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SUPPLEMENTARY MATERIAL
Radical Scavenging Activities of Flavonoids from Roots of Akschindlium
godefroyanum
Kanokkarn Saejuenga* and Natcha Panthamab
a
Program of Science, Faculty of Education, Sisaket Rajabhat University, Sisaket, 33000,
Thailand.; bDepartment of Applied Chemistry, Faculty of Sciences and
Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima, 30000,
Thailand.
*Corresponding author
E-mail address: kanokchem25@hotmail.com
Chemical constituents of crude ethyl acetate of roots of Akschindlium godefroyanum
(Kuntze) H. Ohashi were investigated and seven flavonoids were isolated. Their
structures were identified based on spectroscopic methods as well as comparison with
spectral data reported in literature as six flavanonols and a flavonol including
7,4′-dihydroxy-5,3′-dimethoxyflavanonol (1), neophellamuretin (2), taxifolin (3),
erycibenin D (4), geraldol (5), fustin (6), and garbanzol (7). Compounds 2, 4 and 7
were found in the genus Akschindlium for the first time. Compounds 3, 5 and 6
appeared to have free radical scavenging activities using DPPH assay with IC 50 of 21,
40 and 15 g/mL, respectively.
Keywords: Akschindlium godefroyanum; flavonoids; DPPH.
1. Experimental
1.1 General Procedure
Melting points were determined using Gallenkamp Sanyo. IR spectra were obtained using a
Bruker Tenser 27 spectrophotometer (Bruker, Germany). NMR spectra were recorded on a
Varian Mercury Plus 400 spectrometer (Varian Inc., USA) using CDCl3, CD3OD and
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DMSO-d6 as solvents. The internal standards were referenced from the residue of those
solvents. Column chromatography was carried out on Merck silica gel 60 (230-400 mesh)
(Merck, Darmstadt, Germany). TLC was performed with precoated Merck silica gel 60 PF254
(Merck, Darmstadt, Germany); the spots were visualized under UV light (254 and 365 nm)
and also by spraying with anisaldehyde and then heating until charred. Absorbance at 517 nm
was measured by UV-2450 Shimadzu.
1.2 Plant Material and Extraction Procedure
The roots of A. godefroyanum was collected from Ubolratana district, Khon Kaen province,
Thailand, in December 2012. Its specimen has previously been identified by Prof. Pranom
Chantaranothai, Department of Biology, Khon Kaen University, Thailand where a voucher
(voucher number S. Kanokmedhakul-13) was deposited. Dried roots of A. godefroyanum (4.0
kg) were ground and extracted with n-hexane (15 Lx 3), EtOAc (15 L x 3) and MeOH (15 L
x 3). The solvents were removed under reduce pressure yielding hexane extract 8.1 g, EtOAc
extract 40.0 g and MeOH extract 281.0 g. EtOAc extract (40.0 g) was then separated on a
silica gel flash column chromatography, eluted with gradient system of n-hexane, EtOAc-nhexane, EtOAc and MeOH to give 11 fractions, AG1-AG11. 7,4′-dihydroxy-5,3′dimethoxyflavanonol (1) precipitated as white powder from AG7. Fraction AG3 (3.1 g) was
then separated on a silica gel flash column chromatography, eluted with gradient system of
dichloromethane, EtOAc-dicloromethane, EtOAc and MeOH to give 6 fractions, AG3.1AG3.6. Neophellamuretin (2) precipitated as white powder from AG3.5. AG4 was divided
into 2 fractions, solid fraction (AG4S) and liquid fraction (AG4L). AG4L was then separated
on a silica gel flash column chromatography, eluted with gradient system of n-hexane,
EtOAc-n-hexane, EtOAc and MeOH to give 10 fractions (AG4L.1-AG4L.10). AG4L.4 (2.3
g) was then separated on a silica gel flash column chromatography, eluted with gradient
system of CH2Cl2, MeOH-CH2Cl2 and MeOH yielded 7 fractions (AG4L.4.1-AG4L.4.7).
AG4L.7 (0.066 g) was then separated on Preparative Thin-Layer Chromatography (PLC)
using 7% MeOH: CH2Cl2 as eluent to yield taxifolin (3). AG5 (8 g) was separated on a silica
gel flash column chromatography, eluted with gradient system of CH2Cl2, MeOH-CH2Cl2
and MeOH to give 19 fractions. Erycibenin D (4), geraldol (5) and fustin (6) precipitated as
white powders from AG5.2, AG5.3 and AG5.16, accordingly.
AG5.5 (0.300 g) was
separated on a silica gel flash column chromatography, eluted with a gradient system of 40%
EtOAC: hexane, EtOAc and MeOH to give 12 fractions (AG5.5.1-AG5.5.12). AG5.5.4
(0.1819 g) was then separated on a silica gel flash column chromatography, eluted with 3%
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Acetone: CH2Cl2 and then MeOH to give 16 fractions (AG5.5.4.1-AG5.5.4.16). Garbanzol
(7) was obtained as colorless needles from AG5.5.4.6.
Compounds 1, 3, 5 and 6 were identified by comparison of NMR spectra and physical
properties (m.p., and TLC) with authentic samples. Compounds 2 and 4 were identified by
comparison of NMR spectra with those reported in literature. While compound 7 was
identified by 1D and 2D NMR spectra (DEPT, COSY, HMQC, HMBC and NOESY) since
its NMR data could not be found in literature. All isolated compounds were identified as 7,4′dihydroxy-5,3′-dimethoxyflavanonol (1) (Chaipukdee et al. 2014), neophellamuretin (2)
(Bohlmann et al. 1980), taxifolin (3) (Lee et al. 2011), erycibenin D (4) (Morikawa et al.
2006), geraldol (5) (Lee et al. 2008), fustin (6) (Park et al. 2004) and garbanzol (7) (Oyamada
& Baba 1966).
7,4′-Dihydroxy-5,3′-dimethoxyflavanonol (1): white powder; 1H NMR (400 MHz, DMSOd6): 7.05 (1H, d, J = 1.2 Hz, H-2′), 6.87 (1H, dd, J = 1.2, 8.1 Hz, H-6′), 6.76 (1H, d, J = 8.1
Hz, H-5′), 6.07 (1H, d, J = 1.9 Hz, H-6), 5.91 (1H, d, J = 1.9 Hz, H-8), 5.17 (1H, br s, OH-3),
4.92 (1H, d, J = 11.4 Hz, H-2), 4.36 (1H, d, J = 11.4 Hz, H-3), 3.76 (3H, s, OCH3-3′), 3.74
(3H, s, OCH3-5);
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C-NMR (100 MHz, DMSO-d6):  190.4 (C-4), 165.2 (C-7), 164.2 (C-9),
162.6 (C-5), 147.8 (C-3′), 147.4 (C-4′), 128.8 (C-1′), 121.4 (C-6′), 115.4 (C-5′), 112.7 (C-2′),
102.9 (C-10), 96.0 (C-8), 93.8 (C-6), 83.1 (C-2), 72.9 (C-3), 56.2 (OCH3-5,3′).
Neophellamuretin (2): white powder; 1H NMR (400 MHz, CD3OD):  7.36 (2H, d, J = 8.5
Hz, H-2′,6′), 6.84 (2H, d, J = 8.5, H-3′,5′), 5.96 (1H, s, H-6), 5.12 (1H, m, H-2″), 4.92 (1H, d,
J = 11.6 Hz, H-2), 4.50 (1H, d, J = 11.6 Hz, H-3), 3.12 (2H, m, H-1″), 1.59 (3H, s, H-4″),
1.50 (3H, s, H-5″);
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C-NMR (100 MHz, CD3OD): 197.3 (C-4), 165.0 (C-7), 161.5 (C-5),
159.8 (C-9), 157.7 (C-4′), 130.3 (C-3″), 128.9 (C-2′,6′), 128.2 (C-1′), 122.3 (C-2″), 114.7 (C3′,5′), 107.9 (C-8), 100.4 (C-10), 95.3 (C-6), 83.4 (C-2), 72.3 (C-3), 24.5 (C-4″), 20.9 (C-1″),
16.4 (C-5″).
Taxifolin (3): yellow powder; 1H NMR (400 MHz, CD3OD): 6.97 (1H, br, H-2′), 6.84 (1H,
d, J = 8 Hz, H-6′), 6.80 (1H, d, J = 8 Hz, H-5′), 5.86 (1H, s, H-8), 5.82 (1H, s, H-6), 4.87 (1H,
d, J = 11.4 Hz, H-2), 4.47 (1H, d, J = 11.4 Hz, H-3); 13C-NMR (100 MHz, CD3OD): 196.1
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(C-4), 170.2 (C-7), 163.9 (C-9), 163.0 (C-5), 145.6 (C-4′), 144.9 (C-3′), 128.7 (C-1′), 119.5
(C-6′), 114.5 (C-2′, C-5′), 99.7 (C-10), 96.8 (C-8), 95.8 (C-6), 83.6 (C-2), 72.2 (C-3).
Erycibenin D (4): yellow powder; 1H NMR (400 MHz, CD3OD): 7.72 (1H, d, J = 8.7 Hz,
H-5), 7.12 (1H, d, J = 1.8 Hz, H-2′), 6.98 (1H, dd, J = 1.8, 8.1 Hz, H-6′), 6.83 (1H, d, J = 8.1
Hz, H-5′), 6.53 (1H, dd, J = 2.2, 8.7 Hz, H-6), 6.34 (1H, d, J = 2.18 Hz, H-8), 5.00 (1H, d, J =
11.9 Hz, H-2), 4.54 (1H, d, J = 11.9 Hz, H-3), 3.88 (3H, s, OCH3-3′);
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C-NMR (100 MHz,
CD3OD): 193.1 (C-4), 165.4 (C-7), 163.7 (C-9), 147.5 (C-3′), 146.9 (C-4′), 128.7 (C-5, 1′),
120.8 (C-6′), 114.6 (C-5′), 112.1 (C-10), 111.1 (C-2′), 110.7 (C-6), 102.3 (C-8), 84.3 (C-2),
73.1 (C-3), 55.1 (OCH3-3′).
Geraldol (5): yellow powder; 1H NMR (400 MHz, DMSO-d6):  7.91 (1H, d, J = 8.8 Hz, H5), 7.75 (1H, d, J = 1.9 Hz, H-2′), 7.68 (1H, dd, J = 1.9, 8.5 Hz, H-6′), 6.95 (1H, d, J = 2.1
Hz, H-8), 6.92 (1H, d, J = 8.5 Hz, H-5′), 6.89 (1H, dd, J = 2.1, 8.8 Hz, ), 3.83 (3H, s, OCH33′);
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C-NMR (100 MHz, DMSO-d6): C-4), 162.7 (C-7), 156.8 (C-9), 148.8 (C-
4′), 147.8 (C-3′), 145.3 (C-2), 137.7 (C-3), 126.9 (C-5), 123.0 (C-1′), 121.8 (C-6′), 116.0 (C5′), 115.1 (C-6), 114.6 (C-10), 112.1 (C-2′), 102.5 (C-8), 56.2 (OCH3-3′).
Fustin (6): white powder. 1H NMR (400 MHz, CD3OD); 7.71 (1H, d, J = 8.7, H-5), 6.98
(1H, d, J = 1.1 Hz, H-2′), 6.86 (1H, dd, J = 1.1, 8.1 Hz, H-6′), 6.80 (1H, d, J = 8.1 Hz, H-5′),
6.52 (1H, dd, J = 1.9, 8.7 Hz, H-6), 6.32 (1H, d, J = 1.9 Hz, H-8), 4.93 (1H, d, J = 11.8 Hz, H2), 4.48 (1H, d, J = 11.8 Hz, H-3);
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C-NMR (100 MHz, CD3OD): C-4), 165.4 (C-
7), 163.7 (C-9), 145.7 (C-4′), 144.9 (C-3′), 128.7 (C-5, 1′), 119.6 (C-6′), 114.7 (C-5′), 114.5
(C-2′), 112.0 (C-10), 110.7 (C-6), 102.3 (C-8), 84.2 (C-2), 73.1 (C-3).

Garbanzol (7): white needles; 1H NMR (400 MHz, CD3OD): 7.72 (1H, d, J = 8.7), 7.36 (2H,
d, J = 8.5 Hz, H-2′, 6′), 6.84 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.52 (1H, dd, J = 2.2, 8.7 Hz, H-6),
6.33 (1H, d, J = 2.2 Hz, H-8), 5.00 (1H, d, J = 11.9 Hz, H-2), 4.51 (1H, d, J = 11.9 Hz, H-3);
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C-NMR (100 MHz, CD3OD): C-4), 165.4 (C-7), 163.7 (C-9), 157.7 (C-4′), 129.0
(C-2′, 6′), 128.8 (C-5), 128.1 (C-1′), 114.8 (C-3′, 5′), 112.0 (C-10), 110.8 (C-6), 102.4 (C-8),
84.0 (C-2), 73.1 (C-3).
1.3 DPPH free-radical scavenging activity
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Antioxidant activity of all isolated compounds were determined using DPPH radical
scavenging assays previously described by Bonina et al. (Bonina et al. 2000). Gallic acid was
used as a standard compound. Solutions of compounds were prepared at various concentration
in methanol and 0.1 mL of these solutions were added to 0.9 mL of DPPH solution (DPPH
0.00125 mg in MeOH 50 ml). After 30 minutes of incubation at room temperature in the dark,
the absorbance was measured at 517 nm. All experiments were run in triplicate. Scavenging
activity was calculated by the following equation and shown as IC50 which is the
concentration of compound required to scavenge 50% DPPH radical.
% Radical scavenging = [1- Asample/ ADPPH] x 100
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