The method of quality marker research and quality evaluation of traditional Chinese medicine based on drug properties and effect characteristics

Accepted Manuscript

The method of quality marker research and quality evaluation of traditional Chinese medicine based on drug properties and effect characteristics Tiejun Zhang , Gang Bai , Yanqi Han , Jun Xu , Suxiao Gong , Yazhuo Li , Hongbing Zhang , Changxiao Liu PII: DOI: Reference:

S0944-7113(18)30033-3 10.1016/j.phymed.2018.02.009 PHYMED 52375

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

18 July 2017 16 January 2018 25 February 2018

Please cite this article as: Tiejun Zhang , Gang Bai , Yanqi Han , Jun Xu , Suxiao Gong , Yazhuo Li , Hongbing Zhang , Changxiao Liu , The method of quality marker research and quality evaluation of traditional Chinese medicine based on drug properties and effect characteristics, Phytomedicine (2018), doi: 10.1016/j.phymed.2018.02.009

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The method of quality marker research and quality evaluation of traditional Chinese medicine based on drug properties and effect characteristics

Tiejun Zhanga,*, Gang Baib, Yanqi Hana, Jun Xua, Suxiao Gonga, Yazhuo Lic, Hongbing Zhanga, Changxiao Liuc,**

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of Pharmaceutical Research, Tianjin 300193, People's Republic of China b

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Tianjin Engineering Laboratory of Quality Control Technology of Traditional Chinese Medicine, Tianjin Institute

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Tianjin Key Laboratory of

Molecular Drug Research, Nankai University, Tianjin 300350, People's Republic of China c

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Tianjin Center for New Drug Evaluation and Research, State Key Laboratory of Drug Delivery Technology and

Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, People's Republic of China

*Corresponding author: Tiejun Zhang **Co-corresponding author: Changxiao Liu

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Address: No. 308, An Shan Xi Road, Nan Kai District, Tianjin, P.R. China, 300193

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Tel/Fax number: +86-022-23006848 (TJ. Zhang); +86-022-23006863 (CX. Liu)

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E-mail addresses: [email protected] (TJ. Zhang); [email protected] (CX. Liu)

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Table of contents Introduction................................................................................................................................................................ 5 1 Property and features of function of TCM and its role in quality assessment ......................................................... 5 1.1 The basic properties of TCM ...................................................................................................................... 5 1.1.1 The herbal properties of TCM ........................................................................................................ 5 1.1.2 The biological properties of TCM .................................................................................................. 6

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1.1.3 The chemical nature of TCM .......................................................................................................... 6 1.2 Biological effects of TCM and their characteristics .................................................................................... 7 1.2.1 The dual expression characteristics of traditional efficacy of „drug property‟ and „drug efficacy‟ . 7 1.2.2 Clinical application and effective form of TCM ............................................................................. 7

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1.3 The quality transfer of TCM preparation and in vivo process ..................................................................... 8 1.3.1 The component changes and quality transfer in the preparation process ........................................ 8 1.3.2 Changes of chemical components in drug delivery pathway and in vivo process ........................... 8 2 Research approach and the basis of discovery and determination of Q-markers .................................................... 9

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2.1 Systematic study on the chemical components of TCM based on the transitivity and traceability of quality factors ................................................................................................................................................... 9

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2.1.1 Identification of „original components‟ in herbs ............................................................................. 9 2.1.2 Study on identification of „prototype components‟ in preparation .................................................. 9

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2.1.3 Study on the identification of the „effective components‟ in blood................................................. 9 2.2 Research approach of Q-marker based on chemical component biosynthetic pathway and specificity ...... 9

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2.3 Research approach of Q-marker based on the correlation between chemical component and traditional efficacy ........................................................................................................................................................... 10

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2.3.1 Research method of Q-marker based on component and efficacy ................................................ 10 2.3.2 Characterization and confirmation of effective components based on the „drug property‟ of TCM11 2.3.3 The identification and mode recognition methods of Q-marker based on composition measurability and method specificity ........................................................................................................................... 13

3 Establishment of quality research model based on the core concept of Q-marker and properties of TCM ........... 13 3.1 Research and determination of Q-marker ................................................................................................. 13 3.1.1 Progressive chemical components research of prototype-pharmaceutical process-in vivo process13 3.1.2 Specificity analysis of biosynthetic pathway of chemical components ........................................ 14 2

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3.1.3 Correlation analysis of components-efficacy and determination of effective components based on efficacy expression of TCM .................................................................................................................. 14 3.1.4 Determination of Q-marker based on component testability ........................................................ 14 3.2 Establishment of QC method .................................................................................................................... 14 3.3 Experimental sample collection methods, sampling methods and sample determination ......................... 15 3.3.1 Sample coverage ........................................................................................................................... 15

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3.3.2 Representativeness of sampling .................................................................................................... 16 3.3.3 Transfer of „effectiveness acquisition‟ and „quality factor‟ in TCM production process .............. 16

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Conclusion ............................................................................................................................................................... 17

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ABSTRACT Background: Quality of traditional Chinese medicine (TCM) plays a critical role in industry of TCM. Rapid development of TCM pharmaceutical areas is, however, greatly limited, since there are many issues not been resolved, concerning the quality study of TCM. Hypothesis/Purpose: Core concept of TCM quality as well as the characteristics of TCM were discussed, in order to guide the quality research and evaluation of TCM, further improve the level of TCM quality control.

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Study Design/Methods: In this review, on the basis of systematic analysis of fundamental property and features of TCM in clinical application, the approaches and methods of quality marker (Q-marker) study were proposed through combination of transitivity and traceability of essentials of quality, correlation between chemical ingredients and drug property/efficacy, as well as analysis of endemicity of ingredients sharing similar

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pharmacophylogenetic and biosynthetic approaches.

Results: The approaches and methods of Q-marker study were proposed and the novel integrated pattern for quality assessment and control of TCM was established.

Conclusion: The core concept of Q-marker has helped to break through the bottleneck of the current fragmented

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quality research of TCM and improved the scientificity, integrity and systematicness of quality control.

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Keywords: Quality of TCM; Q-marker; Research idea; Research method; Research approach; Practice

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Abbreviations

TCM, traditional Chinese medicine; QC, quality control; Q-marker, quality marker; LTT, Liujing Toutong

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tabliets; HPLC-Q/TOF-MS, high performance liquid chromatography quadrupole/ time of flight mass spectrometry; GC-MS, gas chromatography mass spectrometry; YHZT, Yuanhu Zhitong dropping pills;

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UPLC-Q/TOF MS, ultra-performance liquid chromatography quadrupole/ time of flight mass spectrometry; SFJD, Shufeng Jiedu capsule; GPCR, G-protein-coupled receptors; PCA, principal component analysis; DFA, discriminant factor analysis; TAS2R10, taste 2 receptor member 10; OR7D4, olfactory receptor family 7 subfamily D member 4; HTR7, 5-hydroxytryptamine receptor 7; D2, dopamine receptor 2; ADRA1A, α1A-adrenoceptor; ADORA1, adenosine A1 receptor; QBD, quality by design

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Introduction Good quality of traditional Chinese medicine (TCM) and its preparations is fundamental of clinical efficacy. Standardization of TCM is now the core of modernization of TCM. Over the past decades, great efforts were made by Chinese scientists in the field of quality control (QC) of TCM, QC was greatly enhanced accordingly. Many issues regarding current quality standard remains, for instance, quality of TCM cannot be properly evaluated. With respect to the problem, a new concept of quality marker (Q-marker) was proposed by Changxiao Liu et al (Liu et

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al. 2016). Since then, quality studies of TCM, in particular Q-marker studies were performed as to establish new research pattern (Zhang et al. 2016a, 2016b; Xiong et al. 2016; Bai et al. 2016; Jiang and Wang 2016). Concerning the core concept of Q-marker, a strategy of quality evaluation and research ideas based on drug property and feature of action were discussed in this paper.

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1 Property and features of function of TCM and its role in quality assessment

1.1 The basic properties of TCM

As one of the conventional therapies, TCMs shares the property of traditional drugs. In the meanwhile, biological features are present since TCMs are mainly sourced from organisms either plants or animals. Hence,

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1.1.1 The herbal properties of TCM

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herbal and biological features are critical in quality evaluation of TCMs.

Herbal features of TCM are reflected as follows: First of all, clinical efficacy of TCMs has been demonstrated

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in long-term applications. The recognition of efficacy and applications are sourced from theory of Chinese medicine and experiences. The conventional efficacy of TCM was therefore passed and developed in clinical

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practice. In the meanwhile, strain and species of TCM are evolutionary, the species of a specific herb used might be different in certain period of time. This may be associated with either knowledge updated/new specie emerges

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or limited natural resources of the original variety/alternative variety. Also, the plants sharing very similar features of morphology and name may lead to incorrectly recorded in literature. In addition, the variety of the same herb used in different regions could be various, the so-called „local prescription‟ was then produced. The original variety of herbs should therefore be verified through analysis of evolutionary development of species of TCM and historical environment of conventional medicine, as well as experience accumulation. Moreover, as one of the key features of conventional medicines, habitat plays a critical role. Habitat of TCM is closely correlated with the quality of TCM. The herbs with good quality were defined as authentic herbs. It is worthy of note that habitat with best quality may not be a fixed place, which also undergo evolutionary development as environmental change and

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deeper understanding. Consequently, evolutionary change of habitat, analysis of rational of authentic herbs, as well as correlation between ecological environment and quality of herbs should be given special consideration in study of Q-marker of TCM. 1.1.2 The biological properties of TCM Quality of a specific herb is also affected by its biological variation, herbs with multiple origins are always from diverse species of plants. The differences, in terms of chemical ingredients, are highly associated with clinical

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efficacy. Single origin herbs are from only one specie though, the intra-species variations occur, leading to heritable change. All changes are then classified into a variety of categories, for instance, subspecies, geography, varieties, locality, variants, ecological cases, chemical type, populations. Polymorphism in medicinal plants is the common problem. Chemical ecotype, as one of the intra-species variations, was associated with biosynthesis of

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secondary metabolites, which were affected by the genes. This may lead to difference, in terms of types of chemical ingredients, absolute and relative content, and eventually quality of herbs. 1.1.3 The chemical nature of TCM

The chemical components of TCM is essential for assessment and QC. The formation, transformation and

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biological response of chemical ingredients of TCM are complicated. Chemical nature of TCM quality should therefore be recognized and assessed through formation, transformation and transfer of entire chemical groups.

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①Formation of chemical substances group -- biosynthetic pathway Bioactive ingredients of TCM are mainly secondary metabolites of medicinal plants, which are produced

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through biosynthesis in vivo and accumulated. According to their origins and geological/ecological courses, specific secondary metabolites are generated in each plant. Raw materials and conditions for bioreaction of

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biosynthesis are provided by environment. The secondary metabolites are adjustment of medicinal plants and surroundings. As known, chemicals like monoterpenes and enol with aromatic scents, stilbene, alkaloids, ferulic

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acid and other phenolic acids are critical for growth, resistance under environmental pressure, insect resistant and allelochemical, respectively (Ye et al. 2012). The quality and efficacy of TCM were determined by those secondary metabolites, in terms of types, contents and proportions. ② Genetic relationships and regularity of chemical ingredients The medicinal plants with close genetic relations share very similar chemical ingredients, the similar efficacy is thus expected. According to systematic evolutionary botany, the correlation between genetic relationship and chemical quality can be attributed to the followings: 1) the class of plants in one unit share the identical ancestral species, most of genus are natural class of group. 2) individual variations in the same class of group are results 6

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from geological and ecological change during the process of systematic development. 3) original ingredients of this genus are identified in those groups closed to ancestral species, the plants are distributed in the center of the origin or the preservation place of the original species; on the contrary, the new ingredients from evolutionary process were found in those groups far from the ancestral ones, which are located either at center of differentiation or on the edge of distribution area. 4) diverse alternative phenomenon was observed in closely related groups as geographic and ecological gradient changed. 5) chemical and efficacy geography of medicinal plants emerges,

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based on the combinations of edicinal plant geography, kinship relation, floristic geography, hypothesis of biosynthetic pathway and ethnopharmacology (Zhang et al. 1996). The analysis of the specificity of chemical ingredients of TCM and prediction of functions, on the basis of theories above, may offer valuable information for drug discovery, also for determination of TCM quality.

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1.2 Biological effects of TCM and their characteristics

1.2.1 The dual expression characteristics of traditional efficacy of ‘drug property’ and ‘drug efficacy’ Drug properties and efficacy are fundamental features of TCM, which describe how TCM are used in treatment of diseases. These two are reflected by the material basis of TCM from diverse aspects (Zhang et al.

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2015; Liu et al. 2015). Characterization of „property-efficacy-material basis‟ and study of correlation are of particular importance in explanation of material basis of drug efficacy, mechanism of action, rationale of

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compatibility, as well as providing guidance in clinical applications. Previous studies under guidance of component-efficacy pattern, has not taken drug property of TCM into

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considerations and the characterization of functional value of TCM is not complete. While the triarchic theory of „property-efficacy-component‟ was believed to be able to explain the material basis and mechanism of TCM by

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integration of multidisciplinary understanding from the basic properties of TCM, basic concepts and terms of TCM theory. It is of great significance to inherit and promote the valuable experience of TCM by restoring and scientific

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explanation of the TCM theory, as well as highlighting the characteristics of TCM theory and eventually providing guidance for clinical practice. Therefore, for quality evaluation of TCM, not only the effective components should be taken into account but also the components of „drug property‟. It is significant to reflect the functional characteristics, quality integrity and „precision quality‟ of TCM. 1.2.2 Clinical application and effective form of TCM In general, TCM is applied as compound prescription in clinical. Effectiveness of TCM is reflected by overall biological effects of entire chemical ingredients, rather than single chemical entity, since single ingredient cannot represent the drug property and efficacy of the herbs, also the efficacy is not simply the sum of all components. 7

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As the matter of fact, „network of efficacy‟ is existing in the interference of pathological course by chemical ingredients, diverse patterns are present among the chemical components. In particular, there is a systematic effect in compound prescription, as well as nonadditivity. For assessment of quality of compound prescription of TCM, compatibility should be taken into consideration. 1.3 The quality transfer of TCM preparation and in vivo process Chemical ingredients of TCM undergo changes, including biosynthesis in medicinal plants and productions,

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acquisition, transfer and biotransformation in vivo. The specific ingredients may also be further metabolized to exert its biological functions. During this process, the quality of TCM is transferred. 1.3.1 The component changes and quality transfer in the preparation process

Most herbs with volatile components may lose its bioactive ingredients in drying. For those rhizomatic TCM,

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contents of soluble ingredients can be lowered by cutting and preparation, while glycosides are degraded to aglycone through enzomolysis in given temperature and humidity.

In addition, many herbs are used after appropriate processing as to either reduce their toxicity, enhance its efficacy or promote decoction. For instance, the ingredients of herbs after wine processing remain unchanged

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though, the cell and tissue structures of herbs altered may boost the transfer procedure, which is beneficial to dissolution of effective components. Also, extraction process is the reacquisition procedure of chemical groups of

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TCM, multiple-components may undergo complicated changes during this process. In the meanwhile, single or mixed decoction may result in different outcomes, solubilization, antagonism, decomposition, precipitation,

quality of TCM.

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complexation observed among those chemicals, thus optimization of extraction is another key factor affecting

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1.3.2 Changes of chemical components in drug delivery pathway and in vivo process TCMs are transferred into human body as specific preparation. The study in vivo is not only the critical

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pathway of correlation between substance and efficacy, but also provide appropriate approach to understand relationship between prototype chemical and substance with biological response, in particular the tracing path. Eventually, this may offer solid evidence to determine the Q-marker.

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2 Research approach and the basis of discovery and determination of Q-markers

2.1 Systematic study on the chemical components of TCM based on the transitivity and traceability of quality factors Chemical ingredients of TCMs undergo biosynthesis in vivo, extraction and purification, as well as absorption and metabolism process, eventually exert efficacy in treatment of disease. Chemical changes and

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transfer in these three steps should be paid more attention, in terms of QC. Based on the transitivity and traceability of quality factors, QC system covering all industrial chain should be established. In this section, chemical components of Liujing Toutong tabliets (LTT) were systematically analyzed as an example (HPLC and GC profiles of LTT and 8 herbs are shown in Fig.S1). 2.1.1 Identification of ‘original components’ in herbs

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Initially, identification of the chemical components of 8 raw herbs in LTT were performed. 162 and 175 chemicals were identified through high performance liquid chromatography quadrupole/ time of flight mass spectrometry (HPLC-Q/TOF-MS) and gas chromatography mass spectrometry (GC-MS), respectively. 2.1.2 Study on identification of ‘prototype components’ in preparation

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Prototype components of LTT were further analyzed by HPLC-Q/TOF-MS. 96 compounds of the preparation identified are mainly isoflavones, coumarins, iridoids and phthalides. Furthermore, the sources of all the

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compounds were categorized by the raw herbs as reference. The material basis of LTT were comprehensively analyzed, providing a reference for further study of investigation of effective components and QC. Several

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standard compounds like specnuezhenide, ligustroflavone, puerarin, isoimperatorin, daidzin and menthone were used in the experiments and the purity of all of these compounds was ＞98%.

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2.1.3 Study on the identification of the ‘effective components’ in blood Finally, 46 compounds were detected in rat plasma after oral administration of LTT by HPLC-Q/TOF-MS, 24

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were identified as prototype and 22 were metabolites. There were 10 isoflavones, 9 phenylpropanoids (including 6 coumarin, 2 lignans and 1 phenylpropionic acid), 2 phthalides, 1 iridoid glycosides, 1 phenylethanol glycoside and 1 other component. The 22 metabolites contained 3 puerarin-related metabolites, 4 ferulic acid-related metabolites, 6 daidzein-related metabolites, 3 3'-methoxypuerarin-related metabolites, 5 genistein-related metabolites and 2 fermononetin-related metabolites. 2.2 Research approach of Q-marker based on chemical component biosynthetic pathway and specificity The Q-marker is the key indicator in quality evaluation of TCM though, the chemical ingredients of TCMs

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are rather complicated. There are diverse compounds in a specific herb, the different herbs may also share the identical chemical ingredients. Thus, one the basis of clear chemical components of TCMs, representative and specific chemicals should be taken as the indicator of QC through analysis of sources and specificity, as to enhance the level of QC. The biosynthetic pathway and specificity analysis of the chemical components of Corydalis Rhizoma in Yuanhu Zhitong dropping pills (YHZT) are taken as an example to clarify the approach of determination of

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Q-marker (HPLC profiles of YHZT and 2 herbs are shown in Fig.S2). As the underground tubers of Corydalis DC., Corydalis Rhizoma contains three main types of alkaloids: the protoberberine alkaloids (e.g., tertiary amines including corydaline and tetrahydropalmatine, quaternary ammonium

including berberine and palmatine), the

protopine alkaloids (e.g., protopine, α-allocryptopine) and the aporphine alkaloids (e.g. D-glaucine,). The

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biosynthetic pathway of the chemical components showed that in the Corydalis DC., the benzylisoquinoline alkaloids may be the precursor of protopine, benzo(c)phenanthridines, aporphines, cularines and other types. The biosynthetic relationship is shown in Fig. 1 (Facchini et al. 2008; Liscombe et al. 2007; Cheng et al. 2014; Dewan et al. 1986; Tani et al. 1974; Takao et al. 1976).

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According to specific analysis of the composition, the protopine alkaloids and the aporphine alkaloids are located downstream of the biosynthetic pathway compared to the protoberberine alkaloids. Therefore, the plant

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specificity of the protopine and aporphine alkaloids is more evident, while protopine, coptisine are considered as the specific components of Papaveraceae species. From the perspective of the component content, corydaline and

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tetrahydropalmatine and others are the main components of Corydalis Rhizoma, in terms of content. By integrating the analysis of biosynthetic pathway and component specificity, tetrahydropalmatine,

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corydaline, coptisine, palmatine, D-tetrahydrojatrorrhizine, dehydrocorydaline and protopine could be taken as the potential Q-markers of Corydalis Rhizoma.

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2.3 Research approach of Q-marker based on the correlation between chemical component and traditional efficacy

2.3.1 Research method of Q-marker based on component and efficacy The rapid screening of component-effect correlation by key pharmacodynamic indicatorwas also a practicable method for screening of TCM effective components. In this study, a bioactivity-integrated method of ultra-performance liquid chromatography quadrupole/ time of flight mass spectrometry (UPLC-Q/TOF MS) combined with NF-κB luciferase reporter assay system was applied to screen the anti-inflammatory ingredients of Shufeng Jiedu capsule (SFJD, HPLC profiles of SFJD and 8 herbs are shown in Fig.S3) ) (Han et al. 2015; Cheng 10

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et al. 2012). By this strategy, 10 compounds of SFJD exhibited a significant NF-κB inhibition effect, including phenylethanoid glycosides (forsythoside E, forsythoside A, verbascoside and isoforsythoside A), iridoid glucosides (hastatoside and verbenalin), lignans (phillyrin), flavonoids (3-hydroxyglabrol and vitexin) and anthraquinones (emodin). To clarify the screening results, the effect of verbenalin, forsythoside A, phillyrin, vitexin and emodin (the purity of these 5 standard compounds was ＞ 98%) on the production of inflammatory markers in LPS-stimulated mouse peritoneal macrophages were measured. The results showed that the pretreatment of these

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five compounds significantly reduced the production of TNF-α and IL-6 in a concentration-dependent manner, which confirmed the anti-inflammatory effect of the five compounds. The data suggested that the five components may be the main Q-markers.

Network pharmacology (Li et al. 2014; Hao and Xiao 2014) combined with G-protein-coupled receptors

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(GPCR) binding experiment was another alternative approach for the screening of TCM effective components (Jastrzebska 2013; Capote et al. 2015). Ten alkaloids prototype components absorbed into blood in Corydalis Rhizoma, 12 coumarins prototype components of Angelicae dahuricae Radix detected in plasma, and 6 metabolites were chosen for network pharmacogy study by integrating research results of previous studies on constituents

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absorbed into blood of YHZT conducted in this lab and published data according to literature (Han et al. 2017a; Zhang et al. 2016c; Li et al. 2014). The prediction results in silico showed that 28 constituents may be able to exert

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bioactivity via regulating 52 proteins and 31 pathways related with hormonal regulation, central analgesia, spasmolysis, inflammation and immunoregulation, further supporting mechanism of action of YHZT with the

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multiple target. Six active ingredients (the purity of standard compounds was＞98%) were further confirmed by GPCR binding experiment and results showed that tetrahydropalmatine, palmatine, glaucine, protopine,

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imperatorin and isoimperatorin may be the main effective components which can be selected as Q-markers (Han et al. 2016a; 2017b).

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2.3.2 Characterization and confirmation of effective components based on the ‘drug property’ of TCM As the fundamental nature of TCM, drug property reflects the essential of efficacy of TCM and taken as the

most important evidence in clinical practice. Therefore, the drug property should be given special consideration in study of material basis of effectiveness of TCM and quality assessment. ① Virtual screening and determination of ‘property (taste)’ material basis based on bionic model and molecular docking The initial definition of five tastes were from actual feeling of taste and smell of TCMs. The method of analyzing the material basis of pungent-taste herbs was established and the taste and smell of herbs, chemical 11

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groups and compounds were characterized by using the electronic nose and electronic tongue in study (Cao et al. 2016). The herb of Corydalis Rhizoma, its chemical groups and single compounds were taken as testing substance, its taste and smell were examined by electronic nose and tongue, and further analyzed by principal component analysis (PCA) and discriminant factor analysis (DFA), the results were shown in Fig. 2 and 3, respectively. As can be seen, alkaloids, especially tertiary alkaloids were identified as the chemicals with pungent and bitter taste, while quarternary alkaloids were chemicals with bitter taste.

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Molecular docking of representative compounds from the pungent, bitter herbs with the function of promoting blood circulation was conducted against taste 2 receptor member 10 (TAS2R10) and olfactory receptor family 7 subfamily D member 4 (OR7D4) established by protein homology modeling, based on the results of electronic nose and electronic tongue tests. By this method, the pungent-taste and bitter-taste material basis were clarified

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from the aspect of taste receptor.

② Screening and determination of ‘efficacy and five tastes’ material basis based on GPCR „Five tastes‟ of TCM has the connotation of the efficacy, such as pungent-taste herbs can spread and promote gas, sweet-taste herbs can nourish and regulate herbal property, sour-taste herbs have the function of astringing and

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checking, bitter-taste herbs have the function of depriving the evil wetness and discharge the defection, salt-taste herbs have the function of lubricant purgation by softening hardness. Drug property theories of four properties and

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five tastes, ascend, descend, sink, float and channel tropism have complicate internal relationship. The stimulation and inhibition effects of LTT and 9 representative compounds (the purity of standard

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compounds was＞98%) on GPCR (Han et al. 2016b), including 5-hydroxytryptamine receptor 7 (HTR7), dopamine receptor 2 (D2), α1A-adrenoceptor (ADRA1A) and adenosine A1 receptor (ADORA1) which were

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closely related with efficacy of pungent and bitter taste herbs, were explored using the fluorescence assay of intracellular calcium ion (the results are shown in Fig. 4, the effect of positive agonists and inhibitors on GPCR

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receptors are shown in Table S1,S2). The results showed that the representative compounds daidzein of isoflavanones (aglycone) in Pueraria

Lobata could inhibit D2 receptor, while puerarin of isoflavanones (glycoside) had no significant inhibitory activity on D2 receptor suggesting that the isoflavanones (aglycone) may be the important effective components for activating collaterals and alleviating pains of Pueraria Lobata. Consequently, they may be the material basis of pungent-taste herbs. Other compounds had no significant effects on D2, ADORA1 and ADRA1A, respectively. However, all representative compounds were demonstrated to be able to activate HTR7 indicating that they may be the effective components and property-flavour material basis. 12

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The effective components and biosynthetic specific components can then be clarified and proposed as QC indicator by these integrated studies.

2.3.3 The identification and pattern recognition of Q-marker based on composition measurability and method specificity

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The measurability of chemical compositions is one of the basic characteristics of Q-marker. Some components associated with efficacy are not suitable as Q-markers due to its lacking of specific determination method. For example, it is hardly to separate, purify and further identify the structure of polysaccharide. Structure specificity of polysaccharide from different herbs remained unclear, as well as their structure-activity relationship.

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Currently, only the content determination method and limits of total polysaccharides were applied. Saponins are hydrolyzed into subprime glycosides or aglycone which are absorbed into the blood and exert biological effects after oral administration. As a result, the content of total saponins, different types of aglycone and characteristic saponins are usually chosen as QC marker. Steroidal saponins can be determined by evaporative light-scattering

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detector since there is no conjugate system in steroidal, which making saponins difficult to be measured by ultraviolet absorption and the end of ultraviolet determination condition is harsh as well.

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The quality difference of TCM is not only reflected on the chemical composition types, but also the difference of chemical composition content and relative proportion between diverse components. In addition, the combined

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effects of multiple component play a prominent role in TCM. In this case, chemical pattern recognition method is proposed to evaluate the quality of TCM. This method can also be applied to analyze spectrum-effect relationship,

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which provide a more effective way for finding and determining Q-marker from complex system of TCM.

3 Establishment of quality research model based on the core concept of Q-marker and properties of TCM

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The establishment of TCM quality research and standards can be divided into three procedures: the

determination of Q-marker, the establishment of QC methods and sample collection and determination, finally establishment of quality standards. 3.1 Research and determination of Q-marker 3.1.1 Progressive study of chemical components from ‘prototype-pharmaceutical process-in vivo’ process The quality research, evaluation, standard establishment and QC of TCM must be based on the systematic analysis of chemical components as to investigate the material basis of efficacy of TCMs. The biosynthesis of

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chemical ingredients of TCM in medicinal plants, acquisition in preparation, transfer, metabolism/exposure in vivo needs to be clarified. This is not only the basis of study of action of mechanism and effectiveness of TCM, but also the approach of transitivity and traceability of quality.

3.1.2 Specificity analysis of biosynthetic pathway of chemical components

identify the representative and specific ingredients of the testing herbs.

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Once the chemical components are confirmed, the specificity of the compounds needs to be analyzed to

Based on the purpose of quality evaluation, the determination of Q-markers should also refer to the followings: 1) it can represent and reflect the commonness of the same type of herbs and also be different from specific ingredients of other herbs. 2) it can reflect the differences in composition of the same type and different

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kinds of herbs, which can be divided into two levels, in terms of species and content. 3) Q-markers and their differences in different herbs should have the relevance and theoretical basis of reliable biosynthetic pathway, tissue of herbs (histochemistry), growing period (harvest period).

3.1.3 Correlation analysis of components-efficacy and determination of effective components based on

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efficacy expression of TCM

Further investigation on the relationship between components and effectiveness is needed to clarify the

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effective components which could be determined as Q-markers. The Q-markers are determined according to the following ideas and methods: the relationship between components and effectiveness must be based on efficacy of

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TCM, the biological nature of „drug property‟ and „drug efficacy‟ should be fully considered in the research methods and evaluation criteria, focusing on the transitivity and traceability of quality elements in the entire

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process of TCM formation and efficacy expression, as well as association with effective components based on the correlation of disease-syndrome-prescription-herb-component-efficiency of the sequential thinking and the overall

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perspective.

3.1.4 Determination of Q-marker based on component testability Component testability is another necessary condition for determining Q-markers. Given other conditions

fulfilled, the testability of the ingredients can be evaluated from different levels, for instance major components, components of a specific class, components and so on according to the structural characteristics of the composition, research basis, methodology and the purpose of quality evaluation. 3.2 Establishment of QC method QC methods should be established once Q-marker is confirmed. Due to the complicated chemical 14

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composition of TCM, there may exist mutual interference between components and the non-deterministic component may exhibit the same or very similar spectral chromatographic behaviors under certain conditions. Therefore, the determination method should have the theoretical basis of the structure specificity. In practice, the comparison of negative samples and blank samples should be properly considered. In the normality of the experiment method, a systematic study of sampling, sample processing, measurement methodology and evaluation methods should be strengthened, and the SOP of each unit should be established.

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3.3 Experimental sample collection methods, sampling methods and sample determination

The coverage of samples and the representativeness of sampling are important factors in the establishment of quality standards. A good quality standard, is not only those QC indicators strongly related to safety and efficacy, but also reflects the real situation of the quality difference of herbs, so as to reasonably evaluating and effectively

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controlling the authenticity, merit and consistency of TCM quality. The raw materials of TCM mostly derived from natural animal and plant organisms, species germplasm (genetic material basis) and differences in the habitat determined the differences in TCM quality. Thus, the coverage and representativeness of the testing sample is critical to the establishment of quality standards.

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3.3.1 Sample coverage

The sample coverage is mainly aimed at the factors such as origins, intraspecific variation, geographical and

① Origin of herbs

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ecological differences and harvest time, which affect the quality of TCM.

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The scope of taxonomy is defined by „species‟ in TCM origin, but in fact the intraspecific variation, which may be manifested in morphological traits and expressed on secondary metabolites, is widespread, and resulting

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different quality differences (Zhang et al. 2004). Therefore, in quality study, the sample to be examined should be considered the actual situation of the intraspecific variation, and the sampling should cover different groups of the

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used species reflecting the magnitude and variation rules of the quality difference. ② Habitat The quality of Chinese herbal medicine largely depends on its origin, the habitat is also one of the basic

properties of Chinese herbal medicine, thus, there are so called „authentic herbs‟. The distribution and spread of medicinal plants have their own natural laws, especially some widely distributed species with wide geographical span of distribution area and greater quality difference. Cultivation of medicinal herbs are also affected by habitat. In recent years, changes in supply and demand of medicinal herbs and the introduction and cultivation of non-authentic estate or non-origin lead to the various quality of cultivated herbs. Therefore, the coverage of the 15

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testing samples is also critical for the quality study. ③ Ecological conditions Ecological factors are vital conditions for the synthesis of active ingredients in TCM. With respect to biosynthesis, not only soil provides raw material sources, but also light, rain, temperature and humidity are reaction conditions. The synthesis of secondary metabolites are very sensitive to ecological factors, since ecological conditions determine the content of effective components and the relative proportion between the

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various components of TCM, and then affect the effectiveness of TCM. Therefore, in quality study, the ecological condition coverage of the testing sample is also significant, which should take the soil of sample origin, altitude, light, slope, ecological communities and other factors into account to ensure the ecological coverage of test sample.

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3.3.2 Representativeness of sampling

The sampling method of the test sample directly determines the authenticity of the results. The distribution of chemical constituents of TCM in different organs, tissues and cells of TCM origin is not homogeneous, and the content of effective components in different organs tissues and cells varies. In the production and clinical

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application of TCM, herbs are used in hundreds of kilograms, while the quality analysis is measured in grams, the representation of sampling is of particular importance regarding how to truly reflect the consistency of sampling

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results and feeding materials.

Sampling of testing samples can be divided into field (or farm) sampling and laboratory sampling. Field

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sampling should generally be based on the standard method of population sampling and field sampling. Laboratory sampling should take account of testing sample for batch, organ, tissue representation, especially the whole herbs.

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As the content in different organs of stem, leaf, flower and fruit is very different, the sampling should be reduced to the true proportion of the plant organs. The distribution of ingredients in the xylem, phloem, parenchyma and

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other tissues is not homogeneous. Difference is also existing in crushing and the size of sample due to the different degree of cell wall lignification and different toughness and mechanical strength of tissue. Therefore, laboratory sampling requires special assurance of consistency of representativeness and uniformity. 3.3.3 Transfer of ‘effectiveness acquisition’ and ‘quality factor’ in TCM production process From the herbs to Chinese medicines and TCM production process, effectiveness of TCM is achieved, as well as the changes and transfer of quality elements. QC methods and determination of quality standards should focus on the whole process of TCM formation and the two-way thinking concept of transitivity and traceability should be considered in the whole process of QC. The pretreatment, extraction, purification, concentration and 16

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drying of the herbs and even the preparation process will result in the acquisition, removal and complex changes in chemical composition. Process route and parameters determine the results, in accordance with the concept of quality by design (QBD), „good‟ QC methods and quality standards should reflect the whole QC and quality traceability and sample determination and sampling should also follow the guidance. As the process of each unit and finished product sample is properly covered, a reliable quality standard is then established according to the measured results.

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3.3.4 Establishment of a new integrated model for quality evaluation of TCM

The new integrated model of TCM quality standard can be established based on the core concept of TCM Q-marker according to the properties and characteristics of TCM. The study of relationship and relevance analysis between TCM chemical components and its biological effect, include „drug property‟ and „drug efficacy‟ of TCM

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in the study of quality evaluation to determine the effective components should be performed, also the specificity analysis of biosynthetic pathway of chemical components to determine quality evaluation index, quality evaluation method and specification of testing samples collected are then established to ensure the representation and coverage of test sample and further conduct sample analysis and finally establish the quality standard according to

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the measured result. The roadmap is shown in Fig. 5.

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Conclusion

There is no doubt that quality is essential for TCM industry. Over the past years, efficacy of TCM were

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questioned. Quality of TCM became the new focus in TCM pharmaceutical field. In the meanwhile, high level of standard of TCM is urgently needed. Research efforts have been made by Chinese scientists in this area though,

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the systematic method and approaches have not been fully established to fulfill the requirement of quality study of TCM, in particular scientific pattern for evaluation and control of TCM. The concept of Q-markers focusing on the

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quality transitivity, traceability, the specificity, differences and dynamic changes of chemical components in the whole process of herbs, production and in vivo process based on specificity of the ingredients with the core of component-efficacy, is conducive to the establishment of the whole process of TCM QC and quality traceability system. In this paper, combined with the basic properties and clinical features of TCM, the integrated model and research pathway of the TCM quality research were proposed which provided new ideas and gave new insight into the TCM quality research. The exploration of the research and method of TCM quality research is a long-term procedure. Q-markers of TCM is not only the core concept of quality evaluation, but also an open concept which need extensive 17

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investigation and exploration by TCM scientific practioners to gradually improve the ideas and methods of quality research, eventually establish a scientific system of quality evaluation and control.

Conflict of interest We confirm that there are no known conflicts of interest associated with this publication and there has been no

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significant financial support for this work that could have influenced its outcome.

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Figure legends

Fig. 1 Biosynthesis relationship of alkaloids in plants from Corydalis DC.

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Fig. 2 Electronic nose results of Corydalis Rhizoma samples

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Fig. 3 Electronic tongue results of Corydalis Rhizoma samples

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Fig. 4 Activation and antagonism of LTT and representative compounds on GPCRs

Fig. 5 The new integrated mode for quality evaluation of TCM

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