[Bile acid derivatives-focused chemical profiling in snake bile].

As a precious traditional Chinese medicine(TCM), snake bile has been widely used in numerous Chinese medicine prescriptions. Bile acid(BA) derivatives have been demonstrated as the primary chemical family in snake bile. In-depth chemical characterization of BAs is of great importance towards the establishment of quality standards and clarification of the effective material basis for snake bile. This study firstly employed ~1H-NMR to preliminarily analyze the chemical profiles of snake bile, an automated fraction collector was subsequently implemented to obtain the fractions-of-interest. The fraction was then concentrated and re-analyzed by LC-MS. Based on ~1H-NMR, BAs were found to be the main components of snake bile, and six BAs including CDCA, CA, TCDCA, TCA, TDCA and GCA were tentatively identified from the representative spectrum with the assistance of literature and reference compounds. Whereas the content of TCA in snake bile was too great, resulting in a great obstacle for the detection of trace components, the automated fraction collector was subsequently implemented to obtain the fractions-of-interest for LC-MS analysis. According to matching MS/MS information and retention time with reference compounds as well as database retrieval, a total of 57 BAs were detected and annotated. Because of the combination of ~1H-NMR and LC-MS platforms, the findings are beneficial for the in-depth characterization of BAs in snake bile, which provides references for the establishment of quality control and evaluation methods of snake bile.

A new bile acid from the traditional chinese medicine shedan.

A new bile acid tauro-16ß-hydroxy-12α-sulfate-5ß-cholenoic acid (1), along with six known ones (2-7), was isolated from the snake bile. Its planar structure and relative configuration were elucidated based on extensive spectroscopic analyses. Moreover, compound 2 showed inhibitory effect on NO production in RAW 264.7 macrophages at non-cytotoxic concentration (20 µM) with inhibitory rate of 69.7%. [Formula: see text].

Simultaneous determination of nine kinds of dominating bile acids in various snake bile by ultrahigh-performance liquid chromatography with triple quadrupole linear iontrap mass spectrometry.

Snake bile is one of the most expensive traditional Chinese medicines (TCMs). However, due to the complicated constitutes of snake bile and the poor ultraviolet absorbance of some trace bile acids (BAs), effective analysis methods for snake bile acids were still unavailable, making it difficult to solve adulteration problems. In present study, ultrahigh-performance liquid chromatography with triple quadrupole linear ion trap mass spectrometry (UHPLC-QqQ-MS/MS) was applied to conduct a quantitative analysis on snake BAs. The mass spectrometer was monitored in the negative ion mode, and multiple-reaction monitoring (MRM) program was used to determine the contents of BAs in snake bile. In all, 61 snake bile from 17 commonly used species of three families (Elapidae, Colubridae and Viperidae), along with five batches of commercial snake bile from four companies, were collected and detected. Nine components, Tauro-3α,12α-dihydroxy-7-oxo-5ß-cholenoic acid (T1), Tauro-3α,7α,12α,23R-tetrahydroxy-5ß-cholenoic acid (T2), taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), cholic acid (CA), Tauro-3α,7α-dihydroxy-12-oxo-5ß-cholenoic acid (T3), and Tauro-3α,7α,9α,16α-tetrahydroxy-5ß-cholenoic acid (T4) were simultaneously and rapidly determined for the first time. In these BAs, T1 and T2, self-prepared with purity above 90%, were first reported with their quantitative determination, and the latter two (T3 and T4) were tentatively determined by quantitative analysis multi-components by single marker (QAMS) method for roughly estimating the components without reference. The developed method was validated with acceptable linearity (r2≥0.995), precision (RSD＜6.5%) and recovery (RSD＜7.5%). It turned out that the contents of BAs among different species were also significantly different; T1 was one of the principle bile acids in some common snake bile, and also was the characteristic one in Viperidae and Elapidae; T2 was the dominant components in Enhydris chinensis. This quantitative study of BAs in snake bile is a remarkable improvement for clarifying the bile acid compositions and evaluating the quality of snake bile.

Rapid identification of bile acids in snake bile using ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Snake bile, a precious traditional Chinese medicine (TCM), was used as the major ingredient of some Chinese patent drugs, such as Shedan Chuanbei powder and Shedan Chenpi powder for hundred years. However, there is still requirement for the comprehensive and definite composition of bile acids in snake bile. In order to rapidly identify the bile acids constituents in snake bile to avoid the adulteration, ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS/MS) has been applied to conduct a qualitative analysis on snake bile acids. ESI ion source was used for mass spectra, and data were collected in both positive and negative ion mode. 16 kinds of reference standards, attributed to free bile acids, taurine- and glycine- conjugated bile acids, were detected and their MS behaviors were summarized. In negative ion mode, the diagnostic ions of free bile acids were obtained via the neutral losses of H2O and CO2 molecules; the diagnostic ions of taurine-conjugated bile acids were at m/z 124.0068 ([C2H6NO3S]-), m/z 106.9803 ([C2H3O3S]-) and m/z 79.9568 ([SO3]-); the diagnostic ion of glycine-conjugated bile acids was at m/z 74.0242 ([C2H4NO2]-). In positive ion mode, dehydration ions, amide bond cleavage ions, and reversed Diels-Alder at A-ring ions were detected in every kind of reference. These reference MS behaviors were used for identifying bile acids without reference standards in snake bile. As a result, totally 15 compounds, including 4 pairs of isomers, were identified by comparing the retention time, exact molecular mass and fragmentation behaviors with reference standards, respectively. Tauro-3ß,7α,12α-trihydroxy-5ß-cholenoic acid, Tauro-△8-3ß,7α,12α- trihydroxy-5ß-cholenoic acid, Tauro-3α,7α,12α,23R-tetrahydroxy-5ß-cholenoic acid, and Tauro-3α,7α-dihydroxy-12-oxo-5ß-cholenoic acid, Taurocholic acid, Glycocholic acid, Taurochenodeoxycholic acid, Taurodeoxycholic acid and Cholic acid were detected as the common bile acids in snake bile. Tauro-△8-3ß,7α,12α-trihydroxy-5ß-cholenoic acid, Tauro-3α,7α,9α,16α-tetrahydroxy-5ß-cholenoic acid, Tauro-3α,12α,17R,22R-tetrahydroxy-5ß-cholenoic acid, and Tauro-△1,8-3ß,7ß,12α-trihydroxy-5ß-cholenoic acid were firstly reported in this study.

Bile acid derivatives-focused chemical profiling in snake bile / 中国中药杂志

As a precious traditional Chinese medicine(TCM), snake bile has been widely used in numerous Chinese medicine prescriptions. Bile acid(BA) derivatives have been demonstrated as the primary chemical family in snake bile. In-depth chemical characterization of BAs is of great importance towards the establishment of quality standards and clarification of the effective material basis for snake bile. This study firstly employed ~1H-NMR to preliminarily analyze the chemical profiles of snake bile, an automated fraction collector was subsequently implemented to obtain the fractions-of-interest. The fraction was then concentrated and re-analyzed by LC-MS. Based on ~1H-NMR, BAs were found to be the main components of snake bile, and six BAs including CDCA, CA, TCDCA, TCA, TDCA and GCA were tentatively identified from the representative spectrum with the assistance of literature and reference compounds. Whereas the content of TCA in snake bile was too great, resulting in a great obstacle for the detection of trace components, the automated fraction collector was subsequently implemented to obtain the fractions-of-interest for LC-MS analysis. According to matching MS/MS information and retention time with reference compounds as well as database retrieval, a total of 57 BAs were detected and annotated. Because of the combination of ~1H-NMR and LC-MS platforms, the findings are beneficial for the in-depth characterization of BAs in snake bile, which provides references for the establishment of quality control and evaluation methods of snake bile.

Quality Analysis of Snake Bile and Its Preparations / 中国药学杂志

OBJECTIVE： To study the bile acid components in snake bile and other animal bile, and establish the quality control method of the snake bile and its preparations. METHODS： Acetonitrile-methanol and 10 mmol•L-1 ammonium acetate was used as mobile phase system and 18 alkyl silane bonded silica gel was used as filling agent. Liquid chromatography-mass spectrometry was used to qualitatively identify and quantitatively analyze the bile acid components in snake bile and other animal bile, and the adulteration control and content determination method of bile acid in snake bile and its preparations were optimized to establish a suitable quality control method of snake bile and its preparations. RESULTS： The examination method was highly specialized and suitable for the quality control of snake bile and its preparations.The linear relationship of sodium taurocholate between 0.302-9.053 μg is good, r=0.999 3, the repeatability test and accuracy test results are good, and the RSD is smaller than 3.0%. CONCLUSION： The established quality control method can guarantee the quality and safety of snake bile and its preparations.

Determination of taurocholic acid in Shedanchuanbei Oral Liquid and snake bile by SPE-HPLC / 中成药

Objective: To establish the method for determination of taurocholic acid in the Traditional Chinese Medicine Shedanchuanbei Oral Liquid and snake bile. Methods: The sample was prepared as mixed solution containing methanol and KH 2PO 4. The mixed solution was injected into Sep Pak C 18 cartridge for the purpose of sample purity. In this processing, the substances which having strong retain action and could harm analytic column were hold in the Sep Pak C 18 cartridge. The eluting solution that the Sep Pak C 18 cartridge had be over loading for taurocholic acid was used as the test solution. The test solution was measured by RP HPLC. The chromatographic conditions were as followed: Supelcosil LC 8 column(150mm?4.6nm,5?m) as analytic column, detect wavelength at 203nm, and MeOH 0.4%KH 2PO 4 mixed solution(56∶44, V/V ) as mobile phase. The inject volume was 50?L. Results: The linear response range of sodium taurocholate was from 0.0253mg?mL -1 to 0.253mg?mL -1 , and the correlation coefficient was 0.9999. The average recovery rate was 101.3%, RSD was 0.40%( n =6). Conclusion: This method was simple, efficient and suitable to the quality control for Shedanchuanbei Oral Liquid and snake bile.