Journal of Ethnopharmacology 143 (2012) 412–423

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Journal of Ethnopharmacology

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Review Compatibility art of traditional Chinese medicine: From the perspective of herb pairs

Shengpeng Wang a,b, Yangyang Hu a,b, Wen Tan a,b,XuWuc, Ruie Chen a,b, Jiliang Cao d, Meiwan Chen a,b,n, Yitao Wang a,b,n a State Key Laboratory of Quality Research in Chinese Medicine, (University of Macau) Macau SAR 999078, China b Institute of Chinese Medical Sciences, University of Macau, Macau SAR 999078, China c Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China d Institute of Medicinal Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China article info abstract

Article history: Ethnopharmacological relevance: Over the past decades, research of traditional Chinese medicine (TCM) Received 11 April 2012 mainly focused on developing potential candidates from Chinese medicinal herbs, while the wisdom of Received in revised form applying these traditional herbs has not been paid as much attention as it deserves. As is well-known, 22 July 2012 multi-herb therapy is one of the most important characteristics of TCM, but the modernization drive of Accepted 23 July 2012 this conventional wisdom has faced many obstacles due to its unimaginable complexity. Herb pairs, the Available online 1 August 2012 most fundamental and the simplest form of multi-herb formulae, are a centralized representative of Keywords: Chinese compatibility. In light of their simplicity and the basic characteristics of complex Traditional Chinese medicine formulae, herb pairs are of great importance in the studies of herb compatibility. Herb pairs Materials and methods: A systematic search of herb pair related research was carried out using multiple Compatibility online literature databases, books and monographs published in the past 20 years. Herb formulae Results: A comprehensive introduction to the compatibility of TCM, the position of herb pairs in TCM and the progresses of several famous herb pairs were provided in this review. Furthermore, the clinical study and the future research trends of herb pairs were also discussed. Conclusions: Herb pairs have played, and may continue to play a key role in full investigation of general herb compatibility for their indispensable position in TCM. Much more research is needed for the standardization, safety evaluation, and mechanism exploration of herb pairs. & 2012 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction ...... 413 2. Herb compatibility and CHM...... 413 2.1. Herb compatibility is a better choice ...... 413 2.2. Herb compatibility is a necessary choice ...... 414 2.3. The incompatibility of CHM...... 414 3. The role of herb pairs in TCM ...... 415 4. Important herb pairs and their scientific rationale...... 416 4.1. Zuojin Wan...... 416 4.2. Danqi Fang ...... 416 4.3. Dangguibuxue Decoction ...... 417 5. Clinical study of herb pairs ...... 417

Abbreviations: ADME, absorption, distribution, metabolism and excretion; APL, acute promyelocytic leukemia; CHM, Chinese ; CYP2E1, Cytochrome P450 2E1; dl-THP, dl-; HIF1a, hypoxia-inducible factor-1alpha; HUVECs, human umbilical vascular endothelial cells; NSCLC, non-small cell cancer; PML-RARa, promyelocytic leukemia retinoic acid receptor a; RRLC, rapid resolution liquid chromatography; SERCA2a, sarco/endoplasmic reticulum ATPase 2a; SFDA, the State Food and Drug Administration; TCM, Traditional Chinese medicine; UPLC-Q-TOF/MS, ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry; VEGF, vascular endothelial growth factor n Corresponding authors at: Institute of Chinese Medical Sciences, University of Macau, Av. Padre Tomas Pereira S.J., Taipa, Macau 999078, China. Tel.: þ853 83974873. E-mail addresses: [email protected] (M. Chen), [email protected] (Y. Wang).

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.07.033 S. Wang et al. / Journal of Ethnopharmacology 143 (2012) 412–423 413

6. Conclusions and perspective ...... 419 Acknowledgments ...... 420 References ...... 420

1. Introduction prescriptions in a flexible way to face the multiple needs of patients. In the procedure of forming a prescription, herb pairs For a long period of time, medical scientists have realized that are always used as the basic units. Actually, although most of the the pathogenesis and progression of diseases are so complicated recorded formulae involve more than two herbs, the interaction that the therapeutic effect of a single drug may be modest and and mutual influence within these formulae can be further ana- hampered by various side effects or resistances in clinic (Keith lyzed and subdivided into several herb pairs (Zhang, 2008). et al., 2005; Zhao et al., 2010). Meanwhile, the pharmaceutical The exact molecular mechanisms of herb pairs are extremely industry has begun to face the challenge of ‘‘more investments, complicated and are still not well elucidated. However, many fewer drugs’’ in drug development (Ruffolo, 2006), which drives famous herb pairs, such as Danggui Buxue Decoction (Astragali many researchers’ attention to the synergistic effects of natural Radix and Angelicae sinensis Radix) and Zuojin Wan (Coptis medicines. Interestingly, to reach the goal of better curative Rhizoma and Euodiae Fructus), have been well studied, and some efficacies and fewer side effects, multi-herb therapy as an essen- possible modes of actions have been hypothesized (Ung et al., tial component of traditional medicine systems has been utilized 2007; Yuan and Lin, 2000). The synergistic effects of herb pairs for thousands of years in China and other countries. In traditional can be achieved by using a pair of herbs with ingredients of Chinese medicine (TCM), drug compatibility has shown its sig- similar therapeutic actions or arising from the potentiation of nificance in long-term clinical practices and many inspiring pharmacokinetic profile, for example one ingredient enhances the experiences have been accumulated. As the marrow of traditional therapeutic effect of another active ingredient by regulating its Chinese culture, TCM has accumulated more than 100, 000 absorption, distribution, metabolism and excretion (ADME) formulae over the past 2000 years (Qiu, 2007), which is quite (Gilbert and Alves, 2003; Ung et al., 2007). Moreover, many different from other traditional herbal therapies across the world. ingredients of herbs are inactive individually but become active It is the essence of syndrome differentiation based on TCM in combination, called coalistic combinations, which are quite theories and this feature has made multi-herb prescription the common in herb pairs (Greco et al., 1995; Kawashima et al., most important characteristic of TCM. Due to its unique philoso- 2004). Peculiarly, recently a holistic systems biology approach has phy and treatment pattern, TCM was identified as one of the been widely applied to address many of the problems encoun- advanced medical sciences until the late 17th century (Yi and tered in TCM research (Buriani et al., 2012; Wang et al., 2009a), Chang, 2004). Until now, Chinese herbal formulae have been and this approach may help to elucidate the mechanism of herb widely explored and their effectiveness are gradually proved by pairs. The mystery of some herb pairs are gradually revealing, in vivo studies and clinical trials in modern times (Chen et al., thus the elucidated mechanism could be applied to more complex 2011; Guo et al., 2012; Lam et al., 2010). In spite of the wide use formulae. Therefore, the study of herb pairs may act as the of herb formulae in ethnopharmacology and in modern pharma- foundation and a pointcut in the investigation of the herb ceuticals, the exact mechanism of most of them is yet to be compatibility. In this review, we offered a comprehensive intro- explored. duction to the compatibility of CHM, the position of herb pairs in In TCM theory, compatibility refers to the combination of two or TCM and the progress of several famous herb pairs. Further, the more herbs based on the clinical settings and the properties of clinical trial and the future research trends of herb pairs were also herbs (Tang, 2003). Studying of the compatibility mechanism of discussed. TCM is a key issue to its modernization, while the research has faced many obstacles due to the unimaginable complexity of multi- herb formulae. As a note of concern, a special group, namely herb 2. Herb compatibility and CHM pairs, is less known to many researchers outside China. Herb pairs, the unique combinations of two relatively fixed herbs in clinic, are In the early-stage medical practices, single herb therapies were the most fundamental and the simplest form of multi-herb therapy. used as predominant remedies for human ailments. With the Herb pairs have played an important role in the development of accumulation of further therapeutic experience, TCM practi- TCM, for their appearance broadened the application of Chinese tioners gradually realized that the pathogenesis of diseases was herbal medicine (CHM) and laid the foundation for the organizing complex and combining diverse medicinal herbs could be much principles of herb formulae. In Treatise on Exogenous Febrile and more efficient (Li and Zhang, 2008). Long-term applications and Miscellaneous Diseases (210 AD), the most important book of herbal their experiences have demonstrated that herb compatibility is formulae, 147 herb pairs were recorded and more than 40 formulae not only a better choice, but also a necessary choice. were organized based on two herbs (Gu, 2005). Until today, in the latest Pharmacopoeia of the People’s Republic of China, many herb 2.1. Herb compatibility is a better choice pair preparations are still routinely recorded (Commission, 2010)(Table 1). Without altering the basic therapeutic features of Many diseases are regulated by multiple pathogenic factors multi-herb formulae, herb pairs as the basic composition units of and thus they are hard to deal with. In these cases, the potency of Chinese herbal formulae are of special clinical significance in TCM a single herb is modest and cannot address the complicated and (Deng et al., 2008a). For example, based on the Persicae Semen multivariate conditions of patients. In order to achieve better (Taoren)–Carthami Flos (Honghua) herb pair, many formulae curative effect, several herbs are combined according to their including Taohong Siwu decoction, Xuefu Zhuyu decoction, Buyang properties so as to extend their abilities. For instance, Yuanhu Huanwu decoction and Fuyuan Huoxue decoction are consequently Zhitong prescription, the combination of Yanhuasuo ( formed, which are all used to activate blood and resolve stasis (Liu Rhizoma) and Baizhi (Angelicae Dahuricae Radix), has been used et al., 2012). Traditional Chinese physicians always modify their to promote blood circulation, alleviate gastralgia, hypochondriac 414 S. Wang et al. / Journal of Ethnopharmacology 143 (2012) 412–423

Table 1 Examples of herb pair prescriptions in the Pharmacopoeia of the People’s Republic of China.

Herb pairs Herbs and original (herb 1 and herb 2) Ratio Preparation Clinical indications Quality control standards prescription (herb 1: herb 2)

Eemiao Wan Cangzhu (Atractylodis Rhizoma), Atractylodes lancea 1:1 Pills Abscesses, eczema, vaginal Berberine hydrochlorideZ3.0 mg/g (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz. and itchy, leucorrhea, and Huangbai (Phellodendri chinensis Cortex), diarrhea caused by Phellodendri chinensis Schneid. dampness-heat, etc. Erzhi Wan Nvzhenzi (Ligustrum Lucidum Fructus), Ligustrum 1:1 Pills Limpness of the waist and Oleanolic acidZ8.0 mg/g Lucidum Ait. and Mohanlian (Ecliptae Herba), Eclipta knees, dizziness, menstrual prostrata L. irregularity, etc. Renshen Renshen ( Radix et Rhizoma), Ginseng 2:3 Capsules Premature of graying hair, 2,3,5,4’-Tetrahydroxystilbene-2- Shouwu C. A. Mey. and Heshouwu (Polygoni Multiflori Radix), forgetfulness, insomnia, O-b-D-glycosideZ2.0 mg/g Jiaonang Polygonum Multiflorum Thnub. inappetence and neurasthenia, etc. Dachuanxiong Chuanxiong (Chuanxiong Rhizoma), Ligusticum 4:1 Oral solution Headache, dizziness, stiff GastrodinZ2.9 mg/ml Koufuye Chuanxiong Hort. and Tianma (Gastrodiae Rhizoma), neck, numbness of upper Gastrodia elata Bl. and lower extremities, etc. Yuanhu Yanhusuo (Corydalis Rhizoma), Corydalis Yanhusuo 2:1 Oral solution, Stomachache, headache, Tetrahydropalmatine Z80 mg/ml, Zhitong Fang W. T. Wang and Baizhi (Angelicae Dahuricae Radix), capsules, soft hypochondriac pain and Z0.15 mg/capsule, Z0.30 mg/soft Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook. f. capsules, dysmenorrhea due to - capsule and Z75 mg/tablet, or Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook. tablets stagnation and blood stasis respectively f. var. formosana (Boiss.) etc. Zuojin Fang Huanglian (Coptis Rhizoma), Coptidis chinensis 6:1 Pills, capsules Hypochondriac pain, bitter Berberine Franch., Coptidis deltoidea C. Y. Cheng et Hsiao or taste and vomiting with hydrochlorideZ31.0 mg/g and Coptidis teeta Wall. and Wuzhuyu (Euodiae Fructus), acid regurgitation, etc. Z30.0 mg/capsule, respectively Euodia rutaecarpa (Juss.) Benth., Euodia rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang or Euodia rutaecarpa (Juss.) Benth. var. bodinieri (Dode) Huang Dangguibuxue Huangqi (Astragali Radix), Astragalus membranaceus 5:1 Oral solution Qi and blood deficient Astragaloside IVZ0.11 mg/ml Koufuye (Fisch.) Bge. or Astragalus membranaceus (Fisch.) Bge. pattern, women during var. mongholicus (Bge.) Hsiao and Danggui (Angelicae menstruation, and sinensis Radix), Angelica sinensis (Oliv.) Diels postpartum blood deficiency, etc.

pain, headache and dysmenorrhea in TCM for hundreds of years immunosuppressive and anti-inflammatory agents (Bao and Dai, (Zhang et al., 2011). Corydalis derived from Corydalis 2011). However, TwHF was historically recognized as a toxic herb Rhizoma possess potent analgesic effects, of which dl-tetrahy- for its frequently occurring side effects, even triptolide and dropalmatine (dl-THP) is the most important compound (Yuan celastrol (especially the former) also have strong side effects et al., 1996). Although the total coumarins and volatile oil as the (Zhou et al., 2012), which is a major barrier to their extensive major active constitutes of Angelicae Dahuricae Radix showed application. Given this, TwHF has long been used in combination rarely analgesic effects, many studies have demonstrated that with Gancao (Glycyrrhizae Radix et Rhizoma) to avoid its unde- both total coumarins and volatile oil could increase the analgesic sirable effects, which may be associated with the antidotal, anti- effect of Corydalis alkaloids. By detecting the dl-THP plasma inflammatory and immune modulation effects of glycyrrhizin concentration of several groups at different time intervals in rats, contained in Gancao (Li and Zhang, 2008; Zhang et al., 2007a). researchers found that co-administration of total coumarins and/ or volatile oil could markedly elevate the plasma concentration of 2.3. The incompatibility of CHM dl-THP when compared with free Corydalis alkaloids (Liao et al., 2010), which indicated that Angelicae Dahuricae Radix may work However, although compatibility is highly advocated in TCM, it as an absorption enhancer in this formula. does not mean that the more complex a formula is, the better it is. Each formula is devised according to the situation of one certain 2.2. Herb compatibility is a necessary choice disease, thus the herbs selected should be compatible with others and help enhance the desired therapeutic effects. Modification of Duality is a nature of most drugs, as all pharmacologically even one herb may alter the balance of the whole formula and active substances produce both wanted and unwanted effects at consequently affect the overall efficacy and safety. Even more the same time, CHM is no exception (Wang et al., 2012b). A single notable is the fact that two herbs used together may reduce or herb often possesses extensive effects, but if some of these effects restrict the therapeutic actions of another, and even produce toxic could not match the situation of a specific disease, it may be effects. These phenomenons were early noticed and traditionally harmful to patients (Zhang, 2008). Moreover, there are many described as ‘‘restraint’’ or ‘‘antagonism’’. Since the Song dynasty poisonous herbs with strong therapeutic actions that are widely (960–1279 AD), eighteen antagonisms and nineteen incompat- used in China. For this concern, skilled TCM practitioners have ibilities have been summarized based on the long-term experi- accumulated rich experiences in counteracting their toxicities or ences of TCM physicians, and the compatibility prohibition was side effects and simultaneously enhancing the therapeutic effects. strictly observed in clinical prescriptions (Table 2). The eighteen For instance, Leigongteng (Tripterygium wilfordii Hook F., TwHF) antagonisms meaning eighteen kinds of mutual antagonism was traditionally used to dispel wind, remove dampness, and compatibilities were firstly recorded in Shennong’s Herbal Classic, relieve arthritis and pain in China. Some promising compounds, and the nineteen incompatibilities were summarized in Jin and including triptolide and celastrol, have been explored as Yuan dynasty. For example, when combined Gancao with either S. Wang et al. / Journal of Ethnopharmacology 143 (2012) 412–423 415

Table 2 Incompatibilities of herb pairs.

Categories of Incompatibility herb pairs incompatibility

Nineteen Puxiao (Mirabilite) Liuhuang (Sulfur) incompatibilities Pishuang (Arsenic trioxide) Shuiyin (Mercury) Lead oxide (Mituoseng) Langdu (Euphorbia ebracteolata Hayata or Euphorbia fischeria Steud.) Qianniuzi (Pharbitis nil (L.) Choisy or Pharbitis purpurea (L.) Voigt) Badou (Croton tiglium L.) Yujin (Curcuma wenyujin Y. H. Chen et C. Ling, Curcuma longa L., Dingxiang (Eugenia caryophyllata Thunb.) Curcuma kwangsiensis S. G. Lee et C. F. Liang or Curcuma phaeocaulis Val.) Xijiao (Rhinoceros unicornis L.) Chuanwu (Aconitum carmichaelii Debx.) Sanleng (Sparganium stoloniferum Buch. –Ham.) Yaxiao (Merabilite) Chishizhi (Halloysitum rubrum) Guangui (Cinnamomum cassia Presl) Wulingzhi (Trogopterori Feces) Renshen ( C. A. Mey.) Eighteen Gancao (Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Gansui (Euphorbia kansui T. N. Liou ex T. P. Wang), Daji (Euphorbia pekinensis antagonisms Glycyrrhiza glabra L.) Rupr.), Haizao (Sargassum pallidum (Turn.) C. Ag. or Sargassum fusiforme (Harv.) Setch.), Yuanhua (Daphne genkwa Sieb. et Zucc.) Wutou (Aconitum carmichaelii Debx. or Aconitum kusnezoffii Beimu (Fritillaria unibracteata Hsiao et K. C. Hisa, Fritillaria cirrhosa D. Don, Reichb.) Fritillaria delavayi Franch., Fritillaria przewalskii Maxim., Fritillaria Taipaiensis P. Y. Li, Fritillaria unibracteata Hsiao et K. C. Hisa var. wabuensis (S. Y. Tang et S. C. Yue) Z. D. Liu, S. Wang et S. C. Chen, or Fritillaria thunbergii Miq.), Gualou (Trichosanthes kirilowii Maxim. or Trichosanthes rosthornii Harms), Banxia (Pinellia ternata (Thunb.) Breit.), Bailian (Ampelopsis japonica (Thunb.) Makino), Baiji (Bletilla striata (Thunb.) Reichb. f.) Lilu ( nigrum L.) Renshen (Panax ginseng C. A. Mey.), Shashen (Adenophorae tetraphylla (Thunb.) Fisch or Adenophorae stricta Miq.), Danshen (Salvia miltiorrhiza Bge.), Xuanshen (Scrophularia ningpoensis Hemsl.), Xixin (Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., Asarum sieboldii Mia. var. seoulense Nakai or Asarum sieboldii Miq.), Shaoyao (Paeonia lactiflora Pall.)

Gansui (Kansui Radix), Daji (Euphorbia pekinensis Radix), Haizao (Sargassum) or Yuanhua (Genkwa Flos), the toxic effects may be enhanced. In recent years, many experiments have manifested that these guidelines are quite rational. Gansui (Kansui Radix) exerts its toxicity mainly through inducing Cytochrome P450 2E1 (CYP2E1) expression and enhancing the activities in rats. When co-administrated with Gancao, its toxicity was significantly enhanced because Gancao is also a predominant CYP2E1 inducer (Dai et al., 2005).

3. The role of herb pairs in TCM

Viewed from the historical angle of the development of herbal Fig. 1. The evolution of the constituting herb number in a herbal formula during therapy, single herb formulae were the original form of herb the past 2300 years. Data collected from Prescriptions form Fifty-two Diseases (283 formulae, around 300 BC), Treatise on Exogenous Febrile and Miscellaneous Diseases prescriptions, and the multi-herb formulae were developed gra- (221 formulae, 210 AD), Monographs of Zhu Danxi (848 formulae, 1281–1358 AD) dually by adding one or more herbs to the pre-existing single herb and current clinical prescriptions (3509 formulae from three hospitals, 2006). formulae (Yi and Chang, 2004). This progress can be best reflected by the changes in the number of constituting herbs in a formula (Fig. 1). In the pioneering Chinese medicinal monograph, Prescrip- prescription. Shennong’s Herbal Classic, the earliest existing mono- tions for Fifty-two Diseases (complied around 300 BC), most graph of CHM, firstly summarized the interactions between two formulae were composed of 1–2 herbs (Yang and Li, 2005). After herbs and identified as ‘‘seven compatibilities’’. The seven aspects 500 years, wrote his representative work, of herbal compatibility, namely single action, mutual reinforce- Treatise on Exogenous Febrile and Miscellaneous Diseases. The ment, mutual assistant, mutual restraint, mutual detoxication, number of the constituting herbs in these newly-established mutual inhibition and mutual antagonism, are a comprehensive formulae were increased to 4.81 (Cheng et al., 2006). In the Yuan summation of the long-term clinical experiences, which still dynasty (1271–1368 AD), the average number of herbs in Zhu affect the cognition of drug interaction in modern medicine. Danxi’s 848 formulae was 6.59. Afterward, more complex for- Actually, no matter how many herbs are chosen to form a mulae appeared, even 40–50 herbs appeared in a single multi- formula, the internal actions remain extremely complicated and herb formula. In the Chinese pharmacopoeia, many herb prescrip- only the mutual influence of two herbs can be observed (Zhang, tions consist of more than twenty herbs, like Qishiwei Zhenzhu 2008). Understanding the mechanism of herb formulae could Wan (70 herbs), Dingkun Wan (27 herbs) and Ershiwuwei start with an analysis of the simplest form of herb compatibility Songshi Wan (25 herbs) (Commission, 2010). because herb pairs possess both the characteristics of complex Therefore, in the evolution of TCM formulae, herb pairs are the formulae and the feature of simplicity to facilitate research. simplest form and also the centralized representative of Chinese Therefore, the study of herb pairs may be the foundation and a herbal compatibility. Although composed of only two herbs, herb pointcut in the full investigation of general herb compatibility pairs ingeniously express the basic idea of the multi-herb (Liang et al., 2012). 416 S. Wang et al. / Journal of Ethnopharmacology 143 (2012) 412–423

4. Important herb pairs and their scientific rationale physicians, involving both pharmacodynamic and pharmacoki- netic mechanism. There were studies reporting that the dissolu- 4.1. Zuojin Wan tion rate of some active ingredients in CR and EF decreased with the regularity that the higher ratio of EF, the lower the rate after Coptis Rhizoma (Huanglian, CR) and Euodiae Fructus (Wuz- combination, perhaps resulting from the formation of macromo- huyu, EF), a famous herb pair which has been widely used for lecular complexes between alkaloids in CR and flavonoids in EF more than one thousand years, was first recorded as Zhuyu Wan (Deng et al., 2008b). For instance, the cumulative contents of in Taiping Holy Prescriptions for Universal Relief in Song Dynasty berberine, palmatine and jatrorrhizin were reduced in different (992 AD) (Zhang and Fan, 2010). According to the theories of TCM, proportions with the ratio of CR decreasing (Cheng et al., 2010). CR and EF, one cold and the other hot in nature, are opposite in However, according to previous studies, the exact interaction nature but check and balance mutually, when clinically combined between CR and EF is still in confusion. to achieve complementary therapeutic actions. In history, this herb pair could be found in various medical books with different 4.2. Danqi Fang ratios, such as Zuojin Wan (6:1, CR:EF, similarly hereinafter) in Danxi’s Experiential Therapy (1281–1358 AD) (Wen et al., 2009), Herb pair containing Salviae miltiorrhizae Radix et Rhizoma Ganlu San (2:1) in General Records of Holy Universal Relief (1111– (Danshen, SM) and Notoginseng Radix et Rhiroma (Sanqi, NRR) 1118 AD) (Tan et al., 2006), Zhuyu Wan (1:1) in Taiping Holy has been widely used to improve coronary and cerebral circula- Prescriptions for Universal Relie, (992 AD) and Fanzuojin Wan (1:6) tion for the therapy of cardiovascular diseases in both China and subsequently derived from Zuojin Wan by TCM physicians, which western countries (Pei et al., 2004). Many preparations of this were ingeniously adjusted to meet the complicated needs in herb pair named Compound Danshen formulae have been mar- clinic. Among more than 10 ratios, Zuojin Wan (ZJW) combined keted for a long time and their application ranked first in market CE and EF at the ratio of 6:1 demonstrated the best efficacy and share among all Chinese medicinal over-the-counter drugs in got the most extensive application in treating gastric diseases. In 2008 (Chu et al., 2011). In 1997, Compound Danshen dripping pill recent years, the rationale of combining these two herbs are (CDDP) as a Chinese medicinal product was unprecedentedly gradually proved and elucidated by experimental studies. listed as an investigational new drug (IND No. 56956) by the Phytochemical research indicated that protoberberine alka- United States Food and Drug Administration (FDA) (Jia et al., loids such as berberine, palmatine, coptisine, epiberberine, jatror- 2012b). Then in 2010, as the major preparation of this herb pair, rhizine and columbamine are the active ingredients of CR (Wang CDDP stepped into phase III clinical trial in US, which was an et al., 2008a), and the effective constitutes in EF are identified as important milestone in the process of TCM modernization. indole and quinolone alkaloids such as evodiamine and rutaecra- Because of its remarkable activities in activating blood circulation, pine (Chiou et al., 2011). Combined as ZJW, CR and EF was applied dilating coronary artery and antagonizing myocardial ischemia to treat gastrointestinal diseases for their properties of stopping (Wei et al., 2007), phytochemical and molecular biological vomiting, decreasing gastric acid, relieving pain and anti-inflam- approaches have been extensively applied to elucidate the ratio- mation, which are associated with their anti-ulcer, anti-acid nale of Salvia Miltrorrhiza pairing with Panax notoginseng in the activities and the inhibitory effect on the growth of Heliobacter past decades. pylori (Zhao et al., 2010). In addition, pharmacologic researches SM, the dry root of Salvia miltiorrhiza Bge., contains both have revealed that ZJW has many other beneficial effects, such as hydrophobic and hydrophilic bioactive components such as anti-tumor, anti-hypertension (Bu et al., 2011) and anti-obesity Danshensu, protocatechuic acid, protocatechuic aldehyde, salvia- (Hu et al., 2009) activities, which may be attributed to the nolic acid B, tanshinones and cryptotanshinone (Li et al., 2008, synergistic effects of CR and EF. For example, over 48 h treatment 2007)(Li et al., 2007)(Ma et al., 2006). Derived from the root of on human hepatocellular carcinoma SMMC-7721 cells, the mix- Panax notoginseng (Burk.) F. H. Chen, NRR contains abundant tures of berberine and evodiamine showed the highest inhibition saponins such as ginsenosides and notoginsenosides (Wan et al., rate (50.00%) as compared with berberine and evodiamine treat- 2006). When the two herbs decoct together for administration, ment individually (20.24% and 16.33%, respectively) (Wang et al., their active components could have better solubility. Saponin is 2008c). On tumor-bearing mice, ZJW also possessed a clear well known as co-solvent which can decrease the surface tension synergistic effect on tumor growth inhibition, lymphocyte pro- of menstruum. Therefore, decocting SM with NRR together, the liferation and inducing apoptosis. Compared with CR and EF saponins of NRR may promote the dissolution of the effective single-treatment groups, the expression of Bax and wild-type ingredients in SM (Zeng et al., 2004). Nowadays, aiming to p53 proteins, the activities of acid phosphatase and alkaline establish a reliable and applicable quality control method, many phosphatase in serum were all much higher (Wang et al., burgeoning methods have been applied to detect the active 2009b). Synergism of CR and EF may also arise from the potentia- constitutions in SM and NRR, such as AOTF-Near infrared spectro- tion of pharmacokinetic parameters. When combined together, scopy (Wang et al., 2008b), capillary electrochromatography (Li both CR and EF alkaloids got better absorption in small intestine, et al., 2007) and multiple columns high performance liquid and the absorption rates would increase once the ratio of CR chromatography (HPLC) (Qian et al., 2008). escalated (Huang et al., 2011). Meanwhile, accumulating experi- CDDP has been used as a classical formula for cardiovascular ments have showed that the ADME feature of this herb pair was diseases for at least 30 years (Wei et al., 2007). Various compound inconsistent at different compatibility ratios. For example, CR–EF Danshen preparations such as Compound Danshen tablets, CDDP, (6:6) was best absorbed in rat jejunum, while good absorption Danqi tablets and Guanxin Danshen tablets are commercially was found in ileum with CR–EF (2:1) (Tu et al., 2011). Moreover, available as a dietary supplement or drug (Prescription/OTC) in recent contradistinction found that berberine had a better absorp- many countries including USA, Singapore, India, Russia, Cuba, and tion, longer half-time and shorter peak-time in ZJW when South Africa (Chu et al., 2011). Pharmacological research indi- compared with other combinations like 2:1 and 1:1 (Wang cated that SM focuses on expanding blood vessels, while NRR et al., 2010). mainly participates in protecting cardiac myocytes. The coopera- The compatibility of CR and EF is not an arbitrary plus of two tion of the two herbs can improve coronary circulation and herbs, but the summarization and sublimation of clinical experi- reduce myocardial ischemia symptom (Zheng et al., 2009). More- ences for the past hundreds of years by various medical over, only when the weight ratio was 5:3 or 1:1 (SM:NRR), the S. Wang et al. / Journal of Ethnopharmacology 143 (2012) 412–423 417 herb pair would have the effect of protecting hypoxia/reoxygena- simultaneously determining the amount of astragaloside IV, tion induced human umbilical vascular endothelial cells calycosin, formononetin, ferulic acid, ligustilide, total saponins, (HUVECs) injury (Zeng et al., 2006). Searching the molecular total flavonoids and total polysaccharides in DBD, researchers mechanism in depth, tanshinone IIA mainly protects cardiac found that AR and AS at a 5:1 was the most appropriate decoction myocytes via inhibiting oxidative stress-induced apoptosis, pro- in dissolving the active components in (Dong et al., 2006). moting angiogenesis and up-regulating vascular endothelial DBT was originally prescribed as a medicinal decoction, but growth factor (VEGF) expression due to the enhancement of today it is also commonly used as a health food supplement for hypoxia-inducible factor-1alpha (HIF1a) mRNA expression (Fu women’s ailments in Asia (Dong et al., 2006). Even many women et al., 2007); salvianolic acid B exerts cardioprotective effects by in menopause are recommended to drink DBD daily to adjust the augmenting VEGF expression, promoting angiogenesis, recovering body functions (Song et al., 2004). In recent years, various the normal expressions of sarco/endoplasmic reticulum ATPase 2a bioactivities related to menopausal symptoms, such as estrogenic (SERCA2a) and phospholamban, simultaneously inhibiting the effect (Gao et al., 2007b), anti-platelet aggregation effect (Zhang activation of platelet during myocardial ischemia and reperfusion et al., 2007b) and immuno-modulatory effect (Kim et al., 2012) (He et al., 2008a, 2008b); ginsenoside Rb1 exerts protective effects have been reported to illustrate the mechanism of DBD. DBD was on myocardial ischemia and reperfusion injury by mediating the applied to cultured Hep3B human hepatocellular carcinoma cells activation of the PI3K pathway and phosphorylation of Akt (Wang to determine its hematopoietic effect in vitro. In agreement with et al., 2008d). All the aforementioned mechanisms illustrate that the ancient formulae, when prepared in a 5:1 ratio of AR and AS, SM–NRR herb pair exerts its effects by the integration of multi- DBD possessed better activity than single AR, AS and a simple target and multi-function, which single herb could not achieve. mixture of AR and AS in stimulating erythropoietin gene expres- However, more than 100 compounds have been isolated and sion and erythropoietin secretion. Both the basal level of erythro- identified in SM and NRR to date (Lu et al., 2011), but only a poietin secretion under normal condition and the hypoxia- fraction of these compounds were confirmed to be responsible for induced erythropoietin production could be significantly their biological effects, understanding the secrets of this herb pair is increased, which indicated that DBD could be used as a health still far from enough. food supplement to prevent anemia and altitude stress (Gao et al., 2008). Meanwhile, researchers have also verified that DBD could 4.3. Dangguibuxue Decoction induce both the proliferation and differentiation of human MG-63 osteoblast cells in a dose-dependent manner. In both MTT and Dangguibuxue decoction (DBD), composed by only Astragali alkalinephosphatase assays, DBD showed stronger activities than Radix (AR, Huangqi) and Angelica sinensis Radix (AS, Danggui), single AR or AS, and the DBD preparation at 5:1 ratio induced the was firstly recorded at the ratio of 5:1 in Nei Wai Shang Bian Huo highest activity in cell proliferation and differentiation (Dong Lun (AD 1247) (Gao et al., 2007a) by Li Dongyuan, one of the four et al., 2006). In addition, in order to decode the mystery of DBD, well-known TCM physicians during the Jin and Yuan Dynasties in pharmacokinetic models were also built up to understand the China. AR was traditionally used to invigorate Qi (the vital energy) main circulating forms in vivo, which may be directly related to while AS can help nourish Blood (the body circulation) (Zhang its therapeutic effects (Li et al., 2009). Using rapid resolution et al., 2007b), thus the two herbs were combined for Qi and Blood liquid chromatography (RRLC)-TOF/MS, 142 metabolites were deficient pattern, clinically in the treatment of anemia and detected and tentatively identified from the drug-containing rat menopausal syndrome (Chen and Gao, 1994). Currently, the bile and plasma samples. Metabolite profiling showed that rat bile rationale of this herb pair are being proved by modern biomedical contained much more DBD-related metabolites than urine, indi- research, because the mixed preparation could increase the cating that after oral administration, DBD was subjected to solubility of active components and also the pharmacological extensive in vivo metabolism to facilitate its excretion from body activities could be enhanced, compared to the single herb (Kim (Li et al., 2011). Therefore, the active compounds and their et al., 2012; Gao and Chen, 2000). Moreover, in agreement with metabolites in plasma may reveal the reason why ancient the ancient theory, many studies also showed that the pharma- physicians prefer to boil AR and AS at particular ratio for oral cological actions of DBD are crucially related to the ratio of the administration. two constituting herbs (Mak et al., 2006), which lead the mechan- ism study of DBD into a hot topic. AR, the dry roots of Astragalus membranaceus (Fisch.) Bge. and 5. Clinical study of herb pairs A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, was proved to contain calycosin, astragaloside I, II, IV, soyasaponin I, A huge number of patients are being treated with TCM formononetin, isomucronulatol, asparagine and polysaccharides prescriptions everyday, which means that a great deal of clinical as its major active components (Sheih et al., 2011). On the other data are generating all the time. There are ethnic considerations hand, AS is derived from the roots of Angelica sinensis (Oliv.) Diels, of studying traditional Chinese formulae in humans first, but the and ligustilide, ferulic acid, senkyunolide-L, butylphthalide and simple fact is that these formulae have been used for thousands of polysaccharides are associated with its pharmacological activities years, and whether studied or not, they will continue to be used in (Wang and Liang, 2010). When the two herbs are boiled together places where TCM is officially recognized (Tang, 2006). To date, for medicinal usages, the active components could get better herb pairs are clinically used not only in the form of traditional dissolution rate and some new compounds may emerge. Until decoction, but also modern preparations such as tablets, capsules, now, many advanced technologies including HPLC-DAD/MS (Wen oral solution and injections. Positive results have been reported in et al., 2008; Wang and Liang, 2010) and ultra-fast HPLC-time-of- patients with gastric ulcer, anemia, migraine, scrotal eczema, and flight mass spectrometry (UPLC-TOF/MS) (Qi et al., 2009) are more. If the application history of herb pairs is merely the applied in analyzing the constitutes of DBD. Since saponins accumulation of experience of traditional physicians, then the account for more than 2% total dry weight of AR (Dong et al., ongoing clinical trials (especially randomized clinical trials) 2003), when AS is boiled together with AR, AR could help increase surely can provide a ground for consensus among professionals. the solubility of the active compounds of AS. For example, the Clinical studies with DBD have demonstrated promising solubility of ferulic acid and ligustilide derived from AS could be results initially in treating anemia, menopausal syndrome and increased by astragaloside (Gao et al., 2007a). Meanwhile, by being auxiliary therapy of cancer patients (Chen et al., 2009; 418 S. Wang et al. / Journal of Ethnopharmacology 143 (2012) 412–423

Table 3 Clinical studies of herb pairs.

Herb 1 (principal ingredients) Herb 2 (principal ingredients) Ratio Patients Treatment Response (EER*) Comments, risk Ref. (Herb1: of bias (high/ (Herb 2) moderate/low)

Cangzhu, Atractylodes lancea Huangbai, Phellodendri chinensis 1:1 32 cases with 150 ml decoction, 94% Not clear (Liu, (Thunb.) DC. or Atractylodes Schneid. (berberine, scrotal bid, followed by procedure 2011) chinensis (DC.) Koidz. phellodendrine, magnoflorine, eczema fumigating- (randomization, (atractylodi, atractylodiol, jatrorrhizine, palmatine, etc.) washing with anesthetization, hinesol, b-eudesmol, dreg decoction, detection), atractylon, etc.) for 2 weeks none-blind; high Sanqi, Panax Notoginseng Yongchongcao, Cordyceps not 2 cancer cases without maintained stable Not randomized (Yoo (Burk.) F. H. Chen militaris Link. (cordycepin, available conventional disease and good study; high et al., (ginsenoside Rb1, Rc, Rg1, adenosine, guanosine, uridine, treatments, 500- quality of life 2011) Rg2, Rg3, Rh, dencichine, cordycepin acid, cordycepin mg capsules, tid, etc.) polysaccharide, etc.) for 17 and 13 months, respectively. Renshen, Panax ginseng C. A. Yinxingye, L. 5: 3 20 healthy capsules highly significant and Double-blind, (Scholey Mey. (ginsenoside Rb1, Rc, (ginkgolides A, B, C, M, J, young containing 320, sustained increase in placebo- and Rg1, Rg2, Rg3, Rh, volatile oil, bilobalide, quercetin, volunteers 640, 960 mg of the number of Serial controlled; low Kennedy, ginseng polysaccharides, kaempferol, isoharmnten) the Ginkgo- Sevens responses 2002) etc.) Ginseng during serial combined arithmetic tasks extracts Huanglian, Coptidis chinensis Wuzhuyu, Euodia rutaecarpa 6:1 60 cases with 250 ml decoction, 85% Randomized (Yin, Franch., Coptidis deltoidea C. (Juss.) Benth., Euodia rutaecarpa gastric ulcer tid, for 6 weeks study, none- 2009) Y. Cheng et Hsiao or Coptidis (Juss.) Benth. var. officinalis blind; moderate teeta Wall. (berberine, (Dode) Huang or Euodia coptisine, worenine, rutaecarpa (Juss.) Benth. var. epiberberine, jatrorrhizine, bodinieri (Dode) Huang palmatine, etc.) (evodiamine, dehydroevodiamine, rutaecarpine, etc.) Huangqi, Astragalus Danggui, Angelica sinensis (Oliv.) 5:1 100 cases of 10 ml oral 47% Not clear of (Liu and membranaceus (Fisch.) Bge. Diels (ferulic acid, ligustilide, anemia solution, bid, for randomization, Xu, 2001) or Astragalus membranaceus butylidenephthalide, 2 months none-blind; low (Fisch.) Bge. var. mongholicus senkyunolide, etc.) 21 breast 200 ml decoction, improved Randomized (Chen (Bge.) Hsiao (Osthole, cancer cases bid, until the immunological study, none- et al., calycosin, astragalosideI, II, before chemotherapy function and blind; moderate 2009) IV, soyasaponin I, chemotherapy stoped enhanced formononetin, chemotherapy isomucronulatol, asparagine, endurance polysaccharides) Huanglian, Coptidis chinensis Muxiang, Aucklandia lappa 4:1 65 cases with 5 tablets, tid, for 75% Randomized (Xia and Franch., Coptidis deltoidea C. Decne. (costunolide, acute diarrhea 7–14 days study, none- Wei, Y. Cheng et Hsiao or Coptidis dehydrocostuslactone, b-costol, blind; moderate 2009) teeta Wall. (berberine, a-cyclocostunilide, etc.) coptisine, worenine, epiberberine, jatrorrhizine, palmatine, etc.) Renshen, Panax ginseng C. A. Fuzi, Aconitum carmichaelii Debx. 1:2 cancer-related 50 ml injection, 70% Randomized (Gu et al., Mey. (ginsenoside Rb1, Rc, (aconitine, mesaconitine, fatigue of 113 q.d., for 3 weeks study, none- 2010) Rg1, Rg2, Rg3, Rh, volatile oil, hypaconitine, benzoylaconine, cases with blind; moderate ginseng polysaccharides, benzoylmesaconine and advanced etc.) benzoylhypaconine, etc) carcinoma 20 cases intravenous protected Randomized (Xia et al., undergoing injection (0.5 ml/ gastrointestinal tract study, none- 2003) cardiac kg), followed by and reduced blind; moderate surgery continuous inflammatory actions infusion of 100 ml injeciton for 2 h Yanhusuo, Corydalis Yanhusuo Baizhi, Angelica dahurica (Fisch. 2:1 176 cases 4 tablets, tid, for 73% No control (Chen W. T. Wang (d-corydaline, ex Hoffm) Benth. et Hook. f. or with migraine 2 months group, not et al., dl-tetrahydropalmatine, Angelica dahurica (Fisch. ex clearly defined 2003) protopine, Hoffm) Benth. et Hook. f. var. outcome, none- 1-tetrahydrocoptisine, formosana (Boiss.) (imperatorin, blind; high 1-tetrahydrocolumbamine, iso imperatorin, oxypeucedanin, d-corybulbine, d-glaucine, cnidilin, oxypeucedanin hydrate, etc.) bergapten, oxypeucedanin, etc.)

n EER: excellent effective rate.

Du et al., 2009; Liu and Xu, 2001). DBD is mainly applied in the with residue stasis-toxin. Clinical studies have found that DBD treatment of Qi and Blood deficient pattern, and patients bearing could markedly accelerate the recovery of immune function that tumors frequently present the syndrome of Qi-Blood deficiency occurs in the early post-operative stages of non-small cell lung S. Wang et al. / Journal of Ethnopharmacology 143 (2012) 412–423 419 cancer (NSCLC) patients (41 cases), and continuously enhance widely known that the concentration of some active ingredients their immunity. DBD also has an anti-tumor action and can in many CHM is lower than their therapeutic dosages. While the accelerate hematopoiesis, protect the function of essential organs, effectiveness of many Chinese herbal formulae is undeniable, it is promote post-operation recovery, thus elevate a patient’s living reasonable to assume that their curative effects may arise from quality and personal confidence in rehabilitation (Du et al., 2009). the synergistic actions of the herbs in a specific formula. Actually, Recently, a systematic summation of randomized controlled trials even if many formulae are proved to be quite efficacious, it may of CDDP was accomplished by our institute (Jia et al., 2012b). By not be possible to fully elucidate the mechanism, particularly for analyzing the 60 randomized controlled trials published between certain complex formulae containing dozens of herbs. In TCM, 1994 (i.e., year of DSP launch) and 2009, we found that CDDP is herb pairs play an indispensable role, not just because of the large apparently more effective than isosorbide dinitrate (a drug quantity and as the basic elements of multi-herb formulae, but routinely prescribed in conventional medicine) in treating angina because herb pairs can reflect the characteristics of complex pectoris, which further confirmed the great potentials of herb pair formulae, and their feature of simplicity can facilitate studies on prescriptions in clinic. mechanism investigation. If the riddle of some herb pairs is Currently, many other clinical trials of herb pair preparations preferentially unraveled, then the elucidated mechanism could are now undergoing in China and many other countries (Table 3). serve as a simpler experimental starting point in studying more However, most these studies were of poor methodology quality complex formulae. This idea may be both logically feasible and and at a high risk of bias, which was associated with a likelihood methodologically operable (Fig. 2). Recently, chemical and mole- of results favoring the trial intervention. The following aspects cular biological approaches have further helped to elucidate the may affect the results. Methodologically, although many studies mechanisms of herb pairs both in material basis and their described the allocating procedure as ‘‘randomly allocating the pharmacological actions. However, several crucial issues in the patients’’, most of them did not provide the details of randomiza- research and application progress do need to be emphasized. tion (Liu, 2011); little study provided any information on allocat- In the tide of the modernization of Chinese medicine, the ing concealment (Yin, 2009); blinding of key research personnel reform of traditional herbal decoction has become a hot topic. A (patients, investigators, and assessors) was used in only few widely approved and extensively applied mode in the medical studies (Scholey and Kennedy, 2002). These factors may result field is the popularization of concentrated granules of a single in bias in sample selection, performance and detection, and easily herb. Currently, over 600 varieties of concentrated granules are led to false-positive findings. Meanwhile, a series of related routinely used in more than 600 hospitals in China (Liu et al., symptoms were selected as the criteria of primary outcome 2011). However, there remains a major concern that this reform instead of some quantitative indicators, which could affect the will lose the advantages of conventional herbal decoction and will outcome assessment. Moreover, primary outcomes were not not ensure the therapeutic effect in clinic. The interactions among clearly defined in some study. Besides, most of these studies the bioactive constituents of herbal formulae are not only limited were carried out in China and published in Chinese, and thus in the process of absorption, metabolism, and enzyme reaction, positive results were more likely to be published than the but also in the procedure of processing, where complicated negative ones. As a whole, for such reasons mentioned above physiochemical reactions may occur (Zhao et al., 2009). In other (obvious limitations in design, serious imprecision and high Asian countries, such as Japan and South Korea, many classical publication bias), the risk of bias of these trials seems inevitable. formulae containing more than two herbs are widely used as To reach a reliable assessment of the efficacious of herb pairs, concentrated granules, which could compensate for the limita- further clinical trials with higher quality should be conducted tions of single herb granules and are actively advocated by many (Flower et al., 2012). Until we do so, it is also essential that we Chinese scientists (Tu and Bi, 2007). As herb pairs play a transi- correctly recognize, evaluate and make full use of these accumu- tional role between single herbs and multi-herb formulae, con- lated clinical experiences. centrated granules of some famous herb pairs could be preferentially explored as a transitional substitute in the reform of traditional herbal decoction. 6. Conclusions and perspective As research progresses, the synergistic mechanisms of some Chinese herbal formulae are being preliminarily elucidated. For In recent years, many promising drugs and candidates have example, Realgar–Indigo naturalis, the combination of Realgar been isolated from CHM, and their mechanisms are being gradu- (tetraarsenic tetrasulfide), Indigo Naturalis, Salviae miltiorrhizae ally understood. Although CHM are being used in large-scale, it is Radix et Rhiroma and Pseudostellariae Radix, was suggested to be

Fig. 2. Herb pairs have acted as a transitionary role in the evolution of multi-herb formulae, and may play a key role in the development of herb formulae R&D. 420 S. Wang et al. / Journal of Ethnopharmacology 143 (2012) 412–423 effective for treating human acute promyelocytic leukemia (APL) and envoy herbs (not all formulae contain all the four grades). The (Xiang et al., 2000). The roles of the active ingredients derived king is the herb possessing primary pharmacological action from Realgar, Indigo Naturalis, and Salviae miltiorrhizae Radix et against the main cause of a disease while the vassal aids the Rhiroma were elucidated, thus the synergy of this formula is now action of the king herb. The assistant herb is chosen to enhance basically understood. Specifically, Realgar directly attacks the the activity of the king and the vassal and always performs a promyelocytic leukemia retinoic acid receptor a (PML-RARa) detoxifying activity; finally the envoy herb helps transport all the oncoprotein and promotes APL cell differentiation. Indirubin and active components to the target organs (Yi and Chang, 2004). A tanshinone IIA, the principal components of Indigo Naturalis and herb pair is constructed based on the comprehensive analysis of Salviae miltiorrhizae Radix et Rhiroma, can enhance Realgar- clinical information, which is the most important principle that induced ubiquitination and the degradation of PML-RARa.In guides the prescription of Chinese herbal formulae (Bao et al., addition, indirubin and tanshinone IIA can also strengthen the 2010). The past decades have witnessed an increasing number of expression of aquaglyceroporin 9, which helps transport Realgar clinical trials on TCM treatment efficacy with unsatisfactory into APL cells and thus strengthens efficacy of the main formula randomized and controlled clinical trial designs. Part of the (Wang et al., 2008a). However, Realgar–Indigo naturalis was just reason is the lack of stratification when using TCM syndrome an isolated incident. Among the 100, 000 recorded formulae, few differentiation (Jiang et al., 2012). Therefore, more efforts should are well studied, and even the mechanisms of herb pairs are not be devoted to substantiate the rationale with biomedical evidence yet elucidated. Over 1,000 herb pairs have been used in China and practise the concept in clinical trials. Meanwhile, it is worth since hundreds of years and their modern preparations are also noting that some clinical trials of the antagonistic herb pairs were widely applied in clinic. For example, the Shenqi Fuzheng injec- also performed in recent years. For example, Gancao and Gansui tion (SFI), isolated from two herbs, Astragali Radix and Codonop- were traditionally believed to be antagonistic, while a clinical trial sis Radix, was primarily approved by the State Food and Drug indicated that the antagonistic herb pairs were effective for Administration (SFDA) in 1999 as an antitumor injection to be cirrhosis ascites cases (Yang et al., 2005). However, in these manufactured and marketed in China (Wang et al., 2012a). Many clinical studies, no safety data were provided. For this reason, in trials have demonstrated that the SFI may play an important role investigating the rationale of antagonistic herb pairs, safety in the treatment of advanced NSCLC via improving tumor should be a prerequisite. ‘‘Safety first’’ has always been our response, performance status, and reducing the toxicity of stan- consistent principle. dard platinum-based chemotherapy (Dong et al., 2010; Qi et al., Shaped by a belief that CHM is efficacious, the mechanism 2011). However, most of these studies were undertaken in China centered approach primarily concerned with the search for the and the mechanisms of action of the injection still remain unclear. molecular, cellular, and pharmacological bases is readily available For these herb pairs with exact curative efficacies, their thera- for CHM research (Tang, 2006). This mode seeks to identify the peutic potential and underlying mechanisms should be paid more active substances of herbs and investigate their mechanism of attention to. action. However, for TCM formulae, especially for those ones In the past ten years, systems thinking has penetrated into the comprising dozens of herbs, to entirely identify their active pharmaceutical and biomedical areas. In the post-genomic era, substances could be fairly difficult. Even if a formula is proved the advances of the ‘‘omics’’ revolution and the methodology to be efficacious, it may not be possible to determine the including genomics, proteomics, and metabolomics technologies mechanism of action and the active substances immediately. have all collectively offered a natural bridge between Chinese and The efficacy driven approach is now widely applied in multi-herb Western medicine (Buriani et al., 2012; Jia et al., 2012a; formulae studies, where the efficacy is firstly to be confirmed Ouedraogo et al., 2012). The practice of TCM is based on a (Tang, 2006). While the ultimate purpose of investigating herb ‘‘holistic’’ philosophy, where Chinese practitioners focus on the pair compatibility is to provide reasonable, secure, and effective inherent balance of the body. Systems biology approaches can indications for pharmacy medication and prescription in actual help identify the therapeutic mechanism of multi-herb formulae, clinical practice, studies on active substances and related including the build-up of pharmacological networks (Jiang et al., mechanisms are still an inevitable issue for further development 2012). As one approach of systems biology, metabonomics adopts of herb formulae. a ‘top-down’ strategy to reflect the function of organisms from terminal symptoms of the metabolic network and understand the metabolic changes of a complete system caused by interventions Acknowledgments in a holistic context. This viewpoint coincides with the holistic thinking of TCM (Wang et al., 2011). Based on the ultra-perfor- Thanks for the financial support of the Research Fund of mance liquid chromatography/quadrupole time-of-flight mass University of Macau (MYRG 208 (Y2-L4)–ICMS11-WYT and spectrometry (UPLC-Q-TOF/MS), the protective effects of Com- UL016/09Y4/CMS/WYT01/ICMS) and the Macao Science and Tech- pound Danshen tablets (a herbal compound preparation consist- nology Development Fund (077/2011/A3) for this research. ing of Salviae miltiorrhizae Radix et Rhiroma, Notoginseng Radix et Rhiroma and Borneolum syntheticum) on myocardial ischemia References were elucidated as reversing potential biomarkers to sham levels, especially for the four metabolites (hypoxanthine, xanthine, Bao, H., Gao, J., Huang, T., Zhou, Z.M., Zhang, B., Xia, Y.F., 2010. Relationship inosine, and allantoin) that lie in the pathway of purine metabo- between syndrome differentiation of traditional Chinese syndrome differen- lism (Lv et al., 2010). 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