Studies On Compatibility Regulation And Effective Substance Of Huangqi-Gegen Herb Pair

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.