PHYTOTHERAPY RESEARCH Phytother. Res. (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5240
REVIEW
The application of metabolomics in traditional Chinese medicine opens up a dialogue between Chinese and Western medicine Hongxin Cao,1,2* Aihua Zhang,1 Huamin Zhang,2 Hui Sun1 and Xijun Wang1* 1
National TCM Key Laboratory of Serum Pharmacochemistry, Key Laboratory of Metabolomics and Chinmedomics, Department of Pharmaceutical Analysis, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040, China 2 China Academy of Chinese Medical Science, Southern Street of Dongzhimen No. 16, Beijing 100700, China
Metabolomics provides an opportunity to develop the systematic analysis of the metabolites and has been applied to discovering biomarkers and perturbed pathways which can clarify the action mechanism of traditional Chinese medicines (TCM). TCM is a comprehensive system of medical practice that has been used to diagnose, treat and prevent illnesses more than 3000 years. Metabolomics represents a powerful approach that provides a dynamic picture of the phenotype of biosystems through the study of endogenous metabolites, and its methods resemble those of TCM. Recently, metabolomics tools have been used for facilitating interactional effects of both Western medicine and TCM. We describe a protocol for investigating how metabolomics can be used to open up ‘dialogue’ between Chinese and Western medicine, and facilitate lead compound discovery and development from TCM. Metabolomics will bridge the cultural gap between TCM and Western medicine and improve development of integrative medicine, and maximally benefiting the human. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: metabolomics; traditional medicine; Western medicine; drug discovery; systems biology; biomarkers.
INTRODUCTION Recent years have seen an explosion in the amount of ‘omics’ data, which has influenced all areas of life sciences including that of drug discovery and development (Kell, 2006). A growing body of evidence now suggests that the notion of ‘one drug-one target’ for one disease does not exert satisfactory therapeutic effects when used in diseases with complex causes. Currently, a paradigm shift is occurring in that there is a new focus on traditional medicine that modulates multiple targets with fewer adverse effects and lower toxicity. Interestingly, traditional Chinese medicine (TCM), which is a unique medical system that assisted the ancient Chinese in successfully fighting the disease, has attracted worldwide interest (Normile, 2003; Zhu et al., 2013). TCM, the ancient medicine popular in China and surrounding areas, has been recognized as a typical representative of complementary and alternative medicine. TCM, accrued a myriad of valuable clinical observations, focuses on the treatment of human disease via the integrity of the close relationship between body and syndrome analysis. The most common clinical practice of TCM is herb combination called formula which consists of several
* Correspondence to: Hongxin Cao, China Academy of Chinese Medical Science, Southern Street of Dongzhimen No. 16, Beijing, 100700, China; Xijun Wang, National TCM Key Laboratory of Serum Pharmacochemistry, Key Laboratory of Metabolomics and Chinmedomics, Department of Pharmaceutical Analysis, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040, China. E-mail: [email protected] (Hongxin Cao); [email protected] (Xijun Wang)
Copyright © 2014 John Wiley & Sons, Ltd.
types of medicinal herbs or minerals, which is quite different from modern medicine. There are more than 10 000 traditional medicines in use for disease therapy in China. A large number of compounds have been isolated from TCM, and most of these resources have not yet been characterized for pharmacological research program aimed at the development of new drugs (Wang et al., 2011). There are several obstacles for the lead compound discovery from TCM. The key issue is that the actual value of TCM has not been fully recognized worldwide due to a lack of scientific approaches to its study. Fortunately, as the post-genome era is approaching, metabolomics has become a hot topic in the research field of life sciences and is also widely used in TCM research (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e;Zhang et al., 2010; Sun et al., 2012a, 2012b); D’Alessandro and Zolla, 2012). Metabolomics has developed in recent years particularly for innovative drug discovery and provides powerful tools for the essence and function of herbal compound recipe (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e). Its property is in concert with the holistic approaches underlying the practice of TCM, suggesting that it is considered to have the potential to and advance revolutionize TCM research (Nicholson and Lindon, 2008; Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). In the systemic context, metabolomics have a convergence with TCM and avoid the complication and tedious processes. As TCM is attractive as pool natural products for medicinal use, increasing attention is being paid to the scientific evaluation of TCM. Using metabolomics is a novel way for the discovery of novel biological active compounds and targets as well as for proving the occurrence of prodrugs (Wilcoxen et al., 2010; Sun Received 30 January 2014 Revised 09 June 2014 Accepted 04 August 2014
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et al., 2013; Zhang et al., 2013a, 2013b). The introduction of the concept of metabolomics, enabling the study of living systems from a holistic perspective, opens up a unique and novel opportunity to reinvestigate TCM (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). The applications of metabolomics strategies to TCM have begun to provide novel insights into the essence and molecular basis of TCM. This review explores some of the most interesting or significant applications of metabolomics as they relate to TCM research and development, attempts have been made to fill the gap between TCM herbs and modern medicine.
ADVANTAGES OF METABOLOMICS Metabolomics represent a global understanding of small-molecule metabolite complement of integrated living systems and dynamic responses to the changes of both endogenous and exogenous factors. Smallmolecule metabolites represent a diverse group of lowmolecular-weight structures including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols and carbohydrates and have an important role in biological systems and represent attractive candidates to understand disease phenotypes (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). It is now finding applications that span almost the full length of the drug discovery and development pipeline, from lead compound discovery to post-approval drug surveillance. The rapid development of analytical platforms, including GC, HPLC, UPLC, CE coupled to MS and NMR spectroscopy, could enable separation, detection, characterization and quantification of such metabolites and related metabolic pathways (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e). Continued development of these analytical platforms will accelerate widespread use of metabolomics into systems biomedicine. Owing to the complexity of the metabolome and the diverse properties of metabolites, the integrated platforms have been frequently used to provide sensitive and reliable detection of thousands of metabolites in a biofluid sample. Metabolomics is the global assessment and validation of endogenous small-molecule metabolites within a biological sample. The field of metabolomics is a rapidly expanding discipline that is being applied to various aspects of biomedicine. Metabolomics has been applied in many fields; one area of considerable interest in metabolomics is that of drug discovery and development (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g).
TCM MEETS METABOLOMICS TCM has been used for thousands of years to treat or prevent diseases. Syndrome and prescription are key issues in TCM; however, vagueness of syndromes and complexity of prescription greatly limited the evaluation to syndromes and effective substance basis of prescription. Therefore, how to solve the evaluation of syndromes and confirming the efficacy material basis in prescription are the current hot issues of international concern. As a new research approach, metabolomics is Copyright © 2014 John Wiley & Sons, Ltd.
the best to fitting the holistic concept of TCM, and it can not only interpret the essence of syndrome but also elucidate the scientific connotation of prescription. Metabolomics is the ultimate phenotyping and has increased the impetus of traditional medicine research. Studies exploring the metabolomic basis of human constitutional types based on TCM and other systems of oriental medicine are becoming popular. Such studies remain important to developing better understanding of scientific validation of TCM. In recent years, metabolomics has played increasingly important roles in TCM research and development (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). Metabolic profiling of biofluids and tissues can provide a panoramic view of abundance changes in endogenous metabolites in monitoring cellular responses to TCM treatments. Precise identification and accurate quantification of metabolites facilitate downstream pathway and network analysis using software tools for the discovery of clinically accessible and minimally invasive biomarkers of TCM efficacy (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). Metabolomic technologies facilitate the systematic characterisation of a drug targets, thereby helping to reduce the typically high attrition rates in discovery projects, improving the overall efficiency of TCM research processes, and will greatly benefit both drug discovery and development of TCM research. It opens up the possibility of studying the effect of complex mixtures in TCM. The most promising use in the near future would be to clarify pathways for the TCM and provide insights into action mechanisms, and enable us to increase research productivity toward TCM. TCM performs treatment based on ZHENG (also known as ‘syndrome’) differentiation, which could be identified clinical special phenotypes by symptoms and signs of patients. With syndrome as the core of diagnosis and therapy in TCM, it has the advantage of collecting macroscopic information of patients for diagnosis. The effectiveness of TCM treatment depends on the accuracy of ZHENG differentiation (Zhang et al., 2013a, 2013b). However, it lacks objectivity and repeatability and caused controversy, because ZHENG classification only depends on observation and clinical experience of TCM doctors. Metabolomics could provide useful tools for exploring the essence of Chinese medicine syndromes (CMS) disease. Following development of metabolomics technologies, it has been more and more applied in study of objectivity differentiating TCM ZHENG and understanding its biological mechanisms. Some works showed that metabolomics is a valuable tool for studying the essence of Chinese medicine’s syndrome theory and therapeutic effect mechanism of TCM (Song et al., 2013a, 2013b). A future hope for the metabolomic approach is the identification of biomarkers and it will help to understand CMS and modernize TCM.
METABOLOMICS IN APPLICATION OF TCM Pharmacological studies Metabolomics approach has been frequently used in pharmacological studies, especially those on TCM. Radix Paeoniae Alba and Radix Paeoniae Rubra are Phytother. Res. (2014)
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popularly used in TCM, and both have hepatoprotective effects. It was applied to study the influence of Radix Paeoniae Alba and Radix Paeoniae Rubra against on acute liver injury (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e). Several potential biomarkers were identified, such as creatine, deoxycholic acid, choline, 5-methylenetetrahydrofolate, folic acid and glycocholic acid. The physiological significance of these metabolic changes was also discussed in details. Jiang et al. reported that his finding to establish a method for detecting both primary and secondary metabolites in Salvia miltiorrhiza and effectively screening potential biomarkers (Jiang et al., 2014). Twenty-six primary and secondary metabolites were identified in samples from different regions. Results revealed that malonate and succinate can be possibly recognized as the key markers for discriminating the geographical origin of S. miltiorrhiza based on the regulation and influence on the root respiratory rates of plants. TCM syndromes and formulas Syndrome and formula (prescription) are two key issues in TCM and the premise research for material basis of TCM. Syndrome is a basic description of the disease in TCM, and prescription is corresponding drugs against syndrome. However, vagueness of syndromes and complexity of prescription greatly limited the evaluation to syndromes and effective substance basis of prescription. As a new research approach, metabolomics is the best to fitting the holistic concept of TCM, and it provides effective research methods for nature of TCM syndrome, modern prescription research and innovative drug design and opens the door for the modernization of TCM industry. A metabolomic strategy was developed to investigate the metabolism characters of ‘Kidney-Yang Deficiency syndrome’ rats (Huang et al., 2013). The disturbed plasma levels of phenylalanine, tryptophan, cholic acid, lysophosphatidylcholines and urinary levels of phenylalanine, hippurate, phenylacetylglycine, N(2)-succinyl-l-ornithine, creatinine, α-ketoglutarate, citrate, phenol sulfate, indoxyl sulfate and cresol sulfate were found and indicated that regulated oxidantantioxidant balance, amino acid metabolism, lipid metabolism, energy metabolism and gut microflora. A strategy of classifying five TCM syndromes in diabetes (Qi-deficiency, Yin-deficiency, Qi- and Yin-deficiency, Damp heat and Blood stasis) was employed based on plasma fatty acid metabolic profiles (Xu et al., 2012a, 2012b). Using orthogonal signal correction-partial least squares method, the five syndromes were obviously distinguished from those of the health control, which confirmed that there existed metabolite differences in different TCM syndromes. Xu et al. using metabolomics technology had discriminated T2DM corresponding to different syndromes (Qi-deficiency, Qi and Yindeficiency and Damp heat) and discover related biomarkers (Xu et al., 2012a, 2012b). The characteristic fatty acids of three TCM syndromes were discovered. The authors drew the conclusion that research into the correlation between syndromes and metabolomics has great clinical significance in terms of the scientific application of TCM treatment based on syndrome differentiation. Totally, the plasma fatty acids of T2DM were up-regulated, while significant differences existed Copyright © 2014 John Wiley & Sons, Ltd.
in different syndromes. Shi Q et al. had discussed the characteristics of plasma metabolites in Coronary heart disease (CHD) patients and CHD Qi deficiency syndrome patients and explore the composition and concentration changes of the plasma metabolomic biomarkers (Shi et al., 2014). The results show that 25 characteristic metabolites related to the CHD patients comparing with the healthy people, and four identifiable variables had significant differences between Qi deficiency and non-Qi deficiency patients. Three TCM syndromes could be effectively separated by the plasma fatty acid profiles and the syndrome-related biomarkers were also screened. Gong M et al. had investigated the metabolic profile of hydrocortisone-induced ‘Kidney-Yang deficiency syndrome’ and the intervention effects of Morinda officinalis (Gong et al., 2012). Principal component analysis (PCA) was employed to process the data from the M. officinalis. Treatment group and the intervention effects of M. officinalis were investigated through the selected potential biomarkers. M. officinalis could effectively alleviate the disturbance of energy and amino acid metabolism and enhance transmethylation, but could not modulate the gut microflora environment. Metabolite profile was used to find out and analyze the metabolic pathway of cardiac blood stasis syndrome by observing the changes of phenotypes intervened by Yangxin Tongmai Recipe (Jian et al., 2012). The drug intervention was highly positively correlated with glycine, malic acid, glutamic acid and glucose, highly correlated with urea and butanedioic acid, but negatively correlated with lysine. According to VIP value, each variable was closely correlated with the drug intervention. Shengxian Decoction (SXT), a classic TCM prescription, consisting of five TCMs: Astragali Radix, Anemarrhenae Rhizoma, Bupleuri Radix, Platycodonis Radix (PG) and Cimicifugae Rhizoma, has been demonstrated to show good therapeutic effects on the cardiovascular system. Metabolomic profiling was adopted to assess the intervention effects of SXT, SXT-PG (SXT lacking PG) and PG, on chronic heart failure (CHF) rats, and to understand the therapeutic mechanisms and synergistic properties of TCM (Zhang et al., 2014). It demonstrated that the administration of SXT, but neither SXT-PG nor PG alone, gave satisfactory curative effects on CHF through partially regulating the perturbed metabolic pathways. Metabolomics seeks to elucidate the therapeutic and synergistic properties and metabolism of prescription and the involved metabolic pathways using modern analytical techniques. Consequently, metabolomic platforms will greatly facilitate the understanding of the action mechanisms of TCM formulae. Either syndrome or prescription is a complex whole system. Departure from the holistic principle, separating the syndrome from the prescription and ignoring the dynamic concepts may possibly lose the advantages of syndrome differentiation and Chinese medicine preparations (Du et al., 2012). Metabolomics bridges a dynamic research method with combination of disease and syndrome. Huang-Lian-Jie-Du-Decoction (HLJDD) is a representative antipyretic and detoxifying recipe with antiinflammatory activity in TCM. To comprehensively and holistically assess its therapeutic effect on ischemic stroke, HLJDD could relieve stroke rats suffering from the ischemia/reperfusion (I/R) injury by ameliorating the disturbance in energy metabolism, membrane and mitochondrial metabolism, etc., alleviating the oxidative Phytother. Res. (2014)
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stress from reactive oxygen species and recovering the destructed osmoregulation(Wang et al., 2013a, 2013b, 2013c). Metabolomic approach was used to profile rheumatoid-arthritis- (RA-) related metabolic changes and to investigate the interventional mechanisms of HLJDD in collagen-induced arthritis rats (Yue et al., 2013a, 2013b). In a study, metabolomic study was developed to obtain a systematic view of the progression of R) and assess the efficacy of HLJDD (Yue et al., 2013a, 2013b). Twenty-four potential biomarkers were identified and primarily involved in 12 metabolism pathways, such as tricarboxylic acids cycle metabolism, lipid metabolism, etc, which revealed a new insight into the RA network in vivo. Taking potential biomarkers found in the study as screening indexes, it revealed that HLJDD could reverse the pathological process of RA through partly regulating the disturbed metabolic pathways. By combining variable importance in the projection values with partial least squares discriminant analysis, 18 potential biomarkers were identified in the plasma samples and primarily involved in glycerophospholipid metabolism, fatty acid metabolism, tryptophan metabolism, etc. HLJDD can potentially reverse the process of RA by partially regulating fatty acid oxidation and arachidonic acid metabolism. Metabolomics approach has been used to characterized the hypertension and intervention effects of Ping Gan prescription. (Jiang et al., 2012). Some significant changes in metabolites such as LysoPC(22:6), LysoPC(20:4), LysoPC(18:1), etc., were identified and associated with the disturbance in sphingolipid metabolism and fat metabolism, which would be helpful to further understand the essence of the hypertension and therapeutic mechanism of Ping Gan prescription. Qin-Re-Jie-Du and Liang-Xue-Huo-Xue are two TCM formulas with putative effects in sepsis treatment. Using a metabolomic approach was to assess their efficacy and investigate their mechanism of action (Li et al., 2013a, 2013b, 2013c, 2013d). As a result, 18 potential metabolite biomarkers involved in multiple metabolic pathways, including increased energy metabolism, fat mobilization and disrupted amino acid metabolism, were identified. An analysis of potential biomarkers verified the holistic effects of the two formulas. An NMR-based metabolomics approach was conducted to holistically explore the effect of Xue-Fu-Zhu-Yu decoction (XFZYD) on high-fat diet induced hyperlipidemia rats (Song et al., 2013a, 2013b). XFZYD could ameliorate hyperlipidemia by intervening in some major metabolic pathways, such as decreasing the accumulation of β-hydroxybutyrate and acetyl-glycoproteins, enhancing glutathione biosynthesis, partially reversing energy and lipid metabolism disturbance. XFZYD could ameliorate the hyperlipidemic symptoms in a global scale and restore the abnormal metabolic state to a near normal level in a time-dependent pattern. Wang X et al. designed to explore globally metabolomic characters of the HQD and the therapeutic effects of Wen-Xin-Formula (Wang et al., 2013a, 2013b, 2013c). Global metabolic profiling with multivariate analysis was performed to discover differentiating metabolites. Seventeen biomarkers were identified, and pathway analysis tools suggest that the glycolysis or gluconeogenesis metabolism, and biosynthesis of unsaturated fatty acids metabolism, etc, were acutely perturbed by HQD. A metabolomic approach was developed to investigate the therapeutic mechanism of Shexiang Baoxin Pill (Xiang et al., 2012). Thirteen related Copyright © 2014 John Wiley & Sons, Ltd.
pathways and four main pathological processes including oxidative injury, energy metabolism dysfunction, amino acid metabolism dysfunction and inflammation are involved in myocardial infarction development. Shengmai injection could normalize the level of plasma endothelin-1, the index related to cardiovascular diseases and sleep disorders, which verified the results of metabolomics (Yan et al., 2011). The regulated metabolites and related metabolic pathways were analyzed, and it was supposed that the effects of Sheng-mai injection involved in the alternation of energy metabolism, lipid metabolism, amino acids metabolism and so on. A metabolomic approach was developed to identify the biomarkers in early period of acute myocardial infarction and reveal the effective mechanism of Shexiang Baoxin Pill (Jiang et al., 2011). The biomarkers include 5-methylcytosine, cystathionine ketimine, 2-oxoadipic acid, thymidine, epinephrine, homocystine, etc. Shexiang Baoxin Pill could offer protective effects for AMI through regulating the pathway of steroid hormone biosynthesis. Shexiang Baoxin Pill showed therapeutic effectiveness on myocardial infarction through regulation of the energy metabolism dysfunction, oxidative injury and inflammation. Metabolomic technique was used to evaluate the pharmacological mechanism in castrated collagen induced arthritis rats treated by Yi Shen Juan Bi Pill (Zhao et al., 2012). Its pharmacological mechanism was partially associated with lipid metabolites involving free fatty acid and lysophosphatidylcholine. Sini decoction (SND) is a well-known formula which has been used to treat myocardial infarction for many years. A urinary metabolomic method was developed to characterize myocardial infarction-related metabolic profiles and delineate the effect of SND on myocardial infarction (Tan et al., 2012). Nineteen potential biomarkers in rat urine were screened out, primarily related to myocardial energy metabolism. It demonstrated that SND administration could provide satisfactory effect on MI through partially regulating the perturbed myocardial energy metabolism. Liu XJ et al. had investigated the antidepressant effects of Xiaoyaosan (XYS) in a chronic unpredictable mild stress (CUMS) depression model (Liu et al., 2012). XYS produced an obvious antidepressant effect. It was found that trimethylamine-N-oxide, alanine, β-hydroxybutyrate, valine, leucine/isoleucine, low-density lipoprotein/very low-density lipoprotein and lipids were lower and phosphatidylcholine, high-density lipoprotein, choline and N-acetyl glycoproteins were higher in CUMS-treated rats, as compared with controls. XYS significantly suppressed behavioral changes and attenuated plasma metabolite changes. The metabolomic characters of the insomnia and the therapeutic effects of SZRD were to be explored globally (Yang et al., 2012). Metabolic profiling was restored to their baseline values after SZRD treatment according to the PCA score plots. Of note, metabolomic approach indicates 20 ions as ‘differentiating metabolites’. The alterations in these metabolites were associated with perturbations in amino acid and fatty acid metabolism, in response to insomnia through immune and nervous system. Qin-Re-Jie-Du and Liang-Xue-Huo-Xue are two TCM formulas with putative effects in sepsis treatment. In a metabolomic study, 18 potential metabolite biomarkers involved in multiple metabolic pathways, including increased energy metabolism, fat mobilization and disrupted amino acid metabolism, were identified (Li et al., 2013a, 2013b, 2013c, Phytother. Res. (2014)
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2013d). An analysis of potential biomarkers verified the holistic effects of the two TCM formulas. Processing mechanism of TCM The mother and lateral root of Aconitum carmichaelii Debx, named ‘Chuanwu’ (CW) and ‘Fuzi’, respectively, has been used to relieve joint pain and treat rheumatic diseases for over 2000 years. However, it has a very narrow therapeutic range, and the toxicological risk of its usage remains very high. The traditional Chinese processing approach, Paozhi (detoxifying measure), can decompose poisonous Aconitum alkaloids into less or nontoxic derivatives and plays an important role in detoxification. Pao zhi is a common traditional approach that usually occurs before most herbs are prescribed whereby during processing, secondary plant metabolites are transformed, thus helping to increase potency, reduce toxicity and alter their effects. Using CW as a model herb, suitable chemical markers are crucial for studying the processing mechanisms of these herbs. In a study, the comprehensive metabolomic characters of CW and prepared CW (ZCW) were investigated to guarantee clinical safety (Li et al., 2013a, 2013b, 2013c, 2013d). Twenty-two key biomarkers responsible for the detoxifying actions of Paozhi were discovered, providing more accurate characterizations of traditional Paozhi detoxification. The difference in metabolomic characters among the crude and processed preparations is still unclear; therefore, Wang X et al. investigate comprehensive metabolomic characters of the crude and its processed products (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e). The underlying regulations of Paozhiperturbed metabolic pathways are discussed according to the identified metabolites, and four metabolic pathways are identified. Fuzi is widely distributed across Asia and North America and has been used to relieve joint pain and treat rheumatic diseases. However, it has very narrow therapeutic ranges, and despite the toxicological risk, its usage remains very high. Paozhi measure is necessary to remove the poisonous. Sun H et al. had designed metabolomics to investigate the difference in metabolomic characters between Fuzi and its processed preparations (Sun et al., 2012a, 2012b). The difference in metabolic profiles between Fuzi and its processed preparations was well observed by the principal component analysis of the MS spectra. Significant changes of 19 metabolite biomarkers were detected in the Fuzi samples and processed preparations. It proves that metabolomic analysis greatly contributes to the investigation of Fuzi metabolism through Paozhi techniques. In TCM practice, white ginseng and red ginseng have traditionally been used for different purposes. In a study, metabolomic approach was developed to evaluate the holistic qualities and to explore characteristic chemical components of commercial white ginseng and red ginseng (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). Metabolomics in drug discovery and development from TCM TCM, relying on natural products, has been playing a very important role in health protection and disease Copyright © 2014 John Wiley & Sons, Ltd.
control for thousands of years in Asia. The key problem in the TCM research is how to use new tools helps revive ancient TCM. Fortunately, the rapid development of new technology platform provides a methodological basis for deep understanding the essence of TCM; therefore, metabolomics may be one of the best methods to study the drug discovery and development process of TCM. In TCM, multicomponent and principally plantderived drugs are used for disease prevention, symptom amelioration and treatment in a personalized manner. Because of their complex composition and consequent multiple targets and treatment objectives, the application of metabolomics seems inherently appropriate and even necessary for the demonstration of their potential efficacy (Pelkonen et al., 2012). Presently, widespread use of this technique would significantly advance drug discovery from TCM by bridging the gap between Chinese and Western medicine. Yinchenhao (YCH, Artemisia annua L), a famous TCM plant, has been used clinically for more than a thousand years to relieve liver diseases in Asia, and its mechanisms are not still completely clear. Metabolomics was performed by UPLC/ESI-Q-TOF/MS combined with pathway analysis and pattern recognition approaches (Sun et al., 2014). The changes in metabolic profiling were restored to their baseline values after YCH treatment according to the score plots. Of note, YCH has a potential pharmacological effect through regulating multiple perturbed pathways to normal state, correlating well to the assessment of biochemistry test. It showed that changed biomarkers and pathways may provide evidence to insight into drug action mechanisms and drug discovery. Another example is a famous TCM herbal formula Yinchenhao Tang (YCHT), whose efficacy in treating liver injury syndrome. YCHT was recorded in ‘Shanghanlun’ that is a classic resource on TCM written by Zhongjing Zhang (150–215 A.D.) (Lv et al., 2008). It consists of Artemisia annua L., Gardenia jasminoides Ellis, and Rheum Palmatum L. and they work together harmoniously to achieve an ideally therapeutic effect for liver disorders. However, its precise mechanism and drug candidates remain very complex and poorly understood. Therapeutic and synergistic effects of YCHT were conducted using biochemistry, pharmacokinetics and systems biology tools, to exploring the key molecular mechanisms. A completely validated method based on HPLC coupled with photodiode array detector was used for controlling quality of medical formula YCHT (Wang et al., 2008a, 2008b, 2008c). As a result, fingerprint chromatogram determined 15 representative general fingerprint peaks. Fingerprinting method was to identify chemical constituents of YCHT in vivo. Forty-five compounds in YCHT and 21 compounds in vivo were identified by the established UPLC-MS method (Wang et al., 2008a, 2008b, 2008c). Metabolomics was incorporated to analyze the pharmacological effects of YCHT and found 32 marker metabolites in vivo. By correlation analysis of these compounds in vivo and marker metabolites, three components (6,7-dimethylesculetin (D), geniposide (G) and rhein (R)) were found as drug candidates. Continuing work was to elucidate drug-interaction property of marker compound D, G and R, based on the pharmacokinetic characteristics. DGR combination could significantly increase the plasma level, slowing the elimination rate than any one or two of the three individual compounds, which may be an indication of a Phytother. Res. (2014)
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synergism (Zhang et al., 2011). Mapping merged major signaling networks; DGR combination exerts a more robust synergistic effect than any one or two of the three individual compounds by hitting multiple targets. They can regulate molecular networks through activating both intrinsic and extrinsic pathways to intensify therapeutic effects (Wang et al., 2013a, 2013b, 2013c) [56]. The biochemical analysis and immunohistochemical assay further support the cooperation of D and G with R in upregulating BCL-2 and down-regulating FAS, with the strongest effect occurring with the DGR combination (6,7-dimethylesculetin, geniposide and rhein) (Wang et al., 2008a, 2008b, 2008c) [57]. At the levels of the proteome and metabolome, the underlying molecular mechanisms were explored, and it was found that DGR activated an array of factors that are involved in energy, amino acid, nucleotide, fatty acid, cofactor and vitamin metabolism. DGR targets not only immunity and metabolism but also key regulatory pathways, thereby helping to restore the normal function. Millennia-old TCM treats disease with many combination therapies involving ingredients used in clinic practice. Warranted or not, enthusiasm for the new techniques and methods is irrepressible. Metabolomics may provide a promising design in natural products and new combination medicine pattern derived from TCM.
FUELLING THE FUTURE TCM have been a rich source of lead drug discovery, based on their capability to create unique and diverse chemical structures. For drug discovery of traditional medicines, we find that these medicines have more implications for drug discovery than just providing new chemical entities. The history of traditional medicines indicates that they depended more on the combination of natural agents than on screening new agents to find new remedies. This phenomenon suggests that shifting the current drug discovery paradigm from ‘finding new drugs’ to ‘combining existing agents’ may be helpful for overcoming the ‘more investment, fewer drugs’ challenge. Metabolomics is a new territory in life science and represents future of biomedicine of the 21st century. Its applicable potentiality in medicine is infinite and will have significant impact on traditional medicine, clinical research and drug development. Metabolomics is expected to play a significant role in bridging the phenotype–genotype gap, since it amplifies changes in the proteome and provides a better representation of the phenotype of an organism than other methods
(Suhre et al., 2011). Metabolomics focuses on interactions of global metabolite analysis, and their functions in the body system, and all the factors affecting one’s health, can be reflected by metabolome. The integral and systematic study of metabolomics is in agreement with TCM theory in nature; consequently, the metabolomics is the best to fit the holistic concept of TCM. Predicting the future is difficult, but it seems possible that regulatory acceptance of metabolomics approach for the study of TCM will not be long delayed. It is expected that current studies and plans employing metabolomics approaches will prove to be positive and informative.
CONCLUSIONS Analyzing and verifying the specific biomarkers, metabolomics enables us to better understand pathological processes, metabolic pathways and drug targets. The metabolome of TCM is particularly a valuable natural resource for the evidence-based TCM. Recent studies suggest that metabolomics for drug discovery and development of TCM will lead to a greater understanding and even greater opportunities. Metabolomics open up ‘dialogue’ between Chinese and Western medicine, it is conceivable that the application of metabolomics technologies will eventually lead to the reconciliation and integration of Chinese and Western medicine. Overall, integration of metabolomics into TCM would make it possible to explore drug discovery and development of TCM, the molecular mechanism of therapeutic effects of herbal medicine with multiple target, and establish a bridge between Chinese and Western medicine.
Acknowledgements This work was supported by grants from the Key Program of Natural Science Foundation of State (grant nos. 90709019, 81173500, 81373930, 81302905, 81102556, 81202639), the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (grant nos. 2011BAI03B03, 2011BAI03B06, 2011BAI03B08), the National Key Subject of Drug Innovation (grant no. 2009ZX09502-005) and the Foundation of Heilongjiang University of Chinese Medicine (grant no. 201209).
Conflict of Interest The authors have declared that there is no conflict of interest.
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Wang X, Yang B, Zhang A, Sun H, Yan G. 2012b. Potential drug targets on insomnia and intervention effects of Jujuboside A through metabolic pathway analysis as revealed by UPLC/ESI-SYNAPT-HDMS coupled with pattern recognition approach. J Proteomics 75(4): 1411–1427. Wilcoxen KM, Uehara T, Myint KT, Sato Y, Oda Y. 2010. Practical metabolomics in drug discovery. Expert Opin Drug Discov 5 (3): 249–263. Wang X, Zhang A, Han Y, et al. 2012c. Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease. Mol Cell Proteomics 11(8): 370–380. Wang R, Xiong AZ, Teng ZQ, Yang QW, Shi YH, Yang L. 2012d. Radix Paeoniae Rubra and Radix Paeoniae Alba Attenuate CCl4-Induced Acute Liver Injury: An Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) Based Metabolomic Approach for the Pharmacodynamic Study of Traditional Chinese Medicines (TCMs). Int J Mol Sci 13(11): 14634–14647. Wang PR, Wang JS, Yang MH, Kong LY. 2013a. Neuroprotective effects of Huang-Lian-Jie-Du-Decoction on ischemic stroke rats revealed by 1H NMR metabolomics approach. J Pharm Biomed Anal 88C: 106–116. Wang X, Wang Q, Zhang A, et al. 2013b. Metabolomics study of intervention effects of Wen-Xin-Formula using ultra highperformance liquid chromatography/mass spectrometry coupled with pattern recognition approach. J Pharm Biomed Anal 74: 22–30. Wang X, Wang H, Zhang A, et al. 2012e. Metabolomics study on the toxicity of aconite root and its processed products using ultraperformance liquid-chromatography/electrosprayionization synapt high-definition mass spectrometry coupled with pattern recognition approach and ingenuity pathways analysis. J Proteome Res 11(2): 1284–1301. Wang X, Lv H, Sun H, et al. 2008a. Quality evaluation of Yin Chen Hao Tang extract based on fingerprint chromatogram and simultaneous determination of five bioactive constituents. J Sep Sci 31(1): 9–15. Wang X, Sun W, Sun H, et al. 2008b. Analysis of the constituents in the rat plasma after oral administration of Yin Chen Hao Tang by UPLC/Q-TOF-MS/MS. J Pharm Biomed Anal 46(3): 477–490. Wang L, Zhou GB, Liu P, et al. 2008c. Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia. Proc Natl Acad Sci U S A 105: 4826–4831. Wang X, Zhang A, Wang P, et al. 2013c. Metabolomics coupled with proteomics advancing drug discovery towards more agile development of targeted combination therapies. Mol Cell Proteomics 12(5): 1226–1238. Xu W, Zhang L, Huang Y, Yang Q, Xiao H, Zhang D. 2012a. Plasma fatty acid metabolic profiles for traditional Chinese medicine syndrome differentiation in diabetic patients using uncorrelated linear discriminant analysis. Se Pu 30(9): 864–869. Xu W, Zhang L, Huang Y, Yang Q, Xiao H, Zhang D. 2012b. Discrimination of type 2 diabetes mellitus corresponding to different traditional Chinese medicine syndromes based on plasma fatty acid profiles and chemometric methods. J Ethnopharmacol 143(2): 463–468. Xiang L, Jiang P, Zhan C, et al. 2012. The serum metabolomic study of intervention effects of the traditional Chinese medicine Shexiang Baoxin Pill and a multi-component medicine polypill in the treatment of myocardial infarction in rats. Mol Biosyst 8(9): 2434–2442. Yang B, Zhang A, Sun H, et al. 2012. Metabolomic study of insomnia and intervention effects of Suanzaoren decoction using ultra-performance liquid-chromatography/electrosprayionization synapt high-definition mass spectrometry. J Pharm Biomed Anal 58: 113–124. Yue R, Zhao L, Hu Y, et al. 2013a. Metabolomic study of collageninduced arthritis in rats and the interventional effects of huang-lian-jie-du-tang, a traditional chinese medicine. Evid Based Complement Alternat Med 2013: 439690. Yue R, Zhao L, Hu Y, et al. 2013b. Rapid-resolution liquid chromatography TOF-MS for urine metabolomic analysis of collageninduced arthritis in rats and its applications. J Ethnopharmacol 145(2): 465–475. Yan B, A JY, Hao HP, et al. 2011. Metabonomic phenotype of "formula corresponding to pattern types" based on "qi and yin deficiency pattern" of myocardial ischemia rat model. Yao Xue Xue Bao 46(8): 976–982. Phytother. Res. (2014)
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Zhang A, Sun H, Wang P, Han Y, Wang X. 2012e. Modern analytical techniques in metabolomics analysis. Analyst 137(2): 293–300. Zhang A, Sun H, Wang P, Han Y, Wang X. 2012f. Recent and potential developments of biofluid analyses in metabolomics. J Proteomics 75(4): 1079–1088. Zhang AH, Sun H, Qiu S, Wang XJ. 2013b. Recent Highlights of Metabolomics in Chinese Medicine Syndrome Research. Evid Based Complement Alternat Med 2013: 402159. Zhang HM, Li SL, Zhang H, Wang Y, Zhao ZL, Chen SL, Xu HX. 2012g. Holistic quality evaluation of commercial white and red ginseng using a UPLC-QTOF-MS/MS-based metabolomics approach. J Pharm Biomed Anal 62: 258–273. Zhang A, Sun H, Yuan Y, Sun W, Jiao G, Wang X. 2011. An in vivo analysis of the therapeutic and synergistic properties of Chinese medicinal formula Yin-Chen-Hao-Tang based on its active constituents. Fitoterapia 82(8): 1160–1168. Zhang F, Zhan Q, Dong X, et al. 2014. Shengxian decoction in chronic heart failure treatment and synergistic property of platycodonis radix: A metabolomic approach and its application. Mol Biosyst 2014;10(8):2055–63. Zhao H, Li J, He X, et al. 2012. The protective effect of yi shen juan bi pill in arthritic rats with castration-induced kidney deficiency. Evid Based Complement Alternat Med 2012: 102641.
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REVIEW
The application of metabolomics in traditional Chinese medicine opens up a dialogue between Chinese and Western medicine Hongxin Cao,1,2* Aihua Zhang,1 Huamin Zhang,2 Hui Sun1 and Xijun Wang1* 1
National TCM Key Laboratory of Serum Pharmacochemistry, Key Laboratory of Metabolomics and Chinmedomics, Department of Pharmaceutical Analysis, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040, China 2 China Academy of Chinese Medical Science, Southern Street of Dongzhimen No. 16, Beijing 100700, China
Metabolomics provides an opportunity to develop the systematic analysis of the metabolites and has been applied to discovering biomarkers and perturbed pathways which can clarify the action mechanism of traditional Chinese medicines (TCM). TCM is a comprehensive system of medical practice that has been used to diagnose, treat and prevent illnesses more than 3000 years. Metabolomics represents a powerful approach that provides a dynamic picture of the phenotype of biosystems through the study of endogenous metabolites, and its methods resemble those of TCM. Recently, metabolomics tools have been used for facilitating interactional effects of both Western medicine and TCM. We describe a protocol for investigating how metabolomics can be used to open up ‘dialogue’ between Chinese and Western medicine, and facilitate lead compound discovery and development from TCM. Metabolomics will bridge the cultural gap between TCM and Western medicine and improve development of integrative medicine, and maximally benefiting the human. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: metabolomics; traditional medicine; Western medicine; drug discovery; systems biology; biomarkers.
INTRODUCTION Recent years have seen an explosion in the amount of ‘omics’ data, which has influenced all areas of life sciences including that of drug discovery and development (Kell, 2006). A growing body of evidence now suggests that the notion of ‘one drug-one target’ for one disease does not exert satisfactory therapeutic effects when used in diseases with complex causes. Currently, a paradigm shift is occurring in that there is a new focus on traditional medicine that modulates multiple targets with fewer adverse effects and lower toxicity. Interestingly, traditional Chinese medicine (TCM), which is a unique medical system that assisted the ancient Chinese in successfully fighting the disease, has attracted worldwide interest (Normile, 2003; Zhu et al., 2013). TCM, the ancient medicine popular in China and surrounding areas, has been recognized as a typical representative of complementary and alternative medicine. TCM, accrued a myriad of valuable clinical observations, focuses on the treatment of human disease via the integrity of the close relationship between body and syndrome analysis. The most common clinical practice of TCM is herb combination called formula which consists of several
* Correspondence to: Hongxin Cao, China Academy of Chinese Medical Science, Southern Street of Dongzhimen No. 16, Beijing, 100700, China; Xijun Wang, National TCM Key Laboratory of Serum Pharmacochemistry, Key Laboratory of Metabolomics and Chinmedomics, Department of Pharmaceutical Analysis, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040, China. E-mail: [email protected] (Hongxin Cao); [email protected] (Xijun Wang)
Copyright © 2014 John Wiley & Sons, Ltd.
types of medicinal herbs or minerals, which is quite different from modern medicine. There are more than 10 000 traditional medicines in use for disease therapy in China. A large number of compounds have been isolated from TCM, and most of these resources have not yet been characterized for pharmacological research program aimed at the development of new drugs (Wang et al., 2011). There are several obstacles for the lead compound discovery from TCM. The key issue is that the actual value of TCM has not been fully recognized worldwide due to a lack of scientific approaches to its study. Fortunately, as the post-genome era is approaching, metabolomics has become a hot topic in the research field of life sciences and is also widely used in TCM research (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e;Zhang et al., 2010; Sun et al., 2012a, 2012b); D’Alessandro and Zolla, 2012). Metabolomics has developed in recent years particularly for innovative drug discovery and provides powerful tools for the essence and function of herbal compound recipe (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e). Its property is in concert with the holistic approaches underlying the practice of TCM, suggesting that it is considered to have the potential to and advance revolutionize TCM research (Nicholson and Lindon, 2008; Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). In the systemic context, metabolomics have a convergence with TCM and avoid the complication and tedious processes. As TCM is attractive as pool natural products for medicinal use, increasing attention is being paid to the scientific evaluation of TCM. Using metabolomics is a novel way for the discovery of novel biological active compounds and targets as well as for proving the occurrence of prodrugs (Wilcoxen et al., 2010; Sun Received 30 January 2014 Revised 09 June 2014 Accepted 04 August 2014
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et al., 2013; Zhang et al., 2013a, 2013b). The introduction of the concept of metabolomics, enabling the study of living systems from a holistic perspective, opens up a unique and novel opportunity to reinvestigate TCM (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). The applications of metabolomics strategies to TCM have begun to provide novel insights into the essence and molecular basis of TCM. This review explores some of the most interesting or significant applications of metabolomics as they relate to TCM research and development, attempts have been made to fill the gap between TCM herbs and modern medicine.
ADVANTAGES OF METABOLOMICS Metabolomics represent a global understanding of small-molecule metabolite complement of integrated living systems and dynamic responses to the changes of both endogenous and exogenous factors. Smallmolecule metabolites represent a diverse group of lowmolecular-weight structures including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols and carbohydrates and have an important role in biological systems and represent attractive candidates to understand disease phenotypes (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). It is now finding applications that span almost the full length of the drug discovery and development pipeline, from lead compound discovery to post-approval drug surveillance. The rapid development of analytical platforms, including GC, HPLC, UPLC, CE coupled to MS and NMR spectroscopy, could enable separation, detection, characterization and quantification of such metabolites and related metabolic pathways (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e). Continued development of these analytical platforms will accelerate widespread use of metabolomics into systems biomedicine. Owing to the complexity of the metabolome and the diverse properties of metabolites, the integrated platforms have been frequently used to provide sensitive and reliable detection of thousands of metabolites in a biofluid sample. Metabolomics is the global assessment and validation of endogenous small-molecule metabolites within a biological sample. The field of metabolomics is a rapidly expanding discipline that is being applied to various aspects of biomedicine. Metabolomics has been applied in many fields; one area of considerable interest in metabolomics is that of drug discovery and development (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g).
TCM MEETS METABOLOMICS TCM has been used for thousands of years to treat or prevent diseases. Syndrome and prescription are key issues in TCM; however, vagueness of syndromes and complexity of prescription greatly limited the evaluation to syndromes and effective substance basis of prescription. Therefore, how to solve the evaluation of syndromes and confirming the efficacy material basis in prescription are the current hot issues of international concern. As a new research approach, metabolomics is Copyright © 2014 John Wiley & Sons, Ltd.
the best to fitting the holistic concept of TCM, and it can not only interpret the essence of syndrome but also elucidate the scientific connotation of prescription. Metabolomics is the ultimate phenotyping and has increased the impetus of traditional medicine research. Studies exploring the metabolomic basis of human constitutional types based on TCM and other systems of oriental medicine are becoming popular. Such studies remain important to developing better understanding of scientific validation of TCM. In recent years, metabolomics has played increasingly important roles in TCM research and development (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). Metabolic profiling of biofluids and tissues can provide a panoramic view of abundance changes in endogenous metabolites in monitoring cellular responses to TCM treatments. Precise identification and accurate quantification of metabolites facilitate downstream pathway and network analysis using software tools for the discovery of clinically accessible and minimally invasive biomarkers of TCM efficacy (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). Metabolomic technologies facilitate the systematic characterisation of a drug targets, thereby helping to reduce the typically high attrition rates in discovery projects, improving the overall efficiency of TCM research processes, and will greatly benefit both drug discovery and development of TCM research. It opens up the possibility of studying the effect of complex mixtures in TCM. The most promising use in the near future would be to clarify pathways for the TCM and provide insights into action mechanisms, and enable us to increase research productivity toward TCM. TCM performs treatment based on ZHENG (also known as ‘syndrome’) differentiation, which could be identified clinical special phenotypes by symptoms and signs of patients. With syndrome as the core of diagnosis and therapy in TCM, it has the advantage of collecting macroscopic information of patients for diagnosis. The effectiveness of TCM treatment depends on the accuracy of ZHENG differentiation (Zhang et al., 2013a, 2013b). However, it lacks objectivity and repeatability and caused controversy, because ZHENG classification only depends on observation and clinical experience of TCM doctors. Metabolomics could provide useful tools for exploring the essence of Chinese medicine syndromes (CMS) disease. Following development of metabolomics technologies, it has been more and more applied in study of objectivity differentiating TCM ZHENG and understanding its biological mechanisms. Some works showed that metabolomics is a valuable tool for studying the essence of Chinese medicine’s syndrome theory and therapeutic effect mechanism of TCM (Song et al., 2013a, 2013b). A future hope for the metabolomic approach is the identification of biomarkers and it will help to understand CMS and modernize TCM.
METABOLOMICS IN APPLICATION OF TCM Pharmacological studies Metabolomics approach has been frequently used in pharmacological studies, especially those on TCM. Radix Paeoniae Alba and Radix Paeoniae Rubra are Phytother. Res. (2014)
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popularly used in TCM, and both have hepatoprotective effects. It was applied to study the influence of Radix Paeoniae Alba and Radix Paeoniae Rubra against on acute liver injury (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e). Several potential biomarkers were identified, such as creatine, deoxycholic acid, choline, 5-methylenetetrahydrofolate, folic acid and glycocholic acid. The physiological significance of these metabolic changes was also discussed in details. Jiang et al. reported that his finding to establish a method for detecting both primary and secondary metabolites in Salvia miltiorrhiza and effectively screening potential biomarkers (Jiang et al., 2014). Twenty-six primary and secondary metabolites were identified in samples from different regions. Results revealed that malonate and succinate can be possibly recognized as the key markers for discriminating the geographical origin of S. miltiorrhiza based on the regulation and influence on the root respiratory rates of plants. TCM syndromes and formulas Syndrome and formula (prescription) are two key issues in TCM and the premise research for material basis of TCM. Syndrome is a basic description of the disease in TCM, and prescription is corresponding drugs against syndrome. However, vagueness of syndromes and complexity of prescription greatly limited the evaluation to syndromes and effective substance basis of prescription. As a new research approach, metabolomics is the best to fitting the holistic concept of TCM, and it provides effective research methods for nature of TCM syndrome, modern prescription research and innovative drug design and opens the door for the modernization of TCM industry. A metabolomic strategy was developed to investigate the metabolism characters of ‘Kidney-Yang Deficiency syndrome’ rats (Huang et al., 2013). The disturbed plasma levels of phenylalanine, tryptophan, cholic acid, lysophosphatidylcholines and urinary levels of phenylalanine, hippurate, phenylacetylglycine, N(2)-succinyl-l-ornithine, creatinine, α-ketoglutarate, citrate, phenol sulfate, indoxyl sulfate and cresol sulfate were found and indicated that regulated oxidantantioxidant balance, amino acid metabolism, lipid metabolism, energy metabolism and gut microflora. A strategy of classifying five TCM syndromes in diabetes (Qi-deficiency, Yin-deficiency, Qi- and Yin-deficiency, Damp heat and Blood stasis) was employed based on plasma fatty acid metabolic profiles (Xu et al., 2012a, 2012b). Using orthogonal signal correction-partial least squares method, the five syndromes were obviously distinguished from those of the health control, which confirmed that there existed metabolite differences in different TCM syndromes. Xu et al. using metabolomics technology had discriminated T2DM corresponding to different syndromes (Qi-deficiency, Qi and Yindeficiency and Damp heat) and discover related biomarkers (Xu et al., 2012a, 2012b). The characteristic fatty acids of three TCM syndromes were discovered. The authors drew the conclusion that research into the correlation between syndromes and metabolomics has great clinical significance in terms of the scientific application of TCM treatment based on syndrome differentiation. Totally, the plasma fatty acids of T2DM were up-regulated, while significant differences existed Copyright © 2014 John Wiley & Sons, Ltd.
in different syndromes. Shi Q et al. had discussed the characteristics of plasma metabolites in Coronary heart disease (CHD) patients and CHD Qi deficiency syndrome patients and explore the composition and concentration changes of the plasma metabolomic biomarkers (Shi et al., 2014). The results show that 25 characteristic metabolites related to the CHD patients comparing with the healthy people, and four identifiable variables had significant differences between Qi deficiency and non-Qi deficiency patients. Three TCM syndromes could be effectively separated by the plasma fatty acid profiles and the syndrome-related biomarkers were also screened. Gong M et al. had investigated the metabolic profile of hydrocortisone-induced ‘Kidney-Yang deficiency syndrome’ and the intervention effects of Morinda officinalis (Gong et al., 2012). Principal component analysis (PCA) was employed to process the data from the M. officinalis. Treatment group and the intervention effects of M. officinalis were investigated through the selected potential biomarkers. M. officinalis could effectively alleviate the disturbance of energy and amino acid metabolism and enhance transmethylation, but could not modulate the gut microflora environment. Metabolite profile was used to find out and analyze the metabolic pathway of cardiac blood stasis syndrome by observing the changes of phenotypes intervened by Yangxin Tongmai Recipe (Jian et al., 2012). The drug intervention was highly positively correlated with glycine, malic acid, glutamic acid and glucose, highly correlated with urea and butanedioic acid, but negatively correlated with lysine. According to VIP value, each variable was closely correlated with the drug intervention. Shengxian Decoction (SXT), a classic TCM prescription, consisting of five TCMs: Astragali Radix, Anemarrhenae Rhizoma, Bupleuri Radix, Platycodonis Radix (PG) and Cimicifugae Rhizoma, has been demonstrated to show good therapeutic effects on the cardiovascular system. Metabolomic profiling was adopted to assess the intervention effects of SXT, SXT-PG (SXT lacking PG) and PG, on chronic heart failure (CHF) rats, and to understand the therapeutic mechanisms and synergistic properties of TCM (Zhang et al., 2014). It demonstrated that the administration of SXT, but neither SXT-PG nor PG alone, gave satisfactory curative effects on CHF through partially regulating the perturbed metabolic pathways. Metabolomics seeks to elucidate the therapeutic and synergistic properties and metabolism of prescription and the involved metabolic pathways using modern analytical techniques. Consequently, metabolomic platforms will greatly facilitate the understanding of the action mechanisms of TCM formulae. Either syndrome or prescription is a complex whole system. Departure from the holistic principle, separating the syndrome from the prescription and ignoring the dynamic concepts may possibly lose the advantages of syndrome differentiation and Chinese medicine preparations (Du et al., 2012). Metabolomics bridges a dynamic research method with combination of disease and syndrome. Huang-Lian-Jie-Du-Decoction (HLJDD) is a representative antipyretic and detoxifying recipe with antiinflammatory activity in TCM. To comprehensively and holistically assess its therapeutic effect on ischemic stroke, HLJDD could relieve stroke rats suffering from the ischemia/reperfusion (I/R) injury by ameliorating the disturbance in energy metabolism, membrane and mitochondrial metabolism, etc., alleviating the oxidative Phytother. Res. (2014)
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stress from reactive oxygen species and recovering the destructed osmoregulation(Wang et al., 2013a, 2013b, 2013c). Metabolomic approach was used to profile rheumatoid-arthritis- (RA-) related metabolic changes and to investigate the interventional mechanisms of HLJDD in collagen-induced arthritis rats (Yue et al., 2013a, 2013b). In a study, metabolomic study was developed to obtain a systematic view of the progression of R) and assess the efficacy of HLJDD (Yue et al., 2013a, 2013b). Twenty-four potential biomarkers were identified and primarily involved in 12 metabolism pathways, such as tricarboxylic acids cycle metabolism, lipid metabolism, etc, which revealed a new insight into the RA network in vivo. Taking potential biomarkers found in the study as screening indexes, it revealed that HLJDD could reverse the pathological process of RA through partly regulating the disturbed metabolic pathways. By combining variable importance in the projection values with partial least squares discriminant analysis, 18 potential biomarkers were identified in the plasma samples and primarily involved in glycerophospholipid metabolism, fatty acid metabolism, tryptophan metabolism, etc. HLJDD can potentially reverse the process of RA by partially regulating fatty acid oxidation and arachidonic acid metabolism. Metabolomics approach has been used to characterized the hypertension and intervention effects of Ping Gan prescription. (Jiang et al., 2012). Some significant changes in metabolites such as LysoPC(22:6), LysoPC(20:4), LysoPC(18:1), etc., were identified and associated with the disturbance in sphingolipid metabolism and fat metabolism, which would be helpful to further understand the essence of the hypertension and therapeutic mechanism of Ping Gan prescription. Qin-Re-Jie-Du and Liang-Xue-Huo-Xue are two TCM formulas with putative effects in sepsis treatment. Using a metabolomic approach was to assess their efficacy and investigate their mechanism of action (Li et al., 2013a, 2013b, 2013c, 2013d). As a result, 18 potential metabolite biomarkers involved in multiple metabolic pathways, including increased energy metabolism, fat mobilization and disrupted amino acid metabolism, were identified. An analysis of potential biomarkers verified the holistic effects of the two formulas. An NMR-based metabolomics approach was conducted to holistically explore the effect of Xue-Fu-Zhu-Yu decoction (XFZYD) on high-fat diet induced hyperlipidemia rats (Song et al., 2013a, 2013b). XFZYD could ameliorate hyperlipidemia by intervening in some major metabolic pathways, such as decreasing the accumulation of β-hydroxybutyrate and acetyl-glycoproteins, enhancing glutathione biosynthesis, partially reversing energy and lipid metabolism disturbance. XFZYD could ameliorate the hyperlipidemic symptoms in a global scale and restore the abnormal metabolic state to a near normal level in a time-dependent pattern. Wang X et al. designed to explore globally metabolomic characters of the HQD and the therapeutic effects of Wen-Xin-Formula (Wang et al., 2013a, 2013b, 2013c). Global metabolic profiling with multivariate analysis was performed to discover differentiating metabolites. Seventeen biomarkers were identified, and pathway analysis tools suggest that the glycolysis or gluconeogenesis metabolism, and biosynthesis of unsaturated fatty acids metabolism, etc, were acutely perturbed by HQD. A metabolomic approach was developed to investigate the therapeutic mechanism of Shexiang Baoxin Pill (Xiang et al., 2012). Thirteen related Copyright © 2014 John Wiley & Sons, Ltd.
pathways and four main pathological processes including oxidative injury, energy metabolism dysfunction, amino acid metabolism dysfunction and inflammation are involved in myocardial infarction development. Shengmai injection could normalize the level of plasma endothelin-1, the index related to cardiovascular diseases and sleep disorders, which verified the results of metabolomics (Yan et al., 2011). The regulated metabolites and related metabolic pathways were analyzed, and it was supposed that the effects of Sheng-mai injection involved in the alternation of energy metabolism, lipid metabolism, amino acids metabolism and so on. A metabolomic approach was developed to identify the biomarkers in early period of acute myocardial infarction and reveal the effective mechanism of Shexiang Baoxin Pill (Jiang et al., 2011). The biomarkers include 5-methylcytosine, cystathionine ketimine, 2-oxoadipic acid, thymidine, epinephrine, homocystine, etc. Shexiang Baoxin Pill could offer protective effects for AMI through regulating the pathway of steroid hormone biosynthesis. Shexiang Baoxin Pill showed therapeutic effectiveness on myocardial infarction through regulation of the energy metabolism dysfunction, oxidative injury and inflammation. Metabolomic technique was used to evaluate the pharmacological mechanism in castrated collagen induced arthritis rats treated by Yi Shen Juan Bi Pill (Zhao et al., 2012). Its pharmacological mechanism was partially associated with lipid metabolites involving free fatty acid and lysophosphatidylcholine. Sini decoction (SND) is a well-known formula which has been used to treat myocardial infarction for many years. A urinary metabolomic method was developed to characterize myocardial infarction-related metabolic profiles and delineate the effect of SND on myocardial infarction (Tan et al., 2012). Nineteen potential biomarkers in rat urine were screened out, primarily related to myocardial energy metabolism. It demonstrated that SND administration could provide satisfactory effect on MI through partially regulating the perturbed myocardial energy metabolism. Liu XJ et al. had investigated the antidepressant effects of Xiaoyaosan (XYS) in a chronic unpredictable mild stress (CUMS) depression model (Liu et al., 2012). XYS produced an obvious antidepressant effect. It was found that trimethylamine-N-oxide, alanine, β-hydroxybutyrate, valine, leucine/isoleucine, low-density lipoprotein/very low-density lipoprotein and lipids were lower and phosphatidylcholine, high-density lipoprotein, choline and N-acetyl glycoproteins were higher in CUMS-treated rats, as compared with controls. XYS significantly suppressed behavioral changes and attenuated plasma metabolite changes. The metabolomic characters of the insomnia and the therapeutic effects of SZRD were to be explored globally (Yang et al., 2012). Metabolic profiling was restored to their baseline values after SZRD treatment according to the PCA score plots. Of note, metabolomic approach indicates 20 ions as ‘differentiating metabolites’. The alterations in these metabolites were associated with perturbations in amino acid and fatty acid metabolism, in response to insomnia through immune and nervous system. Qin-Re-Jie-Du and Liang-Xue-Huo-Xue are two TCM formulas with putative effects in sepsis treatment. In a metabolomic study, 18 potential metabolite biomarkers involved in multiple metabolic pathways, including increased energy metabolism, fat mobilization and disrupted amino acid metabolism, were identified (Li et al., 2013a, 2013b, 2013c, Phytother. Res. (2014)
POTENTIAL APPLICATIONS OF METABOLOMICS IN TCM
2013d). An analysis of potential biomarkers verified the holistic effects of the two TCM formulas. Processing mechanism of TCM The mother and lateral root of Aconitum carmichaelii Debx, named ‘Chuanwu’ (CW) and ‘Fuzi’, respectively, has been used to relieve joint pain and treat rheumatic diseases for over 2000 years. However, it has a very narrow therapeutic range, and the toxicological risk of its usage remains very high. The traditional Chinese processing approach, Paozhi (detoxifying measure), can decompose poisonous Aconitum alkaloids into less or nontoxic derivatives and plays an important role in detoxification. Pao zhi is a common traditional approach that usually occurs before most herbs are prescribed whereby during processing, secondary plant metabolites are transformed, thus helping to increase potency, reduce toxicity and alter their effects. Using CW as a model herb, suitable chemical markers are crucial for studying the processing mechanisms of these herbs. In a study, the comprehensive metabolomic characters of CW and prepared CW (ZCW) were investigated to guarantee clinical safety (Li et al., 2013a, 2013b, 2013c, 2013d). Twenty-two key biomarkers responsible for the detoxifying actions of Paozhi were discovered, providing more accurate characterizations of traditional Paozhi detoxification. The difference in metabolomic characters among the crude and processed preparations is still unclear; therefore, Wang X et al. investigate comprehensive metabolomic characters of the crude and its processed products (Wang et al., 2012a, 2012b, 2012c, 2012d, 2012e). The underlying regulations of Paozhiperturbed metabolic pathways are discussed according to the identified metabolites, and four metabolic pathways are identified. Fuzi is widely distributed across Asia and North America and has been used to relieve joint pain and treat rheumatic diseases. However, it has very narrow therapeutic ranges, and despite the toxicological risk, its usage remains very high. Paozhi measure is necessary to remove the poisonous. Sun H et al. had designed metabolomics to investigate the difference in metabolomic characters between Fuzi and its processed preparations (Sun et al., 2012a, 2012b). The difference in metabolic profiles between Fuzi and its processed preparations was well observed by the principal component analysis of the MS spectra. Significant changes of 19 metabolite biomarkers were detected in the Fuzi samples and processed preparations. It proves that metabolomic analysis greatly contributes to the investigation of Fuzi metabolism through Paozhi techniques. In TCM practice, white ginseng and red ginseng have traditionally been used for different purposes. In a study, metabolomic approach was developed to evaluate the holistic qualities and to explore characteristic chemical components of commercial white ginseng and red ginseng (Zhang et al., 2012a, 2012b, 2012c, 2012d, 2012e, 2012f, 2012g). Metabolomics in drug discovery and development from TCM TCM, relying on natural products, has been playing a very important role in health protection and disease Copyright © 2014 John Wiley & Sons, Ltd.
control for thousands of years in Asia. The key problem in the TCM research is how to use new tools helps revive ancient TCM. Fortunately, the rapid development of new technology platform provides a methodological basis for deep understanding the essence of TCM; therefore, metabolomics may be one of the best methods to study the drug discovery and development process of TCM. In TCM, multicomponent and principally plantderived drugs are used for disease prevention, symptom amelioration and treatment in a personalized manner. Because of their complex composition and consequent multiple targets and treatment objectives, the application of metabolomics seems inherently appropriate and even necessary for the demonstration of their potential efficacy (Pelkonen et al., 2012). Presently, widespread use of this technique would significantly advance drug discovery from TCM by bridging the gap between Chinese and Western medicine. Yinchenhao (YCH, Artemisia annua L), a famous TCM plant, has been used clinically for more than a thousand years to relieve liver diseases in Asia, and its mechanisms are not still completely clear. Metabolomics was performed by UPLC/ESI-Q-TOF/MS combined with pathway analysis and pattern recognition approaches (Sun et al., 2014). The changes in metabolic profiling were restored to their baseline values after YCH treatment according to the score plots. Of note, YCH has a potential pharmacological effect through regulating multiple perturbed pathways to normal state, correlating well to the assessment of biochemistry test. It showed that changed biomarkers and pathways may provide evidence to insight into drug action mechanisms and drug discovery. Another example is a famous TCM herbal formula Yinchenhao Tang (YCHT), whose efficacy in treating liver injury syndrome. YCHT was recorded in ‘Shanghanlun’ that is a classic resource on TCM written by Zhongjing Zhang (150–215 A.D.) (Lv et al., 2008). It consists of Artemisia annua L., Gardenia jasminoides Ellis, and Rheum Palmatum L. and they work together harmoniously to achieve an ideally therapeutic effect for liver disorders. However, its precise mechanism and drug candidates remain very complex and poorly understood. Therapeutic and synergistic effects of YCHT were conducted using biochemistry, pharmacokinetics and systems biology tools, to exploring the key molecular mechanisms. A completely validated method based on HPLC coupled with photodiode array detector was used for controlling quality of medical formula YCHT (Wang et al., 2008a, 2008b, 2008c). As a result, fingerprint chromatogram determined 15 representative general fingerprint peaks. Fingerprinting method was to identify chemical constituents of YCHT in vivo. Forty-five compounds in YCHT and 21 compounds in vivo were identified by the established UPLC-MS method (Wang et al., 2008a, 2008b, 2008c). Metabolomics was incorporated to analyze the pharmacological effects of YCHT and found 32 marker metabolites in vivo. By correlation analysis of these compounds in vivo and marker metabolites, three components (6,7-dimethylesculetin (D), geniposide (G) and rhein (R)) were found as drug candidates. Continuing work was to elucidate drug-interaction property of marker compound D, G and R, based on the pharmacokinetic characteristics. DGR combination could significantly increase the plasma level, slowing the elimination rate than any one or two of the three individual compounds, which may be an indication of a Phytother. Res. (2014)
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synergism (Zhang et al., 2011). Mapping merged major signaling networks; DGR combination exerts a more robust synergistic effect than any one or two of the three individual compounds by hitting multiple targets. They can regulate molecular networks through activating both intrinsic and extrinsic pathways to intensify therapeutic effects (Wang et al., 2013a, 2013b, 2013c) [56]. The biochemical analysis and immunohistochemical assay further support the cooperation of D and G with R in upregulating BCL-2 and down-regulating FAS, with the strongest effect occurring with the DGR combination (6,7-dimethylesculetin, geniposide and rhein) (Wang et al., 2008a, 2008b, 2008c) [57]. At the levels of the proteome and metabolome, the underlying molecular mechanisms were explored, and it was found that DGR activated an array of factors that are involved in energy, amino acid, nucleotide, fatty acid, cofactor and vitamin metabolism. DGR targets not only immunity and metabolism but also key regulatory pathways, thereby helping to restore the normal function. Millennia-old TCM treats disease with many combination therapies involving ingredients used in clinic practice. Warranted or not, enthusiasm for the new techniques and methods is irrepressible. Metabolomics may provide a promising design in natural products and new combination medicine pattern derived from TCM.
FUELLING THE FUTURE TCM have been a rich source of lead drug discovery, based on their capability to create unique and diverse chemical structures. For drug discovery of traditional medicines, we find that these medicines have more implications for drug discovery than just providing new chemical entities. The history of traditional medicines indicates that they depended more on the combination of natural agents than on screening new agents to find new remedies. This phenomenon suggests that shifting the current drug discovery paradigm from ‘finding new drugs’ to ‘combining existing agents’ may be helpful for overcoming the ‘more investment, fewer drugs’ challenge. Metabolomics is a new territory in life science and represents future of biomedicine of the 21st century. Its applicable potentiality in medicine is infinite and will have significant impact on traditional medicine, clinical research and drug development. Metabolomics is expected to play a significant role in bridging the phenotype–genotype gap, since it amplifies changes in the proteome and provides a better representation of the phenotype of an organism than other methods
(Suhre et al., 2011). Metabolomics focuses on interactions of global metabolite analysis, and their functions in the body system, and all the factors affecting one’s health, can be reflected by metabolome. The integral and systematic study of metabolomics is in agreement with TCM theory in nature; consequently, the metabolomics is the best to fit the holistic concept of TCM. Predicting the future is difficult, but it seems possible that regulatory acceptance of metabolomics approach for the study of TCM will not be long delayed. It is expected that current studies and plans employing metabolomics approaches will prove to be positive and informative.
CONCLUSIONS Analyzing and verifying the specific biomarkers, metabolomics enables us to better understand pathological processes, metabolic pathways and drug targets. The metabolome of TCM is particularly a valuable natural resource for the evidence-based TCM. Recent studies suggest that metabolomics for drug discovery and development of TCM will lead to a greater understanding and even greater opportunities. Metabolomics open up ‘dialogue’ between Chinese and Western medicine, it is conceivable that the application of metabolomics technologies will eventually lead to the reconciliation and integration of Chinese and Western medicine. Overall, integration of metabolomics into TCM would make it possible to explore drug discovery and development of TCM, the molecular mechanism of therapeutic effects of herbal medicine with multiple target, and establish a bridge between Chinese and Western medicine.
Acknowledgements This work was supported by grants from the Key Program of Natural Science Foundation of State (grant nos. 90709019, 81173500, 81373930, 81302905, 81102556, 81202639), the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (grant nos. 2011BAI03B03, 2011BAI03B06, 2011BAI03B08), the National Key Subject of Drug Innovation (grant no. 2009ZX09502-005) and the Foundation of Heilongjiang University of Chinese Medicine (grant no. 201209).
Conflict of Interest The authors have declared that there is no conflict of interest.
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