m/z 170.0208 g/mol) and Ellagic acid (m/z 302.0063 g/mol) were found in three fruit extracts (FA, FE, FETC) out of a total of six, three leaf extracts (LA, LE, LM) out of a total of five, and five bark extracts (BA, BE, BETC, BM, EBC) out of a total of eight. Gallic acid, also known as 3,4,5-trihydroxybenzoic acid, is a hydrolysable tannin. Ellagic acid is a dimer of gallic acid formed by oxidative aromatic coupling dimerization. It functions as an antioxidant and can be found in a variety of fruits and leaf extracts. Ursolic acid, a triterpene metabolite, was also present in high concentrations in bark extracts. Ursolic acid has a molecular weight of 456.3609 g/mol and is also known as 3 β,-hydroxy-urs-12-en-28-oic acid. It is a triterpenoid with a C-30 chemical structure made up of isoprenoid units and a pentacyclic loop. Myricetin and quercetine were discovered in some of the bark and leaf extracts as flavonoid compounds. Table 4 summarises the metabolites found in bark, leaf, and fruit extracts.
The correlation matrix of DNA nicking inhibition activity of solvent extracts of T. arjuna. Densiometric analysis showed a highly significant (p < 0.01) positive correlation ( Table 5). The highest correlation with RQ (0.818) is found in RF, followed by band percent

Discussion
The present study's qualitative preliminary phytochemical analysis revealed that solvent extracts of T. arjuna fruit, bark, and leaf contain tannin and phenolics, steroids, alkaloids, flavonoids, and saponin, which are consistent with previous studies 12 . Tannin and phenolics content was found to be higher in Etoh-AB and Meoh-AB. Etoh-AL, Meoh-AL, Meoh-AF, Etoh-AF, Acet-AB, and Acet-AL, which replaced the earlier findings of 19 , demonstrated either reaction against small solvent extracts or less reaction responses. The qualitative phytochemical screening, in vitro antioxidant operation, and liquid chromatography coupled to electrospray-Orbitrapmass spectrometry analyses all complement and confirm the results. Gallic acid and ellagic acids were present in different proportions, leading to antioxidant and DNA nicking inhibition activity. In a solvent extract, the Table 3. Correlation matrix between antioxidants based on the solvent extracts reactions at p < 0.01 level **Correlation is significant at the 0.01 level. www.nature.com/scientificreports/ corresponding region of a specific metabiolite revealed its relative concentration or abundance. Gallic acid, also known as 3,4,5-trihydroxybenzoic acid, is a hydrolysable tannin. Ellagic acid is a dimer of gallic acid formed by oxidative aromatic coupling dimerization. It functions as an antioxidant and can be found in a variety of fruits and leaf extracts. Steroid content was higher in Etoh-AB, Etoh-AF, Meoh-AB, Meoh-AF Acet-AF, Hex-AF, and Etac-AF, which is notable since no studies have highlighted the higher steroid content in T. arjuna Hex-AF. Leaf fractions of acetone and methanol contain more saponin than Etoh-AB, Etoh-AF, and Etoh-AL, which is partially consistent with Kumar et al. 20 , who did not report saponin in water fractions. Hex-AF demonstrated a clear presence of alkaloids among all solvent extracts, providing baseline information on this solvent extract, which is consistent with previous studies 21, 22 that indicated that the fruit of the plant contains more alkaloids and can be extracted using adapted techniques with non-polar solvent rather than serial fractions or defatting herbal materials. In comparison to previous findings 23 , which indicated maximum flavonoid content in methanol fraction of T. arjuna bark, Etoh-AB showed high flavonoid presence among all solvent extracts, followed by Meoh-AB, Etoh-AF, Acet-AB, and so on. This may be due to variations in processing or processing treatment procedures during extraction. In the current analysis, FRAP, ABTS, NO, and DPPH inhibition (%) was used to estimate the antioxidant capacity of selected solvent extracts of T. arjuna, as well as TFC and TPC. The FRAP assay is based on the ability of an antioxidant to reduce a ferric tripyridyltriazine (Fe 3+ -TPTZ) complex to a coloured ferrous tripyridyltriazine (Fe 2+ -TPTZ) complex 23 , which tests the total antioxidant capacity of the material studied. The highest FRAP value was calculated for Etoh-AB, followed by Meoh-AB, Acet-AB, and Dw-AB in bark extracts, and Meoh-AF and Meoh-AL in leaf extracts. Overall, it was greatest in AB, followed by AF and AL. These findings are consistent with those of Kumar et al. 20 stated that alcoholic fractions have the highest FRAP activities, and bark extracts have higher scavenging activity than leaf extracts, but the quantitative result in this study is substituted due to the higher value of FRAP, indicating the efficacy of the solvent extraction. TPC and TFC follow the same pattern, with Etoh-AB having the highest value, followed by Meoh-AB, Acet-AB, and Dw-AB for fruit extracts, Dw-AF, Acet-AF, and Meoh-AF for leaf extracts, and Etoh-AL having the highest value, followed by Meoh-AL, Chlo-AL, and Dw-AL for leaf extracts. According to the analysis of Jayathilake et al., Etoh-AB had the highest TFC and TPC content among all solvent extracts, which arbitrates the free radical scavenging and strong antioxidant activities. Reference 24 proposed a positive, important linear association between antioxidant activity and TPC and TFC contents, suggesting that phenolic compounds and flavonoids were the most abundant antioxidant components in the medicinal herbs studied 2 . According to Fig. 3, Meoh-AB and Etoh-AF have no branching, whereas Etoh-AB and Etoh-AL have the lowest bootstrap value, which may be due to their different responses to antioxidant activities. The current study found that the activities of herbal extracts differ depending on the solvent method used, as well as their response to standard reagents and interactions between solvent extracts. The solvent systems are distributed from right to left based on their polarity and reaction to specifications. Dw-AL connects both parts of the scatter network, indicating that Dw-AL has intermediate activity, and Etoh-AB, Meoh-AB, and Dw-AB are at the extreme left with smaller diameter nodes and connecting edges, indicating their superiority over other solvent extracts, which is consistent with previous studies 19,25 that highlighted the solvent efficiency and polarity for effectivness. Table 3 shows that DPPH, ABTS, and No inhibition (%) have a positive correlation with FRAP and are strongly correlated with each other, elucidating the antioxidant mediated free radical scavenging ability of the solvent extracts, while TFC and TPC are highly correlated with each other and have a substantial positive correlation with NO >ABTS > DPPH > FRAP. Similarly, from Fig. 4, it can be explained that FRAP, DPPH, ABTS, and NO (%) inhibition is positively correlated with Etoh-AB, Meoh-AB, Acet-AB, and Meoh-AL, while TFC and TPC are positively correlated with Etoh-AL, Dw-AF, Meoh-AF, Etoh-AF, and Etoh-AF, respectively, which is consistent with previous studies [24][25][26][27][28] . Antioxidants have antioxidant properties that protect DNA from oxidative damage caused by reactive oxygen species (ROS), which causes structural changes in the three-dimensional structure of DNA. Furthermore, changes in DNA confirmation impact DNA mobility in an electric field. Despite, the fact that plasmid DNA only showed two bands on an agarose gel, it comes in three distinct types. Form I is a supercoiled circular (relaxed) form that migrates faster than other forms. When the supercoiled DNA form is broken, a nicked circular form (form II) is formed. This form migrates much more slowly than another. Another is form III, a linear form that occurs between forms I and II 29 . Plasmid research looks at the conversion of supercoiled plasmid DNA radicals into linear or circular forms 30 . According to the findings, solvent extracts with DNA nicking inhibition activity result in better RF, RQ, band, and lane efficiency (%). Table 2 shows that Etoh-AB, Meoh-AB, and Acet-AB have the highest activity, followed by Etoh-AF, Acet-AF, Chlo-AL, and Etac-AL. Etac-AF, for example, exhibits reasonably similar effects against plasmid DNA nicking, which is consistent with previous studies 11,30 that documented the DNA damage prevention activities of non-polar protic solvents as well, as well as a range of antioxidants present in T. arjuna extracts. Table 5 shows that RF, RQ, lane (%), and band (%) are highly positively correlated to each other, especially RF with RQ and lane (%) with band (%), which may be due to the involvement of these parameters in DNA nicking recovery of linear form of plasmid DNA, which is consistent with previous studies 31, 32 that have reported that the super coiled circular form of DNA migrates faster than other froms. The results of the bibliographic search revealed a substantial increase in research related to the DNA damage prevention activities of the T. arjuna plant, but articles primarily provided densiometric analyses of the DNA nicking inhibition of T. arjuna solvent extracts. DNA damage prevention is regarded as a feature of third line antioxidant defences mediated by the presence of TPC and TFC, which supports the current study results indicating solvent extract having more TFC and TPC encompassing more antioxidant potential thereby proving better free radical scavenging potential congruent with all parameters of the study, which is consistent with previous findings 33  Preparation of solvent extract. Three parts of T. arjuna dry powder were placed in a 1 l capacity conical flask in a 1:5 sample:solvent ratio and held in a shaking incubator at room temperature for 36 h. Following that, the solvent-sample mixture was centrifuged at 6000 rpm for 5 min, and the supernatant was collected and filtered through 110 mm Whatman filter paper and then 90 mm (Whatman no. 1 (40)) filter paper. The residual of the filtration tube and filter paper was put in an aluminium tray and allowed to dry before adding the polar solvent. The filtrate was collected in a beaker and dried under a rotary vacuum evaporator just below the boiling point of the solvent until 1/10th of the original solvent extracts were obtained, which were then stored in amber glass sample vials with holed covers in dark locations. After drying, the solvent extracts were stored in a deep freezer at 4 °C before further use. Both solvent systems were treated in the same way. Fixed volume methods were used for all solvent extracts in in vitro antioxidant studies. In brief, a total of eight different stock concentrations were taken: 25, 50, 75, 250, 500, 750, 1000, and 1250 µg/mL, and 20 µL of each concentration was applied to prepare the sample mixture for analysis.
Qualitative screening of phytochemicals. For qualitative phytochemical analysis, 1 g dry extracts in 100 mL were dissolved in their respective mother solvents to make a 1% stock concentration, and a reaction was set up to determine essential phytochemicals such as phenols, flavonoids, tannin, saponin, alkaloids, and phytosteroids according to standard procedures 35,36 .
In vitro antioxidant activity. Total phenolic compounds. The Folin-Ciocalteu reagent (1:4 dilutions with distilled water) system was used with minor modifications to estimate total phenolic contents (TPC) 37 . In brief, 10 µL of each concentration was taken from the stocks of 25-1250 µg/mL extracts and combined with 1.5 mL of Folin-Ciocalteu reagent and 5.5 mL of triple distilled water in triplicate test tubes labelled as concentration.
The spectrophometer was calibrated at zero with a blank and a control. In the blank, every constituent was pre- www.nature.com/scientificreports/ sent except for the sample, which was replaced with distilled water. The standard was similarly set up in the std labelled test tube, except for the sample. After incubating the reaction mixture at room temperature for 30 min, 1.0 mL of 1 M sodium carbonate was added. The reaction mixture was incubated in a water bath at 40 °C for 20 min before being allowed to cool. In a UV-Vis spectrophotometer, absorbance was measured at 760 nm (Ep-ochTM2 Microplate Spectrophotometer, Biotek, USA). To estimate the TPC of solvent extracts (25-1250 µg/ mL), gallic acid (1 mg/mL) was used as a reference. The results were given in µg/mL Gallic acid equivalents (GAE).
Estimation of total flavonoids contents. The total flavonoid content (µg/mL) was calculated using a modified aluminium chloride (AlCl 3 ) method 38 . 10 L of each concentration (25-1250 µg/mL of extracts) was taken from the stock concentrations and placed in triplicate in a test tube labelled concentration, which was filled with 200 µL distilled water and 150 µL NaNO 2 and held for 10 min at room temperature in a dark spot. After 10 min, 200 µL (10% AlCl 3 ) was added and kept at room temperature in a dark position for another 10 min. Following that, 2 mL (4% NaOH) was applied, and the amount was increased to 5 mL by adding distilled water, and the mixture was incubated for 20 min at room temperature in a dark location. After 20 min, the pink colour produced indicated the presence of flavonoids in the samples, and the OD was measured in a spectrophotometer at 510 nm. To estimate the total flavonoids content of the samples, Quercetin (1 mg/mL) was used as a norm, and TFC was expressed as µg/mL Quercetin equivalents (QE).
DPPH free radical scavenging assay. The free radical scavenging behaviour of the solvent extracts was calculated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, which was slightly changed from 39 . 10 µL of each stock concentration (25-1250 µg/mL of extracts) was taken into triplicate of test tube marked as concentration, then 1 mL freshly prepared DPPH reagent (1 mg/mL in methanol) was applied, and finally, volume was made up 5 mL with distilled water. The control was taken without any samples. The test tubes were incubated at room temperature for 30 min in a dark position before measuring absorbance at 536 nm. The percentage inhibition was determined using the formula below.
A0 is the absorbance of control reaction mixture; A1 is the absorbance of sample.
ABTS assay. The free radical scavenging activity was calculated using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), ABTS radical cation decolorization assay, which was changed slightly from the standard method 40 . In brief, 10 µL of each stock concentration (25-1250 µg/mL of extracts) was taken into triplicate of test tube marked as concentration, to this 3 mL (ABTS prepared with 1:1 of 2.45 mM potassium persulfate, stored in the dark at room temperature for 12-16 h before use, and volume was made up 5 mL adding methanol and adjusting OD 0.734 at 734 nm. The test tube was held at room temperature for 30 min before measuring absorbance at 734 nm. Trolox was used as the norm, and the percentage inhibition was determined using the formula below.
A0 is the absorbance of control reaction mixture; A1 is the absorbance of sample.
NO scavenging activity. Using the Griess IIIosvoy reaction 41 , the Nitric Oxide scavenging activity was determined. In brief, 10 µL of each stock concentration (25-1250 µg/mL of extracts) was taken into triplicate of test tube marked as concentration, to which sodium nitroprusside (10 mM) in phosphate buffered saline was mixed with different concentrations of extract were dissolved and incubated at 30 °C for 2 h. After that, 10-20 µL freshly prepared Griess reagent (1% sulphanilamide in 2.5% phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride in 2.5% phosphoric acid immediately before use) was applied to the reaction mixture, and the absorbance at 546 nm was calculated after 1 h. Gallic acid acid was used as standard. The percentage of no scavenging was estimated as follows.
A0 is the absorbance of control reaction mixture; A1 is the absorbance of sample.
FRAP assay. The ferrous reducing antioxidant assay (FRAP) was performed using a slightly changed standard method 42 . From the stock concentrations (25-1250 µg/mL of extracts), 10 µL was taken from each concentration and 1.5 mL FRAP reagent (10:1:1 of 300 mM Sodium Acetate buffer: 20 mM FeCl3: freshly prepared TPTZ in 40 mM HCl), and finally volume was rendered up to 5 mL by adding distilled water. Similarly, a standard (Ascorbic acid) with varying concentrations (25-1250 µg/mL) was set up in triplicate, and a blank without sample or standards was also taken. After that, the reaction mixture was allowed to incubate for 30 min at room temperature in a dark room. At 593 nm, absorbance was measured. The concentration was given in the form of µM (Fe II)/gm of extracts.
Phytochemical analysis using Liquid chromatography coupled to electrospray-Orbitrapmass spectrometry. 3  www.nature.com/scientificreports/ containing 0.1% formic acid, the flow rate was 0.3 mL/min. The gradient consisted of an isocratic stage of 2 min at 95% phase A, followed by a linear gradient from 5 to 95% phase before the next 18 min and held for 5 min.
The gradient was brought to its initial step in 1 min and kept for 4 min before returning to 100% A for 4 min. An anQ-Exactive plus-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific) equipped with an electrospray source operating in positive and negative ion modes was used for mass spectrometric detection. The mass spectrometer was set to 4.2 kV capillary voltage and 340 °C capillary temperature. The sheath gas and auxiliary gas flow rates were set to 37 and 13 arbitrary units of nitrogen gas, respectively, with the auxiliary gas heater temperature set to 400 °C. At the MS and MS/MS stages, detection was achieved from 100 to 1200 m/z at resolutions of 35,000 and 17,500, respectively. The microscan count was set to one, and the repeat count for dynamic exclusion was set to ten seconds. Thermo Compound Discoverer programme (Version 2.1 SP1 Thermo Fisher Scientific) was used for data analysis, with the workflow Max ID-Detect unknowns with ID using online database searches and the mzCloud search engine.
DNA nicking inhibition. The plasmid DNA pBR322 (Thermo Scientific) was used to investigate the beneficial effect of T. arjuna solvent extracts on hydroxyl radical-mediated DNA damage. First, the extracts were dissolved in 1% tetrahydrofuran (THF) at 50 mg/mL using the fixed dose percentage principle. A reaction mixture (20 µL final volume) of 2.5 µL of 0.25 µg/l plasmid DNA pBR322, 1.5 µL of 1% H2O2, and 16 µL of 50 mg/mL solvent extracts. H2O2 (1%) and tetrahydrofuran (1%) treated plasmid DNAs, tetrahydrofuran (1%) and plasmid DNA and only THF were used as control groups, and the prepared mixture of each solvent extract was incubated at 37 °C for 24 h. 4 µL of bromophenol blue (0.025%) and sucrose (4%) in dH2O loading dye was applied to the mixture and loaded onto the 1% agarose gel. The electrophoresis process was carried out for 45 min at 120 V in the TBE buffer running buffer (pH 8). The Gel was photographed using UV light.
Statistical tools and soft ware's for over all association patterns, significance, networking and ordination scaleogram among solvent extracts based on antioxidant potential and gel quantification and characterization. Using the PAST and Minitab 18 softwares, the significance, networking, and ordination scaleogram among solvent extracts based on antioxidant potential was developed after extracting the values for various antioxidant activities across all association patterns. The data was analysed using Microsoft Excel v.16, and significance was determined using SPSS 20. Paint 3D v.16 was used to edit the images. LAB IMAGE software was used for gel quantification and characterization (Bio-Rad).

Conclusion
Ethanolic and methanol fruit and bark showed that both antioxidants and free radical scavenging substances were abundant. Results from the phytochemical study showed that polar and polar solvent extracts include the bioactive steroids and other concepts. This preliminary investigation of the extracts of T. arjuna's unexplored solvents could provide a starting point for bioactive compounds to design drugs and medicines against geriatric and free radical-caused degenerative disease may lead to an eventual therapeutic intervention.

Statement of plant material used.
The Terminali arjuna used in present study was obtained from the plant located in the campus of ICAR-Central Inland Fisheries Research Institute, Barrackpore, Kolkata. For the same Director ICAR-CIFRI is the competent authority who has granted the permission for carry out the study. The tree is big so cannot be stored as specimen, however, it has census authentication number that been also mentioned in our previous research article 43 . Tree has authentication no 14 and plant still exists, it has been used as per the ethical guidelines and not been damaged or killed.