Asian Pacific Journal of Tropical Medicine (2012)977-985 977

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Document heading doi: Antioxidant activity and identification of bioactive compounds from leaves of Anthocephalus cadamba by ultra-performance liquid chromatography/electrospray ionization quadrupole time of flight mass spectrometry Madhu Chandel1, Upendra Sharma2, Neeraj Kumar2, Bikram Singh2, Satwinderjeet Kaur1*

1Department of Botanical and Environmental Sciences, Nanak Dev University, Amritsar-143005 2Natural Products Division, CSIR-Institute of Himalyan Bioresource Technology (Council of scientific and Industrial research), Palampur, H.P.-176061 India

ARTICLE INFO ABSTRACT

Article history: Objective: Anthocephalus To evaluate the antioxidant potential of different extract/fractions of Received 24 February 2012 cadamba A. cadamba ( ) (Roxb.) Miq. (Rubiaceae) and study the tentative identification of their Received in revised form 30 March 2012 Methods: active constituents. The extract/fractions were screened for antioxidant activity using Accepted 7 April 2012 in vitro Available online 20 December 2012 various assays viz. DPPH assay, ABTS assay, superoxide anion radical scavenging assay, reducing power assay and plasmid DNA nicking assay. Total phenolic content of extract/fractions Keywords: was determined by colorimetric method. An ultra-performance LC-electrospray-quadrupole- Anthocephalus cadamba time of flightA. masscadamba spectrometryResults: method was used to analyse the active constituents of extract/ fractions of . The ethyl acetate fraction was found to be most active fraction Antioxidant activity in all the assays as compared to other extract/fractions. The IC50 value of ethyl acetate fraction UPLC-ESI-QTOF-MS (ETAC fraction) was 21.24 毺g/mL, 1.12 毺g/mL, 9.68 毺g/mL and 57.81 毺g/mL in DPPH assay, ABTS assay, reducing power assay and superoxide scavenging assay respectively. All the extract/ fractions also showed the potential to protect’ the plasmid DNA (pBR322) against the attack of hydroxyl radicals generated by Fenton s reagent. The bioactive compounds wereConclusions: identified by UPLC-ESI-QTOF-MS, by comparing the mass and 毸max with literature values. The potential of the extract/fractions to scavenge different free radicals in different systems indicated that they may be useful therapeutic agents for treating radical-related pathologic damage.

1. Introduction antioxidant research is an important topic. The importance of reactive oxygen species (ROS) and free radicals in cellular damage and the ageing process has attracted increasing In recent times, focus on plant research has increased all attention over the past 20 years[4,5]. ROS, such as superoxide _ over the world and a large body of evidence has collected anion radical (O2 ), hydrogen peroxide (H2O2), hydroxyl • to show immense potential of medicinal used in radical (OH ) and other free radicals are by-products of various traditional systems. Natural plant products are biological metabolism. Within biological systems, there frequently reported as efficient chemopreventive agents. are enzymatic systems and chemical scavengers: dietary Antigenotoxic and antioxidative mechanisms are considered antioxidants (毩- tocopherol, 毬-carotene, ascorbic acid, crucial for the prevention of some degenerative diseases glutathione and uric acid), some hormones (estrogen, [1-3] such as cancer . In food industry and medicinal research, angiotensin) and endogenous enzymes (superoxide dismutase, glutathione peroxidase and catalase), all of them *Corresponding author: Satwinderjeet Kaur, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar-143005 India. are able to remove free radicals formed in cells and thus Tel. 0183 - 2259513, 258802 to 09 Extn.: 3424(Off.) [6,9] Fax: 0183 - 2258819, 2258820 protect against oxidative damage . The oxidation induced E-mail: [email protected]; [email protected] Madhu Chandel et al./Asian Pacific Journal of Tropical Medicine (2012)977-985 978 2.3. Extraction and fractionation by ROS can result in cell membrane breakdown, membrane protein damage and DNA mutation, which can further initiate or propagate the development of many diseases, The leaves were washed with running water to remove such as cancer, liver injury and cardiovascular disease[10- any dust impurities and dried at 40 曟. The material was 12]. The antioxidant phytochemicals from plants, particularly finely powdered and extracted with 95% ethanol under flavonoids and other polyphenols, have been reported to reflux conditions to obtain ethanol extract (ETAC) and inhibit the propagation of free radical reactions, to protect was concentrated under reduced pressure using rotary the human body from disease and longer life expectancy[13- evaporator. ETAC was made aqueous with distilled water 17]. Interest has increased in naturally occurring antioxidants in a separating funnel and further fractionated with organic solvents in order of increasing polarity viz. ethyl acetate and for use in foods or medicinal material as compared to the n E synthetic antioxidants because of their non-side effects -butanol to obtain the fractions, viz., thyl acetate fraction Anthocephalus cadamba (A. cadamba) (EAAC), butanol fraction (NBAC) and remaining water nature[18]. (Roxb.) fraction (WAC) (Figure 1). Miq. (Rubiaceae) is known as wild cinchona and popular “ ” in India as Kadamb . Its bitter and pungent bark is A. cadamba used in ayurvedic medicine for uterine complaints, blood (Leaves powder, 1.4 kg) diseases, leprosy and dysentery. A decoction of the leaves Refluxed with 95% ethanol is recommended as a gargle in cases of stomatitis[19]. A. cadamba Ethanol extract after solvent evaporation The crude extracts from have been shown to (ETAC, 325.4 g) Dissolved in distilled water possess biological activities viz. anti-inflammatory[20], antihepatotoxic activities[21], analgesic activitiy[22], Aqueous ETAC extract × hypoglycemic activity[23], antimicrobial and anthelmintic Ethyl acetate (3 100 mL) A. cadamba activities[24]. The chemical constituents of have been investigated and so far, terpenoids and alkaloids Marc Ethyl acetate fraction [25-30] n × (EAAC, 18 g) have been identified . However, not many reports are -butanol (3 100 mL) available regarding the bioactive phytochemicals from this plant. Keeping this in mind, the present study was planned n Water fraction -Butanol fraction to evaluate the antioxidant potential of extract/fractions A. cadamba (WAC, 40 g) (NBAC, 38.2 g) from leaves of and identification of active Figure 1. Isolation of various extract/fractions from leaves of constituents using UPLC-ESI-QTOF-MS. A. cadamba .

2. Materials and methods 2.4. Phytochemical analysis

2.1. Chemicals 2.4.1. Determination of total phenolic content The total phenolic content of extract/fractions was [31] 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Ferric determined using Folin-Ciocalteu method and gallic acid chloride, L-Ascorbic acid, NADH (Nicotinamide Adenine was used as standard. 100 毺L of extract/fractions was added Dinucleotide), PMS (Phenazine Methosulphate), NBT to 900 毺L of double distilled water followed by the addition (Nitroblue Tetrazolium Chloride) were obtained from of 500 毺L of FC reagent. This was followed by the addition HiMedia Pvt. Limited , India. Potassium persulfate, of 1.5 mL of 20% sodium carbonate. The volume of mixture 10 L ABTS [2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) was made up to m with distilled water and allowed to stand for 2 h. Finally absorbance was taken at 765 nm. diammonium salt], Gallic acid, from Sigma (St. Louis, MO, The phenolic content was calculated as gallic acid (mg/g) USA). Plasmid pBR322 was purchased from Genei Pvt. Ltd., equivalents on the basis of standard curve of gallic acid. Banglore. All other reagents were of analytical grade (AR). 2.5. Antioxidant activity 2.2. Collection of plant material 2.5.1. DPPH-radical scavenging assay A. cadamba T he leaves of were collected from the trees The DPPH radical scavenging assay is commonly employed growing in the campus of Guru Nanak Dev University, in evaluating the ability of antioxidants to scavenge free Amritsar. radicals. The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability. It was carried out by Madhu Chandel et al./Asian Pacific Journal of Tropical Medicine (2012)977-985 979 the method of Blois (1958)[32] with modifications. Different prepared in phosphate buffer pH 7.4) and 1 mL of various concentrations (20-400 g/mL) of extract/fractions of concentrations of the fractions (100, 200, 300, 400 and 500 A. cadamba 毺 leaves were dissolved in methanol and taken 毺g/mL) and the reference compound rutin were mixed and in test tubes in triplicates. Then 2 mL of 0.1 mM methanol the reaction was started by adding 100 毺L of phenazine solution of DPPH was added to each of the test tubes and methosulphate (PMS) solution (60 毺M in phosphate buffer, were shaken vigorously. After 30 min absorbance was taken pH 7.4). The reaction mixture was incubated at 25 曟 for at 517 nm using UV-VIS spectrophotometer. 5 min and the absorbance was measured at 560 nm. The %Radical scavenging activity (%) = [Abs (control) - Abs percentage inhibition was calculated by the formula: × S A (sample) /Abs (control)] 100. uperoxide anion radical scavenging ×activity (%) = [ bs where, Abs (control): Absorbance of DPPH radical + solvent (control) - Abs (sample) /Abs (control)] 100. alone, Where, Abs (control): Absorbance of control, A A Abs (sample): Absorbance of DPPH radical + extract/ bs (sample): bsorbance of sample. fractions. 2.5.5. Plasmid DNA protection assay 2.5.2. ABTS radical scavenging assay To measure the hydroxyl radical scavenging effect of .+ extract/fractions, DNA nicking experiment was performed The spectrophotometric analysis of ABTS scavenging et al according to the protocol of Lee [36]. Plasmid DNA was activity was determined according to the protocol given by ’ et al incubated with Fenton s reagent containing extract/fractions R 33] ABTS e [ . radical cations were produced by reacting and finally the volume of the mixture was raised up to 20 ABTS stock solution (7 mM) and potassium persulfate (2.45 毺L. The mixture was then incubated for 30 min at 37 曟 mM) solution in the ratio of 1: 0.5 and allowing the mixture followed by addition of loading dye. Electrophoresis was to stand in the dark at room temperature for 12-16 h before carried out in TAE (Tris Acetic acid EDTA) buffer and DNA use. The ABTS radical cation solution was diluted with was analyzed followed by ethidium bromide staining. ethanol to an absorbance of (0.70依0.02) at 734 nm. 300 毺L 2.6. UPLC-electrospray ionization-quadrupole time-of- of different concentrations (20-400 g/mL) of the extract/ 毺 flight mass spectrometry fractions were added to the diluted ABTS radical cation solution and absorbance was taken up to 5 min. 2.6.1. Sample preparation

[Abs (control) - Abs (sample) × F UPLC ESI MS % Radical scavenging activity (%) = 100 or - - analysis, samples were prepared in a Abs (control)] mixture of acetonitrile/water (80/20; v/v) and filtered through a 0.22 lm MILLEX GV syringe filter (Millipore, MA, USA) 2.5.3. Reducing power assay prior to inject into the UPLC system. R educing potential of the extract/fractions was determined 2.6.2 UPLC instruments and chromatographic conditions using the method of Oyaizu[34]. Different concentrations of A. cadamba extract/fractions (200-1 000 毺g/mL) of leaves All analysis were performed on Waters Acquity UPLC were dissolved in methanol and taken in test tubes in system (Waters, MA, USA), including binary solvent triplicates. To the test tubes phosphate buffer (pH 6.6, 0.2 M) manager, sample manager, column compartment and photo and 1% Potassium ferricyanide solution was added. These diode array (PDA) detector, connected with Waters Mass Lynx software. An Acquity UPLC BEH C18 column (100 contents were mixed well and were incubated at 50 曟 for 20 × min. After incubation 10% TCA was added and were kept for mm 2.1 mm i.d, 1.7 毺m) also from Waters was used. The centrifugation at 3 000 rpm for 10 min. After centrifugation column temperature was maintained at 30 曟. Samples were 0 05 2.5 mL of supernatant was taken and mixed with double separated using a gradient mobile phase consisting of . % formic acid in water (A) and acetonitrile (B). The gradient distilled water (2.5 mL) and ferric chloride (0.1%). The elution is: 0-0.2 min, 82% A; 0.2-1.4 min, 75% A; 1.4-2.0 O.D (absorbance) was measured spectrophotometrically min, 65% A; 2.2-3.5 min, 65% A, and finally reconditioning at 700 nm. Increase in absorbance of reaction mixture was the column with 82% A for 1 min. The flow rate was set 0.28 interpreted as increase in reducing ability of the sample and -1 mL. min and the injection volume was 1 L. rutin was used as a standard. 毺 2.7. Statistical analysis 2.5.4. Superoxide anion radical scavenging assay

The measurement of superoxide anion scavenging activity The results are presented as the mean 依 standard error of extract/fractions was performed according to the method et al of three experiments. Regression analysis was carried out 1972 [35] of Nishikimi ( ) with slight modifications. About by best fit method and IC50 values were calculated using 1 mL of nitroblue tetrazolium (NBT) solution (156 毺M prepared regression equation. The significance of results was checked P in phosphate buffer, pH 7.4), 1 mL of NADH solution (468 毺M at 曑0.05. Madhu Chandel et al./Asian Pacific Journal of Tropical Medicine (2012)977-985 980 3. Results

100 100 3.1. Total phenolic content (TPC) 80 80 60 60 40 Rutin Rutin Inhibition (%) Inhibition 40 Inhibition (%) Inhibition 20 ETAC 20 ETAC The results of the total phenolic content evaluated using 0 0 20 60 100 200 400 20 60 100 200 400 F C T 1 TPC Concentration ( g/mL) olin- iocalteu method, are shown in able . s of 毺 Concentration (毺g/mL) the extract/fractions was found in the order, ethyl acetate n 100 100 fraction (EAAC) > -butanol fraction (NBAC) > ethanol 80 80 extract (ETAC) > water fraction (WAC). The TPC of the EAAC 60 60 40 Rutin 40 Rutin Inhibition (%) Inhibition Inhibition (%) Inhibition fraction (194.07 依 4.08 mg GAE/g extract) was higher than 20 ETAC 20 ETAC 0 0 those of all other extract/fractions. 20 60 100 200 400 20 60 100 200 400 Concentration (毺g/mL) Concentration (毺g/mL) Table 1 Figure 3. Scavenging effect of extract/fractions from leaves of Total phenolic content (TPC) of extract/fractions from leaves of A. cadamba P＜ A. cadamba. on ABTS radicals. * 0.05.

Extracts/fractions Phenolic content (mg GAE/g of extract) Ethanol extract 149.87依4.40 3.4. Reducing power assay Ethyl acetate fraction 194.07依4.08 n 182 45依4 09 -butanol fraction . . Substances with reducing potential reacts with potassium Water fraction 107.63依8.40 3+ 2+ ferricyanide (Fe ) to form potassium ferrocyanide (Fe ) 3.2. DPPH assay which then reacts with ferric chloride to form ferric ferrous complex that has absorption maximum at 700 nm. The The scavenging effect of crude extract and various fractions increased absorbance of the reaction mixture indicates 50 increased reducing power. The reducing potential of crude based on their IC values was in the order of EAAC (21.24 毺 extract and various fractions was in the order of EAAC > g/mL) > NBAC (36.41 毺g/mL) > ETAC (63.94 毺g/mL) > WAC NBAC > ETAC > WAC (Figure 4). The results were compared (138.66 毺g/mL). The % inhibition values of ETAC, EAAC, NBAC and WAC were 88.06%, 90.03%, 89.24% and 88.63% with the standard compound rutin. at highest tested concentration (400 g/mL) respectively 毺 1.8 2 (Figure ). The results were compared with standard 1.6 compound rutin (IC50=54.05 毺g/mL). 1.4 1.2 Rutin 1.0 ETAC

100 Absorbance 0.8 0.6 EAAC 80 0.4 NBAC 0.2 WAC 60 Rutin 0 ETAC 200 400 600 800 1000 40 EAAC Concentration (毺g/mL)

Scavenging effect(%) NBAC 20 Figure 4. WAC Reducing potential of extract/fractions from leaves of A. cadamba P＜ 0 . * 0.05. 20 60 100 200 400 Concentration (毺g/mL) 3.5. Superoxide scavenging assay Figure 2. Scavenging effect of extract/fractions from leaves of A. cadamba P＜ on DPPH radicals. * 0.05. Superoxide anion is implicated as harmful ROS. It has detrimental effect on the cellular components in a biological

system[37,38]. Although they can not directly initiate lipid 3.3. ABTS radical scavenging assay oxidation, they are potential precursors of highly reactive species such as hydroxyl radical and thus the study of All the extract/fractions showed good potential to scavenge scavenging of this radical is important. Different extract/ ABTS radical cations. The scavenging effect was 95.21%, fractions showed superoxide scavenging activity of 55.18% 95.70%, 94.72% and 81.13% in ETAC, EAAC, NBAC and (ETAC), 72.31% (EAAC), 71.28% (NBAC) and 45.61% (WAC) WAC respectively, at highest tested concentration (Figure 3). at highest tested concentration (500 毺g/mL) (Figure 5). Madhu Chandel et al./Asian Pacific Journal of Tropical Medicine (2012)977-985 981

Rutin was used as standard (IC50=58.75 毺g/mL).

80 70 240 y=0.754x+208.8 250 y=0.16x+180.1 60 *r=0.901 200 *r=0.981 50 200 ETAC 40 160 EAAC 150 30 NBAC 120 Scavenging effect (%) 20 100 WAC 80 10 40 50 Total phenolic content (TPC) 0 Total phenolic content (TPC) 100 200 300 400 500 0 0 0 20 40 60 80 100 120 140 -100 100 300 500 Concentration ( g/mL) 毺 50 50 IC (毺g/mL) IC (毺g/mL) Figure 5. a b Superoxide scavenging potential of extract/fractions from y=-0.039x+190.1 A. cadamba 250 y=-0.144x+197.6 250 leaves of . *r=0.994 *r=0.964 200 200

150 3.6. Plasmid DNA nicking assay 150 100 100

50 50 Total phenolic content (TPC) DNA protective activity was assessed by measuring the Total phenolic content (TPC) 0 0 degree of protection on DNA scission that was induced by the 0 200 400 600 0 1000 2000 3000 ’ IC50 ( g/mL) 50 毺 attack of hydroxyl radicals generated by Fenton s reagent. IC (毺g/mL) d Hydroxyl radical is one of the ROS formed in biological Figure 7. c Correlation between total phenolic content (GAE mg/ Anthocephalus cadamba systems, causing DNA strand breakage, which brings about g of extract) of extract/fractions and their [39,40] carcinogenesis, mutagenesis and cytotoxicity . The antioxidant capacity as determined by (a): DPPH assay; (b): ABTSP protection against the damage caused by hydroxyl radicals ＜assay; (c): reducing power assay; (d): superoxide scavenging assay. * was shown by the agarose gel electrophoresis pattern (Figure 0.05. 6). It is clear from the results that extract/fractions protected 3.8. Identification of bioactive compounds by UPLC the pBR322 plasmid DNA against the DNA damaging effect of hydroxyl radicals. Representative UPLC chromatograms are shown in Figure 8. The retention times (tR), wavelengths of maximum 1 2 3 4 5 6 7 absorbance (毸max), and m/z are listed in Table 2. Peak identification was based on comparision of mass spectra and 毸max from literature. UPLC-ESI-QTOF-MS study of ETAC Nicked circular form A. cadamba Linear form extract from leaves of showed major peaks Supercoiled form at tR 1.21, 2.73 and 3.33 which exhibited the typical UV spectra of chlorogenic acid (m/z=355), dihydrocadambine (m/z=547) and 毬-sitosterol (m/z=411) respectively. The Figure 6. A. cadamba occurrence of peaks with low intensities at tR 1.96 and 2.20 Effect of different extracts/fraction of leaves of have absorption in the UV region at 347 nm (m/z=611) and in DNA protection assay. ’ Lane 1: Negative Control (DW+DNA); Lane 2: Fenton s reagent (FR) 280, 340 nm (m/z=449) respectively, therefore indicating + DNA; Lane 3: pBR322 plasmid DNA+FR +gallic acid (200 毺g/ the identity as rutin and kaempferol 3-O-glucoside (Figure mL); Lane 4: pBR322 plasmid DNA+FR +ETAC (200 毺g/mL); Lane 8a). EAAC fraction showed the high intensity of the peak 5: pBR322 plasmid DNA +FR+EAAC (200 毺g/mL); Lane 6: pBR322 plasmid DNA +FR+ NBAC (200 毺g/mL); Lane 7: pBR322 plasmid corresponding to chlorogenic acid. Other minor peaks were DNA +FR+WAC (200 毺g/mL). of catechin/epicatechin (m/z=291, 毸max=279), feruloyquinic 3.7. Correlation between total phenolic content and acid (m/z=369, 毸max=286, 326) and cadambine (m/z=545, antioxidant activity 毸max=283) (Figure 8b). NBAC and WAC showed the presence of compounds which were qualitatively similar, only the difference in relative abundance of each compound Phenols are very important plant constituents because have been observed. NBAC showed one major peak of of their radical scavenging ability due to their hydroxyl dihydrocadambine at retention time 2.73 (Figure 8c). The [41,42] groups . In the present study a linear correlation peaks with high intensity corresponding to chlorogenic have been observed between total phenolic content and acid, rutin, dihydrocadambine and 毬-sitosterol have been antioxidant activity (Figure 7). Ethyl acetate fraction (EAAC) A. cadamba observed in WAC fraction (Figure 8d). of showed highest scavenging activity in all the assays. The phenolic content was also high in EAAC fraction. Madhu Chandel et al./Asian Pacific Journal of Tropical Medicine (2012)977-985 982 4. Discussion 2.73 2:Diode Array 1.5e-1 (a) ETAC 254 2.91 3.33 Range:1.956e-1 1.25e-1 1.0e-1 1.21 DPPH assay has been widely used to determine the 3.22 7.5e-2 free radical-scavenging activity of various plant extracts 5.0e-2 0.96 1.96 2.50 2.20 [43-48] DPPH 2.50e-2 1.78 and pure compounds . is a stable free radical

0.0 which dissolves in methanol, and its purple colour shows 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.782.96 1:TF MSES+ a characteristic absorption at 517 nm. An antioxidant TIC 1.57e4 when scavenges the free radical by hydrogen donation, the colour changes from purple to light yellow. In the % 3.39 0.87 3.30 2.26 2.54 present study, ethyl acetate fraction was found to be most 3.62 4.02 1.40 1.87 3.84 4.30 4.56 4.64 effective scavenger of DPPH radical among all the fractions 0.31 0.55 et al 7 Time and crude ethanol extract. Zhenbao [49] evaluated the 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Cassia tora antioxidant potential of fractions from and (b)EAAC 2:Diode Array reported ethyl acetate fraction the most effective among all 3.5e+2 Range:4.276e+2 et al [50] 3.0e+2 the fractions. Sun evaluated the antioxidant activity 2.5e+2 Diospyros kaki 2.01 P 2.0e+2 of total flavonoids extract from ersimmon ( 3.05 1.5e+2 2.32 2.73 3.24 L.) leaves and attributed the potent antioxidant activity on 2.20 2.55 1.0e+2 1.55 5.0e+1 the DPPH radical to a direct role in trapping free radicals 0.0 by donating a hydrogen atom. Since the DPPH assay was 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 3.38 1:TOF MS ES+ 2.04 TIC used as a preliminary free radical-scavenging evaluation, 1.25 2.56e3 3.12 we subsequently performed analysis on ABTS radical 3.58 2.85 2.38 3.78 % 2.60 scavenging, reducing power assay, superoxide scavenging 1.60 2.23 4.00 1.00 4.20 and DNA nicking assay to confirm the free radical- 4.42 4.58 0.08 0.38 0.59 scavenging effect from the leaves extract/fractions of Time 8 A. cadamba •+ 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 . ABTS , a protonated radical, has characteristic absorbance maxima at 734 nm which decreases with the 2.73 2:Diode Array 1.0e-1 (c)NBAC 254 [51] H Range:1.40e-1 scavenging of the proton radicals . owever, among 8.0e-2 all the fractions EAAC fraction was most effective based 6.0e-2 on the IC50 value (1.12 g/mL) which was lower than the 4.0e-2 毺 0.96 standard compound rutin (IC50=24.78 g/mL). Similar results 2.0e-2 毺 1.22 1.72 1.95 et al 2.39 were reported by Guo [52] where they evaluated the 0.0 Tuber indicum 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.78 1:TOF MS ES+ antioxidant activity of extract/fractions from TIC n 0.86 1.29e4 where ethyl acetate and -butanol fractions were found to be most effective among all the extract/fractions and % correlated with content of total phenolic and total flavonoid 3.28 1.17 2.63 3.06 3.643.82 4.13 4.28 4.79 I • 1.371.56 1.852.03 2.292.48 which was high in both the fractions. n the present study, 0.21 0.44 + 7 Time it has been observed that the scavenging of ABTS• by the 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 extract/fractions was higher than that• of DPPH . This may• 2:Diode Array + 2.68 3.0e-1 (d)WAC 254 be due to high reactivity of ABTS radicals than DPPH Range:3.05e-1 2.5e-1 1.21 radicals, and unlike the reactions with DPPH radicals 2.0e-1 1.98 3.33 which involve• HAT (hydrogen atom transfer), the reactions 1.5e-1 + 2.40 1.0e-1 2.21 with ABTS radicals involve both HAT and SET (single 3.22 0.97 1.49 1.78 5.0e-2 electron transfer)[53]. Several factors like stereoselectivity 0 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 2.74 of the radicals or the solubility of the extract in different 1:TOF MS ES+ TIC testing systems have been reported to affect the capacity of 2.11e4 extracts to react and quench different radicals[54]. Gramza % et al 3.40 [55] evaluated the ABTS radical scavenging activity of 3.30 0.90 1.28 2.05 2.57 3.11 1.65 2.28 3.59 tea extracts and attributed the scavenging potential to the 1.89 4.93 0.18 0.46 0.75 4.02 4.20 4.534.56 5 Time presence of bioactive compounds catechins and tannins. 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 3 Figure 8. + Representative UPLC chromatograms of extract/fractions Ferric ion (Fe ) reduction is often used as an indicator of A. cadamba from leaves of : (a) ETAC; (b) EAAC; (c) NBAC; (d) WAC. electron donating activity, which is an important mechanism of phenolic antioxidant action[56,57]. The existence of Madhu Chandel et al./Asian Pacific Journal of Tropical Medicine (2012)977-985 983 Table 2 A. cadamba Identified compounds from leaves of . S. No. tR UV 毸max m/z [M+H]+ Tentative identification References 1 1.21 217, 245, 325 355 Chlorogenic acid Broils et al., 1998; Zhao et al., 2011 2 1.55 279 291 Catechin, epicatechin Aaby et al., 2007; Zhao et al., 2011 Abad-Garcia et al., 2009 3 1.96 347 611 Rutin Zhao et al., 2011 4 2.01 286, 326 369 Feruloyquinic acid Dartora et al., 2011 5 2.20 280, 340 449 Kaempferol 3-O-glucoside Zhao et al., 2011; Engels et al., 2011 6 2.32 226, 280 741 Glycosides of flavonoids (on the basis of mass Zhao et al., 2011 fragmentation patteren only) 7 2.55 283 545 Cadambine (indole alkaloid) Handa et al., 1983 8 2.73 285, 326 547 Dihydrocadambine(indole alkaloid) (on the Brown and Fraser, 1974 basis of mass fragmentation patteren only) 9 3.33 220, 327 411 [(M+H)+-4H+] 毬-sitosterol Huang et al., 2007

A. cadamba reductones is the key of the reducing power, which exhibit fractions from leaves of may be attributed to their antioxidant activities through the action of breaking the presence of compounds viz. Chlorogenic acid, Catechin/ the free radical chain by donating a hydrogen atom[58-67]. epicatechin, Rutin, Feruloyquinic acid, Kaempferol 3-O- Reductones are also reported to react with certain precursors glucoside, Glycosides of flavonoids, indole alkaloids and 毬 of peroxide, thus preventing peroxide formation[56,68]. Wang -sitosterol which were identified by UPLC-ESI-QTOF. et al[60] Camellia sinensis evaluated the reducing ability of tea ( In all the assay systems, it was found that EAAC fraction L.) fruit peel extracts using FRAP assay and found showed a higher antioxidant activity followed by NBAC a significant positive correlation between FRAP values and fraction and WAC fraction. The bioactive compounds viz. total flavonoids and concluded that fractions enriched with catechins/epicatechins, feruloyquinic acid and cadambine flavonoids and phenolics, act like ferric-reducing agents which were exclusively present in EAAC fraction might and have a potential to protect humans from oxidative stress contributing the higher antioxidant activity of this fraction. et al[70] induced by excessive ferric ion. Prasad Cinnamomumevaluated The total phenolic content analysis also showed the EAAC antioxidant C. zeylanica activities of different species of fraction with high phenolic content in terms of gallic acid and exhibited a stronger reducing power and equivalents/g of the fraction. The potential of the extract/ attributed the reducing power to the hydroxyl group in fractions to scavenge different free radicals in different assay phenolic compounds which might act as electron donors. systems indicated that they may serve as useful therapeutic The antioxidant activity has been reported to have a direct, agents for treating radical-related pathologic damage. positive correlation with the reducing power[71]. All the Further studies are in progress to isolate the antioxidant extract/fractions showed goodet al scavenging potency towards principles from the extract/fractions using different superoxide radicals. Hu [72] reported that flavonoid spectroscopic techniques. molecule with polyhydroxylated substitution on ring B and a free 3-hydroxyl substitution could present the superoxide Conflict of interest statement scavenging activity. Further the extract/fractions showed 322 the potential to protect the pBR plasmid DNA fromet al the damage caused by hydroxyl radicals. Rajkumar [73] We declare that we have no conflict of interest. evaluatedBergenia the ciliata effect of methanolic and aqueous extracts of rhizome at• 50 毺g concentration on Acknowledgements the protection of DNA againstet al OH radicals generated by photolysis of H2O2. Bibi [74] reported the high DNA 2 2 protection Asteractivity thomsonii against the H O induced damage by the The authors are thankful to UGC (DRS-SAP), New Delhi for extracts of and attributed the DNA protectionin vitro providing financial assistance. Upendra sharma is grateful to activity to the high antioxidant activity. In all the CSIR for SRF. We also thank Shiv Kumar for UPLC analysis. antioxidant assays a significant correlation was observed between total phenolic content and antioxidant activity.et References Our results are in agreement with the reports of Sghaier al[75] where they studied the antioxidantTeucrium and ramosissimum antigenotoxic properties of different extracts of [1] Surh Y, Ferguson LR. 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