探讨“黄芪—葛根”药对配伍规律对药效物质的影响
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摘要
药对配伍规律及其药效物质研究是解决方剂关键问题的突破口,是国家各级科技项目重点支持的领域之一。中药复方(含药对)配伍规律研究的常用方法有文献理论研究、拆方研究、药理效应变化研究、化学成分变化研究、体内化学成分及药代动力学研究及中药血清药物化学研究(即综合方法研究)。配伍规律研究在不同层面和不同角度均取得了显著的进展,但其研究思路与方法尚未成熟,仍存在许多问题,尤其是方法学有待新的突破。对其药效物质基础的研究常用方法有传统的植物化学研究法、药代动力学对血清处理分析的研究法、基于受体理论构建的生物色谱研究法及高通量筛选技术—亲和超滤技术研究法等。
“黄芪-葛根”药对出自《证治汇补》黄芪葛根汤,黄芪补气托表,葛根生津解肌,合用能益气解表,为补气升气药对方。黄芪、葛根单独均有较多有关制剂、化学成分、有效部位或成分、药理作用及临床应用等方面的文献,其药效物质基本明确。黄芪葛根汤联用其他药物临床应用广泛,未见黄芪葛根汤配伍规律影响药效物质相关研究。本课题组前期已探索黄芪散(由本药对加桑白皮组成)改善糖尿病胰岛素抵抗及糖尿病心肌病的作用机理。本文主要在课题前期研究工作及文献调研已探明黄芪、葛根药效物质基础上,以指纹图谱技术为本研究提供方法学保证,从化学成分层面探讨配伍规律；多成分定量从有效成分层面研究其配伍规律影响药效物质的动态过程；研究本药对主要药效物质(总黄酮及总皂苷)的大孔吸附树脂纯化工艺,从有效部位层面探讨本药对配伍规律。目的
1.对中药复方(含药对)配伍规律及其药效物质研究、“黄芪-葛根”药对配伍规律、化学成分及药效物质等进行总结,明确“黄芪-葛根”药对药味层面的配伍规律及黄芪、葛根药效物质。
2.研究黄芪、葛根药材及“黄芪-葛根”药对不同配比的HPLC指纹图谱,考察药材及不同配比药对的异黄酮类成分谱是否因产地、提取溶剂及制备方法的改变而发生较大的变化,从化学成分层面探讨“黄芪-葛根”药对配伍规律对物质基础动态影响。
3.克服药效物质定量研究测定成分单一等问题,建立HPLC-UV测定10种异黄酮类成分及HPLC-ELSD测定黄芪甲苷的方法,考察“黄芪-葛根”药对配伍前后及不同配比时黄酮类及皂苷类成分的溶出率变化情况,从有效成分层面探讨“黄芪-葛根”药对配伍与药效物质动态变化过程,即配伍规律对药效物质动态影响。
4.系统研究影响大孔吸附树脂吸附分离的因素,筛选纯化“黄芪-葛根”药对药效物质(主要为总黄酮和总皂苷)的工艺条件,从有效部位层面探讨“黄芪-葛根”药对配伍规律对药效物质动态影响。
1.检索近10年来国内外有关“中药复方”、“方剂”、“药对”、“配伍规律”、“黄芪”、“葛根”、“化学成分”、“有效部位”、“有效成分”、“黄芪葛根”药对文献,分析综述中药复方配伍规律、药对理论、临床应用、药效物质及实验研究,总结黄芪、葛根品种来源、资源分布、化学成分、药效物质及“黄芪-葛根”药对应用概况、药理作用机理及临床应用。
2.建立“黄芪-葛根”药对及组成药材黄酮类成分指纹图谱测定方法,用HPLC-UV法,Kromasil 100-5 C18色谱柱(4.6 mm X 250 mm,5μm), Phenomenex C18保护柱(4.0mm×3.0 mm)；流动相：甲醇-0.1%枸橼酸溶液,梯度洗脱,流速1.0 mL.min-1,柱温30℃,检测波长250 nm；用HPLC-PDA鉴别色谱峰纯度,参考文献资料及对照品对照法鉴别指认色谱峰；用STATA8.0统计软件分析指纹图谱相关标准化处理数据。
3.黄酮类成分HPLC测定色谱条件同上,黄芪甲苷HPLC测定色谱柱同上,流动相乙腈-水(32：68),流速mL. min-1,漂移管温度(分流)60℃,气体压力25 Psi。测定“黄芪-葛根”药对(2：1)配伍前后不同时间点各成分的溶出量,将“黄芪-葛根”药对配伍合煎样品各成分溶出量按1/0或0/1中所测溶出量折算出理论溶出量；测定“黄芪-葛根”药对不同配比各成分溶出量,同法计算理论溶出量。
4.以“黄芪-葛根”药对总黄酮和总皂苷吸附量为考察指标,根据树脂的吸附和解析能力,从9种国内外常用树脂中筛选出XDA-5、D101、AB-8,对优选的XDA-5吸附树脂绘制吸附等温线、漏出曲线,优化纯化工艺条件,并测定树脂纯化物中有效部位及有效成分含量。结果
1.中药复方配伍规律是中医药理论的精华和中医药现代化的难点,常用方法有文献理论研究、拆方研究、药理效应变化研究、化学成分变化研究、体内化学成分及药代动力学研究及中药血清药物化学研究(即综合方法研究)。药理研究主要是观察复方配伍的药效变化及作用机制,分整方和拆方研究。拆方研究有撤药研究、析因分析、正交设计、均匀设计、聚类分析、基线等比增减设计等方法,析因分析又分为药对研究、药组研究及药性研究。拆方研究在寻找复方增效减毒作用的最佳组合、确定方中主要药物或活性物质、寻找方中药物最佳剂量配比关系、精简方剂等方面取得了确实的成效。但所得结论难以反应原复方的本质内涵,更难以深入揭示复方配伍的普遍规律。药理效应研究从药效学角度对方中各药作用、地位及相互关系进行探讨,证明复方中各药物间的协同或拮抗作用,方中各药具有“君臣佐使”不同地位,一定程度上验证复方组成的合理性及中药配伍应用的优越性,但药效指标选择的局限,难以客观揭示复方组方原理,且中药化学成分研究相对滞后,对复方物质基础与药效之间的关系认识不清,难以从根本上阐明其配伍规律和科学内涵。复方的化学成分是其疗效的根本,已发现大量的活性成分,为解析复方的组成贡献巨大,但复方的化学组成并不代表其在体内发挥生物效应的化学形式,必须结合效应成分数和量的变化说明配伍药物的相互关系。
以药对配伍规律为基础的数据挖掘在发现规律和深入研究方面的突出优势,对药对和方剂的配伍理论产生了积极影响。但目前在药味层次上的数据挖掘虽可发现常用药物组合,难以充分体现药对或方剂的配伍思想。从药性层面进行研究,可发现具有特定功效药对的常见组合模式,这些组合模式能够体现中医的治法治则。
中药药效物质基础是指中药进入人体内作用于多个靶点并产生整体功效的化学成分组,对其主要有两条基本的研究思路：一条是基于西方还原论和微观科学为基础的研究思路,将中药分成不同层次,逐层研究其与药效的关系；另一条则是基于系统论或中药作用的整体性的研究思路。将中药作为一个整体来研究其与药效的关系。在这两条基本思路的主导下,产生了多种不同的研究方法,主要有：传统的植物化学研究方法、药代动力学对血清处理分析的研究方法、基于受体理论构建的生物色谱研究方法及高通量筛选技术——亲和超滤技术研究方法等四类。中药效应物质基础经过几十年的探讨,虽然至今没有一种中药完全弄清其药效物质基础,但在药效物质基础的不同层面上还是取得了很大进展,尤其在有效成分层面。由于对中药效应物质基础理解不同、目标不同、学科背景不同、研究对象不同、采用的方法不同等对中药效应物质基础呈现多元化的趋势。
“黄芪-葛根”药对常用于气阴两虚之高血压、糖尿病、中风后遗症等疾病的治疗。目前,已从黄芪、葛根药材中分离出100余种化合物,其中异黄酮、紫檀烷等黄酮类成分达30多种,已明确药理效应的成分有毛蕊异黄酮及其葡萄糖苷、芒柄花素及其葡萄糖苷、葛根素、大豆苷及其苷元等20多种,这些效应成分具有抗高血压、抗高血脂、抗心律失常、抗心肌缺血、改善微循环障碍、改善胰岛素抵抗等作用。黄芪、葛根总黄酮均有抗氧化、降压、预防心脑血管疾病、雌激素样活性等作用。现代临床“黄芪-葛根”药对联用其它药物治疗脑梗死、血管疾病、冠心病心绞痛、心衰、糖尿病及糖尿病肾病。黄芪散(由此药对加桑白皮组成)可改善改善糖脂代谢、提高胰岛素的敏感性,上调GLUT-4mRNA表达,改善心肌能量代谢；增强心脏抗氧化能力,抑制NF-κB和TNF-α1的表达,减轻心脏和全身炎症反应；降低TGF-β1和SMAD3 mRNA及蛋白表达等多方面的作用。
2.经HPLC-PDA分析色谱峰纯度高,色谱图中各化合物可有效分离；比较乙醇索氏提取、乙醇及30%乙醇及水回流提取等制备方法,选择乙醇回流提取制备供试品溶液；考察流动相组成、洗脱方式、柱温、进样量等,参考文献及对照品对照法鉴定成分：黄芪药材4个、葛根药材6个、黄芪-葛根药对10个,分别为毛蕊异黄酮葡萄糖苷、芒柄花苷、毛蕊异黄酮、芒柄花素、3’-羟基葛根素、葛根素、大豆苷、染料木苷、大豆苷元、染料木素,并在色谱图中确认。对拟定的HPLC条件进行精密度、重现性及稳定性等方法学考察,其RSD值均低于3%,对黄芪、葛根药材10批醇提液、1批水提液,黄芪-葛根药对1：1、2：1、3：1、4：1、5：1及1：2比例水提液进行指纹图谱测定；统计分析结果：色谱指纹图谱相似度均大于0.93,以RRT为变量的指标聚成3类：变量(1-6号吸收峰)聚成一类,变量(7-11号吸收峰)聚成一类,变量(12、13号吸收峰)聚成一类,以PPA为变量的指标聚成3类：变量(1、3-5号吸收峰)聚成一类,变量(6-13号吸收峰)聚成一类,变量(2号吸收峰)聚成一类。主成分分析结果：第一主成分RPC1中1-8及10号吸收峰相关性较强,第二主成分RPC2中9-13号吸收峰相关性较强,第三主成分RPC3中1、5及11号吸收峰相关性较强,其中1、2、5-12号吸收峰分别为3’-羟基葛根素、葛根素、大豆苷、毛蕊异黄酮葡萄糖苷、染料木苷、芒柄花苷、大豆苷元、毛蕊异黄酮、染料木素及芒柄花素。标准化处理分析结果,“黄芪-葛根”药对配比为4：1时1-5及11号吸收峰的A／mg值最高,7、9、10及13号色谱峰在2：1配比时最高,而6、8及12号吸收峰以1：1配比时最高。
3.黄酮类成分的线性范围分别为3'-羟基葛根素76.2～1016 ng、葛根素65.8～1316 ng、大豆苷85.6～1712 ng、毛蕊异黄酮苷33.376～166.88 ng、染料木苷49.98～499.8 ng、芒柄花苷26.176～130.88 ng、大豆苷元29.68～269.8 ng、毛蕊异黄酮44.544～222.72 ng、染料木素48.5～485、芒柄花素26.56～132.8 ng,相关系数(R2)均大于0.9996,平均加样回收率分别为99.27%、102.38%、98.46%、103.06%、101.29%、99.71%、102.28%、97.89%、100.78%及98.37%,其RSD分别为1.77%、2.02%、1.43%、1.41%、1.33%、1.81%、1.32%、0.97%、1.84%及1.79%。黄芪甲苷回归方程y=1.2919x+4.1601,R2=0.9979。“黄芪-葛根”单煎及配伍合煎供试液中,各成分的溶出量在60分钟内逐步升高,之后基本平衡。将“黄芪-葛根”药对配伍合煎样品各成分溶出量按1／0或0／1中所测溶出量折算出理论溶出量,除芒柄花苷配伍后溶出量略有降低之外,其余9种成分合煎时溶出量均比单煎时高。经wilcoson signed-ranktest统计分析,芒柄花苷P>0.05,其余成分P<0.05,说明“黄芪-葛根”药对不同配比各成分的溶出量除芒柄花苷之外的其余9种成分较单一药材具有显著性差异；“黄芪-葛根”药对6种比例配伍10种成分溶出量试验,3'-羟基葛根素、葛根素、大豆苷溶出量实测值较理论值在4：1配伍时增加较多,染料木苷、大豆苷元、毛蕊异黄酮及其苷、芒柄花素及黄芪甲苷在2：1时溶出增加较多,芒柄花苷溶出量的实测值低于理论值。
4.大孔吸附树脂XDA-5型的纯化工艺条件为：取适当浓度的黄芪提取液,上样吸附速度为2 BV／h,饱和吸附后(总黄酮的饱和吸附量为58.83 mg／mL),先以4 BV水洗脱,再分别以4 BV30%、50%及70%乙醇解析,所得纯化物中总黄酮和总皂苷比例不同。中试生产直接70%乙醇洗脱所得树脂纯化物中总黄酮、总皂苷、3’-羟基葛根素、葛根素、大豆苷、染料木苷、芒柄花苷、毛蕊异黄酮苷、大豆苷元及黄芪甲苷含大豆苷元为佐使,揭示其有效部位及有效成分层次的配伍规律。但需要药理效应试验进一步验证。大孔吸附树脂纯化工艺,为黄芪-葛根药对进一步的机理探讨及新药研发提供稳定可控的生产路线。本研究具有较高的理论创新及实际应用价值。
Investigationon the compatibility law and effective substance of Herb-pairs (HP, Yaodui or Duiyao in Chinese) is the key to elueidate the combing foundation of Traditional Chinese medicine formulas (TCMFs), and also the key researching area supported projeet by Chinese government. The common methods to study the compatibility law of TCMFs or HP including document theory research, study on separated complex prescription, pharmacological effects, components diversity, components in vivo or pharmacokinetics and serum pharmacochemistry methods. All these controbuted much to elucidating the compatibility mechanism and variation of efficacy substance(or components in HP) in different points of view and degree. However, up to now, the methodology is still a hot topic of the above researching area. To elucidating effective substance of HP, traditional phytochemistry, serum handling and analysis in pharmacokinetics, biological chromatography based on acceptor and high throughput screening--affinity ultrafiltration.
Huangqi-gegen herb pair (HGHP, a commonly used pharmaceuticals) is from Zhengzhihuibu (by Li Yong-Cui. qing Dynasty), tonifying qi for ascending, and to treat qi deficiency and sinking syndrome. Our previous study showed Huangqisan(HGHP plus sangbaipi) interfering type 2 diabetes mellitus insulin resistance and diabetic cardiomyopathy. This study from the following three aspects conducts the research:i. summarize the data of HP and HGHP. ii.study on compatibility law and effective substance of HGHP. iii.research for purification of effective fraction of HGHP with macroporous resin.
1. Summarized the compatibility law and effective substance of HP, the components and effective compnents of AR and PR, the compatibility law of HGHP. Provide the research thinking and technical route of this study.
2. To research HPLC fingerprint of AR, PR and different compatibility proportion of HGHP, to inspect these inherent ingredients does or not greatly changed due to herbs, extraction, preparation changes. Preliminary investigate HGHP compatibility law dynamic influence on effective substance.
3. To overcome determination single effective component, established 10 isoflavones components HPLC-UV and astragalosideⅣHPLC-ELSD, investigation the dissolved quantity variation of main chemical constituents during pro and post compatibility and different proportion of HGHP, revealed the compatibility law dynamic influence on effective substance of HGHP.
4. Through systematic study many factors of influence of macroporous resin adsorption and separation, screening better macroporous resins of purification of total flavonoids and saponins of HGHP, revealed the compatibility law on effective fraction and effective substance of HGHP.
1. Searched the articles in the past 10 years, found out and summarized the articles with the topic on TCMFs, prescription, HP, compatibility law, AR, PR, HGHP.
2. The HPLC system (Shimadzu, Japen) was equipped with a model 10AT vp plus binary pump controller, a 772i sampler and a SPD-10A vp plus ultraviolet detector, using a Kromasil 100-5 C18 column (250×4.6 mm i.d.; 5μm particle size) connected to a phenomenex security guard column (C18,4.0×3.0 mm), the column temperature was kept constant at 30℃. The mobile phase consisting of 0.1% citric acid in water and methanol using a gradient elution, flow-rate was 1.0 mL.min-1. Aliquots of 10μL were injected into HPLC system for analysis, running 60 minute. Data were collected and analysed using CBM-10A vp plus chemstation software.
3. Flavonoids HPLC condition sup cit, astragalosideⅣHPLC-ELSD condition: chromatographic column sup cit, drift-tube temperature was 60℃, and gas pressure was 25psi. Dissolved quantity of components convert to theory value.
4.Through systematic study many factors of influence of macroporous resin adsorption and separation, screening better macroporous resins of purification of HGHP effective fraction (flavonoids and saponins), reference for development and industrial production of HGHP.
1. HP, which from the previous repeated clinical practice, is clinical trial reports of herbal compatibility. HP is unitary prescription only increase not decrease, reflect the corresponding simply syndrome. HP is the core of TCMFs and in common of a series of TCMFs. HP composed of two single herbal medicines, a special kind of TCMFs, is often used in the TCM. Unique combinations of the traditionally defined TCM herbal properties are frequently used for achieving mutual enhancement, mutual assistance, mutual restraint, mutual suppression or mutual antagonism. The theory of compatibility is the core of the basic theories of TCM. HP is clinical trial reports of herbal compatibility and unitary prescription only increase not decrease, reflect the corresponding simply syndrome. HP is the core of TCMFs and in common of a series of TCMFs. Clarifying the chemical reactions occurring and chemical changes during the compatibility process of two single herbs is useful for elucidating the compatibility mechanism of TCM. Clinical pharmacodynamics of HP relies on its chemical components, so it is important to identify the components in HP and investigate the relationship between the chemical components in HP and its single herbs. Therefore, study and research of TCMFs must based on HP, under the guidance of TCM theory, adopting modern scientific and technology means to study HP of formulas corresponding syndrome, elucidating the compatibility mechanism and variation of efficacy substance (or components in HP).
HGHP, composed of AR and PR, is from Zhengzhihuibu (by Li Yong-Cui. qing Dynasty). HGHP has been widely used in China to treat hypertension of qi-yin asthenia, diabetes due to internal heat and constipation and stroke lingering effects etc. in clinical applications. Most Chinese patent medicines like xintong oral sulution, zhengxintai capsules, zhengxintai tablets etc. contain HGHP with different proportion:1:1,1.18:1, 1.33:1,2:1 and 3.33:1. So far, compounds isolated from AR and PR is more than 100 kinds, which isoflavones up to 30 kinds. The constituents most often associated with its pharmacologic activities are Calycosin, calycosin-7-O-beta-D-glucoside, formononetin, ononin,3'-hydroxypuerarin, puerarin, daidzin, daidzein, genistin and genistein etc., being main isoflavonoids, are displayed beneficial effects such as anti-hypertention, hypolipidemic,anti-arrhythmia, anti-myocardial ischemia, improvement of microcirculaton disturbance, inhibit formation of advanced glycation end products, protective of diabetic insulin resistance etc.. Flavanoids of AR and PR possess antioxidation, decompression, cardiovascular tonic, prevention cardiovascular and cerebrovascular diseases and estrogen activities etc. effects. HGHP used with other medicine treated cerebral infarction, vascular diseases, coronary heart disease, angina pectoris, congestive heart failure, diabetic and diabetic nephropathy, etc.. Huangqi powder (HGHP affiliate sangbaipi) have ameliorate diabetic insulin resistance and diabetic cardiomyopathy effects. Calycosin, formononetin, daidzein and their glucosides, puerarin are active ingredients in AR and PR.
2. AR and PR, commonly used as TCM, are offcially listed in the Chinese Pharmacopoeia. There are lots of studies on components, pharmacological effects and clinical application of single AR and PR. Isoflavones is shared components of two herbs. It is multicomponent complex system. Pharmacodynamic material base is synthetic action of their chemical constituents. There are quite a few HPLC methods with either MS or PAD or UV detection reported in the literature for the identification and quantification of components in single herb of either AR or PR. In addition, there is a recent report on the determination of puerarin (the most abundant isoflavonoid) in PR. From a review of the literature, it was noticed that the efficiency of the assay for quality control of pharmaceuticals is highly dependent upon the selection of the representative markers. As reported in most of the literature for AR and PR, it was readily established that calycosin-7-O-beta-D-glucoside is present in the highest quantity in AR, whilst daidzin is also found in a large quantities in PR. Therefore, the above reported assay, which did not include these active components, could not serve as a sufficient quality control tool for pharmaceuticals containing both AR and PR. A simple and efficient assay method capable of detecting fingerprint and multiple active components from both herbs is essential.
AR and PR (family Leguminosae), commonly used as traditional Chinese medicines, are offcially listed in the Chinese Pharmacopoeia. AR, the dried root of Astragalus membranaceus. var. mongholicus (Bge.)Hsiao. or Astragalus membranaceus (Fisch.)Bge. PR, the dried root of Pueraria lobata (Willd.)Ohwi. Collected 10 batch herbs nationwide. The chromatograms of every peak purity authentication and their maximum absorption in HGHP sample solutions using HPLC-PAD at 190-400 nm. HGHP HPLC fingerprint and multi-components contents determination detected at 250 nm. Compared with ethanol soxhlet, ethanol and 30% ethanol reflux extraction, using ethanol reflux preparate samples. Inspected composition of mobile phase, elution style, column temperature, sample size, According to reference documents and reference substance confrontation method, identified components in chromatogram:4 in AR,6 in PR,10 in HGHP. Precision, repeatability, stability and Accuracy was taken, the RSD was less than 3.0% respectively. Fingerprint similarity greater than 0.93. Shown instrument and detection system for analysis meet the requirment. Sample testing results indicate that materia medica source uniform, quality stable, uniform of dissolution components in different proportion compatibility of HGHP, these inherent ingredients does not greatly changed due to herbs changes and extraction. Normalization analytic result, the A/mg value of No.1-No.5, No.11 chromatographic peak were high HGHP at proportion 4:1, whilst No.7, No.9, No.10 and No.13 were high at 2:1, No.6, No.8 and No.12 were high at 1:1.
3. Working solutions containing all ten reference compounds were prepared by diluting the stock solution to proper concentration in order to draw calibration curves. Each calibration curve contained six different concentrations and was performed in triplicate. All calibration curves were constructed from peak areas of the reference standards versus their concentrations. The limits of detection (LOD) and quantification (LOQ) for each studied component were defined as the lowest concentration of component in the diluted standard solution producing a signal-to-noise ratio (S/N) of 3:1 and 10:1, respectively. Results obtained showed that it's a convenient, accurate and reliable. The method has been successfully applied to the simultaneous determination of 10 isoflavones in HGHP. Concluded with multi-components contents results and correlated data that puerarin was monarch effective component,3'-hydroxypuerarin, daidzin and calycosin-7-O-beta-D-glucoside were subject, genistin and other components were adjuvant and messenger. Dynamic study shown that the dissolution quantity of constituents were higher at 60 minute,9 components post compatibility were higher than those of pro compatibility at different time (significance of statistical analysis). Puerarin, daidzein and astragalosideⅣwere higher at 2:1 proportion.
4. Optimization of purification process conditions, flavonoids and sapoins was taken as the index for investigation, the ability of adsorption and elution, adsorption kinetic curve, adsorption isotherm, leakage curve were detemined. Suitable type macroporous resin owned optimum adsorption and elution parameters. After eluted with 4BV of distilled water and 4BV of 30%,50%,70% ethanol respectively. The contents of total flavonoids, total saponins,3'-hydroxypuerarin, puerarin, daidzin, genistin, ononin, calycosin-7-O-beta-D-glucoside and daidzein were 54.39%,16.28%,6.269%,22.143%, 4.846%,0.874%,0.419%,1.161% and 0.989% in HGHP purified substance, respectively.
1. The compatible principle of monarch, minister, adjuvant and guiding drugs and sever aspects.namely, singular application, mutual reinforcement, mutual assistance, mutual restraint, mutual detoxication, mutual inhibition and incompatibility (except singular application) were main compatibility law of TCMFs. HGHP is mutual reinforcement. Those illustrate that HGHP compatibility law in theory level.
2. AR and PR, commonly used as TCM, are offcially listed in the Chinese Pharmacopoeia. There are lots of studies on components, pharmacological effects and clinical application of AR and PR. Isoflavones is shared components of two herbs. These inherent ingredients does not greatly changed due to herbs changes and extraction (fingerprint similarity greater than 0.93). Those illustrate that HGHP compatibility law and synergistic effect in chemical components level.
3. Concluded with multi-components contents results and correlated data that puerarin was monarch effective component,3'-hydroxypuerarin, daidzin and calycosin-7-O-beta-D-glucoside were subject, genistin and other components were adjuvant and messenger.10 components post compatibility were higher than those of pro compatibility at different time (significance of statistical analysis). Puerarin, daidzein and astragaloside IV were higher at 2:1 proportion. Those illustrate that HGHP compatibility law and synergistic effect in effective components level.
4. Purified substance HGHP, total flavonoids and puerarin were monarch effective component. Total saponins,3'-hydroxypuerarin, daidzin and calycosin-7-O-beta-D-glucoside were subject.genistin and daidzein were adjuvant and messenger. Those illustrate that HGHP compatibility law and synergistic effect in effective fraction and effective components level.
“黄芪-葛根”药对出自《证治汇补》黄芪葛根汤,黄芪补气托表,葛根生津解肌,合用能益气解表,为补气升气药对方。黄芪、葛根单独均有较多有关制剂、化学成分、有效部位或成分、药理作用及临床应用等方面的文献,其药效物质基本明确。黄芪葛根汤联用其他药物临床应用广泛,未见黄芪葛根汤配伍规律影响药效物质相关研究。本课题组前期已探索黄芪散(由本药对加桑白皮组成)改善糖尿病胰岛素抵抗及糖尿病心肌病的作用机理。本文主要在课题前期研究工作及文献调研已探明黄芪、葛根药效物质基础上,以指纹图谱技术为本研究提供方法学保证,从化学成分层面探讨配伍规律；多成分定量从有效成分层面研究其配伍规律影响药效物质的动态过程；研究本药对主要药效物质(总黄酮及总皂苷)的大孔吸附树脂纯化工艺,从有效部位层面探讨本药对配伍规律。目的
1.对中药复方(含药对)配伍规律及其药效物质研究、“黄芪-葛根”药对配伍规律、化学成分及药效物质等进行总结,明确“黄芪-葛根”药对药味层面的配伍规律及黄芪、葛根药效物质。
2.研究黄芪、葛根药材及“黄芪-葛根”药对不同配比的HPLC指纹图谱,考察药材及不同配比药对的异黄酮类成分谱是否因产地、提取溶剂及制备方法的改变而发生较大的变化,从化学成分层面探讨“黄芪-葛根”药对配伍规律对物质基础动态影响。
3.克服药效物质定量研究测定成分单一等问题,建立HPLC-UV测定10种异黄酮类成分及HPLC-ELSD测定黄芪甲苷的方法,考察“黄芪-葛根”药对配伍前后及不同配比时黄酮类及皂苷类成分的溶出率变化情况,从有效成分层面探讨“黄芪-葛根”药对配伍与药效物质动态变化过程,即配伍规律对药效物质动态影响。
4.系统研究影响大孔吸附树脂吸附分离的因素,筛选纯化“黄芪-葛根”药对药效物质(主要为总黄酮和总皂苷)的工艺条件,从有效部位层面探讨“黄芪-葛根”药对配伍规律对药效物质动态影响。
1.检索近10年来国内外有关“中药复方”、“方剂”、“药对”、“配伍规律”、“黄芪”、“葛根”、“化学成分”、“有效部位”、“有效成分”、“黄芪葛根”药对文献,分析综述中药复方配伍规律、药对理论、临床应用、药效物质及实验研究,总结黄芪、葛根品种来源、资源分布、化学成分、药效物质及“黄芪-葛根”药对应用概况、药理作用机理及临床应用。
2.建立“黄芪-葛根”药对及组成药材黄酮类成分指纹图谱测定方法,用HPLC-UV法,Kromasil 100-5 C18色谱柱(4.6 mm X 250 mm,5μm), Phenomenex C18保护柱(4.0mm×3.0 mm)；流动相：甲醇-0.1%枸橼酸溶液,梯度洗脱,流速1.0 mL.min-1,柱温30℃,检测波长250 nm；用HPLC-PDA鉴别色谱峰纯度,参考文献资料及对照品对照法鉴别指认色谱峰；用STATA8.0统计软件分析指纹图谱相关标准化处理数据。
3.黄酮类成分HPLC测定色谱条件同上,黄芪甲苷HPLC测定色谱柱同上,流动相乙腈-水(32：68),流速mL. min-1,漂移管温度(分流)60℃,气体压力25 Psi。测定“黄芪-葛根”药对(2：1)配伍前后不同时间点各成分的溶出量,将“黄芪-葛根”药对配伍合煎样品各成分溶出量按1/0或0/1中所测溶出量折算出理论溶出量；测定“黄芪-葛根”药对不同配比各成分溶出量,同法计算理论溶出量。
4.以“黄芪-葛根”药对总黄酮和总皂苷吸附量为考察指标,根据树脂的吸附和解析能力,从9种国内外常用树脂中筛选出XDA-5、D101、AB-8,对优选的XDA-5吸附树脂绘制吸附等温线、漏出曲线,优化纯化工艺条件,并测定树脂纯化物中有效部位及有效成分含量。结果
1.中药复方配伍规律是中医药理论的精华和中医药现代化的难点,常用方法有文献理论研究、拆方研究、药理效应变化研究、化学成分变化研究、体内化学成分及药代动力学研究及中药血清药物化学研究(即综合方法研究)。药理研究主要是观察复方配伍的药效变化及作用机制,分整方和拆方研究。拆方研究有撤药研究、析因分析、正交设计、均匀设计、聚类分析、基线等比增减设计等方法,析因分析又分为药对研究、药组研究及药性研究。拆方研究在寻找复方增效减毒作用的最佳组合、确定方中主要药物或活性物质、寻找方中药物最佳剂量配比关系、精简方剂等方面取得了确实的成效。但所得结论难以反应原复方的本质内涵,更难以深入揭示复方配伍的普遍规律。药理效应研究从药效学角度对方中各药作用、地位及相互关系进行探讨,证明复方中各药物间的协同或拮抗作用,方中各药具有“君臣佐使”不同地位,一定程度上验证复方组成的合理性及中药配伍应用的优越性,但药效指标选择的局限,难以客观揭示复方组方原理,且中药化学成分研究相对滞后,对复方物质基础与药效之间的关系认识不清,难以从根本上阐明其配伍规律和科学内涵。复方的化学成分是其疗效的根本,已发现大量的活性成分,为解析复方的组成贡献巨大,但复方的化学组成并不代表其在体内发挥生物效应的化学形式,必须结合效应成分数和量的变化说明配伍药物的相互关系。
以药对配伍规律为基础的数据挖掘在发现规律和深入研究方面的突出优势,对药对和方剂的配伍理论产生了积极影响。但目前在药味层次上的数据挖掘虽可发现常用药物组合,难以充分体现药对或方剂的配伍思想。从药性层面进行研究,可发现具有特定功效药对的常见组合模式,这些组合模式能够体现中医的治法治则。
中药药效物质基础是指中药进入人体内作用于多个靶点并产生整体功效的化学成分组,对其主要有两条基本的研究思路：一条是基于西方还原论和微观科学为基础的研究思路,将中药分成不同层次,逐层研究其与药效的关系；另一条则是基于系统论或中药作用的整体性的研究思路。将中药作为一个整体来研究其与药效的关系。在这两条基本思路的主导下,产生了多种不同的研究方法,主要有：传统的植物化学研究方法、药代动力学对血清处理分析的研究方法、基于受体理论构建的生物色谱研究方法及高通量筛选技术——亲和超滤技术研究方法等四类。中药效应物质基础经过几十年的探讨,虽然至今没有一种中药完全弄清其药效物质基础,但在药效物质基础的不同层面上还是取得了很大进展,尤其在有效成分层面。由于对中药效应物质基础理解不同、目标不同、学科背景不同、研究对象不同、采用的方法不同等对中药效应物质基础呈现多元化的趋势。
“黄芪-葛根”药对常用于气阴两虚之高血压、糖尿病、中风后遗症等疾病的治疗。目前,已从黄芪、葛根药材中分离出100余种化合物,其中异黄酮、紫檀烷等黄酮类成分达30多种,已明确药理效应的成分有毛蕊异黄酮及其葡萄糖苷、芒柄花素及其葡萄糖苷、葛根素、大豆苷及其苷元等20多种,这些效应成分具有抗高血压、抗高血脂、抗心律失常、抗心肌缺血、改善微循环障碍、改善胰岛素抵抗等作用。黄芪、葛根总黄酮均有抗氧化、降压、预防心脑血管疾病、雌激素样活性等作用。现代临床“黄芪-葛根”药对联用其它药物治疗脑梗死、血管疾病、冠心病心绞痛、心衰、糖尿病及糖尿病肾病。黄芪散(由此药对加桑白皮组成)可改善改善糖脂代谢、提高胰岛素的敏感性,上调GLUT-4mRNA表达,改善心肌能量代谢；增强心脏抗氧化能力,抑制NF-κB和TNF-α1的表达,减轻心脏和全身炎症反应；降低TGF-β1和SMAD3 mRNA及蛋白表达等多方面的作用。
2.经HPLC-PDA分析色谱峰纯度高,色谱图中各化合物可有效分离；比较乙醇索氏提取、乙醇及30%乙醇及水回流提取等制备方法,选择乙醇回流提取制备供试品溶液；考察流动相组成、洗脱方式、柱温、进样量等,参考文献及对照品对照法鉴定成分：黄芪药材4个、葛根药材6个、黄芪-葛根药对10个,分别为毛蕊异黄酮葡萄糖苷、芒柄花苷、毛蕊异黄酮、芒柄花素、3’-羟基葛根素、葛根素、大豆苷、染料木苷、大豆苷元、染料木素,并在色谱图中确认。对拟定的HPLC条件进行精密度、重现性及稳定性等方法学考察,其RSD值均低于3%,对黄芪、葛根药材10批醇提液、1批水提液,黄芪-葛根药对1：1、2：1、3：1、4：1、5：1及1：2比例水提液进行指纹图谱测定；统计分析结果：色谱指纹图谱相似度均大于0.93,以RRT为变量的指标聚成3类：变量(1-6号吸收峰)聚成一类,变量(7-11号吸收峰)聚成一类,变量(12、13号吸收峰)聚成一类,以PPA为变量的指标聚成3类：变量(1、3-5号吸收峰)聚成一类,变量(6-13号吸收峰)聚成一类,变量(2号吸收峰)聚成一类。主成分分析结果：第一主成分RPC1中1-8及10号吸收峰相关性较强,第二主成分RPC2中9-13号吸收峰相关性较强,第三主成分RPC3中1、5及11号吸收峰相关性较强,其中1、2、5-12号吸收峰分别为3’-羟基葛根素、葛根素、大豆苷、毛蕊异黄酮葡萄糖苷、染料木苷、芒柄花苷、大豆苷元、毛蕊异黄酮、染料木素及芒柄花素。标准化处理分析结果,“黄芪-葛根”药对配比为4：1时1-5及11号吸收峰的A／mg值最高,7、9、10及13号色谱峰在2：1配比时最高,而6、8及12号吸收峰以1：1配比时最高。
3.黄酮类成分的线性范围分别为3'-羟基葛根素76.2～1016 ng、葛根素65.8～1316 ng、大豆苷85.6～1712 ng、毛蕊异黄酮苷33.376～166.88 ng、染料木苷49.98～499.8 ng、芒柄花苷26.176～130.88 ng、大豆苷元29.68～269.8 ng、毛蕊异黄酮44.544～222.72 ng、染料木素48.5～485、芒柄花素26.56～132.8 ng,相关系数(R2)均大于0.9996,平均加样回收率分别为99.27%、102.38%、98.46%、103.06%、101.29%、99.71%、102.28%、97.89%、100.78%及98.37%,其RSD分别为1.77%、2.02%、1.43%、1.41%、1.33%、1.81%、1.32%、0.97%、1.84%及1.79%。黄芪甲苷回归方程y=1.2919x+4.1601,R2=0.9979。“黄芪-葛根”单煎及配伍合煎供试液中,各成分的溶出量在60分钟内逐步升高,之后基本平衡。将“黄芪-葛根”药对配伍合煎样品各成分溶出量按1／0或0／1中所测溶出量折算出理论溶出量,除芒柄花苷配伍后溶出量略有降低之外,其余9种成分合煎时溶出量均比单煎时高。经wilcoson signed-ranktest统计分析,芒柄花苷P>0.05,其余成分P<0.05,说明“黄芪-葛根”药对不同配比各成分的溶出量除芒柄花苷之外的其余9种成分较单一药材具有显著性差异；“黄芪-葛根”药对6种比例配伍10种成分溶出量试验,3'-羟基葛根素、葛根素、大豆苷溶出量实测值较理论值在4：1配伍时增加较多,染料木苷、大豆苷元、毛蕊异黄酮及其苷、芒柄花素及黄芪甲苷在2：1时溶出增加较多,芒柄花苷溶出量的实测值低于理论值。
4.大孔吸附树脂XDA-5型的纯化工艺条件为：取适当浓度的黄芪提取液,上样吸附速度为2 BV／h,饱和吸附后(总黄酮的饱和吸附量为58.83 mg／mL),先以4 BV水洗脱,再分别以4 BV30%、50%及70%乙醇解析,所得纯化物中总黄酮和总皂苷比例不同。中试生产直接70%乙醇洗脱所得树脂纯化物中总黄酮、总皂苷、3’-羟基葛根素、葛根素、大豆苷、染料木苷、芒柄花苷、毛蕊异黄酮苷、大豆苷元及黄芪甲苷含大豆苷元为佐使,揭示其有效部位及有效成分层次的配伍规律。但需要药理效应试验进一步验证。大孔吸附树脂纯化工艺,为黄芪-葛根药对进一步的机理探讨及新药研发提供稳定可控的生产路线。本研究具有较高的理论创新及实际应用价值。
Investigationon the compatibility law and effective substance of Herb-pairs (HP, Yaodui or Duiyao in Chinese) is the key to elueidate the combing foundation of Traditional Chinese medicine formulas (TCMFs), and also the key researching area supported projeet by Chinese government. The common methods to study the compatibility law of TCMFs or HP including document theory research, study on separated complex prescription, pharmacological effects, components diversity, components in vivo or pharmacokinetics and serum pharmacochemistry methods. All these controbuted much to elucidating the compatibility mechanism and variation of efficacy substance(or components in HP) in different points of view and degree. However, up to now, the methodology is still a hot topic of the above researching area. To elucidating effective substance of HP, traditional phytochemistry, serum handling and analysis in pharmacokinetics, biological chromatography based on acceptor and high throughput screening--affinity ultrafiltration.
Huangqi-gegen herb pair (HGHP, a commonly used pharmaceuticals) is from Zhengzhihuibu (by Li Yong-Cui. qing Dynasty), tonifying qi for ascending, and to treat qi deficiency and sinking syndrome. Our previous study showed Huangqisan(HGHP plus sangbaipi) interfering type 2 diabetes mellitus insulin resistance and diabetic cardiomyopathy. This study from the following three aspects conducts the research:i. summarize the data of HP and HGHP. ii.study on compatibility law and effective substance of HGHP. iii.research for purification of effective fraction of HGHP with macroporous resin.
1. Summarized the compatibility law and effective substance of HP, the components and effective compnents of AR and PR, the compatibility law of HGHP. Provide the research thinking and technical route of this study.
2. To research HPLC fingerprint of AR, PR and different compatibility proportion of HGHP, to inspect these inherent ingredients does or not greatly changed due to herbs, extraction, preparation changes. Preliminary investigate HGHP compatibility law dynamic influence on effective substance.
3. To overcome determination single effective component, established 10 isoflavones components HPLC-UV and astragalosideⅣHPLC-ELSD, investigation the dissolved quantity variation of main chemical constituents during pro and post compatibility and different proportion of HGHP, revealed the compatibility law dynamic influence on effective substance of HGHP.
4. Through systematic study many factors of influence of macroporous resin adsorption and separation, screening better macroporous resins of purification of total flavonoids and saponins of HGHP, revealed the compatibility law on effective fraction and effective substance of HGHP.
1. Searched the articles in the past 10 years, found out and summarized the articles with the topic on TCMFs, prescription, HP, compatibility law, AR, PR, HGHP.
2. The HPLC system (Shimadzu, Japen) was equipped with a model 10AT vp plus binary pump controller, a 772i sampler and a SPD-10A vp plus ultraviolet detector, using a Kromasil 100-5 C18 column (250×4.6 mm i.d.; 5μm particle size) connected to a phenomenex security guard column (C18,4.0×3.0 mm), the column temperature was kept constant at 30℃. The mobile phase consisting of 0.1% citric acid in water and methanol using a gradient elution, flow-rate was 1.0 mL.min-1. Aliquots of 10μL were injected into HPLC system for analysis, running 60 minute. Data were collected and analysed using CBM-10A vp plus chemstation software.
3. Flavonoids HPLC condition sup cit, astragalosideⅣHPLC-ELSD condition: chromatographic column sup cit, drift-tube temperature was 60℃, and gas pressure was 25psi. Dissolved quantity of components convert to theory value.
4.Through systematic study many factors of influence of macroporous resin adsorption and separation, screening better macroporous resins of purification of HGHP effective fraction (flavonoids and saponins), reference for development and industrial production of HGHP.
1. HP, which from the previous repeated clinical practice, is clinical trial reports of herbal compatibility. HP is unitary prescription only increase not decrease, reflect the corresponding simply syndrome. HP is the core of TCMFs and in common of a series of TCMFs. HP composed of two single herbal medicines, a special kind of TCMFs, is often used in the TCM. Unique combinations of the traditionally defined TCM herbal properties are frequently used for achieving mutual enhancement, mutual assistance, mutual restraint, mutual suppression or mutual antagonism. The theory of compatibility is the core of the basic theories of TCM. HP is clinical trial reports of herbal compatibility and unitary prescription only increase not decrease, reflect the corresponding simply syndrome. HP is the core of TCMFs and in common of a series of TCMFs. Clarifying the chemical reactions occurring and chemical changes during the compatibility process of two single herbs is useful for elucidating the compatibility mechanism of TCM. Clinical pharmacodynamics of HP relies on its chemical components, so it is important to identify the components in HP and investigate the relationship between the chemical components in HP and its single herbs. Therefore, study and research of TCMFs must based on HP, under the guidance of TCM theory, adopting modern scientific and technology means to study HP of formulas corresponding syndrome, elucidating the compatibility mechanism and variation of efficacy substance (or components in HP).
HGHP, composed of AR and PR, is from Zhengzhihuibu (by Li Yong-Cui. qing Dynasty). HGHP has been widely used in China to treat hypertension of qi-yin asthenia, diabetes due to internal heat and constipation and stroke lingering effects etc. in clinical applications. Most Chinese patent medicines like xintong oral sulution, zhengxintai capsules, zhengxintai tablets etc. contain HGHP with different proportion:1:1,1.18:1, 1.33:1,2:1 and 3.33:1. So far, compounds isolated from AR and PR is more than 100 kinds, which isoflavones up to 30 kinds. The constituents most often associated with its pharmacologic activities are Calycosin, calycosin-7-O-beta-D-glucoside, formononetin, ononin,3'-hydroxypuerarin, puerarin, daidzin, daidzein, genistin and genistein etc., being main isoflavonoids, are displayed beneficial effects such as anti-hypertention, hypolipidemic,anti-arrhythmia, anti-myocardial ischemia, improvement of microcirculaton disturbance, inhibit formation of advanced glycation end products, protective of diabetic insulin resistance etc.. Flavanoids of AR and PR possess antioxidation, decompression, cardiovascular tonic, prevention cardiovascular and cerebrovascular diseases and estrogen activities etc. effects. HGHP used with other medicine treated cerebral infarction, vascular diseases, coronary heart disease, angina pectoris, congestive heart failure, diabetic and diabetic nephropathy, etc.. Huangqi powder (HGHP affiliate sangbaipi) have ameliorate diabetic insulin resistance and diabetic cardiomyopathy effects. Calycosin, formononetin, daidzein and their glucosides, puerarin are active ingredients in AR and PR.
2. AR and PR, commonly used as TCM, are offcially listed in the Chinese Pharmacopoeia. There are lots of studies on components, pharmacological effects and clinical application of single AR and PR. Isoflavones is shared components of two herbs. It is multicomponent complex system. Pharmacodynamic material base is synthetic action of their chemical constituents. There are quite a few HPLC methods with either MS or PAD or UV detection reported in the literature for the identification and quantification of components in single herb of either AR or PR. In addition, there is a recent report on the determination of puerarin (the most abundant isoflavonoid) in PR. From a review of the literature, it was noticed that the efficiency of the assay for quality control of pharmaceuticals is highly dependent upon the selection of the representative markers. As reported in most of the literature for AR and PR, it was readily established that calycosin-7-O-beta-D-glucoside is present in the highest quantity in AR, whilst daidzin is also found in a large quantities in PR. Therefore, the above reported assay, which did not include these active components, could not serve as a sufficient quality control tool for pharmaceuticals containing both AR and PR. A simple and efficient assay method capable of detecting fingerprint and multiple active components from both herbs is essential.
AR and PR (family Leguminosae), commonly used as traditional Chinese medicines, are offcially listed in the Chinese Pharmacopoeia. AR, the dried root of Astragalus membranaceus. var. mongholicus (Bge.)Hsiao. or Astragalus membranaceus (Fisch.)Bge. PR, the dried root of Pueraria lobata (Willd.)Ohwi. Collected 10 batch herbs nationwide. The chromatograms of every peak purity authentication and their maximum absorption in HGHP sample solutions using HPLC-PAD at 190-400 nm. HGHP HPLC fingerprint and multi-components contents determination detected at 250 nm. Compared with ethanol soxhlet, ethanol and 30% ethanol reflux extraction, using ethanol reflux preparate samples. Inspected composition of mobile phase, elution style, column temperature, sample size, According to reference documents and reference substance confrontation method, identified components in chromatogram:4 in AR,6 in PR,10 in HGHP. Precision, repeatability, stability and Accuracy was taken, the RSD was less than 3.0% respectively. Fingerprint similarity greater than 0.93. Shown instrument and detection system for analysis meet the requirment. Sample testing results indicate that materia medica source uniform, quality stable, uniform of dissolution components in different proportion compatibility of HGHP, these inherent ingredients does not greatly changed due to herbs changes and extraction. Normalization analytic result, the A/mg value of No.1-No.5, No.11 chromatographic peak were high HGHP at proportion 4:1, whilst No.7, No.9, No.10 and No.13 were high at 2:1, No.6, No.8 and No.12 were high at 1:1.
3. Working solutions containing all ten reference compounds were prepared by diluting the stock solution to proper concentration in order to draw calibration curves. Each calibration curve contained six different concentrations and was performed in triplicate. All calibration curves were constructed from peak areas of the reference standards versus their concentrations. The limits of detection (LOD) and quantification (LOQ) for each studied component were defined as the lowest concentration of component in the diluted standard solution producing a signal-to-noise ratio (S/N) of 3:1 and 10:1, respectively. Results obtained showed that it's a convenient, accurate and reliable. The method has been successfully applied to the simultaneous determination of 10 isoflavones in HGHP. Concluded with multi-components contents results and correlated data that puerarin was monarch effective component,3'-hydroxypuerarin, daidzin and calycosin-7-O-beta-D-glucoside were subject, genistin and other components were adjuvant and messenger. Dynamic study shown that the dissolution quantity of constituents were higher at 60 minute,9 components post compatibility were higher than those of pro compatibility at different time (significance of statistical analysis). Puerarin, daidzein and astragalosideⅣwere higher at 2:1 proportion.
4. Optimization of purification process conditions, flavonoids and sapoins was taken as the index for investigation, the ability of adsorption and elution, adsorption kinetic curve, adsorption isotherm, leakage curve were detemined. Suitable type macroporous resin owned optimum adsorption and elution parameters. After eluted with 4BV of distilled water and 4BV of 30%,50%,70% ethanol respectively. The contents of total flavonoids, total saponins,3'-hydroxypuerarin, puerarin, daidzin, genistin, ononin, calycosin-7-O-beta-D-glucoside and daidzein were 54.39%,16.28%,6.269%,22.143%, 4.846%,0.874%,0.419%,1.161% and 0.989% in HGHP purified substance, respectively.
1. The compatible principle of monarch, minister, adjuvant and guiding drugs and sever aspects.namely, singular application, mutual reinforcement, mutual assistance, mutual restraint, mutual detoxication, mutual inhibition and incompatibility (except singular application) were main compatibility law of TCMFs. HGHP is mutual reinforcement. Those illustrate that HGHP compatibility law in theory level.
2. AR and PR, commonly used as TCM, are offcially listed in the Chinese Pharmacopoeia. There are lots of studies on components, pharmacological effects and clinical application of AR and PR. Isoflavones is shared components of two herbs. These inherent ingredients does not greatly changed due to herbs changes and extraction (fingerprint similarity greater than 0.93). Those illustrate that HGHP compatibility law and synergistic effect in chemical components level.
3. Concluded with multi-components contents results and correlated data that puerarin was monarch effective component,3'-hydroxypuerarin, daidzin and calycosin-7-O-beta-D-glucoside were subject, genistin and other components were adjuvant and messenger.10 components post compatibility were higher than those of pro compatibility at different time (significance of statistical analysis). Puerarin, daidzein and astragaloside IV were higher at 2:1 proportion. Those illustrate that HGHP compatibility law and synergistic effect in effective components level.
4. Purified substance HGHP, total flavonoids and puerarin were monarch effective component. Total saponins,3'-hydroxypuerarin, daidzin and calycosin-7-O-beta-D-glucoside were subject.genistin and daidzein were adjuvant and messenger. Those illustrate that HGHP compatibility law and synergistic effect in effective fraction and effective components level.
引文
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