Baicalin is a major constituent of antiproliferative effects on CRC cells

Baicalin is a major constituent of antiproliferative effects on CRC cells were verified using an xenograft nude mouse model. of caspase 3, while interactions with residues Leu227 and Asp228 in caspase 9 through its hydroxyl groups. Data from this study suggested that baicalein is usually a potent anticancer metabolite produced from is usually a widely used herbal medicine in the traditional medical systems of China and Japan for a variety of inflammation related illnesses (10C13). The major constituents of this botanical are a group of flavonoid glycosides, including baicalin, and wogonoside, of which baicalin is usually the major constituent in the plant (14,15). is usually most often orally Keratin 8 antibody given. After oral ingestion, the constituents in the plant inevitably come into contact with intestinal microbiota. Many of these constituents could be transformed by the intestinal bacteria before being assimilated (16). As reported before, for natural glycosides such as ginsenosides, the most common metabolic pathway is usually the deglycosylation reaction induced by intestinal bacteria via the stepwise cleavage of the sugar moieties (17C19). After deglycosylation, compared to their parent compounds, the intestinal microbiome metabolites may have more potent biological activity (20C22). Anticancer activities of and its constituents were reported, but previous studies focused more on its natural sourced flavonoid glycosides (23,24). We recently observed that the major constituent of antiproliferative effects on CRC cells were confirmed using an xenograft nude mouse model. Then, we selected HCT-116 colon malignancy cells, which are most sensitive to baicalein treatment, for further mechanistic observations, including cell cycle arrest and apoptosis induction. Due to the fact that caspases are highly conserved in multicellular organisms and function as central regulators of apoptosis, levels of 21849-70-7 IC50 caspase manifestation were subsequently decided. Finally, the possible binding modes of baicalein at the catalytic domains of caspase 3 and 9 were simulated using the receptor-ligand docking analysis. Materials and methods Chemicals and materials All cell culture plasticware were obtained from Falcon Labware (Franklin Lakes, NJ, USA) and Techno Plastic Products (Trasadingen, Switzerland). Trypsin, McCoy’s 5A, Leibovitz’s T-15, RPMI-1640 and DMEM media, and phosphate-buffered saline were obtained from Mediatech, Inc. (Herndon, VA, USA). Penicillin and streptomycin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The MTS assay kit, CellTiter 96 Aqueous Answer Cell Proliferation Assay, was obtained from Promega (Madison, WI, USA). The Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (Rockville, MD, USA). PI/RNase staining buffer was obtained from BD Biosciences Pharmingen (San Diego, CA, USA). Caspase 3 and 9 ELISA kits were obtained from BioVison (Mountain View, CA, USA). Baicalin and wogonoside were obtained from Indofine Chemical Co. Inc. (Hillsborough Township, NJ, USA). Baicalein and wogonin were obtained from Sigma-Aldrich. Herb materials and extraction The roots of were obtained from Chengde (Hebei, China). The voucher samples were deposited at the Tang Center for Herbal Medicine Research at The University or college of Chicago. Dried roots were ground to powder, and the powdered roots were extracted with 70% ethanol for 2 h. 21849-70-7 IC50 The extraction method was boiling under reflux. The filtrate was collected and the extraction process was repeated one more time on the residue. The combined filtrate was condensed under vacuum and lyophilized to yield dried draw out (SbE). Biotransformation of SbE by human fecal microflora Fecal samples were obtained from five adult volunteers, who were non-smokers and had not consumed antibiotics for 3 months before the study. The samples were collected by the donors in plastic cups, and were processed within 30 min of passage. All five fecal samples were mixed and an aliquot of 5 g of the mixed feces was homogenized with 20 ml of phosphate buffer (pH 7.0) to obtain a fecal slurry. The slurry was filtered through muslin to remove particulate material. One microliter of the fecal slurry was mixed with 4 ml anaerobic medium containing 2.5 mg of SbE. They were anaerobically incubated at 37C for 0, 2 or 8 h. Then, 1 ml of reaction mixture was extracted three times with 400 l n-butanol/each time. The combined n-butanol solution was dried under nitrogen steam spray in a water bath (60C). Then the residue was dissolved in methanol. The methanol solution was centrifuged at 17,000 g for 10 min before HPLC analysis. High performance liquid chromatography (HPLC) analysis The HPLC system was a Waters 2960 instrument (Milford, MA, USA) with a quaternary pump, an automatic injector, a 21849-70-7 IC50 photodiode array detector (Model 996), and Waters Empower software for peak identification and integration. The separations were carried out.

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