Traditional Chinese Medicinal (TCM)

well as a lack of demonstration of plausible mechanisms and dosing effects. In short, clinicalbotanical investigations have suffered from the lack of a cohesive research strategy which drawson the expertise of all relevant specialties.With this as background, US and Chinese co-investigators with expertise in Traditional ChineseMedicine (TCM), botany, chemistry and drug discovery, have jointly established a prototypelibrary consisting of 202 authenticated medicinal plant and fungal species that collectivelyrepresent the therapeutic content of the majority of all commonly prescribed TCM herbalprescriptions. Currently housed at Harvard University, the library consists of duplicate or triplicatekilogram quantities of each authenticated and processed species, as well as “detanninized” extractsand sub-fractions of each mother extract. Each species has been collected at 2–3 sites, eachseparated geographically by hundreds of miles, with precise GPS documentation, andauthenticated visually and chemically prior to testing for heavy metals and/or pesticidescontamination. An explicit decision process has been developed whereby samples with the leastcontamination were selected to undergo ethanol extraction and HPLC sub-fractionation inpreparation for high throughput screening across a broad array of biological targets includingcancer biology targets. As envisioned, the subfractions in this artisan collection of authenticatedmedicinal plants will be tested for biological activity individually and in combinations (i.e.,“complex mixtures”) consistent with traditional ethnomedical practice.This manuscript summarizes the rationale, methods and preliminary “proof of principle” for theestablishment of this prototype, authenticated medicinal plant library. It is hoped that thesemethods will foster scientific discoveries with therapeutic potential and enhance efforts tosystematically evaluate commonly used herbal therapies worldwide.Keywords Herbal medicine; Library; Traditional Chinese; Ethnomedicine 1. IntroductionThe topic of whether and how plant based medicines (aka herbal remedies) predictably alterthe natural course of human disease has been an essential and complex aspect of medicinefor thousands of years. By contrast, efforts to systematically apply modern scientificstrategies to prove or disprove the therapeutic value of specific medicinal plants,individually or in complex mixtures, and to optimize their rightful place in modern healthcare, represent a more recent trans-disciplinary challenge.Focusing on Traditional Chinese Medicine (TCM), there was a singular moment in recenthistory when practitioners of TCM and advocates of modern western medicine wereexplicitly called upon to jointly learn from one another, teach one another and, in theprocess, attempt to generate new knowledge for the common good of the next generation.The time was August 1950. The setting was the first National Health Congress of the newlyestablished People’s Republic of China. Chairman Mao Ze Dong spoke on the occasion ofthe proposed establishment of the first five accredited schools of TCM and the need forcollaboration across disparate expert groups. “We should unite all the young andexperienced medical professionals from both Traditional Chinese Medicine and ModernWestern Medicine to form a firmly united front to jointly strive for a great enhancement ofthe people’s health [1]!” Mao’s intention was clear and practical. He sought to proactivelyengage medical experts from both eastern and western traditions to jointly explore what hecalled “The Treasurehouse of Traditional Chinese Medicine,” including its richpharmacopeia.NIH-PA Author Manuscript NIH-PA Author ManuscriptNIH-PA Author Manuscript2. Relevance of herbal and TCM productsRoughly half of all approved prescription drugs are natural products, mostly from plants andmicrobial sources, their semi-synthetic derivatives or fully synthetic analogs [2]. Close to70% of all cancer drugs originated from natural products [3,4]. Therefore, the application ofstate-of-the-art technologies to the systematic evaluation of traditionally used plant basedmedicines (aka ethnobotanicals) remains a highly relevant yet scientifically challenging lineof inquiry.3. Epidemiology and market relevance of herbal and TCM products Herbal medicine use by the American public has increased dramatically over the past two decades. The percentage of US adults reporting the use of herbal (non-vitamin, non-mineral)products to treat or prevent disease increased from 2.5% in 1990 [5] to 12% in 1997 [6] to 14% in 2000 [7] to 19% in 2002 [8] and 18% in 2007 [9]. The estimated out-of-pocket expenditures for herbal therapies by the US adult population in 2007 was $14.8B. This is equivalent to approximately one-third of the total out-of-pocket spending on all prescription drugs ($47.6B) that same year [10].A marketing analysis suggested that sales of TCM herbal products from China increased at an annual rate of 24% between 2004 and 2008 [11]. In 2008, TCM herbal product sales accounted for an estimated 22% of China’s overall healthcare product revenue and were estimated at a value of $26 billion US dollars [11].4. Rationale to build a prototype library based on challenges and lessons learned 4.1. Lessons learned from selected clinical trials In 2003, the NIH’s National Center for Complementary and Alternative Medicine(NCCAM) warned that a lack of reproducibility, quality control and dosage schedulesinvolving natural products might lead to methodologically questionable and/or negativeclinical studies, thereby diminishing opportunities for further investigations ofethnobotanicals [12].By way of example, in 2004 a randomized trial was conducted to test the clinicaleffectiveness of an eight herb Chinese formula, sold under the product name “PC-SPES,” insubjects with advanced prostate cancer [13]. As documented in the medical literature, thiscomplex herbal mixture was found to be clinically superior to the standard, high dose,estrogen salvage protocol in terms of overall reductions in PSA levels and time toprogression of disease for 90 randomized study subjects. However, random testing of theherbal mixture revealed it had been adulterated with small quantities of synthetic estrogenand Coumadin [13]. The authors concluded that the true efficacy of this, and other, herbalmixtures will remain uncertain until the quality, consistency and purity of the naturalproducts under evaluation can be ensured [13]. A review of the medical literature in 2005documented that most publications involving the assessment of herbal therapies in clinicaltrials involved no independent verification of the herbal contents under evaluation [14].The authors of the PC-SPES study also commented on the fact that the levels of estrogenidentified in the commercial PC-SPES products were far too low to have explained theapparent clinical superiority of the PC-SPES therapy as compared with the estrogen salvageprotocol. As such, their comments could be interpreted to raise the possibility, albeit remote,that specific components (i.e. chemical compounds) within the PC-SPES mixture, whenNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptadded to low dose estrogen, might have resulted in an additive or synergistic effect powerfulenough to alter the course of disease in men with advanced prostate cancer.A second case of note involved the evaluation of the herb Echinacea (Echinacea angustifolia DC.) in the prevention of rhinovirus infection (i.e. the common cold). In this study, 437 volunteers were proactively infected with rhinovirus [15]. Subjects were then randomized to four groups. Three of the groups received various preparations and doses of a commonly sold Echinacea extract and one group received a placebo. There were no significant differences across groups with regard to rates of infection, severity of symptoms or viral titers. As such, the trial was considered to have refuted claims of clinical effectiveness of Echinacea . Subsequently, the New England Journal of Medicine published criticisms of the study’s design [16]. These included the suggestion that a different Echinacea species might have been preferable; that the dose used in the study was far too low (by a factor of 6) and that a higher dose might have made this trial more clinically and scientifically relevant [16].A third study involved the evaluation of a popular over-the-counter preparation of the herb saw palmetto (Serenoa repens (W. Bartram) Small) in the treatment of benign prostatic hypertrophy [17]. In this study, 225 subjects were randomized to two groups, one receiving a Saw Palmetto extract in the form of a popular over-the-counter supplement and the other group receiving a placebo. There were no significant differences observed between these two groups in terms of symptomatic improvement. An accompanying editorial [18]commented that the study authors had tested a single, commercially available preparation of saw palmetto, thereby leaving open the possibility that a different preparation might still be effective. Furthermore, these authors contended that in the absence of a plausible mechanism of action, a fair comparison of this herb (or its constituents) to a more conventional FDA approved therapeutic drug, would be problematic if not impossible.Lessons learned from these and other ambitious (and expensive) clinical trials suggest thatfuture human clinical trials involving herbal products must ensure the reproducibility andquality of the intervention materials; and, will require an understanding of mechanisms ofaction and dosing prior to the implementation of new, large scale (and expensive) Phase IIor III clinical trials. The current NIH guidelines involving candidate herbal therapies reflectmany of these hard learned lessons [19] as do the Consort Guidelines for publicationsinvolving randomized controlled trials involving herbal interventions [20].In hindsight, these were methodological inadequacies uncovered by individuals skilled inthe design and conduct of clinical trials. They provided part of the rationale for the studydescribed in this manuscript. What about methodological challenges from the vantage pointof other relevant experts including researchers skilled in botany, chemistry, ethnobotany anddrug discovery?4.2. Lessons learned from the vantage point of drug discovery and ethnobotanyThe current place of natural products in modern drug discovery is inconsistent with theirpast performance and future potential. Natural products have made, and continue to make,substantial contributions both to understanding basic biological processes and treatinghuman disease. If we focus on cancer, natural products from plants have led to frontlinetherapies such as paclitaxel, vinblastine, camptothecin and etoposide [4]. If we look at theimmediate future, geldanamycin analogs – to pick just one example – are being pursued inclinical trials [21,22]. Thus there is a strong scientific argument for continuing to explorenatural products in drug discovery —an argument that is largely unheeded as pharmaceuticalcompanies cut back on, or eliminate, their natural product programs.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptIdentification of natural product-based leads for Western drug discovery has usually resulted from screening of extracts or compounds from diverse biological sources, generally without regard to preexisting knowledge of the therapeutic utility of the producing plant. A good example is the remarkable portfolio of hundreds of thousands of natural products and extracts amassed by the United States National Cancer Institute (NCI) since the inception of its natural product-based efforts in 1955 [23]. Between 1960 and 1982, the NCI screened extracts of 35,000 plant species in collaboration with the U.S. Department of Agriculture (USDA). The strategy adopted was largely one of random selection of a broad range of natural product sources as opposed to selection based on medicinal use, i.e. ethnomedicine [4]. To a large extent, the driving force for NCI’s efforts was biological and geographic diversity rather than pre-existing knowledge of therapeutic utility. Focusing on biological and geographic diversity is a typical paradigm of most natural product drug discovery successes, meaning that the use of natural products for Western drug discovery has largely been one of trial and error. This approach has been referred to as “bio-prospecting.”Paclitaxel, vinblastine and camptothecin were discovered using this approach. Interestingly,Verpoorte has pointed out that there are an estimated 250,000 flowering plant species on earth while as of 2000, fewer than 15,000 (6%) had been screened for biological activity [24].In contrast, many cultures around the world have developed ethnomedical traditions based on therapeutic utility of selected local plants and animals. Such empirical traditions are often hundreds if not thousands of years old, as in the case of TCM for which written records exist going back over 2000 years. Unfortunately, the potential value of ethnomedicines has often been discounted by Western medicine and science, with several identifiable factors accounting for this. First, medical diagnoses in TCM and other ethnomedical systems are often portrayed in ways that are not readily understood by Western clinicians. Second, TCM and other ethnomedicines are often viewed as fundamentally lacking in the mechanistic,scientific bases that usually underpin claims of Western medical efficacy. Third, there hasbeen a lack of rigorous, well-controlled clinical trials demonstrating clinical efficacy (andmechanisms) of TCM and other ethnomedicines. Fourth, existing scientific and clinicalstudies of TCM have often utilized plants that have been quality compromised, may becontaminated with pesticides or heavy metals, may have been botanically misidentified, orare lacking a consistent and reproducible resupply chain. As such, and as noted earlier, priorstudies have frequently been compromised by quality control and botanical authenticationissues, as well as lot-to-lot variability and lack of knowledge of precise growing locationsand conditions, factors that have too often limited reproducibility. Finally, resupply of herbsfor confirmation and follow-up studies is frequently problematic. The limiting factorsmentioned earlier fall into two main categories: variables related to starting materials, andvariables related to execution or interpretation of scientific and clinical studies. While thetwo are interdependent, without addressing the former, there is little value in pursuing thelatter.From the vantage point of ethnobotany, researchers have highlighted the difficulties inreplicating the biological activity of a given plant when attempts are made to repeat anexperiment after subsequent recollection [25]. As such, the challenge of reproducibilityremains a formidable one.There are also challenges in terms of sourcing plant species to be studied. These include:collecting them according to traditional techniques, documenting the precise collection sitesusing GPS technology, authenticating them visually, chemically and, through DNAsequencing, processing and extracting them according to established, traditional andreproducible techniques, storing them properly and so on.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptLastly, there is the inherent conundrum which speaks to the heart of the conceptual difference between traditional (i.e. ethnobotanical) herbal practice and modern (western)drug discovery and therapy. This conundrum can be conveyed through the articulation of two testable hypotheses, specifically; do herbal medicines work, when they work,predominantly because of single chemical compounds, albeit in small quantities? By extension, are herbal medicines and herbal medicine libraries merely repositories for sophisticated “bio-prospecting” in search of novel, and patentable, composition of matter discoveries (aka “new chemical entities”) or derivatives of already known chemical compounds which can be shown to be of new therapeutic application and benefit? One must also consider the fact that herbal therapy as practiced in traditional settings rarely involves the prescription of a single herb at a time. Instead, traditional conceptual frameworks, which preceded contemporary understanding of chemical compounds and their specific effects on precise, biological targets, almost always involve multiple herbs and a presumed additivity and/or synergy of effects on the host subject. The shared view among herbalists, in Asia and elsewhere, has been that a mono-therapy approach will be inferior to a multi-therapy,complex herbal mixture approach [26]. By way of example, a typical TCM prescription routinely includes 8–12 herbs, in varying ratios, with one of the herbs serving as the “king”,a second serving in the capacity of “minister”, a third as “adjutant” and a fourth as the “messenger” [27].Given this disparity of conceptual models, the fundamental (second) testable hypothesis is that herbal therapies work, when they work, due to complex yet predictable effects of multiple compounds within complex mixtures of plant compounds. As evidenced by the near miraculous success of the three drug “AIDS Cocktail,” we now have unequivocal evidence that multi-drug therapy can sometimes be the best – or only –successful therapeutic option [28]. The work of Borisy et al. demonstrated the existence of synergistic effects involving multiple (FDA approved) compounds [29] and the work of Wagner has summarized the existence of synergistic effects involving multiple compounds found in avariety of natural products [30]. Might clinical successes attributed to herbal therapiesinvolve additive and/or synergistic biological effects which can now be more meticulouslydescribed and, ideally, optimized for maximal therapeutic benefit?Lastly, there is the non-technical challenge that Mao attempted to address a half century ago[1]; specifically, can the two schools of medical thought, Eastern and Western (modernbiomedicine) develop shared research strategies which address the above mentioned knownchallenges without compromising the respective beliefs, practices and fundamental tenantsof each tradition? Can ethnobotanists and modern drug discovery experts established aneffective “united front,” and will this exercise result in novel therapeutic discoveries or not?To reframe this question, can an artisan collection of authenticated TCM plants beestablished for the purpose of successful systematic biological evaluation and will such alibrary, when screened strategically, lead to the identification of novel compounds, new usesfor known single compounds and/or their derivatives; and/or novel biological mechanisms?With regard to the second hypothesis mentioned earlier, might there also be a newopportunity to employ contemporary high-throughput screening facilities to search foranticipated as well as unanticipated combinations of single compounds which can be shownto be therapeutically effective due to biological additivity and/or synergy of multiple plantderived compounds? Will combinations with evidence of additivity and/or synergy confirmethnobotanical knowledge about specific plant combinations (i.e. formulas) or might entirelyunanticipated active combinations also be identified?NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript4.3. Considerations in the development of a prototype authenticated TCM plant libraryBetween 2006 and 2010, investigators from Harvard Medical School (HMS), incollaboration with colleagues from the Beijing University of Chinese Medicine (BUCM)and Hong Kong Baptist University (HKBU), embarked on efforts to build a prototypelibrary of 202 botanically authenticated, quality controlled, collection site-documentedChinese medicinal plants. Each plant was to be tested for pesticide and heavy metalcontamination and qualified using standards defined by the Chinese Pharmacopeia [31], anofficial publication of the State Food and Drug Administration (SFDA) of the People’sRepublic of China. Such an artisan library could serve as a necessary prerequisite toreproducible pre-clinical studies of TCM herbs. These are mandatory prerequisites to thesubsequent design and implementation of the next generation of animal and human studiesof herbal remedies. Moreover, the ethnobotanical knowledge of TCM experts is essential toestablish this library, and to efficiently guide drug development experts in their search fornovel single compounds, novel uses of previously identified compounds and hypothetically,novel demonstrations of additivity and/or synergy involving multiple plant compoundswithin plants and within complex mixtures of medicinal plants.In planning for, constructing and evaluating this authenticated TCM herbal library, manyfactors had to be considered including: the selection criteria of medicinal plants to beincluded in the research library; quality assurance including harvest sites and GPSdocumentation; botanical species authentication; collection protocols; plant processingdetails; creation of voucher specimens; testing for pesticides and heavy metal contamination;chemical and quality assessment according to existing standards of the ChinesePharmacopeia; Chinese governmental cooperation, authorization and partnership; storageand shipping logistics of all specimens in China and the US; precise and reproducibleextraction and fractionation procedures; selection of appropriate biological screeningstrategies; establishment of a suitable database and database management system; and,continuous efforts to maintain open communications and opportunities for expandedresearch collaboration among all participating co-investigators.5. Methods(See Fig. 1 for an overview of the methods employed in the creation of this prototype TCMlibrary).5.1. Criteria for inclusionThe selection of plants for the authenticated TCM herbal library was based on two maincriteria: (1) the plants were listed in the official Pharmacopoeia of the People’s Republic ofChina (CP) [31] and, (2) the species were not endangered. This latter criterion is of concernsince the over-harvesting of plants from wild sources for use in TCM is a growing problem[32]. For example, some plants used in Chinese medicine are on the Convention onInternational Trade in Endangered Species of Wild Flora and Fauna (CITES) list, includingfamous medicines such as Mu Xiang (Saussurea costus (Falc.) Lipsch.) and Rou Cong Rong(Cistanche deserticola Y. C. Ma) [33]. The official CP includes synthetic pharmaceuticalmedicines, but also many traditional medicines derived from natural sources. At the timethis study was launched, the most recent edition was the 2005 Chinese Pharmacopeia.The first volume of the 2005 CP includes monographs on Traditional Materia Medica and iscomprised of substances derived from animals, minerals, plants, and fungi. A total of 471monographs based on plant products are included in this volume, comprising 550 differentspecies. The prototype library included a total of 200 species of plants and 2 species offungi, representing more than one-third of all plant species listed in the CP. The 202 selectedNIH-PA Author Manuscript NIH-PA Author ManuscriptNIH-PA Author Manuscriptplants (see Appendix A) are some of the most commonly used in the CP and hence areestimated to represent approximately 75% of all plants used in routine TCM practice.5.1.1. Monograph summarizing relevant information of each plant—Monographs were written for each species included in the project. The goals of the monographs included providing a comprehensive literature review of the traditional uses, as well as a summary of recent information about experimental studies of each plant. In particular, it was envisioned that the monographs would be a useful source of information for screeners of the library.Each monograph included the following sections: names and synonyms of the plant,collection and processing methods, therapeutic indications in TCM and western medicine,types of extracts, major chemical constituents, description of medicinal part of the plant,contraindications, common preparations and inclusion in common TCM formulae, and selected references in the TCM and western literature. The monographs are included in the project database (see section 5.7).5.2. Collection protocol Whenever possible, collection locations were selected from the region that is traditionally known for production of the plant species being collected. Additionally, each plant species was collected from three distinct locations in China separated by hundreds or thousands of miles and usually in different provinces, to ensure that at least one of these plants could meet the appropriate requirements of species identity, quality, and purity, including the absence of contamination by pesticides and/or excessive heavy metals. Plant collection consisted of three separate activities: environmental investigation, bulk harvest, and voucher collection.The environmental investigation included a survey to ensure that the collection area was suitable and capable of yielding enough plant material for the project. The bulk harvest consisted of the harvest of the medicinal part of the plant at the time that the herb is traditionally collected. During the bulk harvest, each sample was collected to give a total of 10 kg dry weight. Following the bulk harvest, all herbs were processed according to thetraditional method as specified by the CP. This typically involved the removal of impuritiessuch as other plant species or soil, followed by drying in the sun for a period of days orweeks. Lastly, vouchers of each sample, consisting of flowering or fruiting material of theplant, were collected according to standard protocols [34]. Vouchers have been stored alongwith a voucher of the bulk harvest medicinal part for future reference. The plant acquisitionteam, organized and overseen by BUCM co-investigators and consultants from HKBU,consisted of at least one Chinese herbal medicine resource expert from each of the 30provinces and autonomous zones where plant acquisition took place. In addition, a 34 personBeijing-based acquisition team consisting of faculty and graduate students of BUCM as wellas 8 additional TCM botanical experts oversaw the quality control and processing of allplant samples collected. Authentication of plant species was overseen by a separate group offaculty with TCM ethnobotanical expertise at HKBU. All steps of plant collection weredocumented using a combination of standardized collection forms, GPS data collection,photographs, and video. (See Appendices B–E for examples of photos imported into study’sdatabase). After harvest and processing, plants were authenticated and tested for quality (seesections 5.3 and 5.4).5.3. Authentication and quality assessmentThe taxonomic identification of each plant was confirmed by multiple experts in China.Plants were authenticated according to morphological and anatomical characteristics [35] bya team at HKBU and examined according to criteria listed in the CP for quality assessmentat BUCM. The quality assessment tests were conducted according to the guidelines andstandards provided in the CP 2005 edition [31]. The CP specifies the method for conductingrelevant tests and provides standard reference values for results. In general, the qualityNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript。