Development of a Quorum Sensing-Mediated Bacterial Autolytic System in Escherichia coli for Automatic Release of Intracellular Products.

Escherichia coli, one of the most efficient expression hosts for recombinant proteins, is widely used in chemical, medical, food, and other industries. De novo engineering of gene regulation circuits and cell density-controlled E. coli cell lysis are promising directions for the release of intracellular bioproducts. Here, we developed an E. coli autolytic system, named the quorum sensing-mediated bacterial autolytic (QS-BA) system, by incorporating an acyl-homoserine lactone (AHL)-based YasI/YasR-type quorum sensing circuit from Pseudoalteromonas into E. coli cells. The results showed that the E. coli QS-BA system can release the intracellular bioproducts into the cell culture medium in terms of E. coli cell density, which offers an environmentally-friendly, economical, efficient, and flexible E. coli lysis platform for production of recombinant proteins. The QS-BA system has the potential to serve as an integrated system for the large-scale production of target products in E. coli for medical and industrial applications.

Biosynthetic mechanism of the yellow pigments in the marine bacterium Pseudoalteromonas sp. strain T1lg65.

The Pseudoalteromonas genus marine bacteria have attracted increasing interest because of their abilities to produce bioactive metabolites. The pigmented Pseudoalteromonas group encodes more secondary metabolite biosynthetic gene clusters (BGCs) than the non-pigmented group. Here, we report a yellow pigmented bacterium Pseudoalteromonas sp. strain T1lg65, which was isolated from a mangrove forest sediment. We showed that the yellow pigments of T1lg65 belong to the group of lipopeptide alterochromides. Further genetic analyses of the alterochromide BGC revealed that the yellow pigments are biosynthesized by aryl-polyene synthases and nonribosomal peptide synthases. Within the gene cluster, altA encodes a tyrosine ammonia acid lyase, which catalyzes synthesis of the precursor 4-hydroxycinnamic acid (4-HCA) from tyrosine in the alterochromide biosynthetic pathway. In addition, altN, encoding a putative flavin-dependent halogenase, was proven to be responsible for the bromination of alterochromides based on gene deletion, molecular docking, and site mutagenesis analyses. In summary, the biosynthetic pathway, precursor synthesis, and bromination mechanism of the lipopeptide alterochromides were studied in-depth. Our results expand the knowledge on biosynthesis of Pseudoalteromonas pigments and could promote the development of active pigments in the future.IMPORTANCEThe marine bacteria Pseudoalteromonas spp. are important biological resources because they are producers of bioactive natural products, including antibiotics, pigments, enzymes, and antimicrobial peptides. One group of the microbial pigments, alterochromides, holds a great value for their novel lipopeptide structures and antimicrobial activities. Previous studies were limited to the structural characterization of alterochromides and genome mining for the alterochromide biosynthesis. This work focused on the biosynthetic mechanism for alterochromide production, especially revealing functions of two key genes within the gene cluster for the alterochromide biosynthesis. On the one hand, our study provides a target for metabolic engineering of the alterochromide biosynthesis; on the other hand, the 4-HCA synthase AltA and brominase AltN show potential in the biocatalyst industry.

Simultaneous determination of four phytoecdysteroids by LC-MS/MS: application to a comparative pharmacokinetic study in normal and adjuvant arthritis rats after oral administration of C. officinalis Kuan phytoecdysteroids extract.

C. officinalis Kuan is the dry root of Cyathula officinalis Kuan. Clinically, it is used for fall and flutter injury, rheumatism and arthralgia. Phytoecdysteroids have significant anti-inflammatory effects, and the phytoecdysteroids present in C. officinalis Kuan exhibit potential for treating rheumatoid arthritis.This study first developed a selective, accurate and efficient LC-MS/MS method for 12-day pharmacokinetic studies regarding the simultaneous determination of cyasterone, 25-epi-28-epi-cyasterone, precyasterone and capitasterone from C. officinalis Kuan phytoecdysteroids extract in normal and adjuvant arthritis rats.An Agilent Eclipse Plus C18 RRHD column (1.8 µm, 50mm × 2.1 mm) with a gradient mobile phase consisting of water (A) and acetonitrile (B) was used for analysis. The mass analysis was performed in an Agilent 6430 QQQ-MS mass spectrometer with positive mode multiple reaction monitoring (MRM).The results indicated that the AUC0-t and AUC0-∞ values of the four phytoecdysteroids in adjuvant arthritis rats were different from those in normal rats on the first day, which could provide a helpful reference for pharmacological and toxicological studies, as well as clinical applications of C. officinalis Kuan in the treatment of rheumatoid arthritis.

1. C. officinalis Kuan is the dry root of Cyathula officinalis Kuan which has been used for the treatment of flapping injury, rheumatism arthralgia, foot flaccidity, and tendon contracture thousands of years in China, and has been officially included in the Chinese Pharmacopoeia.2. A highly accurate, stable, and sensitive ultra-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method was first established and validated for simultaneously determination four phytoecdysteroids: cyasterone, 25-epi-28-epi-cyasterone, precyasterone and capitasterone in normal and adjuvant arthritis rats plasma samples 12 days of continuous gavage of C. officinalis Kuan phytoecdysteroids extract.3. The phytoecdysteroids is the important component of C. officinalis Kuan, which is difficult to separated. And there is no report for the pharmacokinetic study of phytoecdysteroids from C. officinalis Kuan. And the method provides a good reference for the follow-up studies clinical medication of the phytoecdysteroids from C. officinalis Kuan.

Assessing the possibility of China reaching carbon emission peak by 2030 in the context of the COVID-19 pandemic.

At this stage, it is an important goal for China to solve environmental problems to limit the carbon emission peak by 2030 and then to lower its quality gradually thereafter. The outbreak of the COVID-19 pandemic in 2020 seriously impacted all aspects of China's social economy and brought many uncertainties to the realization of the carbon emission peak. Based on the fact, it has important theoretical and practical significance to take the problem of China's carbon emissions during the COVID-19 as the research object. Thus, this paper analyzes the current situation of China's CO2 emissions and finds out that in the context of ongoing COVID-19 response, stimulated by China's economic stimulus policies, CO2 emissions decrease firstly and then gradually rebound. On this basis, the paper constructs a dynamic model of China's CO2 emission system to simulate the change in China's CO2 emissions under different economic stimulus policies. The relevant simulation results demonstrate the following: (1) China cannot realize the CO2 emission goal by 2030 only by adopting traditional economic stimulus policies. (2) The green economic stimulus policy oriented to the development of clean energy will rebound China's CO2 emissions in the short term, but it can effectively reduce CO2 emissions in the long run. The most important factors affecting CO2 emission reduction are industrial energy consumption and industrial structure, while the energy power generation structure and the transportation structure have a relatively weak impact on the emission reduction effect. (3) The green economic stimulus policy combined with economic stimulus measures and a variety of low-carbon measures can enable China to peak CO2 emissions before 2030, with a peak value of 11.059 billion tons. In general, green economic stimulus policies can achieve a win-win situation for China's economic recovery and carbon emission peak.

99mTc-pertechnetate thyroid static scintigraphy unexpectedly revealed ectopic gastric mucosa of upper esophagus.

Technetium-99m (99mTc)-pertechnetate can be taken up by both gastric mucosa and thyroid tissue. Ectopic gastric mucosa of upper esophagus found in thyroid scintigraphy is rare. We reported a 31-year-old woman who underwent 99mTc-pertechnetate thyroid static scintigraphy for abnormal ultrasound findings. A focal uptake lesion was found in her upper chest. Single photon emission computed tomography/computed tomography (SPECT/CT) imaging showed increased uptake in multiple sites of the upper esophagus without significant thickening. Eventually, gastroscopic findings revealed multiple ectopic gastric mucosa of upper esophagus.

Measuring Well-Being of Migrant Gig Workers: Exampled as Hangzhou City in China.

The consistent innovations and applications of information technology drive the vigorous development of the gig economy, and generate gig workers such as food delivery workers and couriers, and make a great contribution to stabilizing employment and increasing income. Gig workers, mostly made up of migrants, and suffer from job and status difficulties. Research on the well-being of migrant gig workers can reveal the practical problems and provide suggestions for narrowing the wealth gap to promote social fairness and justice. Taking Hangzhou city in China as an example, this paper explores the well-being of food delivery workers, couriers, and online car-hailing drivers as representatives of migrant gig workers. Firstly, the relevant data are acquired through the questionnaire. Secondly, the characteristics of this group are analyzed through descriptive analysis, namely: most of them are migrant workers aged between 20 and 39 with low occupation satisfaction due to insufficient social security coverage and limited well-being, despite relatively high income. Based on the analysis of differences in demographic variables and structural equation modeling, the factors affecting the well-being of migrant gig workers are studied, which mainly are occupation satisfaction, social interaction, and social security. The results show that occupation satisfaction is positively affected by family characteristics, social interaction, and social security. In addition, family characteristics and social security positively impact social interaction, but the former has no significant effect on well-being. Finally, this paper enriches the research on the well-being of specific migrant gig workers and gives policy suggestions for enhancing the well-being of migrant gig workers in Hangzhou city from the perspective of optimizing the mechanism, pilot construction, and platform provision.

Generation of a TPH2-EGFP reporter cell line for purification and monitoring of human serotonin neurons in vitro and in vivo.

Generation of serotonin neurons (SNs) from human pluripotent stem cells (hPSCs) provides a promising platform to explore the mechanisms of serotonin-associated neuropsychiatric disorders. However, neural differentiation always yields heterogeneous cell populations, making it difficult to identify and purify SNs in vitro or track them in vivo following transplantation. Herein, we generated a TPH2-EGFP reporter hPSC line with insertion of EGFP into the endogenous tryptophan hydroxylase 2 (TPH2) locus using CRISPR-Cas9-mediated gene editing technology. This TPH2-reporter, which faithfully indicated TPH2 expression during differentiation, enabled us to obtain purified SNs for subsequent transcriptional analysis and study of pharmacological responses to antidepressants. In addition, the reporter system showed strong EGFP expression to indicate SNs, which enabled us to explore in vitro and ex vivo electrophysiological properties of SNs. In conclusion, this TPH2-EGFP reporter cell line might be of great significance for studies on human SN-related development and differentiation, drug screening, disease modeling, and cell replacement therapies.

Derivation of totipotent-like stem cells with blastocyst-like structure forming potential.

It is challenging to derive totipotent stem cells in vitro that functionally and molecularly resemble cells from totipotent embryos. Here, we report that a chemical cocktail enables the derivation of totipotent-like stem cells, designated as totipotent potential stem (TPS) cells, from 2-cell mouse embryos and extended pluripotent stem cells, and that these TPS cells can be stably maintained long term in vitro. TPS cells shared features with 2-cell mouse embryos in terms of totipotency markers, transcriptome, chromatin accessibility and DNA methylation patterns. In vivo chimera formation assays show that these cells have embryonic and extraembryonic developmental potentials at the single-cell level. Moreover, TPS cells can be induced into blastocyst-like structures resembling preimplantation mouse blastocysts. Mechanistically, inhibition of HDAC1/2 and DOT1L activity and activation of RARγ signaling are important for inducing and maintaining totipotent features of TPS cells. Our study opens up a new path toward fully capturing totipotent stem cells in vitro.

Characterization of differentiated autoregulation of LuxI/LuxR-type quorum sensing system in Pseudoalteromonas.

Gram-negative bacteria usually use acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR-type quorum sensing (QS) systems for cell-cell cooperation and/or bacteria-environment communication. LuxI and LuxR are AHLs synthase and receptor, respectively. These two parts could form a positive regulatory feedback loop, controlling various types of group behaviors. However, the autoregulation mechanisms between them are fragmented and could be highly differentiated in different bacteria. Here, we clarified the autoregulation mechanism between LuxI and LuxR in Pseudoalteromonas sp. R3. YasI (LuxI in strain R3) synthesizes two types of AHLs, C8-HSL and 3-OH-C8-HSL. It is worth noting that YasR (LuxR in strain R3) only responds to C8-HSL rather than 3-OH-C8-HSL. YasR-C8HSL can activate the yasI transcription by recognizing "lux box" at yasI upstream. Interestingly, YasR can directly promote the yasR expression with AHL-independent manner, but AHL absence caused by the yasI-deficiency led to the significant decrease in the yasR expression. Further study demonstrated that the yasI-deficiency can result in the decrease in the yasR mRNA stability. Notably, both yasI-deficiency and yasR-deficiency led to the significant decrease in the expression of hfq encoding RNA chaperone. Therefore, it was speculated that not only YasR itself can directly regulate the yasR transcription, but YasR-C8HSL complex indirectly affects the yasR mRNA stability by regulating Hfq.

Analysis of drug resistance and mutation profiles in Mycobacterium tuberculosis isolates in a surveillance site in Beijing, China.

OBJECTIVE: This study analyzed drug resistance and mutations profiles in Mycobacterium tuberculosis isolates in a surveillance site in Huairou District, Beijing, China. METHODS: The proportion method was used to assess drug resistance profiles for four first-line and seven second-line anti-tuberculosis (TB) drugs. Molecular line probe assays were used for the rapid detection of resistance to rifampicin (RIF) and isoniazid (INH). RESULTS: Among 235 strains of M. tuberculosis, 79 (33.6%) isolates were resistant to one or more drugs. The isolates included 18 monoresistant (7.7%), 19 polyresistant (8.1%), 28 RIF-resistant (11.9%), 24 multidrug-resistant (MDR) (10.2%), 7 pre-extensively drug-resistant (XDR, 3.0%), and 2 XDR strains (0.9%). A higher rate of MDR-TB was detected among previously treated patients than among patients with newly diagnosed TB (34.5% vs. 6.8%). The majority (62.5%) of RIF-resistant isolates exhibited a mutation at S531L in the DNA-dependent RNA polymerase gene. Meanwhile, 62.9% of INH-resistant isolates carried a mutation at S315T1 in the katG gene. CONCLUSION: Our results confirmed the high rate of drug-resistant TB, especially MDR-TB, in Huairou District, Beijing, China. Therefore, detailed drug testing is crucial in the evaluation of MDR-TB treatment.

Multiple metabolic parameters and visual assessment of 18F-FDG uptake heterogeneity of PET/CT in advanced gastric cancer and primary gastric lymphoma.

PURPOSE: Advanced gastric cancer (AGC) and primary gastric lymphoma (PGL) are the two most common malignant tumors of the stomach. Conventional imaging examinations have difficulty distinguishing the two. This study explored the values of multiple parameters and visual assessment of 18F-fluorodeoxyglucose(18F-FDG) uptake heterogeneity of positron emission tomography/computed tomography(PET/CT) for differentiating between AGC and PGL. METHODS: This retrospective study included 70 AGC and 26 PGL patients, all of whom had undergone 18F-FDG PET/CT before treatment. We analyzed the differences between AGC and PGL in the distribution of metastatic lesions and multiple metabolic parameters, including the maximum standardized uptake value (SUVmax), SUVmax/maximal thickness(THKmax), metabolic tumor volume and total lesion glycolysis (TLG). In addition, 18F-FDG uptake heterogeneity was visually assessed using a visual scoring method and a method of measuring SUVmax differences (SUVmax-d). RESULTS: The most common metastasis of AGC patients were liver, bone, peritoneal and proximal lymph nodes; PGL patients had fewer peritoneal metastases and lymph node metastasis could appeared to be "skip metastasis." The metabolic parameters-SUVmax, SUVmax/THKmax and TLG-were higher in patients who had PGL, especially in diffuse large B-cell lymphoma (DLBCL). In the visual assessment of 18F-FDG uptake heterogeneity, the measurements of SUVmax-d in PGL were significantly higher than in AGC. Receiver operating characteristics curve analysis suggested that SUVmax has the highest comprehensive diagnostic efficiency due to having the highest value of area under the curve and the highest accuracy (77.2%). CONCLUSION: 18F-FDG PET/CT had a high diagnostic efficiency for discrimination of AGC and PGL, especially between DLBCL and other pathological subtypes. Visual assessment used to evaluate 18F-FDG uptake heterogeneity could help to distinguish the two types of tumors. In addition, our innovative method of measuring the heterogeneity of 18F-FDG uptake-namely, SUVmax-d-could contribute to identification of the two tumor types and should have its significance clarified by future studies.

A Rare Case of Primary Pleura Myxoid Leiomyosarcoma: FDG PET/CT Finding.

Myxoid leiomyosarcoma is a malignant tumor that originates from the mesenchymal tissue with extensive mucoid degeneration. It usually occurs in the uterus; occurrences from other tissues are extremely rare. Here we report the FDG PET/CT findings and clinicopathological of primary pleura myxoid leiomyosarcoma in a 51-year-old man.

Ultrahigh Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry Method for Qualitative and Quantitative Analyses of Constituents of Corydalis bungeana Turcz Extract.

In this study, the constituents of a Corydalis bungeana Turcz extract were qualitatively analyzed using gradient elution with a mobile phase of 0.2% acetic acid and acetonitrile. We obtained comprehensive insight into the constituents of C. bungeana Turcz extracts through the quantitative analysis of 14 compounds by comparison with authentic reference standards, and tentatively identified an additional 44 compounds through electrospray ionization mass spectrometry (ESI-MS) and tandem MS detection. The separation was successfully achieved using an Agilent SB-C18 column (1.8 µm, 150 × 2.1 mm; Agilent, Santa, CA, USA). A tandem quadrupole spectrometer was operated in either full-scan mode or multiple reaction monitoring (MRM) for the qualitative and quantitative analyses of the constituents, respectively. Validation data (inter-day and intra-day combined) for accuracy and precision ranged from -4.80% to 4.73%, and 0.30% to 4.97%, respectively. An ultrahigh performance liquid chromatographic-ESI-tandem MS (UHPLC-ESI-MS/MS) method for qualitative of C. bungeana Turcz (C. bungeana) extract and the quantification of 14 alkaloids, namely, A-N, was developed and validated. Quantitative analysis involved gradient elution with a mobile phase of 0.1% acetic acid and methanol for 45 min. The separation was successfully achieved using a Waters SB-C18 column (1.8 µm, 100 mm × 2.1 mm, Waters, Milford, Massachusetts, USA). The repeatability and stability of the method also met USFDA criteria with CV values lower than 5%. The limit of detection of the 14 alkaloids ranged from 9.74 to 13.00 ng/mL, whereas the linearities of the standard curves were between 0.9991 and 0.9995. In total, 15 commercial samples from 11 different sources were analyzed.

Discovery and Characterization of SY-1365, a Selective, Covalent Inhibitor of CDK7.

Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. SIGNIFICANCE: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.

Ultrahigh-performance liquid chromatography with tandem mass spectrometry for the determination of 10 alkaloids in beagle plasma after the oral administration of the three Coptidis rhizoma extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Coptidis rhizoma (CR) is the dried rhizome of the ranunculaceous plant CR. For decades in China, this plant has been used to treat hypertension, hyperlipidemia, and chronic diarrhea and has been officially included in the Chinese Pharmacopoeia. The present paper presents a review of the pharmacokinetics of CR. AIM OF THE STUDY: The pharmacokinetic studies and differences of 10 alkaloids among Coptis deltoidea C. Y. cheng et Hsiao, Coptis chinensis Franch and Coptis teeta Wall. Are seldom reported. This study is the first to determine corydaline, dehydrocorydaline, tetrahydropalmatine, palmatine, magnoflorine, jatrorrhizine, berberine, worenine, berberrubine, and coptisine, which adopted an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, simultaneously. MATERIALS AND METHODS: Chromatographic separation was performed within 8 min by using an Agilent SB-C18 column (150 mm × 2.1 mm, 1.8 µm) with gradient mobile phase consisting of 0.3% acetic acid water (v/v) and acetonitrile at a flow rate of 0.3 mL/min. Multiple reaction monitoring mode was used to detect the tandem mass spectrum in the positive ionization mode by electrospray ionization source. RESULTS: The method was fully validated to be linear over a wide concentration (r > 0.9916), and the linear concentration range was 0.195-2260 ng/mL. Intra- and interday precisions were below 14.19% and 18.56% for the 10 analytes, respectively. The accuracy ranged from -9.30% to 6.31%. The extraction recovery of the 10 alkaloids and internal standard ranged from 79.76% to 95.37%. Pharmacokinetic comparative study showed that the Cmax and AUC0-∞ values of dehydrocorydaline, tetrahydropalmatine, palmatine, magnoflorine, jatrorrhizine, berberine, worenine, berberrubine, and coptisine increased significantly (p < 0.05), which was different for beagles after oral administration. The results can help determine the mechanism of action and guide clinical application of these three extracts. CONCLUSION: This validated method was successfully applied for the pharmacokinetics study of beagle plasma after oral administration of three CR extract types.

18F-FDG PET/CT for staging and response assessment of primary parotid MALT lymphoma with multiple sites involvement: A case report.

RATIONALE: Mucosa-associated lymphoid tissue (MALT) lymphoma is an extranodal low-grade B cell lymphoma that generally exhibits an indolent clinical course. Currently, the application of F-fluorodeoxyglucose positron emission tomography/computed tomography (F-FDG PET/CT) in MALT lymphoma is still controversial. Herein, we reported a case of using F-FDG PET/CT for staging and response assessment of primary parotid MALT lymphoma with multiple sites involvement. As far as we know, there are no similar case reports have been published before. PATIENT CONCERNS: A 71-year-old woman, who received mass resection twice during the past 2 years due to the repeatedly relapse of facial painless masses and diagnosed as reactive lymphoid hyperplasia by pathologic tests. However, the pathological diagnosis was then changed to primary parotid MALT lymphoma after left parotidectomy operation because of a new mass found in her left parotid. Four months later, the right eyelid of the patient swelled with a blurred vision. Then, F-FDG PET/CT scan was performed for staging, and the imaging results showed an abnormal increase of F-FDG uptake in multiple sites including bilateral ocular adnexal, lungs, pleura, occipital subcutaneous tissue, left kidney, and lymph nodes. DIAGNOSES: The patient was diagnosed as primary parotid MALT lymphoma with Ann Arbor stage of IVA based on the F-FDG PET/CT findings. INTERVENTIONS: The patient received 4 cycles of chemotherapy, followed by a partial metabolic remission (PMR), which was determined by interim F-FDG PET/CT, and finally additional 2 cycles of chemotherapy. OUTCOMES: The follow-up study illustrated that the patient had been alive and doing well at 12 months after chemotherapy. LESSONS: Although MALT lymphoma normally localizes in the primary organs, the involvement of multiple organs and lymph nodes is possible. The use of PET/CT demonstrated significant clinical values in the accurate staging and response assessment of F-FDG-avid MALT lymphoma. It is potentially useful for indicating the progress and transformation of MALT lymphoma, and guidance in localization of pathological biopsy. It is also helpful for clinicians to choose reasonable treatment strategy and improve the prognosis of patients.

A LC-MS/MS method for simultaneous determination of seven alkaloids in rat plasma after oral administration of Phellodendri chinensis cortex extract and its application to a pharmacokinetic study.

A sensitive and rapid liquid chromatography with tandem mass spectrometry method has been developed for simultaneous determination of berberine (I), jateorhizine (II), palmatine (III), tetrahydropalmatine (IV), phellodendrine (V), protopine (VI) and columbamine (VII) in rat plasma after oral administration of Phellodendri chinensis cortex extraction. The plasmas were extracted by liquid-liquid extraction. The tandem mass spectrometric detection was performed in the multiple reaction monitoring mode in the positive ionization. The intra- and interday precisions and accuracies were in range from -12.18 to 13.21%. Mean absolute recoveries of all analytes and internal standard were between 78.6 and 98.9%. The seven alkaloids were proven to be stable during sample storage and analysis procedures. The established method was validated and successfully applied to pharmacokinetics study in rat plasma after oral administration of Phellodendri chinensis cortex extract. The t1/2 of palmatine, columbamine, pellodendrine, berberine, tetrahydropalmaine, jatrorrhizine, and protopine were 5.16, 5.96, 7.18, 19.84, 6.28, 7.08, 6.90 h, respectively. The seven compounds could be rapidly absorbed into blood (time for maximal concentration, 1.80-1.93 h). This study could establish a foundation for further research of Phellodendri chinensis cortex and might provide more useful information to guide the clinical usage.

Simultaneous Determination and Pharmacokinetics Study of Six Triterpenes in Rat Plasma by UHPLC-MS/MS after Oral Administration of Sanguisorba officinalis L. Extract.

A selective and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of ziyuglycoside I (I), 3ß,19α-dihydroxyurs-12-en-28-oic-acid 28-ß-d-glucopyranosyl ester (II), 3ß-[(α-l-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid ß-d-glucopyranosyl ester (III), rosamultin (IV), 1ß-hydroxyeuscaphic acid (V) and alpinoside (VI) in rats after oral administration of Sanguisorba officinalis L. (S. officinalis) extract. The 3ß,19α-dihydroxyurs-12-en-28-oic-acid 28-ß-d-glucopyranosyl ester, 3ß-[(α-l-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid ß-d-glucopyranosyl ester, rosamultin, 1ß-hydroxyeuscaphic acid and alpinoside in rat plasma were the first report in the pharmacokinetics study in the present study. The analytes were quantified using the multiple reaction monitoring (MRM) mode with the electrospray ion source in positive electrospray ionization. Plasma was extracted with ethyl acetate via liquid⁻liquid extraction. Bifendate was used as internal standard (IS). The current method was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery, matrix effect and stability. The lower limits of quantification of ziyuglycoside I, 3ß,19α-dihydroxyurs-12-en-28-oic-acid 28-ß-d-glucopyranosyl ester, 3ß-[(α-l-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid ß-d-glucopyranosyl ester, rosamultin, 1ß-hydroxyeuscaphic acid and alpinoside were 6.1, 4.9, 1.3, 3.8, 1.5 and 5.7 ng/mL, respectively. Intra-day and inter-day precision and the accuracy of the assay were in range from -9.48 to 12.74%. The extraction recoveries of analytes and bifendate (IS) from rat plasma ranged from 77.17% to 92.48%. Six compounds could be rapidly absorbed into blood (Tmax, 0.58⁻1.58 h), and then eliminated relatively slowly (t1/2, 6.86⁻11.63 h). The pharmacokinetic results might contribute to further guide the clinical application of S. officinalis.

An Ultra-High Performance Liquid Chromatography with Tandem Mass Spectrometry Method for Determination of 10 Alkaloids in Beagle Dog Plasma after the Oral Administration of the Corydalis yanhusuo W.T. Wang Extract and Yuanhuzhitong Tablets.

This study has developed a sensitive and simple ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of corydaline, dehydrocorydaline, tetrahydropalmatine, protopine, palmatine, tetrahydroberberine, columbamine, berberine, coptisine and berberrubine in beagle dog plasma after the oral administration of the Corydalis yanhusuo W.T. Wang and Yuanhuzhitong tablets. Chromatographic separation was achieved on an Agilent Eclipse Plus C18 RRHD column (1.8 µm, 50 × 2.1 mm) using a gradient elution program with a mobile phase consisting of acetonitrile and water containing 0.1% formic acid at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection was conducted by multiple reaction monitoring (MRM) mode via an electrospray ionization source in the positive mode. The calibration curves of all analytes showed good linear (r² > 0.9800). The intra-day and inter-day precisions were less than 15% and the accuracies were within ±15%. The extraction recoveries conformed to the acceptable range. And there was no interference of endogenous substances in the sensitive assay method. All analytes were proven to be stable during sample storage and analysis procedures. The pharmacokinetic study indicated that the Yuanhuzhitong tablets could get a better absorption than Corydalis yanhusuo W.T. Wang.

Liquid Chromatography-Tandem Mass Spectrometry Simultaneous Determination and Pharmacokinetic Study of Fourteen Alkaloid Components in Dog Plasma after Oral Administration of Corydalis bungeana Turcz Extract.

A rapid and sensitive Ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of fourteen alkaloids in beagle dog plasma after a single oral dose of the Corydalis bungeana Turcz (C. bungeana) extract selected bifendate as the internal standard (IS). The plasma samples were preprocessed by liquid-liquid extraction (LLE) with aether before separation on an Agilent SB-C18 column (1.8 µm, 150 × 2.1 mm) using a gradient elution program. The mobile phase consists of 0.2% acetic acid and acetonitrile at the flow rate of 0.3 mL/min. In the positive ion mode, the analytes were detected by multiple reaction monitoring (MRM). The results indicated that calibration curves for fourteen analytes have good linearity (R² = 0.9904). The lower limits of quantification (LLOQ) of fourteen alkaloids and IS were all over 4.87 ng/mL and the matrix effects ranged from 94.08% to 102.76%. The mean extraction recoveries of Quality control samples at low (LQC), medium (MQC) and high (HQC) and IS were all more than 78.03%. The intra- and inter-day precision (R.S.D.%) also met the criterion, at the same time the deviation of assay accuracies (R.E) ranged from -13.70% to 14.40%. The Tmax values of fourteen alkaloids were no more than 1 h. The range of Cmax was from 74.16 ± 8.71 to 2256 ± 255.9 ng/mL. The assay was validated in the light of the regulatory bioanalytical guidelines and proved acceptable, which was successfully applied to a pharmacokinetic study of these compounds in beagle dogs after oral administration of Corydalis bungeana Turcz extract.