Ethiopian Journal of Science and Sustainable Development e-ISSN 2663-3205 ASTU Volume 7 (1), 2020 Journal Home Page: www.ejssd.astu.edu.et Research Paper Chemical Constituents of the Roots Extract of Dryopteris schimperiana and Evaluation for Antibacterial and Radical Scavenging Activities Bayan Abdi, Emebet Getaneh, Temesgen Assefa, Aman Dekebo, Hailemichael Tesso, Teshome Abdo, Yadessa Melaku Department of Applied Chemistry, School of Applied Natural Science, Adama Science and Technology University, P.O.Box 1888, Adama, Ethiopia Article Info Abstract Keywords: Dryopteris schimperiana, NMR antibacterial activity antioxidant. Dryopteris schimperiana (Dryopteridaceae) is traditionally used in folk medicine of Ethiopia against bacteria and internal parasites. In view of its traditional use and absence of scientific reports, an attempt was made to explore the chemical constituents, antibacterial and radical scavenging activities of the solvent extracts of the root of Dryopteris schimperiana. In this regard, the root was successively extracted with n-hexane, CHCl3 and methanol to afford 5 g (2%), 2 g (0.8%) and 26.6 g (10.6%), respectively. The n-hexane and CHCl3 extracts showing similar TLC profile were mixed and fractionated over silica gel column chromatography which led to the isolation of two compounds identified as heptacosanol (1) and isorhmentin (2). The structures of the isolated compounds were accomplished using spectroscopic methods including UV-Vis, IR and NMR. To the best of our knowledge, these compounds have not been reported from the genus Dryopteries. The extracts and isolated compounds were evaluated for their antibacterial activities using agar well diffusion method against two Gram-positive bacteria (Staphylococcus aureus and Bacillus subtitis) and two Gramnegative bacteria (E. coli and Salmonella). The results showed that the n-hexane extract, methanol extracts and isolated compounds were active against all tested bacteria, with compound 2 shown to have an inhibition zone of 17 mm which is comparable with the positive control. Furthermore the extracts and isolated compounds were assessed for their radical scavenging activity using DPPH assay. Isorhmentin (2) displayed pronounceable % radical scavenging activity (82.8%) compared with ascorbic acid indicating its strong ability to act as radical scavenger. This study demonstrated that the antibacterial and radical scavenging activity of the root of D. shimperiana is accounted to the presence of isorhamnetin. Therefore, the biological activity displayed by the constituents of the roots of D. shimperiana corroborates the traditional use of this plant against bacteria. 1. Introduction human diseases remains widespread in developing countries. The recent reports of World Health Organization (WHO) indicated that 70 to 90% of world population especially from developing countries uses plant remedies for their health care. The principal components responsible for the traditional use of these Plants have been known from prehistoric times to treat a wide array of diseases affecting human beings and livestocks. Out of a quarter of a million identified plants in the world about one fourth has at one time or other used by some people or cultures for medicinal purpose. The use of traditional medicine for treating  Corresponding author, e-mail: yadessamelaku2010@gmail.com https://doi.org/10.20372/ejssdastu:v7.i1.2020.153 1 © 2020 Adama Science & Technology University. All rights reserved Bayan Abdi et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020 specimen GM005. The sample was washed with tap water and dried under shade at the laboratory of Department of Chemistry, Adama Science and Technology University. plants are accounted to the presence of secondary metabolites including flavonoids, alkaloids, terpenoids, anthraquinone, lignans, etc (Chanda et al., 2006). Therefore, plants are useful to humans as a source of bioactive pharmaceuticals. Although many have been treated by conventional pharmaceutical approaches, there is a growing interest in the use of natural products by the general public. At the present, many scientists are interested to evaluate the chemical constituents of plants used traditionally by peoples for medicinal purpose. Some plant species belonging to the genus Dryopteries are among medicinal plants that are traditionally used for the treatment of wide array of diseases. Pharmacologically, some species in the genus Dryopteris were reported as an effective antibacterial agent against various bacterial pathogens including Staphylococcus aureus, Bacillus subtitis, E. coli, Salmonella typhus and Pseudomonas aeruginosa. Some species in this genus were shown to have various biological activities including anthelmintic, antiviral, antitumor, antimicrobial, antioxidative and antiinflammatory (Han et al., 2015). Previous phytochemical investigation of some species in Dryopteries has resulted in the isolation of flavonoids, anthraquinones (Kwanzoquinone A, hydroxychryphanol, Kwanzoquinone (B6, C7, D8, E9, F10)) and alkaloids (Harborne, 1966; Min et al., 2010; Imperato, 2006; Imperato, 2007). Dryopteris schimperiana is traditionally used in the folk medicine of Ethiopia against bacterial infections in addition to its use to remove internal parasitic worms. Despite the extensive popular use of this plant as a remedy against bacteria, there is no information describing the chemical constituents, antibacterial and radical scavenging activities of the roots of D. schimperiana. Therefore in this paper, we report for the first time the chemical studies, antibacterial and radical scavenging activities of the root extracts of D. schimperiana. 2.2. Instruments Melting point was determined in capillary tube with a Thiele tube melting point apparatus. Analytical TLC was run on a 0.25 mm thick layer of silica gel GF254 (Merck) on aluminum plate. Spots were detected by observation under UV light (254 nm) followed by dipping in vanillin/H2SO4. Column chromatography was performed using silica gel (230-400 mesh) Merck. Solvent was freed using rotavapor. Infrared (IR) spectra were obtained on Perkin-Elmer 65FT ((IR νmax KBr 4000-400 cm-1) infrared spectrometer using KBr pellets. 1D NMR were obtained on Brucker Avance instrument NMR machine (Bruker Avance 400 NMR spectrometer) at the Chemistry Department of Addis Ababa University. 2.3. Extraction and Isolation The ground root of D. schimperiana (250 g) was successively extracted on maceration with each 1.5 L of n-hexane, CHCl3 and MeOH for 72 hrs. The extracts were filtered and concentrated using rotary evaporator to give their corresponding extracts. The profile of each extract was subsequently analyzed by TLC. The hexane and CHCl3 extracts which displayed similar TLC profile were mixed. The combined extract (7 g) was dissolved and fractionated over silica gel (150 g) column chromatography using n-hexane:EtOAc:MeOH of increasing polarities as eluent to afford 92 fractions (Table 1), each 50 mL. Samples showing similar spots on their TLC profiles were mixed. Fractions 14-18 which showed one spot on TLC were combined and dried to afford compound 1. On the other hand, fraction 25 after rechromatographed over silica gel column chromatography afforded compound 2. 2. Material and Methods 2.4. Phytochemical screening tests 2.1. Plant material collection and authentication Qualitative phytochemical screening of the n-hexane and methanol extracts of the root of D. shimpiriana were done following standard procedures to detect the presence or absence of secondary metabolites including flavonoids, saponins, phenolics, terpenoids, steroids, coumarins, anthraquinones, alkaloids, phenolics and The roots of D. schimperiana were collected from Kaka mountain Lemu Bilbilo District, Arsi Zone, Oromia Region, Ethiopia. The plant material was identified by a botanist and deposited at the National Herbarium of Addis Ababa University with voucher 2 Bayan Abdi et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020 equal volume of concentrated H2SO4 was added by sides of the test tube. The upper layer turns red and H2SO4 layer showed yellow with green fluorescence. This indicates the presence of steroids (Balick, 1996). tannins (Balick, 1996). The detailed experimental procedure used to analyze each phytochemicals is presented as follows: Flavonoids (Alkaline reagent test): Each 0.25 g crude extracts of n-hexane and methanol were treated with ethyl acetate (10 mL) and heated for 3 min using steam bath. The mixture were filtered, and mixed with ammonia (1 mL) solution. Formation of intense yellow color ratifies the presence of flavonoids (Balick, 1996). Coumarins: To 2 mL (1 mg/mL) of the extract was added 3 mL of 10% NaOH. Formation of yellow color indicates the presence of coumarins (Balick, 1996). Anthraquinones: The extracts (1 mg/mL) were boiled with 10% HCl for few min. in water bath and filtered. The filtrate was allowed to cool and equal volume of CHCl3 was added to the filtrate. Few drops of 10% ammonia were added to the mixture and heated. The formation of rose-pink color was taken as an indication for the presence of anthraquinones (Balick, 1996). Table 1: Column chromatographic fractionation of the combined hexane and chloroform extracts of the root of D. shimpiriana Eluent Ratio Fractions n-hexane 100% F1-F4 n-hexane/EtOAc “ “ “ “ “ “ “ 98:2 96:4 90:10 80:20 75:25 70:30 60:40 50:50 F5- F7 F8-F10 F11-F13 F14-F18 F19-F26 F27-F31 F32-F33 F34-F37 Alkaloids (Wagner’s test): The extracts (1 mg/mL) were dissolved individually in dilute HCl and filtered. Filtrates were treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids (Balick, 1996). 2.5. Antibacterial Activity The methanol extract, n-hexane extract and the two isolated compounds were evaluated for their in vitro antibacterial activity using disc diffusion method against two Gram positive bacterial pathogens (Staphylococcus aureus and Bacillus subtilis) and two Gram negative bacteria (Escherichia coli, and Salmonella thyphimurium). The bacterial cultures were inoculated into the Muller Hinton Agar (MHA). Approximately, 20 mL of sterile MHA were poured into sterile culture plates and allowed to set wells of about 6 mm in diameter which were punched on the plates. The tests were conducted at 5 mg/mL for both crude extracts and isolated compounds. Bacterial concentration was prepared at 1.3×108 CFU/mL. The plates were incubated at 37°C. The antibacterial activity of the plant extracts and compounds were evaluated by measuring the zone of inhibition against the test organism after 24 hr (Tiwari, 2011). The results were calculated as averages of triplicate tests. The zone of inhibitions in all cases were includes the diameter of the wells. DMSO was used as negative control during the whole test on bacteria while amoxil was used as positive control. Saponins (Froth test): Each 0.25 g crude extracts of nhexane and methanol were dissolved and diluted with distilled water (10 mL) and shaken for 15 min. Formation of layer of foam indicates the presence of saponnins (Balick, 1996). Phenolics (Ferric chloride test): Each 0.1 g crude extracts of n-hexane and methanol were treated with few drops of 2% of FeCl3 and the formation of bluish green to black color indicates the presence of phenols (Balick, 1996). Test for tannins (Gelatin test): To the crude extracts 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins (Balick, 1996). Terpenoids (Salkowski test): The extracts (5 mg) were mixed with 2 mL of chloroform and filtered. The filtrates were treated with few drops of concentrated H2SO4, shaken and allowed to stand. Appearance of golden yellow color indicates the presence of terpenoids (Balick, 1996). Steroids (Salkowski test): One mL of the extracts (1 mg/mL) was dissolved in 10 mL of chloroform and 3 Bayan Abdi et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020 and CHCl3. This indicates that the secondary metabolites present in the roots of D. shimpiriana are mainly polar. 2.6. DPPH Radical Scavenging Assay The radical scavenging assay of the extract and constituents of the roots of Dryopteris shimpiriana were assessed using DPPH according to the following procedure (Rivero-Perez et al., 20017). In view of this, the hexane extract was dissolved in methanol to afford 1 mg/mL. It was serially diluted in methanol to give concentration of 500, 250, 125 and 62.5 µg/mL. To 1 mL of each concentration, 4 mL DPPH (0.04%DPPH in MeOH) was added to make 100, 50, 25 and 12.5 µg/mL solutions. This was repeated for the methanol extracts and isolated compounds. Then all the samples prepared were incubated in an oven at 37oC for 30 min. and then absorbance was recorded at 517 nm using UV-Vis spectrophotometer. The experiments were done triplicates and results were reported as averages of the triplicates. The percentage inhibition was calculated using the formula (Boakye-Yiadom, 1997). 3.1. Phytochemical Screening The combined n-hexane and chloroform extracts, and methanol extracts of the root of Dryopteris shimpiriana was screened for its secondary metabolites including alkaloids, saponins, terpenoids, phenolics, tannins, anthraquinones, flavonoids, steroids and coumarins with the results depicted in Table 2. As revealed from Table 2, the n-hexane/CHCl3 and methanol extract of the root of Dryopteris shimpiriana contains secondary metabolites including alkaloids, terpenoids, anthraquinones, and flavonoids. However, neither steroids nor tannins were detected in the nhexane/CHCl3 and methanol extracts. Flavonoids are classes of natural products that are used as an essential component in a variety of nutraceutical, pharmaceutical, medicinal and cosmetic applications. This is attributed to their anti-oxidative, anti-inflammatory, antimutagenic and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme function (Aoki et al., 2000). Therefore, the presence of flavonoids in this plant is significant as the plant may serves as anti-oxidative, anti-inflammatory, antimutagenic and anti-carcinogenic properties. Hitherto, secondary metabolites including saponins, flavonoids, alkaloids, anthraquinones, and terpenoids exert antimicrobial activity through different mechanism (Ghamba et al., 2014). Therefore, the presence of one or more of these secondary metabolites may account for the traditional use of this plant against bacteria. . (A control - A sample) ×100 A control Where A control was the absorbance of the DPPH solution and A sample was the absorbance in the presence of sample in DPPH solution. Samples were analyzed in triplicate. Ascorbic acid was used as positive control. (%) inhibition = 3. Results and Discussion The roots were successively extracted with nhexane, CHCl3 and MeOH to afford 5 g (2%), 2 g (0.8%) and 26.7 g (10.6%) crude extracts, respectively. The latter extract was found to be red in color while the first two were black. As clearly observed, the extract yield obtained using MeOH was higher compared to n-hexane Table 2: Phytochemical screening results of the roots extracts of Dryopteris shimpiriana Secondary metabolites Test/reagents Hexane and CHCl3 extract Methanol extract Alkaloids Saponins Terpenoids Wanger test Froth test Salkowskis test + + + + + + + + + + - + + + + - Phenolics Tannins Anthraquinones Coumarins Flavonoids Steroids (+) indicates presence Ferric chloride test Gelatin test 10%HCl & then NH3 10%NaOH Alkaline test Salkowskis test (-) indicates Absence 4 Bayan Abdi et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020 due to methylene carbons in the aliphatic regions at δC 32.8, 31.9, 29.7, 29.6, 29.4, 29.3, 25.7 and 22.7. The intense signal observed at δC 29.7 is due to the presence of seventeen overlapping methylene carbons which agreed well with the 1H-NMR. Furthermore, the carbon resonance at δ 14.1 is characteristic signal for terminal methyl group. The above NMR spectral data generated indicated that compound 1 is a long chain alcohol named as heptacosanol whose structure is shown in Figure 1. 3.2. Characterization of isolated compounds Two compounds were isolated from the combined nhexane and chloroform extracts of the root of Dryopteris shimpiriana. The detailed structure elucidations of these compounds are presented below. Compound 1 (12 mg) was isolated as white solid melting at 82-83oC (Lit. 81-82oC) (Koay, C.Y., et al., 2013). The UV-Vis spectrum (CHCl3) showed absorption maxima neither in the ultra-violet nor in the visible region indicating the absence of conjugated chromophore. The IR spectrum displayed absorption bands at 3400 and 2950 cm-1 due to hydroxy and aliphatic C-H stretching, respectively. The 1H-NMR spectrum (CDCl3) demonstrated a triplet signal at δ 3.66 (2H, J = 6.8 Hz) assigned to methylene protons on oxygenated carbon. The spectrum also displayed another signal at δH 1.61 (4H, m) which were assigned to four protons on two methylene carbons next to carbon bearing oxygen. The presence of many methylene protons integrating for 46 hydrogens is evident at δH 1.27 (46H, brs). This clearly indicates the presence of overlapping signals due to many methylenes in the structure of the compound. The upfield signal at δH 0.89 (3H, t, J = 6.8 Hz) is diagnostic for the presence of terminal methyl group. The 13C-NMR spectrum with the aid of DEPT-135 showed the presence of ten well resolved carbon signals of which nine are due to methylenes and one is methyl group. The downfield signal observed at δC 63.1 is ascribed to an oxygenated methylene carbon. This agreed very well with the data obtained from the proton NMR spectrum. The spectrum also displayed signals 27 1 OH 1 Figure 1: Structure of heptacosanol (1) Comparison of the NMR spectral data of compound 1 with those reported in the literature for 1-heptacosal (Koay et al., 2013) was in close agreement (Table 3). This compound was previously reported from the leaves of Strobilanthes crispus (Koay et al., 2013) but has not been reported from any species of Dryopteris. Compound 2 (17 mg) was isolated as a yellow solid from the combined n-hexane and CHCl3 extract of the root of Dryopteris shimpiriana. The UV-Vis spectrum (MeOH) showed absorption maxima at 271 and 338 nm suggesting the presence of a flavonoid chromophore. The IR spectrum displayed an absorption band at 1646 cm-1 attributable to an α, β-unsaturated carbonyl. The presence of C-O and C-H stretching were evident from the observed absorption bands at 1090 and 2924 cm-1, respectively. Table 3: 1H- and 13C-NMR spectral data of compound 1 with those reported in the literature for heptacosanol, δ in ppm and J in Hz NMR spectral data of compound 1 1 H-NMR Data reported for 1-heptacosanol (Koay et al., 2013) 13 1 C-NMR H-NMR 13 C-NMR 3.66 (2H, t, J = 6.60, H-1) 63.1 (C-1) 3.64 (2H, t, J = 6.30, H-1) 63.1 (C-1) 1.61 (4H, m, H-2&3) 32.8 (C-2) 1.57 (4H, m, H-2&3) 32.8 (C-2) 1.27(46H, br s, H-4 to H-26) 31.9 (C-3) 1.25(48H, br s, H-3 to H-26) 31.9 (C-3) 0.89 (3H, t, J = 6.8, H-27) 29.7-29.3(C4-24) 29.7-29.3(C4-24) 25.7 (C-25) 25.7 (C-25) 22.6 (C-26) 22.6 (C-26) 14.1 (C-27) 0.88 (3H, t, J = 6.60, H-27) 5 14.1(C-27) Bayan Abdi et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020 1 The H-NMR spectrum showed signals in the aromatic region at δH 6.45 (1H, br s) and 6.61 (1H, br s) which are apparently assigned to protons on A ring of flavonoid skeleton. The proton signals at δH 7.13 (1H, d, J = 8.4 Hz), 7.76 (1H, unresolved dd, J = 8.4 Hz) and 7.85 (1H, bro s) indicates ABX muntiplicity pattern suggesting a trisubsitituted B-ring of the flavonoid skeleton. The signal due to the presence of one methoxy protons is evident at δH 3.98 (3H, s). The proton decoupled 13C-NMR spectrum with the aid of DEPT-135 displayed the presence of five methine, one methyl and ten quaternary carbons (Table 4). The presence of α, β-unsaturated ketone is evident from the appearance of the carbonyl carbon signal at δC 178.2 (C4). The spectrum showed the presence of seven quaternary carbon signals in the oxygenated aromatic/olefinic regions at δC 163.7 (C-7), 161.0 (C-5), 156.5 (C-2), 156.2 (C-9), 144.8 (C-4̍), 144.4 (C-3̍), 120.9 (C-1̍) and 137.9 (C-3). Two typical sp2 quaternary carbon signals were observed at δC 137.9 and 104.4 attributed to C-3 and C-5a carbons of flavonoid skeleton. The presence of five methine signals are evident at δC 121.3 (C-6̍), 114.9 (C-2̍), 114.8 (C-5̍), 98.4 (C-6) and 93.5 (C-8). The latter two signals are characteristics of C-6 and C8 of the ring A of flavonoid skeleton, respectively, suggesting meta oxygenation substitution pattern of ring A. The up field signal at δc 59.4 is typical for the presence of only one methoxyl in the structure of the compound. The above spectral data and comparison with literature suggests that the compound is identical with isorhamnetin (2, figure 2, Table 2) (José et al., 2012). 6' 8 HO O 2 1' 4' OH O 4 6 OH OH O 2 Figure 2: Structure of isorhamnetin (2) The 13C-NMR spectral data of compound 2 was also compared with the literature reported for isorhamnetin with the data presented in Table 4. 3.3. Antibacterial Activity The antibacterial activity of the hexane extract, methanol extract and the two isolated compounds from the roots of Dryopteris shimpiriana were investigated using agar well diffusion method, against some selected human pathogens such as E. coli, Bacillus subtilis, S. aureus and Salmonella typhimurium (Table 5). The antibacterial activity of extracts and isolated compounds against bacterial pathogens using agar well diffusion method is expressed using inhibition zones. Antibacterial activity (x) is then characterized and classified based on the inhibition growth zone diameters and described as slight (x< 4 mm diameter), medium (x = 4-8 mm), high (x = 8-12 mm), and very high (x> 12 mm) (Obdak et al., 2017). In view of this, the extracts and isolated compounds assessed in the present study showed considerable antibacterial activity against three bacterial starins i.e S. aureus, E. coli and S. thyphlomurium compared with the positive and negative controls. The activities displayed by both Table 4: NMR spectral comparison of isorhamnetin isolated from Dryopteris shimpiriana and one that reported in literature (José et al., 2012) S No 2 3 4 5 6 7 8 8a 13 C-NMR data of 2 156.5 137.9 178.2 161.0 98.4 163.7 93.5 156.2 Literature reported for isorhamnetin 156.7 135.2 177.5 160.8 99.2 162.7 94.4 155.9 S No 5a 1̍̍̍ˈ 2ˈ 3ˈ 4ˈ 5ˈ 6ˈ OCH3 6 13 C-NMR data of 2 104.4 120.9 114.9 144.4 144.8 114.8 121.3 59.4 Literature reported for isorhamnetin 105.5 120.9 114.6 146.3 149.5 113.5 122.1 59.3 Bayan Abdi et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020 Table 5: Zone of bacterial growth inhibition diameter (mm) Sample (5 mg/mL) Hexane extract Methanol extract Compound 1 Compound 2 Amoxil DMSO Zone of inhibition diameter (mm) S. aureus B. subtitus E. coli S. thyphlomurium 13.0 ± 0.42 10.0 ± 0.5 12.0 ± 0.4 17.0 ± 0.2 23.0 ± 0.4 - 9.0 ± 0.3 9.0 ± 0.38 NT 9.6 ± 0.13 20.0 ± 0.5 - 10.3 ± 0.42 10.0 ± 0.51 13.6 ± 0.2 12.6 ± 0.3 20.0 ± 0.34 - 9.0 ± 0.51 9.6 ± 0.2 NT 10.6 ± 0.24 18.0 ± 0.2 - Results are reported as M± SD; NT: not tested; Amoxil and DMSO were used as positive and negative controls, respectively compared with compound 1 and the two extracts, and comparable with the positive control. Furthermore the antibacterial activity displayed by the extracts and isolated constituents of the root of D. shimpiriana substantiates the traditional use of this plant against bacteria. compounds are relatively better than the extracts. Compound 1 and 2 showed promising antibacterial activity against E. coli and S. aureus with zone of inhibition of 13.6 ± 0.2 and 17.0 ± 0.2, respectively, compared to amoxil (20.0 ± 0.34 and 23.0 ± 0.4, respectively) suggesting the activity displayed by the extracts of the root of D. shimpiriana is likely accounted to the known flavonoid, isorhamnetin. The activity displayed by the two extracts is likely accounted to the presence of various secondary metabolites including saponins, flavonoids, alkaloids, anthraquinones, and terpenoids. This agrees very well with the previous report which indicated as these secondary metabolites exert antimicrobial activity via different mechanism (Ghamba, et al., 2014). Analysis of the antibacterial activity revealed as compound 1 and 2 were both active against all tested bacteria. The activities displayed by both compounds are relatively better than the extracts. Especially noteworthy is the very high activity of compound 2 (17 mm) shown against S. aureus. The result is superior 3.4. Radical scavenging activity DPPH radical scavenging assay is a simple method for finding antioxidants by measuring absorbance at 517 nm due to the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical. Those compounds that exhibit antioxidant activity change the purpled colored DPPH reagent to yellow in addition to their ability of lowering the absorbance at 517 nm. This phenomenon results due to the quenching of the DPPH radical and formation of DPPH-H that would lower absorption. In this work, the extracts and isolated compounds from the roots D. shimpiriana were evaluated using DPPH assay (Table 6). Table 6: Results of radical scavenger activity of extracts and isolated compounds Samples %DPPH inhibition at 100(µg/mL) 50 (µg/mL) 25 (µg/mL) 12.5 (µg/mL) Hexane/CHCl3 extract 60.3±0.05 54.1±0.01 47.5±0.08 41.2±0.02 MeOH extract 63.9±0.01 51.6±0.01 44.7±0.07 36.5±0.10 Compound 1 44.7±0.03 40.0±0.05 33.3±0.01 25.7±0.02 Compound 2 82.8±0.01 77.1±0.01 71.4±0.02 63.8±0.03 Ascorbic acid 90.0±0.02 7 Bayan Abdi et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (1), 2020 The % DPPH inhibition shown by the methanol extract and the combined n-hexane/CHCl3 extract is 63.9 and 60.3%, respectively. The activity displayed by the extract is accounted to the presence of flavonoids which was confirmed by the alkaline test. On the other hand, compound 2 displayed pronounceable free radical scavenging activity with an average percent inhibition of 82.8, 77.1, 71.4, and 63.8% at 100, 50, 25 and 12.5 μg mL-1, respectively. The result is significant compared to ascorbic acid which inhibited the radical by 90% at 100 μg mL-1. Further confirmation of the antioxidant activity of compound 2 was made due to the observed immediate discoloration of the purple DPPH solution to yellow. Therefore, the radical scavenging activity of the roots of D. schimperiana is accounted to the presence of the known flavonoids, namely, isorhamnetin (2). 4. Conclusions In conclusion, phytochemical screening analysis conducted on roots extract of Dryopteris schimperiana revealed the presence of alkaloids, terpenes, anthraquinone, flavanoids, and tannins. The combined n-hexane and CHCl3 extract after silica gel column chromatography furnished two compounds identified as spectroscopic methods as heptacosanol (1) and isorhamnetin (2). The extracts of the roots of D. shimeriana were shown to contain compounds with wide-spectrum antibacterial activity, capable of inhibiting the growth of Grampositive and negative bacteria. 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