Fitoterapia 81 (2010) 524–527

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Alkaloids from Sophora flavescens Aition

Xiu-Jin Liu a,b, Mei-Ai Cao a, Wen-Hai Li a, Cheng-Shuo Shen a,
Shi-Qiang Yan a,⁎, Cheng-Shan Yuan a,⁎
a
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China
b
Fujian Fukang Pharmaceutical Co. LTD., Fuzhou 350002, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Although the quinolizidine alkaloids and flavonoids, the main active components of the
Received 28 October 2009 traditional Chinese medicine Sophora flavescens, have been largely investigated, a new matrine
Accepted in revised form 29 December 2009 alkaloid derivative 9α-hydroxy-7,11-dehydromatrine (1) and a rare 1,4-diazaindan-type
Available online 15 January 2010
alkaloid flavascensine (17), together with 15 known alkaloids, were isolated from S. flavescens.
The structures were established on the basis of spectroscopic techniques.
Keywords: © 2010 Elsevier B.V. All rights reserved.
Sophora flavescens
Leguminosae
Alkaloids
9α-hydroxy-7,11-dehydromatrine
Flavascensine

1. Introduction 2. Experimental

Sophora flavescens, also referred to as ‘Kushen’, is an 2.1. Genernal

important source of alkaloids and is distributed throughout
China [1,2]. S. flavescens has antibacterial, antipyrotic, antipy-
retic, antiarrhythmic, antiasthmatic, antiulcerative, anti-HBV
and antineoplastic effects and is used to treat jaundice,
leukorrhea, carbuncles, pyogenic infections of the skin, scabies,
enteritis as well as dysentery [1,3]. A phytochemical investiga-
tion on the constituents of S. flavescens was carried out and led
to the isolation of a new quinolizidine alkaloid, 9α-hydroxy-
7,11-dehydromatrine (1) and a rare 1,4-diazaindan-type
alkaloid, flavascensine (17), together with 15 known quinoli-
zidine alkaloids (2–16) (Fig. 1). In this paper, we describe the
isolation and structural elucidation of the new compounds.
⁎ Corresponding authors. State Key Laboratory of Applied Organic
Chemistry, College of Chemistry and Chemical Engineering, Lanzhou
University, Lanzhou 730000, PR China. Tel.: +86 931 8914178; fax: +86
931 8915557.
E-mail address: yuancs@lzu.edu.cn (C.-S. Yuan).
Optical rotations were measured on a perkin-Ekmer Model
341 polarimeter. IR spectra were recored on a Nicolet NEXUS
670 FT-IR spectrometer over the range of 400–4000 cm− 1. UV
spectra were obtained on a Shimadzu UV-260 spectrometer.
NMR spectra were conducted on a Varian Mercury-300BB or
Varian Mercury-400BB NMR spectrometer (δ in ppm rel. to
TMS, J in Hz). EI-MS data were recorded on a HP 5988A-GC/
MS instrument, in m/z (rel.%). HR-EI-MS and HR-ESI-MS
determinations were run on a Bruker APEX II mass spectrom-
eter. Sephadex LH-20 (Amersham Pharmacia Biotech), RP-18
silica-gel (150–200 mesh, Merck), MCI gel CHP20P (75–
150 μm, Mitsubishi Chemical), silica-gel (200–300 mesh,
Qingdao Marine Chemical Factory) were used for Column
chromatography (CC). Analytical and preparative TLC was
performed on silica-gel plates (GF254 10–40 μm, Qingdao
Marine Chemical Factory). Analytical TLC was provided to
follow the separation and check the purity of isolated
compounds. Spots on the plates were observed under UV
light and visualized by spraying them with 5% H2SO4 in
C2H5OH (v/v), followed by heating or spraying with Dragen-
dorff's reagent directly.

0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2010.01.008
 X.-J. Liu et al. / Fitoterapia 81 (2010) 524–527
Fig. 1. Structures of compounds 1–17.
2.2. Plant materials
The dry roots of S. flavescens Aition were purchased from
Focitang Medical Company, Lanzhou, Gansu province, P. R.
China, in Apr. 2005, and identified by Prof. Pei-Jun Yu, the
Second Affiliated Hospital of Lanzhou University. The voucher
specimen (No. 200504SF) is deposited in the Institute of
Organic Chemistry, Lanzhou University.
2.3. Extraction and isolation
The dried, chipped roots of S. flavescens (10.0 kg) were
extracted with acetone/H2O (7:3, 10 l×7 d×3) at room temper-
ature. The combined extracts were evaporated in vacuo to afford
a black residue (1500 g). The residue was suspended in warm
water (1.5 l) and then extracted successively with ethyl acetate
(1.5 l×3) and n-butanol (1.5 l×3), and concentrated to give
residue A (185 g) and B (580 g), respectively. The latter was
resolved in warm water (1.0 l), acidified with 1 mol/l HCl to pH
4–5, extracted with CHCl3 (1.0 l×3). The aqueous layer was
neutralized with 1 mol/l NaOH to pH 9–10 and extracted with
CHCl3 (1.0 l×3) once again and concentrated in vacuo to obtain
the crude base (55.0 g).
The crude base (55.0 g) was subjected to the silica-gel
chromatograph column (CC) (800 g silica-gel; CHCl3/CH3OH/
28% NH4OH, 50:1:0.1→2:1:0.1) to afford six fractions (Fr. 1–6).
Fr. 2 was chromatographed on silica-gel CC using petroleum
ether/Me2CO/28% NH4OH (20:1:0.2→1:1:0.2) to give 17 (4 mg),
2 (N1 g) and 6 (N1 g). Fr. 3 was separated by silica-gel CC eluting
525
with CHCl3/Me2CO/28% NH4OH (50:1:0.2→2:1:0.2) to obtain 11
(54 mg), 15 (27 mg) and crude 8. The crude 8 was further
purified on a Sephadex LH-20 column (CHCl3/MeOH, 2:1) to give
pure 8 (13 mg). Fr. 4 was subjected to silica-gel CC with
petroleum ether/EtOAc/CH3OH (12:12:1→1:1:1) to furnish 4
subfractions (Fr. 4 A–D). Fr. 4 A was rechromatographed on Si-gel
CC eluenting with CHCl3/EtOAc/28% NH4OH (10:1:0.2→1:1:0.2)
and preparative TLC (CHCl3/Me2CO/28% NH4OH (2:1:0.2)) to
afford 7 (18 mg). Fr. 4 B was purified on silica-gel CC with CHCl3/
Me2CO/28% NH4OH (10:1:0.2→0:1:0.2) to provide 3 (6 mg) and
4 (26 mg). Fr. 4 C was subjected to silica-gel CC (EtOAc/Me2CO/
CH3OH/28% NH4OH (50:10:1:0.1→1:1:1:0.1)) to yield 5 (9 mg),
14 (23 mg), 16 (47 mg) and crude 1. The pure 1 (5 mg) was
obtained after further purification on a Sephadex LH-20 column
(CHCl3/MeOH, 2:1) and submitted to preparative TLC (CHCl3/
EtOAc/28% NH4OH (1:1:0.1) and then subjected to RP-18 silica-
gel (150–200 mesh) eluting with H2O/MeOH (5:1 →1:1). Fr. 4 D
was re-separated by silica-gel CC (EtOAc/MeOH, 20:1 →0:1) and
further purified on MCI gel CHP20P (H2O/MeOH, 5:1 →1:3) to
give 12 (31 mg) and 13 (17 mg). Portion of Fr. 5 was purified on
Sephadex LH-20 column (CHCl3/MeOH, 2:1) to afford 9 (87 mg).
10 (N1 g) was obtained after recrystallization of Fr. 6 from
Me2CO.
2.4. Spectral data of compounds
(−)-9α-hydroxy-7,11-didehydromatrine (1): colorless oil.
[α]20
D : −52° c=0.6, CH3OH . UV (CH3OH, nm): λ (log ε)=238.2
(4.15). IR (KBr): 3406, 2932, 2863, 2804, 2752, 1631, 1437, 1398,
526 X.-J. Liu et al. / Fitoterapia 81 (2010) 524–527

1 13
1247, 1179, 1139, 1056 and 730. H and C NMR spectral data: see Table 2
1 13 1 13

Table 1. HR-EI-MS: 261.1595 ([M–H]+, C15H21N2O+ H, CNMR and HMBC Data for 17 ( H: 300 MHz, C: 75 MHz; DMSO-d6; δ in
2 ; calc.
ppm, J in Hz).
261.1598), 243.1491 ([M–H2O–H]+, C15H19N2O+), 218.1421
([M–C3H7–H]+, C12H14N2O+ 2 ), 204.1267 ([M–C3H5O–H] ,
+
No δ(H) δ(C) (DEPT) HMBC (H to C)
C12H16N2O+), 190.1238 ([M–C4H8O]+, C11H14N2O+).
20
2 59.75 (s)
Flavascensine (17): faint yellow powder. [α]D : −28° 3 1.54 (br t, 11.1) 43.54 (t) C-2, C-3a, C-8, C-9
(c = 0.48, CH3OH). UV (CH3OH, nm): λ (log ε) = 295.1 (4.02). 2.01 (dd, 11.1, 6.9) C-3a, C-7a, C-9
IR (KBr): 3422, 2967, 2929, 2363, 1643, 1562, 1409, 1222, 5 57.70 (s)
1 13
6 196.47 (s)
1170 and 1101. H and C NMR spectral data: see Table 2. HR-
7 92.96 (s)
ESI-MS: 209.1648 ([M + H]+, C12H21N2O+, calc. 209.1648). 8 1.28 (s) 29.43 (q) C-2, C-3, C-9
9 1.19 (s) 27.93 (q) C-2, C-3, C-8
3. Results and discussion 10 1.09 (s) 27.64 (q) C-5, C-6, C-11
11 1.04 (s) 23.30 (q) C-5, C-6, C-10
12 1.44 (s) 8.49 (q) C-6, C-7, C-7a
Compound 1, a colorless oil, showed a positive reaction
3a 4.10 (dd 11.1, 6.9) 51.96 (d) C-3, C-7a
with Dragendorff's reagent, indicating it to be an alkaloid. Its 7a 164.48 (s)
IR spectrum (KBr) exhibited strong absorption bands for OH
group (3406 cm− 1), a lactam-CO (1631 cm− 1) and a trans-
quinolizidine moiety (2863, 2804 and 2752 cm− 1) [4]. The methine proton bearing a hydroxyl group, and the resonances
UV spectrum showed an absorption band at 238.2 nm log at δ(H) 2.86 (1H, dd, J = 12.3, 5.1Hz) and 3.13 (1H, dd,
ε = 4.15 , supporting the presence of a double bond conju- J = 10.5, 3.0 Hz) due to the Ha-8 and Hb-10 were respectively
gated to a lone pair of nitrogen in the molecule [5]. The HR-EI- downfielded for 0.84 and 0.22 ppm, compared to those of 7
1 1
MS displayed the quasi-molecular [M–H]+ peak at m/z [5]. Analysis the H– H COSY and HSQC spectra correlations
261.1595, suggesting the molecular formula C15H22N2O2 with indicated the presence of –CH2–CH(O)–CH2–, which was well
6° of unsaturation. Meanwhile, HR-EI-MS exhibited frag- agreed with the spin system given by the 1D-TOCSY
13
ment ion at m/z 243.1491, corresponding to [M–H2O–H]+, experiments. Its C NMR and DEPT spectral data (Table 1)
inferring the presence of a hydroxyl group in the molecule. The exhibited the presence of 15 carbons including nine methy-
fragment ion peak at m/z 218.1421, due to [M–C3H7–H]+, is lenes, three methines and one lactam-CO quaternary carbon
typical of the matrinoid skeleton [6]. as well as two olefinic quaternary carbons. Moreover, a trans-
1
The H NMR spectrum (CDCl3) of 1 (Table 1) showed the quinolizidine absorption bands were observed in the IR
characteristic signals of a matrine-type alkaloid at δ(H) 4.31 spectrum implying that the double bond should be only
(1H, dd, J = 12.0, 2.4 Hz, H α-17) and 3.17 (1H, br t, located at the C-7 and C-11 [4]. In addition, the C-9 methylene
J = 12.0 Hz, Hβ-17) [5]. Moreover, the 1H NMR spectrum of signal at δ(C) 24.54 in 7 was replaced by a oxygenated
1 was very similar to that of leontalbinine (compound 7), methine carbon δ(C) 67.10 in 1, and the signals at δ(C) 38.49
except for an additional signal at δ(H) 3.74 (1H, dddd, (C-8, CH2) and 63.94 (C-10, CH2) were downfielded 12.76 and
J = 12.3, 10.5, 5.1, 3.0 Hz, H-9) which was assigned to a 6.86 ppm, respectively, which suggested the hydroxy group
was located at C-9. The conclusion was further confirmed by
the correlations in the HMBC experiment (Table 1): H-4 to C-
Table 1
1 13 1 13 17; H-6 to C-10 and C-11; H-8 to C-6, C-10 and C-11; H-9 to C-
H, C NMR and HMBC Data for 1 ( H: 300 MHz, C:75 MHz; CDCl3; δ in ppm,
J in Hz). 10; H-10 to C-2; H-12 to C-7; H-13 to C-11 and C-15; H-14 to
C-12; H-17 to C-6, C-11 and C-15.
No δ(H) δ(C) (DEPT) HMBC (H to C) In addition, the IR spectrum exhibited a trans-quinolizi-
2 2.81 (d, 12.3) 55.15 (t) C-3, C-4 dine moiety (2863, 2804 and 2752 cm− 1), so H-6 was in α-
2.10–2.18 (m) orientation [4]. The large coupling constants observed from
3 1.46-1.54 (m) 21.41 (t) C-5 H-9 to both Hβ-8 (J = 12.3 Hz) and Hβ-10 (J = 10.5 Hz)
1.62–1.66 (m)
indicated that the H-9 was in β-orientation [5]. The NOE
4 1.75–1.78 (m) 26.66 (t) C-2, C-6
1.66–1.69 (m) C-3, C-5, C-17
experiment was performed to determine the configuration of
5 1.89–1.94 (m) 31.80 (d) C-6, C-7, C-10 H-5. The signal of H-5 was enhanced by 4.22% when
6 2.28 (d, 4.8) 60.63 (d) C-7, C-10, C-11, C-17 irradiation at Hα-17, showing that H-5 had the same α-
7 111.21 (s) orientation. From the above-mentioned information, the
8 1.87 (t, 12.3) 38.49 (t) C-6, C-7, C-9, C-10, C-11
structure of 1 was unambiguously elucidated as 9α-hy-
2.86 (dd, 12.3, 5.1)
droxy-7,11-dehydromatrine.
9 3.74 (dddd, 12.3, 10.5, 5.1, 3.0) 67.10 (d) C-10
10 2.13 (t, 10.5) 63.94 (t) C-2, C-6, C-8, C-9
Compound 17 was isolated as a faint yellow powder
−1
3.13 (dd, 10.5, 3.0) ([α]20
D : − 28°, c = 0.48, CH3OH). N–H (3422 cm ), car-
11 130.62 (s) bonyl (1643 cm− 1) as well as C=C (1562 cm− 1) absorp-
12 2.66 (dt, 11.1, 5.4) 24.70 (t) C-7, C-11, C-13, C-14 tions were observed in the IR spectrum (KBr). HR-ESI-MS
2.32 (dd, 11.1, 3.9)
showed the [M + H]+ ion peak at m/z 209.1648, consistent
13 1.78–1.82 (m) 19.52 (t) C-12, C-14, C-15
1.69–1.75 (m) C-11, C-12, C-14, C-15 with the molecular formula C 12 H 20 N 2 O with 4° of
14 2.48 (br t, 6.0) 32.67 (t) C-12, C-13, C-15 unsaturation.
1
15 168.68 (s) The HNMR spectrum (DMSO-d6) of 17 (Table 2) exhibited
17 4.31 (dd, 12.0, 2.4) 40.65 (t) C-5, C-6, C-11, C-15 five methyl singlets at δ(H) 1.04, 1.09, 1.19, 1.28 and 1.44,
3.17 (br t, 12.0) C-5, C-6, C-15
which were in agreement with resonances at δ(C) 23.30 (q),
 X.-J. Liu et al. / Fitoterapia 81 (2010) 524–527
Fig. 2. Partial structures from two-dimensional NMR for 17.
13
27.64 (q), 27.93 (q), 29.43 (q) and 8.49 (q) in the C NMR
spectrum. A methine δ(H) 4.10 (1H, dd, J = 11.1, 6.9 Hz)
bearing a nitrogen atom and the residual two protons δ(H)
1.54 (1H, br t, J = 11.1 Hz) and 2.01 (1H, dd, J = 11.1, 6.9 Hz)
also could be found in the 1HNMR spectrum on the basis of
the chemical shifts, splitting patterns and coupling constants.
Besides five CH3 groups, the another seven carbon signals
13
were observed in the C NMR (DEPT) spectrum (Table 2) as
follows: a CH2 signal δ(C) 43.54, a CH linked to a nitrogen
atom δ(C) 51.96, five quaternary C-atoms including two C-
atoms adjacent to nitrogen at δ(C) 57.70 and 59.75, an
olefinic C-atom linked to a nitrogen at δ(C) 164.48 as well as
an α,β-unsaturated carbonyl δ(C) 196.47. Additionally, the
position of the methyl at δ(C) 8.49 was presumed to be
located at the α-C of the α,β-unsaturated carbonyl group
considering the pronounced upfield chemical shift, which
was further proved by the key HMBC correlations: H-12 to C-
6, C-7, and C-7a.
Carefully analysis of HMBC correlations (Table 2) resulted
in two substructures A and B (Fig. 2). Substructure A was
assembled by means of HMBC correlations H-10 to C-5, C-6
and C-11; H-11 to C-5, C-6 and C-10; and H-12 to C-6, C-7 and
C-7a. Substructure B was assembled on the basis of HMBC
correlations H-3a to C-3; Ha-3 to C-3a and C-9; Hb-3 to C-2, C-
3a, C-8 and C-9, H-8 to C-2, C-3 and C-9; and H-9 to C-2, C-3
and C-8. The key correlations in the HMBC spectrum H-3a to C-
7a as well as Hb-3 to C-7a indicated that C-3a was connected
with C-7a. In addition, the chemical shifts of C-2 and C-3a
showed that the other nitrogen was linked to each C-atom.
Consequently, the two substructures could be assembled into
a structure undoubtedly as 2,2,5,5,7-pentamethyl-
1,2,3,3a,4,5- hexahydro-6H-pyrrolo[3,2-b]pyridin-6-one.
In the NOE difference spectrum, when irradiation at H-3a
caused the enhancement for Ha-3, H-9 and H-11 by 5.39%,
3.11% and 7.04%, respectively, indicating that H-3a, Ha-3, H-9
and H-11 were laid on the same side (α) of the molecule.
Similarly, the signal for H-8 was enhanced by 4.52% upon
irradiation of Hb-3, suggested the Hb-3 and H-8 were β-
orientation. Thus, compound 17 was deduced as (3aα)-
2,2,5,5,7- pentamethyl-1,2,3,3a,4,5- hexahydro-6H-pyrrolo
[3,2-b]pyridin-6-one, and named flavascensine. It is a rare
1,4-diazaindan-type alkaloid and there is only one analog
which has been reported in the previous study [7].
527
The structures of the known compounds 2–16 were
determined by comparison of their physical and spectral
data with those published in the literatures. They were
identified as (+)-matrine (2) [8], (−)-sophoridine (3) [9],
(+)-isomatrine (4) [8], (+)-allomatrine (5) [8], (−)-sopho-
carpine (6) [9], (+)-7,11-dehydromatrine (leontalbinine) (7)
[5], (+)-sophoramine (8) [8], (+)-oxymatrine (9) [10], (+)-
oxysophocarpine (10) [11], (+)-5α-hydroxymatrine [(+)-
sophoranol] (11) [12], (+)-9α-hydroxymatrine (12) [6],
(−)-9α-hydroxysophocarpine (13) [9], (−)-9α-hydroxyso-
phoramine (14) [5], (−)-14β-hydroxymatrine (15) [13], and
(−)-N-methylcytisine (16) [14].
Acknowledgements
This work was supported by the National Basic Research
Program (973 Program) of China (2007CB108903) and the
National Natural Science Foundation of China (No. 20621091
QT Program & J0730425).
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