Eisenberg et al. vivo animal models for the particular disease being targeted, needed to exist, in order to ensure that any lead compounds identified in initial screening campaigns could be furthe developed pharmacologically to a point where drug development value could be firmly established. Fifth, assays needed to offer the possibility of eventual patent-protected licensable opportunities(compound, target, or both), since it was recognized that eventual development of lead compounds would depend on interest by the pharmaceutical industry, which would likely require appropriate intellectual property protections allowing for a return n investment Guided by the above five principles, the first several thousand fractions were screened across a range of assays representing a variety of therapeutic areas. Although beyond the scope of this manuscript, active screens included targets in the following areas: oncology (angiogenesis, tumor-specific differential cytotoxicity, and cell survival pathways), anti infectives(bacterial, anthrax), anti-virals(HIv-1), diabetes and neurodegeneration (Alzheimer's disease, Parkinsons disease). Other therapeutic areas under consideration included assays for novel targets in arthritis, cancer(and non-cancer) stem cells, cardiovascular disease, anti-infectives (Methicillin-resistant Staphylococcus aureus, tuberculosis, influenza A/HINI), hepatitis C, immuno-oncology approaches, inflammation, multiple sclerosis, and pain 5.9. Description of selected (initial)screens involving cell based and whole organism(e.g Zebra fish)assays Human fibroblasts and cancer cell lines [LNCaP, PC3 (Prostate), MCF7, MDA-231 ( Breast, A549 (Lung), BJ Fibroblasts] were propagated using standard conditions as suggested by ATCC. Cell proliferation and/or survival were determined by using Cell Titer lo(Promega)in a 384-well format assay as previously described [40] For zebra fish screening, fractions were delivered robotically to 96 well plates. Four embryos were added per well by pipette at roughly 6 h post fertilization to allow early development to occur before treatment. More than 1000 extract fractions were initially screened at nominal concentrations(based on the assumption of a pure compound of Mw from 6 h post fertilization to 72 h post fertilization. All wells were examined by lighr s 500)on embryos in 10% Hanks Saline with 0.003% phenyl thiourea in water, obse microscope to look for signs of altered development and impaired angiogenesis Fractions that showed grossly defective development were not further analyzed 5.9.1. LC/Ms and NMR techniques for compound characterization-LC/MS was performed on an Agilent 6130 Single Quadrupole system. NMR spectra of active compounds were obtained on a Varian 600 MHz spectrometer. Compounds were identified either using authentic samples or comparing their chemical shifts with those reported in the 9 5.9.2. International collaboration agreements-Agreements covering the scope of work, ownership of materials and intellectual property, sharing of potential financial revenues and co-authorship of academic publications were negotiated by representatives of the participating US and Chinese Academic Institutions. These were also reviewed by representatives of the Chinese Ministry of Health, Chinese Ministry of Education, State Administration of TCM, Chinese Fda and the chinese ministry of Science and Autho
vivo animal models for the particular disease being targeted, needed to exist, in order to ensure that any lead compounds identified in initial screening campaigns could be further developed pharmacologically to a point where drug development value could be firmly established. Fifth, assays needed to offer the possibility of eventual patent-protected licensable opportunities (compound, target, or both), since it was recognized that eventual development of lead compounds would depend on interest by the pharmaceutical industry, which would likely require appropriate intellectual property protections allowing for a return on investment. Guided by the above five principles, the first several thousand fractions were screened across a range of assays representing a variety of therapeutic areas. Although beyond the scope of this manuscript, active screens included targets in the following areas: oncology (angiogenesis, tumor-specific differential cytotoxicity, and cell survival pathways), antiinfectives (bacterial, anthrax), anti-virals (HIV-1), diabetes and neurodegeneration (Alzheimer’s disease, Parkinson’s disease). Other therapeutic areas under consideration included assays for novel targets in arthritis, cancer (and non-cancer) stem cells, cardiovascular disease, anti-infectives (Methicillin-resistant Staphylococcus aureus, tuberculosis, influenza A/H1N1), hepatitis C, immuno-oncology approaches, inflammation, multiple sclerosis, and pain. 5.9. Description of selected (initial) screens involving cell based and whole organism (e.g. Zebra fish) assays Human fibroblasts and cancer cell lines [LNCaP, PC3 (Prostate), MCF7, MDA-231 (Breast), A549 (Lung), BJ Fibroblasts] were propagated using standard conditions as suggested by ATCC. Cell proliferation and/or survival were determined by using Cell Titer Glo (Promega) in a 384-well format assay as previously described [40]. For zebra fish screening, fractions were delivered robotically to 96 well plates. Four embryos were added per well by pipette at roughly 6 h post fertilization to allow early development to occur before treatment. More than 1000 extract fractions were initially screened at nominal concentrations (based on the assumption of a pure compound of MW 500) on embryos in 10% Hank’s Saline with 0.003% phenyl thiourea in water, observing from 6 h post fertilization to 72 h post fertilization. All wells were examined by light microscope to look for signs of altered development and impaired angiogenesis. Fractions that showed grossly defective development were not further analyzed. 5.9.1. LC/MS and NMR techniques for compound characterization—LC/MS was performed on an Agilent 6130 Single Quadrupole system. NMR spectra of active compounds were obtained on a Varian 600 MHz spectrometer. Compounds were identified either using authentic samples or comparing their chemical shifts with those reported in the literature. 5.9.2. International collaboration agreements—Agreements covering the scope of work, ownership of materials and intellectual property, sharing of potential financial revenues and co-authorship of academic publications were negotiated by representatives of the participating US and Chinese Academic Institutions. These were also reviewed by representatives of the Chinese Ministry of Health, Chinese Ministry of Education, State Administration of TCM, Chinese FDA and the Chinese Ministry of Science and Technology. Eisenberg et al. Page 11 Fitoterapia. Author manuscript; available in PMC 2012 January 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Eisenberg et al. 6. Results 6.1. Current status of the authenticated TCM library The initial plans of the participating co-investigators involved the collection of 202 individual TCM species in triplicate and the completion of all aspects of the methods described earlier as applied to all collected specimens. As envisioned, this would have >E9 resulted in an estimated 10.000 fractions. a total of 3709 fractions from 80 TCM authenticated species have been created and screened to date. Consequently, the results reported here are those that have been obtained based on work completed thus far At least one bulk sample has been collected from each of 202 species of TCM plants and fungi. Of these, 186 species have been authenticated according to macroscopic and microscopic characteristics, 158 species have been tested for quality according to standards in the CP [31] and 129 species have been tested for heavy metals and pesticides. The bulk samples of 136 species and voucher samples of 79 species have been delivered to HMS with the remaining samples in China. All of the data and images related to collections authentication, extraction, and fractionation, are currently stored in the projects TM-CtS database for future reference [39] The mean number of fractions per sample is 45(median 47; range 19-52). In addition to the fractions that were eluted from the HPLC column, the total number of fractions per plant always included four additional fractions that represent the crude extract, the desalting and defatting washes of the crude extract, and the cleaned extract prior to fractionation For example, the m number of fractions for one plant, 52, is equal to these four fractions P9z plus a maximum of 48 possible fractions that are eluted from the HPLC fractionation column. These four additional fractions were included in the screening library. See Table 2 for a summary of the status of the library to date 6. 2. Summary of plant species included Almost all (-9g%)of the plants included in the prototype TCM library are flowering plants The library includes 2 species of gymnosperm and 2 species of fungi. Of the flowering plants, the most commonly represented families are: Asteraceae, (10.7%), Rosaceae, (5.9%) Lamiaceae, (5.4%), Fabaceae, (4.9%), and Liliaceae, (4.9%) Of the bulk samples in the library, the majority come from underground parts of plants(42. 4%), including bulbs, rhizomes, roots, and tubers. Other medicinal plant parts in the collection, consistent with TCM practice, include fruit and/or seed(29.8%), leaf and/or stem(17. 1%), flower(5.4%) d whole plant (4.4%). The names of all species included in the library appear in Appendix In terms of the natural geographic range of species in the collection, 17% of the species in the collection are endemic to China, as compared with estimates of 30-50% species endemic to China as a whole [41]. The most common distribution pattern of species in the collection 9 is one that covers multiple countries in Asia(56%). For example, Schisandra chinensi (Turcz. )Baill. occurs in China, Japan, Korea, and Russia [41]. In addition, roughly 5-10% of the plants in the collection are originally exotic introductions from countries outside of summary of native range distributions of plants in the prototype TCM library is shha y iE China. For example, fennel(Foeniculum vulgare Mill. )and pomegranate(Punica granatum L )are plants in the CP that are originally from the Mediterranean region of Europe n In Fig he plants in the library have been collected from a wide geographical range of sites hroughout China, including sites in 21 of the 23 provinces and 4 out of 5 autonomous Autho
6. Results 6.1. Current status of the authenticated TCM library The initial plans of the participating co-investigators involved the collection of 202 individual TCM species in triplicate and the completion of all aspects of the methods described earlier as applied to all collected specimens. As envisioned, this would have resulted in an estimated 10,000 fractions. A total of 3709 fractions from 80 TCM authenticated species have been created and screened to date. Consequently, the results reported here are those that have been obtained based on work completed thus far. At least one bulk sample has been collected from each of 202 species of TCM plants and fungi. Of these, 186 species have been authenticated according to macroscopic and microscopic characteristics, 158 species have been tested for quality according to standards in the CP [31] and 129 species have been tested for heavy metals and pesticides. The bulk samples of 136 species and voucher samples of 79 species have been delivered to HMS, with the remaining samples in China. All of the data and images related to collections, authentication, extraction, and fractionation, are currently stored in the project’s TM-CTS database for future reference [39]. The mean number of fractions per sample is 45 (median 47; range 19–52). In addition to the fractions that were eluted from the HPLC column, the total number of fractions per plant always included four additional fractions that represent the crude extract, the desalting and defatting washes of the crude extract, and the cleaned extract prior to fractionation. For example, the maximum number of fractions for one plant, 52, is equal to these four fractions plus a maximum of 48 possible fractions that are eluted from the HPLC fractionation column. These four additional fractions were included in the screening library. See Table 2 for a summary of the status of the library to date. 6.2. Summary of plant species included Almost all (~99%) of the plants included in the prototype TCM library are flowering plants. The library includes 2 species of gymnosperm and 2 species of fungi. Of the flowering plants, the most commonly represented families are: Asteraceae, (10.7%), Rosaceae, (5.9%), Lamiaceae, (5.4%), Fabaceae, (4.9%), and Liliaceae, (4.9%). Of the bulk samples in the library, the majority come from underground parts of plants (42.4%), including bulbs, rhizomes, roots, and tubers. Other medicinal plant parts in the collection, consistent with TCM practice, include fruit and/or seed (29.8%), leaf and/or stem (17.1%), flower (5.4%), and whole plant (4.4%). The names of all species included in the library appear in Appendix A. In terms of the natural geographic range of species in the collection, 17% of the species in the collection are endemic to China, as compared with estimates of 30–50% species endemic to China as a whole [41]. The most common distribution pattern of species in the collection is one that covers multiple countries in Asia (56%). For example, Schisandra chinensis (Turcz.) Baill. occurs in China, Japan, Korea, and Russia [41]. In addition, roughly 5–10% of the plants in the collection are originally exotic introductions from countries outside of China. For example, fennel (Foeniculum vulgare Mill.) and pomegranate (Punica granatum L.) are plants in the CP that are originally from the Mediterranean region of Europe. A summary of native range distributions of plants in the prototype TCM library is shown in Fig. 4. The plants in the library have been collected from a wide geographical range of sites throughout China, including sites in 21 of the 23 provinces and 4 out of 5 autonomous Eisenberg et al. Page 12 Fitoterapia. Author manuscript; available in PMC 2012 January 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript