Asian Pacific Journal of Tropical Medicine (2012)977-985 977 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm 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, Guru Nanak Dev University, Amritsar-143005 India 2Natural Plant 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 plants 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 Mumbai, 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