全 文 ：Iridoid Glycosides from Buddleja lindeyana
LU Jiang-hai1 , PU Xiao-ping1 , TU Guang-zhong2 , and ZHAO Yu-ying1＊
(1 .School of Pharmaceutical Sciences , Peking University , Beijing 100083 , China;
2.Beijng Institute of Microchemistry , Beijing 100091 , China)
Abstract:Aim　To study the chemical constituents of Buddleja lindeyana.Methods　The constituents were separat-
ed and purified by different methods of chromatography , and their structures were elucidated by IR , MS and NMR.Results
Three iridoid glycosides and two other compounds were isolated from Buddleja lindeyana.Their structures were elucidated to
be 6-O-feruloylajugol (1), erythro-6-oxo-4′-(3-methoxy l-4-hydroxyphenylglycol-8″)-feruloylaj-ugol (2), threo-6-oxo-
4′-(3-methoxyl-4-hydroxyphenylglycol-8″)-feruloylajugol(3), tetra-cosanoic acid 2 , 3-dihydroxypropyl ester(4), and ga-
lactitol(5).Conclusion　All the compounds have been isolated from this plant for the first time.Compounds 1 , 2 and 3
have protective effect agains MPP+-induced apoptosis.
Key words:iridoid gly coside;Buddleja lindeyana ;6-O-feruloylajugol
CLC number:R284.1;R284.2;R285.5　　　Document code:A　　Article ID:1003-1057(2004)3-151-04
Introduction
Buddleja lindleyana , a plant of the family Buddleiaceae ,
is widely used in southwest region of China as an anti-inflamma-
tory medicine for treating swelling , pain of lungs , and skin
wounds.Many plants of this family are reported to possess anti-
inflammatory , antibacterial and cytotoxic properties , which are
usually attributed to flavonoids , sesquiterpenes and phenylpro-
panoid glycosides
[1-4] .But reports on the chemical constituents
and bioactivities of Buddleja lindeyana are scarcely available in
literature.In our recent studies , five compounds were isolated
from the plant.On the basis of spectroscopic evidence , their
structures were identified as 6-O-feruloylajugol (1), erythro-6-
oxo-4′-(3-methoxyl-4-hydroxyphenylglycol-8″)-feruloylajugol(2),
threo-6-oxo-4′-(3-methoxyl-4-hydroxyphenylglycol-8″)-feruloyla-
jugol(3), tetracosanoic acid 2 ,3-dihydroxy propylester(4), and
galactitol(5), which were isolated from this plant for the first
time.Their anti-apoptosis activity was studied , of which com-
pounds 1 , 2 and 3 can protect the mesencenphalic neurons from
the toxicity of 1-methyl-4-phenylpyri-dinium ion(MPP+).
Received date:2004-03-11.
Foundat ion item:National Natural Science Foundation of China
(20172004)
＊Corresponding author:Tel 86-10-82801592 , Fax 86-10-82801592
E-mail nmechem@mail.bjmu.edu.cn
Result and Discussion
The whole plant of Buddleja lindeyana was powdered
and extracted with 95%EtOH.The extract was suspend-
ed in water and extracted successively with petroleum
ether , EtOAc , and n-BuOH.The n-BuOH soluble part
was separated by a combination of D101 macroporous resin ,
silica gel , Rp-18 silica gel and Sephadex LH-20 column
chromatographies to obtain compounds 1-5.
Compound 1 was obtained as an amorphous white
powder , The 1H NMR spectrum exhibited the proton signal
at δ5.50(1H , d , J =2Hz , H-1), two cis-olefinic pro-
tons at δ6.22(1H , d , J =4.5 Hz , H-3), 4.97(1H ,
d , J =4.5 Hz , H-4), which were characteristic of H-3 ,
H-4 and H-1 of an iridoid moiety , other protons at 1.97-
2.58 , a methyl group at δ1.38(3H , s), as well as an
anomeric proton of sugar at δ4.65(1H , d , J =7.5 Hz).
The
13
C NMR spectrum of 1 showed the corresponding car-
bon signals at δ93.4 , 140.1 , 104.6 , four carbon signals
at δ51.6 , 47.9 , 39.4 , 26.1 , and an anomeric carbon at
δ99.4 , which suggested that 1 is an iridoid glycoside.
Furthermore , 1H NMR spectrum gave a typical ABX system
at δ7.17(1H , br.s), 7.07(1H , br.d , J =8.1 Hz),
6.8(1H , d , J =8.1Hz), methoxy group at δ3.88(3H ,
s), and trans-olefinic protons at δ7.64 , 6.40(each 1H ,
d , J =15.9 Hz), indicating that 1 has a feruloyl struc-
ture.In the 13 C NMR spectrum , all signals of 1 are in
151Journal of Chinese Pharmaceutical Sciences 2004 , 13(3)
Figure 1　Structures of compounds 1-3
good agreement with those of 6-O-feruloylajugol[ 3] .Thus
compound 1 was established as 6-O-feruloylajugol.As
above , the structures of 2-5 were identified on the basis
of physical and chemical properties , spectral analysis , and
comparison with those in literature
[ 3 , 5, 6] .
Experimental
Apparatus and reagents
Melting points were determined on an X4 apparatus
and uncorrected.1H and 13C NMR , DEPT were recorded
with a Bruker AM-500 instrument.MS was taken onAEI-
MS-50 spectrometer.Silica gel was purchased from Ma-
rine Chemical Factory in Qingdao.Sephadex LH-20
(Chemical Reagent Factory , Tianjin)was used.
Extraction and isolation
The plant was collected in Jingzhai , Guangxi Autono-
mous Region of China , and identified by Prof.Chen Hu-
biao , and a specimen was deposited in the Department of
Natural Medicines , Peking University .
The whole plant of B .lindeyana (13.5 kg)was
powdered and extracted with 95%EtOH.The extract was
suspended in water and extractee with petroleum ether ,
EtOAc, and n-BuOH successively.The EtOAc extract
(57 g)was separated by silica gel column chromatography ,
eluted with petroleum ether-acetone (99∶1※20∶80)to
afford 26 fractions.Fr.13-14 were subjected to silica gel
column chromatography , eluted with petroleum ether-ace-
tone(5∶1)to give Compound 4(20 mg).The n-BuOH
extract(300 g)was fractionated by CC on silica gel , using
CHCl3-MeOH-H2O(65∶35∶10 , lower layer)as eluent to
give 14 fractions.Fr.9-13 were chromatographed on a
Rp-18 silica gel column (30%MeOH-H2O)to give 22
fractions.After evaporation , Fr.6 yielded compound 5
(50 mg).Fr.17-18(2 g)were separated by the combi-
nation of Rp-18 silica gel column (30% MeOH-H2O),
Sephadex LH-20 (50%MeOH-H2O)and PTLC [ CHCl3-
MeOH-H2O(65∶35∶10 , lower layer)] to afford compound
1(20 mg), and a mixture of 2 and 3(80 mg).
Identification
Compound 1 　Amorphous white powder , 1H NMR
(300MHz , CD3OD)δ:5.50(1H , d , J =2.0Hz , H-1),
6.22(1H , d , J =4.5 Hz , H-3), 4.97(1H , d , J =4.5
Hz , H-4), 2.90(1H , br.dd , J =9.5 , 2.0 Hz , H-5),
4.92(1H , m , H-6), 2.23(1H , dd , J =14.0 , 6.0Hz , H-
7-A), 1.97(1H , dd , J =14.0 , 6.0 Hz H-7-B), 2.58
(1H , dd , J =9.5Hz , 2 Hz , H-9), 1.38(3H , s , H-10);
7.17(1H , br.s , H-2′), 6.80(1H , d , J =8.1 Hz , H-
5′), 7.07(1H , br.d , J =8.1 Hz , H-6′), 7.64(1H , d ,
J =15.9 Hz , H-7′), 6.40(1H , d , J =15.9 Hz , H-8′),
3.88(3H , OCH3);4.65(1H , d , J =7.5 Hz , glc-H-1).
13
C NMR(75MHz , CD3OD)(see Table 1).All data were
identical with those of 6-O-feruloylajugol.
　　Compound 2　Amorphous white powder , 1H NMR
(300 MHz , (CD3)2CO):δ5.50(1H , d , J =2.0 Hz ,
H-1), 6.21(1H , d , J =4.5 Hz , H-3), 4.97(1H ,d ,
J =4.5Hz , H-4), 2.92(1H , d , J =9.5 Hz , H-5),
4.92(1H , m , H-6), 2.33 (1H , dd , J =14.0 , 6.0
Hz , H-7-A), 1.99 (1H , dd , J =14.0 , 4.0 Hz ,H-7-
B), 2.57(1H , d , J =9.5 Hz , H-9), 1.38 (3H , s ,
H-10);7.17(1H , br.s , H-2′), 6.96(1H , d , J =
8.1 Hz , H-5′), 7.07(1H , br.d , J =8.1Hz ,H-6′),
7.64(1H , d , J =15.9Hz , H-7′), 6.41(1H , d , J =
15.9 Hz , H-8′);δ7.02(1H , br.d , J =2.0 Hz , H-
2″), 6.71(1H , d , J =8.1Hz , H-5″), 6.84(1H , br.
d , J =8.1 Hz , H-6″), 4.89 (1H , d , J =5.5 Hz , H-
7″), 4.49 (1H , m , H-8″), 3.82 (3H , s , OCH3),
3.79(3H , s , OCH3);4.66(1H , d , J =7.5 Hz , glc-
H-1).13C NMR(CD3COCD3)(see Table 1).All data
were identical with those of erythro-6-O- 4′-(3-methoxyl-
4-hydroxyl phenyl-8″)-feruloylajugol.
Compound 3　Amorphous white powder , 1H NMR
(300 MHz , (CD3)2CO):δ5.50(1H , d , J =2.0 Hz ,
H-1), 6.21 (1H , d , J =4.5 Hz , H-3), 4.97 (1H ,
dd , J =4.5 Hz , H-4), 2.93(1H , d , J =9.5 Hz , H-
5), 4.94 (1H , m , H-6), 2.24 (1H , dd , J =14.0
Hz , 6 Hz , H-7-A), 1.99(1H , dd , J =14.0 , 4.0 Hz ,
152 Journal of Chinese Pharmaceutical Sciences 2004 , 13(3)
H-7-B), 2.57(1H , d , J =9.5 , 2.0 Hz , H-9), 1.38
(3H , s , H-10), 7.22(1H , br.s , H-2′), 7.05 (1H ,
d , J =8.1 Hz , H-5′), 7.10(1H , dd , J =8.0 , 2.0
Hz , H-6′), 7.62 (1H , d , J =15.9 Hz , H-7′), 6.43
(1H , d , J =15.9 Hz , H-8′);7.02(1H , d , J =2.0
Hz , H-2″), 6.75(1H , d , J =8.1 Hz , H-5″), 6.86
(1H , dd , J =8.1 Hz , 2 Hz , H-6″), 4.89(1H , d ,
J =5.5 Hz , H-7″), 4.49(1H , m , H-8″), 3.89(3H ,
s , OCH3), 3.81(3H , s , OCH3), 4.67 (1H , d , J =
8.1 Hz , glc-H-1).13 C NMR (75 MHz , (CD3)2CO)
(see Table 1).All data were identical with those of threo-
6-O-4′-(3-methoxyl-4-hydroxyl phenyl-8″)-feruloylajugol.
Table 1　13C NMR data of Compounds 1-3(CD3OD)
No. 1 2 3
1 93.4 93.7 93.7
3 140.1 141.4 141.4
4 104.6 104.8 104.8
5 39.4 39.2 39.7
6 80.3 80.7 80.7
7 47.9 48.2 48.2
8 79.1 79.4 79.4
9 51.6 51.9 51.9
10 26.1 26.4 26.4
1′ 127.1 129.9 129.8
2′ 111.6 112.2 112.2
3′ 149.6 148.6 149.3
4′ 150.9 151.8 152.3
5′ 116.5 117.6 117.8
6′ 124.2 123.9 123.8
7′ 146.9 146.5 146.5
8′ 115.5 115.9 116.1
9′ 169.1 169.1 169.1
OCH3 56.4 56.9 56.9
1″ 134.1 134.2
2″ 112.6 112.6
3″ 147.4 147.3
4″ 151.9 152.1
5″ 117.2 117.4
6″ 121.1 121.4
7″ 74.4 73.8
8″ 85.8 86.5
9″ 62.7 62.3
OCH3 56.6 56.8
Glc 1 99.4 99.7 99.7
2 74.8 75.1 75.1
3 78.2 78.2 78.2
4 71.7 71.7 72.1
5 78.1 78.4 78.2
6 62.9 63.2 63.2
　　Compound 4　Amorphous white powder , mp 86-
87 ℃, EIMS m z:443[M+1] +.1H NMR(CDCl3):
2.37(2H , t , J =7.5Hz , H-2), 1.65(2H , m , H-3),
1.32-1.28(42H , br.s), 0.87(3H , t , H-24);4.74
(1H , dd , J =11 Hz , 4.7 Hz , H-1α), 4.66(1H , dd ,
J =11.0 Hz , 6.3 Hz , H-1β), 4.26(1H , m , H-2′),
4.15(2H , d , J =5.0 Hz , H-3′).13 C NMR:174.37
(C-1), 34.2(C-2), 25.0(C-3), 29.7-29.1(C-4-C-
21), 32.0(C-22), 22.7(C-23), 14.13(C-24), 65.2
(C-1′), 70.87 (C-2′), 63.35 (C-3′).All data were
identical with those of tetracosanoic acid 2 , 3-di-
hydroxypropyl ester.
Compound 5 　Needle crystal (MeOH), mp
170 ℃, IR (cm-1):3 280 , 2 930 , 1 455 , 1 427 ,
1 280 , 1 090 , 1 020 , 930 , 887 , 705 , 630.Rf value
and mp were identical with those of an authentic sample ,
galactitol.All data were identical with those of galactitol.
Assay for anti-apoptotic activity of Buddleja
lindleyana
Cell culture was performed according to the method
used by Mochizuki
[ 7] .The test was conducted on control
group , MPP+ model group , positive drug group , and
three iridoid glycosides groups.The cell viability was de-
termined using a modified MTT
[ 14]
assay.In brief , mese-
ncephalic neurons were seeded in 96-well plates at a den-
sity of 1×104 cells per well.The cultures were grown for
4 d , and then medium was changed to that containing 40
μg·mL-1 concentration of three iridoid glycosides.After 6
h , 100 μm MPP+ was added.After incubation for up to
48 h , MTT [ 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-dipheny-
ltetrazolium bromide] solution (5 μg·mL-1 in DMEM)
was added to the 96-well plates and the cells were incubated
Table 2　Protective effect of iridoid glycosides against MPP+-
induced apoptosis
Group Concentrat ion(μg·mL-1) Optical density( x±s)
Cont rol 1.14±0.02
MPP+ 0.78±0.02
EGF 0.87±0.02＊
Compound 1 40 1.12±0.10＊
Compounds 2+3 40 1.25±0.06＊
Signif icance:＊P<0.05 vs MPP group;n=3;EGF was used as the positive
control
153Journal of Chinese Pharmaceutical Sciences 2004 , 13(3)
for 4 h at 37 ℃.After the medium was removed , the
cells and dye crystals were solubilized by adding 200 μL
of DMSO , and the absorption was measured at 570 nm
(540 nm as a reference)with a model 550 microplate
reader(Bio-Rad).The absorption was larger and cell vi-
ability was higher.As a positive control , 100 ng·mL-1 of
epidermal growth factor(EGF)was used see (Table 2).
Conclusion
Compounds 1 , 2 and 3 have the anti-apoptotic activi-
ty , and can protect the mesencenphalic neurons from the
toxicity of 1-methyl-4-phenylpyridinium ion(MPP+).
Acknowledgement
This project was supported by National Natural Sci-
ence Foundation of China(No.20172004).
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33.
醉鱼草中的环烯醚萜苷
陆江海1 , 蒲小平1 , 涂光忠2 , 赵玉英1
(1.北京大学药学院 , 北京 100083;2.北京微量化学研究所 , 北京 100091)
摘要:目的　研究醉鱼草(Buddleja lindeyana)的活性成分。方法　应用多种色谱技术分离纯化 , 用 IR ,MS 和 NMR等谱学
方法解析化合物结构 。结果与结论　从醉鱼草(Buddleja lindeyana)的全草分离到 5 个化合物 , 其中三个为环烯醚萜苷。它们
的结构为:6-氧-阿魏酰筋骨草苷(1), 赤式-6-氧-4′-(3-甲氧基-4-羟基苯丙三醇-8″)-阿魏酰筋骨草苷(2), 苏式-6-氧-4′-(3-甲氧
基-4-羟基苯丙三醇-8″)-阿魏酰筋骨草苷(3), 二十四烷酸-α-单甘油脂(4), 半乳糖醇(5)。上述成分均为首次从醉鱼草中得
到。且化合物 1 , 2 和 3对神经细胞有保护作用。
关键词:醉鱼草;环烯醚萜苷;6-氧-阿魏酰筋骨草苷
154 Journal of Chinese Pharmaceutical Sciences 2004 , 13(3)