Insights from molecular investigations of traditional Chinese herbal stroke medicines: Implications for neuroprotective epilepsy therapy

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Epilepsy & Behavior 8 (2006) 350–362

Review

Insights from molecular investigations of traditional Chineseherbal stroke medicines: Implications for neuroprotective

epilepsy therapy

Nikolaus J. Sucher *

Division of Neuroscience, Department of Neurology, Children’s Hospital and Harvard Medical School, Boston, MA, USA

Received 21 November 2005; accepted 25 November 2005

Abstract

Traditional Chinese herbal medicine is the most widely practiced form of herbalism worldwide. It is based on a sophisticated system ofmedical theory and practice that is distinctly different from orthodox Western scientific medicine. Most traditional therapeutic formu-lations consist of a combination of several drugs. The combination of multiple drugs is thought to maximize therapeutic efficacy by facil-itating synergistic actions and ameliorating or preventing potential adverse effects while at the same time aiming at multiple targets.Orthodox drug therapy has been subject to critical analysis by the ‘‘evidence-based medicine’’ movement, and demands have been madethat herbal medicine should be subject to the same kind of scrutiny. However, evaluation of the effectiveness of herbal medicines can bechallenging, as their active components are often not known. Accordingly, it may be difficult to ensure that an herbal preparation used inclinical trials contains the components underlying its purported therapeutic effect. We reasoned that the identification of actions of herbalmedicines at well-defined molecular targets and subsequent identification of chemical compounds underlying these molecular effectsmight serve as surrogate markers in the hypothesis-guided evaluation of their therapeutic efficacy. A research program was initiatedto characterize in vitro molecular actions of a collection of 58 traditional Chinese drugs that are often used for the treatment of stroke.The results indicate that these drugs possess activity at disparate molecular targets in the signaling pathways involved in N-methyl-D-aspartate (NMDA) receptor-mediated neuronal injury and death. Each herbal drug contains diverse families of chemical compounds,where each family comprises structurally related members that act with low affinity at multiple molecular targets. The data appear tosupport the multicomponent, multitarget approach of traditional Chinese medicine. Glutamate release and excessive stimulation ofNMDA receptors cause status epilepticus-induced neuronal death and are involved in epileptogenesis. Therefore, these results are alsorelevant to the development of antiepileptogenic and neuroprotective therapy for seizures. The combination of principles of modernmolecular medicine with certain ideas of traditional empirical Chinese medicine may be beneficial in translational medicine in general.� 2005 Elsevier Inc. All rights reserved.

Keywords: Traditional Chinese medicine; Translational medicine; N-methyl-D-aspartate receptor; Patch clamp; Excitotoxicity; Flavonoids; Magnesium;PDZ domain; Epilepsy; Stroke

1. Introduction

An increasing number of patients and medical practitio-ners in the industrialized world use herbal medicines as asupplement to or substitute for prescription drugs [1,2].Herbal medicines are often considered to be a gentle and

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safe alternative to synthetically manufactured drugs. Inter-estingly, more than half of the medically important phar-maceutical drugs are either natural products orderivatives of natural products [3–5]. At the same time,most of the world’s population living in developing coun-tries depends on herbal medicine as the primary source ofhealth care. In many cases, the use of herbal drugs andbelief in their effectiveness are rationalized based on theirlongstanding use as folk medicines. In this context, increas-ing interest has been aroused in Western countries in the

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N.J. Sucher / Epilepsy & Behavior 8 (2006) 350–362 351

practice, philosophy, and science of traditional medicine inChina [6]. Traditional Chinese herbal medicine is the mostwidely practiced form of herbalism worldwide. It is basedon a sophisticated system of medical theory and practicethat is distinctly different from orthodox Western scientificmedicine. The increasing popularity of ‘‘alternative’’ formsof therapy has led to demands that herbal medicine be sub-ject to scientific scrutiny [7,8]. Orthodox ‘‘scientific’’ medi-cal practice itself has been subjected to critical analysisrecently by the so-called ‘‘evidence-based medicine’’ move-ment. Patients may simply want to know whether herbaltherapy works and whether it is safe to use. Cliniciansand scientists may also want to understand the underlyingmechanism. Unfortunately, however, the answers to thesequestions are not straightforward. They often depend onwho is answering, what particular type of inquiry is con-ducted, and what endpoint is accepted [9]. Although ithas been pointed out previously that there is no essentialdifference in the research methods that can be used to testbotanical or pharmaceutical effectiveness [7], evaluation ofthe effectiveness of herbal medicines in practice can be verydifficult, as often neither their biological targets nor thechemical compounds acting at these targets are known[8,10]. The biologically active compounds are mostly sec-ondary metabolites, which plants and microorganisms pro-duce in response to specific challenges and insults, forexample, in defense against herbivores and pathogensand as signaling compounds to increase their chance ofreproduction [11]. Accordingly, it is not easy to ensure thata given herbal preparation used in an in vivo experiment orcontrolled clinical trial actually contains the active ingredi-ent(s) underlying its purported therapeutic effect.

In an attempt to address some of these issues, a fewyears ago we initiated a research program aimed at thecharacterization of molecular targets and effects of herbalmedicines. We reasoned that the identification of effectsof herbal medicines at well-defined molecular targets andsubsequent identification of chemical compounds underly-ing or contributing to these effects might be useful for thehypothesis-guided evaluation of their therapeutic efficacy.For example, an herbal drug’s activity in certain highly spe-cific molecular assays might be used as a means of initialquality control. A drug’s activity in such assays and the tis-sue concentration in animals or patients of previously iden-tified compounds mediating these effects may then bemonitored and correlated with its experimentally or clini-cally observed efficacy. The availability of such ‘‘surrogate’’markers will allow investigation of whether a given molec-ular activity is necessary and/or sufficient for its therapeuticeffects and whether drug mixtures have additive or syner-gistic effects.

In this research, we chose to investigate Chinese herbalmedicines that are often used for the treatment of acutestroke [12]. Despite tremendous progress in molecular neu-roscience over the last 25 years or so, there is a great needfor the development of pharmacological stroke treatments.To date, there are no successful therapeutic interventions

available for the majority of stroke patients [13–15]. Intra-venous thrombolytic therapy with recombinant tissue plas-minogen activator (rtPA), a major milestone in thedevelopment of effective treatments for ischemic stroke,can be used only within 3 hours after the onset of ischemia,and all patients with hemorrhagic stroke must be excluded.These requirements limit rtPA therapy to only 1 to 2% ofall stroke patients [16]. At the same time, the aging of thegeneral population in many countries, including the UnitedStates, is contributing to an increase in the number ofstroke patients.

Traditional Chinese medicine (TCM) stroke therapy hasa long history. The famous Chinese physician ZhangZhongjing described the symptoms of acute stroke about2000 years ago. In 1995, the State Administration ofTCM of the People’s Republic of China issued standardsfor the diagnosis of stroke and the evaluation of the effica-cy of treatments [17]. Many Chinese medicine practitionersfirmly express the conviction that herbal therapy is effectivein stroke patients, greatly reducing the occurrence of a last-ing handicap. The therapeutic efficacy of these traditionalChinese stroke drugs appears to be supported by recentclinical studies [18].

Here, I present an introduction to the principles of Chi-nese herbal medicine and then review the design and resultsof our studies, and provide some perspective on lessonslearned from this work. Many aspects of this work maybe of interest to workers in epilepsy, as mounting evidenceindicates that the pathways involved in neuronal injury anddeath in stroke are involved in status epilepticus-inducedneuronal death [19] and at least some of the same pathwaysmight play a role in epileptogenesis [20–24]. An introduc-tion to traditional Chinese stroke therapy can be foundin Gong and Sucher [12]. An excellent source of detailedand authoritative information regarding the theory andpractice of TCM has been published [25].

2. Traditional Chinese medicine

A system of medical thought and practice that is dis-tinctly different from Western medicine has been developedand used in China for thousands of years [25]. The theoryand practice of this unique form of medicine are an integralpart of Chinese culture where it is accordingly simplyreferred to as Chinese medicine (zhong yi in Chinese), asopposed to Western medicine (xi yi). The main principlesof Chinese medicine were first published in the textbookHuang di nei jing some 2500 years ago [26], although somebasic ideas may date back to the origins of Chinese civiliza-tion some 5000 years ago.

In recent years, a drive to ‘‘modernize’’ this ancient formof medicine in China has been gaining momentum [27,28].In this context, it is now promoted worldwide as traditionalChinese medicine and often referred to using the acronymTCM. The modernization of TCM began as a government-sanctioned program in the 1950s following the establish-ment of modern China in 1949 and has accelerated since

352 N.J. Sucher / Epilepsy & Behavior 8 (2006) 350–362

Deng Xiao Ping instituted the reform and opening policiesthat have been transforming China in the past two decades.The modernization program encompasses the introductionof a legal framework for the regulation of TCM, the crea-tion of government-run TCM hospitals, the establishmentof TCM universities, the protection and cultivation of nat-ural resources, the reformation of the production and dis-tribution of medicines based on the traditional materiamedica, and increased support for basic and clinicalTCM research [29]. Although the motivations and stakesof various parties involved in the modernization of TCMvary widely, it is commonly acknowledged that TCM holdsthe promise of considerable economic rewards if it can gainrecognition as an officially sanctioned complementary andalternative mode of therapy in the industrialized nations.At the same time, however, some practitioners of Chinesemedicine are weary of the destruction of its philosophicalcore at the hands of regulators [27].

One of the defining principles of Chinese medicine is itsgeneral view of the body as an integral whole that is closelyconnected with the surrounding outside world. The organsinside of the body are themselves interconnected via aninterlacing network of ‘‘channels’’ and ‘‘collaterals.’’ Dis-eases can be caused by endogenous as well as exogenousfactors. Chinese medicine emphasizes prevention of diseasethrough a healthy lifestyle.

The diagnosis in Chinese medicine is ‘‘syndrome’’based. A particular syndrome (zheng) is characterizedby a patient’s signs and symptoms, the location of theunderlying ‘‘lesion,’’ its etiology, and the specific rela-tionship between the patient’s resistance and the patho-logical agents [25]. Thus, a syndrome is the concrete(physical) manifestation of a fundamental pathologicalprocess convoluted with incidental aspects (e.g., thepatient’s physical and psychological constitution, thephysical and social environment). As such, these ‘‘syn-dromes’’ normally do not, in general, correspond to thedisease classifications of Western scientific medicine.Moreover, Chinese medicine is ‘‘personalized’’ medicinein the sense that each patient is treated with a specificbut varying combination of drugs chosen on the basisof the particular presenting syndrome (symptom com-plex) observed at a given point in time, rather than anosologically defined disease.

Chinese medicine practitioners use mainly three thera-peutic modalities: acupuncture [30], moxibustion [25], andherbal medicine [31]. Acupuncture has gained wide popu-larity in Western countries. In this therapeutic procedure,stainless-steel needles are inserted into specific acupuncturepoints over the entire body based on the underlying pathol-ogy. Moxibustion is much less well known in the West,although historically it predates the development of acu-puncture. It involves the burning of dried bundles of mug-wort (Artemisia vulgaris) over certain regions (includingacupuncture points) of the body to stimulate the flow ofblood and qi. By far the oldest, most commonly practicedand important form of therapy in Chinese medicine,

however, is pharmacotherapy using drugs derived fromplant, animal, and mineral sources [25].

3. The Chinese materia medica and traditional

pharmacotherapy

Herbal pharmacotherapy is as old as Chinese medicineitself [32]. The Chinese materia medica has grown substan-tially over the last two millennia from a few hundred tomore than 10,000 animal, mineral, and plant sources ofdrugs. For example, an authoritative 10-volume compendi-um of the Chinese materia medica lists more than 11,228plant species, 1150 animal species (including human), and74 minerals as sources of drugs with indications for theiruse [33]. The official pharmacopoeia of the People’s Repub-lic of China lists information on some 530 traditional drugsincluding some 460 herbal drugs, more than 40 drugs ofanimal origin, human hair, and placenta, and some 30 min-erals [34]. Individual herbal drugs can be derived frommore than one species of a specific genus. For example,the drug Ramulus Uncariae cum uncis (Gouteng) can bederived from Uncaria rhynchophylla, U. macrophylla,U. sessilifrucus, U. hirsute, and U. sinensis, four species ofthe gambir plant, a woody vine with hooklike thorns thatbelongs to the family Rubiaceae (madder family) [34].There are 34 Uncaria species worldwide, 13 of which arefound in China [35,36].

The drugs that a patient takes home from the pharmacybased on a doctor’s prescription are not ‘‘fresh,’’ but ‘‘pro-cessed.’’ Herbs and other natural materials become drugsonly after processing. The specific processing proceduresplay an important role in Chinese medicine [25]. Processingcan prevent or abate adverse effects of some drugs thatwould be toxic in unprocessed form. It can enhance orreduce the potency of drugs and even change their thera-peutic effect. Processing is also used to make drugs easierto store and prepare and to make them more palatableand more convenient to take for the patient. Methods ofprocessing include cutting, cleaning, rinsing, grounding,softening, parching (baking until the drugs change colorto yellow or brown or become charred), roasting, steaming,and boiling [25].

Most prescriptions consist of a combination of severaldrugs. In fact, combination therapy is a fundamental prin-ciple of Chinese medicine. The combination of multipledrugs in complex formulations (and the presence of multi-ple active compounds even in single herbs) is thought tomaximize therapeutic efficacy by facilitating synergisticactions (mutual reinforcement) of the drugs and ameliorat-ing (mutual restraint) or preventing (mutual detoxification)a drug’s potential adverse effects at the same time as target-ing one or several pathophysiological mechanisms (mutualassistance) [12]. Some drugs may be weakened (mutualinhibition) in their therapeutic effect when they are com-bined; others may become outright poisonous or cause seri-ous adverse effects in combination, which must be avoided[25].


There are some 230 well-known medicinal formulas inChinese herbal medicine that contain from 1 to 17 differentdrugs (Fig. 1). They are often classified into 22 groups fol-lowing a scheme first proposed in the book Yi fang kao

published at the end of the 16th century [25]. For example,one class of prescriptions is used to treat wind syndromes.Wind syndromes can be due to exogenous or endogenouswind. The prescriptions used to treat wind syndromes acteither to dispel exogenous wind or to calm endogenouswind. Stroke and certain forms of seizures belong to thewind-related syndromes. The composition of each formulahas been determined largely empirically, taking intoaccount the experience gained by observing the effects ofsingle herbs. Each formula, however, is thought to conformto the ancient principle known as jun chen zuo shi. Jun (rul-er or monarch in English) corresponds to the principalingredient that is directed at the main cause and/or symp-toms of the disease. Chen (minister, official) designatesdrugs that are directed at both the underlying cause(s) ofthe disease and the accompanying symptoms and compli-cations. Other drugs ‘‘assist’’ (zuo) the leading drugs inachieving their curative effects by treating any secondarysymptoms of the disease and by counteracting any poten-tial adverse effects of the primary drugs. Finally, the shi

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Fig. 1. Quantitative analysis of traditional Chinese formulations andpatent medicines. Top: Frequency distribution of the number of drugs in233 well-known formulations: ‘‘How many different drugs are combined inan average formulation?’’ The median is 6 drugs. The mean is 6.6 ± 3.5drugs per formulation. Inset: Frequency distribution for 319 Chinesepatent medicines. The median is 7. The mean is 8.3 ± 6.6. Bottom:Frequency distribution of the number of formulations containing aspecific drug: ‘‘How many formulations contain a specific drug?’’ Themedian for each drug is 1; i.e., half of 410 drugs are present in only oneformulation. The mean is 3.6 ± 6.1 formulations. Inset: Distribution for880 drugs used in patent medicines. The median is 1 formulation. Themean is 4.2 ± 8.5 formulations.

(enabling) drugs direct the action of all other drugs intothe right ‘‘channels’’ and ensure, together, that these drugsdo not exceed the patient’s capacity to cope with theiraction. The practical realization of these principles isrevealed by a quantitative analysis of the composition ofwell-known traditional formulations (Fig. 1). On average,each traditional formulation contains 7 ± 3 of more than400 different drugs. More than half of these drugs areunique to a particular prescription; the remaining drugsare used in more than one formulation (7 ± 8, n = 190).A similar pattern is observed in patent medicines (Fig. 1).

Following the doctor’s instructions, patients preparetheir medicines at home as a decoction by first soakingand then boiling the drugs in water. The patient drinksthe decoction warm. Its often bitter taste can make itsingestion an exercise in self-control. Some drugs, e.g., lico-rice (Gancao), are specifically added to prescriptions tomake them more palatable.

4. Rationale for molecular studies of traditional Chinese

herbal medicines

Our molecular studies were initially motivated by thenotion that the practical experience with traditionalChinese herbal therapy of stroke might provide a basisfor the discovery of natural products that could serve asleads for the development of effective and safe neuroprotec-tive drugs [12,37]. The distinguishing characteristic of thisdrug discovery program was its focus on a small numberof herbal drugs that were selected based on their estab-lished use in traditional stroke therapy as documented inboth historical and modern Chinese medical and scientificliterature (reviewed in Refs. [12,37]).

Databases of the scientific literature in China containtens of thousands of articles relevant to traditional Chinesemedicine [38]. The overwhelming majority of articles are,not surprisingly, written in Chinese, although some provideEnglish abstracts. A large number of articles are devoted tothe phytochemistry of Chinese medicinal plants. Tens ofthousands of different compounds have been isolated fromthese plants, and not all these compounds are (well) knownin the West. There is also a considerable body of work ded-icated to investigations of the biological effects of crudedrugs and isolated components. Last but not least, thereare many articles devoted to the clinically observed thera-peutic effects of traditional Chinese drugs. The design,analysis, and interpretation of many clinical investigationsof traditional medicines in the Chinese literature appear tobe perplexing and surprising [39]. It is often difficult toevaluate the results as evidence of the efficacy of TCM ther-apy [40]. Nonetheless, literature searches can help identifytraditional Chinese medicines with documented clinicaleffects, which can than be tested selectively in relevantmolecular assays. One difficulty in the evaluation of clinicalarticles is the fact that there is normally no ‘‘one-to-one’’correspondence between the traditional syndrome-baseddiagnosis and the Western disease-based diagnosis. For


example, there is no term that corresponds directly to epi-lepsy. Although the ancient TCM literature contains pre-cise descriptions of acute events that can be clearlyidentified as seizures (referred to as xian), seizure-likesymptoms are sometimes interpreted as signs of psychosis(dian or kuang) [41]. In our work, the search was facilitatedby the fact that there exists a TCM syndrome called windstroke (zhong feng) that can be considered synonymouswith stroke.

The choice of molecular assays was based on currentunderstanding of the pathophysiology of neuronal injuryduring the reperfusion phase subsequent to acute ischemicstroke [42]. Over the past decade, many of the mechanismsinvolved in the cascade of events leading to neuronal injuryand death in acute stroke and status epilepticus have beendiscovered, suggesting multiple well-defined molecular tar-gets for therapeutic intervention [16,19,42–45]. A great dealof attention has focused on five key mechanisms: (1) activa-tion of receptors for excitatory amino acids, (2) calciuminflux, (3) generation of free radicals (e.g., nitric oxide),(4) a form of programmed cell death similar to apoptosis,and (5) inflammation. Release of the excitatory amino acidglutamate, the predominant excitatory neurotransmitter inthe brain, and subsequent activation of N-methyl-D-aspar-tate (NMDA) receptors (NMDARs) are the key eventstriggering an increase in intracellular calcium and genera-tion of free radicals leading to either fulminant cell death,necrosis, or a delayed form of cell death, apoptosis[42,44–47].

NMDARs are a subclass of ionotropic glutamate recep-tors that are normally involved in neuronal differentiation,migration, synapse formation, and the shaping of axonaloutgrowth patterns during the development of the centralnervous system [48]. Stimulation of NMDARs is thoughtto be a necessary condition for the generation of someforms of memory. Excessive activation of NMDARs, how-ever, is thought to be an important step in a common path-way leading to degeneration of neurons in stroke andprolonged seizures (e.g., during status epilepticus) and anumber of other acute and chronic brain diseases such asHuntington’s disease, Alzheimer’s disease, and amyotro-phic lateral sclerosis [19,45]. Too little NMDAR activity,in contrast, is thought to be the basis of some of the devas-tating symptoms of schizophrenia [49]. Disappointingly,however, NMDAR antagonists, a class of drugs with prov-en neuroprotective activity in animal stroke models, eitherfailed or had to be withdrawn from clinical testing due tounacceptable adverse effects in patients [14,50].

Although a number of factors contributed to the appar-ent clinical failure of the NMDAR antagonist drugs[14,51], the disappointment has been such that some haveconcluded that the concept of NMDAR antagonism isintrinsically not a valid therapeutic approach [52]. Ironical-ly, there is now good evidence that the therapeutic effects ofthe drug memantine, which has been used for the treatmentof Parkinson’s disease for about two decades in Europe,are most likely due to its NMDAR antagonist activity

(for a review, see [52]). Furthermore, accumulating evi-dence indicates that synaptic and extrasynaptic NMDARsdiffer in their subunit composition and their respective con-tribution to excitotoxicity [53]. Therefore, giving up onNMDA antagonists may have been premature. NMDARsmight still prove to be a valid target for neuroprotectivetherapy provided that drugs can be developed that are spe-cific for certain NMDAR subtypes or that only reduceNMDAR activity without shutting it off completely [54].

We reasoned that if NMDAR antagonism was a mech-anism of action of traditional Chinese drugs used for thetreatment of acute stroke, then those drugs and, by impli-cation, potentially single active compounds might possesssuch an advantageous pharmacodynamic profile. We iden-tified 58 traditional medicines that are commonly used inprescriptions for therapy of the acute phase of stroke(Table 1), and we set out to identify the action of thesedrugs on the NMDAR and NMDAR-linked downstreampathways leading to neuronal injury and death [55]. Wefurthermore purified single chemical compounds from tra-ditional Chinese drugs interacting with these targets.

5. The NMDAR as a target of traditional Chinese medicines

We screened aqueous extracts of traditional Chinesedrugs for their ability to abate NMDA-induced neurotox-icity in primary cultures of mouse cortical neurons. In par-allel with the cytotoxicity experiments, we applied thewhole-cell patch-clamp technique in mouse cortical neuro-nal cultures to screen for NMDAR antagonist activity inthe extracts [56]. Data from patch-clamp experiments usingTCM extracts revealed that aqueous extracts of RadixScutellariae baicalensis (Huangqin), Radix Stephaniaetetrandrae (Fangji), Radix Salviae miltiorrhizae (Danshen),and Ramulus Uncariae cum uncis (Gouteng) partiallyblocked NMDA-evoked currents. Blockade of theNMDA-evoked currents in the presence of the extracts ofS. baicalensis, S. tetrandra, and S. miltiorrhiza was volt-age-dependent and showed a negative slope conductancereminiscent of the effect of Mg2+ ions, an NMDAR chan-nel blocker. Indeed, atomic absorption spectrophotometryrevealed the presence of free and chelated Mg2+ in theseextracts. We then demonstrated that removal by ion-ex-change chromatography of the free Mg2+ from the aqueousDanshen extract abolished the previously observed block-ade of the NMDA-induced currents [57].

In contrast, the Uncaria extract blocked NMDA-evokedcurrents even at depolarized potentials and abrogatedNMDA-induced neuronal death. Gouteng is known forits antispasmodic, sedative, anticonvulsive, and antihyper-tensive properties and has been used in TCM for the treat-ment of asthma, epilepsy, and cardio- and cerebrovasculardisorders including ischemic and hemorrhagic stroke [58].In Japan, U. sinensis is the main medicinal plant ofChoto-san, a decoction that was shown to be effective inthe treatment of patients with vascular dementia [59]. Con-sistent with our findings, Lee and colleagues reported that a

Table 1Traditional Chinese medicines used in stroke therapy

Medicinal name Scientific name Pinyin/Chinese name

Aloe Aloe vera Luhui

Bombyx batryticatus Bombyx mori Jiangcan

Bulbus Fritillariae thunbergii Fritillaria thunbergii Zhebeimu

Caulis Bambusae in taeniam Bambusa tuldoides Zhuru

Caulis Polygoni multiflori Polygonum multiflorum Shouwuteng

Cornu Saigae tataricae Saiga tatarica Lingyangjiao

Flos Albiziae Albizia julibrissin Hehuanhua

Flos Carthami Carthamus tinctorius Honghua

Flos Chrysanthemi indici Chrysanthemum indicum Yejuhua

Flos Daturae Datura metel Yangjinhua

Folium Apocyni veneti Apocynum apocyni veneti Luobuma

Fructus Forsythiae Forsythia suspensa Lianqiao

Fructus Tribuli Tribulus terrestris Jili

Herba Ajugae AJuga decumbens Jingucao

Herba Asari Asarum heterotropoides Xixin

Herba Asari Asarum heterotropoides var. mandshuricum Xixin

Herba Centellae Centella asiatica Jixuecao

Herba Ephedrae Ephedra sinica. Mahuang

Herba Equisetihiemalis Equisetum hiemale Muzei

Herba Erigeron Erigeron breviscapus Dengyexixin

Herba Leonuri Leonurus heterophyllus Yimucao

Herba Menthae Mentha haplocalyx Bohe

Lasiosphaera seu Calvatia Lasiosphaera fenzlii Mabo

Lumbricus Pheretima asiatica Dilong

Periostracum cicadae Cryptotympana pustulata Chantui

Poria Poria cocos Fuling

Radix Acanthopanacis senticosi Acanthopanax senticosus Ciwujia

Radix Achyranthis bidentatae Achyranthues bidentata Niuxi

Radix Angelicae pubescentis Angelica pubesens Duhuo

Radix Bupleuri chinensis Bupleurum chinense Chaihu

Radix Gentianae macrophyllae Gentiana macrophylla Qinjiao

Radix Ledebouriellae Ledebouriella divaricata Fangfeng

Radix Paeoniae rubra Paeonia lactiflora Chishao

Radix Polygalae Polygala tenuifolia Yuanzhi

Radix Puerariae Pureraria lobata Gegen

Radix Rehmanniae Rehmannia glutinosa Dihuang

Radix Salviae miltiorrhizae Salvia miltiorrhiza Danshen

Radix Scutellariae Scutellaria baicalensis Huangqin

Radix Stephaniae tetrandrae Stephania tetrandra Fangji

Ramulus Mori Morus alba Sangzhi

Ramulus Uncariae cum uncis Uncaria rhynchophylla Gouteng

Rhizoma Acori tatarinowii Acorus tatarinowii Shichangpu

Rhizoma Arisaematis Arisaema erubescens Tiannanxing

Rhizoma Cimicifugae foetidae Cimicifuga goetida Shengma

Rhizoma Gastrodiae Gastrodia elata Tianma

Rhizoma Ligustici Ligusticum chuanxiong Chuanxiong

Rhizoma Notopterygii Notoptergium incisium Qianghuo

Rhizoma Paridis Paris polyphylla var. yunnanensis Chonglou

Rhizoma Pinelliae Pinellia ternata Banxia

Rhizoma Sparganii Sparganium stoloniferum Sanling

Rhizoma Typhonii Typhonium giganteum Baifuzi

Romulus Loranthis Loranthus parasiticus Sangjisheng

Scolopendra Scolopendra subspinipes Wugong

Scorpio Buthus martensi Quanxie

Semen Persicae Prunus Persica Taoren

Semen Plalycladi Platycladus orientalis Baiziren

Semen Xanthi sibricum Xanthium sibiricum Cangerzi

Semen Ziziphi spinosae Ziziphus jujuba var. spinosa Suanzaoren


methanol extract of U. rhynchophylla reduced NMDA-mediated excitotoxicity in cultured hippocampal slicesand blocked NMDA-induced whole-cell currents in acutelydissociated hippocampal neurons [60].

To identify the chemical compounds underlying theobserved NMDAR antagonist activity of the aqueousUncaria extract, we employed two approaches. In thefirst approach, we tested the NMDAR antagonist


activity of several chemical compounds that were previ-ously isolated from Uncaria species [36,61]. These com-pounds were the alkaloids rhynchophylline andisorhynchophylline and the phenolic compounds (flavo-noids) catechin, epicatechin, hyperin, and caffeic acid,which had been shown previously to protect culturedcerebellar granule cells from glutamate-induced death[62,63]. Rhynchophylline and isorhynchophylline wereshown to be low-affinity noncompetitive NMDARantagonists [64]. Although both rhynchophylline andisorhynchophylline blocked NMDA-induced whole-cellcurrents to some degree, only isorhynchophylline(100 lM) significantly reduced NMDA-induced celldeath under our experimental conditions. The phenoliccompounds reduced neither NMDA-induced cell deathnor NMDA-induced whole-cell currents (Sun andSucher, unpublished observations).

In the second approach, we undertook the bioactiv-ity-guided isolation of the active principle from theextract. We used the whole-cell patch-clamp techniqueto record NMDA-induced currents and NMDA-in-duced cytotoxicity in cultured mouse cortical neuronsas bioassays during the fractionation of Gouteng. Thecytotoxicity data were concordant with the patch-clampdata at all stages of the fractionation; i.e., there was alinear correlation between the observed inhibition ofNMDA-induced whole-cell currents by a certain frac-tion and its protective effect against NMDA-inducedcell death. Some fractions that had no effect onNMDA-induced currents proved to be toxic to the cul-tures, in contrast to whole Gouteng extract. Surprising-ly, however, the bioactivity-guided purification of theNMDAR antagonist from Gouteng (5 kg) yielded noalkaloids, but did yield the surfactant sodium dodecylsulfate (SDS; �30 mg). The compound reversiblyblocked NMDA-induced currents in a dose-dependentfashion and reduced NMDA-induced toxicity in mousecortical neurons (24.6 ± 11.2% compared with control,P < 0.01) [55]. SDS is a widely used synthetic anionicsurfactant that can act as a wetting agent by loweringthe surface tension of aqueous solutions. SDS is usedin detergents, shampoos, creams, lotions, medical prep-arations, toothpaste, and even some foods. SDS andrelated anionic surfactants are high-production-volumechemicals in Europe and the United States and arecommon environmental pollutants worldwide. It isnot known whether SDS in Gouteng from Jiangsuprovince was derived from an exogenous source, suchas environmental pollution or contamination duringhandling and processing (possibly in conjunction withthe plant’s inability to metabolize the compound), orfrom an as yet undiscovered endogenous syntheticpathway. In any case, it will be interesting to furtherinvestigate the molecular action of this drug at theNMDA receptor and evaluate the therapeutic potentialof SDS or SDS derivatives with NMDAR antagonistactivity.

6. Protein interaction domains as targets of traditional

Chinese medicines

Functional NMDARs are composed of an essentialNR1 subunit, one or more NR2 subunits (NR2A-D),and, in some cases, additional NR3 subunits (NR3A-B)[48]. Individual NR subunits, in turn, associate with addi-tional proteins to form a large, dynamic, postsynapticreceptor complex [65]. For example, it has been shownthat neuronal nitric oxide synthase (nNOS), the enzymeresponsible for the generation of nitric oxide, is locatedclose to the NMDAR complex through its interactionwith one of multiple postsynaptic density 95(PSD-95)/disclarge/zonula occludens-1 (PDZ) domains of PSD-95,which in turn binds NR2 subunits through another ofits PDZ domains. nNOS is activated by an increase inthe local concentration of calcium, which enters the cellthrough the ion channel that is part of NMDAR, result-ing in an increase in the production of nitric oxide (NO).NO can, in turn, act as a neurotoxin when it combineswith superoxide, resulting in the formation of peroxyni-trite (ONOO) [66].

It was previously shown that deletion of PSD-95 dis-sociates NMDAR activity from NO production, sup-pressing excitotoxicity but leaving NMDAR activityunaffected [67,68]. Thus, disruption of the interactionbetween nNOS and PSD-95 appears to represent apotential therapeutic approach aimed at abatingNMDAR-mediated neurotoxicity [68]. Along these lines,we screened the original 22 traditional Chinese medicinalherbs based on the hypothesis that they might containsmall compounds that might be capable of binding tothe second PDZ domain of PSD-95, thereby disruptingits interaction with nNOS [69]. Such compounds mightbe useful in the further development of therapeutic strat-egies based on the disruption of PDZ-based interactionsof proteins [70].

We performed nuclear magnetic resonance (NMR)-based chemical shift perturbation experiments to monitorthe binding of compounds from herbal extracts to 15N-la-beled PDZ2 of PSD-95. The exquisite sensitivity of bind-ing-induced chemical shift changes of PDZ2 of PSD-95allowed us to discover PDZ-binding components in aque-ous herbal extracts by simply comparing the 1H–15N spec-tra of extract-added PDZ2 with that of free PDZ2. Usingthis approach, we found that the aqueous extract of RadixScutellariae induced significant chemical shift changes inPSD-95 PDZ2. The NMR titration experiment demon-strated that the compounds specifically bound to thenNOS/NR2B binding pocket of PDZ2. Four flavones—baicalin, norwogonoside, oroxylin A-glucuronide (alsoknown as oroxyloside), and wogonoside—were isolatedand found to account for the PDZ binding activity of theextract. Interestingly, however, the structural perturbationof PDZ2 by any single compound was considerably lessthan that observed with the crude extract, even when itwas added at 10-fold molar excess [69].

Fig. 2. Formulation of Tianma Gouteng Yin (see also Table 2). ConchaHaliotidis (1), Ramulus Uncariae cum uncis (2), Herba Taxilli (3), CaulisPolygoni multiflori (4), Radix Scutellariae (5), Radix Achyranthis biden-tatae (6), Fructus Gardeniae (7), Rhizoma Gastrodiae (8), Poria cum lignohospite (9), Cortex Eucommiae (10), and Herba Leonuri (11).


These data describe the effect of flavonoids at a novelmolecular target, adding to the long list of biologicalactions of these phenolic compounds [71]. Thus, flavonoidsexemplify how members of a specific family of phytochemi-cals can interact with multiple targets. For example, baica-lin and baicalein might reduce the production of nitricoxide via its PDZ binding action, while at the same timescavenging superoxide radicals, which would prevent theformation of highly toxic peroxynitrite from the reactionof nitric oxide with superoxide [72,73]. A plethora of otherpreviously described molecular effects of these flavonoidsmight further contribute to their reported neuroprotectiveeffects [72–75].

7. Caspases as targets of traditional Chinese stroke drugs

Several lines of evidence indicate that the demise ofneurons during the reperfusion phase of stroke is theresult of activation of cell death programs such as apopto-sis [47,76]. Apoptosis is mediated by activation of specificmembers of the family of cysteinyl aspartate-specific pro-teinases termed caspases. A number of in vivo studies havedemonstrated the activation of caspases during cerebralischemia and infarction [77–83]. Proapoptotic stimuli firstlead to the activation of so-called initiator caspases suchas caspase-8, which in turn cleave inactive proenzymeforms of so-called effector caspases such as caspase-3and caspase-7. Once active, the effector caspases cleave alarge number of cellular proteins, leading to the ‘‘clean’’disintegration of affected cells. Caspase-3-deficient micewere found to be more resistant to ischemic stress andshowed a significant reduction in stroke volume [84]. Cur-rent research is aimed at the development of specific cas-pase inhibitors as therapeutic agents [85].

We investigated whether or not traditional Chinesestroke drugs might possess caspase inhibitory activity[86]. We screened the aqueous extracts for their ability toinhibit caspase-8 activity using 19F NMR spectroscopy tomonitor the chemical shift of the trifluoromethyl substitu-ent on the coumarin moiety of the tetrapeptide IETD-AFC on cleavage between D and AFC by the caspase.We found that extracts of forsythia fruit (Fructus Forsyth-iae, Lianqiao in Chinese) and fleeceflower stem (CaulisPolygoni multiflori; Shouwuteng) inhibited the activity ofcaspase-8 with IC50 values of 2.8 and 4.3 mg/L, respective-ly. Extracts prepared from the horn of the antelope Saigatatarica Linnaeus (Cornu Saigae tataricae; Lingyangjiao),the red peony root (Radix Paeoniae rubrae; Chishao),and the scorpion Buthus martensi (Scorpio; Quanxie)showed less potent inhibitory activity, with IC50 values of11.8, 96.8, and 8.8 mg/L, respectively. We also investigatedwhether the extracts of Fructus Forsythiae and CaulisPolygoni multiflori possessed similar inhibitory activitytoward the effector caspases caspase-3 and caspase-7. Fluo-rescence-based enzymatic assays revealed that both TCMdrugs also blocked caspase-3 and caspase-7, although theIC50 of both extracts with caspase-3 was �2.5 times higher.

8. Conclusions

The results of our studies indicate that Chinese herbalmedicines possess activity at multiple molecular targets inthe glutamate receptor-triggered signaling pathways lead-ing to neuronal injury and death. Individual herbal drugscontain diverse families of chemical compounds, whereeach family comprises structurally related members thatact with low affinity at multiple molecular targets. In addi-tion, we have shown that some flavonoids might target animportant class of protein interaction domains [87,88].Rapidly accumulating evidence indicates that spatiallyand temporally regulated protein–protein interactions arethe basis of the metabolic and signaling networks that pro-vide the cell with both the stability and flexibility to per-form its functions and respond to changes in itsenvironment [89–92]. Together, the data and a large bodyof evidence in the literature appear to support the multi-component, multitarget, and network-oriented approachof traditional Chinese pharmacotherapy and provide amodern interpretation of the principle of jun chen zuo shi.This approach is exemplified by the traditional formulationTianma Gouteng Yin (Fig. 2). This well-known prescriptionis used by TCM to calm excessive endogenous wind, whichcan be a pathogenic factor in hypertension, stroke, or sei-zures [25]. The prescription is a combination of 11 differentdrugs (Table 2). Rhizoma Gastrodiae, Ramulus Uncariaecum uncis, and Concha Haliotidis are the principal drugsthat are directly aimed at the excessive wind; the otherdrugs fulfill ancillary functions including promotion of cir-culation and diuresis (Herba Leonuri) and relief of mentalstress (Cortex Eucommiae and Herba Taxilli). Results from

Table 2Composition of the traditional Chinese prescription Tianma Gouteng Yin

Drug Scientific name Chinese Chemical constituents

Caulis Polygoni multiflori Polygonum multiflorum Shouwuteng Anthraquinones: chrysophanol, emodin, rhein, physcionConcha Haliotidis Haliotis versicolor Shijueming Amino acids

Inorganic: Al, Ca, Cl, Cr, Cu, Fe, I, K, Mg, Mn, Na, Ni, P, S, Sr, Ti, V, ZnPhenolic compounds: batatasin-IIIPolysaccharides: chitin

Cortex Eucommiae Eucommia ulmoides Duzhong (Tri)terpenoids: betulin, betulic acid, ursolic acidFructus Gardeniae Gardenia jasminoides Shanzhi Sterols: b-sitosterol, stigmasterol, daucosterolHerba Leonuri Leonurus heterophyllus Yimucao Alkaloids: L-stachydrine and leonurine

Flavonoids: rutin, quinqueloside, genkwanin, quercetin, quercitrin, isoquercitrin, hyperoside, apigenin,kaempferolPhenolic compounds: caffeic acid(Di)terpenoids: leocardin

Herba Taxilli Taxillus chinensis Sangjisheng Flavonoids: avicularin, quercetinPoria cum ligno hospite Poria cocos Zhufushen Fatty acids: caprylic acid, undecanoic acid, lauric acid, dodecenole acid, palmitic acid

Glucans: pachyman(Tri)terpenoids: tumulosic acid, eubricoic acid, pinicolic acid, pachymic acid

Radix Achyranthis bidentatae Achyranthis bidentata Niuxi Sterols: ecdysterone, inokosterone.Radix Scutellariae Scutellaria baicalensis Huangqin Flavonoids: baicalin, wogonin, norwogonin, oroxylin, scutellarin. MgRamulus Uncariae cum uncis Uncaria rhynchophylla Gouteng Alkaloids: akuammigine, corynantheine, corynoxeine, hirsuteine, isorhynchophylline, isopteropodine, pteropodine,

rhynchophylline, scopoletin, vallesiachotamine, yohimbineFlavonoids: catechine, epicatechine, hyperine(Tri)terpenoids: ursolic acid

Rhizoma Gastrodiae Gastrodia elata Tianma Phenolic compounds: vanillin

358N.J.Sucher

/Epilep

sy&

Behavio

r8(2006)350–362


our investigations reveal that this decoction contains drugsacting as NMDAR antagonists (Ramulus Uncariae cumuncis and Radix Scutellariae), a drug acting as an antago-nist at PDZ binding domains (Radix Scutellariae), and adrug with caspase inhibitory activity (Caulis Polygoni mul-tiflori). Thus, the decoction contains drugs with actions atseveral levels of the signaling pathway involved in neuronalinjury and death. The data further suggest that inhibitionof NMDARs may be part of the therapeutic action of thisdecoction. Mg2+, rhynchophylline, isorhychophylline, andSDS act as noncompetitive, low-affinity NMDAR antago-nists. Their mode of action may explain the absence of seri-ous adverse effects related to NMDR blockade byformulations containing these compounds. At the sametime, rhynchophylline and isorhynchophylline have beenreported to interact with, in addition to NMDARs, a con-siderable number of other molecular targets [58,93,94]. ATianma Gouteng decoction would furthermore contain alarge number of other structurally diverse chemical com-

O

O

OHOH

HH

H

HO

NH

N

O

H

O

O O

H

OH

OH

OH HH

NHCCH3H

OH

CH2OH

O

OOH

HH

NHCCH3H

OH

CH2OH

O

O

Chrysophanol

Rhynchophylline

Chitin

Betulin

Fig. 3. Examples of the structural variety of chemical compounds isolated froman anthraquinone from Polygonum multiflorum. Stigmasterol is a phytosterol frBaicalin is a flavonoid from Scutellaria baicalensis. Rhynchophylline is an alkGastrodia elata. Chitin is a polyglucan from Concha haliotidis. Palmitic acid is

pounds such as amino acids, alkaloids, anthraquinones,fatty acids, glucans, flavonoids and other phenolic com-pounds, polysaccharides, and terpenoids, in addition to aconsiderable number of inorganic compounds and tracemetals (Fig. 3). Thus, it is very likely that the decoctionmight afford additional beneficial therapeutic effects as anantioxidant and anti-inflammatory, as well as by actingon a considerable number of other neuronal and nonneuro-nal targets that have yet to be identified.

Together these data indicate that the reductionisticapproach of our studies proved useful for the identificationof molecular targets and some of the active chemical con-stituents of herbal drugs mediating these effects. Althoughthese investigations point to possible mechanistic explana-tions for known therapeutic effects of these drugs, they donot by themselves provide any direct evidence in thisregard. The molecular effects described here may, however,serve as useful surrogate markers in pharmacokinetic, met-abolic, and/or pharmacodynamic animal experiments and

OH

HO

H

H

H

HO

O

H

O

O

OH

HO

OH O

OO O

H

OHH

OH

COOH

Stigmasterol

Vanillin

Palmitic acid

Baicalin

the ingredients of Tianma Gouteng Yin (see also Table 2). Chrysophanol isom Gardenia jasmonides. Betulin is a triterpenoid from Eucommia ulmoides.aloid from Uncaria rhynchophylla. Vanillin is a phenolic compound froma fatty acid from Poria cocos.


clinical studies. Importantly, however, these results alsosuggest that a reductionistic approach in clinical trials orin vivo animal experiments might be prone to failure, asonly the combination of several drugs might prove to beeffective clinically. It is interesting to note in this contextthat the failure of several large-scale clinical trials with sin-gle drugs acting as NMDAR antagonists, including magne-sium ions [95], has prompted clinical and preclinicalresearchers in the West to suggest that successful stroketherapy may necessitate the development and use of combi-nation therapies such as the combination of thrombolysisand neuroprotection [13,43,46,96]. Combination therapyhas become accepted practice in the treatment of a numberof chronic diseases including type 2 diabetes [97], hyperten-sion [98], and epilepsy [99].

We initially approached the Chinese materia medica asa vast but largely untapped source of natural products inthe hope of discovering novel compounds serving as leadsfor the development of Western-style drugs [8,10]. It is,however, clear that the empirical development of Chineseherbal medicine over at least some 2000 years has resultedin a multidrug, multitarget approach. It is our belief thatthis may well be a major legacy of Chinese medicine, withtradition pointing the way to a network-oriented approachto pharmacotherapy in the future [100]. It may be prudentto consider this approach in the current attempts to trans-late the molecular and mechanistic knowledge gained frombasic research into successful therapeutic regimens. Thefurther development of translational medicine may benefitfrom the combination of principles of modern molecularmedicine with certain ideas of traditional empirical Chi-nese medicine.

Acknowledgments

I thank Dr. M. Carles and Dr. Jiayi Zhou for helpfuldiscussions and critical reading of the manuscript.

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Insights from molecular investigations of traditional Chinese herbal stroke medicines: Implications for neuroprotective epilepsy therapy