Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 9, Number 1, April 2020 | Pages: 1-6 | DOI: 10.14421/biomedich.2020.91.1-6 ISSN 2540-9328 (online) Antioxidant Activities of the Leaf Extract and Fractions of Dryopteris filix-mas (L.) Schott could be Attributed to The Abundance of Polyphenol Compounds Earnest Oghenesuvwe Erhirhie1,2,*, Emmanuel Emeka Ilodigwe1, Daniel Lotanna Ajaghaku3, Blessing Ogechukwu Umeokoli4, Peter Maduabuchi Eze5, Festus Basden Chiedu Okoye4 1Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria. 2Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chukwuemeka Odumegwu Ojukwu University, Igbariam, Nigeria. 3Department of Pharmacology, Faculty of Pharmaceutical Sciences, Enugu State University of Science and Technology, Enugu State, Nigeria. 4Department of Pharmaceutical and Medicinal Chemistry Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria. 5Department of Environmental Health Science, Faculty of Health Sciences and Technology, Nnamdi Azikiwe University, Nnewi campus, Anambra State, Nigeria. Corresponding author* erhirhieochuko@yahoo.com, Tel: +234-7060434974 Manuscript received: 17 August, 2019. Revision accepted: 06 February, 2020. Published: 10 April, 2020. Abstract Dryopteris filix mas (D filix-mas) is wildly used in ethnomedicine for the management of rheumatoid arthritis, wounds and other diseases. We investigated the anti-oxidant activities of its leaf extract, and chromatographic fractions. The ethanol leaf extract was partitioned into four fractions; n-hexane, ethyl acetate, n-butanol and water. Ferric reducing anti-oxidant power (FRAP), 1, 1-diphenyl-2-picrylhydrazil (DPPH) and nitric oxide (NO) scavenging in vitro assays were carried out on the extract and fractions at 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL. The most active fraction (ethyl acetate fraction) was further purified using chromatographic techniques to isolate its major compound whose structure was elucidated using ID nuclear magnetic resonance (NMR) and mass spectrometry. The ethyl acetate fraction produced the highest free radical scavenging activity among the other fractions. The fraction (VLC-E7) from which the bioactive compound, quercetin-3-O-αL-rhamnopyranoside, was isolated had the best FRAP and DPPH scavenging activities with EC50 and IC50 values of 88.81 ± 3.41 and 26.87 ± 0.24 respectively more than the ethyl acetate fraction. This study revealed that the polyphenol flavonoid, quercetin-3-O-αL-rhamnopyranoside could be responsible for antioxidant activity of ethno-medicinal property of D filix-mas leaf. Keywords: Dryopteris filix-mas; wounds; rheumatoid arthritis; quercetin-3-O-αL-rhamnopyranoside; anti-oxidant properties INTRODUCTION Free radicals are constantly produced in all normal living cells (Kumar et al., 2014). Imbalance between the production of these radicals and endogenous antioxidants creates a state of oxidative stress with its associated damaging effects on endogenous molecules such as lipids, proteins and nucleic acids (Yan et al., 2015). Unbridled oxidative stress has been reported to be implicated in several chronic and degenerative diseases such as diabetes, rheumatoid arthritis, cancer, neurodegenerative diseases, atherosclerosis, ischemic heart disease, ageing among others (Khatoon et al., 2013). In attempt to ameliorating oxidative damages caused by free radicals, synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ) had been developed. However, these synthetic antioxidants left much to be desired due to their side effects, such as liver toxicity, carcinogenicity as well as their high cost and inaccessibility (Deepa et al., 2014). These limitations have inspired the search for more effective antioxidant from natural source, especially medicinal plants, which are biodegradable, less toxic, affordable and accessible (Ezeja et al., 2015; Ahlem et al., 2015). D filix-mas is an evergreen fern belonging to the family of Dryopteridaceae. Male fern, worm fern, aspidium and shield fern are common names assigned to it. It grows between 60-150 cm high and it is habitat to stream and moist environments (Uwumarongi et al., 2016). It is native to Europe, Asia, and North America. Its leaves are bipinnated and consist of 20-35 pinnae on each side of the rachis (Fig 1). Its stalks are covered with orange-brown scales (Bafor et al., 2017). The leaf decoction is ethno medicinally used in rheumatoid arthritis, wounds, bleeding disorders, ulcers, worm infestation and malaria (Tagarelli et al., 2010; Erhirhie et al., 2019). It is also reported as one of the ferns with https://doi.org/10.14421/biomedich.2020.91.1-6 2 Biology, Medicine, & Natural Product Chemistry 9 (1), 2020: 1-6 useful secondary metabolites against chronic diseases and aging (Valentyna et al., 2017). Previous studies have found that it possesses antihelmintic activity (Urban et al., 2014), anti- microbial activity (Mandal and Mondal, 2014), anti- diarrheal (Uwumarongi et al., 2016), uterine relaxant (Bafor et al., 2017) and anti-inflammatory (Erhirhie et al., 2019) activities. A preliminary study by Sekender et al, (2012) revealed that it possesses anti-oxidant activity. In this present study, we evaluated the antioxidant activity of the leaf extract and fractions. The bioactive compound responsible for its antioxidant activity was isolated and characterized. Figure 1. D. Filix-mas photograph. METHODOLOGY Materials Visible Spectrophotometer (721g, Zhejiang, China), Thermostatic water bath (Equitron Mumbai India), Analytical weighing balance (Ohaus Corp. NJ USA), Quercetin (Institute of Pharmaceutical Biology and Biotechnology, Heinerich-Heine University, Dusselforf, Germany), 2,2-Diphennyl-1-picrylhydrazyl, DPPH (Sigma Aldrich), Ascorbic acid (Sigma- Aldrich). Procedure Plant collection, extraction, fractionation and purification of active compound Plant collection, authentication, extraction, fractionation, chromatographic separation, and structural elucidation techniques were carried out using similar method as described in our earlier study (Erhirhie et al., 2019). The purification and structural elucidation was based on bioactivity guided antioxidant screening of liquid-liquid fractions (n-hexane, ethyl acetate, butanol, and water fractions), vacuum liquid chromatographic fractions and Sephadex fractions. DPPH scavenging assay The test was carried out based on the method described by Ajaghaku et al (2017). The reaction mixture contained 0.1 mL of various concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL) of sample, 0.1 mL of 0.6 mMol of DPPH and 0.8 mL of methanol. The mixture was incubated in the dark for 30 minutes at room temperature. The absorbance of the sample was measured at 517 nm against blank (methanol) using a spectrophotometer. Ascorbic acid and quercetin were used as standards. A tube containing 0.1 mL of DPPH solution and 0.9 mL of methanol served as control. Experiments were carried out in duplicate. Free radical scavenging activities of each sample were determined as follows: 𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = (𝐴𝐶– 𝐴𝑆)/𝐴𝐶) × 100 AC : Average absorbance of control AS : Average absorbance of sample A graph of percentage inhibition against concentration was plotted and the concentration that produced 50% inhibition (IC50) was extrapolated using a regression analyses equation. Ferric reducing antioxidant power (FRAP) assay FRAP assay was carried out following the method described by Habibur et al, (2013). Two hundred and fifty microliter (0.25 mL) of various concentrations, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL of samples were mixed with 0.625 mL of phosphate buffer and 0.625 mL of 1% potassium ferricyanide [K3FeCN6]. The mixtures were heated at 50oC for 20 minutes. Then, 0.625 mL of 10% trichloroacetic acid (TCA) was added and the mixtures were centrifuged at 3000 rpm for 10 minutes. From the upper layer, 0.625 mL was pipetted and mixed with 0.625 mL of distilled water and 0.125 mL of 0.1% (w/v) ferric chloride (FeCl3) solution. Absorbances of the mixtures were measured at 700 nm against air using a spectrophotometer. Ascorbic acid and quercetin were used as standards. Tests were performed in duplicate and percentage inhibition was calculated using the formula below. % Inhibition = Average absorbance of sample−Average absorbance of blank 1 x 100 1 Erhirhie et al. – Antioxidant Activities of the Leaf Extract and Fractions … 3 A graph of percentage inhibition against concentration was plotted and the effective concentration (EC50) was extrapolated using a regression analyses equation. Nitric oxide scavenging activity This assay was carried out following the method described by Ezeja et al., (2015). Two milliliter of 10 mM sodium nitroprusside prepared with phosphate buffer saline (pH 7.4) was mixed with 0.5 mL of samples at various concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL). The mixture was incubated at room temperature for 150 minutes. Thereafter, 0.5 mL of the reaction mixture was withdrawn and mixed with 0.5 mL of Griess reagent (1% sulphanilamide + 0.1% naphthylethylenediamine dichloride + 3% phosphoric acid) was added to each test tube. The absorbance of the pink chromophore formed was measured at 540 nm. Ascorbic acid and quercetin were used as standards. Assays were carried out in duplicates. Percentage inhibition was calculated using the formula below Abs Control−Abs Test Abs Control × 100 1 Inhibitory concentration, IC50 was estimated from graph of % inhibition against concentration using regression analyses equation Statistical analysis Results were presented as mean ± standard error of mean (SEM) using Statistical Package for Social Science (SPSS, version 20). Calculation of fifty percent inhibitory concentration (IC50) of the extracts and fractions was carried out using regression equation in Microsoft Excel, 2010. RESULTS DPPH, scavenging activity of extract and fractions In DPPH scavenging assay, 50% inhibitory concentration (IC50) of 134.12 ± 2.88, 94.97 ±0.14, 63.91 ± 1.29, 62.09 ± 0.21and 45.72 ± 0.16 µg/mL were recorded in n-hexane, water fractions, extract, butanol and ethyl acetate fractions respectively. The ethyl acetate fraction produced the lowest IC50 value among other samples, except ascorbic acid and quercertin which showed IC50 values of 12.26 ± 0.20 µg/mL and 10.46 ± 0.15 µg/mL respectively (Table 1). Inhibition against DPPH at 100 µg/mL by VLC fractions Ethyl acetate VLC fractions, VLC-E1 (78.68%), VLC- E3 (52.33%), VLC-E7 (72.38%), VLC-E6 (73.84%), VLC-E14 (65.60%), VLC-E8 (73.08%), VLC-E10 (74.81%), VLC-E12 (72.09%), VLC-E16 (69.67%) produced more than 50 % inhibition against DPPH (Table 2). Table 1. Free radical (DPPH) scavenging activities of extract and fractions of D filix-mas. DPPH, scavenging activity, IC50(ug/ml) Extract 63.91 ± 1.29 n-hexane fraction 134.12 ± 2.88 Ethyl-acetate fraction 45.72 ± 0.16 Butanol fraction 62.09 ± 0.21 Water fraction 94.97 ±0.14 Ascorbic acid 12.26 ± 0.20 Quertcetin 10.46 ± 0.15 Values are expressed as mean ± Standard error of mean. Table 2. DPPH scavenging activities of ethyl acetate vacuum liquid chromatographic fractions of D filix-mas. Solvent ratio Sample code Inhibition against DPPH (%) Ethyl acetate fraction 73.26 N(500): E (0) VLC-E1 78.68 N(450): E (50) VLC-E2 19.38 N(400): E(100) VLC-E9 18.12 N(350): E(150) VLC-E13 23.06 N(300):E(200) VLC-E4 28.00 N(250): E(250) VLC-E11 36.34 N(200):E(300) VLC-E3 52.33 N(150):E(350) VLC-E15 16.86 N(100):E(400) VLC-E5 12.79 N(50): E(450) VLC-E17 37.79 N(0):E(500) VLC-E7 72.38 D(500):M(0) VLC-E6 73.84 D(450):M(50) VLC-E14 65.60 D(350):M(150) VLC-E8 73.06 D(250):M(250) VLC-E10 74.81 D(100):M(400) VLC-E12 72.09 D(0):M(500) VLC-E16 69.67 N = n-hexane, E = ethyl acetate, M = methanol. Ferric reducing antioxidant power (FRAP), Nitric oxide (NO) and DPPH scavenging activities of extract selected fractions. FRAP activity In FRAP assay, quercetin and ascorbic acid produced the lowest EC50 values followed by VLC-E7, ethyl acetate fraction, extract and butanol fraction. In nitric oxide scavenging assay, ascorbic acid exhibited the lowest IC50 value (14.74 ± 0.11 µg/ml) followed by ethyl acetate fractions (15.38 ± 3.65 µg/ml) and butanol fractions (15.45 ± 2.72 µg/ml), followed by VLC-E7 fraction (16.66 ± 0.48 µg/ml), quercetin (18.14 ± 2.57 µg/ml) and extract (1854.60 ± 200.25 µg/ml). In DPPH assay, quercetin and ascorbic acid produced the lowest IC50 values (11.50 ± 0.08 and 14.19 ± 0.43µg/mL) followed by VLC-E7 (26.87 ± 0.24 µg/mL), ethyl acetate fraction (50.25 ± 0.40 µg/mL), butanol fraction (62.09 ± 0.21 µg/mL) and crude extract (63.91 ± 1.29 µg/mL) (Table 3). 4 Biology, Medicine, & Natural Product Chemistry 9 (1), 2020: 1-6 DPPH, scavenging activity of Sephadex fractions DPPH scavenging activities of samples tested decreased in the following order; Quercetin (10.46 ± 0.15 µg/mL) > Ascorbic acid (12.26 ± 0.20 µg/mL) > SPH-E3 (26.35 ± 1.38 µg/mL) > VLC-E7 (26.87 ± 0.24 µg/mL) > SPH- E6 (33.44 ± 0.38 µg/mL) > SPH-E5 (37.30 ± 0.30 µg/mL) > SPH-E7 (46.95 ± 0.61 µg/mL) > SPH-E4 (86.10 ± 7.69 µg/mL) (Table 4). Table 3. Ferric reducing antioxidant power (FRAP), Nitric oxide (NO) and DPPH scavenging activities of extract and fractions of D filix-mas. Sample FRAP, EC50 (µg/mL) Nitric oxide, IC50 (µg/mL) DPPH, IC50 (ug/mL) Extract 1854.60 ± 200.25 38.51 ± 2.23 63.91 ± 1.29 Ethyl acetate fraction 461.75 ± 22.91 15.38 ± 3.65 50.25 ± 0.40 Butanol fraction 1911.03 ± 137.44 15.45 ± 2.72 62.09 ± 0.21 VLC-E7 88.81 ± 3.41 16.66 ± 0.48 26.87 ± 0.24 Ascorbic acid 69.34 ± 2.41 14.74 ± 0.11 14.19 ± 0.43 Quercetin 30.81 ± 0.86 18.14 ± 2.57 11.50 ± 0.08 FRAP: Ferric reducing antioxidant power. Values are expressed as mean ± Standard error of mean. Table 4. DPPH scavenging activity, of Sephadex (SPH) fractions of D filix-mas. DPPH, scavenging activity, IC50( µg/mL) VLC-E7 26.87 ± 0.24 SPH-E1 - SPH-E2 22.10 ± 0.62 SPH-E3 26.35 ± 1.38 SPH-E4 86.10 ± 7.69 SPH-E5 37.30 ± 0.30 SPH-E6 33.44 ± 0.38 SPH-E7 46.95 ± 0.61 Ascorbic acid 12.26 ± 0.20 Quercetin 10.46 ± 0.15 Values are expressed as mean ± Standard error of mean. DISCUSSION It is well established that by-products of oxygen metabolism produce free radicals such as reactive oxygen and nitrogen species which results to cellular damage and pathogenesis of several diseases such as cancer, cardiovascular diseases, diabetes, neurodegenerative diseases among others (Li et al., 2016). There is a revitalization of interest in the search for natural anti-oxidant that could ameliorate reactive oxygen species which are implicated in numerous disease conditions (Ahlem et al., 2015; Deepa et al. 2014). In this study, we investigated the antioxidant properties of the extract and fractions of D filix-mas and also elucidated the major compound responsible for its antioxidant activity using in vitro bioassay guided isolation approach. DPPH test is a widely acceptable and reproducible assay for screening potential antioxidants (Patel et al., 2010). This method involves the ability of anti-oxidants to donate electrons to DPPH thereby causing discoloration of the reaction mixture from deep violet color to yellow color (Patel et al., 2010). The yellow color change observed in this study substantiates the ability of the extract and fractions of D filix-mas to scavenge free radicals generation by DPPH. This further suggests that the constituents present in the extract and fractions could ameliorate oxidative damages by free radicals. Reduction of ferric (Fe3+) to ferrous (Fe2+) ions is a documented method of determining antioxidants with reducing power, capable of breaking free radical chain progression in lipid peroxidation (Habibur et al., 2013). From this study, reduction of iron (III) complex to the ferrous form, iron (II) could be a possible antioxidant mechanism as a result of the presence of reducing agents in the extract and fractions of D filix-mas. Phenolic compounds such as quercetin has been reported to prevent free radicals by forming complex and chelating metal ions such as iron and cupper, thereby preventing auto-oxidative damage to the living system (Nimse and Pal, 2015). Nitric oxide generated following incubation of sodium nitroprusside with phosphate buffer in the presence of oxygen is known to cause toxicity to biomolecules (Deepa et al., 2013). The scavenging capacities of the extract and fractions against nitric oxide generation suggest that D filix-mas could prevent nitric oxide generation in various disease conditions. Polyphenols such as quercetin has been reported to interact with nitric oxide synthase resulting in modulation of nitric oxide production (Hussain et al., 2016). The roles of phenolic compounds from medicinal plants as supplement in the treatment of oxidative stress and free radicals mediated tissue damage cannot the overemphasized (Li et al., 2016). Flavonoids and phenolic acids from polyphenols have been reported to play a significant role in scavenging free radicals and prevention of oxidative damage to cells. Phenolic compounds have capacity to neutralize several forms of oxidizing free radicals due to their electron donating abilities (Habibur et al., 2013; Ajaghaku et al., 2017). Ojo et al., (2013) reported that flavonoids inhibit lipid peroxidation in various biological systems. Presence of hydroxyl groups in flavonoids allows them to disconnect free radical chain reactions via the formation of intramolecular hydrogen bonds (Monika et al., 2011). Erhirhie et al. – Antioxidant Activities of the Leaf Extract and Fractions … 5 From the forgoing, antioxidant properties elicited by the extract and fractions of D filix-mas could be attributed to presence of flavonoids. This is substantiated by presence of the flavonoid, quercitrin - as the major compound in the HPLC chromatogram of the extract and fractions of D filix-mas as documented in our earlier study (Erhirhie et al., 2019). The ethyl acetate VLC fraction (VLC-E7), where the compound, quercetin-3-O-αL-rhamnopyranoside was isolated exhibited the highest anti-oxidant activity compared to the extract and other fractions. In line with this finding, quercetin-3-O-αL-rhamnopyranoside isolated from other medicinal plants was found to elicit significant antioxidant activity (Zhang et al., 2014). It is possible that possible that quercetin-3-O-αL- rhamnopyranoside may be the key antioxidant compound of D filix-mas. Quercetin, the most widely distributed flavonoid in nature, existing mostly in its glycoside form quercitrin has been reported to possess various properties such as antioxidant, anti-inflammatory, neuroprotective, antiviral, anticancer, hepatoprotective, cardioprotective, antimicrobial and anti-obesity properties (Maalik et al., 2014). Mir, et al., (2017) reported that flavonoids from Tridax procumbens possess antioxidant properties. Phenolic compounds, catechin, epicathechin, dihydroquercetin isolated from Alchornea floribunda leaf were also found to elicit antioxidant properties (Ajaghaku et al., 2017). In this study, the ability of extract and fractions of D filix- mas to scavenge generation of free radicals from DPPH, nitric oxide as well as its chelating capacity could be attributed to the phenolic compound, quercetin- 3-O-αL-rhamnopyranoside, which was postulated to be responsible for its anti-inflammatory activity as earlier reported (Erhirhie et al., 2019). 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