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 Autho5.6.1. 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￾herb 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