Supporting information
The extraction and isolation processes of MHBFC
9 kg cut crude drugs of Millettia pulchra (Benth.) Kurz var. Laxior (Dunn) Z. Wei
(Papilionaceae) was extracted with 72 L of 60% EtOH by microwave-assisted extraction for
20 min. x 2 times at 55°C as reported [1]. The filtered solvents were combined and
concentrated under reduced pressure to yield 1.5 L of crude extract. After washing with 3 L of
petroleum ether (PE) to remove pigments，the crude extract was further partitioned with 4.5 L
of ethyl acetate (AcOEt). The AcOEt layer was concentrated and dried in vaccum to obtain a
dry residue (100 g). This dry residue was fractionated by column chromatography (CC) using
a silica gel H-packed column (10 cm × 120 cm) with a gradient system of PE/AcOEt (98:2)
to PE/AcOEt (0:100) to obtain 269 fractions Fr.Gp.1 (F1- F269) (one fraction per 0.5 L of
eluant). Then, all fractions were combined according to thinlayer chromatography (TLC)
analysis and afforded fractions A–M. Antioxidative activities of all prefractionational
fractions were evaluated to sieve the further purified constituents [2]. Fr.H indicated strong
antioxidative effect, so Fr.H (5.5 g) was further fractioned by CC (275 g, ф5 cm x 60 cm),
eluted with a gradient of PE-AcOEt (96:4, 92:8, 88:12) to divided into frs.H1-H3. Fr.H1 (2.8 g)
was subjected to another CC fractionation (silica gel-H, ф2.5 cm x 60 cm), eluted with a
gradient of PE-AcOEt (100:0, 98:2, 92:8) to afford frs.H1a-H1c. Fr.H1a (700 mg) was further
re-crystallized by CHCl3/MeOH (50:50). Finally, 12 mg of MHBFC (Rf=0.59 with
PE-EtOAc=4:1 as developer) was obtained by preparative liquid chromatography purification
using an ODS-BP column (10 cm × 250 cm) at 3 mL/min with acetonitrile/0.2% acetic acid
(85:15) as mobile phase, λ 238 nm, and tR 34.10-35.20 minute.
References
1.
Jian, J., Lin, X., Huang, R., et al. (2009). The study on total flavonoids extraction of
Yulangsan. Lishizhen Med. Materia Med. Res., 8: 1998-1999
1
2.
Jian J., Li, Y., Jiang, W., et al. (2009). The effects of two chalcone monomers from
Yulangsan on scavenging free radicals. Chin. J. Gerontology, 18: 2353-2354.
Table 1S 1H- and 13C- NMR spectroscopic data of MHBFC (CDCl3, 500 MHz)
δC
Position
1
141.8
10
2
7.57(overlapped) 128.6
11
3
7.57(overlapped) 130.6
12
117.0
4
7.57(overlapped) 128.6
13
154.8
5
7.57(overlapped) 130.6
14
8.2(d)
145.7
6
7.57(overlapped) 128.6
15
7.2(d)
110.0
7
158.2
16
104.2
17
8.17(s)
150.0
174.2
18
3.95(s)
60.2
Position
8
9
δH
7.77(s)
2
δH
δC
122.0
8.17(s)
128.4
119.8
Table 2S Bond lengths [nm] and angles [°] of MHBFC.
Bond
Bond length
Bond
Bond length
C(1)-C(6)
1.392(2)
C(18)-H(18A)
0.9800
C(11)-C(10)-C(9)
116.80(12)
C(1)-C(2)
1.395(2)
C(18)-H(18B)
0.9800
C(12)-C(11)-C(10)
122.56(13)
C(1)-C(7)
1.4845(19)
C(18)-H(18C)
0.9800
C(12)-C(11)-H(11)
118.7
C(2)-C(3)
1.383(2)
O(1)-H(1)
0.8400
C(10)-C(11)-H(11)
118.7
C(2)-H(2)
0.9500
Bond
C(11)-C(12)-C(13)
116.21(13)
C(3)-C(4)
1.389(2)
C(6)-C(1)-C(2) 118.99(13)
C(11)-C(12)-H(12)
121.9
C(3)-H(3)
0.9500
C(6)-C(1)-C(7) 119.40(13)
C(13)-C(12)-H(12)
121.9
C(4)-C(5)
1.382(2)
C(2)-C(1)-C(7) 121.60(12)
O(3)-C(13)-C(12)
126.00(12)
C(4)-H(4)
0.9500
C(3)-C(2)-C(1) 120.53(13)
O(3)-C(13)-C(16)
110.09(13)
C(5)-C(6)
1.387(2)
C(3)-C(2)-H(2) 119.7
C(12)-C(13)-C(16)
123.91(13)
C(5)-H(5)
0.9500
C(1)-C(2)-H(2) 119.7
C(15)-C(14)-O(3)
112.71(13)
C(6)-H(6)
0.9500
C(2)-C(3)-C(4) 120.11(15)
C(15)-C(14)-H(14)
123.6
C(7)-O(1)
1.3035(17)
C(2)-C(3)-H(3) 119.9
O(3)-C(14)-H(14)
123.6
C(7)-C(8)
1.3833(19)
C(4)-C(3)-H(3) 119.9
C(14)-C(15)-C(16)
105.78(13)
C(8)-C(9)
1.4195(19)
C(5)-C(4)-C(3) 119.66(14)
C(14)-C(15)-H(15)
127.1
C(8)-H(8)
0.9500
C(5)-C(4)-H(4) 120.2
C(16)-C(15)-H(15)
C(9)-O(2)
1.2776(17)
C(3)-C(4)-H(4) 120.2
C(13)-C(16)-C(17)
118.85(13)
C(9)-C(10)
1.4838(19)
C(4)-C(5)-C(6) 120.43(14)
C(13)-C(16)-C(15)
105.93(12)
C(10)-C(17)
1.3987(19)
C(4)-C(5)-H(5) 119.8
C(17)-C(16)-C(15)
135.21(13)
C(10)-C(11)
1.4105(18)
C(6)-C(5)-H(5) 119.8
O(4)-C(17)-C(16)
118.54(12)
C(11)-C(12)
1.380(2)
C(5)-C(6)-C(1) 120.26(14)
O(4)-C(17)-C(10)
122.42(12)
C(11)-H(11)
0.9500
C(5)-C(6)-H(6) 119.9
C(16)-C(17)-C(10)
119.03(12)
C(12)-C(13)
1.381(2)
C(1)-C(6)-H(6) 119.9
O(4)-C(18)-H(18A)
109.5
C(12)-H(12)
0.9500
O(1)-C(7)-C(8) 121.65(13)
O(4)-C(18)-H(18B)
109.5
C(13)-O(3)
1.3671(17)
O(1)-C(7)-C(1) 115.55(12)
H(18A)-C(18)-H(18B) 109.5
C(13)-C(16)
1.3949(19)
C(8)-C(7)-C(1) 122.80(12)
O(4)-C(18)-H(18C)
C(14)-C(15)
1.338(2)
C(7)-C(8)-C(9) 120.37(13)
H(18A)-C(18)-H(18C) 109.5
C(14)-O(3)
1.3779(19)
C(7)-C(8)-H(8) 119.8
H(18B)-C(18)-H(18C) 109.5
C(14)-H(14)
0.9500
C(9)-C(8)-H(8) 119.8
C(7)-O(1)-H(1)
C(15)-C(16)
1.437(2)
O(2)-C(9)-C(8)
C(15)-H(15)
0.9500
O(2)-C(9)-C(10) 117.02(12)
C(16)-C(17)
1.3972(19)
C(8)-C(9)-C(10) 122.21(12)
C(17)-O(4)
1.3784(16)
C(17)-C(10)-C(11)119.42(13)
C(18)-O(4)
1.4219(19)
C(17)-C(10)-C(9) 123.74(12)
Bond angle
120.69(13)
Bond
C(13)-O(3)-C(14)
C(17)-O(4)-C(18)
3
Bond angle
127.1
109.5
109.5
105.49(11)
114.70(11)
MKL(9kg)
extracted by microwave-assisted method,
using 72L 60% EtOH for 2 times
Crude extract (1.5L)
washed with 3L PE to remove pigments,
then partitioned with 4.5 L AcOEt
Dry residue (100 g)
separated by using a silica gel H-packed column
(10 cm × 120 cm) with a gradient system of
PE/ AcOEt (100:0) to PE-AcOEt (60:40)
269 Fractions
combined according to TLC analysis
Fr. A-M
sieved by guiding with antioxidative activity
Fr. H
F1
F2
separated by using a silica gel H
column (5 cm × 60 cm) with
PE/AcOEt (96 : 4)
F3
separated by using a silica gel H column
(2.5 cm × 60 cm) with PE/ AcOEt (92: 8)
Crystal
recrystalized by CHCl3 / MeOH (50:50)
purified by PHPLC with acetonitrile/ 0.2% acetic acid (85:15) as
mobile phase at 3 mL/ min by monitoring at 238 nm ,
tR34.10-35.20 min.
MHBFC (12mg)
Figure 1S The extraction and isolation processes of MHBFC.
Figure 2S The chemical structure of MHBFC.
Figure 3S Molecular stereo configuration and crystal packing of MHBFC.