系统生物学与中药质量控制与安全

Journal of Ethnopharmacology 126 (2009) 31–41

Contents lists available at ScienceDirect

Journal of

Ethnopharmacology

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j e t h p h a r

m

Review

Quality and safety of Chinese herbal medicines guided by a systems biology perspective

Jiangshan Wang a ,c ,e ,Rob van der Heijden c ,Shannon Spruit b ,Thomas Hankermeier c ,Kelvin Chan d ,Jan van der Greef b ,c ,e ,Guowang Xu a ,e ,??,Mei Wang b ,e ,?

a

CAS Key Laboratory of Separation Science for Analytical Chemistry,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,116023Dalian,China b

SU BioMedicine and TNO,Quality of Life,Utrechtseweg 48,3700AJ Zeist,The Netherlands c

Division of Analytical Biosciences,Leiden Amsterdam Center for Drug Research,Leiden University,P.O.BOX 9502,2300RA Leiden,The Netherlands d

Department of Pharmacy &Research Institute in Healthcare Science,School of Applied Sciences,University of Wolverhampton,Wolverhampton WV11LY,UK e

Sino-Dutch Centre for Preventive and Personalized Medicine,Utrechtseweg 48,3700AJ Zeist,The Netherlands

a r t i c l e i n f o Article history:

Received 2April 2009

Received in revised form 15July 2009Accepted 18July 2009

Available online 13 August 2009Keywords:

Chinese materia medica Systems biology Systems toxicology Metabolomics Aconitum roots

a b s t r a c t

Chinese herbal medicines,often referred as Chinese materia medica (CMM),are comprised of a complex multicomponent nature.The activities are aimed at the system level via interactions with a multitude of targets in the human body.This review aims at the toxicity aspects of CMM and its preparations at the different steps of production;harvesting,processing and the ?nal formulation.The historic perspective and today’s issues of the safety of CMM are introduced brie?y,followed by the descriptions of the toxic CMM in the current Chinese Pharmacopoeia (2005).Subsequently,several aspects of safety are illustrated using a typical example of a toxic CMM,Aconitum roots,and some recent ?ndings of our own research are included to illustrate that proper processing and multi-herbs formulation can reduce the level of toxic components.This also explains that in CMM,some herbs,such as Aconitum ,Ephedra species are never used as single herb for intervention and that aconite is only used when it is processed and in combination with speci?c matched other herbs.The formulation principle of multi-herbs intervention strategy is a systems approach for the treatment and prevention of disease.In this light,the role of systems toxicology in the safety and quality of Chinese herbal medicine is proposed as a promising method.Moreover the principles of practiced-based and evidence-based research are discussed from a symbiotic perspective.

? 2009 Elsevier Ireland Ltd. All rights reserved.

Contents 1.

Introduction (32)

1.1.Chinese materia medica and underlying principles..........................................................................................321.

2.Global interest and challenges ...............................................................................................................322.

The past and present safety evaluation of CMM.....................................................................................................332.1.Historical safety evaluation of CMM..........................................................................................................332.2.Present safety evaluation of CMM............................................................................................................

33

Abbreviations:CM,Chinese medicine;CMM,Chinese materia medica or Chinese medicinal materials;LD 50,the median lethal dose for 50%of the tested subjects;DI-ESI-MS,direct infusion electrospray ionization mass spectrometry;MALDI-MS,matrix assistant laser desorption ionization mass spectrometry;HPLC–MS,high performance liquid chromatography mass spectrometry;PCA,principle component analysis;PLS-DA,partial least squares-discriminant analysis;SCN,sodium channel;Na(v)I Alpha,Voltage-gated sodium channel type I alpha protein (encoded by the gene SCN1A);ASCT2(SLC1A5),amino acid transport system;ENaC,epithelial Na(+)channel;RAI,(or PPP1R13L protein)-protein phosphatase 1,regulatory (inhibitor)subunit 13like;GRO3,an early growth response transcription factor that is essential for the development of muscle spindles;XRCC1,X-ray repair cross complementing protein 1,involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation;NMDA receptors,N-methyl-d -aspartate receptors (a class of ionotropic glutamate receptors characterized by af?nity for N-methyl-d-aspartate);Annexin A1,Protein of the annexin family exhibiting lipid interaction and steroid-inducibility;AMPA receptors,Cell surface proteins that bind glutamate and directly gate ion channels in cell membranes.AMPA receptors were originally discriminated from other glutamate receptors by their af?nity for the agonist AMPA;SLICK,a type of rapidly gating sodium-activated channel.

?Corresponding author at:SU BioMedicine and TNO,Quality of Life,Utrechtseweg 48,3700AJ Zeist,The Netherlands.Tel.:+31306944848;fax:+31306944040.??Corresponding author at:CAS Key Laboratory of Separation Science for Analytical Chemistry,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,116023Dalian,The Netherlands.Tel.:+8641184379530;fax:+8641184379559.

E-mail addresses:xugw@https://www.360docs.net/doc/d310651945.html, (G.Xu),mei.wang@tno.nl (M.Wang).0378-8741/$–see front matter ? 2009 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jep.2009.07.040

32J.Wang et al./Journal of Ethnopharmacology126 (2009) 31–41

3.CMM and constituents with known side effects in the Chinese Pharmacopoeia (33)

4.Application of systems toxicology in toxicity investigations (34)

5.From pharmacovigilance to mechanisms of toxicity (35)

6.The toxicity of Aconitum species (36)

6.1.Origin of the CMM from the Aconitum species (36)

6.2.Toxic constituents of A.carmichaeli and quality control (36)

6.3.Processing and formulations (36)

6.4.Biological effects of Aconitum alkaloids (36)

7.Conclusion (39)

Acknowledgements (40)

References (40)

1.Introduction

1.1.Chinese materia medica and underlying principles

Chinese herbal medicine is often referred as Chinese mate-ria medica(CMM).The use of CMM in the practice of Chinese medicine(CM)has a history of several thousands of years and has developed into a unique holistic health care system for the pre-vention,diagnosis and treatment of diseases.The human body is a complex and highly interconnected system,which is dynami-cally regulated within boundaries,the so-called homeostasis.The strategy of CM is based on supporting the self-healing capabilities of the body(salutogenesis)to recover from an imbalanced state. This approach is a?ne-tuned at the individual level by optimiz-ing each formula and is as such not addressing a disease with a single chemical compound or aiming at relieving a single symp-tom.In practice a Chinese medicine practitioner uses a unique detailed diagnostic procedure,not familiar to Western medicine,in order to prescribe a personalized mixture of CMM that the patient would typically take orally as an aqueous decoction.In the for-mula,each herb contains many compounds that possess concerted actions(Chan and Lee,2002;Chan,2005).A successful formula organizes these concerted actions derived from different herbs to create holistic,multi-target,multi-dimensional pharmacological actions to achieve personalized therapy.Such an approach is not yet embedded in Western medicine,but the advance of pharma-cogenomics and systems biology have created the building blocks to bridge this CMM approach with modern pharmacology(Wang et al.,2005a;Van der Greef et al.,2006;Van der Greef and McBurney, 2005).

1.2.Global interest and challenges

Currently,the practice of CMM is not only popular in China and some Asian regions;but also it is appreciated worldwide.A large-scale study of the Chinese population of Taiwan showed that from1996to2001,more than60%of all subjects have used Chi-nese medicine during the6-year interval.CMMs(85.9%)were the most common modality(Chen et al.,2007).It was estimated that more than1.5billion people all over the world are trusting in the ef?cacy and safety of Chinese medicine(Hosbach et al.,2003).In the‘Western world’,the term‘alternative’medicine is now often replaced by‘complementary’medicine.This emphasizes the com-plimentary nature of both developed medical systems.The latest term‘integrative’medicine underpin the gain that can be obtained when Western and complementary modalities are used together (Wang et al.,2005a).Parallel to this increased popularity,clini-cal pharmacological interest in the quality,safety and ef?cacy of CMM and herbal remedies in general have also grown.An aque-ous decoction from a mixture of CMM can be viewed as a source of many chemical entities,and side effects are inevitable espe-cially when not taken according to the‘CM practitioners’https://www.360docs.net/doc/d310651945.html,pared with the safety of pharmaceutical medicines,the inves-tigation of toxicity of CMM is far more dif?cult(Chan,2005).An estimated number of11,145medicines retrieved from medicinal plants are known in China(Huang et al.,2004).Their ef?cacy and toxicity are mostly based on historical,long term clinical experi-ence instead of modern scienti?c evaluation.A challenge is the variation in chemical contents caused by the variation of growth during the day–night rhythm,seasonal change,age and polymor-phisms within one species(Zhu,1998;Chang et al.,2006).The variation in chemical contents contributes to varying observations of potential toxicity of CMMs.Furthermore,the physiological status of the body plays a role in the toxicity as it may reduce or pro-mote toxicity by molecular interactions.Moreover,the causes of adverse reactions associated with CMM are complex and diverse (Ko,2004).Important to note is that given the personalized char-acter of CMM,different scienti?c methods need to be applied rather than the golden standard randomized,cross-over,double blinded clinical trial designs.Moreover one could argue that a prac-tical evaluation over many more patients than normally used in clinical trials yields a better insight.Typically clinical trials for Western medicine are started with evidence for possible posi-tive action in human,but only10%of the new chemical entities tested pass the clinical trial research often with toxicology as major stumble block.After the introduction into the market the practice-based regime is entered and a signi?cant number of newly introduced drugs need to be withdrawn because of serious side effects.In principle the sequence of practice-based and evidence based research is reversed in the current research.However,the lack of large scale comparisons between doctors and the variability of the herbal materials as well as the production,create a challenge for a more meta-analysis view.The limited knowledge collected today makes it dif?cult to diagnose the toxic effects derived from herbal medicines in general(Chan et al.,2005).On the other hand modern Chinese medicines produced under good manufacturing practice(GMP)and distributed through hospital channels provided a clear view on the ef?cacy and safety of a number of formulas.Tra-ditional knowledge on the practice of processing of crude CMM and the knowledge of the optimal combination of herbs in for-mulas to limit or reduce toxicity are as important as the ef?cacy issue.

In recent years,modern chemical,physical and biological meth-ods are applied for the investigation of the toxicity of CMM and signi?cant progress has been achieved.Poisonous compounds from individual toxic CMMs have been identi?ed and the mech-anisms of toxicity are under scienti?c investigations.Despite the great progress,safety of CMM is still a dif?cult task,partly due to the complexity of CMM but also due to the lack of appro-priate approaches within toxicity research of complex herbal mixtures.In this paper,we will give a general introduction on the safety of CMM.Furthermore,the systems toxicology approach is addressed as a promising tool to investigate CMM toxic-ity.

J.Wang et al./Journal of Ethnopharmacology126 (2009) 31–4133

2.The past and present safety evaluation of CMM

2.1.Historical safety evaluation of CMM

The toxicity of potent CMM was recorded in the earliest Chi-nese Pharmacopoeia(ShenNong BenChao Jing)in the East Han dynasty(25–220AD)(Liu et al.,2003;Liang and Gao,1992),a total of365CMMs derived from plants,animals and minerals were recorded as medicines and classi?ed into superior,average and inferior medicines.In the inferior category125medicines were con-sidered to be toxic or with side effects.Cautions on these medicines were recommended:to be used only for treatment of speci?c dis-eases in combination with other speci?c CMM;to be processed before using it as formulation;that the dose to be increased gradu-ally to avoid strong toxic effects;that they should not be used alone or for a long period.

Further development,based on experience,of this traditional document concerned the description of toxicity by the introduc-tion of a scale of toxicity ranking from high,medium,small to minor toxicity.Experience in using toxic CMM was further recorded and during the Ming dynasty(1368–1644AD)in the more compre-hensive and famous Chinese Pharmacopoeia–BenChao GangMu –appeared with a systematic review on toxicity of CMM by inclu-sion of a special chapter about toxic herbs.In this chapter,the toxic CMM recorded in previous documents were summarized system-atically.The use of toxic CMM and methods to reduce the toxicity were also covered(Liu et al.,2003).

2.2.Present safety evaluation of CMM

The bene?ts and health risks of all medicines,apart from clearly indicated principles regarding dose-related therapeutic and toxic effects,depend on how they are used and prescribed for public use. In a report of the World Health Organization(WHO),it is described that in the United States of America(USA),annually more than 1.5million people are seriously injured and hospitalized because of prescription drugs making adverse reactions a major cause of death(Patwardhan,2005).Efforts in drug discovery and develop-ment cannot guarantee complete safety of medications introduced by pharmaceutical companies,and about51%of the drugs approved by Food and Drug Administration(FDA)have serious adverse effects not detected prior to their approval(Patwardhan,2005).There-fore,the safety and risks associated with medical intervention is an issue across all categories of health care(Patwardhan,2005). This expands to food safety.In the USA,the Center for Disease Control and Prevention(CDC)estimated that among the country’s 290million residents,food borne diseases cause approximately 76million illnesses annually,including325,000hospitalizations and5000deaths(De Waal and Robert,2005).The safety is also a common concern in all kinds of herbal medicines,including Arabic herbal medicines,Ayurvedic herbal medicines and Kampo medicines(Saad et al.,2006;Ikegami et al.,2004;De Smet,2004). In conjunction to this increased popularity,clinical pharmacologi-cal interest in the safety of CMM has also emerged,especially after recognizing the so-called Chinese herb-induced nephropathy.This in fact is aristolochic acid-induced nephropathy.Nephropathy,a rapid progressive form of interstitial renal?brosis,was observed in patients who had taken a slimming regimen containing a mix-ture of pharmaceutical drugs and CMM that contained aristolochic acid.This incident was identi?ed?rst in Belgium(Vanherweghem et al.,1993).This unfortunate medical disaster is a typical case of inappropriate application of CMM;in the incident the aristolochic acid came from Aristolochiae fangchi Radix(Guang fangji)and not Stephenia tetrandra Radix(Fangji)that should have been added to the CMM drugs.The medical public has hence become more focused on the potential toxicity of CMM progressively(Chan,2005).The causes of adverse reactions associated with CMM are revealed in the literature,including variability in active/toxic ingredients,use of inherent toxic herbs,overdose,drug–herb interactions,coexist-ing diseases,and idiosyncratic reactions(Ko,2004;Tomlinson et al.,2000).

Quality control is another major issue of CMM that is related to adverse reactions,such as contamination(pesticides,heavy metals, microbes etc.),adulteration,misidenti?cation,improper process-ing and preparation(Chan,2005;Ko,2004;Maxion-Bergemann et al.,2006).With respect to the frequency of adverse reactions,a study carried out in Hong Kong showed that adverse events due to CMM accounted for0.2%of hospital admissions(3patients in1701 admissions)during an8-month period,and the frequency was far below that for Western pharmaceuticals at which was about4.4% (Bensoussan et al.,2000).A further investigation through com-prehensive practitioner survey in Australia showed that0.16%of Chinese medicine(including acupuncture and CMM)consultations resulted in an adverse event whilst pharmaceutical drug related hospital admissions in Australia is5.7%(Bensoussan et al.,2000). Similar investigations performed in the United Kingdom indicated that the most common adverse events associated with CMM were diarrhoea,fatigue,and nausea.No serious adverse events were reported(MacPherson and Liu,2005).The complexity of toxico-logical events associated with herbal medicines makes it necessary to have a multidisciplinary team of experts to examine suspicious reports.A total of20reports of suspected herbal poisoning were collected from the public hospitals in Hong Kong during a2-year period.In10patients,the adverse events were unlikely to be related to herbal medicines(Chan et al.,2005).In conclusion CMM is not absolutely safe,but its toxicity should not be exaggerated based on incidents of inappropriate use.The tendency to use a reductionistic view and extrapolate toxicity of components to the formula level neglecting synergetic safety effects is a major source of misunder-standing today.Formulas can only be evaluated in the context of the whole,but studies on single herbs or components can be helpful for

a scienti?c understanding of the toxic and synergetic effects.

3.CMM and constituents with known side effects in the Chinese Pharmacopoeia

All medicines when used inappropriately may turn bene?-cial pharmacological actions into toxicity.From the statements of Paracelsus in the?fteenth century and well-established knowl-edge in modern toxicology it is realized that dosage determines the toxicity of compounds.This is why Table1contains informa-tion about the compound dose which causes lethality or speci?c effects.Therefore,herbs regarded as nontoxic may have poten-tial to cause adverse events.Ginseng,is a well-known safe tonic herb and classi?ed as superior herb traditionally.However,it in higher doses produced in some people nervousness and eleva-tion of mood with occasional depression.Ginkgo biloba possesses platelet aggregation inhibiting effects that may be related to the side effect of spontaneous hemorrhage(Tomlinson et al.,2000). On the other hand,Papaver somniferum is seen as highly toxic,but its seed(poppy seed)does not contain the toxic opium alkaloids in suf?cient high amounts to cause severe toxicity(Dewick,1997). In an early survey of48potent or poisonous CMMs available for CM practice in the Hong Kong market(Xu and Chan,1994),the LD50was determined in mg per kg of these CMMs in mice as a measure of toxicity with microscopic records and thin-layer chro-matograms as the guidance for quality measure.Experiments on mice were carried out to determine the acute toxicity of32plant herbs,9animal products and6mineral products in total.For some of the CMMs,different experiments were made on the unprocessed and processed products for comparison.The results of the exper-

34

J.Wang et al./Journal of Ethnopharmacology 126 (2009) 31–41

Table 1

Main toxic compounds and their toxic https://www.360docs.net/doc/d310651945.html,pound Administration LD 50(mg/kg)Main effects

References

Aconitine Intraperitoneal 0.27Arrhythmias

Dong et al.(1981)Intravenous 0.10Convulsion or effect on seizure threshold.Friese et al.(1997)Mesaconitine Intraperitoneal 0.21No record

Hikino et al.(1977)Intravenous 0.068Convulsion or effect on seizure threshold.Friese et al.(1997)Hypaconitine

Oral

5.8No record Bisset (1981)

Intraperitoneal 1.10Arrhythmias Dong et al.(1981)

Subcutaneous 1.9Ataxia Murayama et al.(1991)Intravenous 0.47No record Bisset (1981)Talatisamine

Intravenous 116No record Bisset (1981)Benzoylhypaconine

Intraperitoneal 120No record Bisset (1981)Oral

830No record Bisset (1981)Subcutaneous 130No record Bisset (1981)Higenamine

Intravenous 58.9No record Bisset (1981)Intraperitoneal 300No record Bisset (1981)oral

3350No record Bisset (1981)Karacoline Intraperitoneal 298No record Bisset (1981)Intravenous 51.5No record Bisset (1981)

Beiwutine

Intraperitoneal

0.42

Arrhythmias

Dong et al.(1981)

iments indicate that:(1)The toxic doses of some potent CMM were under 10g/kg:semen Strychni (Ma Qian Zi),Radix et Rhizoma Sinopodophylli (Tao Er Qi),Fructus bruceae (Ya Dan Zi),Fructus Cro-tonis (Ba Dou),and Semen armeniacae amarum ,preparatum (Bei Xing or Ku Xing Ren,processed);(2)Among the animal products,the toxicity of Mylabris (Ban Mao)and Venenum bufonis (Chan Su)was extremely high (<1g/kg),the toxicity of the other animal prod-ucts could not be detected due to insolubility of the materials;and (3)Among the mineral products,the toxic doses of Chalcan-thitum (Dan Fan),Calomelas (Qing Fen)and Natrii sulhas (Mang Xiao)were found between 1and 10g/kg;the values of LD 50of the other mineral products could not be obtained due to insolubility of materials.Subsequent to this publication the Hospital Authority in Hong

Kong commissioned a Clinical Toxicology Task Force for Chi-nese Medicine to monitor poison cases related to CMM (Hong Kong Hospital Authority,2002).

In this section,the focus is on CMM with recorded toxicity in the latest issue of the Pharmacopoeia of the People’s Republic of China (Chinese Pharmacopoeia (CP),2005).Among the of?cial CMMs included in the CP 2005,59herb derived products are classi?ed as having toxic effects,and which are considered by the Chinese Pharmacopoeia Commission as of?cial drugs based on long term ‘trial &error’practice.Isolation and identi?cation of bioactivity of constituents from toxic herbs are the basis to further research into mechanisms of toxicity involved (Van der Heijden et al.,2004).For example,Aconitum species used in CMM are one of the major causes leading to toxicity,diterpenoid alkaloids isolated from Aconitum

Fig.1.A classi?cation of components from 59toxic CMMs (Number of compounds is 121).

species with pharmacological activities provide the bases to inves-tigate and understand the toxicity (Ameri,1998).The identi?cation of a nephric toxic compound,aristolochic acid,is revealing the toxi-city mechanism and many similar herbal medicines containing the same constituent with similar toxicity have been discovered sub-sequent to the tragic case on Guang fangji (Aristolochiae fangchi ).However,until now knowledge of the constituents of toxic herbs is limited.There are a total of 121toxins that have been reported,and are derived from the known 59toxic CMM in the CP (Dewick,1997;Zhu,1998).Considering the huge number of chemical entities in these herbs,the existing knowledge is limited.These chemicals can be classi?ed into the following categories:alkaloids,terpenes,coumarins and proteins (Fig.1).In terms of knowing the identities of the constituents of herbal medicines,large scale of basic elucidation is needed (Stone,2008).

4.Application of systems toxicology in toxicity investigations

All medicinal products used for human intervention should have proven quality,safety and ef?cacy (QSE).In Western medicine sev-eral factors are taken in account that might in?uence the ?nal drug response.Differences in drug metabolizing enzymes can give an increase or decrease in active or toxic constituents and differences in transporters will affect the bioavailability of drugs in cells,while different drug targets will affect the ef?cacy.Small molecules and proteins can also in?uence the ?nal effect by interaction with the drug and unknown off-targets effects are often only revealed in larger clinical settings.Generally,the above-mentioned factors con-tribute to drug response of CMM.Problems and dif?culties arise in the quality assurance of herbal medicinal products because they contain many unidenti?ed chemical entities in the ?nished prod-ucts and the actual bioactive components are seldom known.This is in principle not different from food science related challenges.Recent advances in analytical chemistry and related disciplines pro-vide opportunities to elucidate the complex chemical compositions of active compounds in CM herbs (Wang et al.,2009).

The current paradigm in evaluating drug safety is the applica-tion of data obtained from animals with extrapolation to possible risks in humans.The sensitivity of animals to detect potential dam-age induced by an intervention,the translational issues as well as variation within human subjects (gender,age,location,culture,etc.)decrease the reliability of risk assessment (Waring and Ulrich,2000).It is not surprising that drugs that have passed extensive safety testing and used in the market for years still can result in health care disasters with subsequent withdrawal from human use

J.Wang et al./Journal of Ethnopharmacology 126 (2009) 31–4135

(Couzin,2005).Understanding the mechanism is the key to improve the safety of drugs (Stevens,2006).The ?rst step is the integration of new advanced technologies and insights in life sciences,into the existing drug development framework.The past decade has witnessed an explosion in the number and variety of techniques available for bio-molecular analysis at different levels providing the opportunity to understand life via system based thinking (Van der Greef et al.,2007,2004).The shift from histopathology towards molecular toxicology as an early assessment of drugs has obtained considerable attention (Waring and Ulrich,2000).Toxi-cogenomics is explored for detection drug induced transcription changes in gene expression.New technologies,such as toxico-proteomics and metabolomics,offer additional complementary approaches and provide a deeper and holistic insight in systems tox-icology (Adourian et al.,2008).Metabolomics focuses on metabolic responses expressed in accessible biological ?uids (e.g.plasma and urine)offering the potential to associate molecular phenotype with the physiological state (Van der Greef et al.,2006).Upon its emergence as metabolite (or body ?uid)pro?ling combined with pattern recognition (Van der Greef et al.,1983a;Van der Greef and Leegwater,1983b ),metabolomics became a valuable technique in toxicology (Van der Greef et al.,1983a;Van der Greef and Leegwater,1983b;Lindon et al.,2003).Inspired by the successful applications,metabolomics was applied in the toxicity of CMM (Chan et al.,2008;Chen et al.,2008,2006).One of our studies also demonstrated that metabolomics was a sensitive approach.Doxorubicin (DOX)is used in the treatment of a variety of human neoplasms.However,the clinical use of DOX is limited by the development of a dose-dependent cardiotoxicity and animal studies reveal potential liver and kidney lesions.A urine metabolite pro?ling based systems tox-icology approach was utilized for the investigation of drug induced pathology.Multivariate analysis,partial least squares-discriminant analysis (PLS-DA),was applied to mine useful information from pro?ling data.PLS-DA is a supervised chemometric method,which builds a regression model for maximum separation between pre-de?ned classes and variables responsible for class differences are highlighted by their coef?cients in loading plot.Urine metabo-lites pro?ling by ultra performance liquid chromatography–mass spectrometry (UPLC–MS)contains information of several hundreds endogenous metabolites,providing a broad view on changes at the systems phenotype level.This method was applied to urine sam-ples of rats exposed to single dosages of DOX (5,10or 20mg/kg)prior and at three time points after dosage (24h,48h and 96h).Clinical chemistry and histopathology cannot detect signi?cant changes in animals dosed at the 5mg/kg level.However,by apply-ing metabolite pro?ling in combination with multivariate analysis changes induced by DOX after dose (Fig.2A)are detected.The observed changes re?ect potential biomarkers (cholic acid,hippuric acid and tryptophan etc.)and insights in the mechanistic studies (Fig.2B).

5.From pharmacovigilance to mechanisms of toxicity

Herbal medicines are complex to evaluate with regards to toxicity and ef?cacy effects using conventional methodological approaches.This complexity includes chemical complexity,in par-ticular in prescriptions containing multiple herbs,the lack of known synergetic active ingredients,the risk of contaminants such as pesticides,heavy metals and addition of other ingredients (some-times pharmaceuticals),deterioration and variation in composition (Chan,2005).Moreover interactions between CMM and pharma-ceutical drugs need to be addressed (Chan and Cheung,2000).On the other hand the knowledge obtained during long periods of use from direct empirical practice requires consideration in toxicity

research.

Fig.2.(A)Score plot of partial least squares-discriminant analysis (PLS-DA).Each spot in the score plot represents an individual rat.The animals before dosing and three time points after dosing can be separated clearly based on urine metabolites determined by UPLC-MS.Square:pre-dose,circle:24h after dose,triangle:48h after dose,cross:96h after dose.Each group contains 5rats.PLS-DA is a data analysis tool for classi?cation in metabolomics.(B)Loading plot of PLS-DA.In the loading plot,each point presents a metabolite (labeled by m/z )detected by UPLC–MS.The metabolites located away from the center have signi?cant impact on classi?cation of the different groups of rats in the score plot and can be assigned as potential biomarkers.

Therefore,the toxicity evaluation of existing CMM or established CMM is guided by re-evaluation of experience and by linking this to recognized mechanisms to obtain a better risk-bene?t consider-ation.The ?rst level is the collection of the data from documents and pharmacovigilance.Many established CMMs with toxicity have been recorded through generations of use and more cases related to toxic effects will be enhanced by improved modern pharma-covigilance.This information is directly obtained from practical use by human subjects and reported by general CM practioners or hospitals.The second level is the analysis and discovery of toxic compounds within suspected CMM.In many of the toxic herbal medicines,the components responsible for toxicity have been found and can be utilized to perform quality control and for further in depth research of toxic mechanisms (such as aconitine and aristolochic acid).The third level is to utilize the ancient ‘wis-dom’for toxicity control.For instance Ma-huang (Ephedra ),a CMM derived from the Ephedra species used to treat asthma,nose and lung congestion and fever with anhidrosis,has recently been used in high amounts for weight reduction resulting in many cases of poisoning.Such practices deviate from the traditional use of Ma-huang as documented in the Chinese literature.This is considered as an unprofessional practice of CM (Lee et al.,2000).During long

36J.Wang et al./Journal of Ethnopharmacology126 (2009) 31–41

period of time,for safety considerations,many methods have been developed to reduce the toxicity,mainly in the processing and for-mulation.

Processing of herbs comprises of the techniques used for trans-forming crude CMM into a form for clinic prescription,including cleaning,drying,boiling,steaming and frying,etc.Formulation is referred to as composition of multiple CMM for clinical use, water based extraction(decoction)etc.The principles that after the processing and formulation,the toxicity or different application ef?cacy of CMMs is reduced or altered are fundamental in Chinese medicine practice.This approach is illustrated in the following sec-tion by our research work on the toxic CMM,Aconitum root.The most important issue to realize in this context when compared to Western medicine principles is that for safety evaluation the whole formulation needs to be considered as synergetic effects in for-mula’s are used not only to enhance the pharmacological principles but especially also to enhance the safety effects.So in depth analyt-ical procedures reveal excellent information on toxic components but the context determines whether the toxicity is maintained in a formula.

6.The toxicity of Aconitum species

6.1.Origin of the CMM from the Aconitum species

Radix Aconitum refers to the root part of the medicinal plant that belongs to the genus of the family Ranunculaceae and is comprised of about400species of which166are endemic in China(http://www.e?https://www.360docs.net/doc/d310651945.html,/?ora page.aspx??ora id=2).The most well-known species is Aconitum napellus(Monk’s hood wolfsbane,aconite).Among them,only two species are used in CMM;Aconitum carmichaeli Debx and Aconitum kusnezof?Reichb. The herbal medicines in the China Pharmacopoeia are Radix A. carmichaeli(Chuanwu,from the major root of the species A. carmichaeli Debx.)and its processed form Radix Aconitum Preparata (Zhichuanwu),Radix A.Kusnezof?i(Caowu,from the root of A. Kusnezof?i Reichb),and its processed form Radix A.Kusnezof?i Preparata(Zhicaowu),Folium A.Kusnezof?i(Caowuye)and the processed Radix Aconitum Lateralis Preparata(Fuzi,the lateral root of the species A.carmichaeli Debx.)(Zhu,1998;Chinese Pharmacopoeia Commission,2005).

6.2.Toxic constituents of A.carmichaeli and quality control

In the early ShengNong BenCao Jing,Radix A.carmichaeli (Chuanwu)was already recorded as a high risk medicine.Diter-pene alkaloids are major chemical components of Radix A. carmichaeli.These alkaloids share a common C19norditerpenoid skeleton and can be divided into four types according to the degree of esteri?cation and substitutes at C8and C14posi-tions.Aconitum alkaloids without any ester side chain at C8and C14are non-ester alkaloids(NEAs).Normally,the C14position is occupied by a benzoyl group and further classi?cation can be made according to the chemical group substituted at the C8position namely,monoester-diterpenoid aconitines(MDAs), diester-diterpenoid aconitines(DDAs)and lipoalkaloids with the substitution of hydroxyl,acetyl and fatty acid acyl,respectively.The diversity of chemical structures leads to different biological effects (Ameri,1998).Table1lists the known compounds from Radix A. carmichaeli and their LD50values on mice(database of ChemIDPlus, https://www.360docs.net/doc/d310651945.html,/chemidplus/).In general,traditional oral dosing is much safer than other routes of application.The toxi-city might be derived from many compounds,in the case of Radix A. carmichaeli,the alkaloids aconitine,mesaconitine and hypaconitine are held responsible for the toxic effects(Zhu,1998;Ameri,1998).Chemical constituents pro?ling is a suitable approach.With the pro?ling approach,as many as possible components are determined simultaneously and provide a method to characterize the quality of herbal medicine in a holistic perspective.Especially,the pro?ling approach based on mass spectrometry is helpful for active/toxic components screening and monitoring(Wang et al.,2009).By applying the pro?ling method described by Wang et al(2009),alka-loids pro?ling based on direct infusion electrospray ionization mass spectrometry(DI-ESI-MS),matrix assistant laser desorption ioniza-tion mass spectrometry(MALDI-MS)and high performance liquid chromatography mass spectrometry(HPLC–MS)reveals that there are several tens of alkaloids detected in processed Radix Aconitum Lateralis Preparata collected from different drug stores(Fig.3A–C). For each speci?c alkaloid,signi?cant variation in content between different samples can be observed.However,focusing on one or several alkaloids is not enough to provide a holistic view.Principle component analysis(PCA)is a multivariate method which presents the chemical constituents in a multivariate way.PCA was used to ?nd the similarity in alkaloids content between different samples by the score plot(Fig.3D)and the speci?c alkaloids responsible for the similarity were revealed by loading plot(Fig.3E).

6.3.Processing and formulations

The proper traditional application of Radix Aconitum Lateralis Preparata means processing by boiling,steaming prior to use in the clinic.In CMM only processed form is used.Additionally,the herbal drug is only used in a multi-herbal formulation.The knowledge from empirical practice indicates that the processing and formu-lation have a great impact on the safety aspects of the Aconitum CMM.A logic way to elucidate the detoxi?cation is to examine if after processing and formulation,the content of the toxic alka-loids(aconitine,mesaconitine and hypaconitine)is dramatically changed.Therefore,we studied the content of alkaloids in the Aconi-tum root before and after the processing and formulation by LC-MS. The raw materials were processed,mainly by steaming,according to the method described in CP2005.The processed roots were for-mulated with Glycyrrhiza uralensis and dry ginger roots and boiled (Chinese Pharmacopoeia Commission,2005).After that,content of alkaloids in the raw materials,processed roots and the formulation was determined by HPLC–MS(Wang et al.,2009).

As shown in Fig.4the content of3toxic markers(aconitine, mesaconitine and hypaconitine)are responsible for arrhythmias decreased,this is consistent with previous studies(Zhu,1998; Ameri,1998).These results indicated that the toxic alkaloids were hydrolyzed to low toxic alkaloids(Ameri,1998).Currently, in Chinese Pharmacopoeia,the content of aconitine should be below0.17mg/g in the Radix Aconitum Lateralis Preparata(Chinese Pharmacopoeia Commission,2005).The limitation of all toxic alka-loids(aconitine,mesaconitine and hypaconitine)is still under investigation.

6.4.Biological effects of Aconitum alkaloids

It is known that the Aconitum alkaloids play a major role in the toxicity(Table1),and the degree of toxicity may be indicated by the LD50values.An approach for elucidating the underlying mechanism could be performed by mapping the toxic chemicals into a biological pathway context.The toxicity of the typical poi-sonous alkaloid aconitine was investigated by the Metacore system (GeneGo Inc,St Joseph,MI).Metacore is an integrated software suite for functional analysis of experimental data.The scope of data types includes microarray and SAGE gene expression,SNPs and CGH arrays,proteomics,metabolomics,pathway analysis,Y2H and other custom interactions.MetaCore TM is based on a proprietary manually created database of human protein–protein,protein–DNA

J.Wang et al./Journal of Ethnopharmacology 126 (2009) 31–4137

Fig.3.Alkaloids pro?ling of A.carmichaeli by ESI-MS (A),LC–MS (B),MALDI-MS (C),PCA score plot (PC1vs.PC2)based on LC–MS measurement (D)and loading plot (E).Each spot in the score presents a sample;the distance between samples indicates their similarity in chemical constituents,i.e.shorter distance indicates higher similarity.The loading plot can be used to ?nd components which are responsible for the similarity.For example,the samples in the square of score plot are similar in chemical constituents,which is mainly because they have higher content of components in the square of the loading plot.

Table 2

Possible targets and its corresponding diseases from Metacore.Channels Corresponding disease

References

Na(v)I Alpha

Epilepsy,seizures,migraine spasms and abnormalities.Lossin et al.(2002);Dichgans et al.(2005);Weiss et al.(2003);Ceulemans et al.(2004);Wallace et al.(2003)SCN 3A No corresponding disease

SCN 2A

Epilepsy,epilepsy of the frontal lobe and abnormalities.Maurer-Morelli et al.(2006)

Tetrodotoxin-resistant Na(I)channel

Several cardiac diseases,including tachycardia,

arrhythmia’s,bradycardia and ventricular ?brillation

Gouas et al.(2005);Makiyama et al.(2005);Wolf and Berul (2006);Itoh et al.(2005)

38J.Wang et al./Journal of Ethnopharmacology

126 (2009) 31–41

Fig.4.The reduction in content of three toxic alkaloids during processing and for-mulation.The contents of three toxic alkaloids in raw material,processed root and formulation are normalized by the content in the raw material.The typical chemical reaction responsible for reduction of toxicity is the hydrolysis of aconitine.

and protein compound interactions,metabolic and signaling path-ways and the effects of bioactive molecules in gene expression (https://www.360docs.net/doc/d310651945.html,/metacore.php ).By using pathway anal-ysis tool integrated in Metacore,Fig.5shows that administration of aconitine may result in a cascade of biological events.Aconitine

has a direct link with 4types of ion channels (Fig.5and Table 2).Na(v)I Alpha is the voltage-gated sodium channel type I alpha (encoded by the gene SCN1A).SCN2A and SCN3A are voltage-gated sodium channels.They are transmembrane glycoprotein complexes and responsible for the generation and propagation of action poten-tials in neurons and muscle.Tetrodotoxin-resistant Na(I)channel is a novel sodium channel and is suggested to be associated with central sensitization in chronic neuropathic pain states.Within the Metacore system the channels connected to related diseases are shown in Table 2.It can be seen from the three channels that there is a direct link between aconitine and its toxic effects,mainly arrhyth-mias.The approach of quality control linking chemical entities with toxicity control is of interest for future consideration for potentially toxic herbs.Deeper insight into the mechanism of the bioactiv-ity of a wide spectrum of Aconitum alkaloids could be helpful to understand this.

We applied the Metadrug software (GeneGo Inc,St Joseph,MI)to deduce mechanism of action of small compounds from manually created networks and analyse molecular inter-actions between given compounds and drug targets using the Elsevier MDL Discovery Gate and GeneGo databases (https://www.360docs.net/doc/d310651945.html,/metacore.php ),to study the mecha-nism and bioactivity pro?le of some Aconitum alkaloids (aconitine,beiwutine,benzoylaconine,benzoylhypaconine,higenamine,hypaconitine,mesaconitine).The data generated results that show the activity to serotonin histamine and dopamine receptors to be the common bio-activities of these alkaloids.In addition,the structural diversity of the compounds results also in other effects such as those affecting the receptor ligands (annexin A1and GRO3),generic binding protein (XRCC1),and ligand-gated ion channel (NMDA receptors,AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)receptors,kainate receptors).It shows that aconitine in?uences in?ammation reactions through binding receptor ligands (annexin A1and GRO 3)and this can in?uence transmission of nerve impulse through action on the serotonin,the histamine and the dopamine receptor (Table 3

).

Fig.5.The result of the Metacore analysis of the compound aconitine.

J.Wang et al./Journal of Ethnopharmacology126 (2009) 31–4139

Table3

The targets of several alkaloids found by Metadrug.

Target Aconitine Beiwutine Benzoylaconine Benzoylhypaconine Higenamine Hypaconitine Mesaconitine

Annexin A1+

GRO3+

Serotonin receptors+++++++

Histamine receptors+++++++

Dopamine receptors+++++++

XRCC1+++++

NMDA receptors+

AMPA receptors+

Kainate receptors+

+indicates there is an action of compound on target.

Using MetaDrug TM software:this software deduces toxicity and mechanism of action of small compounds from manually curated networks,using the Elsevier MDL Discovery Gate and GeneGo databases(https://www.360docs.net/doc/d310651945.html,/metadrug.php).

In the development of synthetic drugs,the general way to increase therapeutic effects and decrease the toxicity is by molecu-lar structure modi?cation.This method has proven to be effective, but at present it has become more and more dif?cult to?nd an effec-tive drug with little side effects.By contrast,in the development of future CMM another approach is followed by changing the relative quantities of the groups of CMM constituents and using the syner-getic effects to enhance the desired intervention pro?le.Normally, the active compounds from a CMM herb have similar structures which can lead to similar bioactivity.For example,many of the alka-loids from Radix A.carmichaeli share a common C19norditerpenoid skeleton.Therefore,from a group of compounds with similar chem-ical structures(which is common for active compounds from herbal medicine),each of them may have diverse biological activities and some of the functions are shared and others are speci?c to a given structure.In the best situation,the mutual functions are therapeu-tically related,and the additional biological functions derived from speci?c compounds are connected to adverse or toxic effects.The combined usage of these chemically similar compounds results in an accumulated therapeutic effect,while the adverse effects are reduced resulting in safer medicine.In the case of the Aconitum alkaloids the reduction or complete elimination of aconitine,hypa-conitine and mesaconitine is useful which was commonly used by processing and formulation in traditional use of CMM.According to the actual effect of the processed aconite there is less decrease in analgesic activity but there is a decrease in toxicity after pro-cessing(Liou et al.,2005).When the therapeutic effects are derived from a speci?c function related to a certain compound,the Western medicine approach is to purify the active compound,which is nor-mally the method new drugs from herb or other natural sources are found.To decipher the mechanism of an ancient formulae used in CM practice;it is possible to elucidate the composite formulae with the aim to control toxicity or increase the ef?cacy.The prescription or Fu-Fang(multiple herbal formulas)may contain antagonistic or synergistic CMM.In the latter case,the dose of the toxic herb can be reduced.

7.Conclusion

In the present review,the safety of CMM and CMM products, and factors modulating their toxicity has been considered.These factors exert effects on the?nal reactions of the body to the herbal medicines because of the many compounds within multi-herbal preparations or in single herbal preparations.The59toxic herbs, which were identi?ed and discussed shows that toxicological infor-mation about these CMM is limited.The alkaloids and terpenes were the two major classes of toxins.The advances of analytical technology have also provided better methodology for the qual-ity control of complex CMM.The new approaches outdate when applicable the commonly used measurement of some typical or major compounds only within one speci?c species.It was shown that the processing of the CMMs and the use of herbal mixtures in formulas are both important in toxicity assessment and may open up new approaches for toxicity control.For Aconitum roots it was shown how biochemical and bio-statistical analysis provides detailed information about toxicity and suggesting hints about the responsible molecular mechanisms.The present review proposes a systems biology approach for screening extracts of CMM indi-vidually or in combinations to detect biomarkers for bioactivity in cell lines,animal models,and subsequently in human volunteers via clinical studies.The data obtained can be linked and analyzed by means of megavariate data analysis.The latter is a powerful technique that analyses data sets with a large number of vari-ables and correlates for instance metabolomics?ngerprints with a certain phenotype of bioactivity,toxicity or ef?https://www.360docs.net/doc/d310651945.html,ing these biomarkers or biomarker?ngerprints,it is possible to offer better quality assurance for herbal formulations,to relate bioactivity in animal models and human subjects and to link CMM compositions to bioactivity and to optimize CMM formulae to identify toxicity of certain formulations(Wang et al.,2005a).This concept is illus-trated in Fig.6,a system has been developed and patented(Wang et al.,2005b)based on monitoring biological effects with a biomarker pro?le,enabling opportunities to not only discover effects or pro-vide claim support but especially to detect synergetic effects,which are the main principles in herbal medicine.

Finally it is mandatory to understand that Chinese herbal medicine should be investigated and evaluated understanding the principles by which it has been developed.This implies that for-mulas

and not constituents much be researched alone,one cannot investigate building blocks and extrapolate the information to the whole as at each level of complexity new emerging properties are formed.Moreover the realization that practice-based knowl-edge is essential also in Western medicine for?nal evaluation of the risks and bene?ts of a patient,but that scienti?c evidence is

Fig.6.The concept of linking biological activities with multi-component herbal mixture by using bio-statistics.

40J.Wang et al./Journal of Ethnopharmacology126 (2009) 31–41

mandatory for a better guidance and understanding of the future of our medicine provides a balanced view and opening to fuse the knowledge from the science evolved from different philosophical backgrounds.In Western medical science we follow the route form data to information to knowledge and?nally to wisdom,today we have the chance to explore the reversed-route starting out with wisdom and trying to discover the underlying mechanism-based principles.

Acknowledgements

We are grateful for Dr.Suzan Wopereis for her kind help in con-ducting the Metacore and Metadrug analysis,Dr.Andre Schram for valuable discussion.The Sino-Dutch center for Preventive and Per-sonalized Medicine is jointly supported by the China International Science and Technology Cooperation Program(2007DFA31060)and National Key Technologies R&D Program(2006BAI11B07)from the Ministry of Science and Technology of China,the Netherlands Genomics Initiative(NGI),the Netherlands Metabolomics Center and TNO.

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