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 Authoassessment 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