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

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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 Biology, Faculty of Mathematics and Natural Sciences, Sebelas DPPH radical scavenging ability (%) = (1- [Asample / Acontrol]) x 100 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 45C. 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 80C 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 4C 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 20C 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. Briefly, 0.2 ml of extract was mixed with The concentration of total flavonoids in the extracts was determined 0.25 ml of potassium phosphate buffer (0.2 M and pH 6.6) and 0.25 sing an assay modified from Zou et al. (2004). Plant extract (0.2 ml) ml of potassium ferricyanide (1% w/v). The mixture was incubated

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Table 1. Total phenolic and total flavonoid contents of the aqueous extracts of S. willdenowii leaves and stems.

Total phenolics (mg gallic acid equivalents Total flavonoids (mg quercetin equivalents Type of extract /g dry matter) /g dry matter) Leaf Stem Leaf Stem Fresh + heat 7.74 ± 0.18a, A 2.56 ± 0.03a, B 6.93 ± 0.07a, A 1.32 ± 0.04a, B Dried + heat 5.23 ± 0.21b, A 2.10 ± 0.06b, B 4.98 ± 0.14b, A 1.54 ± 0.06b, B Fresh 6.22 ± 0.21c, A 1.77 ± 0.01c, B 3.16 ± 0.03c, A 0.65 ± 0.04c, B

Data are mean  SE values (n=3). Different lowercase letters indicate significant differences within a column when three types of either leaf or stem extracts were compared by the Fisher’s Least Significant Difference (LSD) test (P < 0.05). Different uppercase letters indicate significant differences within a row when leaf and stem extracts prepared the same way were compared by Student’s t test (P < 0.05).

at 50C for 20 min. After adding in 0.25 ml trichloroacetic acid (10% “Fresh” leaf extracts, respectively. Among stem extracts, w/v), the mixture was centrifuged at 3000 rpm for 10 min. The upper total flavonoid content of “Fresh + Heat” extract was 14% layer of the solution (0.5 ml) was then mixed with 0.15 ml of lower than that in the “Dried + Heat” extract, but was deionized water and 0.15 ml of FeCl3 (0.1% w/v). After keeping the mixture at room temperature for 10 min, its absorbance was read at 103% higher compared with “Fresh” extract. 700 nm. Results are expressed in g ascorbic acid equivalents/mg dry matter, calculated from a standard curve prepared with 0 to 40 + g/ml ascorbic acid. The second method used to assess the DPPH and ABTS radical scavenging activity reducing power of the extracts was the FRAP assay (Benzie and Strain, 1996). FRAP reagent was prepared fresh by mixing acetate All leaf and stem extracts exhibited concentration- buffer (300 mM, pH 3.6), 2,4,6-tripyridyl-s-triazine (10 mM), and dependent DPPH radical scavenging activities (Figure 1A FeCl3.6H2O (20 mM) in a 10:1:1 (v:v:v) ratio and then pre-warmed to 37C before use. For measurements, 0.2 ml of extract was mixed and B). Notably, all leaf extracts exhibited greater DPPH with 1.2 ml of FRAP reagent and then incubated at 37C for 5 min. radical scavenging activities compared with stem extracts. Absorbance of the mixture was then read at 593 nm. Reducing Among leaf extracts, “Fresh + Heat” extract showed power is presented in mmole Fe2+ equivalents, calculated from a significantly higher activity than the other two extracts in standard curve prepared with 0 to 0.40 mM FeSO4.7H2O. concentration range 0.5 to 3 mg/ml (P < 0.05). DPPH

radical scavenging activities of “Dried + Heat” and “Fresh” Data analysis extracts were not statistically different over the whole concentration range examined. Among stem extracts, Data reported are mean ± standard errors obtained from three “Fresh” extracts at concentration 1 mg/ml and higher replicate extractions. For ease of comparison, concentrations of showed significantly lower DPPH radical scavenging “Fresh + Heat” and “Fresh” extracts were expressed in mg dry matter per volume, calculated based on the percentage of water activity than the other two extracts (P < 0.05). “Dried + content determined after oven-drying the plant material. Statistical Heat” and “Fresh + Heat” stem extracts were generally analysis was performed using SAS (Version 9.2). Data were similar in their DPPH radical scavenging response curves. analyzed by the ANOVA test and means of significant differences Concentration-dependent ABTS+ radical scavenging were separated using Fisher’s Least Significant Difference test at activities were observed in all extracts (Figures 2A and B). the 0.05 level of probability. For comparison of only two means, All leaf extracts showed higher ABTS+ radical scavenging Student’s t test was used at the 0.05 level of probability. activities than the stem extracts. At extract concentration 5 mg dry matter/ml, radical scavenging activities of leaf RESULTS extracts ranged between 73 and 98%, but those of stem extracts were only between 35 and 53%. “Fresh + Heat” Total phenolic and total flavonoid contents leaf extract showed the highest ABTS+ radical scavenging activity, whereas “Fresh” leaf extract Overall, total phenolic contents of leaf extracts were 2.5 consistently exhibited the lowest scavenging activity. to 3.5-fold higher compared with stem extracts (Table 1). Among stem extracts, “Fresh” extract showed the lowest Among leaf extracts, total phenolic content of “Fresh + level of ABTS+ radical scavenging activity, while “Fresh + Heat” extract was 48% and 24% higher than that of Heat” and “Dried + Heat” stem extracts are not “Dried + Heat” and “Fresh” extracts, respectively. Among statistically different from one another over the stem extracts, “Fresh + Heat” extract had the highest total concentration range tested. phenolic content, being at least 22% higher compared with other stem extracts. Leaf extracts contained 3.2 to 5.3-fold higher levels of DPPH EC50 and TEAC values total flavonoids compared with stem extracts (Table 1). Total flavonoid content of “Fresh + Heat” leaf extract was Overall, DPPH EC50 values of stem extracts are 1.9 to 39 and 119% greater compared with “Dried + Heat” and 3.5-fold higher compared with leaf extracts (Table 2). In

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Figure 1. DPPH radical scavenging activities of three types of aqueous extracts of leaves; A and stems; B of S. willdenowii, compared with ascorbic acid and Trolox. Data are mean  SE values (n=3).

both leaf and stem extracts, EC50 values of “Fresh” another, each about half of that of “Fresh” extract. TEAC extracts were the highest. Among leaf extracts, “Fresh + values of leaf extracts were 2.8 to 3.5-fold greater than Heat” extract had the lowest DPPH EC50 value. Among those of stem extracts (Table 2). TEAC values of leaf stem extracts, EC50 values of “Fresh + Heat” and “Dried extracts were in the following order: “Fresh + + Heat” extracts were not significantly different from one Heat” >“Dried + Heat” > “Fresh”, where TEAC value of

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Figure 2. ABTS radical cation scavenging activities of three types of aqueous extracts of leaves; A and stems; B of S. willdenowii. Data are mean  SE values (n=3).

“Fresh + Heat” extract was 1.9-fold higher compared with dependent increase in FRAP values (Figure 3A and B). “Fresh” extract. Among stem extracts, TEAC values of Leaf extracts overall had higher FRAP values than stem both “Fresh + Heat” and “Dried + Heat” extracts were not extracts. Among leaf extracts, FRAP values of “Fresh + statistically different from one another, both about 1.7-fold Heat” extracts were consistently the highest, higher compared with “Fresh” extract. whereas“Fresh” extracts was the lowest. Among stem extracts, FRAP values of “Fresh + Heat” and “Dried + Heat” extracts were not significantly different from one Ferric reducing ability another over the whole concentration range tested, whereas FRAP values of “Fresh” extracts were All leaf and stem extracts showed concentration- consistently the lowest. Besides FRAP assay, ferric

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Table 2. Antioxidant capacity of the aqueous extracts of S. willdenowii leaves and stems based on DPPH and ABTS radical scavenging assays.

DPPH EC50 TEAC (mmole Trolox equivalents Type of extract (mg dry matter/ml) /100 g dry matter) Leaf Stem Leaf Stem Fresh + heat 1.39 ± 0.09a, A 4.28 ± 0.10a, B 8.63 ± 0.32a, A 2.47 ± 0.18a, B Dried + heat 2.08 ± 0.04b, A 3.92 ± 0.12a, B 7.66 ± 0.20b, A 2.73 ± 0.26a, B Fresh 2.40 ± 0.02c, A 8.34 ± 0.14b, B 4.52 ± 0.15c, A 1.57 ± 0.12b, B

DPPH EC50, concentration of extract required to scavenge 50% of DPPH radicals. TEAC, Trolox equivalent antioxidant capacity, determined by means of the ABTS radical cation scavenging assay using Trolox as standard. Data are mean  SE values (n=3). Different lowercase letters indicate significant differences within a column when three types of either leaf or stem extracts were compared by the Fisher’s Least Significant Difference (LSD) test (P < 0.05). Different uppercase letters indicate significant differences within a row when leaf and stem extracts prepared the same way were compared by Student’s t test (P < 0.05).

reducing ability was also determined using the method of Oyaizu (1986) (Table 3). Both assays indicated that ferric reducing ability of leaf extracts was at least 2-fold higher compared with stem extracts. Both assays also found that among leaf extracts, “Fresh + Heat” extract had the highest ferric reducing ability, while “Fresh” extract had the lowest.

Correlation between total phenolic contents and antioxidant capacities

Radical scavenging activities and FRAP values were both strongly correlated with the total phenolic contents of leaf extracts (Table 4). High R2 values for DPPH radical scavenging activity (0.95 - 0.99) and ABTS+ radical scavenging activity (0.89 - 0.99) indicate that at least 89% of the free radical scavenging activities detected can be attributed to total phenolic contents. On the other hand, high R2 values (0.99) indicate that almost all ferric reducing potential of the leaf extracts can be accounted for by their total phenolic contents.

DISCUSSION

In this study, two strategies were adopted to ensure the relevance of our observations to the uses of S. willdenowii in traditional medicine and cooking. First, three types of extracts were prepared to reflect the uses of S. willdenowii as raw vegetable (“Fresh” extract; no heat treatment), cooked vegetable and fresh herb infusion or decoction (“Fresh + Heat” extract; with heat treatment) and dried herb infusion or decoction (“Dried” + Heat; with heat treatment) (Setyawan, 2009). Secondly, extraction was carried out using water to reflect the most likely form of solvent used in preparing S. willdenowii in traditional medicine and cooking.

Figure 3. Ferric reducing potential of three types of aqueous Our results clearly demonstrated the antioxidant extracts of leaves; A and stems; B of S. willdenowii, expressed potential of S. willdenowii, evidenced by its free radical as FRAP values. Data are mean  SE values (n=3). scavenging and ferric reducing abilities. The total

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Table 3. Ferric reducing potential of the aqueous extracts of S. willdenowii leaves and stems.

2+ Reducing power (g ascorbic acid equivalents/ FRAP values (mmole Fe equivalents/ Type of extract mg dry matter) 100 g dry matter) Leaf Stem Leaf Stem Fresh + heat 3.24  0.10a, A 0.85  0.01a, B 4.94  0.09a, A 1.97  0.03a, B Dried + heat 3.13  0.10a, A 1.46  0.21b, B 3.62  0.06b, A 1.77  0.09a, B Fresh 2.76  0.02b, A 0.70  0.03a, B 2.49  0.03c, A 0.93  0.03b, B

FRAP, Ferric reducing antioxidant power.Data are mean  SE values (n=3). Different lowercase letters indicate significant differences within a column when the three different types of leaf or stem extracts were compared by Fisher’s least significant difference (LSD) test (P < 0.05). Different uppercase letters indicate significant differences within a row when leaf and stem extracts prepared the same way were compared by Student’s t test (P < 0.05).

Table 4. Correlation analysis between total phenolic contents of leaf extracts and three antioxidant parameters.

Correlation of determination (R2) Type of extract DPPH radical scavenging ABTS+ radical scavenging Ferric reducing antioxidant activity activity power (FRAP) values Fresh + heat 0.95 0.89 0.99 Dried + heat 0.99 0.90 0.99 Fresh 0.99 0.99 0.99

R2 values presented above are all statistically significant (P < 0.05).

phenolic content, TEAC value and FRAP value of S. 2007; Pham-Huy et al., 2008) and antioxidant therapy willdenowii “Fresh + Heat” leaf extract, although lower, has been considered “a promising avenue for treatment” are comparable to those we calculated from values (Pham-Huy et al., 2008). Our findings of antioxidant reported for S. tamariscina methanolic extract, which are activities in S. willdenowii extracts, therefore, support the 11.18 mg gallic acid equivalents/g dry matter, 15.51 use of the plant in traditional medicine. Moreover, when mmole Trolox/100 g dry matter and 15.04 mmole consumed as vegetable, S. willdenowii may contribute to Fe2+/100 g dry matter (Gan et al., 2010), respectively. total intake of dietary antioxidants. On the other hand, our Interestingly, the “Fresh + Heat” leaf extract also had observation of the lower antioxidant capacity of stem higher total phenolic content and FRAP value than the extracts compared with leaf extracts implies that aqueous extracts of at least 14 Chinese medicinal plants exclusion of stem materials may increase the yield of (Wong et al., 2006). We also detected higher levels of antioxidant activity for a given quantity of S. willdenowii total flavonoids in the leaf than in the stem extracts, dry matter used. consistent with previous observation on Selaginella sinensis (Zhao et al., 2010). The genus Selaginella is a rich source of biflavonoids (Kim and Park, 2002; Conclusions

Setyawan, 2011). Amentoflavone, the most common This study has provided evidence for potent antioxidant biflavonoid of Selaginella, exhibits a broad range of activity in the aqueous extracts of S. willdenowii. Total medicinal properties, including antioxidant, anticancer, phenolic and flavonoid contents as well as antioxidant antimicrobial, antiviral and anti-inflammatory (Setyawan, activities were all more pronounced in the leaf extracts, 2011). Free radical scavenging activities and ferric although detectable in stem extracts. Correlation analysis reducing ability of S. willdenowii leaf extracts were indicated that total phenolic content was likely a main strongly correlated with total phenolic contents. These determinant of antioxidant activity in S. willdenowii. relationships imply that water-soluble phenolics Overall, our findings affirm the value of S. willdenowii as compounds, including flavonoids, are potentially a key a medicinal herb, in addition to its value as a source of determinant of antioxidant properties in S. willdenowii leaf food antioxidants when used as vegetable. extracts. Correlation among these parameters has also been reported for the aqueous extracts of other medicinal plants (Wong et al., 2006; Guo et al., 2008). ACKNOWLEDGEMENTS Antioxidants are crucial to eliminating reactive oxygen species, which are prevalent in many diseases and This research was supported by the UTAR Research health conditions (Finkel and Holbrook, 2000; Valko et al., Fund.

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