This manuscript summarizes our rationale for the development of a library of authenticated medicinal plants, methods used, the status of this library and preliminary research conducted to date. The discussion will be limited to lessons learned based on the work completed thus far, followed by a preliminary list of researchable hypotheses and proposed next steps.
Importantly, through the present collaborative study, US and Chinese co-investigators have jointly proposed and implemented a potentially reproducible strategy to identify, collect, authenticate, quality control, extract and pre-fractionate medicinal (TCM) plant species for the purpose of systematic biological, i.e. pre-clinical, evaluation using state of the science drug discovery technologies. While expensive and complex, this methodological approach has the potential to address many of the criticisms aimed at previous investigations involving herbal (natural) products and sets the stage for future pre-clinical research investigations involving medicinal plants.
US and Chinese co-investigators with expertise in (TCM), botany, chemistry and drug discovery, have jointly established a prototype library consisting of 202 authenticated medicinal herbal and fungal species that collectively represent the therapeutic content of the majority of all commonly prescribed TCM herbal prescriptions. Currently housed at HMS, the library consists of duplicate or triplicate kilogram quantities of each authenticated and processed species, as well as “detanninized” extracts and sub-fractions of each mother extract. Importantly, each species has been collected at two or three unique sites, each separated geographically by hundreds of miles, with precise GPS documentation of each collection location as well as information about the seed origins of plant. Each of the plant samples has been authenticated visually and chemically prior to testing for contamination by heavy metals and/or pesticides. An explicit decision process has been developed whereby samples of each species with the least contamination were selected to undergo ethanol extraction and reproducible HPLC sub-fractionation procedures in preparation for high throughput screening across a broad array of biological targets including, but not limited to, targets relating to cancer biology. (See Appendix G for a graphic summary of methods used to develop this prototype library).
In the process of developing this prototype TCM library, we tested all collected specimens for their heavy metal and/or pesticide content. A detailed summary of these findings and their clinical significance is beyond the scope of this article and is being reported in a separate manuscript [38
Preliminary biological screening of this library has led to the following observations. We have been able to document evidence of biological activity in a variety of screens ranging from standard cell based growth assays to screens involving whole animals (i.e. zebra fish). As anticipated, the fractionation process has resulted in many fractions that are dominated by single compounds and this has greatly facilitated the process of identifying active components in a given screen. The finding that TRP channel agonists may alter angiogenesis is provocative but has yet to be reduced to a molecular mechanism. Indeed, the cellular targets are still unknown as we might have obtained the observed effects by stimulating channels on the endothelial cells or some other cell type that plays a role in angiogenesis.
Given that the work presented here only includes preliminary screens and compound characterization, a number of testable hypotheses remain unanswered. First, is the question of how this library, developed according to a systematic protocol differs from comparable libraries consisting of TCM plant species, extracts and fractions elsewhere. As examples, at least five such collections of research TCM extracts are known to exist throughout East Asia and these include the: (1) TCM natural product library at the Dalian Institute of Chemical, Physics, Chinese Academy of Sciences; (2) Kunming Institute of Botany, the Chinese Academy of Sciences; (3) Beijing Tong Ren Tang Chinese Medicine Co. Ltd.; (4) Shanghai Institute of Materia Medica; and (5) Sun Ten Pharmaceuticals Co. Ltd. and Sun Ten Phytotech in Taiwan. While several of these collections include a minority of samples which were individually grown and/or collected by the named entity mentioned earlier, the majority of plant samples in these extract collections were purchased on the open market and almost certainly came from multiple collection sites (i.e. numerous farms and/or wild sites) with uncertain (and heterogeneous) collection and processing procedures. As such, these other established TCM library collections are less stringent with regard to quality assurance and the potential for reproducibility as compared with the present library.
As noted in the Introduction, any study of botanical medicines must adhere to strict and repeatable methods for ensuring the quality of the botanical product. It has been well documented that the bioactive ingredients of plant-based medicines vary in ways that have pharmacological consequences [42
]. This variation can exist within a single population of one named species [45
]. Furthermore, plant chemistry is known to vary according to many factors, including ontogeny [46
], geography [47
], and season [49
], even within the same genetic individual. Consequently, one plant-based medicine may consist of multiple genotypes, even within one species. Each genotype may consist of multiple chemotypes, depending on the place where they live and the time of collection. Efforts to rigorously test the biological activity of plant-based medicines and reliably produce standardized plant extracts for clinical use must account for the combined possibility of genetic and chemical variability within a single botanical medicine [51
]. Therefore, it is of the utmost importance to ensure that the botanical provenance and details of the collection and processing are recorded in detail and to the maximal extent possible. Every step in the creation of this prototype library, from collection to extraction and fractionation has been stored in a database created specifically for this project (i.e. the TM-CTS) [39
]. While these steps maximize the prospect of demonstrating reproducibility of biological effects from compounds derived from specific plant collections, explicit proof of reproducibility remains another critically important next step.
The larger question, of course, is whether the effort and expense required to create such a library of authenticated medicinal plants will result in scientific discoveries that justify the investment. Some would argue that this investment, while risky, is justifiable and essential to the comprehensive and authoritative assessment of commonly used medicinal plants worldwide. In their important review of this subject, Schmidt and colleagues [52
] remind us that most medical systems developed prior to the 20th Century relied almost exclusively on multicomponent medicines obtained from natural sources. Today, however, the modern pharmaceutical industry relies almost exclusively on single-ingredient drugs, typically referred to as new chemical entities (NCE’s). However, the rate of NCE discovery has slowed in recent decades [53
]. In addition more diseases are being treated with combinations of single-component drugs. These combination therapies are designed to lower the incidence of resistance or target several pathological processes simultaneously. They are particularly important in treating infectious diseases such as HIV [28
], tuberculosis [55
], malaria [56
] and complex chronic diseases like cancer [57
] and metabolic syndrome [58
By contrast, traditional herbal medicinal systems, including those of China, India, Africa, the Asian Pacific and Hawaii classically relied on complex mixtures of plants to treat common symptoms and diseases. Schmidt et al. contend that traditional medicinal use of multicomponent botanicals may be better suited to prevent or control complex multi-factorial diseases as compared with the efficacy of single active ingredient pharmaceuticals [52
]. This conceptual model inextricably leads to a testable hypothesis, namely, that multicomponent botanical therapies (MCBTs), if standardized using unequivocal botanical identification techniques and quality assurance methods, can be shown to contain individual chemical compounds which, when combined in specific ratios, result in predictable additive and/or synergistic biological effects capable of altering the natural course of established disease. How then might we get from our current state of knowledge to a confirmation or rejection of this testable hypothesis?
The specific methods detailed in this manuscript, which can be used to expand the entire library of TCM medicinal plants or, alternatively, to build new parallel libraries of medicinal plants from other cultures and continents (e.g., those of India, Africa, the Mideast, Hawaii, etc.) begin to address issues pertaining to standardization and quality control of MCBTs. The next logical challenge is to use such potentially reproducible libraries to produce evidence of: (1) novel single compounds (i.e. NCE’s) with demonstrable provocative biological effects; or, (2) single compounds which are already known but which can be shown through HTS screening to possess here-to-fore unknown biological properties; or (3) evidence of entirely novel biological mechanisms; and (4) evidence that single compounds of interest, from this library (aka, “singletons”), when paired with one or more additional compounds from the same plant, a different plant in a MCBT (as is the case in most traditionally used herbal formulas and “patent medicines”) or hypothetically, in combination with a small molecule which has already been recognized as an FDA approved drug, will result in additive or synergistic effects. Such combination studies could readily be performed under automated HTS conditions using well-established techniques for combination-based screening based on the combination index methodology originally established by Chou and Talalay [59
The present library sets the stage for these subsequent investigations. Initial screening of the 3709 fractions from 80 authenticated TCM plant species has resulted in a spectrum of leads, the majority of which remain in early stages of investigation for their reproducibility, relative potency, pharmacokinetics, safety and therapeutic potential as prospective single compound drug candidates. While we remain intrigued by the potential of combinatorial studies, due to funding challenges, we have yet to initiate combinatorial studies to explore the existence of additivity or synergy of biological effects involving lead candidate compounds within the TCM library and/or with FDA approved small drug molecules.
Several implications of this ongoing work are noteworthy. First, the successful academic partnerships between US and Chinese institutions and co-investigators have resulted in a series of agreements which are consistent with the Convention on Biological Diversity [60
] and demonstrate the willingness of multiple international stakeholder groups to collaborate across professional disciplines and international boundaries. These agreements and working relationships properly acknowledge the indigenous and traditional medical knowledge of our TCM co-investigators and their home country, China. Indeed, the ethnobotanical expertise of our Chinese colleagues has been a prerequisite to the implementation of this prototype library, and will be essential for the continued, targeted scientific evaluation of this library over the coming years. Future investigations of medicinal plants in other countries may choose to refine this collaborative strategy for use in their respective international settings.
Second, in spite of the fact that there has been a relative decline in natural product-based drug discovery by most major pharmaceutical companies, the history of modern drug discovery and the relatively high proportion of FDA approved prescription drugs derived from plants. “....begs the question of whether plant (based compounds), secondary metabolites and related synthetic compounds perform better as drugs than randomly synthesized compounds [52
].” For this reason, we plan to continue investigations of this prototype library in search of compounds and/or combinations of compounds with therapeutic potential.
Third, while the therapeutic value of MCBT’s has not, as of yet, been firmly established, an important hypothetical adjunct favoring them is that they have the potential “…to provide combination therapies which can simultaneously target various elements of human diseases, providing efficacy and safety unmatched by NCE’s.” This multi-targeted “birdshot” approach may provide a viable alternative to the “silver bullet” NCE approach better designed to work with one symptom or pathogen at a time [52
There is also the undeniable realization that generations of traditional herbal practitioners who serve as repositories of millennia of empirical ethnobotanical knowledge are thinning in numbers. Unless reproducible research strategies are soon established to promote thoughtful, culturally sensitive and scientifically authoritative evaluation of herbal medicine systems, and evidence of their value, we may witness a decline in the number of well-educated trainees interested in maintaining these ethnomedical traditions worldwide. As a result, we may diminish opportunities for scientific discovery and the perpetuation of ethnobotanical study as a direct result of our failed collective stewardship of this shared global heritage.
The challenge to “form a firmly united front” [1
] by engaging experts in both traditional and modern medicine is still timely today. The difference is that we now have far more powerful technologies to illuminate our shared path towards this goal.
Only time will tell if the methods proposed here to establish a testable library of authenticated, commonly used medicinal plants will result in the discovery of new knowledge and the advent of novel therapeutic options. We will continue screening the existing library as resources allow. It is encouraging that the NIH and NCCAM have listed “Research on Complementary and Alternative Medicine pharmacologic interventions,” which presumably includes TCM and other herbal therapies, as a prioritized strategic objective for the next five years of NIH funding [61
]. It is hoped that this prototype TCM library as well as other natural product repositories with similar features, will enhance ongoing efforts to systematically evaluate commonly used herbal therapies worldwide.
An anonymous Chinese proverb says: “The methods used by one may be faulty. The methods used by many will be better.”