Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2011, Article ID 784826, 10 pages doi:10.1093/ecam/nep155 Original Article Therapeutic Potential of Polar and Non-Polar Extracts of Cyanthillium cinereum In Vitro Gunjan Guha, V. Rajkumar, R. Ashok Kumar, and Lazar Mathew School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore 632 014, Tamil Nadu, India Correspondence should be addressed to R. Ashok Kumar, ashoku [email protected] Received 27 January 2009; Accepted 8 September 2009 Copyright © 2011 Gunjan Guha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cyanthillium cinereum (Less.) H. Rob. (Asteraceae) has been traditionally known for its medicinal properties, all aspects of which are yet to be exploited. This study was aimed at investigating the therapeutic potential of polar (methanolic and aqueous) and nonpolar (hexane and chloroform) crude extracts of the whole plant. Several parameters including free-radical (DPPH•, •+ • ABTS ,H2O2 and OH) scavenging, reducing power, protection of DNA against oxidative damage, cytotoxicity, inhibition of oxidative hemolysis in erythrocytes, total phenolic content and inhibition of lipid peroxidation were examined. All the free- radical generating assay models demonstrated positive scavenging efficiency with differential but considerable magnitudes for the four extracts. However, only the hexane extract showed significant H2O2 scavenging effect. Lipid peroxidation was estimated by thiobarbituric acid-malondialdehyde (MDA) reaction, and a high degree of inhibition was shown by all the extracts. Reducing power of the polar extracts was higher than the non-polar ones. All extracts showed a concentration-dependent increase in phenolic contents. Oxidative damage to erythrocytes was hindered by all extracts in diverse degrees. XTT assay showed that all extracts have mild cytotoxic property. The aqueous extract evidently demonstrated protective effect on pBR322 plasmid DNA against oxidative breakdown. These results suggested the potential of C. cinereum as medicine against free-radical-associated oxidative damage and related degenerative diseases involving metabolic stress, genotoxicity and cytotoxicity. 1. Introduction medicinal value in diverse traditional usage in different nations, and also gets recognition in the Ayurvedas [3]. Atoms or molecules containing one or more unpaired elec- The whole plant is used in decoction or infusion to treat trons are termed as free radicals which are accountable for fever [4]. It provides remedy for spasms of the urinary tissue degeneration by means of DNA and protein damage bladder and strangury, and is often combined with quinine and lipid peroxidation. Oxidative stress associated with free to treat malaria [3]. Sesquiterpene lactones, which possess radicals is involved in the pathophysiology of aging and antimalarial activity, have been isolated from the plant [5]. various age-related ailments such as cataracts, atherosclero- Cyanthillium cinereum has therapeutic potentials against sis, diabetes, Alzheimer’s disease, and so forth. The extent asthma [6], cancer [7], cholera, colic pain, cough, diarrhea, of damage caused by free radicals might be mitigated dysentery, impotency and night-blindness [4]. The seeds are through supplementation with one or more antioxidants [1]. used as a source of alexipharmic and anthelmintic drugs, Various compounds with differential antioxidant properties and as an alterative in leprosy and chronic skin diseases are found in floral resources which are considered to have [3]. Cyanthillium cinereum leaves have analgesic, antipyretic high potential in the context of therapeutic approaches to and anti-inflammatory effects [8]. Paste of stem/bark is used encounter and prevent free radical damage. Diverse medic- to heal cuts, while flowers are traditionally used to treat inal plants have been screened and assessed for properties in conjunctivitis [3], arthritis [9] and rheumatism [10]. Root antagonism to free-radical-induced oxidative stress [2]. infusion is used as an antidote to scorpion sting and snake Cyanthillium cinereum (Less.) H. Rob. (synonym: Ver- venom [3]. nonia cinerea (Linn.) Less.), commonly known as little The present study is aimed at estimating the diverse ironweed, is a common annual weed (Asteraceae) with a therapeutic potentials of non-polar (hexane and chloroform) wide range of geographical distribution. The plant has great and polar (methanolic and aqueous) extracts of C. cinereum 2 Evidence-Based Complementary and Alternative Medicine (whole plant) in vitro with respect to antioxidant and free- USA) with methanol as blank. Trolox was used as positive radical-scavenging properties, inhibition of lipid peroxida- control. tion, cytotoxicity and protection from DNA and cell damage. The level of percentage inhibition of DPPH• by the different extracts was calculated according to the following 2. Methods formula: A − A = C × 2.1. Chemicals and Other Reagents. 1,1-Diphenyl-2-picryl- % Radical scavenging A 100, (1) hydrazyl (DPPH), thiobarbituric acid (TBA), ethylene C diamine tetraacetic acid (EDTA), gallic acid, ascorbic acid, where AC is the absorbance of the control and A is trichloroacetic acid (TCA), phenazine methosulfate (PMS) the absorbance of sample. Percentage scavenging was also (also known as N-methylphenazonium methosulfate), evaluated in Trolox equivalence. dimethyl sulfoxide (DMSO), Dulbecco’s phosphate buffered saline (PBS) (Ca2+/Mg2+ free) and L-15 (Leibovitz) cell 2.5. ABTS Radical Scavenging Activity. ABTS assay was culture medium (with l-glutamine) were purchased from performed according to the protocol of Arnao et al. [12]. Himedia Laboratories Pvt. Ltd. (India). 2,2-Azinobis-3- The stock solution was prepared by mixing equal volumes of ethylbenzothiazoline-6-sulfonic acid (ABTS) and Trolox • 7.4 mM ABTS + solution and 2.6 mM potassium persulfate (6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid) solution followed by incubation for 12 h at room tempera- were purchased from Sigma Aldrich Chemical Co. ture in dark. Working solution was prepared freshly before (Milwaukee, WI, USA). XTT {2,3-bis(2-methoxy-4-nitro- each assay by diluting the stock solution by mixing 60 ml 5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium methanol to 1 ml stock solution. Different concentrations of hydroxide} was obtained from Sigma Chemical Co. (St. the phyto-extracts were taken in test tubes. Three milliliters Louis, MO, USA). Folin-Ciocalteau reagent was procured • of ABTS + working solution was added to each tube and from Sisco Research Lab (India). The remaining chemicals allowed to react for 2 h in dark. Absorbance was taken at and solvents used were of standard analytical and HPLC 734 nm. Percentage radical scavenging activity was calculated grades, respectively. pBR322 was purchased from Medox using a similar formula as employed for the DPPH assay, and Biotech India Pvt. Ltd (India). MDA-MB-435S (human plotted along with Trolox equivalence. breast carcinoma) cell line was obtained from National Centre for Cell Science (Pune, India). 2.6. Lipid Peroxidation Inhibition Efficiency. Inhibition effi- 2.2. Plant Material. Cyanthillium cinereum (whole plant) ciency of lipid peroxidation inhibition (LPI) was estimated was collected in the month of July 2007 from Vellore district by TBA assay [13]. A 6-week-old female Wistar albino rat (12◦55N, 79◦11E), Tamil Nadu, India, and identified at weighing approximately 150 g was sacrificed under general Botanical Survey of India, Southern Circle, Coimbatore, ethereal anesthesia and its liver was excised. In total, 10% India (BSI/SC/5/07-08/Tech.-523, 13 July 2007). Healthy (w/v) liver homogenate was prepared in Dulbecco’s PBS 2+ 2+ plants were screened and thoroughly washed. The cleansed (Ca /Mg free) (pH 7.4), and centrifuged at 3000 rpm × plants were freeze-dried for 2 months at –80◦Cina (503 g) for 15 min to obtain a clear supernatant. Diverse .TM ◦ concentrations of each extract were taken in different test MDF-U32V V.I.P Series 86 C UltraLow Temperature ◦ Freezer (Sanyo Biomedical, IL, USA). The dried plants were tubes and evaporated to dryness at 80 C. One milliliter powdered for the preparation of extracts. of 0.15 M potassium chloride and 0.5 ml of the obtained supernatant were added to each tube. Lipid peroxidation was initiated by the addition of 100 μl of 0.2 mM ferric chloride 2.3. Preparation of Extracts. Fifty grams of whole plant ◦ powder was serially extracted with hexane, chloroform, and incubated at 37 C for 30 min. Two milliliters of ice-cold methanol and water using Soxhlet apparatus. These crude 0.25 N-hydrochloric acid containing 15% TCA and 0.38% ◦ TBA was added to stop the peroxidation reaction, followed extracts were concentrated at 40 Cunderreducedpres- ◦ sure (hexane: 360 mbar; chloroform: 474 mbar; methanolic: by an incubation of 1 h at 80 C. The samples were brought 337 mbar; aqueous: 72 mbar) with Rotavapor R-215 (BUCHI¨ down to room temperature and centrifuged at 7500 rpm × Labortechnik AG, Switzerland) to yield dry extracts. The (3144 g) for 15 min. Absorbance of the supernatant was final quantities of the hexane, chloroform, methanolic and measured at 532 nm. aqueous extracts obtained were 2.22, 0.9, 9.79 and 5.21 g, Percentage LPI was calculated by the following formula: respectively. AC − A %LPI=