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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- Optical rotations were measured on a perkin-Ekmer Model
retic, antiarrhythmic, antiasthmatic, antiulcerative, anti-HBV 341 polarimeter. IR spectra were recored on a Nicolet NEXUS
and antineoplastic effects and is used to treat jaundice, 670 FT-IR spectrometer over the range of 400–4000 cm− 1. UV
leukorrhea, carbuncles, pyogenic infections of the skin, scabies, spectra were obtained on a Shimadzu UV-260 spectrometer.
enteritis as well as dysentery [1,3]. A phytochemical investiga- NMR spectra were conducted on a Varian Mercury-300BB or
tion on the constituents of S. flavescens was carried out and led Varian Mercury-400BB NMR spectrometer (δ in ppm rel. to
to the isolation of a new quinolizidine alkaloid, 9α-hydroxy- TMS, J in Hz). EI-MS data were recorded on a HP 5988A-GC/
7,11-dehydromatrine (1) and a rare 1,4-diazaindan-type MS instrument, in m/z (rel.%). HR-EI-MS and HR-ESI-MS
alkaloid, flavascensine (17), together with 15 known quinoli- determinations were run on a Bruker APEX II mass spectrom-
zidine alkaloids (2–16) (Fig. 1). In this paper, we describe the eter. Sephadex LH-20 (Amersham Pharmacia Biotech), RP-18
isolation and structural elucidation of the new compounds. 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
⁎ Corresponding authors. State Key Laboratory of Applied Organic compounds. Spots on the plates were observed under UV
Chemistry, College of Chemistry and Chemical Engineering, Lanzhou
University, Lanzhou 730000, PR China. Tel.: +86 931 8914178; fax: +86
light and visualized by spraying them with 5% H2SO4 in
931 8915557. C2H5OH (v/v), followed by heating or spraying with Dragen-
E-mail address: yuancs@lzu.edu.cn (C.-S. Yuan). 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) 524–527 525

Fig. 1. Structures of compounds 1–17.

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

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],
Fig. 2. Partial structures from two-dimensional NMR for 17.
(−)-9α-hydroxysophocarpine (13) [9], (−)-9α-hydroxyso-
phoramine (14) [5], (−)-14β-hydroxymatrine (15) [13], and
13
27.64 (q), 27.93 (q), 29.43 (q) and 8.49 (q) in the C NMR (−)-N-methylcytisine (16) [14].
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)
Acknowledgements
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
This work was supported by the National Basic Research
the chemical shifts, splitting patterns and coupling constants.
Program (973 Program) of China (2007CB108903) and the
Besides five CH3 groups, the another seven carbon signals
13 National Natural Science Foundation of China (No. 20621091
were observed in the C NMR (DEPT) spectrum (Table 2) as
QT Program & J0730425).
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 References
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