Antioxidant Properties of Aqueous Extracts of Selaginella Willdenowii
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Journal of Medicinal Plants Research Vol. 6(7), pp. 1289-1296, 23 February, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1376 ISSN 1996-0875 ©2012 Academic Journals Full Length Research Paper Antioxidant properties of aqueous extracts of Selaginella willdenowii Tsun-Thai Chai* and Fai-Chu Wong Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, 31900 Kampar, Malaysia. Accepted 12 December, 2011 Selaginella willdenowii is a plant used as traditional medicine and vegetable in some Asian countries. Unlike other medicinally important Selaginella species, little is known about the pharmacological properties of S. willdenowii. This study was therefore conducted to evaluate the antioxidant properties of aqueous extracts of leaves and stems of S. willdenowii, both fresh and dried prepared with or without heat treatment. Total phenolic and flavonoid contents of the leaf extracts were consistently higher compared with stem extracts. Total phenolic and flavonoid contents of hot water extracts of fresh leaves were up to 4.4-fold and 10.7-fold higher, respectively, when compared with all other extracts. Radical scavenging and ferric reducing activities of the leaf extracts were also consistently higher compared with stem extracts. Trolox equivalent antioxidant capacity (TEAC) and ferric reducing antioxidant power (FRAP) of hot water extracts of fresh leaves were up to 5.5-fold and 5.3-fold higher, respectively, when compared with all other extracts. Correlation analysis revealed that total phenolic content is likely a key determinant of the radical scavenging and ferric reducing abilities in the leaf extracts. Overall, our findings affirm the value of S. willdenowii as a medicinal herb as well as a source of dietary antioxidant. Key words: Ferric reducing antioxidant power, flavonoids, free radical scavenging activities, phenolics, Selaginella willdenowii, trolox equivalent antioxidant capacity. INTRODUCTION Selaginella willdenowii (Desv.) Baker is a lycophyte S. willdenowii infusion is administered to treat high fever native to Malaysia, Indonesia and Myanmar (Valdespino, and its ashes are used as liniment for backache (Khare, 1993). The plant is used as traditional medicine in several 2007). Asian countries. In Malaysia, S. willdenowii leaf decoction The use of S. willdenowii in traditional medicine is not is used to treat wounds (Hanum and Hamzah, 1999), surprising as many other Selaginella species are also high fever and backache, as well as being used as tonic used as medicinal herbs in different countries. For medicine (Eswani et al., 2010). In Brunei, it is used to example, in China, Selaginella doederleinii is used in treat gastric pains and infections of urinary tracts (Haji anticancer treatment (Lee and Lin, 1988). In India, Mohiddin et al., 1992). In Indonesia, it is used to treat Selaginella tamariscina var. pulvinata and Selaginella wounds, menstrual pains (Setyawan, 2009) and skin involvens are used to prepare life-prolonging tonics diseases, in addition to being consumed as vegetable (Khare, 2007). In Mexico, Selaginella lepidophyll is used (De Winter and Jansen, 2003; Setyawan, 2009). In India, to treat several ailments, including kidney stone, gastric ulcer and rheumatism (De Winter and Jansen, 2003). The uses of various Selaginella species in traditional medicines in Asia, Africa and Latin America have been *Corresponding author. E-mail: [email protected]. Tel: recently reviewed (Setyawan, 2009). +6054688888. Ext: 4516. Fax: +6054661676. An earlier report of the isolation of anticancer Abbreviations: DPPH, 1,1-diphenyl-2-picrylhydrazyl; ABTS, biflavonoids from S. willdenowii (Silva et al., 1995) 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); FRAP, implies significant bioactive potential in the species. ferric reducing antioxidant power; TEAC, trolox equivalent However, unlike other Selaginella species, very little antioxidant capacity. information can be found in the literature about the 1290 J. Med. Plants Res. antioxidant activity or other bioactive potential of S. was added to 0.15 ml of NaNO2 (5% w/v) and the mixture was willdenowii. Antioxidant potential of other medicinally incubated at room temperature for 6 min. Next, 0.15 ml of important Selaginella species, for example, S. AlCl3.6H2O (10% w/v) was added to the mixture, which was then left at room temperature for 6 min. Next, 0.8 ml of NaOH (10% w/v) was tamariscina (Kyung Ae and Sang Kook, 1999; Gan et al., added and the absorbance of the mixture was read at 510 nm after 2010), Selaginella bryopteris (Sah et al., 2005), S. standing at room temperature for 15 min. For the blank, the extracts involvens, and S. delicatula (Gayathri et al., 2005; Seong were replaced with water. To correct for background absorbance, et al., 2008) have already been verified. Therefore, the each sample measurement was accompanied with a simultaneous aim of this study was to assess the total phenolic and control reaction in which AlCl3.6H2O was replaced with water. A flavonoid contents, free radical scavenging capacity, and standard curve was prepared from 0 to 500 g/ml quercetin dissolved in 80% ethanol. Total flavonoid content was expressed in reducing power of S. willdenowii. To our knowledge, this mg quercetin equivalents/g dry matter. paper represents the first report of antioxidant activity in S. willdenowii extracts. Determination of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability MATERIALS AND METHODS DPPH radical scavenging activity of the extracts was assessed as Plant materials described previously with modifications (Lim and Quah, 2007). To 1 ml of DPPH (0.10 mM in methanol), 1 ml of extract was added. The S. willdenowii plants were collected from a Malay village located at mixture was left in the dark for 30 min before its absorbance was read at 517 nm. A blank was prepared for each sample in which the the boundary of Bujang Melaka Forest Reserve, Perak, Malaysia, in May 2011. The plant material was authenticated by Professor Dr DPPH solution was replaced with methanol. DPPH radical Ong Hean Chooi at Institute of Biological Sciences, University of scavenging ability (%) was calculated as follows: Malaya, Malaysia and Ahmad Dwi Setyawan at Department of DPPH radical scavenging ability (%) = (1- [A / A ]) x 100 Biology, Faculty of Mathematics and Natural Sciences, Sebelas sample control Maret University, Indonesia. Voucher specimens were deposited at Department of Chemical Science, Universiti Tunku Abdul Rahman. where Acontrol is the absorbance of control reaction (without plant extract) and Asample is the absorbance in the presence of a plant extract. Ascorbic acid and Trolox were used as references. Results Preparation of aqueous extracts are also presented as EC50 values, which represent concentrations of extracts required to scavenge 50% of DPPH radicals. The aerial plant parts were separated into leaves and stems after they were collected. Leaf and stem materials were each divided into three lots to be used in three replicate extractions. Three types of Determination of 2,2’-azino-bis(3-ethylbenzthiazoline-6- aqueous extracts were prepared, designated “Dried + Heat”, “Fresh sulphonic acid) (ABTS) radical cation scavenging ability + Heat” and “Fresh” respectively. To prepare the “Dried + Heat” extract, plant material was first oven-dried for 72 h at 45C. The ABTS radical cation (ABTS+) scavenging activity of the extracts was dried material was pulverized in a Waring blender and then determined as described in Re et al. (1999) with modifications. To extracted with autoclaved deionized water at a 1:10 (dry weight: prepare the ABTS+ stock solution, an equal volume of ABTS volume) ratio at 80C for 4 h (Wong and Kitts, 2006). The heat- solution (8 mg/ml) was first mixed with potassium persulfate (1.32 incubated homogenate was vacuum-filtered and the filtrate was mg/ml). The mixture was kept in the dark for 12 h at room centrifuged at 9000 rpm and 4C for 15 min. The supernatant temperature. Then, an ABTS+ working solution was prepared by obtained was immediately aliquoted (500 l each) and stored at - diluting the ABTS+ stock solution with potassium phosphate buffer 20C until used. “Fresh + Heat” extract was prepared as described (100 mM, pH 7.4) to obtain an absorbance of 0.700 0.005 at 734 above, except that fresh material was homogenized in a Waring nm. For measurements, 0.1 ml of extract was added to 1 ml of blender, added with deionized water at a 1:4 (fresh weight: volume) ABTS+ working solution. The mixture was kept in the dark for 10 ratio prior to heat treatment. “Fresh” extract was prepared as for the min before its absorbance was read at 734 nm. ABTS+ radical “Fresh + Heat” extract, but without any heat treatment. scavenging ability (%) was calculated as follows: + ABTS radical scavenging ability (%) = (1- [Asample / Acontrol]) x 100 Determination of total phenolic content Where A is the absorbance of control reaction (without plant The concentration of total phenolics in the extracts was determined control extract) and A is the absorbance in the presence of a plant using a Folin-Ciocalteu colorimetric assay (Waterhouse, 2001). A sample extract. Relative antioxidant capacities of the extracts are also mixture of extract (0.2 ml), deionized water (0.8 ml) and Folin- presented as TEAC values (mmole Trolox equivalents/100 g dry Ciocalteu reagent (0.1 ml) was first incubated at room temperature matter), calculated from a standard curve prepared with 0 to 0.25 for 3 min. Next, 0.3 ml of Na CO (20% w/v) was added and the 2 3 mM Trolox. mixture was incubated at room temperature for 120 min. Absorbance of the mixture was read at 765 nm. A standard curve was prepared from 0 to 100 mg/l gallic acid. Total phenolic content was expressed in mg gallic acid equivalents/g dry matter. Determination of ferric reducing ability Ferric reducing capacities of the extracts were assessed with two Determination of total flavonoid content methods. The first method was based on Oyaizu (1986), using ascorbic acid as reference.