摘要
在中医药整体的现代研究框架中,中药质量一直是急需攻克的重大问题之一。目前应用较广泛的中药质量评价方法是对已知的一、二个主要成分或有效成分作为指标成分予以检测控制。但中药发挥功效可能是药材或成方所含物质群整体作用的结果,传统的中医理论和医疗实践对现行中药质量控制的模式提出了挑战,即测定任何一种活性成分都难以体现其内在的质量。
中药指纹图谱技术是目前能够为国内外广泛接受的一种质量评价模式,它是对中药材或中成药进行适当处理后,采用一定的分析手段,得到的能够标示该中药材或中成药特性的共有峰的图谱。该法提供的信息量大,能够反映中药的整体性和模糊性,是对中药质量进行有效控制的可行方法之一。
本文主要以广藿香、砂仁药材及藿香正气制剂为研究对象,探索指纹图谱这一新质控技术在中药质量研究中的应用。
1.广藿香GC-MS特征指纹图谱“数字化标准”的构建与应用
(1)在导师课题组前期研究的基础上,结合对文献数据的挖掘,建立了石牌、高要、海南藿香“特征指纹图谱数据信息”。(2)为了验证其实用性,本文对24批次市售药材、3种含广藿香的复方药物进行分析,分别与石牌、海南、高要广藿香“数字化”信息进行相似度比对。(3)采用GC与GC-MS同步分析10批次样品,以考察在GC的适用性。
结果表明在24批次商品药材中,22批次可以确定产地,提示“数字化标准”可用于药材的鉴别;在3个中药复方中,藿香正气超临界提取物中的藿香为海南产,藿香正气滴丸、藿香正气软胶囊与海南藿香数字化模式的相似度最接近;在相同的色谱条件下,广藿香挥发油GC-MS指纹特征图谱能在气相色谱中体现,其GC-MS指纹特征图谱在GC中具有推广运用价值。
2.阳春砂仁GC-MS特征指纹图谱“数字化标准”的构建与应用
(1)采用GC-MS分析20批次阳春砂仁的主要成分,同时对1985年以来国内主要研究单位对16批次阳春砂仁的文献报道进行数据挖掘。结果表明20批次阳春砂仁均含有α-蒎烯、茨烯、β-蒎烯、β-月桂烯、柠檬烯、芳樟醇、樟脑、异龙脑、龙脑、乙酸龙脑酯等10个主要特征成分,合计相对含量达86.35%±5.69%,具有一定代表性;近20年来国内10余个主要研究单位报道的阳春砂仁大都含有以上10个特征成分,且相对含量也近似。以此10个特征指标成分计,本文分析样品的均值与文献报道样品的均值相似度为0.990(夹角余弦法),由此合并数据处理以建立具有广泛代表性的阳春砂仁GC-MS特征指纹图谱“数字化”信息。
(2)“数字化”信息在药材质量评价中的应用表明,约85%以上阳春产砂仁的质量比较均匀稳定,与16个文献报道阳春砂仁相似度比对的结果显示基本与原文作者的结论基本一致;在药材品种鉴定中的应用表明,文献报道的10个非阳春砂仁中仅绿壳砂与阳春砂仁非常相似,而缩砂、长太砂、华山姜、山姜、长序砂仁等与阳春砂仁通过数字化模式可以明显区分开来。提示阳春砂仁GC-MS特征指纹图谱数字化信息模式使在不同时间段(1985~2007)、不同仪器、色谱柱、色谱条件下的分析结果可以相互比对,适用于药材的质量评价与鉴定。
(3) GC的适用性研究表明,采用GC与GC-MS分别分析13批次阳春砂仁挥发油,同一批次的阳春砂仁挥发油两者图谱从外观来看非常相似,13批阳春砂仁样品GC与GC-MS特征指标成分群的相似度在0.994~1.000(夹角余弦法)之间,GC分析结果与数字化模式的相似度为0.986~0.998之间,在夹角余弦相似度天然波动范围内。
3.藿香正气制剂特征指纹图谱的构建
(1)藿香正气制剂GC-MS特征指纹图谱的构建采用GC-MS法对32批不同剂型、不同厂家、不同批号的藿香正气制剂样品进行分析,结果表明这些样品中含有柠檬烯、紫苏酮、β-广藿香烯、β-榄香烯、反式丁香烯、α-愈创木烯、刺蕊草烯、α-广藿香烯、δ-愈创木烯、β-桉油醇、广藿香醇、广藿香酮、十六酸、油酸和亚油酸、厚朴酚、未鉴定1、未鉴定2、和厚朴酚等18个主要成分。这18个主要成分的出峰先后顺序及相对含量具备特征性,可作为藿香正气制剂内在质量评价和鉴定的依据。
(2)藿香正气制剂HPLC特征指纹图谱的构建采用HPLC对29批藿香正气制剂样品进行分析,并采用“中药色谱指纹图谱相似度评价软件”对不同剂型、不同厂家、不同批号的藿香正气制剂进行相似度对比。结果表明90%的藿香正气制剂质量较好且均匀稳定,夹角余弦相似度均大于0.90;同一厂家不同批次间样品相似度较高,不同厂家产品质量存在较大差异;藿香正气丸的4个生产厂家之间相似度在0.744~0.948之间;藿香正气胶囊的5个生产厂家相似度在0.853~0.984之间;藿香正气软胶囊的2个生产厂家相似度为0.939;藿香正气水的3个生产厂家相似度在0.905~0.938之间;相同处方不同剂型产品存在较大差异性,丸剂与胶囊样品的相似度为0.880;软胶囊与水剂、片剂、滴丸的相似度在0.763~0.959之间。为藿香正气制剂质量控制研究提供了一定参考。
4.藿香正气制剂制备工艺过程中指纹图谱的追踪分析
通过对藿香正气制剂各个剂型制备工艺过程中水提、醇提、成品各步骤色谱图的比较分析,提示成品特征峰基本为处方药材各特征峰的加和,并未产生新峰。
(1)藿香正气丸结果表明:①丸剂各药材特征峰峰面积虽然在工艺过程中均有不同程度的降低,但40个特征峰最后的转移率平均值达到76.14%,转移率较高;②已鉴定的6个特征成分能够完全保留,且转移率为69.13~86.93%之间;③市售藿香正气丸成分保留的分析结果也表明,80%藿香正气丸能够保留药材中的大部分特征峰,与藿香正气散的相似度比对结果大于0.90:综上所述,藿香正气丸的制备工艺较为合理,能够保留大部分的特征成分;但从丸剂挥发油GC-MS分析结果来看,丸剂中的挥发油成分保留结果不太理想,提示丸剂制备工艺中应改进挥发油的提取方法。
(2)藿香正气胶囊胶囊与丸剂是同一处方,结果表明:①胶囊成品的平均转移率较丸剂低10.28%;②已鉴定的6个特征成分只保留了其中的4个,转移率为56.75~74.94%;③市售藿香正气胶囊的成分保留分析结果也表明,藿香正气胶囊的成分保留情况较丸剂差;但从胶囊挥发油GC-MS指纹图谱分析结果来看,胶囊中的挥发油成分保留结果较丸剂好,提示胶囊的制备工艺需进一步的改进。
(3)藿香正气软胶囊、水、片剂三者属同一处方,且制备工艺相似,结果表明:①这3种剂型的制剂成品转移率结果比较接近,分别为70.74%、67.50%、70.35%;②市售藿香正气软胶囊、水、片剂成分保留分析结果也表明,这三种制剂均能较好的保留处方药材中的特征成分,提示这三种剂型的制备工艺较为合理;③从挥发油GC-MS指纹图谱分析结果来看,软胶囊的挥发油成分保留最为完整,片剂中的挥发性成分的含量会随着储存时间的延长而减少;藿香正气水因其辅料中含有乙醇,所以检出成分较少。提示藿香正气水与藿香正气片的挥发油保留都有待于进一步的改进。
本实验通过建立藿香正气制剂的药材、半成品及成品的指纹图谱,为藿香正气制剂各处方药材的提取方法及提取率提供了一定数据,亦为藿香正气制剂的工艺研究提供了一定的参考。
Chinese medicine is a treasure of Chinese nation,which has a long history, with the exact efficacy,low toxicity.There is an increasing attention to the world.In the modern study framework of the Chinese medicine,the quality has been in urgent need of one of the major problems to overcome.At present,the wider application of traditional Chinese medicine quality evaluation method is known for one or two major components of the active ingredient or ingredients to be detected as a control indicator.However,the efficacy of traditional Chinese medicine to play is the result of the hole medicinal substances.The traditional Chinese medicine theory and medical practice challenges to the existing pattern of quality control,that is,any kind of determination of the active ingredients are not on their intrinsic quality.
Fingerprinting of Chinese medicine is widely accepted at home and abroad to a quality evaluation model.The amount of information provided by the Fingerprinting,and to reflect the holistic medicine and ambiguity.It is an effective and feasible method to the quality control of the Chinese medicine.
The research is to explore the application of this new fingerprint technology which could be used for the quality control of traditional Chinese medicine.
1.Patchouli characteristics of GC-MS fingerprint "digital standards" Construction and Application
(1) In the tutor group the basis of preliminary studies,combined with the literature data mining,the establishment of Shipai,Gaoyao,Hainan Agastache "the characteristics of fingerprint data."(2) In order to verify its usefulness,the paper sold a batch of 24 ingredients,three kinds of compound drugs containing patchouli analysis with Shipai,Hainan,Gaoyao patchouli "digital" information than similarity right.(3) The use of GC and GC-MS analysis of 10 batches of samples simultaneously in order to study the applicability of the GC.
The results show that the medicinal product in the 24 batches,22 batches of origin can be identified,suggesting that the "digital standards" can be used for the identification of medicinal herbs;in 3 Chinese herbal compound, the Zhengqi supercritical fluid extract of Agastache rugosa in Hainan production,Zhengqi pills,capsules and soft Zhengqi Digital Hainan Huoxiang closest similarity model;in the same chromatographic conditions,patchouli essential oil from the characteristics of GC-MS fingerprint patterns can be reflected in gas chromatography,the GC-MS fingerprinting patterns in the GC with the promotion of use of the value.
2.Villosum characteristics of GC-MS fingerprint "digital standards" Construction and Application
(1) using GC-MS analysis of 20 batches of the major components of Amomum villosumAt the same time,since 1985,major research units of the 16 batches of Amomum villosum data mining reported in the literature.The results showed that 20 batches of Amomum villosum containα-pinene,Mainzene,β-pinene,β-myrcene,limonene,linalool,camphor,isoborneol,borneol,bornyl acetate 10 the main features of a component of the total relative content of 86.35%±5.69%,with a certain representation;the past 20 years more than 10 major domestic research units Amomum villosum are reported to contain characteristics of more than 10 ingredients,and relatively similar content. Features of this 10 component indicators,the analysis of samples in this article reported in the literature of the mean and the mean similarity of samples is 0.990(angle cosine law),which combined data processing in order to establish a broadly representative Amomum villosum characteristics of GC-MS fingerprint of the "digital" information.
the digitalized characteristic fingerprint information pattern of volatile oil of Amomum villosum indicated in quality evaluate of medical material that the quality of 85%Amomum villosum was stable.The similarity result of literature data as the same as original writer.It was displayed in variety certification of medical material that only Amomum villosum Lour.var.xanthioides T.L.Wu et Senjen is very alike with Amomum villosum and it can be distinguished between Amomum xanthioidis semen、Lout.、Alpinia chinenses、Alpinia japonica、Amomum thyrsodlum and Amomum villosum by the digitalized characteristic fingerprint information pattern.The digitalized characteristic fingerprint information pattern of Amomum villosum can be used to compare the result between different time、machines、columns and conditions. The method is reliable to evaluate quality of Amomum villosum.
(3) GC applicability of studies have shown that the use of GC and GC-MS analysis of 13 separate batches of essential oil from Amomum villosum,Amomum villosum batches of the same volatile oil from the appearance of the two maps look very similar,13 samples approved Amomum villosum GC and GC-MS characteristics of indicators of the similarity in base composition of 0.994 -1.000(angle cosine law) between,GC analysis of model results and the similarity of the figures for 0.986-0.998 between the angle cosine similarity in the scope of natural fluctuations.
3.Zhengqi agents fingerprint characteristics and application of Construction
(1) agents Zhengqi GC-MS fingerprint mapping method using GC-MS batch of 32 different formulations,different manufacturers,different batches of samples preparation Zhengqi analysis,results showed that the samples contain limonene,purple Su-one,β-patchouli ene,β-elemene,trans-caryophyllene,α-Guaiene,gill Turcz ene,α-patchouli en-,δ-Guaiene,β-E oil alcohol, patchouli alcohol,ketone patchouli,palmitic acid,oleic acid and linoleic acid,magnolol did not identify 1,no identification of 2,and Magnolol major components,such as 18.This is the 18 major components of the peak and the relative content of the order in which they have the characteristic can be used as Zhengqi preparation and identification of the intrinsic quality of the basis for evaluation.
(2) agents Zhengqi HPLC fingerprint characteristics of the construction of 29 approved the use of HPLC Zhengqi preparation of samples for analysis and the use of "similarity of chromatographic fingerprints of Chinese medicine evaluation software" of different forms,different manufacturers,different batches of Zhengqi contrast agents for similarity.The results showed that 90% of the Zhengqi agents and even better quality and stability,angle cosine similarity are greater than 0.90;the same manufacturer in different batches of high similarity between samples from different manufacturers there is a big difference in the quality of products;Zhengqi 4 pills similarity between manufacturers in between 0.744 -0.948;Zhengqi 5 capsule manufacturers in the similarity between 0.853-0.984;Zhengqi soft capsule manufacturer of two similarity to 0.939;Zhengqi water three manufacturers similarity in the 0.905 -0.938;different formulations of prescription products the same as there is a big difference,pills and capsules of similar samples to 0.880;soft capsules and liquid,tablets,pills in the similarity between 0.763-0.959.Zhengqi quality control of preparations for the study provides a reference.
4.Zhengqi process of preparation of the follow-up analysis of fingerprint Zhengqi agents through various forms during the preparation of aqueous, ethanolic,finished all the steps of a comparative analysis of chromatograms, suggesting that the basic product for the characteristic peaks of the characteristic peaks of prescription medicines and the increase did not generate a new peak.
(1) pill Zhengqi results showed that:①Fengfeng pill characteristics of the area of medicinal herbs in the process although in varying degrees in the lower,but the final 40 of the transfer characteristic peak rate reached 76.14 percent on average,a higher transfer rate;②has identified six characteristics of the components can be fully retained,and the transfer rate of 69.13-86.Between 93%;③Pill Zhengqi components market to retain the results of the analysis also shows that 80%of Zhengqi medicine pills to retain most of the characteristic peaks with Zhengqi scattered than the results of the similarity is greater than 0.90;To sum up,the preparation of pills Zhengqi more reasonable,able to retain most of the characteristics of composition;pills from volatile oil GC-MS analysis,the pills in the retention of volatile oil components are not too satisfactory results,suggesting that pill Preparation process should be improved methods of volatile oil extraction.
(2) Zhengqi Capsule capsule and prescription pills are the same,the results showed that:①the average capsule finished pills low transfer rate i0.28%;②has identified six characteristics of the components of which only the four, the transfer rate of 56.75-74.94%;③Zhengqi capsuleingredients market to retain the results of the analysis also shows that the composition of capsule Zhengqi reservations worse than the pill;from the capsules of volatile oil GC-MS fingerprint analysis,the capsules of volatile oil components in the retention of results than the pill,and prompted the preparation of capsules for further improvement.
(3) Zhengqi soft capsule,water,three belong to the same prescription tablet and is similar to the preparation process,results showed that:①This form three kinds of agents results finished transfer rate close to 70.74%, respectively,67.50%,70.35%;②market Zhengqi soft capsule,water,tablets ingredients to retain the results of the analysis also shows that these three agents are able to better retain the characteristics of prescription medicines in the composition,suggesting that these three forms of the preparation process more reasonable;③from the volatile oil GC-MS fingerprint analysis, the soft capsule composition of the volatile oil to retain the most complete, tablets of the content of volatile components as the extension of storage time reduced;Zhengqi in water because of its accessories containing ethanol,so fewer components detected.Zhengqi prompted Zhengqi water and the preparation of films are to be further improved.
This experiment Zhengqi agents through the establishment of medicinal materials,semi-finished and finished products fingerprint for the preparation of the prescription medicines Zhengqi extraction methods and the extraction rate to provide some data,the process for preparation Zhengqi study provides some reference.
引文
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