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鬼箭锦鸡儿中紫檀烷类化合物抗真菌活性研究(英文)



全 文 :中国现代应用药学杂志 2009 年 9 月第 26 卷第 9 期 Chin JMAP, 2009 September, Vol.26 No.9 ·691·
·论 著·

Antifungal Activity of Pterocarpans from Caragana jubata (pall.) Poir.

SONG Ping1, YANG Xinzhou2, YU Jun1 (1.Department of Chemistry, Qinghai Nationalities Institute, Xining 810007, China;
2.Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Basel CH-4056, Switzerland)

ABSTRACT: OBJECTIVE To study the antifungal activity of pterocarpans from Caragana jubata (pall.) poir.. METHODS
The compounds have been separated and purified by polyamine resin and silica gel column chromatography as well as the
preparative HPLC method from the active fraction. The minimum inhibitory concentration(MIC) were measured by broth
microdilution method according to document M27-A published by the National Committee for Clinical Laboratory Standards
(NCCLS). RESULTS The CHCl3 extract of the MeOH extract prepared from the whole plant of Caragana jubata (pall.)
Poir.exhibited good antifungal activity. Bioassay-directed fractionation led to the purification of five pterocarpans, 3-methoxy
maackiain (1), maackiain (2), 3-methoxy-9-hydroxy pterocarpan (3), 3,9-dimethoxy pterocarpan (4) and 3-methoxy-4,9-dihydroxy
pterocarpan (5), in which compounds 2 showed potential antifungal activity against three Candida strains at the level of MICs
6.25-25 μg·mL-1. CONCLUSION All of the compounds were isolated from the title plant for the first time. And biological
validation showed pterocarpans were responsible for antifungal activity.
KEY WORDS: Caragana jubata (pall.) Poir.; Pterocarpans; antifungal activity

鬼箭锦鸡儿中紫檀烷类化合物抗真菌活性研究

宋萍 1,杨新洲 2,于军 1 (1.青海民族学院化学系,西宁 810007;2.巴塞尔大学生物制药学院药物科学系,巴塞尔 CH-4056,瑞士)

摘要:目的 研究鬼箭锦鸡儿中紫檀烷类化合物的抗真菌活性。方法 在生物活性指导下,利用聚酰胺、硅胶常规色谱和制
备型高效液相色谱方法进行化合物分离,采用波谱技术和化学方法鉴定化合物的结构。最低抑菌浓度(MIC)测定参照美国国
家临床实验室标准化委员会(NCCLS)推荐的 M27-A 方案中的微量肉汤稀释法。结果 鬼箭锦鸡儿整株植物氯仿部分显示好
的抗真菌活性,活性追踪分离得到五个紫檀烷类化合物,分别是 3-甲氧基高丽槐素(1)、高丽槐素(2)、3-甲氧基-9 羟基紫檀
烷(3)、3,9-二甲氧基紫檀烷(4)和 3-甲氧基-4,9 二羟基紫檀烷(5),其中化合物 2 面对三种念珠菌菌株,显示出潜在的抗真
菌活性,最低抑菌浓度范围为 6.25~25 μg·mL-1。结论 所有化合物均为首次从该植物分离得到,并且生物活性结果确认紫檀
素类化合物是有效的抗真菌成分。
关键词:鬼箭锦鸡儿;紫檀烷;抗真菌活性
中图分类号:R284.1; R284.2; R285.5 文献标志码:B 文章编号:1007-7693(2009)09-0691-04

Caragana jubata (pall.) Poir. which is subordinate
to Caragana Fabr. in a perennial leguminous bush
endemic to the northwest of China. It is one of the
oldest medicinal plants used in traditional Tibetan
medicines, widely distributed in Qinghai, Tibet,
Sichuan, Gansu, Ningxia, Xinjiang and Inner
Mongolia provinces, where they live in Shady slope or
half-shade slope at an altitude of 3 000-4 700 m[1-3].
The whole plants of Caragana jubata (pall.) Poir.
have been long used to treat some cardiovascular
diseases, such as atherosclerosis, hyperlipidemia,
hypertension, blood circulation disorder, blood stasis
and so on, and it is also used to treat arthritis and
abnormal menstruation, and relieve muscle pain[4-6].
However, the limited chemical principles, resveratrol,
Cassigarol E, scirpusin B, a few volatile oil and
flavonoids were isolated from C. jubata (pall.) Poir[7-8].
As part of our in vitro antimicrobial screening efforts,
the CHCl3 fraction of the methanol extract from the
whole plants of C. jubata (pall.) Poir. showed potential
antifungal activity against three Candida strains,
Candida albicans, C. krusei, and C. parapsilosis.
Bioassay-guided fractionation led to the purification
of five pterocarpan, 3-methoxy maackiain (1),
maackiain (2), 3-methoxy-9-hydroxy pterocarpan (3),
3,9-dimethoxy pterocarpan (4) and 3-methoxy-4, 9-
基金项目:国家自然科学基金项目(30660221);青海省自然科学基金项目(2006-N-554)
作者简介:宋萍,女,教授 Tel: (0971)8800307 E-mail: spzhe@126.com
·692· Chin JMAP, 2009 September, Vol.26 No.9 中国现代应用药学杂志 2009 年 9 月第 26 卷第 9 期
dihydroxy pterocarpan (5). All of the compounds were
firstly isolated from this plant, and maackiain (2)
showed potential antifungal activity against three
Candida strains at the level of MICs 6.25-25 μg·mL-1.
1 Instrument and experiment material
1.1 Instruments and regents
DL-CJ-2N Ultraclean working table,MJ-180B
Fungi incubators, Electronic Analytical Balance,
Abbott Bacterial Analyzer,DK-98-1 Electron constant
temperature water bath boiler.
Column chromatography (CC), silica gel
(200-300 and 300-400 mesh), polyamide resin
(100-200 mesh), Analytical TLC (precoated silica gel
plates, GF-254), Preparative and Semi-preparative
HPLC system (two PrepStar SD-1 solvent delivery
modules, a ProStar UV-Vis 320 detector and a ProStar
701 Fraction Collector), a LiChrospher 100 RP-18
column (220 mm×25 mm, 12 μm), Perkin-Elmer 341
polarimeter, Bruker DRX-400 MHz spectrometer.
Fluconazole and Chloroamphenicol were pur-
chased from Three-dimensional Shanghai pharmacy
Ltd.(batch number 20041104 and 20060309), and all
other regents were of analytical grade.
1.2 Used microorganisms
1.2.1 Bacterial strains Klebsislla pneumoniae (Kp)
was obtained as hospital isolates from Huashan
Hospital, Shanghai, Staphylococcus aureus ATCC
25923 (Sa), S. epidermidis ATCC 26069 (Se), Bacillus
sublitis ATCC 6633 (Bs), Escherichia coli ATCC
25922 (Ec), Cryptococcus neoformans (Cn) were
purchase from the National Institute for the Control
of Pharmaceutical and Biological Products (NICPBP,
Beijing, China).
1.2.2 Fungal strains Candida albicans ATCC 64550
(Ca), C. krusei ATCC 6258 (Ck), C. parapsilosis ATCC
22019 (Cp), Torulopsis glabrata (Tg) were purchase from
the National Institute for the Control of Pharmaceutical
and Biological Products (NICPBP, Beijing, China).
1.3 Plant material
The whole plants of Caragana jubata (pall.) Poir.
were collected in September 2006 in Guo-luo county,
Qinghai province, PR China and identified by Prof. Jie
Duo, Qinghai Institute of Tibetan Medicine. A voucher
specimen (No. 2006CJP09) is deposited in the
Herbarium of Qinghai Nationalities Institute, Xining,
Qinghai province, PR China.
2 Methods and results
2.1 Extraction and isolation
Air-dried whole plants of Caragana jubata (pall.)
Poir. (1 kg) were ground and then percolated with
methanol. The concentrated extract was suspended in
H2O and partitioned successively with hexane, CHCl3,
EtOAc and n-BuOH to afford Hexane fraction (34 g),
CHCl3 fraction (76 g), EtOAc fraction (45 g), n-BuOH
fraction (59 g) and water fraction (31 g), respectively.
20 g of the CHCl3 fraction was subjected to CC over
polyamide resin (500 g) eluted with 5 % and 95 %
aqueous EtOH. The 95% EtOH fraction (12 g) was
subjected silica gel column eluted gradiently with
CHCl3/MeOH. 1.3 g of the evaporated residue from
CHCl3/MeOH elution was subjected to preparative
HPLC (CH3CN in H2O from 15 % to 80 %) to yield 1
(10 mg) and 2 (105 mg), respectively. 0.97 g of the
evaporated residue from CHCl3/MeOH elution was
subjected to preparative HPLC (CH3CN in H2O from
25 % to 100 %) to yield 3 (54 mg), yield 4 (5 mg) and
yield 5 (13 mg), respectively. The purity of the
isolated five compounds was above 95% (HPLC).
2.2 Identification
3-Methoxy maackiain (1) colorless crytals
(MeOH); mp, 169-169 ℃ ESIMS m/z 321 [M+Na]+,
299 [M+H]+; 1H-NMR (CDCl3): δ 7.40 (1H, d, J = 8.6
Hz), 6.72 (1H, s), 6.61 (1H, dd, J = 8.6, 2.6 Hz), 6.42
(1H, d, J = 2.6 Hz), 6.40 (1H, s), 5.90 (1H, brs), 5.85
(1H, brs), 5.46 (1H, d, J = 6.9 Hz), 4.19 (1H, dd, J =
10.9, 4.9 Hz), 3.78 (3H, s), 3.61 (1H, t, J = 10.9 Hz),
3.46 (1H, m). The above data are consistent with those
of the litereture’s[9-10].
Maackiain (2) colorless crytals (MeOH); mp,
179-181 ℃; ESIMS m/z 307 [M+Na]+, 285 [M+H]+;
1H-NMR (CDCl3): δ 7.40 (1H, d, J = 8.5 Hz), 6.73
(1H, s), 6.57 (1H, dd, J = 8.5, 2.6 Hz), 6.41 (1H, s),
6.40 (1H, d, J = 2.6 Hz), 5.90 (1H, brs), 5.87 (1H, brs),
5.43 (1H, d, J = 7.0 Hz), 4.18 (1H, dd, J = 11.0, 5.0
Hz), 3.61 (1H, t, J = 11.0 Hz), 3.42 (1H, m). The
above data are consistent with those of the
litereture’s[9-10].
3-Methoxy-9-hydroxy-pterocarpan (3) colorless
crytals (CHCl3); mp, 64-65 ; E℃ SIMS m/z 293
[M+Na]+, 271 [M+H]+; 1H-NMR (CDCl3): δ 7.39 (1H,
d, J = 8.4 Hz), 7.17 (1H, d, J = 8.8 Hz), 6.57 (1H, dd,
J = 8.4, 2.6 Hz), 6.46 (1H, d, J = 8.8, 2.3 Hz), 6.43
(1H, d, J = 2.6 Hz), 6.40 (1H, d, J = 2.3 Hz), 5.46 (1H,
d, J = 6.6 Hz), 4.21 (1H, dd, J = 10.4, 4.3 Hz), 3.79
(3H, s), 3.60 (1H, t, J = 10.4 Hz), 3.56 (1H, m). The
above data are consistent with those of the
中国现代应用药学杂志 2009 年 9 月第 26 卷第 9 期 Chin JMAP, 2009 September, Vol.26 No.9 ·693·
litereture’s[11].
3,9-Dimethoxy-pterocarpan (4) colorless oil;
ESIMS m/z 307 [M+Na]+, 285 [M+H]+; 1H-NMR
(CDCl3): δ 7.42 (1H, d, J = 8.2 Hz), 7.12 (1H, d, J =
8.8 Hz), 6.62 (1H, dd, J = 8.2, 2.6 Hz), 6.47 (1H, d, J =
8.8, 2.3 Hz), 6.45 (1H, d, J = 2.6 Hz), 6.42 (1H, d, J =
2.3 Hz), 5.50 (1H, d, J = 6.6 Hz), 4.21 (1H, dd, J =
10.2, 4.4 Hz), 3.78 (3H, s), 3.76 (3H, s), 3.60 (1H, t, J =
10.2 Hz), 3.57 (1H, m). The above data are consistent
with those of the litereture’s[11].
3-Methoxy-4,9-dihydroxy pterocarpan (5) color-
less crystal; mp, 173~ 175 ℃; ESIMS m/z 309
[M+Na]+, 287 [M+H]+; 1H-NMR (CDCl3): δ7.45 (1H,
d, J = 8.4 Hz), 6.98 (1H, d, J = 8.6 Hz), 6.69 (1H, dd,
J = 8.4, 2.5 Hz), 6.77 (1H, d, J = 8.6 Hz), 6.48 (1H, d,
J = 2.5 Hz), 5.51 (1H, d, J = 6.7 Hz), 4.23 (1H, dd, J =
10.2, 4.3 Hz), 3.81 (3H, s), 3.62 (1H, t, J = 10.2 Hz),
3.55 (1H, m). The above data are consistent with those
of the litereture’s[12].
2.3 Experiment Methods
The antimicrobial activity was determined using
broth dilution techniques as previously described[13-14].
2.4 Screening for antimicrobial activity
The antimicrobial activity was determined using
broth dilution techniques as previously described[13-14].
The solutions (maximum concentration) of the
compounds (i.e. the compounds that induced zones of
inhibition) were prepared in DMSO, serially (2-fold)
diluted and 0.5 mL of each dilution was introduced
into a test tube containing 4.4 mL of Selenite broth;
then 0.1 mL of bacteria suspension (5×105 cfu·mL-1)
was added and the mixture was homogenized. The
total volume of the mixture was 5 mL, with the test-
compound concentrations in the tube ranging from
200 to 6.25 μg·mL-1 and those of the standard
compounds, i.e. chloroamphenicol and fluconazole,
ranging from 256 to 2.0 μg·mL-1 and 200 to 1.56
μg·mL-1, respectively. After 24 h of incubation at 37 ℃,
the MIC values were reported as the lowest
concentration of anti-microbial that prevented visible
growth. Results and Compound structural formula as
follows Tab 1 and Fig 1.
Tab 1 Antimicrobial activity of five fractions and compounds 1-5 in MIC values (μg·mL-1)
表 1 5 个组分和 1~5 化合物的抗菌活性最低抑菌浓度值 (μg·mL-1)
Sa Se Es Ec Kp Ca Ck Cp Tg Cn
Fractions
Hexane 200 100 >200 >200 200 >200 >200 >200 >200 >200
CHCl3 >200 >200 100 >200 >200 25 50 25 100 >200
EtOAc >200 200 >200 >200 200 200 >200 200 >200 >200
n-BuOH >200 200 200 100 200 200 100 200 >200 >200
Water 200 200 >200 100 >200 200 >200 200 >200 >200
Compounds 1 200 200 >200 >200 >200 50 25 25-50 200 100
Compounds 2 200 100 >200 >200 >200 12.5 6.25 25 50 50-100
Compounds 3 >200 >200 200 >200 >200 100 100 50 >200 >200
Compounds 4 >200 >200 >200 >200 >200 50 100 50 >200 200
Compounds 5 >200 >200 >200 >200 >200 25 50 50 200 200
Chloroamphenicol 4.0 4.0 8.0 2.0
Fluconazole 1.56 50 1.56 6.25-12.5 50
Note: MIC was defined as the lowest concentration that inhibited visible growth. Chloroamphenicol and Fluconazole were used as positive control agents
注:MIC 定义为最低抑菌生长浓度;氯霉素和氟康唑被用于阳性对照药剂

Fig 1 Structures of pterocarpans compounds 1-5
图 1 1~5 紫檀烷化合物的结构
3 Discussion
The CHCl3-soluble fraction of the MeOH extract
prepared from the whole plant of Caragana jubata
(pall.) Poir. exhibited potentially antifungal activities
against three Candida species (C. albicans, C. krusei,
C. parapsilosis). The bioassay-guided isolation of the
CHCl3-soluble fraction yielded five pterocarpans. The
pterocarpan analogues are the major class of
constituents of Caragana jubata (pall.)Poir., in
which compounds 2 and 3 occurred especially as
major second metabolites with high contents in the
whole plants. All the isolates were tested on
antimicrobial assays, and some of the pterocarpan
derivatives described in this work are markedly potent
in vitro against three pathogenic fungal strains. The
antifungal activity of compounds 1 and 2 against C.
·694· Chin JMAP, 2009 September, Vol.26 No.9 中国现代应用药学杂志 2009 年 9 月第 26 卷第 9 期
krusei was even more than that of the known antibiotic
agent, fluconazole. Antifungal results of compounds
1–5 and their structural features have inferred the
preliminary patterns of structure-activity relationship.
Compounds 1-5 with the pterocarpan skeleton are
more or less active, in which compounds 1 and 2
bearing a 8,9-methylenedioxy group are more active
than compounds 3-5 with a hydroxyl or methoxy
group at position 9 and 10. If the hydroxyl group at
position 3 is substituted by the methoxy group,
compounds 1, 3 and 4 becomes less active. Compound
5 with an additional hydroxyl group at position 4
showed stronger activity than 3.

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收稿日期:2008-10-31



穿膜肽 R6 促进低分子肝素大鼠肠道吸收的研究

吕慧侠,张振海,孙博,周建平*(中国药科大学药剂教研室,南京 210009)

摘要:目的 研究穿膜肽 R6 促进低分子肝素大鼠肠道吸收的作用。方法 以用药前后血液凝固时间的变化为指标,采用
大鼠十二指肠给药的方法评价 R6 对低分子肝素的促吸收作用,采用肠袢法研究 R6 的主要促吸收部位。结果 R6 作为吸
收促进剂,低分子肝素十二指肠给药后凝血时间显著延长,R6 在十二指肠、空肠、回肠部位均显示促吸收作用,且在回
肠部位的促吸收效果最显著。结论 R6 能够显著促进低分子肝素的大鼠肠道吸收。
关键词:低分子肝素;穿膜肽;六聚精氨酸
中图分类号:R944.9 文献标志码:A 文章编号:1007-7693(2009)09-0694-04

Intestinal Absorption Studies on Low Molecular Weight Heparin in Rats Using Cell-penetrating Peptide
R6 as Absorption Enhancer

LV Huixia, ZHANG Zhenhai, SUN Bo, ZHOU Jianping* (Department of Pharmaceutics, China Pharmaceutical
University, Nanjing 210009, China)

ABSTRACT: OBJECTIVE To study the intestinal absorption of low molecular weight heparin (LMWH) in rats using
cell-permeable peptides R6 as absorption enhancer. METHODS The intestine absorption enhancing effect of R6 on LMWH
作者简介:吕慧侠,女,讲师 *通信作者:周建平,男,教授 Tel: (025)83271272 E-mail: zhoujpcpu@yahoo.cn