全 文 ：引种栽培品药用大黄中蒽醌类成分分析
康毅华1 ,陈智岩1 ,王振月1 ,王谦博2 ,唐先明1 ,周哓慧1
(1.黑龙江中医药大学 ,黑龙江 哈尔滨 150040;2.黑龙江省药品检验所 ,黑龙江 哈尔滨 150010)
摘　要:目的:分析引种栽培品药用大黄中蒽醌类成分的情况 。方法:采用高效液相色谱法及薄层色谱
法测定 ,色谱柱为 Thermo C18柱(250mm×4.6mm),流动相为甲醇:0.1%磷酸水=70:30;柱温:室温;流
速:1ml min;检测波长:254nm。展开剂:石油醚(60 ～ 90℃)-甲酸乙酯-甲酸(15:8:1)的上层溶液;检测
波长:365nm。结果:引种栽培品药用大黄中蒽醌类成分含量高于原产地甘肃药用大黄对照药材。
关键词:栽培品药用大黄;蒽醌类成分;高效液相色谱法;薄层色谱法
中图分类号:R927.2　　　文献标识码:A　　　文章编号:1002-2392(2007)03-0030-02
收稿日期:2007-02-13　　修回日期:2007-04-30
作者简介:康毅华(1954-),女 ,高级实验师 ,主要从事中药化学方面的
研究。
　　大黄为蓼科植物药用大黄(Rheum officinale Baill.)
的干燥根及根茎 。大黄是我国传统药材之一 ,并为我
国重要的出口商品。大黄在植物泻下药中占有重要地
位 ,世界上大多数国家的药典均有收载 。现代药理实
验研究表明[ 1] :大黄具有泻下 、抗菌 、抗病毒 、利胆 、收
敛 、消炎 、止血等多方面的作用 。其发挥作用的活性物
质主要为蒽醌类衍生物。因此 ,蒽醌类成分含量的高
低在很大程度上决定着大黄的品质[ 2] 。本课题对栽培
品药用大黄进行了定性定量研究 ,为其替代野生药用
大黄药材提供了实验基础 。
1　实验材料
1.1　药品与试剂
药用大黄药材的干燥根 ,甘肃中医学院的种子 ,引
种栽培于黑龙江中医药大学药用植物园;药用大黄对
照药材 、大黄素 、大黄酸 、大黄素甲醚 、白藜芦醇由中国
药品生物制品检定所提供;甲醇为 Dikma公司产品 ,色
谱纯;磷酸 、石油醚 、甲酸 、甲酸乙酯 、乙醚 、三氯甲烷均
为分析纯;水为娃哈哈纯净水;硅胶 H(中国青岛海洋
化工集团公司),羧甲基纤维素纳(上海化工厂)。
1.2　仪器与设备
美国 Waters 高效液相色谱仪(Waters 600 型泵 ,
2487型紫外检测器 , Empower色谱工作站);Thermo C18
分析柱(250mm ×4.6 mm , 5μm);Dikma 微孔滤膜
(0.45μm);瑞士 Mettler AE240 电子天平;ZF-Ⅰ型三
用紫外分析仪。
2　方法与结果
2.1　栽培品药用大黄的薄层鉴别
2.1.1　对照品溶液的制备　分别精密称取大黄酸 、大
黄酚 、大黄素 、白藜芦醇对照品适量 , 加甲醇制成每
1ml含 1mg 的溶液 ,混合均匀作为对照品溶液 。
2.1.2　供试品溶液及对照药材溶液的制备　取药用
大黄药材粉末(过四号筛)0.1g ,加甲醇 20ml ,浸泡 1h ,
滤过 ,取滤液 5ml ,蒸干 ,残渣加水 10ml使溶解 ,再加盐
酸1ml ,加热回流 30min ,立即冷却 ,用乙醚分2次振摇提
取 ,每次20ml ,合并乙醚液 ,蒸干 ,残渣加三氯甲烷 1ml使
溶解 ,作为供试品溶液。同法制备对照药材溶液。
2.1.3　薄层鉴别　照药典薄层色谱法(附录 Ⅵ B)试
验[ 3] ,吸取上述 3种溶液各 4μl ,分别点于同一以羧甲
基纤维素纳为黏合剂的硅胶 H 薄层板上 ,以石油醚
(60 ～ 90℃)-甲酸乙酯-甲酸(15:8:1)的上层溶液为
展开剂 ,展开 ,取出 , 晾干 , 置紫外光灯(365nm)下检
视 ,再置氨蒸气熏后 ,观察(结果见图1)。
图 1　大黄薄层检测结果
1.栽培品药用大黄;2.药用大黄对照药材;3.对照品混合溶液
S1 白藜芦醇;S2 大黄酚;S3 大黄素;S4 大黄酸
2.1.4　结果　供试品在与对照药材相应位置上显示
四个橙黄色荧光斑点;在与对照品白藜芦醇相应位置
上显示淡蓝色荧光斑点;在与对照品大黄酸 、大黄酚 、
大黄素相应位置上显示橙黄色荧光斑点 。置氨蒸气中
熏后 ,除白藜芦醇斑点外均变成红色。
从薄层检视结果中可以看出 ,栽培品药用大黄中
不仅含有大黄酸 、大黄素 、大黄酚等蒽醌类成分 ,还含
有对照药材中无显示的二苯乙烯类成分白藜芦醇 。
2.2　高效液相法测定栽培品药用大黄中蒽醌类成分
的含量情况
·30· ACMP.Aug , 2007 , Vol.35 , No.3
2.2.1　色谱条件　甲醇:0.1%磷酸水=70:30;柱温:
室温;流速 , 1ml min;进样量 , 20μl;检测波长:254nm。
对照品溶液及供试品 、对照药材溶液的色谱图见图 2 、
图3 。
图 2　标准品混合溶液
1.大黄素;2.大黄酚;3.大黄素甲醚
2.2.2　标准曲线的绘制　分别精密称取大黄素甲醚 、
大黄素对照品适量 ,加甲醇制成每 1ml含 0.05mg的溶
液 ,大黄酚制成每 1ml含 0.2mg的溶液 ,然后分别吸取
2μl 、4μl 、6μl 、8μl 、10μl标准溶液 ,注入高效液相色谱仪 ,
进行测定 。最后 ,以进样量X(μg)为横坐标 ,峰面积为
纵坐标(Y)绘制标准曲线 ,并得出回归方程 。
2.2.3　供试品溶液及对照药材溶液的制备　精密称
取栽培品药用大黄粉末(过四号筛)0.15g ,置具塞锥形
瓶中 ,精密加入甲醇 25ml ,称定重量 ,加热回流 1h ,放
冷 ,再称定重量 ,用甲醇补足减失的重量 ,摇匀 ,滤过。
精密量取续滤液 5ml , 置烧瓶中 ,挥去溶剂 ,加 8%盐
酸溶液 10ml ,超声处理 2min ,再加三氯甲烷 10ml ,加热
回流 1h ,放冷 ,置分液漏斗中 ,用少量三氯甲烷洗涤容
器 ,并入分液漏斗中 ,分取三氯甲烷层 ,酸液再用三氯
甲烷提取 3次 ,每次 10ml ,合并三氯甲烷液 ,减压回收
溶剂至干 ,残渣加甲醇溶解 ,转移至 10ml量瓶中 ,加甲
醇至刻度 ,摇匀 ,滤过 ,取续滤液 ,即得。同法制备药用
大黄对照药材溶液。
图 3　供试品及对照药材溶液谱图(A.栽培品药用大黄溶液;B.药用大黄对照药材溶液)
2.2.4　精密度试验　精密吸取混合对照液 20μl ,进
样 ,连续测定 6 次 , 求得峰面积值的相对标准偏差
(RSD)分别为大黄素甲醚 0.6%,大黄素1.5%,大黄酚
1.7%,结果表明仪器精密度良好 。
2.2.5　稳定性试验　精密吸取混合对照液 20μl ,进
样 ,分别于 1h 、2h 、3h 、4h 、5h时重复上述操作 ,计算各
成分峰面积相对标准偏差(RSD)为:大黄素甲醚
1.9%、大黄素 1.3%、大黄酚 0.8%。结果表明混合对
照品溶液在5h内基本稳定 。
2.2.6　加样回收试验　精密称取栽培品药用大黄粉
末一定量 ,加入一定量混合对照品溶液 ,按游离供试品
溶液方法制备供试品溶液 ,采用上述色谱条件测定 , 3
种蒽醌类成分的回收率分别为:大黄素甲醚 95.1%、
大黄素97.3%、大黄酚 95.5%。
2.2.7　结果　栽培品药用大黄中大黄素和大黄素甲
醚的含量低于大黄对照药材 ,但大黄酚的含量高于大
黄对照药材(见表 1)。
3　讨论
上述实验结果可以看出 ,引种栽培品药用大黄薄
层色谱中除检测出与对照药材相同的蒽醌类成分外 ,
还检测出二苯乙烯类成分白藜芦醇的斑点;高效液相
表 1　药用大黄中各成分的线性关系及含量
成　分 回归方程 相关系数(r)
线性范围
(μg)
样品含量
(%)
对照药材
含量(%)
大黄素 y=1.060×106x-1.101×104 0.9991 0.10-0.50 0.65 0.54
大黄酚 y=2.061×106x-1.740×105 0.9998 0.40-2.00 1.16 0.67
大黄素甲醚 y=6.027×105x-1.134×105 0.9995 0.10-0.50 0.33 0.20
色谱检测结果表明 ,引种栽培品药用大黄中大黄素 、大
黄酚及大黄素甲醚的含量都明显高于药用大黄对照药
材中相应成分的含量 。对从甘肃中医学院引种栽培的
药用大黄药材的定性定量研究为药用大黄药材的引种
栽培提供了科学依据 ,也为保护野生药用大黄资源提
供了有效的方法 。
参考文献:
[ 1] 　李秀才.大黄的研究进展[ J] .中国药学杂志 , 1998 , 33(10):581-
584.
[ 2] 　魏玉辉 ,武新安 ,陈岚 ,等.大黄蒽醌类成分含量测定方法实验研
究[ J] .兰州大学学报(医学版), 2005 ,31(1):13-15.
[ 3] 　国家药典委员会.中华人民共和国药典(一部)[ S] .北京:化学工
业出版社 , 2005 , 17-18.
·31·中医药学报 2007 年第 35卷第 3 期
ABSTRACTS FROM ORIGINAL ARTICLES
Proliferation effect of Ferulic acid on endothelial cells ECV304 of
human umbilical vein
WANG Jun1 、2 , YUAN Zhuo1 , ZHANG Jun-ping1
Abstract:Objective:To observe the proliferation effect of ferulic acid
on the endothelial cells ECV304 of human umbilical vein , so as to find
a candidate way for therapeutic angiogenesis.Method:We incubated
cells with FA(102 ng ml , 103 ng ml , 104 ng ml)for 24 h after synchro-
nizing the ECV304 at G0 , BrdU-ELISA assay was performed to test
the DNA synthesis.The expression of VEGF mRNA by RT-PCR was
also observed.Result:Compared with control , different concentration of
ferulic acid can improve the BrdU incorporation and enhance the ex-
pression of VEGF mRNA with a dose-dependent manner raging from
102 ng ml to 104 ng ml.Conclusion:Ferulic acid can significantly im-
prove the proliferation of ECV304 and play an important role in angio-
genesis.
Author s address:1.Tianjin University of Traditional Chinese Medi-
cine , Tianjin 300193;2.Beijing University of Chinese Medicine , Bei-
jing 100029
Key words:Ferulic acid;endothelial cells;proliferation;VEGF
(Original article on page 4)
Study of Fermented Red Yeast Rice extract on osteoblast in vitro
YE Bin , YANG Zhong-lin
Abstract:Objective:Study on the effect of the extracts of fermented
red yeast rice(RYR)on cell proliferation and differentiation in cultured
rat osteoblast.Method:The effects of the extracts on proliferation and
differentiation were studied respectively by using MTT and ALP assays
in newborn rat calvarial (ROB) cell.The relationships between cell
number and the absorbance of MTT and ALP assays were examined.
Result:The aqueous extract concentration of 10-2 and 10-3 mg ml
showed the effects on proliferation in cell(P <0.05), and 10-3 mg ml
of aqueous extract showed the effect on differentiation in cell(P <0.
05);the 95% alcohol extract concentration of 10-2 and 10-3mg ml
both showed significant effects on proliferation and differentiation in cell
(P <0.05 or P<0.01).Conclusion:Both of the aqueous extract
and the 95% alcohol extract can promote proliferation and differentia-
tion of ROB cell in vitro , and the 95% alcohol extract showed a signifi-
cant effect.
Author s address:Key Lab for Modern Traditional Chinese Medicine ,
Ministry of Education , China Pharmaceutical University , Nanjing
210038 , China
Key words:Fermented Red Yeast Rice;cell culture;osteoblast;osteo-
porosis
(Original article on page 10)
Effects of Marrow-supplementing and Blood-engendering Gran-
ule on vla-6 cd49f in patients of chronic aplastic anemia
SUN Wei-zheng1 , WANG Jin-huan2 , SUN An-tao2
Abstract:Objective:To Observe the expression level of VLA-6 CD49f
in CAA patients before and after treatment by marrow-supplementing
and blood-engendering granule(MSABE).Method:Forty-five cases
of CAA were divided into two groups randomly-twenty-five patients
of the treating group were treated with MSABE and twenty of the control
group were treated with Zaizhang Shengxue tablet(ZZSXT).We ex-
plored flow cytometry(FCM)to detect forty-five patients the expres-
sion of related adhesion moleculesVLA-6 CD49f on hematopoietic stem
and progenitor cell(HSPC)before and after treatment.Result:The lev-
els of VLA-6 CD49f was lower than that in the normal controlled group
before treatment , but after treatment it got elevated.The enhancement
in experimental group was superior to that in control group , and the ad-
vancement in Yang deficiency group was superior to that in Yin defi-
ciency group.Conclusion:There was abnormal expression of bone mar-
row VLA-6 CD49f in CAA patients.MSABE can adjust the expression
level of marrow mononuclear cell to improve hematopoiesis in bone mar-
row .MSABE is an effective drug to treat CAA , and is worthy of further
study and development.
Author s address:1.Affiliated No 1 Hospital of Heilongjiang Univer-
sity of Chinese Medicine , Harbin 150040;2.Heilongjiang University of
Chinese Medicine , Harbin 150040
Key words:Anemia;Aplastic;Adhere;VLA-6 CD49f(Original article on page 19)
Determination of amino acids in Pheretima aspergillum by pre -
column derivatization of HPLC
PEI Fu-cheng1 , LI Chang-xin1 , REN Gui-ping2
Abstract:Objective:To determine the contents of the amino acids and
study the characters of Pheretima aspergillum(E .Perrier).Methods:
The samples of Pheretima aspergillum(E .Perrier)were derived with o
-phthaldialdehyde (OPA) and 9 - fluorenylmethyl chloroformate
(FMOC)in pre-column , and separated by HPLC.Seventeen kinds
of amino acids were determined with the ration of peak area by external
standard calibration.Results:All kinds of amino acids can be well sepa-
rated in 26 min , the concentration of amino acids and peak areas achie-
ved a nice linear relations (r is higher than 0.995).Conclusion:The
method is sensitive , accurate, simple , and reliable for the determination
of amino acids in Pheretima aspergillum(E.Perrier).
Author s address:1.The Second Traditional Chinese Medicine Work-
shop of Harbin Pharmaceutical Group ,Harbin 150078 , China;2.North-
east Agricultural University ,Harbin 150030 , China
Key words:Pheretima aspergillum(E.Perrier);amino acid;HPLC;pre
-column derivatization
(Original article on page 26)
Analysis of Anthraquinones in cultivated Rheum officinale Baill
KANGYi-hua1 , CHEN Zhi-yan1 , WANG Zhen - yue1 , WANG
Qian-bo2 , TANG Xian-ming1 , ZHOU Xiao-hui1
Abstract:Objective:To analysis the Anthraquinone content in cultivat-
ed Rheum officinale Baill.Method:The HPLC and TLC methods were
employed.The samples were separated by isocratic elution on Thermo
-C18 Hypersil column(250mm×4.6mm , 5μm), and then , with the
methanol-0.1% phosphoric acid solution as mobile phase , detected
wave at UV254nm and the flow rate 1.0ml min.The developing agent
was upper stratum liquor of petroleum benzin(60～ 90℃)- ethyl for-
mate-formic acid(15:8:1).The samples were detected at UV365nm.
Results:The Anthraquinone contents of cultivated Rheum officinale
Baill.exceeded the standard sample of Rheum officinale Baill.from
Gansu Province.
Author s address:1.Heilongjiang University of Chinese Medicine.
Harbin150040 , China;2.Heilongjiang Institute for Drug Control.Harbin
150010 , China
Key words:cultivated Rheum officinale Baill.;Anthraquinones;
HPLC;TLC
(Original article on page 30)