A total of 18 batches of HQT were included in the present study. Four batches coded as PHY906-6, 7, 8, 10 were manufactured with PhytoCeutica's proprietary SOP. Eight batches of HQT were purchased from Sun Ten Pharmaceutical Co. LTD in Taiwan and designated as HQT-E, F, G, H, I, J, K and L. Six batches of HQT were obtained from various vendors (Chung Song Zong, Ko Da, Min Tong, Sheng Chang, Sheng Foong, Kaiser; Taiwan) who did not provide quality information, and were labeled as HQT-CSZ, KD, MT, SC, SF and KP3. The proprietary standard operating procedures (SOP) by PhytoCeutica for PHY906 used hot water extraction (80°C) of four herbs, namely Scutellaria baicalensis Georgi (S), Paeonia lactiflora Pall. (P), Glycyrrhiza uralensis Fisch. (G) and Ziziphus jujuba Mill. (Z) (ratio 3:2:2:2). The hot water extraction is then spray dried with insoluble dextran into a granulated powder, packaged and stored in foil containers at 4°C.
Chemical standards including baicalin (S), baicalein (S), wogonin (S), scutellarin (S), glycyrrhizin (G), ononin (G), liquiritin (G), liqiritigenin (G), paeoniflorin (P) and albiflorin (P), were obtained from Chromadex (USA). Apigenin and formic acid were obtained from Sigma-Aldrich (USA). Solvents were of LC/MS grade from JT Baker (USA).
Dried PHY906 or HQT powder (100 mg) was dissolved in one mL of 80°C water. The mixture was vortexed for one minute, placed in an 80°C water bath for 30 additional minutes with one minute of vortexing for every ten minutes. The sample was then cooled in a water bath of ambient temperature for five minutes, centrifuged for ten minutes at 10,000 rpm (Eppendorf Model 5810R, USA) and the resulting supernatant was filter (0.2 μm) sterilized. For subsequent LC/MS analysis, a 20 μL aliquot of this light brown extract was diluted with 980 μL of water. The final nominal concentration after extraction and dilution was 2 mg of dry weight PHY906 or HQT powder extract per mL of water. For biological experiments, the 100 mg/mL nominal concentration solution stock was diluted in the appropriate buffer or medium to the required final concentration.
High-performance liquid chromatography (HPLC) was performed with a Waters (USA) CapLC XE Pump equipped with a CapLC autosampler and a Waters (USA) CapLC 2996 Photodiode Array Detector. The eluents were (A) 100% water with 0.1% formic acid and (B) 100% acetonitrile with 0.1% formic acid and the column was a Waters Atlantis dC18 3 μm 0.3 mm × 150 mm NanoEase column (USA). The column was heated to 40°C and was preceded by a 0.5 μm precolumn frit. Gradient elution from 0 to 50% B over 70 minutes at 8 μL/min was used with an initial hold of five minutes. The column was then ramped to 95% B over four minutes, held in place for two minutes and returned to initial conditions over two minutes. Total run time was 120 minutes. Electrospray ionization was performed on a Micromass (UK) Q-Tof-II mass spectrometer. Samples (0.5 μL) were introduced without splitting into the electrospray interface through a 60 μm stainless steel capillary tube. A positive capillary voltage of 3.25 kV was used in positive ion mode and a negative capillary voltage of 3.25 kV was used in negative ion mode. The electrospray source was heated to 80°C and the desolvation gas (N2) was heated to 150°C at a flow rate of 400 L/hr. The Q-Tof was scanned from 50-2000 amu over one second. The resolution of the instrument under these conditions was ~10,000. For exact mass measurements, a reserpine lock mass ([M+H] of 609 amu) was introduced at the electrospray interface allowing mass measurements to be within 0.0002 amu. With external standards, mass accuracy to 0.002 amu was routine with experimental and theoretical mass matching accuracy of 20 ppm or better.
Cell culture for gene expression studies
Three cell lines, namely Jurkat (ATCC no TIB-152), KB (ATCC no CCL-17) and HepG2 (ATCC no HB-8065), were selected for the experiments. HepG2 was selected for three reasons: (1) the cell line is stable, robust and well characterized; (2) the number of differentially expressed genes in HepG2 is generally observed to be higher than in the other two cell lines and (3) the liver is considered the primary drug-metabolizing organ for oral drugs. The HepG2 hepatocellular carcinoma cell line was cloned and a cell-bank created. A strict set of SOPs were developed to ensure reproducible growth characteristics including passage number and cell density. A HepG2 sub-clone cell was thawed with three passages to 80% confluency in 10% FBS complete MEME media at 37°C with 5% CO2. Computed IC50 values (concentration required to inhibit cell growth by 50%) were based on three independent experiments comparing a 72-hour exposure of the cells to eight concentrations ranging from 0.001 to 10 mg/mL of the PHY906-6 extract with control untreated cells. Cells were stained with 0.5% methylene blue, lysed with 1% sarcosine and cell viability determined by UV/VIS absorbance at A595.
Three independent experiments were performed on the HepG2 cells treated with one IC50 dose of the herbal extract or control buffer for 24 hours. At this time point, 100% of the cells were still viable. RNA was collected for gene profiling. GeneChip hybridization experiments with Affymetrix Human genome chip U133A (USA) were carried out at the Affymetrix Resource Laboratory, Yale University School of Medicine, USA. Data were processed with Microarray Suite 5.0 (Affymetrix, USA) software to generate a list of candidate genes for further investigation.
Quantitative real-time polymerase chain reaction (qRT-PCR) experiments
Selected gene probes were purchased as Assays-on-Demand from Applied Biosystems (USA) to confirm and quantify the candidate genes identified in the GeneChip experiments.
PHY906-6, 7, and 8 and HQT-F were compared for their effectiveness in potentiating the antitumor activity of the cancer chemotherapy drug CPT-11 or Camptosar® (Pfizer, USA). Female BDF-1 mice (Charles River Laboratories, USA) of 4-6 weeks old (16-20 grams) implanted with murine Colon 38 colorectal cancer cells (National Cancer Institute, USA) were used in the experiments. Colon 38 cells were grown in RPMI 1640 medium (JRH Biosciences, USA) supplemented with 10% fetal bovine serum and 100 μg/ml kanamycin. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2:95% air. For studies of the effects of PHY906 on antitumor efficacy and toxicity, Colon 38 cells (1-2 × 106 cells in 0.1 ml phosphate-buffered saline, PBS) were transplanted subcutaneously (sc) into the BDF-1 mice. The length and width of the tumors were measured with a sliding caliper. The tumor size (S) was estimated according to the formula as follows:
where L is length, W is width.
After 10 to 14 days, mice with tumor sizes of 150-300 mm3 were selected. Treatment groups consisted of five mice each. Tumor size, body weight and mortality of the mice were monitored daily. Mice were sacrificed when the tumor size reached 10% of the body weight.
PHY906 was administered per oral (po) whereas Camptosar® was administered intraperitoneally (ip). PHY906 was given twice daily (bid) at approximately 10 am and 3 pm. On days when Camptosar® was also administered, PHY906 was given 30 minutes earlier. Unless otherwise indicated, dosages were 500 mg/kg for PHY906 and 360 mg/kg for Camptosar®. Mice in the control groups were administered a vehicle of either PBS (ip) or water (po). All animal studies were conducted at the Yale University Animal Facility and approved by the Institutional Animal Care and Use Committee.
Pattern comparison by R value and Phytomics Similarity Index (PSI)
The linear correlation R value is a standard statistical method  used to compare two datasets and to compare the absolute intensity or value of each of the collected (N) data points. These data points can be either ion current spectral intensities collected by LC/MS, UV-VIS or relative gene expression level values determined by qRT-PCR. The R value varies between -1.0 (perfect anti-correlation) and 1.0 (perfect correlation) and is a measure of the similarity of the two sets of intensities. The Phytomics Similarity Index (PSI) is also a statistical method that compares the fingerprint patterns by computing a correlation value not of the intensities of the N peaks but rather on the ratio data computed for each of the N data points with each of the other (N-1) data points. Using these (N-1) ratio values in the computation for each of the N data points provides the similarity of that peak in relation to all of the other peaks in the fingerprint pattern (PCT US02/34121) The ratio information is incorporated into the analysis as it provides relative information between various peak intensities reflecting the importance of the balance of the compound amounts (or gene expression levels). As an example, the integrated ion counts for each of the N peaks (mass and retention time) are extracted from the overall spectra of two different batches (A and B). These N ion intensities, representing the chemical fingerprint of each batch, are placed, conceptually, along the diagonal of a matrix of dimension N × N and the ratios of the intensities are placed in the assigned Mi,j (i ≠ j and i, j ≤ N) off-diagonal matrix locations. There are, therefore, a total of N(N-1)/2 unique non-diagonal elements describing the full set of intensity ratio information between all of the peaks with each peak contributing (n-1) ratios. Matrices A and B were respectively designated as MA and MB. Each column/row in MA and MB may be represented by the vectors as follows:
The linear correlation is then computed using all of the columns or rows in both matrices.
The correlation value R for each column i.e. peak, can be obtained with the standard Pearson coefficient or the Spearman ranked coefficient . The result of this analysis is a vector of R scores, where each vector element corresponds to a data point (e.g. MS peak, or gene) that is common to both datasets. While each data point (i) has its own correlation score, Ri, the average of all of the individual R scores produces a diagnostic single value for similarity defined as the PSI. In this example, the PSI score would range between 0.0 (complete dissimilarity) to 1.0 (complete identity) to -1.0 (perfect anti-correlation). The individual PSI values can be weighted by a variety of factors including intensity, slope or biological importance. A weighting function found to be valuable is the individual peak slope calculated from plotting (n-1) ratios for peak i batch A to the equivalent (n-1) ratios for peak i in batch B. Highly similar batches tend to have PSI values greater than 0.85 with only a few outliers at lower PSI values. Batches that have poor similarity tend to have PSI values less than 0.75 with a greater number of individual outliers at lower PSI values. The PSI algorithm along with tools for filtering and sorting the LC/MS data were implemented in the software package PhytomicsQC™.