Novel cytotoxic steroidal glycosides from the roots of Liriope muscari.

The present study was designed to investigate the chemical constituents and bioactivities of the roots of Liriope muscari (Decne.) L.H. Bailey. The compounds were isolated through various chromatography techniques, including silica gel, Sephadex LH-20, and semi-preparative HPLC. The structures were elucidated by infrared (IR), mass spectrometric (MS), 1D- and 2D-NMR analyses in comparison with reference data. In addition, the cytotoxicity of these compounds against human breast cancer MDA-MB-435 cells was evaluated by the MTT assay. Two new steroidal glycosides, 25(R, S)-ruscogenin-1-O-[ß-D-fucopyranosyl (1→2)]-[ß-D-xylopyranosyl(1→3)]ß-D-glucopyranoside (Liriopem I, 1) and 25(R, S)- ruscogenin-1-O-[ß-D-fucopyranosyl (1→2)]-[ß-D-xylopyranosyl(1→4)]-ß-D-fucopyranoside (Liriopem II, 2 and two known compounds LM-S6 (3) and DT-13 (4) were isolated and identified. Liriopem I(1), liriopem II(2) and DT-13 (4) showed remarkable cytotoxicity with IC50 values being (0.58 ± 0.08), (0.05 ± 0.10), and (0.15 ± 0.09) µg·mL(-1), respectively. In summary, compounds 1 and 2 identified in the present study exerted cytotoxicity against breast cancer cells, providing a basis for future development of these compounds as novel anticancer agents.

Determination and fingerprint analysis of steroidal saponins in roots of Liriope muscari (Decne.) L. H. Bailey by ultra high performance liquid chromatography coupled with ion trap time-of-flight mass spectrometry.

Liriope muscari (Decne.) L. H. Bailey is a well-known traditional Chinese medicine used for treating cough and insomnia. There are few reports on the quality evaluation of this herb partly because the major steroid saponins are not readily identified by UV detectors and are not easily isolated due to the existence of many similar isomers. In this study, a qualitative and quantitative method was developed to analyze the major components in L. muscari (Decne.) L. H. Bailey roots. Sixteen components were deduced and identified primarily by the information obtained from ultra high performance liquid chromatography with ion-trap time-of-flight mass spectrometry. The method demonstrated the desired specificity, linearity, stability, precision, and accuracy for simultaneous determination of 15 constituents (13 steroidal glycosides, 25(R)-ruscogenin, and pentylbenzoate) in 26 samples from different origins. The fingerprint was established, and the evaluation was achieved using similarity analysis and principal component analysis of 15 fingerprint peaks from 26 samples by ultra high performance liquid chromatography. The results from similarity analysis were consistent with those of principal component analysis. All results suggest that the established method could be applied effectively to the determination of multi-ingredients and fingerprint analysis of steroid saponins for quality assessment and control of L. muscari (Decne.) L. H. Bailey.

Chinese medicine syndrome distribution of chronic hepatitis B virus carriers in immunotolerant phase.

OBJECTIVE: To explore Chinese medicine (CM) syndrome distribution of chronic hepatitis B virus (HBV) carriers in immunotolerant phase (ITP). METHODS: One hundred and eighty-five chronic HBV carriers in ITP, seen in the Third Affiliated Hospital of Sun Yat-sen University from May 2009 to December 2010, were admitted in an observational study under the guidance of CM. Patients' CM symptoms and signs, demographics, liver biochemistries, and qualitative HBV DNA were recorded in the questionnaires. CM syndromes were then differentiated to 15 detailed types and analyzed by generalization. Lastly, the location, pathogenic factors and nature of the disease were also assessed. RESULTS: When CM syndrome patterns were differentiated to 15 types, there were 27 (15%) no syndrome cases, 94 (50%) single syndrome cases and 64 (35%) compound syndromes cases. The main detailed syndromes included Liver (Gan)-qi depression (LQD), Kidney (Shen)-qi deficiency (KQD), Spleen (Pi)-qi deficiency (SQD) and Kidney-yang deficiency (KYAD). After CM syndromes generalized to five types, their frequency was Spleen-Kidney deficiency (SKD)>LQD>inner dampness-heat retention (IDHR)>Liver-Kidney deficiency (LKD)>blood stasis blocking collateral (BSBC). SKD and LQD occupied 64%. The disease location included Liver, Gallbladder (Dan), Spleen, Stomach (Wei) and Kidney. The pathogenic factors were mainly qi stagnation, qi deficiency, yang deficiency, concurrently dampness-heat and blood stasis. The deficiency syndrome was more than excess syndrome in its nature. CONCLUSIONS: Most of chronic HBV carriers in ITP have their CM syndrome, and the most common types are SKAD, LQD. This study suggests that the natural history may be improved through breaking the state of immune tolerance or shorten the time of ITP by strengthening Spleen-Kidney and reliving Liver qi.

[Effects of glycyrrhizin on the expression of hepatitis B virus and Toll like receptors 2,4 in HepG2.2.15 cells expressing low HBsAg].

OBJECTIVE: To investigate the effects of glycyrrhizin (GL) on the expression of hepatitis B virus e antigen (HBeAg), HBV DNA, Toll-like receptors 2,4 (TLR2,4) and proliferation of cells in HepG2.2.15 cell line. METHODS: Real-time PCR examined HBV DNA, ELISA examined HBsAg, HBeAg and MTT examined the proliferation of cells. FCM examined the positive percent of cells expressing TLR2,4 before and after stimulated with GL, in contrast to the blank group. RESULTS: The expression of HBsAg was low in the cell line, so e antigen was studied. The total HBeAg mean was significantly difference on the second day after stimulated (P<0.01), but only in the group with 400 microg/ml HBeAg decreased significantly in contrast to the blank group (P<0.05), the group with 800 microg/ml increased significantly in contrast to the other groups (P<0.01). The total HBV DNA mean on the third day after stimulated was significant, only the group with 50 microg/ml decreased in contrast to the blank group, but P>0.05, the other four groups increased. The intensity of TLR4 expression in the cells was significantly higher than that of the control group (P<0.05), both of total mean TLR2,4 increased significantly (P<0.01). The four groups except the group with 200 microg/ml increased significantly in no dose-dependent manner (P<0.05). GL in three goups under 200 microg/ml all could make cells proliferate, but only 200 microg/ml group had significant difference compared to the blank group (P<0.05). Both 400 and 800 microg/ml groups inhibited the growth of cells (P<0.01). The proliferation of cells were notably negative correlated with the expression of HBeAg, HBV DNA (P<0.05). CONCLUSION: The study suggestes GL could inhibit or promote HBV DNA replicating and e antigen secreting in mutative HepG2.2.15 cell line, the correlation between the proliferation of cells and the both are negative. GL could upregulate TLR2,4 in no dose-dependent manner. Influencing HBV maybe have no correlation to up regulating TLR2,4 by GL at least in vitro.

[The clinical study on decompensatory cirrhotic patients treated by Bie Jia Jian].

OBJECTIVE: To observe the clinical curative effect on decompensatory cirrhotic patients treated by Bie Jia Jian. METHODS: 98 decompensatory cirrhotic patients were randomly divided into two groups: 49 patients in treatment group and 49 in control group. Both groups were treated with the same western medicine of protecting and supporting liver. Except that, treatment group were treated by Bie Jia Jian. RESULTS: The Contents of AST, ALT, total bilirubin (TB), direct bilirubin (DB), hyaluronic acid (HA), Laminin (LN) , procollagen III (pc III), and type IV collagen (IV.C) in both groups decreased after treatment, and prothrombin time activity (PTA) increased. Among them, the decrease of TB, DB, HA, LN, PC-III and IV-C, and the increase of PTA in treatment group were more obvious than those in control group (P < 0. 05). CONCLUSION: Bie Jia Jian is effective in treating decompesatory cirrhotic patients.

Traditional Chinese medicine syndromes of chronic hepatitis B with precore mutant.

AIM: This study aims at exploring the distribution of TCM syndromes in CHB patients with HBV pre-core mutation (1896) and the relationship between pre-core mutation and T lymphocytes subgroup, through which to provide objective data on clinical syndrome differentiation of TCM, and further to suggest the therapeutic principle and guide clinical treatment. METHODS: One hundred and forty CHB patients were evenly divided into two study groups, HBV pre-core mutant group and HBV pre-core wild-type group. Besides, 30 healthy blood donors were selected as a healthy control group. HBV-labeled compound, T lymphocytes subgroup, and HBV-DNA pre-core mutant were tested in the study groups. T lymphocytes subgroup were also tested in the control group. All the patients were both diagnosed by syndrome differentiation of TCM and western medicine. RESULTS: The most common syndrome in mutant group was damp-heat combined with blood stasis, and the most common syndrome in the wild-type group was damp-heat stasis in the middle-jiao. There were more cases of medium and severe hepatitis in mutant group than that in wild-type group. The content of CD4+ lymphocytes and CD4+/CD8+ ratio were decreased gradually (healthy control group>wild-type group>mutant group). In the wild-type group, severe and medium CHB patients had considerably lower level of them than mild CHB patients. However, in the mutant group, the opposite result appeared. Meanwhile, the content of HBV-DNA in mutant group was higher than that in wild-type group. CONCLUSION: Damp, heat, toxin and blood stasis were the basic pathogens of CHB, whether pre-core mutant or not. CHB with precore mutant may lead to more severe hepatitis. The decreased content of CD4+ lymphocytes and ratio of CD4+/CD8+ may be taken as one of the indices in confirming the deficiency syndrome of CHB patients with pre-core mutation.