Eisenberg et al. Lastly, there is the inherent conundrum which speaks to the heart of the conceptual difference between traditional (ie. 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? B 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 al ways 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 variety of natural products[30]. Might clinical successes attributed to herbal therapies ummarized the existence of synergistic effects involving multiple compounds found in evolve additive and/or synergistic biological effects which can now be more meticulously described 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(modern biomedicine) develop shared research strategies which address the above mentioned known challenges without compromising the respective beliefs, practices and fundamental tenants of each tradition? Can ethnobotanists and modern drug discovery experts established an effective"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 be established for the purpose of successful systematic biological evaluation and will such a 9 with regard to the second hypothesis mentioned earlier, might there also be a new opportunity to employ contemporary high-throughput screening facilities to search for anticipated as well as unanticipated combinations of single compounds which can be shown to be therapeutically effective due to biological additivity and/or synergy of multiple plant derived compounds? Will combinations with evidence of additivity and/or synergy confirm ethnobotanical knowledge about specific plant combinations (i.e. formulas)or might entirely unanticipated active combinations also be identified? Autho
Lastly, 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 a variety of natural products [30]. Might clinical successes attributed to herbal therapies involve additive and/or synergistic biological effects which can now be more meticulously described 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 (modern biomedicine) develop shared research strategies which address the above mentioned known challenges without compromising the respective beliefs, practices and fundamental tenants of each tradition? Can ethnobotanists and modern drug discovery experts established an effective “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 be established for the purpose of successful systematic biological evaluation and will such a library, when screened strategically, lead to the identification of novel compounds, new uses for 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 new opportunity to employ contemporary high-throughput screening facilities to search for anticipated as well as unanticipated combinations of single compounds which can be shown to be therapeutically effective due to biological additivity and/or synergy of multiple plant derived compounds? Will combinations with evidence of additivity and/or synergy confirm ethnobotanical knowledge about specific plant combinations (i.e. formulas) or might entirely unanticipated active combinations also be identified? Eisenberg et al. Page 6 Fitoterapia. Author manuscript; available in PMC 2012 January 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
4.3. Considerations in the development of a prototy pe authenticated TCM plant library Between 2006 and 2010, investigators from Harvard Medical School(HMS),in collaboration with colleagues from the Beijing University of Chinese Medicine(BUCM) and Hong Kong Baptist University(HKBU), embarked on efforts to build a prototype library of 202 botanically authenticated, quality controlled, collection site-documented Chinese medicinal plants. Each plant was to be tested for pesticide and heavy metal contamination and qualified using standards defined by the Chinese Pharmacopeia [31],an official publication of the State Food and Drug Administration (SFDA)of the Peoples Republic of China. Such an artisan library could serve as a necessary prerequisite to reproducible pre-clinical studies of TCM herbs. These are mandatory prerequisites to the subsequent design and implementation of the next generation of animal and human studies of herbal remedies. Moreover, the ethnobotanical knowledge of TCM experts is essential to establish this library, and to efficiently guide drug development experts in their search for novel single compounds, novel uses of previously identified compounds and hypothetically novel demonstrations of additivity and/or synergy involving multiple plant compounds hin plants and within complex mixtures of medicinal plants In planning for, constructing and evaluating this authenticated TCM herbal library, many factors had to be considered including: the selection criteria of medicinal plants to be ncluded in the research library; quality assurance including harvest sites and gPs documentation; botanical species authentication; collection protocols; plant processing details; creation of voucher specimens; testing for pesticides and heavy metal contamination; chemical and quality assessment according to existing standards of the Chinese P9z Pharmacopeia; Chinese governmental cooperation, authorization and partnership; storage and shipping logistics of all specimens in China and the US; precise and reproducible extraction and fractionation procedures; selection of appropriate biological screening strategies; establishment of a suitable database and database management system; and continuous efforts to maintain open communications and opportunities for expanded research collaboration among all participating co-investigators 5. Methods (See Fig. I for an overview of the methods employed in the creation of this prototype TCM library) 5.1. Criteria for inclusion The selection of plants for the authenticated TCM herbal library was based on two main criteria:(1)the plants were listed in the official Pharmacopoeia of the People's republic of hina( CP)[31] and, (2)the species were not endangered. This latter criterion is of concerr since 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 on International Trade in Endangered Species of Wild Flora and Fauna(CITES)list, including famous medicines such as Mu Xiang (Saussurea costus(Falc )Lipsch )and Rou Cong ro ( Cistanche deserticola Y C Ma)[33]. The official CP includes synthetic pharmaceut ong 于9 is study was launched, the most recent edition was the 2005 Chinese Pharmacopea he medicines, but also many traditional medicines derived from natural At the tin The first volume of the 2005 CP includes monographs on Traditional Materia Medica and comprised of substances derived from animals, minerals, plants, and fungi. A total of 471 monographs based on plant products are included in this volume, comprising 550 different species. The prototype library included a total of 200 species of plants and 2 species of fungi, representing more than one-third of all plant species listed in the CP. The 202 selected Autho
4.3. Considerations in the development of a prototype authenticated TCM plant library Between 2006 and 2010, investigators from Harvard Medical School (HMS), in collaboration with colleagues from the Beijing University of Chinese Medicine (BUCM) and Hong Kong Baptist University (HKBU), embarked on efforts to build a prototype library of 202 botanically authenticated, quality controlled, collection site-documented Chinese medicinal plants. Each plant was to be tested for pesticide and heavy metal contamination and qualified using standards defined by the Chinese Pharmacopeia [31], an official publication of the State Food and Drug Administration (SFDA) of the People’s Republic of China. Such an artisan library could serve as a necessary prerequisite to reproducible pre-clinical studies of TCM herbs. These are mandatory prerequisites to the subsequent design and implementation of the next generation of animal and human studies of herbal remedies. Moreover, the ethnobotanical knowledge of TCM experts is essential to establish this library, and to efficiently guide drug development experts in their search for novel single compounds, novel uses of previously identified compounds and hypothetically, novel demonstrations of additivity and/or synergy involving multiple plant compounds within plants and within complex mixtures of medicinal plants. In planning for, constructing and evaluating this authenticated TCM herbal library, many factors had to be considered including: the selection criteria of medicinal plants to be included in the research library; quality assurance including harvest sites and GPS documentation; botanical species authentication; collection protocols; plant processing details; creation of voucher specimens; testing for pesticides and heavy metal contamination; chemical and quality assessment according to existing standards of the Chinese Pharmacopeia; Chinese governmental cooperation, authorization and partnership; storage and shipping logistics of all specimens in China and the US; precise and reproducible extraction and fractionation procedures; selection of appropriate biological screening strategies; establishment of a suitable database and database management system; and, continuous efforts to maintain open communications and opportunities for expanded research collaboration among all participating co-investigators. 5. Methods (See Fig. 1 for an overview of the methods employed in the creation of this prototype TCM library). 5.1. Criteria for inclusion The selection of plants for the authenticated TCM herbal library was based on two main criteria: (1) the plants were listed in the official Pharmacopoeia of the People’s Republic of China (CP) [31] and, (2) the species were not endangered. This latter criterion is of concern since 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 on International Trade in Endangered Species of Wild Flora and Fauna (CITES) list, including famous medicines such as Mu Xiang (Saussurea costus (Falc.) Lipsch.) and Rou Cong Rong (Cistanche deserticola Y. C. Ma) [33]. The official CP includes synthetic pharmaceutical medicines, but also many traditional medicines derived from natural sources. At the time this 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 is comprised of substances derived from animals, minerals, plants, and fungi. A total of 471 monographs based on plant products are included in this volume, comprising 550 different species. The prototype library included a total of 200 species of plants and 2 species of fungi, representing more than one-third of all plant species listed in the CP. The 202 selected Eisenberg et al. Page 7 Fitoterapia. Author manuscript; available in PMC 2012 January 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Eisenberg et al. plants(see Appendix A)are some of the most commonly used in the CP and hence are estimated 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, ontraindications, 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 the traditional method as specified by the CP. This typically involved the removal of impurities uch as other plant species or soil, followed by drying in the sun for a period of days or weeks. Lastly, vouchers of each sample, consisting of flowering or fruiting material of the plant, were collected according to standard protocols [34]. Vouchers have been stored along with a voucher of the bulk harvest medicinal part for future reference. The plant acquisition team, organized and overseen by BUCM co-investigators and consultants from HKB onsisted of at least one Chinese herbal medicine resource expert from each of the 30 provinces and autonomous zones where plant acquisition took place. In addition, a 34 person Beijing-based acquisition team consisting of faculty and graduate students of BUCM as well as 8 additional TCM botanical experts oversaw the quality control and processing of all plant samples collected. Authentication of plant species was overseen by a separate group of faculty with TCM ethnobotanical expertise at HKBU. All steps of plant collection were documented using a combination of standardized collection forms gPs data collection photographs, and video. (See Appendices B-E for examples of photos imported into studys 9 database). After harvest and processing, plants were authenticated and tested for quality(see sections 5.3 and 5.4) 5.3. Authentication and quality assessment The taxonomic identification of each plant was confirmed by multiple experts in China Plants were authenticated according to morphological and anatomical characteristics [35]by a team at HKBU and examined according to criteria listed in the CP for quality assessment at BUCM. The quality assessment tests were conducted according to the guidelines and standards provided in the CP 2005 edition [31]. The CP specifies the method for conducting elevant tests and provides standard reference values for results. In general, the quality Autho
plants (see Appendix A) are some of the most commonly used in the CP and hence are estimated 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 the traditional method as specified by the CP. This typically involved the removal of impurities such as other plant species or soil, followed by drying in the sun for a period of days or weeks. Lastly, vouchers of each sample, consisting of flowering or fruiting material of the plant, were collected according to standard protocols [34]. Vouchers have been stored along with a voucher of the bulk harvest medicinal part for future reference. The plant acquisition team, 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 30 provinces and autonomous zones where plant acquisition took place. In addition, a 34 person Beijing-based acquisition team consisting of faculty and graduate students of BUCM as well as 8 additional TCM botanical experts oversaw the quality control and processing of all plant samples collected. Authentication of plant species was overseen by a separate group of faculty with TCM ethnobotanical expertise at HKBU. All steps of plant collection were documented using a combination of standardized collection forms, GPS data collection, photographs, and video. (See Appendices B–E for examples of photos imported into study’s database). After harvest and processing, plants were authenticated and tested for quality (see sections 5.3 and 5.4). 5.3. Authentication and quality assessment The taxonomic identification of each plant was confirmed by multiple experts in China. Plants were authenticated according to morphological and anatomical characteristics [35] by a team at HKBU and examined according to criteria listed in the CP for quality assessment at BUCM. The quality assessment tests were conducted according to the guidelines and standards provided in the CP 2005 edition [31]. The CP specifies the method for conducting relevant tests and provides standard reference values for results. In general, the quality Eisenberg et al. Page 8 Fitoterapia. Author manuscript; available in PMC 2012 January 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Eisenberg et al. 9 assessment tests include methods for determining the identity and purity of the plant. For the purposes of the quality assessment tests, the identity of the plant is determined using methods such as qualitative thin-layer chromatography (TLC)and reactivity tests. There are also quantitative tests to determine the concentration of certain chemical ingredients. For example, the CP stipulates that licorice root(Glycyrrhiza glabra l )must have at least 2.0% glycyrrhizic acid by dry weight [31]. Lastly, some of the quality assessment tests relate to >E9 determination of impurities in the plants. For example, many species require a test of water and ash content. The test of water content is to determine how well the sample may b preserved, and the test of ash content provides information about the amount of solid impurities, such as soil, that are included with the plant sample Samples which did not meet quality assessment tests were considered to be unsuitable for subsequent study as they d not meet established and reproducible standards of quality assurance. The list of quality assessment tests stipulated by the CP is provided in Table 1. The methods used for deciding on the results of quality assessment tests and whether the results would necessitate plant ecollection are summarized in Fig. 2. 5.4. Testing for heavy metals and pesticides Following standards established by NSF International and American Standards Institute (ANSI) Standard 173 for acceptable metal levels in dietary supplements [36], all plants were tested for heavy metal and pesticide content, including five heavy metals(As, Cd, Cr, Pb and Hg)and pesticide residue There is currently considerable variation between different countries for testing pesticides in dietary supplements [37], and typically only a few pesticides are routinely examined. A comprehensive approach to pesticides testing was used in this project, and a broad screen including 162 pesticide residues was employed. More details pertaining to these research methods are reported separately [38] 5.5. Shipping, import, export and maintenance of plant materials The logistics involved in the dried plant material supply chain, from when the material was transferred from BUCM to HMS possession, included abiding by the export/import regulations for all countries involved, freight forwarders, and insurance to protect the esources. Prior to shipment, it was necessary to apply for and obtain the appropriate import permits from the United States Department of Agriculture Animal and Plant Inspection Services(USDA APIs). With the USDA APIS permit in hand, agreements were executed. At certain steps of the project, it was necessary that the dried plant material bulk shipments be maintained in a pest-free, climate-controlled storage facility. For example, the samples were held in China prior to shipment to the United States. After the samples arrived in the United States they were placed in storage. In both instances, bulk samples were maintained in a special storage facility with controlled temperature and humidity. The samples currently maintained in the United States are kept at 20C and 50% relati humidity in a state-of-the-art facility 9 5.6. Selecting one of three samples for initial extraction and fractionation Each species in the library was collected from three separate locations, but only one sample per species was to be initially extracted and fractionated for high throughput screening Samples were selected for extraction and fractionation using two main criteria: (1)absence or lowest values of heavy metal and pesticide contamination among samples collected for each species; and(2)ease of collection. Heavy metal and pesticide amounts were determined as described elsewhere [38]. Ease of collection was judged by collection experts at BUCM. The explicit decision tree used to make determinations is summarized in Fig. 3 Autho
assessment tests include methods for determining the identity and purity of the plant. For the purposes of the quality assessment tests, the identity of the plant is determined using methods such as qualitative thin-layer chromatography (TLC) and reactivity tests. There are also quantitative tests to determine the concentration of certain chemical ingredients. For example, the CP stipulates that licorice root (Glycyrrhiza glabra L.) must have at least 2.0% glycyrrhizic acid by dry weight [31]. Lastly, some of the quality assessment tests relate to determination of impurities in the plants. For example, many species require a test of water and ash content. The test of water content is to determine how well the sample may be preserved, and the test of ash content provides information about the amount of solid impurities, such as soil, that are included with the plant sample. Samples which did not meet quality assessment tests were considered to be unsuitable for subsequent study as they did not meet established and reproducible standards of quality assurance. The list of quality assessment tests stipulated by the CP is provided in Table 1. The methods used for deciding on the results of quality assessment tests and whether the results would necessitate plant recollection are summarized in Fig. 2. 5.4. Testing for heavy metals and pesticides Following standards established by NSF International and American Standards Institute (ANSI) Standard 173 for acceptable metal levels in dietary supplements [36], all plants were tested for heavy metal and pesticide content, including five heavy metals (As, Cd, Cr, Pb and Hg) and pesticide residues. There is currently considerable variation between different countries for testing pesticides in dietary supplements [37], and typically only a few pesticides are routinely examined. A comprehensive approach to pesticides testing was used in this project, and a broad screen including 162 pesticide residues was employed. More details pertaining to these research methods are reported separately [38]. 5.5. Shipping, import, export and maintenance of plant materials The logistics involved in the dried plant material supply chain, from when the material was transferred from BUCM to HMS possession, included abiding by the export/import regulations for all countries involved, freight forwarders, and insurance to protect the resources. Prior to shipment, it was necessary to apply for and obtain the appropriate import permits from the United States Department of Agriculture Animal and Plant Inspection Services (USDA APIS). With the USDA APIS permit in hand, service agreements were executed. At certain steps of the project, it was necessary that the dried plant material bulk shipments be maintained in a pest-free, climate-controlled storage facility. For example, the samples were held in China prior to shipment to the United States. After the samples arrived in the United States they were placed in storage. In both instances, bulk samples were maintained in a special storage facility with controlled temperature and humidity. The samples currently maintained in the United States are kept at 20 °C and 50% relative humidity in a state-of-the-art facility. 5.6. Selecting one of three samples for initial extraction and fractionation Each species in the library was collected from three separate locations, but only one sample per species was to be initially extracted and fractionated for high throughput screening. Samples were selected for extraction and fractionation using two main criteria: (1) absence or lowest values of heavy metal and pesticide contamination among samples collected for each species; and (2) ease of collection. Heavy metal and pesticide amounts were determined as described elsewhere [38]. Ease of collection was judged by collection experts at BUCM. The explicit decision tree used to make determinations is summarized in Fig. 3. Eisenberg et al. Page 9 Fitoterapia. Author manuscript; available in PMC 2012 January 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Eisenberg et al. 5.6.1. Extraction and fractionation procedures-Between 1 and 4 kg of the herb samples selected for extraction and fractionation were subjected to industrial-scale grinding, followed by extraction in 95%(V/v)ethanol for 30 min with sonication, using a solvent-to- be extracted. Tannins were removed from the resulting ethanol extracts via polyamide an herb volume ratio of 7: 1. Using this method, compounds with a broad range of polarity column chromatography(polyamide: extract=10: 1), followed by desalting and defatting on HP-20 resin(HP-20: extract=50: 1). The HP-20 column was washed with water to remove salts, then with ethanol, and finally with 50% ethanol/dichloromethane to remove fatty acids. The resulting ethanol eluate was subjected to large-scale reverse-phase C-18 HPLC chromatography, with step-wise elution using 2% ethanol increments from 0 to 100% in 70 min and then 100% ethanol for 26 min, resulting in approximately 48 fractions per extract Target quantities were 15 mg eluted material per fraction: 15 mg or less of each fraction was then dried in 4 ml glass vials for future formatting for screening. For each fraction for which more than 15 mg of material was obtained, the extra material was dried in appropriately ized glass vials. All dried fractions were stored at -20C until prepared for screening or used for further purification steps 5.6.2. Preparing extract fractions for screening-To re-suspend fractions, 100% DMSO was added to each dried fraction in 4 ml vials for a final concentration of 15 mg/ml DMSO was added using the Biomek FX(Beckman Coulter, Inc )automated pipetting work station, allowing 352 fractions to be processed in each plating session. Small (10 mm) Teflon-coat stir bars (V&P Scientific)were added to each 4 ml glass vial and vials were stirred overnight at room temperature. Fractions that did not go into solution by stirring were and-pipetted briefly the next morning to fully re-suspend them. Fractions were aliquoted by P9z hand into 96-deep well plates(VWR 4002-011). To make plates for screening, extracts were re-formatted from the 96-well master plates into 384-well plates(ABgene AB-1056)using a Velocity-ll V-Prep(Agilent) automated pipetting work station. All plated extracts were ored at-20°C 5.7. Construction of electronic database In order to meet the data requirements of this initiative, a novel database was designed to organize and store data related to all aspects of plant collections, authentication, shipping processing and biological screening. The database, called the"Traditional Medicine ollection Tracking System (TM-CTS), was designed to support the daily needs of the project in the context of the international collaboration between investigators in the United States and China, and to include information related to all aspects of the project from plant collection through extraction and fractionation. In addition the database was constructed to work in concert with the screening database used in the project. The TM-CTS is described in a separate paper 391 5.8. Initial screening strategies extracts were based on five major themes. First, assays needed to represent therapeutic areas 于9 of high unmet medical need. Second, assays were to represent the most"cutting-edge biological advances in their respective therapeutic areas, in recognition of the fact that the goal was to identify and develop high-impact new drugs with therapeutic efficacies or scopes going beyond existing medicines. Third, assays needed to be carried out on small enough scales to support high throughput screening(HTS)in 96-or 384-well plate formats, and to not require unreasonably large amounts of material. More specifically, targeted assay volumes ranged from 0. 1 to 1.0 uL per assay point, with occasional exceptions made for 8- 10 uL per assay point for certain cell-based assays considered to be of uniquely high value Fourth, sufficient down-stream assay capabilities, particularly the existence of appropriate in Autho
5.6.1. Extraction and fractionation procedures—Between 1 and 4 kg of the herb samples selected for extraction and fractionation were subjected to industrial-scale grinding, followed by extraction in 95% (v/v) ethanol for 30 min with sonication, using a solvent-toherb volume ratio of 7:1. Using this method, compounds with a broad range of polarity can be extracted. Tannins were removed from the resulting ethanol extracts via polyamide column chromatography (polyamide: extract=10:1), followed by desalting and defatting on HP-20 resin (HP-20: extract=50:1). The HP-20 column was washed with water to remove salts, then with ethanol, and finally with 50% ethanol/dichloromethane to remove fatty acids. The resulting ethanol eluate was subjected to large-scale reverse-phase C-18 HPLC chromatography, with step-wise elution using 2% ethanol increments from 0 to 100% in 70 min and then 100% ethanol for 26 min, resulting in approximately 48 fractions per extract. Target quantities were 15 mg eluted material per fraction: 15 mg or less of each fraction was then dried in 4 ml glass vials for future formatting for screening. For each fraction for which more than 15 mg of material was obtained, the extra material was dried in appropriately sized glass vials. All dried fractions were stored at −20 °C until prepared for screening or used for further purification steps. 5.6.2. Preparing extract fractions for screening—To re-suspend fractions, 100% DMSO was added to each dried fraction in 4 ml vials for a final concentration of 15 mg/ml. DMSO was added using the Biomek FX (Beckman Coulter, Inc.) automated pipetting work station, allowing 352 fractions to be processed in each plating session. Small (10 mm) Teflon-coat stir bars (V&P Scientific) were added to each 4 ml glass vial and vials were stirred overnight at room temperature. Fractions that did not go into solution by stirring were hand-pipetted briefly the next morning to fully re-suspend them. Fractions were aliquoted by hand into 96-deep well plates (VWR 4002-011). To make plates for screening, extracts were re-formatted from the 96-well master plates into 384-well plates (ABgene AB-1056) using a Velocity-11 V-Prep (Agilent) automated pipetting work station. All plated extracts were stored at −20 °C. 5.7. Construction of electronic database In order to meet the data requirements of this initiative, a novel database was designed to organize and store data related to all aspects of plant collections, authentication, shipping, processing and biological screening. The database, called the “Traditional Medicine Collection Tracking System (TM-CTS),” was designed to support the daily needs of the project in the context of the international collaboration between investigators in the United States and China, and to include information related to all aspects of the project from plant collection through extraction and fractionation. In addition, the database was constructed to work in concert with the screening database used in the project. The TM-CTS is described in a separate paper [39]. 5.8. Initial screening strategies Strategies used for screening the initial collection of several thousand pre-fractionated extracts were based on five major themes. First, assays needed to represent therapeutic areas of high unmet medical need. Second, assays were to represent the most “cutting-edge” biological advances in their respective therapeutic areas, in recognition of the fact that the goal was to identify and develop high-impact new drugs with therapeutic efficacies or scopes going beyond existing medicines. Third, assays needed to be carried out on small enough scales to support high throughput screening (HTS) in 96- or 384-well plate formats, and to not require unreasonably large amounts of material. More specifically, targeted assay volumes ranged from 0.1 to 1.0 μL per assay point, with occasional exceptions made for 8– 10 μL per assay point for certain cell-based assays considered to be of uniquely high value. Fourth, sufficient down-stream assay capabilities, particularly the existence of appropriate in Eisenberg et al. Page 10 Fitoterapia. Author manuscript; available in PMC 2012 January 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript