New Pyrroline Isolated from Antarctic Krill-Derived Actinomycetes Nocardiopsis sp. LX-1 Combining with Molecular Networking.

Antarctic krill (Euphausia superba) of the Euphausiidae family comprise one of the largest biomasses in the world and play a key role in the Antarctic marine ecosystem. However, the study of E. superba-derived microbes and their secondary metabolites has been limited. Chemical investigation of the secondary metabolites of the actinomycetes Nocardiopsis sp. LX-1 (in the family of Nocardiopsaceae), isolated from E. superba, combined with molecular networking, led to the identification of 16 compounds a-p (purple nodes in the molecular network) and the isolation of one new pyrroline, nocarpyrroline A (1), along with 11 known compounds 2-12. The structure of the new compound 1 was elucidated by extensive spectroscopic investigation. Compound 2 exhibited broad-spectrum antibacterial activities against A. hydrophila, D. chrysanthemi, C. terrigena, X. citri pv. malvacearum and antifungal activity against C. albicans in a conventional broth dilution assay. The positive control was ciprofloxacin with the MIC values of <0.024 µM, 0.39 µM, 0.39 µM, 0.39 µM, and 0.20 µM, respectively. Compound 1 and compounds 7, 10, and 11 displayed antifungal activities against F. fujikuroi and D. citri, respectively, in modified agar diffusion test. Prochloraz was used as positive control and showed the inhibition zone radius of 17 mm and 15 mm against F. fujikuroi and D. citri, respectively. All the annotated compounds a-p by molecular networking were first discovered from the genus Nocardiopsis. Nocarpyrroline A (1) features an unprecedented 4,5-dihydro-pyrrole-2-carbonitrile substructure, and it is the first pyrroline isolated from the genus Nocardiopsis. This study further demonstrated the guiding significance of molecular networking in the research of microbial secondary metabolites.

Decreased PKG transcription mediated by PI3K/Akt/FoxO1 pathway is involved in the development of nitroglycerin tolerance.

Phosphoinositide 3-kinase (PI3K)/Akt plays a pivotal role in the vascular response. The present study is to determine whether PI3K/Akt pathway in vascular smooth muscle cells is involved in nitroglycerin (NTG) tolerance and the underlying mechanism. Nitrate tolerance of porcine coronary arteries in vitro was induced by incubation of NTG (10-5 M) for 24 h. Nitrate tolerance in vivo was obtained by subcutaneous injection of mice with NTG (20 mg kg-1, tid, 3 days) and the aortas were used. Protein levels of total and phosphorylated Akt, forkhead box protein O1 (FoxO1), and cGMP-dependent protein kinase (PKG) were determined by western blot analysis. Isometric vessel tension was recorded by organ chamber technique. PKG mRNA was determined by real-time PCR. The cellular translocation of FoxO1 was observed by immunofluorescence. Reactive oxygen species (ROS) level was measured by DHE staining. The vascular relaxation to NTG was significantly inhibited in in vivo and in vitro NTG tolerant arteries. Meanwhile, the protein level of phosphorylated Akt at Ser473 was increased in the tolerant arteries. The attenuated relaxation and the augmented Akt-p were ameliorated by LY294002, a specific inhibitor of PI3K. The protein and mRNA expression of PKG were significantly down-regulated in NTG tolerant arteries, which were reversed by LY294002. The level of phosphorylated FoxO1 at Ser256 and its translocation from the nucleus to the cytosol were both increased in NTG tolerance and were also inhibited by LY294002. ROS production was significantly increased in NTG tolerant arteries, which was not be affected by LY294002 but inhibited by N-acetyl-L-cysteine. In conclusion, the present study suggests that PI3K/Akt in vascular smooth muscle is involved in the development of NTG tolerance via inhibiting PKG transcription and the effect is mediated by FoxO1.

Predictive role of UCA1-containing exosomes in cetuximab-resistant colorectal cancer.

BACKGROUND: Primary or acquired resistance to cetuximab often occurs during targeted therapy in metastatic colorectal cancer (mCRC) patients. In many cancers, the key role of the long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) in anticancer drug resistance has been confirmed. Emerging evidence has shown that specific exosomal lncRNAs may serve as meaningful biomarkers. In this study, we hypothesize that exosomal UCA1 might predict the response to cetuximab in CRC patients. METHODS: First, acquired cetuximab-resistant cell lines were generated, and UCA1 expressions in these cells and their exosomes were compared. We also systematically evaluate the stability of exosomal UCA1. Thereafter, the predictive value of exosomal UCA1 in CRC patients treated with cetuximab was evaluated. Finally, through cell apoptosis assays and immunofluorescence staining, we analyzed the role of UCA1-containing exosomes in conferring cetuximab resistance. RESULTS: UCA1 expression was markedly higher in cetuximab-resistant cancer cells and their exosomes. Exosomal UCA1 was shown to be detectable and stable in serum from CRC patients. In addition, circulating UCA1-containing exosomes could predict the clinical outcome of cetuximab therapy in CRC patients, and UCA1 expression was considerably higher in the progressive disease/stable disease patients than in the partial response/complete response patients. Furthermore, exosomes derived from cetuximab-resistant cells could alter UCA1 expression and transmit cetuximab resistance to sensitive cells. CONCLUSIONS: We discovered a novel role of UCA1-containing exosomes, showed their capability to transmit drug resistance and investigated their potential clinical use in predicting cetuximab resistance.

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q-TOF-MS/MS combined with UPLC/QQQ-MS/MS.

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4-dihydroxybenzoic acid, salidroside, p-coumaric acid-4-O-ß-d-glucopyranoside, bergeninum, 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, syringate, 6''-O-galloylsalidroside, rhodiosin, rhodionin and kaempferol-7-O-α-l-rhamnoside, were simultaneously quantified by the developed ultra-performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB-C18 column (3.0 × 100 mm, 1.8 µm) with linear gradient elution of methanol-0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R, 0.9979-0.9997), precision (RSD, 1.3-4.7%), repeatability (RSD, 1.7-4.9%), stability (RSD, 2.2-4.9%) and recovery (RSD, 0.6-4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.

Research on the change of chemical composition in productive process of Re Du Ning injection by HPLC/Q-TOF MS.

A high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) was developed for the analysis of chemical composition change in the production process of Re Du Ning injection, a Chinese medicine preparation with a combination of Lonicera japonica Thunb., Gardenia jasminoides Ellis and Artemisia annua L. A total of 90 compounds from raw materials-intermediates-Re Du Ning injection were detected; among them, 55 compounds were identified or tentatively characterized, and the characteristic ions of different types of compounds were described. Based on these studies, the different types of compounds in the various process routes were analyzed. A total of 28 compounds, including seven iridoid glycosides and six monoterpenes from G. jasminoides Ellis, five iridoid glycosides, nine phenolic acids and one unknown compound from L. japonica Thunb., were transferred to Re Du Ning injection, and two unknown compounds were generated in the production process of Re Du Ning injection. The results indicated that the Chinese Medicine Pharmaceutical process control is very important. This method could provide some reference for other Chinese medicine preparations.

[Metabolism of six saponins by rat intestinal bacteria in vitro].

To investigate the metabolism of six saponins by rat intestinal bacteria in vitro.Six saponins, including notoginsenoside R1, ginsenoside Rg1, ginsenoside Rg2, ginsenoside Re, ginsenoside Rd and ginsenoside Rb1, were incubated for 8 and 24 h with rat intestinal bacteria under anaerobic environment, respectively. After the samples were precipitated by acetonitrile and extracted with ethyl acetate, LC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites. The potential metabolites in rat feces were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results showed that six saponins could be easily metabolized by rat intestinal bacteria. Notoginsenoside R1 was mainly metabolized into five metabolites, and it's metabolic pathway was notoginsenoside R1→ginsenoside Rg1→ginsenoside Rh1 and ginsenoside F1→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rg1 was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Rg1→ginsenoside Rh1 and ginsenoside F1→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rg2 was mainly metabolized into two metabolites, and it's metabolic pathway was ginsenoside Rg2→ protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Re was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Re→ginsenoside Rg2→ginsenoside F1→protopanaxatriol→dehydrogenated protopanaxatriol. Ginsenoside Rd was mainly metabolized into four metabolites, and it's metabolic pathway was ginsenoside Rd→ginsenoside Rg3 and ginsenoside F2→ginsenoside Rh2→protopanaxadiol. Ginsenoside Rb1 was mainly metabolized into five metabolites, and it's metabolic pathway was ginsenoside Rb1→ginsenoside Rd→ginsenoside Rg3 and ginsenoside F2→ginsenoside Rh2→protopanaxadiol. In summary, six saponins could be quickly metabolized by rat intestinal bacteria in vitro. Their major metabolic pathways were deglycosylation and dehydrogenation.

[Determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials by LC-MS/MS].

To develop a LC-MS/MS method for the determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials, the column was Agilent ZORBAX Eclipse plus C18 (3.0 mm x 50 mm, 1.8 µm), and the mobile phase consisted of methanol-water (containing 0.2% formic acid) (95:5) at a flow rate of 0.5 mL · min(-1). The multiple reaction ion monitoring (MRM) with an ESI interface in the negative ion mode was selected. The results showed that the linear ranges of five kinds of ginkgolic acids were in the range of 0.2-36.0 µg · L(-1) (r ≥ 0.999 5). The lowest limit of quantification (LOQ) of ginkgo acid C13: 0, C15:1, C17:2, C15:0 and C17:1 were 0.18, 0.18, 0.21, 0.10 and 0.20 µg · L(-1), respectively. The average recovery was between 73.28% and 87.56%, and the average content of total ginkgolic acids in three batches of samples was in the range of 0.023-0.028 µg · g(-1), which was much lower than 2 µg · g(-1) prescribed in drug registration standards. This method is simple and rapid with high sensitivity, which can be used for the determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials.

[Study on limit detection of flavones in diterpene ginkgolides meglumine injection materials by LC-MS and HPLC-DAD].

Limit test of flavones in diterpene ginkgolides meglumine injection materials by UV-Vis and HPLC-DAD method was studied in this essay. The HPLC-DAD method has lower LOD (about 1% of the UV-Vis), that is, the sensitivity is higher than UV-Vis method. Through the analysis of the kinds of flavonoids ingredients in the samples by LC-MS, the three compounds with highest contents are kaempferol, quercetin and isorhamnetin. Kaempferol, quercetin and isorhamnetin were chosen as reference compounds for HPLC analysis, and the HPLC separation analysis was carried on an Agilent Eclipse plus C18 column (4.6 mm x 250 mm, 5 µm) with methanol and water containing 0.4% phosphoric acid (50: 50) as mobile phase, and the flow rate was 1.0 mL x min(-1). The detection wavelength was set at 360 nm. This method has good specificity, precision and reproducibility. The LODs of quercetin, kaempferide and isorhamnetin were 27.6, 22.3, 29.5 µg x L(-1). The average recovery was 87.9% (RSD 3.3%), 91.7% (RSD 3.1%), 88.3 (RSD 1.3%) for quercetin, kaempferide and isorhamnetin, respectively. Based on the 10 batches of sample results and sensitivity of different HPLC, the content of total flavonoids ingredients of diterpene ginkgolides meglumine injection materials was limited no more than 2 x 10(-5). This method is simple, quick and has good maneuverability, and could be used to the limit test of flavonoids in the diterpene ginkgolides meglumine injection materials.

[Similarity between leaves of Nauclea officinalis and stems of Nauclea officinalis].

The study is to develop a method to determine 3 batches leaves of Nauclea officinalis and stems of N. officinalis by HPLC. The differences between strictosamide contents and fingerprints was compared, then chromatographic peak of fingerprints was validated with the assistance of LC-MS. The strictosamide contents in stems of N. officinalis were higher than leaves of N. officinalis. The main chemical composition in leaves of N. officinalis and stems of N. officinalis were alkaloid which revealed by LC-MS. There are 7 chemical compositions were same between them, but the chemical composition in leaves of N. officinalis is more than stems of N. officinalis. This provides a scientific basis for the development of the potential medicinal value of leaves of N. officinalis and the sustainable utilization of N. officinalis.

[Quantitative and qualitative evaluation on tablets of Ginkgo biloba leaves using fingerprint and LC-MS analysis].

A reasonable method for the quality control of tablets of Ginkgo biloba leaves was established in this paper. The total flavonol glycosides and terpene lactones of G. biloba tablets were quantified by HPLC. Totally, 16 batches of the commercially available tablets of G. biloba leaves were determined. Among of them, 2 batches were unqualified in the content of total flavonol glycosides, and 3 batches were unqualified in the content of terpene lactones. A validated HPLC fingerprint method was established to evaluate the commercially available tablets of G. biloba leaves with the assistance of LC-MS. Sixteen batches showed the similarity of 0.763-0.989. There were 31 fingerprint chromatogram peaks were identified as flavonoids compositions by LC-MS. This provides a research idea for the quality control of tablets of G. biloba leaves.

Dihydroartemisinin accentuates the anti-tumor effects of photodynamic therapy via inactivation of NF-κB in Eca109 and Ec9706 esophageal cancer cells.

BACKGROUND: Photodynamic therapy (PDT) is a new treatment for esophageal cancer which has been shown to be effective in the elimination of tumor. However, PDT could induce the activation of nuclear factor-kappa B (NF-κB) in many photosensitizers based PDT, which plays a negative role in PDT. In addition, our previous results have shown that dihydroartemisinin (DHA), which was the most potent one of artemisinin derivatives, has anticancer activity in esophageal cancer cells. METHODS: Cell viability was determined by MTT analysis, and apoptosis was evaluated by flow cytometry. Nuclear extract was obtained for determining NF-κB DNA-binding activity, while total protein extract obtained for downstream gene expression by western blot. RESULTS: We demonstrated DHA enhanced PDT-induced growth inhibition and apoptosis in both human esophageal cancer cell lines Eca109 and Ec9706 in vitro. The mechanism was at least partially due to DHA deactivated PDT-induced NF-κB activation, so as to decrease tremendously the expression of its target gene Bcl-2. CONCLUSION: Our results demonstrate that DHA augments PDT-induced growth inhibition and apoptosis in esophageal cancer cells, and that inactivation of NF-κB activity is a potential mechanism by which DHA sensitizes esophageal cancer cells to PDT-induced growth inhibition and apoptosis.

[Rapid identification of six chemical constituents in Guizhi Fuling capsule by DART-Q-TOF-MS].

In order to establish a rapid method for identifying six constituents in Guizhi Fuling capsule, Q-TOF with DART ion source was used to perform the direct analysis of compounds in Guizhi Fuling capsule. The DART sampler delivery rate was 0.2 mm s(-1). The temperature of helium gas of DART was 450 degrees C. The capillary voltage was kept at 1 000 V. The temperature of the drying gas of Agilent 6538 Q-TOF MS was set at 350 degrees C. The flow rate of the drying gas of MS was set at 3.5 L x min(-1). The MS scan range was m/z 50-1 000. Based on accurate mass measurements and the elemental compositions of the product ions and fragmentation patterns of reference conpounds, six components, amygdalin, paeonol, paeoniflorin, cinnamic acids, gallic acid, benzoic acid were identified rapidly. The method can rapidly identify six chemical constituents in three batch of Guizhi Fuling capsule. The DART-Q-TOF-MS method is simple, rapid and specific and it can be used for rapid identification and characterization of compounds in traditional Chinese medicines.

[Quality evaluation of guizhi fuling capsule using self-control method of reference Chinese medicine preparation].

Taking guizhi fuling capsule (GZFL) for instance, a new method about reference Chinese medicine preparation which was used as standard substance for the quality evaluation of complex Chinese medicine preparation by the fingerprint of reference preparation instead of standard fingerprint was proposed. It could eliminate the errors from different instruments, chromatographic columns and solve the problem of similarity matching in the absence of standard fingerprint. The qualification of reference GZFL was evaluated according to the quality control method of GZFL from Chinese Pharmacopoeia. Then multiple batches of GZFL were estimated, taking fingerprint of reference preparation and standard fingerprint as references, respectively, at different instruments and chromatographic columns. Finally, the packaging and expiration date for reference GZFL were confirmed according to the results of stability investigation. The results indicated that the fingerprint of reference GZFL could be used to assess the quality of GZFL better than standard fingerprint. The data of accelerated stability and long-term stability test demonstrated that reference GZFL was stable in the conditions of double blister package. Therefore, reference GZFL can be used as standard substance in quality control of GZFL.

Inhibition of signal transducer and activator of transcription 3 and cyclooxygenase-2 is involved in radiosensitization of cepharanthine in HeLa cells.

OBJECTIVES: To investigate the radiosensitizing effects of cepharanthine (CEP) in the human cervical adenocarcinoma HeLa cell line and to examine the underlying mechanisms. MATERIALS/METHODS: Survival of HeLa cells after treatment with or without ionizing radiation (IR) and CEP administration was investigated. MTT assays and apoptosis analysis were used to assess cytotoxicity. Nude mouse xenografts were established to evaluate the antitumor effects of CEP and IR in vivo. Expression of signal transducer and activator of transcription 3 (STAT3) and its downstream signaling molecules as well as cyclooxygenase-2 (COX-2) were examined by Western blot analysis. RESULTS: Clonogenic assays showed that treatment with CEP and IR resulted in significant radiosensitization. Cepharanthine and IR treatment achieved maximum cytotoxic effects on HeLa cells with regard to apoptosis induction. Cepharanthine efficiently decreased IR-induced STAT3 and COX-2 activation. The STAT3 target genes, including the antiapoptotic Bcl-2 and the cell cycle regulator c-Myc, were decreased concomitantly. In vivo administration of CEP (20 mg/kg every 2 days) combined with radiation in HeLa xenografts enhanced tumor growth delay and apoptosis (indicated by activated caspase-3 Western blot analysis), with reduced expression of STAT3, Bcl-2, c-Myc, and COX-2. CONCLUSIONS: Cepharanthine was shown to induce radiation sensitization in HeLa cells in vitro and in vivo. The inhibitory effects of CEP on STAT3 signaling pathway and COX-2 help us to better understand the radiosensitization of CEP.

A polymorphism at the 3'-UTR region of the aromatase gene is associated with the efficacy of the aromatase inhibitor, anastrozole, in metastatic breast carcinoma.

Estrogen-related genes and the fat mass and obesity-associated (FTO) gene play a critical role in estrogen metabolism, and those polymorphisms are associated with a poor prognosis in breast cancer. However, little is known about the association between these polymorphisms and the efficacy of anastrozole. The aim was to investigate the impact of the genetic polymorphisms, CYP19A1, 17-ß-HSD-1 and FTO, on the response to anastrozole in metastatic breast carcinoma (MBC) and to evaluate the impact of those polymorphisms on various clinicopathologic features. Two-hundred seventy-two women with hormone receptor-positive MBC treated with anastrozole were identified retrospectively. DNA was extracted from peripheral blood and genotyped for five variants in three candidate genes. Time to progression was improved in patients carrying the variant alleles of rs4646 when compared to patients with the wild-type allele (16.40 months versus 13.52 months; p = 0.049). The rs4646 variant alleles were significantly associated with longer overall survival (37.3 months versus 31.6 months; p = 0.007). This relationship was not observed with the rs10046, rs2830, rs9926298 and rs9939609 polymorphisms. The findings of this study indicate that rs4646 polymorphism in the CYP19A1 gene may serve as a prognostic maker of the response to anastrozole in patients with MBC who are treated with anastrozole.

Genus Acrostalagmus: A Prolific Producer of Natural Products.

Acrostalagmus is known for its ability to produce numerous bioactive natural products, making it valuable in drug development. This review provides information on the sources, distribution, chemical structure types, biosynthesis, and biological activities of the compounds isolated from the genus Acrostalagmus in the family Plectosphaerellaceae from 1969 to 2022. The results show that 50% of the compounds isolated from Acrostalagmus are new natural products, and 82% of the natural products derived from this genus are from the marine Acrostalagmus. The compounds isolated from Acrostalagmus exhibit diverse structures, with alkaloids being of particular importance, accounting for 56% of the natural products derived from this genus. Furthermore, within the alkaloid class, 61% belong to the epipolythiodioxopiperazine family, highlighting the significance of epipolythiodioxopiperazine as a key characteristic structure within Acrostalagmus. Seventy-two percent of natural products derived from Acrostalagmus display bioactivities, with 50% of the bioactive compounds exhibiting more significant or comparable activities than their positive controls. Interestingly, 89% of potent active compounds are derived from marine fungi, demonstrating their promising potential for development. These findings underscore Acrostalagmus, particularly the marine-derived genus Acrostalagmusas, a valuable source of new bioactive secondary metabolites, and emphasize the vast resource importance of the ocean.

The evaluation and implementation of Direct Analysis in Real Time quadrupole time-of-flight tandem mass spectrometry for characterization and quantification of geniposide in Re Du Ning Injections.

RATIONALE: The Direct Analysis in Real Time (DART) ionization source coupled with a quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) system has the capability to desorb analytes directly from samples from complex Chinese herbal preparations without sample cleanup or chromatographic separation. METHODS: In this work, a method based on DART/Q-TOF MS/MS has been developed for rapid determination of geniposide present in 'Re Du Ning Injections', a Chinese herbal preparation. The method has been evaluated for both qualitative and quantitative analysis of geniposide in Re Du Ning Injections. RESULTS: Variables including polarity for ion detection, DART gas heater temperature, matrix effect and sample presentation speed were investigated. The quantitative method was validated with respect to linearity, sensitivity, repeatability, precision and accuracy by using both internal and external standards. A comparison of the results obtained using the DART-based method was made with those obtained using a conventional High-Performance Liquid Chromatography/Diode-Array Detector (HPLC/DAD) by analyzing geniposide in four batches of Re Du Ning Injections. CONCLUSIONS: The DART/Q-TOF MS/MS-based method provides a rapid, efficient and powerful method to analyze compounds from complex Traditional Chinese Medicines with limited sample preparation thus reducing time and complexity of quality control for those materials.

Identification and quantification of free radical scavengers in the flower buds of Lonicera species by online HPLC-DPPH assay coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Flos Lonicerae, derived from the flower buds of several medicinal Lonicera species, is a commonly used herbal medicine with multiple pharmacological activities, one of the major ones being antioxidant activity. In this study, free radical scavengers in the flower buds of six Lonicera species were screened, identified and quantified by online HPLC-DPPH (1,1-diphenyl-2-picrylhydrazyl) assay coupled with LC quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). The antioxidants were firstly screened from the complex plant matrix by the online HPLC-DPPH assay. Then the active compounds were identified by LC Q-TOF MS/MS, and the possible fragmentation pathways were proposed. The reactivity of antioxidants available was investigated using an internal standard method by online LC assay. The contents of 12 antioxidants were also determined or estimated by HPLC coupled with diode array detector. The total antioxidant capability determined by the online method was used as the marker to evaluate the quality of Flos Lonicerae. The results were important to clarify the material basis and therapeutic mechanism of Flos Lonicerae.

[Analysis of the antimycobacterial activities of rifabutin and the relationship between drug-resistance and rpoB mutations of Mycobacterium tuberculosis].

OBJECTIVE: To compare the antimycobacterial activities of rifampicin (RFP) and rifabutin (RBT), and to evaluate the correlation between RBT resistance and genetic alterations in the rpoB gene. METHODS: The microplate-based alamar blue assay (MABA) method was performed to detect the antimycobacterial activities of RFP and RBT in 168 strains of Mycobacterium tuberculosis (M. tuberculosis). Meanwhile, we also analyzed the 81 bp core region of rpoB gene by DNA sequencing. The rate of gene mutations was analyzed by chi-square test. RESULTS: RBT was sensitive for all of the 66 RFP-sensitive strains with no mutations in 81 bp core region of rpoB gene. But of the 102 RFP-resistant strains, 76 strains were also resistant to RBT. Cross resistance between RFP and RBT was 74.5% (76/102). Alterations at codons 516, 526, 531 in the rpoB gene correlated with resistance to both RFP and RBT. While point mutations at codons 511 and 533 possibly influenced the susceptibility to RFP but not to RBT. The mutation rate (92.1%, 70/76) of rpoB gene of RBT-resistant strains was significantly higher than that (23.9%, 22/92) of RBT-sensitive strains (χ(2) = 78.12, P < 0.05). CONCLUSIONS: RBT was more active against M. tuberculosis as compared to RFP. The RFP-resistant strains with MIC ≤ 4 mg/L were still susceptible to RBT. Our results suggest that analysis of genetic alterations in the rpoB gene is useful for predicting RFP-resistance, and may have implications for evaluating RBT-resistance.

Characterization and identification of saponins in Achyranthes bidentata by rapid-resolution liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

A rapid-resolution liquid chromatography (RRLC) method coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) has been developed for analysis of oleanane-type triterpenoid saponins in Achyranthes bidentata. Collision-induced dissociation techniques were used to fragment the precursor molecular ions and the resulting product ions. A retro-Diels-Alder rearrangement from the oleanane aglycone skeleton in the MS/MS process yielded characteristic fragment ions in positive ion mode. These characteristic ions were helpful in predicting the aglycone structure. Losses of monosaccharide sequences, presence of sugar-chain fragment ions, and cleavage of CO(2) were observed for important information on sugar types and attachment sequences. Fragmentation rules of three major groups of saponins from A. bidentata were summarized, and the possible fragmentation pathways were proposed. A total of 22 compounds including both the target and unknown oleanane-type triterpenoid saponins were rapidly screened and predicted in the herbal extract by the developed method. The RRLC-Q-TOF MS/MS method has provided a powerful approach for rapid separation, target screening and structural elucidation of oleanane-type saponins, and also opened perspectives for similar studies on other herbal medicines.