2014 Volume 62 Issue 2 Pages 191-195
Three new glycosides: thotneoside A (quercetin 3-O-(6″-O-phenylacetyl)-β-D-galactopyranoside) (1), thotneoside B (quercetin 3-O-(6″-O-phenylacetyl)-β-D-glucopyranoside) (2) and thotneoside C (3-methyl-2-butenoic acid 1-O-β-D-glucopyranoside) (3), together with nine known compounds; quercetin (4), quercetin 3-O-β-D-galactopyranoside (5), quercetin 3-O-(6″-O-galloyl)-β-D-galactopyranoside (6), quercetin 3-O-β-D-galacturonopyranoside (7), quercetin 3-O-β-D-glucuronopyranoside (8), quercetin 3-O-α-L-rhamnopyranoside (9), rutin (10), quercetin 3-O-α-L-arabinopyranoside (11) and 2,4,6-trihydroxyacetophenone 2-O-β-D-glucopyranoside (12) have been isolated from the shade dried leaves of Aconogonon molle, commonly known as “Thotne″ in Nepal. The structures were elucidated on the basis of chemical and spectroscopic methods. All of these compounds were isolated for the first time from A. molle and their in vitro antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay. Quercetin (4) and its glycosides (1–2, 5–11) showed potent free radical scavenging activity.
Aconogonon molle (D. DON) H. HARA (syn. Polygonum molle D. DON) (Polygonaceae) is a perennial herb, 1.2−2.5 m tall. It is locally called as “Thotne” in Nepal and distributed throughout Nepal, Bhutan, India, Indonesia, Myanmar, Sikkim, Thailand, South and South East Asia between 1200–2400 m. Traditionally in Nepal, tender shoots are used in diarrhea and young shoots are eaten as vegetable and pickles. The whole plant is astringent.1,2) In China, it is used for carbuncle, swollen abscess, fistula and scrofula.3) Although different classes of secondary metabolites such as flavonoids, chalcones, anthraquinones, naphthoquinones, sesquiterpenoids, lignans, coumarins, stilbene glycoside, acetophenone glycosides4) etc. were reported from different Polygonum species, but no previous chemical investigation was performed on title plant. Thus, the chemical analysis and biological activity evaluation on this plant is important from the aspects of natural product and food chemistry. Therefore, present study was aimed for detail chemical analysis of leaves of A. molle and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of the isolated compounds.
Successive extraction of the shade dried leaves (270 g) of A. molle with 70% MeOH and MeOH gave total 35.6 g of extract. The water soluble fraction of the extract was applied to repeated column chromatography (CC) on MCI gel CHP20P, Sephadex LH-20, octadecyl silica (ODS) and silica gel to isolate the compounds 1–12 (Fig. 1).
Compound 1 was obtained as pale yellow amorphous powder, [α]D21 −11.6° (c 0.90, pyridine : H2O=8 : 2). Its molecular formula was determined as C29H26O13 on the basis of a high resolution (HR)-FAB-MS peak of [M-H]− at 581.1290 (calcd for C29H25O13, 581.1295). The UV spectrum of compound 1 showed absorption maxima (λmax) at 361 and 258 nm suggesting a flavone derivative. Compound 1 was positive towards ferric chloride reagent and gave pink color with magnesium-hydrochloric acid (Mg-HCl) reagent, suggesting a flavonol derivative. The 1H-NMR spectrum of compound 1 (Table 1) showed proton signals due to a hydrogen bonded OH group at δH 12.65 (s), an ABX system; δH 7.65 (1H, dd, J=2.1, 8.2 Hz), 7.53 (1H, d, J=2.1 Hz), 6.82 (1H, d, J=8.2 Hz) and two meta coupled aromatic protons at δH 6.42 (1H, d, J=2.1 Hz), δH 6.19 (1H, d, J=2.1 Hz) which were assignable to 5,7-disubstituted A ring and 1,3,4-trisubstituted B ring of flavonol backbone.5) The signals; a multiplete at δH 7.19–7.20 (3H, m), a doublets of doublet at δH 7.02 (2H, dd, J=2.1, 8.2 Hz) and a singlet at δH 3.31 (2H, s) were assignable to phenylacetyl moiety.6) Moreover the proton signals at δH 5.37 (1H, d, J=7.6 Hz), 4.07 (1H, dd, J=7.9, 11.3 Hz), 3.98 (1H, dd, J=4.3, 11.3 Hz), 3.40–3.63 (4H) were assignable to the monosaccharide moiety and the coupling constant (J=7.6 Hz) of anomeric proton suggested the β-configuration. The 13C-NMR spectra of compound 1 showed signals equivalent to total 29 carbons, in which 15 carbon signals (Table 1) were assignable to a 3-O-substituted quercetin moiety5); and eight signals (170.6 (C=O), 133.9 (C), 129.0 (CH×2), 128.1 (CH×2), 126.6 (CH), 38.4 (CH2)) were assignable to a phenylacetyl moiety.6) The remaining six signals (101.5 (CH), 72.7 (CH×2), 70.8 (CH), 68.2 (CH), 63.7 (CH2)) for a monosaccharide revealed the β-galactopyranosyl moiety.5) The complete assignment of the proton and carbon atoms, their positions, the linkage of the galactopyranoside moiety and phenylacetyl group in the compound 1 were determined on the basis of chemical shifts, 1H–1H correlation spectroscopy (COSY), 1H-detected heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond connectivity (HMBC) spectra (Fig. 2). Acid hydrolysis of compound 1 afforded D-galactose confirmed on the basis of optical rotation, [α]D20 +60.0° and co-TLC with authentic sample. The absolute configuration of D-galactopyranosyl moiety in the compound 1 was further decided on the basis of Klyne’s rule7) by comparing the molecular rotation [M]D of compound 1 with that of methyl-β-D-galactopyranoside. The [M]D is calculated by the formula: ([M]D=([α]D× molecular weight (MW)/100).7) The [α]D of methyl-β-D-galactopyranoside was reported as −16.0° (c 1.5, MeOH),8) from which the [M]D was calculated to be −31.0°. The [M]D of compound 1 was found to be −67.5° whose sign was similar to that of methyl-β-D-galactopyranoside ([M]D−31.0°) and quercetin 3-O-β-D-galactopyranoside (5) ([M]D −123.9°). Thus, from the physical and spectral data, the structure of compound 1 was elucidated as quercetin 3-O-(6″-O-phenylacetyl)-β-D-galactopyranoside and named as thotneoside A.
| Position | 1 (in DMSO-d6) | 2 (in DMSO-d6) | 3 (in D2O) | |||
|---|---|---|---|---|---|---|
| 1H | 13C | 1H | 13C | 1H | 13C | |
| 1 | 167.0 | |||||
| 2 | 156.1a) | 156.2a) | 5.71, s | 113.8 | ||
| 3 | 133.3 | 133.0 | 164.4 | |||
| 4 | 177.3 | 177.3 | 1.87, s | 27.3 | ||
| 5 | 161.1 | 161.1 | 2.06, s | 20.5 | ||
| 6 | 6.19, d (2.1) | 98.6 | 6.19, d (2.1) | 98.6 | ||
| 7 | 164.2 | 164.1 | ||||
| 8 | 6.42, d (2.1) | 93.4 | 6.42, d (2.1) | 93.5 | ||
| 9 | 156.2a) | 156.3a) | ||||
| 10 | 103.7 | 103.7 | ||||
| 1′ | 120.9 | 120.9 | ||||
| 2′ | 7.53, d (2.1) | 115.7 | 7.54, d (2.1) | 116.1 | ||
| 3′ | 144.8 | 144.7 | ||||
| 4′ | 148.4 | 148.4 | ||||
| 5′ | 6.82, d (8.2) | 115.1 | 6.85,d (8.2) | 115.0 | ||
| 6′ | 7.65, dd (2.1, 8.2) | 121.9 | 7.56, dd (2.1, 8.2) | 121.4 | ||
| C5-OH | 12.65, br s | 12.64, br s | ||||
| Gal/Glc-1 | 5.37, d (7.6) | 101.5 | 5.43, d (7.6) | 100.7 | 5.47, d (7.9) | 93.7 |
| Gal/Glc-2 | 3.40–3.63 | 70.8 | 3.37–3.61 | 73.8b) | 3.40, dd (7.9, 9.2) | 72.2 |
| Gal/Glc-3 | 3.40–3.63 | 72.7 | 3.37–3.61 | 76.1 | 3.49, t (9.2) | 75.8 |
| Gal/Glc-4 | 3.40–3.63 | 68.2 | 3.37–3.61 | 69.7 | 3.49, t (9.2) | 69.4 |
| Gal/Glc-5 | 3.40–3.63 | 72.7 | 3.37–3.61 | 73.9b) | 3.38, m | 77.0 |
| Gal/Glc-6 | 4.07, dd (7.9, 11.3) 3.98, dd (4.3, 11.3) | 63.7 | 4.19, dd (2.1, 11.9) 3.95, dd (6.4, 11.9) | 63.3 | 3.79, dd (2.1, 12.5) 3.62, dd (5.5, 12.5) | 60.7 |
| 1‴ | 133.9 | 134.0 | ||||
| 2‴ | 7.02, dd (2.1, 8.2) | 129.0 | 7.02, dd (2.1, 7.3) | 129.1 | ||
| 3‴ | 7.19–7.20 | 128.1 | 7.18–7.20 | 128.1 | ||
| 4‴ | 7.19–7.20 | 126.6 | 7.18–7.20 | 126.6 | ||
| 5‴ | 7.19–7.20 | 128.1 | 7.18–7.20 | 128.1 | ||
| 6‴ | 7.02, dd (2.1, 8.2) | 129.0 | 7.02, dd (2.1, 7.3) | 129.1 | ||
| 7‴ | 3.31, s | 38.4 | 3.29, s | 38.5 | ||
| 8‴ | 170.6 | 170.6 | ||||
a), b) Assignments with same superscript may be reversed in same column.
Compound 2 was obtained as yellow amorphous powder, [α]D21 −21.1° (c 0.16, pyridine) Its molecular formula was determined as C29H26O13 on the basis of a HR-FAB-MS peak of [M+Na]+ at 605.1302 (Calcd for C29H26O13Na, 605.1271). The UV spectrum of compound 2 also showed absorption maxima (λmax) at 361 and 258 nm suggesting a flavone derivative. Compound 2 was positive towards ferric chloride test and gave pink color with magnesium-hydrochloric acid (Mg-HCl) reagent, suggesting a flavonol derivative. All the 1H- and 13C-NMR data of compound 2 were similar to compound 1, except for the 1H- and 13C-NMR signals of sugar moiety (Table 1). The 1H-NMR of sugar moiety of 2 appeared at δH 5.43 (1H, d, J=7.6 Hz), 4.19 (1H, dd, J=2.1, 11.9 Hz), 3.95 (1H, dd, J=6.4, 11.9 Hz), 3.37–3.61 (4H) and the coupling constant (J=7.6 Hz) of anomeric proton suggested the β-configuration of sugar moiety. In the 13C-NMR spectra, signals for sugar moiety appeared at δC 100.7, 76.1, 73.9, 73.8, 69.7, 63.3, which were assignable to β-glucopyranosyl moiety.5,6,9) Acid hydrolysis of compound 2 afforded D-glucose as confirmed on the basis of optical rotation, [α]D20 +49.1° and co-TLC with authentic sample. The absolute configuration of D-glucopyranosyl moiety in compound 2 was further supported by the application of Klyne’s rule,7) as the sign for the molecular rotation ([MD]D) of 2 (−122.8°) is similar to the sign of methyl-β-D-glucopyranoside (−66.3°).10,11) Thus, the structure of compound 2 was elucidated as quercetin-3-O-(6″-O-phenylacetyl)-β-D-glucopyranoside and named as thotneoside B.
Compound 3 was obtained as colorless gum, [α]D21 −32.2° (c 0.90, H2O). Its molecular formula was determined as C11H18O7 on the basis of a HR-FAB-MS peak of [M+Na]+ at 285.0954 (calcd for C11H18O7Na, 285.0950). The UV spectrum of compound 3 showed absorption maxima (λmax) at 235 nm suggesting a α, β-unsaturated carbonyl group. The 1H-NMR spectrum showed at δH 5.71 (1H, s) assignable to olefinic singlet. The signals at δH 5.47 (1H, d, J=7.9 Hz), δH 3.49 (2H, t, J=9.2 Hz), δH 3.40 (1H, dd, J=7.9, 9.2 Hz), δH 3.38 (1H, m), δH 3.79 (1H, dd, J=2.1, 12.5 Hz) and δH 3.62 (1H, dd, J=5.5, 12.5 Hz) were assignable to the monosaccharide moiety. The signal at δH 5.47 (J=7.9 Hz) was assignable to the anomaric proton. Two singlet at δH 2.06 (3H, s) and δH 1.87 (3H, s) were assignable to two methyl groups. The 13C-NMR spectrum of compound 3 showed total 11 carbon signals (δC 167.0, 164.4, 113.8, 93.7, 77.0, 75.8, 72.2, 69.4, 60.7, 27.3, 20.5). Of them, six signals at 93.7, 77.0, 75.8, 72.2, 69.4, 60.7 were similar with β-glucopyranosyl5) parts of tiglic acid 1-O-β-D-glucopyranoside12) and supported by 1H-NMR data. The signal at 93.7 was assignable to anomaric carbon and coupling constant of anomaric proton (J=7.9) was in good agreement with a β-configuration of sugar moiety. Furthermore, in the 13C-NMR spectrum, there were an ester carboxyl carbon at δC 167.0, two olefin signals as methine carbon at δC 113.8 and a tetrasubstituted carbon at δC 164.4 and two methyls at δC 27.3 and 20.5 were assignable to senecioyl group.13) Acid hydrolysis of compound 3 afforded D-glucose as confirmed on the basis of optical rotation, [α]D20 +44.3° and co-TLC with authentic sample. The absolute configuration of D-glucopyranosyl moiety in compound 3 was further confirmed by the application of Klyne’s rule,7) as the sign of molecular rotation of compound 3 (−84.3°) was similar to the molecular rotation ([M]D) of methyl-β-D-glucopyranoside (−66.3°).10,11) Thus, the structure of the compound 3 was elucidated as 3-methyl-2-butenoic acid 1-O-β-D-glucopyranoside and named as thotneoside C.
The structures of known compounds were elucidated on the basis of their physical and spectral data and comparison with literature as quercetin (4),5) quercetin 3-O-β-D-galactopyranoside (5),14) quercetin 3-O-(6″-O-galloyl)-β-D-galactopyranoside (6),15) quercetin 3-O-β-D-galacturonopyranoside (7),16) quercetin 3-O-β-D-glucuronopyranoside (8),15) quercetin 3-O-α-L-rhamnopyranoside (9),17) rutin (10),18) quercetin 3-O-α-L-arabinopyranoside (11),19) 2,4,6-trihydroxyacetophenone 2-O-β-D-glucopyranoside (12).20)
All of the isolated compounds were tested for their in vitro antioxidant activity towards DPPH free radical scavenging assay. The 50% inhibition concentration (IC50) for DPPH radical-scavenging activity were calculated and given in Table 2. The results were compared with Trolox as positive control (IC50, 96.1 µM). The free radical scavenging activity of quercetin 3-O-(6″-O-galloyl)-β-D-galactopyranoside (6) was strongest (IC50, 24.6 µM) followed by quercetin (4) (IC50 37.7) and other quercetin glycosides (1, 2, 5, 7–11). In flavonoids, the presence of an ortho-dihydroxyl group in the B ring is shown to be effective in free radical scavenging21) and the enhancement of scavenging activity of 6 might be due to the presence of galloyl group. These results also indicated that a the substitution at C3–OH by glycosyl moiety reduced the free radical scavenging activity. These findings were similar to the previous reports regarding the free radical scavenging activity of flavonoids.22,23) Thus, the presence of such strong antioxidants in the leaves of A. molle might be able to protect against oxidative damage.
| Compounds | IC 50 (µM) |
|---|---|
| 1 | 45.9 |
| 2 | 45.5 |
| 3 | >3816.0 |
| 4 | 37.7 |
| 5 | 51.4 |
| 6 | 24.6 |
| 7 | 48.6 |
| 8 | 52.3 |
| 9 | 53.5 |
| 10 | 49.8 |
| 11 | 48.6 |
| 12 | >3030.0 |
| Trolox | 96.1 |
Optical rotations were measured with a JASCO DIP-1000KUY polarimeter. NMR spectra were measured on a JEOL α-500 spectrometer (1H: 500 MHz and 13C: 125 MHz). Chemical shifts are given in ppm with reference to TMS. Mass spectra were recorded on JEOL JMS 700 MStation mass spectrometer. Column chromatography (CC) was carried out with silica gel 60 (0.040–0.063 mm, Merck), MCI gel CHP20P (75–150 µm, Mitsubishi Chemical Industries Co., Ltd.), Sephadex LH-20 (Amersham Pharmacia Biotech) and Chromatorex ODS (30–50 µm, Fuji Silysia Chemical Co., Ltd.). TLC was performed on precoated silica gel 60 F254 plates (0.2 mm, aluminum sheet, Merck). Authentic samples of sugars were obtained from Sigma-Aldrich Chemie, Germany.
Plant MaterialThe fresh leaves of A. molle were collected from Nagarkot (1654 m), Bhaktapur, Nepal in August 2012 and identified by Prof. Takashi Watanabe, Kochi University of Technology, Japan. The voucher specimen (Voucher No.: KUNP20120825-1) was deposited on Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
Extraction and IsolationThe shade dried leaves (270 g) were extracted successively with 70% MeOH (4.5 L) at 55°C (5 h) and room temperature (about 20°C, for 6 d), and MeOH (4.5 L) at 55°C (5 h) and room temperature (about 20°C, for 22 h). The combined extract was evaporated under reduced pressure to give 35.6 g extract. Then 32.1 g extract was suspended in water (500 mL) to give water soluble (28.0 g) and water insoluble fraction (4.1 g). The water soluble fraction (28.0 g) was subjected on MCI gel CHP20P CC and eluted successively with water, 40%, 60%, 80% and 100% MeOH to give eleven fractions (1–11). Fraction 3 (2.4 g, 40% MeOH eluate) was further subjected to MCI gel CHP20P CC (10–40% MeOH) to give ten subfractions (3-1–3-10). Fraction 3-2 (322 mg, 10% MeOH eluate) was subjected on Sephadex LH-20 CC (40% MeOH) to afford compound 7 (61 mg). Fraction 3-4 (170 mg, 20% MeOH eluate) was subjected on ODS CC (10–18% MeOH) to give five subfractions (3-4-1–3-4-5). Fraction 3-4-2 (70 mg, 10% MeOH eluate) was applied on Sephadex LH-20 CC (40% MeOH) to afford compound 3 (21 mg). Fraction 3-6 (410 mg, 30% MeOH eluate) was subjected on Sephadex LH-20 CC (75% MeOH) to give four subfractions (3-6-1–3-6-4). Then fr. 3-6-3 (221 mg) was subjected on ODS CC (18–27% MeOH) to give seven fractions (3-6-3-1–3-6-3-7). Fraction 3-6-3-2 (19 mg, 24% eluate) was subjected on silica gel (6 : 4 : 1, CHCl3 : MeOH : H2O) to afford compound 12 (6 mg). Fraction 3-6-3-5 (158 mg, 27% eluate) was subjected on Sephadex LH-20 CC (60% MeOH) to affords compounds 7 (13 mg) and 8 (55 mg). Fraction 4 (4.9 g, 60% MeOH eluate) was subjected to MCI gel CHP20P CC (10–50% MeOH) to give eleven subfractions (4-1–4-11). Fraction 4-6 (1575 mg, 40% MeOH eluate) was subjected on Sephadex LH-20 CC (60% MeOH) to give eight subfractions (4-6-1–4-6-8). Fraction 4-6-3 (145 mg, 60% MeOH eluate) was successively subjected on Sephadex LH-20 CC (MeOH) and ODS CC (30% MeOH) to afford compound 10 (18.9 mg). Fraction 4-6-4 (497 mg, 60% MeOH eluate) was successively subjected on Sephadex LH-20 CC (MeOH) and ODS CC (30% MeOH) to afford compounds 5 (67 mg) and 9 (156 mg). Fraction 4-6-6 was obtained as compound 6 (66 mg). Fraction 5 (529 mg, 60% MeOH eluate) was subjected on Sephadex LH-20 CC (MeOH) to afford compound 11 (148 mg). Fraction 7 (400 mg, 60% MeOH eluate) was subjected on Sephadex LH-20 CC (MeOH) to give eight subfractions (7-1–7-8). Fraction 7-2 (40 mg) subjected on ODS CC (60% MeOH) to give four subfractions (7-2-1–7-2-4). Fraction 7-2-3 (16 mg) subjected on ODS CC (55% MeOH) to afford compound 2 (4.1 mg). Fraction 7-7 was obtained as compound 4 (28 mg). Fraction 8 (253 mg, 80% MeOH eluate) was subjected on Sephadex LH-20 CC (MeOH) to give four subfractions (8-1–8-4). Fraction 8-2 (59 mg) subjected on ODS CC (60% MeOH) to give five subfractions (8-2-1–8-2-5). Fraction 8-2-2 was further subjected on Sephadex LH-20 CC (MeOH) to afford compound 1 (17 mg).
Thotneoside A (1): Pale yellow amorphous powder. [α]D21 −11.6° (c 0.90, pyridine: H2O (8 : 2)); UV λmax (MeOH) nm (log ε): 361(4.12), 258 (4.20). HR-FAB-MS peak of [M−H]− at 581.1290 (Calcd for C29H25O13, 581.1295). 13C- and 1H-NMR data are given in Table 1.
Thotneoside B (2): Pale yellow amorphous powder. [α]D21 −21.1° (c 0.16, pyridine); UV λmax (MeOH) nm (log ε): 361(4.12), 258 (4.20) HR-FAB-MS peak of [M+Na]+ at 605.1302 (Calcd for C29H26O13Na, 605.1271). 13C- and 1H-NMR data are given in Table 1.
Thotneoside C (3): Colorless gum. [α]D21 −32.2° (c 0.90, H2O); UV λmax (MeOH) nm (log ε): 235(2.95). HR-FAB-MS peak of [M+Na]+ at 285.0954 (calcd. for C11H18O7Na, 285.0950). 13C- and 1H-NMR data are given in Table 1.
Known compounds: quercetin (4), quercetin-3-O-β-D-galactopyranoside (5): [α]D21 −21.3° (c 0.25, pyridine), quercetin-3-O-(6″-O-galloyl)-β-D-galactopyranoside (6): [α]D21 −42.5° (c 0.44, pyridine), quercetin-3-O-β-D-galacturonopyranoside (7): [α]D21 −36.6° (c 0.51, H2O), quercetin-3-O-β-D-glucuronopyranoside (8): [α]D21 −8.6° (c 0.44, pyridine), quercetin-3-O-α-L-rhamnopyranoside (9): [α]D21 −146.7° (c 1.0, pyridine), rutin (10): [α]D21 −26.1° (c 0.72, pyridine), quercetin-3-O-α-L-arabinopyranoside (11): [α]D21 −138.3° (c 0.41, pyridine), 2,4,6-trihydroxyacetophenone 2-O-β-D-glucopyranoside (12): [α]D21 −57.6° (c 0.27, CH3OH).
Acid Hydrolysis of Compounds 1, 2 and 3Compound 1 (5.0 mg) in 2 M HCl (2.0 mL), 2 (2.0 mg) in 2 M HCl (1.0 mL) and 3 (2.0 mg) in 2 M HCl (1.0 mL) were separately heated at 70°C for 3 h in a sealed tube. Reaction mixtures were separately extracted with EtOAc and separated into EtOAc and aq. layers. From the aq. layer of compounds 1, 2 and 3; D-galactose {1.1 mg, [α]D20 +60.0° (c 0.11, H2O)}, D-glucose {0.7 mg, [α]D20 +49.1° (c 0.07, H2O)} and D-glucose {1.1 mg, [α]D20 +44.3° (c 0.11, H2O)} were obtained, respectively. The co-TLC in silica gel together with the authentic samples was performed using CHCl3–MeOH–H2O (6 : 4 : 1, v/v/v) and n-BuOH–AcOEt–H2O (5 : 1 : 4, v/v/v, upper phase) as the developing solvents. Aqueous H2SO4 (10%) was used as the detection reagent. Galactose was detected from compound 1 and glucose was detected from compounds 2 and 3.
Antioxidative ActivityThe DPPH radical scavenging activity of 1–12 was measured by the method as described by Li and Seeram24) with slight modifications. Briefly, 80 µL of each compound at various concentrations (in dimethyl sulfoxide (DMSO) : EtOH=1 : 1) was mixed with 40 µL of MES buffer (200 mM, pH 6.0) and 40 µL of DPPH solution (800 µM in EtOH) in a 96-well plate. The reaction mixture was shaken vigorously and left for 30 min. at room temperature in the dark. The anti-oxidative activity corresponding to the scavenging of DPPH radicals was measured at 510 nm with UV spectrophotometer using following formula: Radical scavenging activity (%)=100×(A−B)/A. Where, A is the control absorbance of DPPH radicals without samples and B is the absorbance after reacting with samples. Trolox was used as the positive control. The result is expressed as mean of three experiments. From these data, curve was plotted and concentration (µM) of the sample required for 50% reduction of the DPPH radical absorbance (IC50) was calculated.
The authors would like to thank the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan for the scholarship to Khem Raj Joshi and Hari Prasad Devkota. We are grateful to Ms. Teruo Tanaka and Mr. Toshiyuki Iriguchi of Institute of Resource Development and Analysis, Kumamoto University for measurement of NMR and mass spectra, respectively. This work was supported in part by Program for Leading Graduate Schools “HIGO” (Health Life Science: Interdisciplinary and Glocal Oriented), MEXT, Japan.
