PREVENTION OF DNA SUGAR, HUMAN PERIPHERAL LYMPHOCYTES AND ERYTHROCYTES DAMAGES FROM FREE RADICAL INDUCED OXIDATION BY NATURAL ANTIOXIDANTS

Objective: The present study focused on the identification of particular extract which shows signification protection of DNA sugar against excessive oxidation. Methods: The different extracts (water, alcohol, alcohol: water, and hexane) of Agathi seeds ( Sesbania grandiflora Linn) were evaluated using various antioxidant and other relevant assays like DNA sugar protection and antioxidant activities. Results: The alcohol: water (1:1) extract of S. grandiflora Linn seeds showed the highest antioxidant and free radical scavenging activity. It inhibited membrane lipid peroxidation by 55% at 50 µ g/ml, scavenged approximately 69% of hydroxyl and 1,1-diphenyl-2-picrylhydrazayl radicals at 2–3 fold lower concentrations compared to the other extracts. In addition, the alcohol: water extract inhibited ferrous sulfate: ascorbate-induced sugar oxidation of DNA and also showed non-toxic nature against lymphocytes. Conclusion: These results establish the antioxidant potential of the extract, which could be used as natural antioxidant source.


INTRODUCTION
Natural diet is rich sources of active compounds polyphenols, carotenoids, flavonoids, vitamins, proteins, polysaccharides, and certain important trace metals such as zinc and selenium [1][2][3]. These play a major role as an antioxidant source in reduce cellular damages [4]. Antioxidants are man-made or natural substances, diet high in green leafy vegetables and fruits, which are good sources of antioxidants, have been found to be healthy [5]. Antioxidant substances could be natural or synthetic. Natural antioxidants are obtained from natural sources are safe, non-toxic, inexpensive, and have been used in food, cosmetics, and other related industries. On the other hand, synthetic antioxidants are substances created from chemical process; in excessive usage, they are reported as toxic [6]. Hence, day by day, researchers are in the search of new source of natural antioxidants.
The plant Agathi (Sesbania grandiflora) belongs to the family Fabaceae of genus Sesbania and species Grandiflora. It is widely grown in India, Indonesia, Myanmar, the Philippines, Thailand, and South-East Asian countries. The leaves are bitter are rich in Vitamin C, calcium, sterols, saponin, quercetin, myricetin, and other chemical antioxidants [7]. It is reported that, the seeds of S. grandiflora rich with natural antioxidant Vitamin E and Phytocerol contents. Hence, herein we made an attempt to find the DNA sugar protectant and non-toxic nature of antioxidant content of ethanol-water extract of S. grandiflora [8].

METHODS
Agathi seeds were obtained from authentic source, Bangalore Karnataka state, India. 100 g of well dried Agathi seeds were grounded into a fine powder using a domestic electric grinder.

Preparation of extracts of agathi seeds
One gram of the Agathi seeds powder was added to 100 ml of doubledistilled water, ethyl alcohol: water (1:1), ethyl alcohol, and hexane. The ethyl alcohol and hexane were analytical grade. The solutions were homogenized and the resultant suspension was centrifuged using refrigerated centrifuge for 10 min at 4°C. The supernatants were filtered using Whatman No. I filter paper. The water extract of Agathi seeds powder (Ethanol-water Agathi seed extract) was lyophilized, ethyl alcohol-water (1:1) extract and hexane extracts were rotary evaporated and concentrated. Similarly, other solvent extracts the ethyl alcohol extract of Agathi seeds and the hexane extract of Agathi seeds were concentrated separately using rotary evaporator. Each dried extract (was dissolved mg) in 0.1 ml of the respective extracting solvent or solvent mixture and made up to 10 ml with water. The solution was filtered using glass wool followed with 0.45 µm microbial filter and stored at 4°C for further studies.

Estimation of protein content
The total protein content of the crude extract was determined as per the standard protocol [9]. Various concentrations of bovine albumin (0-100 g/mL) or agathi leaves extr act at the concentr ati on r angi ng fr om 0 to 20 L wer e added to s er i es of tubes and the volume was made up to 100 L wi th 0.15M NaCl. 1 mL Br adfor d's r eagent was added to all the tubes and mixed well. The absorbance was measured at 595 nm. The concentration of the protein in the samples was determined from the calibration curve.

Estimation of total sugar
The total sugar of the agathi ethanol-water extract was estimated by the phenol-sulphuric acid method [10]. Different aliquots of the extract Omkar et al.
(0-25 L) wer e made up to 1mL wi th di s ti lled water . To thi s 1 mL of 5% phenol and 5 mL of concentrated sulphuric acid were added keeping the mixture ice-cold water bath. Orangishred color developed was read at 520 nm immediately. The sugar concentration of the extract was calculated according to the standard glucose calibration curve.

Determination of total phenol content
The total phenolic content of the agathi ethanol-water extract was determined according to the method of Folin-Ciocalteu reaction [11] with appropriate modifications, using gallic acid as standard. An ali quot of the s amples (10-40 L) was mi xed wi th 50% Foli n-Ci ocalteu reagent; the volume was made up to 1mL with methanol: water mixture (50:50 v/v). Further, the mixture was then allowed to stand for 10min at room temperature followed by the addition of 20% Na 2 CO 3 . Further, the absorbance was measured at 725 nm. Results were expressed as milligrams of gallic acid equivalents per gram.

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
DPPH radical scavenging activity was assessed as per the standard method [12]. DPPH, α-tocopherol, and Ascorbic acid were procured from Sigma (St. Louis, USA). Different extracts of Agathi seeds at various concentrations ranging from 10 to 100 µg, were mixed in 1 ml of freshly prepared 0.5 mM DPPH ethanolic solution and 2 ml of 0.1 M acetate buffer at pH 5.5. The resulting solutions were then incubated at 37°C for 30 min and measured colorimetrically at 517 nm in a Shimadzu ultraviolet (UV)-1601 spectrophotometer (Tokyo, Japan). Standard antioxidants such as α-tocopherol and Ascorbic acid, all at 400 µM and 100 µM respectively, were used as positive controls under the same assay conditions. Negative control was without any inhibitor or extract. Lower absorbance at 517 nm represents higher DPPH scavenging activity. Percent DPPH radical scavenging activity of the extracts was calculated accordingly from the decrease in absorbance at 517 nm in comparison with the negative control.

% Viability
Number of viable cells Total number of cells 1 u 00 0

Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity was determined using Deoxyribose assay [13] with some modification. The reaction mixture contained FeCl 3 and as cor bate (100 M ) , H 2 O 2 ( 1 mM ) , EDTA (100 M ) , 2-deoxy-D-ribose (2.8mM), and 1mL of 0.1mM potassium phosphate buffer (pH 7.4) mixed in various concentrations of Agathi seeds extract (50−400 g/mL) . The r eacti on mi xtur e was i ncubated for 1 h at 37°C. The reaction was terminated by adding 1 mL each of Trichloroacetic acid (2.8%) and Thiobarbituric acid (0.5%); this mixture was placed in boiling water bath for 15 min. After cooling, the reaction mixture was centrifuged for 5 min at 5000 rpm. The control was without any test compound and the readings were taken at 535 nm. The percentage hydroxyl radical scavenging activity was determined by comparing with control. Decreased absorbance of the reaction mixture indicated decreased oxidation. Consider the following: Co n ntrol 1 u 00

Determination of antioxidant activity using erythrocyte ghost and linolenic acid micelles
The erythrocyte membranes (ghosts) preparation was carried out as per the standard procedure [14]. In brief, fresh venous human blood samples were drawn with ACD anticoagulant (acid citrate dextrose) mixed well and refrigerated centrifuged, the obtained pellet was was hed 3 to 5 ti mes us i ng i s otoni c phos phate buffer s ali ne (PBS 5 mM , pH 7.4, and 150 mM NaCl) . Fur ther , the cell pellet was s us pended i n hypotoni c (PBS 5 mM , pH 7.4 at 4°C) for hemolys i s to take place. Fur ther , contents wer e r efr i ger ated centr i fuged at 12, 000 r pm for 20 mi n. The obtai ned er ythr ocytes wer e s epar ated fr om plas ma and buffy coat again washed with fresh hypotonic phosphate buffer and centr i fuged at 1500 r pm to r emove unlys ed RBC cells . The membr anes wer e di s per s ed pale yellowi s h pi nk "ghos t" s us pended i n i s otoni c 5 mM phos phate buffer , pH 7.4. By Br adfor d's method, the pr otei n content of ghos t was es ti mated [6]. Ghos t s us pens i on (200 μg) and linolenic aci d (1.8 μmole) were subjected to peroxidation by Fenton reactants (fer r ous s ulfate and as cor bi c aci d) (10:100 μmole) in a final volume of 0.5 mL Tr i s -buffer ed s ali ne (TBS 100 mM , pH 7.4, and 0.15 M NaCl) wi th i ncr eas i ng concentr ati on of agathi s eeds extr act (10 to 50 μg); the contents wer e i ncubated for an hour and 1% TBA was added. Fi nally, the contents wer e kept i n a boi li ng water bath for 15 mi n and then cooled, centrifuged to remove precipitate if any. The color developed was r ead at 535 nm us i ng UV vi s i ble s pectr ophotometer .

% inhibition
Absorbance Absorbance Absorbance Control T est Co n ntrol 1 u 00

Isolation of lymphocyte and their protection
Human peripheral lymphocytes were isolated according to the standard protocol [15]. Fresh venous blood (10 mL) mixed with four volumes of hemolyzing buffer (150 mM NH 4 Cl in 10 mM tris buffer, pH 7.4) and mixed well. The contents were incubated for 30 min at 4°C and centrifuged at 1200 rpm for 20 min and the supernatant was discarded. The pellet was washed twice to thrice with 10 mL of 250 mM m-inositiol in 10 mM phosphate buffer, pH 7.4, and re-suspended in the same solution. The cell viability was determined by tryphan dye blue exclusion method. Percentage viability was calculated as follows;

% Viability
Number of viable cells Total number of cells 1 u 00 0

Determination of DNA sugar damage by spectrophotometric method
Fenton's r eactants i nduced oxi dati ve DNA s ugar damage was determined according to the standard protocol [16]. In brief, the reaction mixture in a total volume of 1 mL containing 1 mg calf thymus DNA was treated with Fe 3+ (10 mM ) , EDTA (10 mM ) and H 2 O 2 ( 2 mM ) without or with various concentrations of the extract (10-50 µg) in potas s i um phos phate buffer (20 mM , pH 7.4) . As cor bi c aci d (10 mM ) was added to the r eacti on mi xtur e and was i ncubated at 37°C for 1 h i n water bath wi th s haker . To 1 mL of the above mi xtur e 1 mL of Tr i chlor o aceti c aci d and 1 mL of 1% TBA wer e added and boi led for 20 mi n. The contents were cooled and the pink color absorbance was read s pectr ophotometr i cally at 523 nm.

% inhibition
Absorbance Absorbance Absorbance Control T est Co n ntrol 1 u 00

Statistical analysis
Statistical analysis was done in SPSS (Windows Version 10.0.1 Software Inc., New Yor k) us i ng a one-s i ded s tudent's t-tes t. All r es ults r efer to means±SD. p>0.05 was considered as statistically significant when compared to relevant controls.

RESULTS AND DISCUSSION
The different extracts of Agathi seeds were subjected to proximate analysis and DPPH radical scavenging activity. The proximate analysis showed that the ethanol-water extract contains Polyphenols, proteins, and negligible amount of sugars (data not shown). The antioxidant and DNA protective nature and non-toxic nature to cells study results are as follows.

DPPH radical scavenging effect
The DPPH radical scavenging activity of ethanol-water extract of Agathi seeds on Fe 3+ dependent hydroxyl radical generation was reported and confirmed using a direct approach [17,18]. The results obtained are shown in Table 2, the ethanol-water extract of Agathi seeds exhibited powerful DPPH radical scavenging activity of 88% at 25 µg, which was much more than water, ethanol, hexane extract (at 100 µg dosage each), which showed DPPH radical scavenging activity of 40%, 41%, 51%, and 46%, respectively. The well-known antioxidants such as alphatocopherol (85.5 µg) and ascorbic acid (100 µg) showed 82 and 78% DPPH radical scavenging activity respectively. The results indicate that ethanol-water extract is a powerful free radical scavenger compared to other extracts and known antioxidants. Hence, for further studies only ethanol-water extraction of Agathi seeds considered.

Hydroxyl radical scavenging activity
The short-lived hydroxyl radicals are highly reactive of all the reduced form of dioxygen and in excess, initiate cell damage in in vivo model [19,20]. The antioxidant effect of Agathi seeds extracts on hydroxyl radicals generated by Fe 3+ ions was measured by the extent of deoxyribose degradation, which is an indicator of TBA-MDA adducts formation. Among the various extracts tested (Fig. 1). The ethanolwater extract showed maximum hydroxyl radical scavenging activity by 69% at 50 µg which is comparatively good when compared to standard antioxidant Ascorbic acid (400 µM) and alpha-tocopherol (400 µM) which were 75% and 71% respectively. This implies that ethanol-water extract could be an effective hydroxyl radical scavenger.

Inhibitory effect of AWEC on fenton reactant-induced DNA sugar damage
To find the chelating and the inhibitory effect of ethanol-water extract of Agathi seeds against iron-dependent oxidation of calf thymus DNA sugar was tested by a TBARs assay. As shown in Fig. 2, the extract offered effective inhibition by 55% at 50 µg against ferrous sulfate: ascorbate-induced [16]. DNA sugar damage using erythrocyte ghost as a source of lipids, when compared to Ascorbic acid (400 µM) and Alpha-tocopherol (400 µM), showed 62% and 71% respectively. When Linolenic acid micelle is used as a source of lipids, the maximum protection provided by the ethanol-water extract is 55% when compared to Ascorbic acid (400 µM) and Alpha-tocopherol (400 µM), showed 66% and 71%, respectively. Similar studies have reported that extracts of Curry leaves, Turmeric, Coleus aromaticus leaves extract exhibit chelating effect of ferrous ions and a reducing capacity [16,19,21,22].

Lymphocytes protection studies
We also investigated the lipid peroxidation induced cell death by Fenton reactants (Table 2). Treatment with Fenton reactants on lymphocyte cells significantly showed cell toxicity. The maximum cell death was induced by Fenton reactant noticed at 30 min. The cell death induced by the Fenton reactant is alone about 35%. The protection offered by ethanol-water extract was 96% at both 0.6 and 0.8 mg/ml. This will show the protective nature and non-toxic nature of the extract. The high percentage of viable cell clearly indicates that ethanol-water extract of Agathi seeds is non-toxic protein with no cytotoxicity toward human lymphocytes (Table 1).

CONCLUSION
This work highlights the importance of curry leaves which have been traditionally used in the Indian culinary system from time immemorial, as a rich source of antioxidants. More specifically, the extraction of curry leaves in a 1:1 ethyl alcohol: water mixture, compared to other solvent systems, showed maximum antioxidant and free radical scavenging activities under in vitro conditions. Thus, the ethyl alcohol: water (1:1) extract of curry leaves could be a very good lead for the extraction of an effective natural nutraceutical or antioxidant drug. Further investigation into ethyl alcohol: water (1:1) extract of curry leaves for its lead active compounds and in vivo, antioxidant mechanisms are warranted.