摘要
蒙古黄芪(Astragalus membranaceus (Fisch.)Bge. var. mongholicus (Bge.) Hsiao)为我省道地药材,是重要益气药。现代药理学研究证实,黄芪具有抗病毒感染、保护心脏及肾脏、双向调节血糖、抗缺氧、抗自由基氧化、抗衰老、抗肿瘤、增强免疫力、对中枢神经的双向调节作用、利尿、降压等功效。黄芪主要含有黄芪苷Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ等多种皂苷,毛蕊异黄酮、槲皮素、异黄烷、芒柄花素等黄酮类成分还有黄芪多糖Ⅰ等多糖成分,及多种氨基酸和微量元素。高效液相色谱(High-Performance Liquid Chromatography, HPLC)具有灵敏、快捷、重现性好等优点,已经被越来越广泛的应用于中药的质量控制等方面。目前HPLC中由于紫外检测器其高灵敏度和稳定性,在高效液相色谱中应用最广泛。但它只适用于能够吸收紫外和可见光的物质。由于许多物质并不含特征紫外吸收基团,没有紫外或可见光吸收,或紫外-可见光吸收弱,例如皂苷类、糖类、萜类、甾体类、氨基酸类和脂肪酸类等化合物没有紫外吸收或者仅有末端吸收,无法或很难采用HPLC-UV检测器进行检测。蒸发光散射检测器(evaporative light-scattering detector, ELSD)给高效液相色谱的检测技术带来了活力,这种检测器的主要特点是：可以测定许多没有紫外-可见光吸收的物质,适用于梯度洗脱,对温度的要求不苛刻,且比示差折光检测器灵敏度高,不依赖样品的光学性质,比紫外检测器更适合此类中药的分析及质量控制。
本文主要以HPLC-ELSD为分析手段,对山西道地药材黄芪中皂苷类成分提取工艺及其体外代谢进行了初步研究。
1.山西道地药材蒙古黄芪中黄芪甲苷含量测定方法研究
采用高效液相色谱-蒸发光散射检测(HPLC-ELSD)法建立测定山西道地药材蒙古黄芪中黄芪甲苷含量的方法。流动相为乙腈-水,梯度洗脱,柱温：25℃；进样量：20μL；流速：1mL/min, ELSD检测器漂移管温度：60℃,气流速：2.0L/min,增益：8。系统性试验中,色谱柱理论塔板数按黄芪甲苷计算为127001,分离度为2.25,拖尾因子为1.02,表明对称性良好,保留时间为35.58 min。通过对方法的线性、精密度、回收率、稳定性等的考察,结果表明：黄芪甲苷在7.5～750μg/mL范围内线性关系良好R2=0.9972,日内、日间精密度均良好,平均回收率在98.0%～102.0%之间,稳定性良好,该方法操作简便,采用梯度洗脱分离度好,操作简便,适用于山西道地药材蒙古黄芪中黄芪甲苷的研究。
2.蒙古黄芪中皂苷类成分提取工艺的优化
优选黄芪中黄芪甲苷的醇-氨提取工艺,以黄芪甲苷的含量为指标,采用L9(34)正交试验筛选黄芪的最佳提取工艺条件。优化的提取工艺为甲醇：氨水为9：1,溶剂总量150mL,超声40min,提取3次。
3.蒙古黄芪中皂苷类成分在大鼠肝微粒体体外代谢研究
本实验采用高效液相-蒸发光散射检测器为检测手段,对黄芪中皂苷类提取物的大鼠肝微粒体体外代谢进行了初步的研究,对于科学认识中药的安全性、临床合理用药及安全性评价等具有一定作用。
4.蒙古黄芪提取物与黄芪注射液的特征图谱研究
本实验以HPLC-ELSD梯度洗脱法对黄芪药材及市售黄芪注射液进行了特征图谱研究,从图谱中可以看出,虽然都出现了相应的主成分峰,但是峰面积比值不尽相同。说明中药注射剂必须在固定原药材产地及提取工艺等前提下,才可能保证中药注射剂的质量稳定可控,为黄芪药材及含黄芪主成分注射剂研究提供了一定的科学依据和参考。
Astrgalus membranaceus(Fisch.)Bge. var. mongholicus(Bge.)Hsiao is famous-region drug in shanxi province. It can benefit vital energy in our body. Modern pharmacology studies confirmed that, Astrgalus membranaceus(Fisch.)Bge. var. mongholicus(Bge.)Hsiao has the following effect including:anti-virus infection, protecting the heart and kidney, Bi-directional regulation of blood sugar, Anti-hypoxia, anti-free radical oxidation, anti-aging, anti-tumor, enhance immunity, bi-directional regulatory role of the central nervous system, diuretic, hypertension, etc. Astragalus contains mainly astragalinⅠ,Ⅱ,Ⅲ,Ⅳ,Ⅴand many other saponins, calycosin, quercetin, isoflavans, formononetin and other flavonoids astragalus polysaccharideⅠand other polysaccharide components, and variours amino acids and trace elements. High-performance liquid chromatography has been widely used in quality control of traditional medicine due to its high sensitivity, UV detector has the characteristic of the high sensitivity and stability, and widely used in high performance liquid chromatography. But it can apply only to ultraviolet and visible light absorption substances. Because Many of substances, do not contain UV-absorbing group, they have no absorption of ultraviolet or visible light, or UV-visible light weak absorption, such as saponins, sugars, terpenoids, steroid, amino acids and fatty acids and other compounds, they are difficult to apply the HPLC-UV detector for detection. Evaporative light scattering detector's advanced characeristics bring high-performance liquid chromatography detection techniques energy, the main characteristics of this detector are that: the matter without UV-visible light absorption can be measured, application in the gradient elution, can work in not harsh temperature, and higher Sensitivity than a differential refractive index detector. ELSD do not rely on the optical properties of the sample and the ELSD detector is more suitable for such analysis and quality control of these traditional Chinese medicines.
By way of HPLC-ELSD, This paper preliminary studies the extraction technology and the metabolism in vitro of saponins active ingredient in Astragalus membranaceus (Fisch.)Bge. var. mongholicus (Bge.) Hsiao.
1. Study on analytical method for determination of the content of astragaloside IV in Astragalus membranaceus (Fisch.)Bge. var. mongholicus (Bge.) Hsiao.
To establish analytical method that determines the content of astragaloside IV in Astragalus membranaceus(Fisch.)Bge. var. mongholicus(Bge.) Hsiao by used of HPLC-ELSD. The Diamonsil C18 column was used; and the mobile phase was a mixture of acetonitrile—water; temperature of column was 25℃; injection volume:20μL; ELSD detector drift tube temperature:60℃, gas flow:2.0L/min, gain:8. In the system Applicability Test, The column number of theoretical plates in accordance with terms astragaloside IV 351,083, separating degree was 4.23, tailing factor was 1.02, indicating a good symmetry, retention time of 35.58 min. By the study on linear, precision, stability, recovery, etc, the study results show that Linear range of astragaloside IV concentration was 7.5～750μg/mL R2= 0.9972, showing a good linear relationship, the RSD of precision was favorable, the mean recovery was between 98.0～102.0%, good stability and the HPLC-ELSD method established in this paper was simple for determination of astragoloside IV in Astragalus membranaceus (Fisch.)Bge.var.mongholicus (Bge.)Hsiao.
2. Study on optimization of extraction process or Astragalus membranaceus(Fish.) Bge.var.mongholicus (Bge.)Hsiao
To optimize the extraction technology for Astragalus membranaceus(Fish.) Bge.var.mongholicus (Bge.)Hsiao dissolved in methanol-ammonia water. The process was studied by orthogonal design L9 (34) with the extraction rate of astragoloside IV as criteria. The optimum extracting condition was:adding amount of solvent (methanol:ammonia water is 9:1) ultrasound extraction for 40 min(totally extracting for 3 time).
3. Study on metabolism in vitro in rat liver microsomes for Astragalus membranaceus(Fish.) Bge.var.mongholicus (Bge.)Hsiao
In this study, a preliminary study on metabolism of the extract of Astragalus membranaceus(Fish.) Bge.var.mongholicus (Bge.)Hsiao in rat liver microsomes by HPLC-ELSD, it has certain role in study on Astragalus membranaceus(Fish.) Bge.var.mongholicus (Bge.)Hsiao, Scientific understanding of the safety of Chinese medicine, Clinical medication, and safety evaluation etc.
4. Study on the atlas of extract in Astragalus membranaceus(Fish.) Bge.var.mongholicus (Bge.)Hsiao and Radix Astragali Injection
In this experiment, to study the atlas of extract in Astragalus membranaceus(Fish.) Bge.var.mongholicus (Bge.)Hsiao and Commercially available injection made of Astragalus using gradient elution by HPLC-ELSD method. The atlas show that, there are corresponding main component peaks, but the number of the peak and peak area ratio between them were not the same. Illustrated that the quality, stability, controllability of the traditional chinese medicine injection can be assured only on condition that a fixed place of origin of Raw medicinal herbs and extraction process control, provide certain scientific basis and reference for the studies of Astragalus herbs and injection products containing the main ingredient Astragalus.
引文
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