Synthesis and immunosuppressive activity of l-rhamnopyranosyl flavonoids

Article abstract of DOI:10.1039/b604521a

Astilbin, a flavonoid isolated from different plants, shows diverse biological activities. This paper reports the synthesis and immunosuppressive activity of seven analogues of astilbin, which may shed light on the structure-activity relationship of the c

Full text of DOI:10.1039/b604521a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and immunosuppressive activity of L-rhamnopyranosyl flavonoids
Xiaoliang Yang,a Yang Sun,b Qiang Xu*b and Zijian Guo*a
Received 29th March 2006, Accepted 4th May 2006
First published as an Advance Article on the web 8th June 2006
DOI: 10.1039/b604521a
Astilbin, a flavonoid isolated from different plants, shows diverse biological activities. This paper
reports the synthesis and immunosuppressive activity of seven analogues of astilbin, which may shed
light on the structure–activity relationship of the compounds. The following glycosyl flavonoids,
6-a-L-rhamnopyranosyloxyflavanone (20), 3-a-L-rhamnopyranosyloxyflavone (22),
3-a-L-rhamnopyranosyloxyflavanone (24), 3-a-L-rhamnopyranosyloxychromanone (26),
4-a-L-rhamnopyranosyloxychromanol (27), 7-hydroxy-3-a-L-rhamnopyranosyloxyflavanone (30) and
4ꢀ-hydroxy-3-a-L-rhamnopyranosyloxyflavanone (32) were prepared respectively by glycosylation of
6-hydroxyflavanone (1), 3-hydroxyflavone (2), 3-hydroxyflavanone (5), 3-hydroxychromanone (8),
4-chromanol (9), 7-benzyloxy-3-hydroxyflavanone (12), 4ꢀ-benzyloxy-3-hydroxyflavanone (15). Among
them, compounds 5, 8, 12 and 15 were synthesized from flavanone (3), 4-chromanone (6),
7-hydroxyflavanone (10) and 4ꢀ-hydroxyflavanone (13) respectively. Similar to astilbin (4), compounds
22, 24, 26, 30 and 32 significantly inhibited the single mixed lymphocytes reaction (sMLR) and
enhanced the apoptosis of spleen cells isolated from mice with sheep red blood cell-induced
delayed-type hypersensitivity respectively. However, compound 20 only showed a slight tendency to
inhibit sMLR at higher concentration. Both compounds 20 and 27 did not influence the cell apoptosis.
These data suggest that the following factors play essential roles in determining the biological activity
of the flavonoids: the position at which the sugar is linked to the flavone, the presence of carbonyl on
C-4 and phenol hydroxyl group in A or B ring. However, the presence of a B ring is unfavorable for the
biological activity and the double bond at C(2)–C(3) in C-ring shows little effect on the activity.
dysfunction of liver-infiltrating cells contained, and the rhamnose
Introduction
may be an essential requirement for the dysfunction.9 It has been
There is now overwhelming evidence from diverse studies that
confirmed that the activity of astilbin is mainly targeted to the
activated T lymphocytes.11,12
flavonoid glycosides, as well as their aglycones, exhibit significant
biological activities such as antitumor and antimicrobial activity,
radical-scavenging properties and immunoactivity coupled with
low toxicity.1–5 Therefore, their use as potential therapeutic com-
pounds against a variety of diseases is of great interest.6 More than
one thousand flavonol O-glycosides have so far been isolated. And
there are many variations in both the nature of the sugar moiety
and its position of attachment to the aglycone. About 80% of
flavonol O-glycosides have a sugar linkage at the 3-OH.
Astilbin, a flavanoid with a rhamnose, representing a typical
structure of a flavonol 3-O-glycoside, has been isolated from the
root of Astilbe Odontophylla Miquel,7 the bark of Hymenaea
martiana8 and from the rhizome of Smilax glabra Roxb.9 This
compound has recently been proven to exhibit some important
bioactivities including antioxidant and aldose reductase inhibitory
effects.10 Although the mechanism of action has yet to be deter-
mined at the molecular level, both astilbin and dihydroquercetin
have been applied to the assay of hepatotoxicity caused by
nonparenchymal cells, where astilbin was shown to prevent the
hepatocyte damage from nonparenchymal cells by inducing the
Despite the biological importance of flavonol glycosides, the
synthesis of these compounds appears to be rare.13–24 Major
advances in the synthesis of flavonoid glycosides have been made
by the development of the Koenigs–Knorr method,25 in which
the key-step of most syntheses involves the condensation of a
flavonoid aglycon with a per-O-acetylglycosyl halide. However,
due to the presence of a high concentration of silver salts, the final
workup for this method is complicated.26 The Zemplen–Farkas
synthesis,27 which involves the use of a per-O-acetyl glycosyl
bromide in a homogeneous alkaline medium, is more convenient
from this point of view, but the yields generally remain low
probably owing to the partial hydrolysis of per-O-acetylglycosyl
bromide during the course of the reaction. With the development
of various glycosylation procedures and sophisticated protecting
group strategies, other methods are reported in the synthesis of
7-O-glucosides28 and astilbin.13,29
The aim of the present study is to develop an al-
ternative strategy for the efficient synthesis of glycosyl
flavonoids starting from readily available 6-hydroxyflavanone, 3-
hydroxyflavone, 3-hydroxyflavanone, chromanone, 4-chromanol,
7-hydroxyflavanone, 4ꢀ-hydroxyflavanone and rhamnose, and to
investigate the biological activity of the compounds synthesized
on T lymphocytes. This work may be important for the under-
standing of the structure–activity relationship of astilbin-related
species.
aDepartment of Chemistry, State Key Laboratory of Coordination Chemistry,
Nanjing University, Nanjing, 210093, China. E-mail: zguo@nju.edu.cn;
Fax: +86 2583314502; Tel: +86 2583594549
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing Univer-
sity, Nanjing, 210093, P. R. China. E-mail: qiangxu@jlonline.com; Fax: +86
2583597620; Tel: +86 2583597620
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Results and discussion
Chemistry
With the commercially available glycosyl acceptors 1, 2, 9 and
the synthesized glycosyl acceptors 5,30 8,31 12 and 15,32 the
strategy adopted for the synthesis of the glycosides involves
the following steps: first, the synthesis of the glycosyl donors
(Scheme 1), and then the glycosylation of 6-hydroxyflavanone,
3-hydroxyflavone, 3-hydroxyflavanone, 3-hydroxychromanone, 4-
chromanol, 7-benzyloxy-3-hydroxyflavanone or 4ꢀ-benzyloxy-3-
hydroxyflavanone, and finally the deprotection of the acetyl of
the sugars and the benzyl of the flavanone (Scheme 2).
Scheme 1 Conditions and reagents: a: (Ac)2O, pyridine, b: NH3 (gas),
THF and CH3OH (2 : 1), c: CCl3CN, DBU, CH2Cl2.
In the first step, the 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl
trichloroacetimidate (18)33 was directly attained by base-
catalyzed reaction of 2,3,4-tri-O-acetyl-rhamnopyranose (17)
with trichloroacetonitrile. The commonly used bases were
NaH, K2CO334 or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), with
NaH35 being used more often. In this work, DBU was chosen
as the catalyst because its boiling point is low and it can
be removed easily at the end of the reaction. It was found
that compound 18 was unstable in the course of purification
and easily decomposed in the moist atmosphere. For example,
when compound 18 was purified by column chromatography
◦
using EtOAc–petroleum ether (bp, 60–90 C) as the eluent, only
its decomposed product 1-hydroxyrhamnose (17) was obtained,
Therefore, the corresponding 1-hydroxy sugars were treated with
CCl3CN in the presence of cat. DBU in dry CH2Cl2 under
N2 protection, and the a imidate of target compound can be
synthesized in excellent yields (>98%). Further experiment showed
that compounds 18 synthesized by this method can be used directly
without purification.
The glycosylation of acceptors 1, 2, 5, 8 or 12 with rhamnosyl
imidates was carried out in dry CH2Cl2 with cat. TMSOTf to
afford the acetyl protected flavone derivatives 19, 21, 23, 25 and 28
(Scheme 2). This reaction required considerable optimization as
summarized in Table 1. The optimal activator is TMSOTf.36 High
yield synthesis of the desired glycoside in a short reaction time was
achieved when the reaction was conducted at −20 ◦C followed by
gradual warming up to room temperature (Table 1). Concerning
the synthesis of glycosides 19 with acceptor 1, a higher yield was
originally anticipated due to the lack of internal hydrogen bonding
between the C(3)-hydroxyl group and C(4) carbonyl in acceptors
2, 5 and 8,13 which would lower the reactivity. However, it turned
out not to be the case and both glycosyl acceptors 1 and 2 gave
poor results mainly due to the presence of phenolic hydroxyls
as well as the reaction temperature rather than the internal
hydrogen bond of the glycosyl acceptor. Phenolic hydroxyls are
weaker nucleophiles compared to aliphatic hydroxyls, therefore
they usually require activation. Thus, the glycosylation product
Scheme 2 Conditions and reagents: a: TMSOTf, CH2Cl2, −20–0 ◦C, b:
NH3 (gas), CH3OH, c: 10% Pd/C, cyclohexene, EtOH.
was designed to be obtained by using the “inverse procedure” of
the Schmidt reaction,37 where acceptors1 and 2 shouldbeactivated
firstly by a catalytic amount of TMSOTf before the addition of
donor 18. It should be noted that the “inverse procedure” of
the Schmidt reaction was developed to improve the glycosylation
efficiency by activating the less reactive acceptors or by preventing
the decomposition of the highly reactive donors. However, this
work demonstrated that it is not an efficient method for the
glycosylation of phenolic hydroxyls.
The conversion of acetyl protected flavone derivatives 19, 21, 23
or 25 and 28 to final target molecules was initially attempted by
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Table 1 Temperature effect on glycosidation
Glycosidation product (yield, %)a
Entry Acceptor Donor
−78 ◦C–rt
−20 ◦C–rt
1
2
3
4
1
2
5
8
18
18
18
18
19 (33.8)
21 (24.3)
—
19 (62.3)
21 (50.7)
23 (76.1)
25 (84.5)
—
a Isolated yields.
removing the acetyl groups under basic conditions such as with
CH3ONa–CH3OH.38 However, the sugar moieties were cleaved
under these conditions. For example, in the case of compound
22, the isolated adduct was 3-hydroxyflavone. The cleavage of
sugar was effectively suppressed when NH3 (gas) was employed
in CH3OH at room temperature. This procedure provided target
compound 22 with 83.6% yield. The optimization of reaction time
is crucial and the best reaction time was found to be within 2 h.
Longer reaction time was found to cause partial cleavage of the
sugar moieties.
Seven novel glycosyl flavonoids as well as some precursors were
synthesized. The new compounds were fully characterized by 1H
NMR, 13C NMR, 2D H–H COSY, 2D C–H COSY, IR, and
1
EA. The selected H and 13C NMR data on the novel glycosyl
flavonoids are listed in Tables 2 and 3.
The configurations of the glycoside adducts were mainly de-
1
termined by H NMR, and discussed based on the mechanism
of glycosylation. According to the mechanism of the glycosy-
lation reaction which has been studied by Kochetkov and co-
workers,39 the normal glycosidic products were mainly derived
from the corresponding sugar 1,2-orthoester intermediates, which
are classical glycosyl donors used in the construction of 1, 2-trans-
glycosidic linkages in glycosylation with donors carrying C-(2)
acetyl protecting groups.40 Then the rhamnosides compounds 19,
21, 23, 25 and 28 should have the a configuration.
1
The H NMR spectra confirmed that the configuration at the
anomeric center in compounds 19, 21, 23, 25 and 28 was a with
the coupling constant of the anomeric proton signal being J = ca.
1.2 Hz. These data were in agreement with the mechanism of the
glycosylation reaction.40
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Table 3 Selected 13C NMR data on novel glycosyl flavonoids
20
22
24
26
27
30
32
DMSO-d6
MeOH-d4
MeOH-d4
MeOH-d4
DMSO-d6
DMSO-d6
Acetone-d6
C-2
C-3
C-1ꢀ
C-2ꢀ
C-3ꢀ
C-4ꢀ
C-5ꢀ
C-6ꢀ
—
—
—
—
81.87, 81.46
83.12, 82.86
101.48, 101.33
70.87, 70.67
71.14, 70.92
72.71, 72.16
69.62, 69.09
17.06, 16.91
73.54, 72.46
70.08, 69.17
101.00, 99.69
71.06, 70.84
71.16, 70.99
72.74, 72.70
69.72, 69.47
17.07, 16.74
62.27, 62.18
29.11, 27.46
98.11, 97.72
71.90, 71.59
68.96
73.30, 73.25
71.90, 71.59
17.94, 17.77
82.81, 82.66
78.05, 76.35
101.70, 101.21
70.96, 70.88
71.24, 71.00
72.49, 71.97
69.75, 69.45
18.60
83.30,82.90
78.46, 76.67
101.22, 101.06
70.93, 70.71
71.54, 71.35
72.81, 72.43
69.51, 69.04
17.59, 17.51
100.01
70.98
71.27
72.65
70.41
18.74
102.35
71.84
71.05
71.33
70.95
18.28
1
According to the H NMR spectral data (Table 2), the con-
figuration at C(2)–C(3) in compounds 24, 29, 30 and 32 can be
determined to be a trans-configuration with two diastereomers.
For compound 24, the H-(2) and H-(3) signals appeared in pairs
at d 5.33 (0.45 H, d, J = 11.30 Hz), d 5.24 (0.55 H, d, J = 11.90 Hz)
and d 4.69 (0.45 H, d, J = 11.30 Hz), d 4.73 (0.55 H, d, J = 11.90
Hz), respectively. For 29, the H-(2) and H-(3) signals appeared in
pairs at d 5.24 (0.34 H, d, J = 11.21 Hz), d 5.19 (0.67 H, d, J =
11.73 Hz) and d 4.68 (0. 67 H, d, J = 11.81 Hz), d 4.62 (0.34 H, d,
J = 11.24 Hz) respectively. The coupling constants of the signals
(J > 6 Hz) suggested that both of them adopt a trans-configuration
at C(2)–C(3).
process of separation by column chromatography. However, the
configuration on C(2)–C(3) was changed from cis- for 23 and 28
to trans- for 24 and 29 (Scheme 4); whether the transformation
derived from the reaction under basic conditions or from the
separation by column chromatography was unknown.
All the diastereomers of astilbin have been isolated and their
chirality has been proposed,41–43 and it is noteworthy that each
isomer has diagnostic signals in the 1H-NMR spectra. When both
C-2 and C-3 have an (S) configuration, anisotropy of the benzene
ring (B-ring) causes upfield shifts of H-5ꢀ and H-6ꢀ, and that of
carbonyl group at C-4 causes downfield shifts of H-1ꢀ and H-2ꢀ. In
case of a (2R,3R) configuration, the contrary effects are expected
to produce the signals shown in Scheme 3.
Scheme 4
Biological activities of the compounds
The biological activity of compounds 20, 22, 24, 26, 27, 30 and
32 was evaluated with their effects on single mixed lymphocytes
reaction and apoptosis of spleen cells, and compared with that
of astilbin. The former assay was to examine the proliferation
of the lymphocytes from BALB/c mice under the stimulation of
the mitomycin C-treated spleen cells from C57BL/6 mice. In the
latter experiment, the spleen cells were isolated from mice with
a footpad reaction induced by sheep red blood cells. These two
assays were aimed at evaluating the effects of these compounds
on the T lymphocyte function since the single mixed lymphocytes
reaction is usually used for evaluating lymphocyte proliferation
and the main effector cells involved in the footpad reaction are
mainly CD4+ T cells.
Scheme 3
1
Comparing the H NMR signals of compounds 24, 29, 30, 32
with that of astilbin, the aglycone chirality of 24 was assigned as a
mixture of (2S,3S) and (2R,3R) with a ratio of 1.2 : 1, the aglycone
chirality of 29, 30 and 32 was assigned as a mixture of (2S,3S) and
(2R,3R) with a ratio of 2 : 1 respectively.
From the 1H NMR data of these compounds, we can conclude
that the configuration at the anomeric center was not affected nei-
ther during the conversion of acetyl protected flavone derivatives
19, 21, 23, 25 and 28 to the target molecules 20, 22, 24, 26 and 29
using NH3 (gas) in CH3OH at room temperature nor during the
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As shown in Fig. 1, astilbin and compounds 22, 24, 26, 30 and
32 significantly inhibited the single mixed lymphocytes reaction
compared with the control. Compound 20 tended to inhibit the
single mixed lymphocytes reaction at higher concentrations, but
not significantly. Compound 27 did not have the tendency.
Among them, compounds 22 and 24, which are 3-O-flavone gly-
coside and 3-O-flavanone glycoside respectively, showed a similar
activity. This finding suggests that the double bond at C(2)–C(3)
in C-ring is not important for their biological activities. However,
compounds 20 and 27, which are 6-O-flavanone glycoside and
4-O-chromanol glycoside respectively, did not show a significant
effect in both assays. Therefore, the linking position of sugar and
the carbonyl on C(4) are more important for the activities. In
addition, the phenolic hydroxyls on the flavanone may contribute
to the effect of the compounds since the activity of compounds 30
and 32 seems to be more remarkable than of compounds 22 and
24. Finally, lacking B ring on C(2) seems favorable to the activity
since compound 26 showed a significant biological effect in both
assays.
Conclusion
Fig. 1 Effect of compounds on single mixed lymphocytes reaction. The
lymphocytes (5 × 105) from BALB/c mice were incubated with the lympho-
cytes (5 × 105) from C57BL/6 mice, which had been treated with mitomy-
cin C (500 mg mL−1) for 1 h in the presence or absence of the various
concentrations of compounds at 37 ◦C in 5% CO2 for 72 h. The proli-
feration of lymphocytes was measured by the MTT method. The experi-
ment was repeated three times. *P < 0.05 vs. control (Student’s t test).
In conclusion, seven flavonoids of rhamnose were synthesized
and characterized. The biological data showed that compounds
22, 24, 26, 30 and 32 significantly suppressed single mixed
lymphocytes reaction and induced the apoptosis of spleen cells
isolated from mice with sheep red blood cell-induced delayed-type
hypersensitivity. The activity of these compounds is comparable
to that of astilbin. However, the activity of compounds 20 and
27 is much less pronounced, suggesting that the linking position
of the sugar to the flavone and the carbonyl on C(4) are very
important, while the double bond at C(2)–C(3) in the C ring
does not have a major impact on the biological activity. The
B ring of astilbin appears unnecessary for the activity. These
results will be important for the rational design of astilbin-type
immunosuppressive agents.
As shown in Fig. 2, astilbin and compounds 22, 24, 26,
30 and 32 significantly increased the apoptotic cells of spleen
cells activated by sheep red blood cell-induced delayed-type
hypersensitivity reaction in a dose-dependent manner (Fig. 2).
However, compounds 20 and 27 failed to cause the apoptosis.
Experimental
Materials and methods
All experiments dealing with air- and moisture-sensitive com-
pounds were conducted under an atmosphere of dry nitrogen.
˚
Dichloromethane was distilled from CaH2 and stored over 4 A
molecular sieves. For thin-layer Chromatography (TLC) analysis,
Merck precoated plates (silica gel GF254) were used. Solvents
such as acetic anhydride, pyridine, petroleum ether, ethyl acetate,
tetrahydrofuran (THF) and methanol are all analytical grade and
used as received. The 6-hydroxyflavanone, 3-hydroxyflavone, fla-
vanone, trichloroacetonitrile, TMSOTf and DBU were purchased
from Aldrich.
Fig. 2 Effect of compounds on the apoptosis of spleen cells isolated
from mice with sheep red blood cell-induced delayed-type hypersensitivity.
BALB/c mice were sensitized by s.c. injecting 40 lL of 2.5 × 108 mL−1
sheep red blood cells in saline into the left footpad. Five days later, they
were challenged by s.c. injecting 40 lL of 2.5 × 109 mL−1 of the cells in
saline into the right footpad. Six hours later, spleen cells were isolated and
incubated with the compounds at 37 ◦C for 12 h. *P < 0.05, **P < 0.01
vs. control (Student’s t test).
Melting points were determined with a ‘Mel-Temp’ apparatus.
The IR spectra were recorded on a Hitachi 260–01 spectrometer in
KBr discs. The 1H NMR and 13C NMR spectra were recorded with
a Bruker DPX 300 spectrometer. The thin-layer chromatography
(TLC) was performed on silica gel GF254 by iodo gas or UV
detection. Column chromatography was conducted by elution of
a column (8 × 100 mm, 16 × 240 mm, 18 × 300 mm) and EtOAc–
petroleum ether (bp, 60–90 ◦C) as the eluent. Optical rotations
These results suggest that the compounds having the aglycone of
astilbin show an inhibitory activity against T lymphocytes through
decreasing the proliferation and inducing the apoptosis of T cells.
were measured at 20
digital polarimeter, using a 10 cm, 10 ml cell.
2
◦C with a Perkin Elmer Model 343
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20.99, 17.52, 17.23. IR (KBr); cm−1 2984.81, 1749.66, 1693.84,
1609.02, 1576.71, 1466.32, 1370.97, 1311.21, 1225.54, 1139.16,
1080.47, 1039.88, 982.78, 918.12, 883.86, 839.61, 763.54, 699.29.
Elemental anal. for C27H28O10: Found (calculated) (%): C, 63.22
(63.28); H, 5.51 (5.47).
Preparation of the compounds
Glycosylation
The acceptor 2 (1.12 g, 0.005 mol) and compound 18 (2.39 g,
0.0055 mol) were dried together under high vacuum for 2 h, then
dissolved in anhydrous CH2Cl2 (60 ml). TMSOTf (50 ll) was added
dropwise at −20 ◦C under N2 protection. The reaction mixture was
stirred for 3 h, during which time the temperature was gradually
raised to ambient temperature. Then the mixture was neutralized
with Et3N and concentrated under reduced pressure to give a
syrupy residue, which was purified by column chromatography
(3 : 2 petroleum–EtOAc) to give compound 21.
3 - (2,3,4 - Tri -O-acetyl-a-L-rhamnopyranosyl)-oxy-chromanone
◦
1
(25). White solid. Mp 48–50 C; [a]2D0 −7.9 (c 1.0, MeOH), H-
NMR (300 MHz, CDCl3): d 7.89 (d, 1 H, J = 7.88 Hz), 7.48 (t, 1 H,
J = 7.78 Hz), 7.05 (t, 1 H, J = 7.50 Hz), 6.98 (d, 1 H, J = 8.37 Hz),
5.33–5.29 (m, 2 H, H-2ꢀ and H-3ꢀ), 5.06 (t, 1 H, J = 9.57 Hz, H-4ꢀ),
4.95 (bs, 1 H, H-1ꢀ), 4.49–4.38 (m, 3 H, H-2 and H-3), 4.27–4.21
(m, 1 H, H-5ꢀ), 2.14 (s, 3 H), 2.03 (s, 3 H), 1.97 (s, 3 H), 1.10 (d, 3
H, J = 6.24 Hz, H-6ꢀ); 13C-NMR (75 MHz, CDCl3): d 189.56,
170.46, 170.39, 170.21, 161.56, 136.70, 127.95, 122.28, 120.31,
118.20, 97.20, 74.13, 71.15, 70.08, 69.35, 69.10, 67.50, 21.16, 21.12,
21.01, 17.47. IR (KBr); cm−1 2986.35, 2938.81, 1749.42, 1702.85,
1607.74, 1579.83, 1479.26, 1466.95, 1371.50, 1325.63, 1288.13,
1222.53, 1146.72, 1095.90, 1048.31, 982.85, 937.69, 909.02, 835.76,
763.80. Elemental anal. for C29H30O12, found (calculated) (%), C,
57.72 (57.80); H, 5.72 (5.50).
6-(2,3,4-Tri-O-acetyl-a-L-rhamnopyranosyl)-oxy-flavanone (19).
Yellow powder. Mp 67–69 ◦C; [a]2D0 −7.6 (c 1.0, MeOH), 1H-NMR
(300 MHz, CDCl3): d 7.63 (t, 1 H, J = 2.5 Hz), 7.50–7.40 (m, 5 H),
7.27–7.23 (m, 1 H), 7.03 (d, 1 H, J = 9.0 Hz), 5.52–5.44 (m, 4 H),
5.17 (t, 1 H, J = 9.9 Hz), 4.02–3.97 (m, 1 H), 3.13–2.86 (m, 2 H),
2.21 (s, 3 H), 2.07 (s, 3 H), 2.04 (s, 3 H), 1.23 (d, 3 H, J = 6.2 Hz);
13C NMR (75 MHz, CDCl3): d 191.91, 170.46, 170.40, 170.34,
157.73, 150.65, 150.59, 139.06, 139.04, 129.24, 129.19, 126.52,
121.52, 119.91, 113.02, 112.99, 96.55, 96.53, 80.13, 80.12, 71.29,
69.97, 69.23, 67.69, 44.91, 44.89, 21.26, 21.16, 21.11,17.82. IR
(KBr); cm−1 2984.91, 2939.81, 1750.13, 1693.00, 1616.93, 1484.09,
1437.13, 1371.72, 1278.03, 1248.04, 1223.08, 1129.91, 1056.22,
984.37, 905.02, 828.42, 813.72, 762.52, 699.87. Elemental anal.
for C27H28O10: found (calculated) (%): C, 63.31 (63.28); H, 5.40
(5.47).
7-Benzyloxy-3-(2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-oxy-
◦
flavanone (28). White solid. Mp, 56–57 C; [a]2D0 −8.3 (c 1.0,
1
MeOH), H NMR (300 MHz, CDCl3): d 7.92–7.87 (m, 1 H),
7.60–7.35 (m, 10 H), 6.78–6.73 (m, 1 H), 6.65 (m, 1 H), 5.51 (s,
0.66 H, J = 0.91 Hz, H-2), 5.40 (s, 0.34 H, J = 1.01 Hz, H-2),
5.17–4.91 (m, 5 H, CH2, 0.66 H-1ꢀ, H-2ꢀ, H-3ꢀ and 0.34 H-4ꢀ), 4.78
(t, 0.66 H-4ꢀ, J = 4.97 Hz), 4.12 (m, 1 H, H-3), 4.07 (d, 0.34 H-1ꢀ,
J = 0.93 Hz), 3.80–3.75 (m, 0.34 H-5ꢀ), 2.15–1.94 (9.66 H, 3 Ac,
0.66 H-5ꢀ), 1.09 (d, 1 H, J = 6.22 Hz), 0.78 (d, 2 H, J = 6.22 Hz);
13C NMR (125 MHz, CDCl3): d 187.39, 187.21, 170.35, 170.28,
170.24, 170.18, 170.13, 169.88, 166.20, 166.10, 163.77, 163.71,
136.30, 136.19, 136.16, 135.45, 129.92, 129.85, 129.24, 129.20,
129.14, 129.08, 128.78, 128.75, 127.93, 127.90, 127.12, 126.99,
113.56, 113.09, 112.19, 102.29, 102.24, 98.58, 95.31, 82.23, 81.82,
77.50, 73.76, 71.18, 70.86, 70.83, 70.76, 69.53, 69.25, 69.24, 69.13,
67.52, 66.65, 21.13, 21.05, 17.53, 17.30; IR (KBr); cm−1 3096.20,
3062.34, 2984.50, 2850.12, 1750.27, 1684.15, 1609.52, 1575.81,
1498.60, 1445.92, 1371.15, 1244.31, 1224.20, 1173.76, 1137.26,
1038.89, 971.89, 901.62, 837.35, 785.42, 739.74, 698.81, 604.58;
Elemental anal. for C34H34O11, found (calculated) (%), C, 66.06
(66.02); H, 5.58 (5.50).
3-(2,3,4-Tri-O-acetyl-a-L-rhamnopyranosyl)-oxy-flavone (21).
◦
White powder. Mp 66–68 C; [a]2D0 −12.1 (c 1.0, MeOH), 1H
NMR (300 MHz, DMSO-d6): d 8.10 (d, 1H, J = 7.5 Hz), 7.92
(m, 2 H), 7.83 (t, 1 H, J = 7.8, 7.1 Hz), 7.71 (d, 1 H, J = 8.2, 7.8
Hz), 7.61 (m, 3 H), 7.51 (dd, 1 H, J = 7.5, 7.1 Hz), 5.54 (s, 1 H),
5.11 (dd, 1 H, J = 3.0, 10.2 Hz), 4.81 (t, 1 H, J = 10.0 Hz) 3.25
(m, 1 H), 2.11 (s, 3 H), 1.98 (s, 3 H), 1.96 (s, 3 H), 0.74 (d, 3 H,
J = 6.1 Hz); 13C NMR (75 MHz, DMSO-d6): d 174.22, 170.48,
170.37, 170.27, 157.78, 155.74, 137.21, 135.21, 131.96, 130.86,
129.66, 129.46, 126.20, 125.86, 124.19, 119.33, 98.59, 70.10,
69.35, 69.15, 68.59, 21.35, 21.31, 21.23, 17.57. IR (KBr); cm−1
3063.18, 2985.54, 2938.81, 1751.49, 1644.46, 1619.09, 1568.46,
1467.79, 1393.82, 1370.89, 1223.39, 1201.32, 1135.65, 1055.19,
982.20, 965.61, 905.07, 795.59 763.12, 698.81. Elemental anal. for
C27H26O10: found (calculated) (%): C, 63.59 (63.53); H, 5.10 (5.10).
Removal of the acetyl groups of the sugars
3-(2,3,4-Tri-O-acetyl-a-L-rhamnopyranosyl)-oxy-flavanone (23).
Compound 21 was dissolved in a saturated solution of NH3 (gas)
in MeOH (20 ml). After 48 h at rt, the reaction mixture was
concentrated, and the residue was purified by chromatography (1 :
3 petroleum ether–EtOAc) to afford compound 22.
◦
White solid. Mp 68–70 C; [a]2D0 −3.5 (c 1.0, MeOH), H-NMR
(300 MHz, CDCl3): d 7.96–7.92 (m, 1 H), 7.61–7.54 (m, 3 H), 7.50–
7.34 (m, 3 H), 7.14–7.08 (m, 2 H), 5.53 (d, 0.34H, J = 2.0 Hz, H-2,),
5.42 (d, 0.66 H, J = 1.7 Hz, H-2,), 5.15–4.99 (m, 2 H, H-2ꢀ and H-
3ꢀ), 4.96–4.89 (t, 0.66 H, H-4ꢀ), 4.90 (d, 0.34 H, J = 1.1 Hz, H-1ꢀ),
4.81–4.74 (t, 0.34 H, J = 9.9 Hz, H-4ꢀ), 4.20–4.17 (m, 1.66 H, H-3
and 0.66 H-1ꢀ), 3.73–3.68 (m, 0.66 H, H-5ꢀ), 2.13–2.07 (m, 0.34 H,
H-5ꢀ), 2.05–1.92 (9 H), 1.03 (d, 2 H, J = 6.2 Hz, H-6ꢀ), 0.78 (d, 1
H, J = 6.2 Hz, H-6ꢀ); 13C-NMR (75 MHz, CDCl3): d 189.12,
188.69, 170.27, 170.19, 170.16, 170.12, 170.08, 169.87, 161.71,
161.62, 137.14, 137.03, 136.22, 135.38, 129.28, 129.19, 129.09,
128.81, 128.16, 128.07, 127.20, 127.00, 122.53, 122.43, 119.56,
119.11, 118.69, 118.66, 98.54, 95.35, 81.87, 81.46, 77.80, 73.90,
71.13, 70.77, 69.55, 69.32, 69.20, 69.08, 67.61, 66.75, 21.09, 21.02,
1
6-a-L-Rh◦amnopyranosyloxyflavanone (20). Yellow powder.
1
Mp 71–73 C; [a]2D0 −10.5 (c 1.0, MeOH), H NMR (300 MHz,
DMSO-d6): d 7.54 (d, 2 H, J = 7.7 Hz), 7.48–7.41 (m, 4 H), 7.31–
7.27 (m, 1 H), 7.06 (d, 1 H, J = 9.0 Hz), 5.64–5.58 (m, 1 H, H-2),
5.32 (d, 1 H, J = 1.2 Hz, H-1ꢀ), 5.05 (d, 1 H, OH), 4.88 (d, 1 H, OH),
4.75 (d, 1 H, OH), 3.85 (m, 1 H, H-2ꢀ), 3.66–3.63 (m, 1 H, H-3ꢀ),
3.52–3.46 (m, 1 H, H-5ꢀ), 3.34–3.17 (m, 1 H, H-4ꢀ), 2.84–2.79 (m, 2
H, H-3), 1.11 (d, 3 H, J = 6.1 Hz); 13C NMR (75 MHz, DMSO-d6):
d 192.30, 157.20, 157.18, 151.21, 151.17, 139.81, 129.39, 127.43,
127.12, 127.07, 121.63, 120.15, 112.83, 100.01, 79.74, 79.71, 72.65,
2488 | Org. Biomol. Chem., 2006, 4, 2483–2491
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71.27, 70.98, 70.41, 44.29, 18.74. IR (KBr); cm−1 3406.51, 2931.66,
1689.38, 1617.28, 1484.49, 1437.19, 1280.05, 1219.42, 1197.94,
1123.74, 1060.04, 1020.05, 982.68, 911.92, 808.63, 760.72, 698.22.
Elemental anal. for C21H22O7: found (calculated) (%): C, 65.12
(65.28); H, 5.72 (5.70).
914.09, 779.16. Elemental anal. Found (calculated) (%), C, 55.80
(55.87); H, 5.40 (5.26).
4-a-L-Rhamnopyranosyoxychromanol (27). White solid. Mp
◦
125–127 C; [a]2D0 −5.8 (c 1.0, MeOH), 1H-NMR (300 MHz,
CDCl3): d 7.21 (m, 2 H), 6.85 (m, 2 H), 5.01 (d, 1 H, J = 0.86 Hz,
H-1ꢀ), 4.98 (d, 0.66 H, J = 0.84 Hz, H-1ꢀ), 4.70 (m, 0.33 H, H-4),
4.64 (m, 0.66 H, H-4), 4.32–4.21 (m, 2 H, H-2), 3.84–3.81 (m, 1 H,
H-2ꢀ), 3.75–3.67 (m, 2 H, H-3ꢀ and H-5ꢀ), 3.47 (t, 1 H, J = 9.36 Hz,
H-4ꢀ), 2.16–2.03 (m, 2 H, H-3), 1.39–1.22 (m, 3 H, H-6ꢀ); 13C-
NMR (300 MHz, CDCl3): d 155.33, 155.19, 131.37, 130.89, 130.55,
130.19, 121.76, 120.68, 120.31, 120.22, 117.53, 117.40, 98.11 (C-1ꢀ),
97.72 (C-1ꢀ), 73.30 (C-4ꢀ), 73.25 (C-4ꢀ), 72.20 (C-5ꢀ), 72.02 (C-5ꢀ),
71.90 (C-2ꢀ), 71.59 (C-2ꢀ), 68.96 (C-3ꢀ), 68.05 (C-4), 67.99 (C-4),
62.27 (C-2), 62.18 (C-2), 29.11 (C-3), 27.46 (C-3), 17.94 (C-6ꢀ),
17.77 (C-6ꢀ). IR (KBr); cm−1, 3385.61, 3083.14, 3041.59, 2975.32,
2926.50, 1610.54, 1584.97, 1489.45, 1455.09, 1381.98, 1360.76,
1270.02, 1254.03, 1224.59, 1120.28, 1097.20, 1060.89, 1081.98,
1015.46, 981.51, 912.31, 835.93, 806.56, 754.64. Elemental anal.
for C15H20O6, found (calculated) (%): C, 60.83 (60.81); H, 6.78
(6.76).
3-a-◦L-Rhamnopyranosyloxyflavone (24). Yellow powder. Mp
96–98 C; [a]2D0 −14.6 (c 1.0, MeOH), 1H NMR (300 MHz, DMSO-
d6): d 8.12 (d, 1 H, J = 7.7 Hz), 7.91 (m, 2 H), 7.83 (t, 1H, J =
8.2, 7.2 Hz), 7.77 (d, 1 H, J = 8.2 Hz), 7.58 (m, 3H), 7.51 (t, 1 H,
J = 7.7, 7.2 Hz), 5.38 (d, 1 H, J = 1.1 Hz), 4.05 (brs, 1 H), 3.45
(dd, 1 H, J = 2.9, 9.2 Hz), 3.14 (t, 1 H, J = 9.4 Hz), 2.91 (m, 1
H), 0.75 (d, 3 H, J = 6.1 Hz); 13C NMR (75 MHz, DMSO-d6):
d 174.57, 157.55, 155.68, 138.02, 135.05, 131.74, 131.27, 129.69,
129.31, 126.07, 125.87, 124.27, 119.31, 102.35, 71.84, 71.33, 71.05,
70.95, 18.28. IR (KBr); cm−1 3422.65, 3064.11, 2920.93, 2930.99,
1637.47, 1616.67, 1561.11, 1467.37, 1396.97, 1203.44, 1134.14,
1059.28, 945.84, 914.98, 760.15, 695.90; MS: 385.0 (M + 1), 407.1
(M + Na), 790.9 (2M + Na). Elemental anal. For C21H20O7: found
(calculated) (%): C, 65.27 (65.63); H, 5.30 (5.21).
3-a-◦L-Rhamnopyranosyloxyflavanone (24). White solid. Mp
82–84 C; [a]2D0 −11.5 (c 1.0, MeOH), 1H-NMR (300 MHz, MeOH-
d4): d 7.86–7.83 (m, 1 H), 7.59–7.53 (m, 3 H), 7.48–7.40 (m, 3 H),
7.13–7.03 (m, 2 H), 5.33 (d, 0.45 H, J = 11.3 Hz, H-2), 5.24 (d,
0.55 H, J = 11.9 Hz, H-2), 5.14 (d, 0.55 H, J = 1.4 Hz, H-1ꢀ), 4.73
(d, 0.55 H, J = 11.9 Hz, H-3), 4.69 (d, 0.45 H, J = 11.3 Hz, H-3),
4.32–4.20 (m, 0.45 H, H-5ꢀ), 4.05 (dd, 0.55 H, J = 1.7, 3.2 Hz,
H-2ꢀ), 3.91 (d, 0.45 H, J = 1.3 Hz, H-1ꢀ), 3.68 (dd, 0.45 H, J = 3.3,
9.6 Hz, H-3ꢀ), 3.47 (dd, 0.45 H, J = 1.6, 3.2 Hz, H-2ꢀ), 3.36–3.28
(m, 1 H, 0.45 H-4ꢀ and 0.55 H-3ꢀ), 3.19 (t, 0.55 H, J = 9.6 Hz,
H-4ꢀ), 2.00 (m, 0.55 H, H-5ꢀ), 1.18 (d, 1.35 H, J = 6.2 Hz, H-
6ꢀ), 0.82 (d, 1.65 H, J = 6.2 Hz, H-6ꢀ); 13C-NMR (300 MHz,
MeOH-d4): d 193.50, 191.66, 161.54, 161.32, 137.69, 136.92,
136.71, 136.55, 129.34, 129.24, 128.75, 128.73, 127.78, 127.68,
127.21, 127.06, 122.14, 120.42, 120.08, 117.99, 117.96, 101.48,
101.33, 83.37, 83.00, 79.25, 76.90, 72.71, 72.16, 71.14, 70.92, 70.87,
70.67, 69.62, 69.09, 17.06, 16.91. IR (KBr); cm−1 3420.98, 2931.80,
1689.90, 1607.95, 1467.08, 1382.48, 1311.80, 1231.90, 1136.59,
1100.63, 1061.22, 1028.71, 980.16, 838.96, 796.89, 761.66, 696.77.
Elemental anal. for C21H22O7: Found (calculated) (%): C, 65.37
(65.28); H, 5.85 (5.70).
Removal of the benzyl on the flavanone44
Compound 28 (0.067 g, 0.1362 mmol) was dissolved in dry
EtOH (10 ml) and cyclohexene (3.4 ml), 0.67 g 10% Pd/C was
added and the suspension was stirred under 62 C for 4 h (TLC
monitoring). The catalyst was removed by filtration and the filtrate
was processed by evaporation to dryness. The residue was purified
by chromatography (1 : 2 petroleum ether–EtOAc) to afford
compound 29.
◦
7-Benzyloxy-3◦-a-L-rhamnopyranosyloxyflavanone (29). White
solid. Mp 74–76 C; [a]2D0 −10 (c 1.0, MeOH); 1H NMR (300 MHz,
CDCl3): d 7.83 (d, 1 H, J = 8.79 Hz, 0.34 H), 7.77 (d, 0.66 H, J =
8.82 Hz), 7.54–7.36 (m, 10 H), 6.70–6.67 (m 1 H), 6.53–6.50 (m, 1
H), 5.26 (s, 0.66 H-1ꢀ), 5.24 (d, 0.34 H, J = 11.21 Hz, H-2), 5.19
(d, 0.66 H, J = 11.73 Hz, H-2), 5.04 (s, 2 H), 4.66–4.59 (dd, 1
H, J = 11.24, 11.81 Hz, H-3), 4.24–4.18 (m, 0.34 H-5ꢀ and 0.66
H-2ꢀ), 3.86–3.60 (m, 0.34 H-1ꢀ, 0.34 H-2ꢀ and 0.34 H-3ꢀ), 3.41–
3.34 (m, 0.66 H-3ꢀ and 0.34 H-4ꢀ), 3.25 (t, 0.66 H-4ꢀ, J = 9.51
Hz), 1.91–1.82 (m, 0.66 H-5ꢀ), 1.22 (d, 1 H, J = 6.08 Hz), 0.83
(d, 2 H, J = 6.00 Hz); 13C NMR (125 MHz, CDCl3): d 192.12,
190.44, 165.99, 165.87, 163.49, 163.34, 137.46, 136.55, 136.11,
136.08, 129.90, 129.73, 129.47, 129.22, 129.16, 129.11, 128.74,
127.97, 127.86, 127.74, 114.43, 114.03, 111.80, 111.68, 102.14,
102.10, 100.93, 100.40, 83.63, 83.24, 78.17, 76.38, 73.24, 72.59,
71.68, 70.81, 70.75, 69.47, 68.79, 17.89, 17.82. IR (KBr): cm−1
3423.61, 3064.93, 3033.98, 2972.47, 2931.63, 1686.45, 1609.39,
1574.29, 1498.08, 1443.76, 1364.45, 1253.97, 1227.92, 1168.14,
1102.35, 1061.08, 1026.76, 981.19, 836.26, 757.39, 739.42, 697.32;
Elemental anal. for C28H28O8, found (calculated) (%), C, 68.30
(68.29); H, 5.75 (5.69).
3-a-L-Rhamnopyranosyloxychromanone (26). White solid. Mp
◦
1
60–62 C; [a]2D0 −10.4 (c 1.0, MeOH-d4), H-NMR (300 MHz,
CDCl3): d 7.84 (dd, 1 H, J = 1.57, 7.88 Hz, H-5), 7.55 (t × d,
1 H, J = 1.69, 7.86 Hz, H-7), 7.06 (t, 1 H, J = 7.52 Hz, H-6),
7.01 (dd, 1 H, J = 8.41, 8.40 Hz, H-8), 5.11 (d, 0.66 H, J =
1.34 Hz, H-1ꢀ), 4.97 (d, 0.34 H, J = 1.17 Hz, H-1ꢀ), 4.61–4.37
(m, 3 H, H-2 and H-3), 3.94–3.92 (dd, J = 1.73, 3.29 Hz, 0.66
H, H-2ꢀ), 3.87 (dd, J = 1.69, 3.35 Hz, 0.34 H, H-2ꢀ), 3.75–3.62
(m, 2 H, H-3ꢀ and H-5ꢀ), 3.43 (t, 0.66 H, J = 9.49 Hz, H-4ꢀ), 3.37
(t, 0.34 H, J = 9.58 Hz, H-4ꢀ), 1.32 (d, 2 H, J = 6.20 Hz), 1.16
(d, 1 H, J = 6.21 Hz); 13C-NMR (300 MHz, CDCl3); d 191.20,
190.47, 161.92, 161.75, 136.64, 136.45, 127.28, 127.20, 121.85,
121.72, 120.13, 119.91, 117.89, 117.84, 100.99, 99.69, 73.54, 72.74,
72.70, 72.46, 71.16, 71.06, 70.99, 70.84, 70.08, 69.72, 69.47, 69.17,
17.07, 16.74. IR (KBr); cm−1 2954.59, 2900.47, 1757.31, 1737.02,
1688.30, 1605.37, 1478.19, 1466.47, 1380.48, 1327.47, 1293.77,
1236.39, 1217.27, 1153.05, 1122.09, 1079.61, 1037.58, 954.70,
7-Hydroxy-3-a-L-rhamnopyranosyloxyflavanone (30). White
◦
solid. Mp 105–107 C; [a]2D0 −9 (c 1.0, MeOH); 1H NMR
(300 MHz, DMSO-d6): d 7.61–7.40 (m, 6 H), 6.50 (d, 1 H, J =
8.34 Hz), 6.28 (d, 1 H, J = 8.13 Hz), 5.42 (d, 0.34 H, J = 9.41
Hz), 5.28 (d, 0.66 H, J = 11 27 Hz), 4.95 (s, 0.66 H-1ꢀ), 4.68 (d,
0.66 H, J = 11.32 Hz), 4.64 (d, 0.34 H, J = 9.46 Hz), 3.90–2.94
(m, 0.34 H-1ꢀ, H-2ꢀ, H-3ꢀ, H-4ꢀ and 0.34 H-5ꢀ), 2.02–1.85 (m, 0.66
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H, H-5ꢀ), 1.01 (d, 1 H, J = 5.46 Hz), 0.68 (d, 2 H, J = 5.43 Hz);
13C NMR (125 MHz, CDCl3): d 191.41, 189.44, 167.63, 167.23,
163.54, 163.22, 138.28, 137.58, 129.68, 129.61, 129.52, 129.46,
129.22, 129.10, 128.57, 128.45, 112.75, 112.67, 112.40, 112.19,
103.29, 103.25, 101.96, 101.70, 101.21, 82.81, 82.66, 78.05, 76.35,
72.49, 71.97, 71.24, 71.00, 70.96, 70.88, 69.75, 69.45, 18.60. IR
(KBr); cm−1 3405.80, 2976.13, 2932.18, 2856.87, 1676.63, 1610.19,
1498.95, 1466.61,1453.52, 1360.98, 1262.99, 1166.58, 1098.79,
1061.74, 1036.02, 980.30, 913.18, 841.79, 812.52, 751.82, 698.65;
Elemental anal. for C21H22O8, found (calculated) (%), C, 62.66
(62.69); H, 5.62 (5.47).
Single mixed lymphocytes reaction46
The lymphocytes (5 × 105) from BALB/c mice were incubated
with the lymphocytes (5 × 105) from C57BL/6 mice, which had
been treated with mitomycin C (500 mg mL−1) for 1 h in the
presence or absence of the various concentrations of compounds
at 37 ◦C in 5% CO2 for 72 h. The proliferation of lymphocytes was
measured by MTT method. The OD540 values were determined
by an ELISA reader. Stimulation index was calculated as following
formula: stimulation index = (ODsample − ODC57BL/6)/ODBALB/c. The
experiment was repeated three times.
4ꢀ-Hydroxy-3-◦a-L-rhamnopyranosyloxyflavanone (32). White
solid. Mp 89–91 C; [a]2D0 −10 (c 1.0, MeOH); 1H NMR (300 MHz,
acetone-d6): d 7.85–7.80 (m, 1 H), 7.63–7.56 (m, 1 H), 7.45–
7.38 (m, 1 H), 7.16–7.02 (m, 2 H), 6.93–6.86 (m, 2 H), 5.31 (d,
0.34 H, J = 11.22 Hz), 5.23 (d, 0.66 H, J = 11.72 Hz), 5.14
(d, 0.66 H-1ꢀ, J = 0.85 Hz), 4.77 (d, 0.66 H, J = 11.72 Hz),
4.72 (d, 0.34 H, J = 11.22 Hz), 4.22–4.16 (m, 0.34 5ꢀ-H), 4.03–
4.00 (m, 0.34 H-1ꢀ, 0.66 H-2ꢀ), 3.70–3.56 (m, 0.34 H-3ꢀ), 3.53–
3.12 (m, 0.34 H-2ꢀ, 0.66 H-3ꢀ and H-4ꢀ), 2.28–2.21 (m, 0.66 H-
5ꢀ), 1.13 (d, 1 H, J = 6.20 Hz), 0.88 (d, 2 H, J = 6.16 Hz);
13C NMR (125 MHz, CDCl3): d 193.51, 191.60, 161.61, 161.43,
158.55, 136.74, 136.55, 129.32, 128.51, 127.75, 127.36, 127.18,
122.13, 122.06, 120.33, 119.47, 118.15, 118.13, 115.66, 101.22,
101.06, 83.30, 82.90, 78.46, 76.67, 72.81, 72.43, 71.54, 71.35, 70.93,
70.71, 69.51, 69.04, 17.59, 17.51. IR (KBr); cm−1 3406.63, 2925.55,
2854.36, 1694.34, 1607.90, 1579.01, 1519.23, 1465.25, 1370.05,
1323.59, 1278.52, 1232.14, 1172.83, 1146.97, 1104.31, 1060.79,
1033.02, 979.28, 912.45, 873.71, 832.64, 813.70, 762.45, 692.17;
Elemental anal. for C21H22O8, found (calculated) (%), C, 62.71
(62.69); H, 5.55 (5.47).
Apoptosis measurement
Sheep red blood cell-induced delayed-type hypersensitivity was
induced in BALB/c mice. Mice were sensitized by injecting 40 lL
of 2.5 × 108 mL−1 sheep red blood cells in saline into the left
footpad. Five days later, they were challenged by painting 40 lL
of 2.5 × 109 mL−1 of the cells in saline into the right footpad.
Six hours later, spleen cells were isolated and incubated with the
compounds at 37 ◦C for 12 h, then were used for apoptosis assay.
Spleen cell apoptosis was measured by flow cytometry as
previously described.47 Briefly, spleen cells were suspended in
0.1 mol L−1 citrate buffer (pH 7.2) containing 0.1% Triton X-100
and incubated at 37 ◦C for 30 min. The tubes were then vortexed
and centrifuged and the resultant pellets were washed and stained
with 10 lg mL−1 propidium iodide in the citrate buffer at room
temperature. The percentage of apoptotic nuclei was analyzed by
FACScan cytofluorimeter (Becton Dickinson).
Acknowledgements
We are grateful for financial support from the National Sci-
ence Foundation of China (Nos 20231010, 20228102, 30370351,
30230390 and 20572043).
Biological assays
Animals
Female BALB/c and C57BL/6 mice (SPF, 18–22 g) were obtained
from the Experimental Animal Center of Shanghai (Shanghai,
China). They were maintained with free access to pellet food and
water in plastic cages at 21 2 ◦C and kept on a 12 h light–dark
cycle. Animal welfare and experimental procedures were carried
out strictly in accordance with the guide for the care and use
of laboratory animals (National Research Council of USA, 1996)
and the related ethical regulations of our university. All efforts were
made to minimize animal’s suffering and to reduce the number of
animals used.
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