Anticancer Effects of Ursi Fel Extract and Its Active Compound, Ursodeoxycholic Acid, in FRO Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is one of the most fatal human malignancies. Ursi Fel (UF) is the bile of a brown bear that has been traditionally used for heat clearance and toxin relief in Korean and Chinese medicines. In this study, we determined the anticancer effects of a UF extract and its active compound, ursodeoxycholic acid (UDCA), in FRO human ATC cells. FRO cells were treated with UF extract and UDCA at different concentrations for various durations. Cell viability was measured using an MTT assay. Cell apoptosis was investigated by flow cytometric analysis following Annexin V and propidium iodide (PI) staining, and Hoechst staining was used to observe nuclear fragmentation. The expression of pro-apoptotic (Bax, caspase-3, cytochrome c, and PARP), anti-apoptotic (Bcl-2), and angiogenetic (TGF-ß, VEGF, N-cadherin, and sirtuin-1) proteins and the phosphorylation of Akt and mechanistic target of rapamycin (mTOR) were determined by western blot analysis. Treatment with UF extract at 10, 25, and 50 µg/mL and UDCA at 25, 50, and 100 µM/mL significantly inhibited the growth of FRO cells in a dose-dependent manner. Flow cytometry and Hoechst staining revealed an increase in the apoptosis of FRO cells mediated by UF extract and UDCA in a dose-dependent manner. UF extract (25 and 50 µg) and UDCA (50 and 100 µM) significantly increased the expression of Bax, caspase-3, cytochrome c, and PARP and inhibited the expression of Bcl-2, TGF-ß, VEGF, N-cadherin, and sirtuin-1 in FRO cells. Furthermore, UF extract and UDCA treatment stimulated Akt phosphorylation and inhibited mTOR phosphorylation in these cells. These results indicate that UF extract and UDCA exert anticancer properties in FRO cells by inducing apoptosis and inhibiting angiogenesis via regulating the Akt/mTOR signaling pathway.

Bile acids with a cyclopropyl-containing side chain. 3. Separation, identification, and properties of all four stereoisomers of 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid.

3 alpha,7 beta-Dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid (CUDCA) (2a), a side-chain cyclopropylog of ursodeoxycholic acid (UDCA) was shown to be a mixture of four stereoisomers (CUDCA A-D). The 22S,23R, and 22R,23S diastereoisomers have been separated, their respective configurations assigned by 13C NMR spectroscopy, and original synthetic schemes for their preparation elaborated. Moreover, theoretical models of the structure of UDCA and CUDCA A-D were built by using molecular computer graphic techniques. It was shown that the four diastereoisomers greatly differ in hydrophilicity, in critical micellar concentration (CMC) in water, and exhibit a different interaction with intestinal bacterial enzymes. It was also shown that CUDCA A-C are not conjugated with glycine or taurine in the liver, while CUDCA D is secreted into bile predominantly as taurine and glycine conjugate.

HPLC study of the impurities present in different ursodeoxycholic acid preparations: comparative evaluation of four detectors.

The use of HPLC with different detectors has been investigated for the analysis of bile acid impurities present in four different commercially available ursodeoxycholic acid preparations. The bile acids were efficiently separated by C18 reversed-phase HPLC using methanol-water (3:2, v/v) as the mobile phase. The detectors used for bile acid detection were: UV at 200 nm refractive index (RI) and an evaporative light scattering mass detector (ELSD II). A prederivatization method with the formation of a fluorescent naphthacyl ester has also been used. GC-MS analysis of Me-TMS bile acid derivatives was included as a reference method. The four ursodeoxycholic acid samples were 98-99% pure. The main impurities present in the samples were chenodeoxycholic acid and to a lesser extent lithocholic acid. Only one sample was found to be almost 100% pure using all the detectors. Significant agreement of the data was found between RI, ELSD II detectors and the fluorescent method; the UV detector was unsuitable for use in this method. The analytical performances of the four detectors for bile acid analysis are reported and discussed. When the four-detector data were compared with the GC-MS method, reasonable agreement resulted. Discordant results were found in the quantitation of trace impurities like lithocholic acid and/or other minor bile acids present in amounts less than 0.1%.

Characterization of sarcosylsarcoursodeoxycholic acid formed during the synthesis of sarcoursodeoxycholic acid.

This report describes the isolation of sarcosylsarcosine conjugate of ursodeoxycholic acid (UDCA) formed during the synthesis of sarcoUDCA by the mixed anhydride method. The compound was characterized by its chemical ionization mass spectrum. The diamino acid conjugate was formed only when the free amino acid was used for conjugation. This was confirmed by the isolation of glycylglycoUDCA during the conjugation of UDCA with free glycine but not with glycine ethyl ester hydrochloride. Pure sarcoUDCA was prepared by conjugation of UDCA with sarocisine methyl ester hydrochloride while sarcoUDCA on further reaction with the protected sarcosine derivative gave pure sarcosylsarcoUDCA in 52% yield.

Thin-layer chromatographic separation of conjugates of ursodeoxycholic acid from those of litho-, chenodeoxy-, deoxy-, and cholic acids.

Separation of the glycine and taurine conjugates of ursodeoxycholic acid from those of lithocholic acid, chenodeoxycholic acid, deoxycholic acid, and cholic acid by thin-layer chromatography is described. Thus, on running a silica gel G plate first in a solvent system of n-butanol-water 20:3 and then in a second solvent system of chloroform-isopropanol-acetic acid-water 30:20:4:1, all the above-mentioned conjugated bile acids are separated from one another. The application of this method to study the change in the biliary bile acid conjugation pattern in ursodeoxycholic acid-fed gallstone patients is described.