Flavonoids in the poisonous plant oxytropis falcata

Article abstract of DOI:10.1021/np100339u

Three new flavonoids, oxytropisoflavans A (1) and B (2) and (6aR,11aR)-3,8-dihydroxy-9,10-dimethoxypterocarpan (3), together with 30 known flavonoids (4-33), were isolated from the aerial parts and roots of Oxytropis falcata. The absolute configurations of 3 and C-3 in 1 and 2 were deduced by circular dichroism. The structure of flavonoid 2 was confirmed by single-crystal X-ray diffraction analysis and that of flavonoid 3 by total synthesis of its racemate. Oxytropisoflavan A (1) is an unprecedented chalcan-isoflavan biflavonoid, whereas oxytropisoflavan B (2) possesses a rare modified A-ring. Pterocarpan 3 has good radical-scavenging activity in the 1,1-diphenyl-2- picrylhydrazyl (DPPH) assay.

Full text of DOI:10.1021/np100339u

1
398
J. Nat. Prod. 2010, 73, 1398–1403
Flavonoids in the Poisonous Plant Oxytropis falcata
†
,‡
†,‡
,†,‡
Wen-Hao Chen, Rui Wang, and Yan-Ping Shi*
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu ProVince, Lanzhou Institute
of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China, and State Key Laboratory of Applied
Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou UniVersity, Lanzhou 730000, People’s Republic of China
ReceiVed May 19, 2010
Three new flavonoids, oxytropisoflavans A (1) and B (2) and (6aR,11aR)-3,8-dihydroxy-9,10-dimethoxypterocarpan
(
3), together with 30 known flavonoids (4-33), were isolated from the aerial parts and roots of Oxytropis falcata. The
absolute configurations of 3 and C-3 in 1 and 2 were deduced by circular dichroism. The structure of flavonoid 2 was
confirmed by single-crystal X-ray diffraction analysis and that of flavonoid 3 by total synthesis of its racemate.
Oxytropisoflavan A (1) is an unprecedented chalcan-isoflavan biflavonoid, whereas oxytropisoflavan B (2) possesses a
rare modified A-ring. Pterocarpan 3 has good radical-scavenging activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH)
assay.
Flavonoids are a group of ubiquitous and diverse molecules
produced via the phenylpropanoid pathway in higher plants, and
about 2% of all the photosynthesized carbon is converted into
1
flavonoids. They play a variety of roles in plants including
protection against UV damage and phytopathogens (e.g., phytoal-
exins in legumes), acting as pigments or copigments in influencing
flower color, modulating auxin distribution, and as signal molecules
2
to symbiotic microbes.
Flavonoids often possess antioxidant activity, and the potential
health benefits of fruit, vegetables, green tea, and red wine might
3
-5
partly be because of this property.
which are limited primarily to the Leguminosae, exhibit estrogenic
In addition, the isoflavonoids,
6
,7
and anticancer activity and, in common with the flavonoids, are
also receiving considerable attention as health-promoting “nutra-
ceuticals”.
Plants of the genera Astragalus and Oxytropis (both Leguminosae)
containing the indolizidine alkaloid swainsonine are commonly
known as locoweeds. Consumption of the aerial plant parts of many
locoweed species causes significant economic loss to cattle, sheep,
8
and horses. O. falcata Bunge, known as a toxic plant, is commonly
regarded as “the king of herbal medicine” in Tibetan medicine.
Alcohol extracts of O. falcata mixed with other medicinal materials
(
Qingpenggao) have been patented as a treatment for pain and
9
arthritis. It is a common paradigm in the search for new drugs in
human medicine to investigate constituents of plants that have an
ethnopharmacological basis for their use. Recently, a comprehensive
review described the therapeutic potential of the toxic components
Results and Discussion
The EtOH extracts of the aerial parts and roots of O. falcata
1
0
were suspended in H O and then partitioned successively with
2
of poisonous plants.
petroleum ether, EtOAc, and n-BuOH, respectively. Both petroleum
In this regard, a systematic investigation of the composition of
this poisonous plant has been conducted. We recently reported the
ether-soluble fractions were combined, because they exhibited
similar constituents on TLC. The organic-soluble fractions were
further purified by Si gel CC and PTLC to yield three new
flavonoids, oxytropisoflavans A (1) and B (2) and (6aR,11aR)-3,8-
dihydroxy-9,10-dimethoxypterocarpan (3).
1
1
occurrence of pendulone and a series of 24-hydroxyoleanane-
1
2
13
type triterpenoids and N-benzoylindole analogues from O.
falcata. In this study we describe the isolation and characterization
of three new flavonoids, oxytropisoflavans A (1) and B (2) and
Oxytropisoflavan A (1) was obtained as an optically active, pale
(6aR,11aR)-3,8-dihydroxy-9,10-dimethoxypterocarpan (3), together
32 9
brown oil, with an elemental formula of C32H O determined by
+
with 30 known flavonoids (4-33) from the aerial parts and roots
of O. falcata. The radical-scavenging activity against DPPH and
cytotoxic activity against the human promyelocytic leukemic (HL-
HRESIMS (m/z 583.1944, [M + Na] ). There was evidence of
hydroxy and aromatic ring absorptions in the IR spectrum at 3363,
-1
1510, and 1463 cm .
1
3
6
0) cell line of the new compounds are also described.
The C NMR spectrum of 1 (Table 1) displayed signals for 32
3
2
carbons, including eight sp carbons, 13 quaternary sp carbons with
2
eight linked to an oxygen atom, and 11 tertiary sp carbons. The
H NMR spectrum of 1 (Table 1) showed four aliphatic signals at
*
To whom correspondence should be addressed. Tel: +86-931-4968208.
1
Fax: +86-931-8277088. E-mail: shiyp@licp.cas.cn.
†
δ
4.40 (1H, d, J ) 5.2 Hz, H-R), 4.68 (1H, m, H-ꢀ), 2.73 (1H,
Lanzhou Institute of Chemical Physics.
Lanzhou University.
H
‡
dd, J ) 4.4, 14.0, H-γa), and 2.61 (1H, dd, J ) 7.2, 14.0, H-γb),
1
0.1021/np100339u  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/04/2010

Journal of Natural Products, 2010, Vol. 73, No. 8 1399
Table 1. H (400 MHz) and 13C (100 MHz) NMR Data of 1 in
1
a
6
Acetone-d
no.
2
δ
H
mult (J in Hz)
4.23 dd (3.2, 10.0),
.96 t (10.0)
δ
C
HMBC (HfC)
C-4, 9
69.7 t
3
3
4
3.48 m
2.90 dd (11.2, 15.6),
32.6 d
30.4 t
C-2, 3, 5, 9, 10, 1′
C-4, 7, 9, R
2
.78 dd (4.0, 15.6)
5
6
7
8
9
1
1
2
3
4
5
6
6.85 s
132.9 d
120.1 s
155.2 s
104.1 d
154.1 s
113.5 s
121.1 s
148.4 s
136.3 s
152.0 s
103.7 d
121.7 d
47.7 d
6.33 s
C-6, 7, 9, 10
0
′
′
′
′
′
6.50 d (8.8)
6.83 d (8.8)
4.40 d (5.2)
4.68 m
C-1′, 3′, 4′
C-2′, 4′
′
Figure 2. CD spectra of 1-3 and 6.
R
ꢀ
γ
C-5, 6, 7, 1′′, 2′′, 6′′, γ
C-1′′, 1′′′
74.4 d
41.4 t
2.73 dd (4.4, 14.0),
C-R, ꢀ, 2′′′, 6′′′
moiety seemed to be pentaoxygenated, with four aromatic protons
2
.61 dd (7.2, 14.0)
resonating at δ
H
6.85 (1H, s, H-5), 6.33 (1H, s, H-8), 6.50 (1H, d,
1
2
3
4
5
6
1
2
3
4
5
6
3
4
7
2
′′
120.4 s
155.2 s
102.9 d
156.8 s
106.9 d
130.9 d
130.5 s
130.5 d
115.0 d
155.8 s
115.0 d
130.5 d
60.1 q
J ) 8.8, H-5′), and 6.83 (1H, d, J ) 8.8, H-6′), reminiscent of
′′
15,16
(
3R)-(-)-isomucronulatol (6).
Further supporting evidence came
′′
6.36 d (2.4)
C-1′′, 2′′, 4′′, 5′′
13
from C NMR data, which showed a close resemblance with those
of propterol B (5) and (3R)-(-)-isomucronulatol (6).
′′
′′
6.24 dd (2.4, 8.4)
7.02 d (8.4)
C-1′′, 3′′,
C-R, 2′′, 4′′
′′
Connectivities of the two substructures of compound 1 were
3
′′′
established by analysis of the HMBC spectrum as follows: The J
′′′
7.02 d (8.4)
6.72 d (8.4)
C-γ, 3′′′, 4′′′, 6′′′
C-1′′′, 2′′′, 4′′′, 5′′′
HMBC cross-peaks from H-R to C-2′′/C-6′′/C-5/C-7, as well as
′′′
2
the J HMBC cross-peaks from H-R to C-1′′/C-6, indicated the
′′′
connection between C-6 and C-R. This was corroborated by
the observation of NOESY correlations between H-R and the
aromatic protons H-5 and H-6′′. The positions of the other functional
′′′
6.72 d (8.4)
7.02 d (8.4)
3.78 s
3.81 s
9.63 s
7.94 s
5.13 s
8.44 s
8.01 s
C-1′′′, 3′′′, 4′′′, 6′′′
C-γ, 2′′′, 4′′′, 5′′′
C-3′
C-4′
C-6, 7, 8
C-1′, 2′, 3′
C-R, ꢀ, γ
C-1′′, 2′′, 3′′
C-3′′, 4′′, 5′′
C-3′′′, 4′′′, 5′′′
1
′′′
′-OCH
′-OCH
-OH
′-OH
3
1
1
3
55.4 q
groups were assigned by H- H COSY, HSQC, HMBC, and
NOESY (Figure 1) experiments, which resulted in the assignment
of all proton and carbon signals of 1 (Table 1).
The CD data of compound 1 (Figure 2) exhibited a strong
positive Cotton effect for the L transition region (260-300 nm),
indicating the 3R absolute configuration for 1. Such an allocation
ꢀ
-OH
2
4
4
′′-OH
′′-OH
′′′-OH
a
1
18
b
8.07 s
Assignments were based on DEPT135, 1H- H COSY, HSQC, and
is strongly supported by the same Cotton effect in the CD spectrum
1
6
of (3R)-(-)-isomucronulatol (6) (Figure 2). It should be empha-
sized that although the chiral chalcan unit in compound 1 is chirally
perturbed by the isoflavan unit and may contribute to the lower
HMBC experiments.
characteristic of a substituted propyl group in the chalcan-ꢀ-ol
structure, and five aliphatic signals at δ
1
4
1
18
H
4.23 (1H, dd, J ) 3.2,
wavelength L
a
transition region (220-260 nm), the diagnostic
1
1
0.0, H-2a), 3.96 (1H, t, J ) 10.0, H-2b), 3.48 (1H, m, H-3), 2.90
b
L transition region originates mainly from the isoflavan-type
(
1H, dd, J ) 11.2, 15.6, H-4a), and 2.78 (1H, dd, J ) 4.0, 15.6,
H-4b), typical of an isoflavan nucleus.
chromophore. The absolute configurations of C-R and C-ꢀ remain
to be defined.
1
5,16
These observations and
the molecular mass of 560 suggested that compound 1 could be a
biflavonoid possessing a chalcan-ꢀ-ol unit connected to an isoflavan
The EIMS of compound 1 exhibited prominent fragment peaks
at m/z 450, 423, 302, 107, and 180 (base peak) originated from the
fission of the R,C-1′′-, R,ꢀ-, R,C-6-, and ꢀ,γ-linkage and a
characteristic retro-Diels-Alder-type cleavage of the isoflavan
skeleton (Scheme 1). Consequently, the structure of oxytropisofla-
van A (1) was characterized as (3R)-(-)-isomucronulatol-(6fR)-
propterol B.
1
1
unit. From analysis of the H- H COSY NMR experiment, the
chalcan unit of 1 appeared to be 2′′,4′′,4′′′-trihydroxylated (propterol
1
7
B, 5), as indicated by signals at δ
6
6
H
6.36 (1H, d, J ) 2.4, H-3′′),
.24 (1H, dd, J ) 2.4, 8.4, H-5′′), 7.02 (3H, d, J ) 8.4, H-6′′, 2′′′,
′′′), and 6.72 (2H, d, J ) 8.4, H-3′′′, 5′′′), while the isoflavan
1
1
Figure 1. Key H- H COSY, HMBC, and NOE correlations of 1-3.

1
400 Journal of Natural Products, 2010, Vol. 73, No. 8
Chen et al.
Scheme 1. EIMS Fragmentation Pattern of 1
Table 2. H and 13C NMR Data of 2 and 3a
1
b
c
2
3
no.
δ
H
mult (J in Hz)
δ
C
no.
δ
H
mult (J in Hz)
δ
C
2
3
4
5
6
7
4.29 dd (4.5, 9.9), 3.68 t (9.9)
3.91 m
73.2 t
29.7 d
39.5 t
36.6 t
33.5 t
199.1 s
1
2
3
4
4a
6
7.33 d (8.8)
6.56 dd (2.4, 8.8)
132.3 d
109.8 d
159.1 s
103.1 d
156.9 s
66.0 t
d
2.02 m
1.92 m
d
6.36 d (2.4)
2.67 m, 2.15 br d (17.5)
4.27 dd (5.2, 10.8),
.67 t (10.8)
3
8
9
1
1
2
3
4
5
6
3
4
2
1
5.24 s
108.3 d
175.5 s
65.9 s
120.6 s
149.0 s
136.8 s
152.4 s
104.0 d
122.3 d
60.9 q
6a
6b
7
8
9
10
10a
11a
11b
3.55 m
41.1 d
125.0 s
106.0 d
144.8 s
140.0 s
138.4 s
143.8 s
78.4 d
0
6.64 s
′
′
′
′
′
6.47 d (8.7)
6.80 d (8.7)
3.67 s
3.75 s
8.98 s
5.48 d (6.8)
′
112.0 s
60.6 q
59.7 q
′-OMe
′-OMe
′-OH
0-OH
9-OMe
10-OMe
3.78 s
3.89 s
56.3 q
5.59 s
a
1
1
b
at 300 MHz for H and 75 MHz for 13C. c In
1
Assignments were based on DEPT135, H- H COSY, HSQC, and HMBC experiments. In DMSO-d
6
1
13
d
6
acetone-d at 400 MHz for H and 100 MHz for C. Signals overlapped.
It is important to emphasize that compound 1 is the first example
of a chalcan-isoflavan biflavonoid from natural sources and also
the first report of the existence of a natural biflavonoid in the genus
Oxytropis. Propterol B (5) and (3R)-(-)-isomucronulatol (6) were
isolated for the first time from the genus Oxytropis.
carbonyl group. The structure of the compound was established
from its H and C NMR data (Table 2), which were compared to
1
13
those of (3R)-(-)-isomucronulatol (6). The assignments of protons
1
1
and carbons were done via 2D NMR ( H- H COSY, HSQC, and
HMBC) and DEPT experiments. It is structurally related to 6. Ring
A of 2 contained an R,ꢀ-unsaturated carbonyl carbon at C-7 (δ
199.1) and the C-8 (δ 108.3), C-9 (δ 175.5) double bond
characteristic of the ring A partial structure in 11b-hydroxy-11b,1-
Oxytropisoflavan B (2) was obtained as optically active, colorless
crystals, with an elemental formula of C17
H
20
O
6
determined by
+
HRESIMS (m/z 321.1326, [M + H] ). An IR hydroxy peak was
-1
-1
13
19
observed at 3333 cm , The absorption at 1710 cm and C NMR
peak at δ 199.1 implied the presence of an R,ꢀ-unsaturated ketone
dihydromedicarpin. Inspection of the HMBC spectrum (Figure
C
1) allowed for a complete assignment of all signals in this partial

Journal of Natural Products, 2010, Vol. 73, No. 8 1401
2
ZnCl -catalyzed condensation of substituted pterocarpans has been
developed by Trivedi and co-workers through the reaction of
2
-alkoxy-1,4-benzoquinones with appropriately substituted 2H-
27,28
chromenes.
To verify the structure of 3, we attempted a total
synthesis of (()-3 by formal [3+2] cycloaddition reaction of 2,3-
alkaloxy-1,4-benzoquinone with 2H-chromene using ZnCl as
2
catalyst.
The synthesis of (()-3 is depicted in Scheme 2, starting from
commercially available resorcinol (I) and pyrogallol (IV). Initially,
2
9
I was treated with propargyl bromide to furnish II, which was
subjected to intramolecular cyclization using PtCl
generate 2H-chromene (III). On the other hand, pyrogallol (IV)
Figure 3. ORTEP drawing of 2.
2
at 100 °C to
3
0
structure. The HMBC experiment showed that H-4 (δ 2.02, 2H,
m) was related to C-1′ (δ 120.6) and C-2 (δ 73.2) and H-5 (δ 1.92,
was first methylated with dimethyl sulfate in acetone to give 1,2,3-
3
1
trimethoxylbenzene (V), which upon oxidation with H O using
2
2
32
2
H, m) to C-6 (δ 33.5), C-7 (δ 199.1), C-9 (δ 175.5), and C-10 (δ
5.9).
3 6
K Fe(CN) in HOAc gave 2,3-dimethoxy-1,4-benzoquinone (VI).
6
Finally, a cycloaddition reaction between III and VI, employing
In the NOESY experiment, H-3 (δ 3.91, 1H, m) showed
ZnCl as catalyst, afforded (()-3 with an overall of 3.2%. The
2
correlations with HO-2′ (δ 8.98, 1H, s), HO-10 (δ 5.59, 1H, m),
and H-6′ (δ 6.80, 1H, d, J ) 8.7 Hz). These correlations suggested
both the location and the R-orientation of the hydroxy group at
C-10. The X-ray crystallographic analysis of compound 2 (Figure
structures of the synthetic compounds were elucidated mainly on
1
13
the basis of comparison of their H and C NMR spectra
2
9-32
(Supporting Information) with reported data.
On the basis of NMR, EIMS, CD, optical rotation data, and
comparison with reported data, the known flavonoids were identified
3
) clearly established its structure. The CD spectrum of compound
3
3
17
2
(Figure 2) matched very well with that of (3R)-(-)-isomucronu-
as (6aR,11aR)-vesticarpan (4), propterol B (5), (3R)-(-)-
1
16,18
15,16 34
latol (6) at the L
b
transition region.
The (3R,10S) absolute
isomucronulatol (6),
mucronulatol (7), (3R)-7,2′,3′-trihydroxy-
35 11,36
configuration of 2 could therefore be assigned. Thus, the structure
of oxytropisoflavan B (2) was established as (3R,10S)-10-hydroxy-
4′-methoxyisoflavan (8), pendulone (9),
and flavonoids 10-33
(see Supporting Information). A flavonoid such as 31 was isolated
for the first time from the genus Oxytropis. The accumulation of
the phytoalexins medicarpin and maackiain (32) has been partly
interpreted as the resistance of chickpea (Leguminosae family) to
Ascochyta rabiei (a phytopathogenic deuteromycete parasite of
5
,10-dihydroisomucronulatol.
Compound 2 is the first natural product with an isoflavan skeleton
possessing an R,ꢀ-unsaturated ketone carbonyl group in ring A. A
similar compound was formed with protein preparation when
2
0
37
incubated in the presence of NADPH.
chickpea). Flavonoids except for 7, 10-12, 16-18, 20, and 32
Compound 3 was obtained as a pale brown oil and was optically
were isolated for the first time from O. falcata.
2
0
active ([R]
D
-155, c 0.8, acetone). In its HRESI mass spectrum,
The new flavonoids (1-3) were evaluated for their growth
inhibition against the HL-60 cell line by the MTT method and free
radical scavenging activities using the DPPH test. However, none
of them exhibited significant activity on HL-60 cells with IC50
values over 50 µM. In the DPPH test, flavonoids 1 and 2 lacked
significant activity, with IC20 values more than 50 µM. Compound
3 showed potent antioxidant activity, with an IC20 value of 3.8 µM,
when compared with the IC20 ) 7.6 µM of the reference, Vitamin
E (VE).
the protonated molecular ion was observed at m/z 317.1016 [M +
+
H] (calcd for C17
formula C17 . The H NMR and H- H COSY spectra showed
four aliphatic protons in a single spin system at δ 4.27 (1H, dd,
J ) 5.2, 10.8 Hz), 3.67 (1H, dd, J ) 10.8 Hz), 3.55 (1H, m), and
.48 (1H, d, J ) 6.8 Hz), attributed to CH -6, H-6a, and H-11a of
17 5
H O , 317.1020), corresponding to the molecular
1
1
1
16 5
H O
H
5
2
a pterocarpan skeleton, respectively (Table 2). The presence of a
pterocarpan skeleton was supported by the 13C NMR spectrum,
which showed the corresponding carbons at δ
(
C
66.0 (C-6), 41.1
C-6a), and 78.4 (C-11a). In the H NMR spectrum further signals
were observed that showed the presence of two methoxy groups
3.78, 3.89), an ABX aromatic proton spin system (δ 6.36,
.56, 7.12), and a single aromatic proton (δ 6.64). The spin systems
were assigned by comparison of the NMR data with those of
In conclusion, the coexistence of flavonoids 2 and 6 in O. falcata
further confirmed the biotransformation indicated by the related
compounds obtained from the catabolism of the phytoalexin
medicarpin by the chickpea pathogenic strains. The presence of
the same substituents in equivalent positions in 3 and pendulone
(9) and the co-occurrence of these compounds in O. falcata
exemplify the close biosynthetic relationship between pterocarpans
and isoflavonoids. This is the first report showing the potential of
pterocarpans as a class of natural antioxidant.
21
1
2
0
(
δ
H
H
6
H
22
(6aR,11aR)-3,8-dihydroxy-9-methoxypterocarpan. The ABX spin
system corresponds to A-ring protons with the biogenetically
expected oxygenation at C-3, and this was confirmed by the HMBC
experiment. In the HMBC spectrum (Figure 1), correlations of the
methoxy protons (δ
1
H
3.78, 3.89) with the signals at δ
38.4, together with the aromatic proton (δ 6.64, H-7) with the
41.1 (C-6a), 144.8 (C-8), and 140.0 (C-9), allowed
C
140.0 and
Experimental Section
H
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer model 341 polarimeter with a 1 dm cell. Melting
points were determined on an X-4 Digital Display micro-melting point
apparatus, uncorrected. UV spectra were measured on a Shimadzu UV-
signals at δ
C
placement of the hydroxy group at C-8 and methoxy groups at C-9
and C-10, respectively.
Pterocarpans are found in nature only in a 6a,11a-cis configu-
ration. In agreement with this are the coupling constant between
H-6a and H-11a (J ) 6.8 Hz) and the comparison with the literature
2
60 spectrophotometer. IR spectra were obtained on a Nicolet NEXUS
670 FT-IR spectrometer. CD spectra was recorded on an Olis DSM
1000 spectrophotometer. NMR spectra were recorded on Varian
INOVA-300 and INOVA-400 spectrometers. Chemical shifts are given
on the δ (ppm) scale using TMS as internal standard. EIMS and
HRESIMS were carried out on a VG ZABHS mass spectrometer and
a Bruker APEX II mass spectrometer, respectively. The X-ray diffrac-
tion data were collected on a Bruker Smart Apex CCD diffractometer,
and the structure was solved by direct methods using Bruker SHELXS-
2
3
values (J ) 6.6 Hz for cis and 13.4 Hz for trans). The absolute
configuration at C-6a and C-11a was assigned as R from its negative
2
0
optical rotation ([R]
D
-155) and the negative Cotton effect (∆ε -
24,25
4
7.7) at 228 nm (Figure 2).
Thus, compound 3 was character-
ized as (6aR,11aR)-3,8-dihydroxy-9,10-dimethoxypterocarpan.
Pterocarpans have been gaining considerable attention on the
9
7. DPPH was obtained from Sigma-Aldrich Chemical Co. (St. Louis,
26
account of their wide range of biological activities. In the synthesis
of pterocarpans, it is essential to use methodologies that allow
varying the substituents at rings A and D. Recently, a one-step,
MO). Silica gel (200-300 mesh) used for column chromatography and
silica GF (10-40 µm) for TLC were both supplied by the Qingdao
2
54
Marine Chemical Factory, Qingdao, People’s Republic of China. TLC

1
402 Journal of Natural Products, 2010, Vol. 73, No. 8
Chen et al.
a
Scheme 2. Synthesis of (()-3
a
Reagents and conditions: (a) propargyl bromide (1 equiv), K
2
CO
3
(3 equiv), acetone, reflux, 22 h (60%); (b) PtCl
2
(0.05 equiv), toluene, 100 °C, 15 h (20%); (c)
(
CH
3
O)
2
SO
2
(6 equiv), K
2
CO
3
(10 equiv), acetone, reflux, 6 h (93%); (d) K
3
Fe(CN) (0.5 equiv), H O (2.2 equiv), HOAc, rt, 24 h (40%); (e) ZnCl (1.8 equiv), DCM,
6
2
2
2
rt, 2 h (72%).
was detected at 254 nm, and spots were visualized by spraying with
% H SO in C OH (v/v) followed by heating.
Plant Material. Oxytropis falcata was collected from Sunan County,
Gansu Province, People’s Republic of China, in June 2006, and
identified by adjunct Prof. Huan-Yang Qi. A voucher specimen (No.
ZY-0601) has been deposited in the State Key Laboratory of Applied
Organic Chemistry, Lanzhou University, People’s Republic of China.
Extraction and Isolation. Plant material (aerial parts 3.5 kg, roots
3
over Si gel (CHCl -MeOH, 8:1) followed by recrystallization afforded
5
2
4
2
H
5
24 (9 mg) and 29 (7 mg). However, the same strategy used in the
separation of the n-BuOH-soluble fraction (9 g) of the roots of O. falcata
resulted in no flavonoids.
2
0
Oxytropisoflavan A (1): pale brown oil; [R] -28 (c 0.7, acetone);
D
-1
IR (neat) νmax 3363, 2922, 1510, 1463, 1096 cm ; UV (MeOH) λmax
(log ε) 212 (1.63), 282 (0.27) nm; CD (MeOH, ∆ε) λmax 286 (+11.5),
1
13
261 (-22.7), 249 (-20.8) nm; H and C NMR data, see Table 1;
EIMS (relative abundance) m/z 552 (0.3), 450 (4), 432 (11), 423 (2),
302 (59), 240 (33), 239 (32), 180 (RDA, 100), 107 (46); HRESIMS
0
.5 kg) was air-dried, ground, and exhaustively extracted with 95%
EtOH at room temperature. Each part of the plant was processed
separately; the EtOH extracts of the aerial parts and roots of O. falcata
m/z 583.1944 (calcd for C32
32 9
H O +Na, 583.1939).
2
0
were suspended in H
2
O and partitioned with petroleum ether, EtOAc,
Oxytropisoflavan B (2): colorless, needle-like crystal (MeOH); [R]
D
and n-BuOH, successively. Both petroleum ether-soluble fractions were
combined because they exhibited similar constituents on TLC [Si gel;
petroleum ether-EtOAc (3:1 v/v)], to give the crude petroleum ether-
soluble fraction. The petroleum ether-soluble fraction (aerial parts 65 g
+150 (c 0.2, MeOH); IR (neat) νmax 3333, 2926, 1710, 1618, 1602,
-1
1180 cm ; UV (MeOH) λmax(log ε) 199.9 (1.08), 258.5 (0.50); CD
1
(MeOH, ∆ε) λmax 302 (+24.9), 250 (+21.9), 213 (-40.5) nm; H and
1
3
C NMR data, see Table 2; EIMS (relative abundance) m/z 320 (18),
+
roots 15 g) was subjected to Si gel column chromatography (CC)
eluting with petroleum ether-EtOAc (40:1) followed by stepwise
302 (6), 292 (8), 261 (15), 193 (83), 192 (64), 180 (RDA 17), 179
(40), 167 (55), 128 (100); HRESIMS m/z 321.1326 (calcd for C17H O ,
21 6
321.1333).
addition of EtOAc to yield six fractions [Frp1 (40:1, 10 g), Frp2 (20:
1
1
, 15 g), Frp3 (10:1, 8 g), Frp4 (5:1, 5 g), Frp5 (2:1, 9 g), Frp6 (0:1,
7 g)]. Fraction Frp1 mainly contained fatty materials, while the last
(6aR,11aR)-3,8-Dihydroxy-9,10-dimethoxypterocarpan (3): pale
20
brown oil; [R]
D
-155 (c 0.8, acetone); IR (neat) νmax 3387, 2940, 1621,
-1
fraction (Frp6, eluted with EtOAc) was a complex mixture. Fraction
Frp2 was chromatographed on silica gel with a petroleum ether-EtOAc
gradient system to give four subfractions (Frp2.1-Frp2.4). Further
purification of subfraction Frp2.1 through repeated cheomatography
with petroleum ether-EtOAc (35:1) and petroleum ether-acetone (20:
1470, 1158, 1097 cm ; UV (MeOH) λmax (log ε) 213 (1.98), 286 (0.43)
1
13
nm; CD (MeOH, ∆ε) λmax 291 (+10.4), 228 (-47.7); H and C NMR
data, see Table 2; HRESIMS m/z 317.1016 (calcd for C17H O ,
17 5
317.1020).
Crystallographic Data of 2. Crystallographic data for the structure
of oxytropisoflavan B (2) have been deposited with the Cambridge
Crystallographic Data Center as supplementary publication number
CCDC 763261. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
+44(0)1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
1
) as eluants over Si gel yielded 10 (1 g) and 11 (50 mg). Frp2.2 was
subjected to Si gel CC with petroleum ether-EtOAc (3:1) as eluant
followed by recrystallization from acetone to yield 16 (6 g). Frp2.3
and Frp2.4 were separated using the same procedure as Frp2.1 to afford
2
0 (50 mg), 12 (130 mg), and 21 (4 mg), respectively.
The EtOAc-soluble fraction (100 g) of the aerial parts of O. falcata
DPPH Scavenging Assay. To determine the antioxidant activity of
the new compounds, DPPH radicals were used. In the radical form,
this molecule has an absorbance at 517 nm that disappears with
acceptance of an electron from an antioxidant compound to become a
was subjected to Si gel CC, using a step gradient-elution technique,
employing mixtures of CH Cl -EtOAc and MeOH as solvents, to
afford five fractions (Fre1 (0:1), 23 g; Fre2 (9:1), 18 g; Fre3 (4:1),
5 g; Fre4 (2:1), 16 g; Fre5 (MeOH), 20 g) according to TLC analysis.
Fre1 was rechromatographed with CHCl
2
2
3
8
1
stable diamagnetic molecule. The method described by Chen et al.
3
-EtOAc (50:1) followed by
was used with some modifications. For each compound, different
concentrations were tested. Aliquots of 30 µL of an EtOH solution
containing each new compound were added to 3 mL of a 100 µM EtOH
solution of DPPH. Absorbance at 517 nm was determined after 30 min
at room temperature, and the percent antiradical activity (AA) was
calculated using the following formula:
petroleum ether-actone (30:1) to give 17 (35 mg), 14 (12 mg), 13 (23
mg), and 4 (7 mg), respectively. Fre2 was subjected to Si gel CC
(
CHCl
compounds were eluted in the following order: 7 (9 mg), 6 (5 mg), 9
21 mg), 3 (10 mg), and 2 (18 mg). Likewise, a similar isolation
3
-EtOAc, 20:1) to provide 25 (41 mg) and 26 (60 mg). The
(
procedure adopted for the EtOAc-soluble fraction (10 g) of the roots
of O. falcata afforded 19 flavonoids; the pure compounds were isolated
in the following order: 8 (4 mg), 33 (2 mg), 6 (540 mg), 15 (4 mg), 19
AA% ) 100-{[(Abssample-Absblank) × 100]/Abscontrol}
(
(
8 mg), 18 (4 mg), 32 (28 mg), 9 (16 mg), 14 (8 mg), 28 (7 mg), 30
3 mg), 31 (18 mg), 3 (12 mg), 22 (4 mg), 23 (4 mg), 27 (6 mg) 2 (6
EtOH (3 mL) was used as a blank. A 100 µM DPPH EtOH solution
(3 mL) was used as a negative control. The IC20 value is the
concentration of test sample required to scavenging 20% DPPH free
radicals. VE was used as a positive control.
mg), 5 (1 mg), and 1 (8 mg).
The n-BuOH-soluble fraction (18 g) of the aerial parts of O. falcata
was subjected to Si gel CC, using a step gradient-elution technique,
Assessment of HL-60 Cell Viability. The HL-60 cell viability was
assessed using the MTT colorimetric assay, which is based on the
reduction of MTT by the mitochondrial succinate dehydrogenase of
employing mixtures of CHCl
fractions (Frn1 (20:1), 5 g; Frn2 (10:1), 5 g; Frn3 (4:1), 3 g; Frn4 (2:
), 2 g; Frn5 (MeOH), 2 g) according to TLC analysis. Fraction Frn2
was chromatographed on Si gel with a CHCl -MeOH gradient system
to give three subfractions. Repeated chromatography of subfraction 2
3
-MeOH as solvent, to afford five
3
9
1
intact cells to a purple formazan product. Briefly, aliquots of HL-60
4
3
cells containing 5 × 10 /mL were added to each well of 96-well flat-
microtiter plates and incubated with various concentrations of com-

Journal of Natural Products, 2010, Vol. 73, No. 8 1403
pounds. Six replicate wells were used in each point in the experiments.
After different exposure times, MTT solution (5 mg/mL in PBS) stored
at 4 °C in the dark was added to each well and plates were incubated
for 4 h at 37 °C. Extraction buffer [10% SDS-0.1 M HCl, 1:1 (v/v)]
was added. After an overnight incubation at 37 °C, the optical densities
at 570 nm were measured by using a Bio-Rad 550 ELISA microplate
reader.
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(
(
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Acknowledgment. This project was financially supported by the
Foundation for the National Key Technology Research and Develop-
ment Program of China (No. 2007BAI37B05) and National Natural
Science Foundation of China (No. NSFC 20621091).
4
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Supporting Information Available: Names and structures of
flavonoids 10-33; IR, HR-ESIMS, 1D/2D NMR spectra of flavonoids
(
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(
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NP100339U