Discussion on the Quality Evaluation Method of Traditional Chinese Medicine Compound Preparations

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1. Guangxi University of Chinese Medicine, Nanning 530200, China; 2. Guangxi Zhuang Yao Medicine Center of Engineering and Technology, Nanning 530200, China

Abstract As an important representative of the modernization of the Chinese medicine industry, traditional Chinese medicine (TCM) compound preparations are an indispensable part of the development of TCM. The complex ingredients and unclear action mechanism of TCM compound preparations restrict the development of TCM. In line with this problem, the modern TCM research has made extensive remarkable achievements. From the single indicator in the past to multiple indicator, the research on the combination of TCM fingerprints and pharmacodynamics also has made great progress. The introduction of quality evaluation methods such as the "quantitative analysis of multi-components by a single marker", "dose-effect" indicator, "Bioactive Equivalent Combinatorial Components" (BECCs) and TCM quality markers have brought new ideas for quality control of TCM compound preparations.

Key words Traditional Chinese medicine (TCM), Compound preparations, Quality evaluation method

1 Introduction

In order to promote the sustainable and healthy development of China’s traditional Chinese medicine industry, relevant departments have issued policies and regulations such asLawofthePeople’sRepublicofChinaonTraditionalChineseMedicineandOutlineoftheStrategicPlanontheDevelopmentofTraditionalChineseMedicine, which has created a good development environment for the TCM big health industry[1]. TheChinesePharmacopoeiahas been published for 10 editions. The TCM quality control has gone through three stages of development: simple appearance identification, microscopic identification and chromatographic analysis. From the detection of a single component to the detection of multiple components, the quantitative indicators are also developing from the indicator components to the active components[2]. The Volume I ofChinesePharmacopoeia(2015 edition) improved the research ideas, evaluation methods and models of the quality standards of TCM complex systems, and strengthened the overall control of the TCM quality[3].

As an important representative of the modernization of the Chinese medicine industry, traditional Chinese medicine (TCM) compound preparations are an indispensable part of the development of TCM. In recent years, the continuous development of TCM has gradually made the quality control of traditional Chinese medicine compound preparations turning into exploration and research in a deeper and wider field. Further cross-integration of multi-disciplinary and multi-disciplinary fields, research and innovation of new technologies and methods have brought new ideas to the quality control of TCM compound preparations[4]. The efficacy of TCM e compound is the result of the comprehensive action of its components, and it is the embodiment of multi-component, multi-target and multi-channel interaction. In order to fully reflect the complexity and integrity of traditional Chinese medicine compound, the quality control method of traditional Chinese medicine compound is developing from single component research to group component research, from "micro analysis" to "macro analysis", gradual combination of qualitative and quantitative analysis of material components and pharmacodynamic activity, which have laid an important foundation for the quality control and comprehensive evaluation of traditional Chinese medicine compound preparations. At present, the problems of the effectiveness and safety of traditional Chinese medicine compound are still not well solved, and there are various methods for comprehensive quality evaluation and control of traditional Chinese medicines. In this study, we collected and sorted out relevant data in recent years, and summarized the comprehensive analysis methods of the quality evaluation of several traditional Chinese medicine compound preparations from the traditional Chinese medicine fingerprint, "quantitative analysis of multi-components by a single marker", "dose-effect" indicator, "Bioactive Equivalent Combinatorial Components" (BECCs) and TCM quality markers, to provide certain references for further research.

2 Evaluation method based on traditional Chinese medicine fingerprint

The fingerprint of traditional Chinese medicine is a semi-quantitative identification technology. Compared with the measurement mode of single active component, it contains more information, is multi-level, quantifiable, comprehensive, and more scientific and reasonable evaluation method. Traditional Chinese medicine fingerprints can effectively study the quality control of traditional Chinese medicine compound preparations, so as to evaluate the quality and stability of compound preparations[5]. For the quality control research on traditional Chinese medicine compound preparations, the detection method used should not only reflect rich identification information, but also have the ability to adapt to the characteristics of multi-components of traditional Chinese medicine compound. As a suitable research model, traditional Chinese medicine fingerprint is based on the comprehensive study of chemical composition, emphasizing the identification of the common peak assignment of the fingerprint and the similarity evaluation of the fingerprint, which can not only characterize the chemistry of the traditional Chinese medicine compound, but also fully reflect the content of each component. Through such model, the quality of the traditional Chinese medicine compound can be comprehensively and concretely described, so as to make a scientific and reasonable evaluation[6]. At present, if the traditional Chinese medicine compound contains a variety of unclear active components, it is able to clarify the active components by establishing its fingerprint, which makes the quality control of Chinese medicine compound significantly improved.

Using HPLC, Wang Xuelietal.[7]established the fingerprint information of Antai Pills, and preliminarily clarified its chemical basis. Also using UPLC, Sun Dongmeietal.[8]established a fingerprint of Fufang Qima Capsule, identified 22 common peaks, and found that there was no significant difference between sample batches. This method provides a reference for quantitative control study of compound preparations containing ASTRAGALI RADIX, GASTRODIAE RHIZOMA, CITRI GRANDIS EXOCARPIUM, and CHUANXIONG RHIZOMA. Using capillary zone electrophoresis, Sun Guoxiangetal.[9]established a capillary electrophoresis fingerprint of Xiaoyao Pill, optimized the experimental conditions by square optimization method, confirmed 21 common peaks in the samples, and verified the quality stability of Xiaoyao Pills between different batches by the system fingerprint quantification method. The fingerprint of traditional Chinese medicine has created a new chapter in the quality control of traditional Chinese medicine. With the development of science and the innovation of hardware equipment, various combined technologies have been widely applied to study the pharmacodynamic substance basis of traditional Chinese medicine compound preparations[10]. Using HPLC-QTOF/MS technology, Han Yanqietal.[11]carried out a qualitative study on 51 chemical components in Yuanhu Zhitong Prescription. Yang Hetal.[12]combined the GC-MS with chemometrics to study the fingerprint of compound Shengui Hair-growth Tincture.

3 Evaluation method based on fingerprint and multiple indicator quantitative determination

Traditional Chinese medicine has used the quality control model of western medicine for many years to control the quality of traditional Chinese medicine through qualitative and quantitative detection of one or two indicator components in traditional Chinese medicine, which does not conform to the complex system of traditional Chinese medicine as a multi-component, and multi-target interaction when exerting pharmacodynamic effects, and the efficacy of the traditional Chinese medicine compound is affected by the content and proportion of each component. Therefore, it is necessary to select multiple indicator components or active ingredients for quantitative determination, especially chemical components associated with the efficacy. The fingerprint of traditional Chinese medicine has become a feasible quality evaluation mode in identifying the authenticity of Chinese medicine products and evaluating the quality consistency and stability. It is necessary to combine the overall identification of the fingerprint with the quantitative analysis of the multi-indicator components, that is, to include the qualitative function of the fingerprint and the quantitative function of the key components, so as to realize the comprehensive and effective quality control of the traditional Chinese medicine products. Wangetal.[13]studied the fingerprint of Xiaokeqing Granule and quantitatively determined the content of five components in the compound by the external standard method. According to their study, the amount of each component was uniform and stable in the 28 batches of samples, and the comprehensive examination of the fingerprint and quantitative results can make an overall evaluation of the quality of the medicine. Zhao Zhiguoetal.[14]established the fingerprint of Qianliexin Capsule and identified eight components in the compound extract. The quantitative results of multiple indicators showed that the linear relationship of each component was good, and the similarity of the 10 batches was in the range of 0.999 6 and 1.000 0. Through the fingerprint analysis of Qianliexin Capsule combined with multiple indicator quantitative analysis, they provided a more comprehensive method and technical support for the quality control and evaluation of the preparation.

4 Evaluation method based on quantitative analysis of multi-components by a single marker

Considering that multi-indicator quantitative analysis is restricted by the supply and demand of reference materials in actual production and scientific research, and the cost of multi-indicator testing is high[15], Wang Zhiminetal.[16]proposed the quantitative analysis of multi-components by a single marker for quality control of traditional Chinese medicine. Through determining only one component that is easily obtained from the reference substance, and by the proportional relationship between the functional components of the traditional Chinese medicine, it is expected to realize the simultaneous determination of other components that have no reference substance or are difficult to supply. Under the condition that the reference substance is insufficient, this method realizes the multi-component quantitative and multi-indicator quality control of traditional Chinese medicine compound, and effectively improves the analysis efficiency and reduces the detection cost. Deng Junjie[17]studied the single marker multi-component determination method of Zilongjin Tablet, and taking Tanshinone IIA as the internal reference substance, determined the content of four active ingredients in the sample using the single marker multi-component determination method and external standard method. The results showed that the amount of RAD of the main components measured by the two methods was lower than 0.5%, proving that the single marker multi-component determination method was feasible and further ensuring the efficacy of Zilongjin Tablet. Taking the Schisandrin as the internal standard, He Chunxietal.[18]established a single marker multi-component determination method for the six components of Wuzi Yanzong Pill. Through calculating the content of each component in Wuzi Yanzong Pill, and comparing with the measured value of the external standard method, the results indicate that there is no significant difference between the two methods. Therefore, the single marker multi-component determination method can effectively make quantitative determination of the various components of Wuzi Yanzong Pill. There are problems of many varieties of traditional Chinese medicine compound preparations but fewer types of indicator components. The single marker multi-component determination method mainly selects active components of sovereign medicinal, but seldom monitors components of ministerial medicinal[19], so this method still needs tackling some problems in the development of quality control of traditional Chinese medicine compound preparations.

5 Evaluation methods related to the basis of pharmacodynamic substances

5.1 Spectrum-effect relationship quality control mode of traditional Chinese medicineThe fingerprint has the characteristics of strong characteristic, large volume of information and high specificity, which can comprehensively reflect the type and quantity of chemical components contained in the traditional Chinese medicine compound. However, the fingerprint of traditional Chinese medicine has certain limitations in characterizing the relationship between chemical components and pharmacodynamics. It reflects the chemical information of the traditional Chinese medicine component, but can not reflect whether the chemical component is the pharmacological component and the degree of association with the pharmacodynamic effect, thus it can not reflect the efficacy of traditional Chinese medicine. In other words, only using fingerprints to evaluate the quality of traditional Chinese medicine products, it is difficult to achieve the purpose of ensuring the effectiveness of traditional Chinese medicines. In China, Li Rongetal.[20]first proposed the creation of traditional Chinese medicine spectrum effect in China, and used fingerprint as a research basis, combined with drug efficacy evaluation, to explore the relationship between the active components of traditional Chinese medicine and clinical drug effects reflected by the fingerprint. The study of spectrum-effect relationship makes the efficacy of traditional Chinese medicine ingredients be reflected in the fingerprint, and reflect the relationship between fingerprint characteristics and drug efficacy, and clarify the basis of pharmacodynamics, so as to control the quality of traditional Chinese medicine more reasonably[21]. Using HPLC, Hu Fangetal.[22]established the fingerprint of Buzhong Yiqi Pill. They carried out mouse experiments on different batches of Buzhong Yiqi Pill, and measured the body mass and organ index of the mice to establish a spectrum-effect correlation analysis. Correlation analysis between fingerprint and pharmacodynamic data showed that each chromatographic peak had a certain correspondence with the qi-tonifying effect of the preparation, and hesperidin and formononetin contributed a lot to the qi-tonifying effect. Using HPLC, Cheng Lingetal.[23]established the fingerprint of Liuwei Wuling Tablet, and carried outinvitroactivity measurement to establish the spectrum-effect relationship. The results showed that the fingerprints contained 23 common peaks. Through bivariate correlation analysis, it was found that the anti-hepatic fibrosis function of Liuwei Wuling Tablet is the result of multi-component interaction. SCHISANDRAE CHINENSIS FRUCTUS, LIGUSTRI LUCIDI FRUCTUS, FORSYTHIAE FRUCTUS, and CURCUMAE RHIZOMA are main sources of pharmacodynamic substances. The spectrum-effect relationship study provides a reference for clarifying the pharmacodynamic substance basis and improving the quality standards for the preparation.

5.2 Dose-effect indicator quality control modeThe dose-effect indicator quality control mode adopts the strategy of "systematic stratification screening" as a whole or system, that is, the solvent system separation method combined with the pharmacodynamic experiment, to initially determine an effective extract which can represent the overall prescription effect. On this basis, based on the "genetic diagnosis and treatment" strategy, taking the spectrum-effect relationship of traditional Chinese medicine as a breakthrough, the research was carried out on effective substance identification and quality control of traditional Chinese medicine. Taking the whole of traditional Chinese medicine as the research object, based on the research of "spectrum-effect" relationship, the solvent component separation method ("knock out" method) to obtain the target component, and the target component was established by establishing an efficient, sensitive and economical evaluation method. The parallel comparison and identification were carried out for the biological activity or efficacy of the target component, the negative sample and the whole Chinese medicine, to reflect the key components of the efficacy of the traditional Chinese medicine. Then, using the "knock-in" method, the target components of different doses were added to the negative sample, the equivalent effect comparison was carried out with the overall pharmacodynamic action or toxicity of the traditional Chinese medicine, and the "dose-effect" relationship of the target component was established, so as to scientifically and reasonably establish a limited range of pharmacodynamic components closely related to the safety and effectiveness of traditional Chinese medicine[24]. Zhang Tiantian[25], Li Junxian[26], Kong Weijun[27], and He Jing[28]applied the "knock-out" strategy to the "dose-effect" of key pharmacodynamic components of Chinese herbal medicines such as LONICERAE JAPONICAE FLOS, COPTIDIS RHIZOMA, BOVIS CALCULUS, and CURCUMAE LONGAE RHIZOMA. In the relationship research, the related methods and theories of quality standards based on biological evaluation of ISATIDIS RADIX, CURCUMAE LONGAE RHIZOMA, COPTIDIS RHIZOMA, and PUERARIAE LOBATAE RADIX were established. This method always regards traditional Chinese medicine as a whole, is related to the efficacy of medicine, pays attention to the relationship of multi-component integration of traditional Chinese medicine, and can rapidly and accurately identify the key efficacy substances of traditional Chinese medicine. However, it is not applicable to the traditional Chinese medicines that have unclear efficacy and the efficacy can not be quantified or ingredients are too complex.

5.3 Quality control mode of bioactive equivalent combinatorial componentsAmong numerous chemical components of traditional Chinese medicine, research on those components that can reflect the efficacy of traditional Chinese medicine is fundamental to the basic research of traditional Chinese medicine. When using traditional Chinese medicine to treat diseases, not all ingredients have efficacy. Instead, there is a group of components with clear chemical structure and clear content, which have the same efficacy as the original Chinese medicine, namely, bioactive equivalent combinatorial components. The bioactive equivalent combinatorial components can provide indicator group for the quality control method of traditional Chinese medicine, and can also provide a combination of lead compounds for the research of synthetic drugs. Liu Petal.[29]found a group of 18 chemical components in the Fufang Danshen Prescription (accounting for 15.01% of the original prescription). They found that the group showed, at the cell and animal levels, the same anti-myocardial ischemia effect as the compound Danshen prescription, proving that the bioactive equivalent combinatorial components can be used as the "labeled dose" of the active ingredient of this preparation. However, the same drug can treat different diseases, and at this time, the pharmacodynamic substance basis is bioactive equivalent combinatorial components. For example, the bioactive equivalent combinatorial components of Fufang Danshen Dripping Pill include six components[30], which are different from 18 components that have anti-inflammatory and anti-ischemic effects. Due to the limitations of modern technology, there are still many shortcomings in the development of bioactive equivalent combinatorial components. However, through continuous exploration and research, the technical methods of the bioactive equivalent combinatorial components can be gradually improved, showing a broad application prospect.

6 Evaluation method based on Q-marker

Academician Liu Changxiao proposed the quality marker (Q-marker) of traditional Chinese medicine[31], that is, the Chinese medicine Q-marker is a chemical substance which is inherent in or processed in Chinese herbal medicines and closely related to the efficacy of traditional Chinese medicine. It is used as the representative of the effectiveness and safety of traditional Chinese medicine for quality control. It can be found that the necessary condition as a Q-marker is a substance closely related to the efficacy of traditional Chinese medicine, which further links the effectiveness, material basis and quality control components of traditional Chinese medicine, and is actually manifesting the effectiveness and safety of the quality standard of traditional Chinese medicine from the material basis of the medicinal materials. The proposal of Q-marker promoted the construction of quality control and quality traceability system for traditional Chinese medicine, and provided a new perspective for the quality control and evaluation of traditional Chinese medicine compound. Besides, it will play an important role in the future research on quality control of traditional Chinese medicine and compound preparation. Based on the research mode of traditional Chinese medicine Q-marker, using UPLC-Q-Exactive combined technology, negative ion monitoring mode, and with the aid of full scan, Zhou Xiujuanetal.[32]carried out a rapid analysis of the components contained in Qingreiling Granules. They identified 32 chemical components in this compound, and 11 components such as baicalin, wogonoside, and forsythiaside A can be used as representative Q-markers for Qingreiling Granules. Using UPLC-Q-TOF-MS, Wang Qiongjunetal.[33]confirmed 22 chemical components in Shuanghuanglian Preparation, and using HPLC-DAD simultaneous quantitative detection, they determined 22 main active components in the preparation, through the combination of analysis of chemical components and pharmacological and pharmacological activities, they studied Q-marker of Shuanghuanglian Preparation, which provided a new method for systematic evaluation of the quality of the preparation. Zhang Tiejunetal.[34]proposed a research path for the quality of traditional Chinese medicines based on the "five principles", namely, the specificity, effectiveness, measurability, mass transfer and traceability, and compound compatibility environment of components; they carried out the research from these five principles and successfully applied them to the Q-marker study of Shufeng Jiedu Capsule and Yuanhu Zhitong Dripping Pill, which provided systematic research ideas and methods for the perspective of the integrity of Chinese medicine Q-markers.

7 Conclusions and prospects

In summary, the evaluation methods of various Chinese medicine compound quality control have been innovated in their theories, developed and improved in practice, which provides a source of vitality for the scientific evaluation of the quality of traditional Chinese medicine. The research field of quality control of traditional Chinese medicine compound preparations is cutting-edge and challenging. Based on the substance of traditional Chinese medicine quality, the method of determining the content of single or certain indicator components to evaluate the quality of traditional Chinese medicine has its limitations, which is contrary to the TCM theory. As a important quantitative evaluation method for the multi-indicator quality control of Chinese medicine, the single marker multi-component determination method has certain limitations. For example, this method focuses on the quantitative aspects of traditional Chinese medicine. At present, many scholars have combined the spectrum-effect relationship with single marker multi-component determination method[35], which is related to the pharmacodynamic relationship from both qualitative and quantitative perspectives, to make more systematic evaluation of the quality of traditional Chinese medicine. The application and rapid development of traditional Chinese medicine fingerprints reflect the trend of overall quality management of traditional Chinese medicines, and conform to the characteristics of the overall characterization of traditional Chinese medicine quality control, and can effectively control the quality of traditional Chinese medicine. The ideal state of quality control of traditional Chinese medicine is to analyze and test the pharmacodynamic components under the condition that the research on the pharmacodynamic substances of traditional Chinese medicine is basically clear. However, the basic research of pharmacodynamic substances is very difficult. It is still difficult to scientifically and accurately clarify the material basis for efficacy of a traditional Chinese medicine. The spectrum-effect relationship, dose-effect indicator, and bioactive equivalent combinatorial component quality control mode can combine different research ideas, which enriches the content of basic research on Chinese medicine pharmacodynamics. The production of Q-markers of traditional Chinese medicine has brought new ideas to the analysis of multi-category components of complex systems, and provided a new perspective and mode for quality control of traditional Chinese medicine.

In order to take up a large share in the international pharmaceutical market, traditional Chinese medicine products must take the road of characteristic quality control under the conditions of fully embodying the characteristics of traditional Chinese medicine, which has far-reaching significance for achieving the goal of internationalization and modernization of Chinese medicine. Therefore, only through combining different research ideas and methods and techniques, learning from each other’s strengths, will there be a substantial breakthrough; through formulating and improving the comprehensive evaluation of the quality of traditional Chinese medicine, it is able to truly grasp the connotation of traditional Chinese medicine and realize the long-term development of traditional Chinese medicine and pharmacy. In short, the research trend of the quality evaluation method of traditional Chinese medicine compound preparations will evidently walk to multi-disciplinary and multi-field penetration and integration, comprehensive analysis of multiple indicators, and scientific and comprehensive evaluation of the quality of traditional Chinese medicine compound preparations can better ensure the safety and effectiveness of using medicines.