[Content determination of seven active components of Eucommiae Cortex in aortic vascular endothelial cells of spontaneously hypertensive rats by UPLC-MS/MS].

In the present study, the contents of seven active components [genipinic acid(GA), protocatechuic acid(PCA), neochlorogenic acid(NCA), chlorogenic acid(CA), cryptochlorogenic acid(CCA),(+)-pinoresinol di-O-ß-D-glucopyranosid(PDG), and(+)-pinoresinol 4'-O-ß-D-glucopyranoside(PG)] of Eucommiae Cortex in aortic vascular endothelial cells of spontaneously hypertensive rats(SHR) were simultaneously determined by ultra-high liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The qualified SHR models were selected. The primary aortic endothelial cells(VECs) of rats were separated and cultured by ligation and adherence, followed by subculture. After successful identification, an UPLC-MS/MS method for simultaneously determining the contents of GA, PCA, NCA, CA, CCA, PDG, PG in seven components of Eucommiae Cortex in VECs was established, including specificity, linearity, matrix effect, recovery, accuracy, precision and stability. The established method had the lo-west limit of quantification of 0.97-4.95 µg·L~(-1), accuracy of 87.26%-109.6%, extraction recovery of 89.23%-105.3%, matrix effect of 85.86%-106.2%, and stability of 86.00%-112.5%. Therefore, the established accurate UPLC-MS/MS method could rapidly and simultaneously determine the contents of the seven active components of Eucommiae Cortex in VECs of SHRs, which provided a refe-rence for the study of cellular pharmacokinetics of active components of Eucommiae Cortex extract.

Clinicopathological Characteristics and Prognosis in Endometrial Cancer With Bone Metastasis: A SEER-Based Study of 584 Women.

PURPOSE: Bone metastasis from endometrial cancer (EC) is rare and poorly described. The purpose of the present study was to investigate the correlation between the clinically accessible factors and survival time among EC patients with bone metastasis. PATIENTS AND METHODS: We retrospectively identified and reviewed EC patients with bone metastasis from 2010 to 2016, based on the Surveillance, Epidemiology and End Results (SEER) database. Univariable and multivariable Cox regressions were applied to evaluate the effects of clinical variables on survival. Kaplan-Meier plots were used to visually demonstrate the correlation between independent risk factors and survival. RESULTS: Clinical data of 584 EC patients with bone metastasis from the SEER database were analyzed. EC patients with bone metastasis experienced extremely poor survival, with 1-year overall survival (OS) and cancer-specific survival (CSS) rates 33.8 and 35.8%, respectively. Variables associated with OS and CSS in the univariable analysis included race, tumor grade, tumor subtype, tumor size, lung, liver and brain metastases, surgery, radiotherapy, and chemotherapy. In the multivariable analysis, tumor grade, tumor subtype, liver and brain metastases, local surgery, and systemic chemotherapy remained independent risk factors for OS and CSS. However, local radiotherapy was an independent predictor of OS, not CSS. CONCLUSIONS: We identified several factors affect the survival of EC patients with bone metastasis, which is useful for clinicians to assess patients' outcomes. Our study supports surgery and radiotherapy of primary EC, and systemic chemotherapy for prolonging survival among EC patients with bone metastasis, which lays a solid foundation for defining optimal treatment strategy in this specific cohort.

2-methylbenzoyl berbamine, a multi-targeted inhibitor, suppresses the growth of human osteosarcoma through disabling NF-κB, ERK and AKT signaling networks.

Osteosarcoma is the most common malignant bone tumor in children and young adults, and it has a survival rate of only 60% with current cytotoxic chemotherapy combined with aggressive surgery. The aim of this study was to evaluate the therapeutic efficacy of the berbamine derivative 2-methylbenzoyl berbamine (BBD24) for osteosarcoma in vitro and in vivo. We used human osteosarcoma cell lines, primary osteosarcoma cells and mouse models to evaluate the inhibitory effects of BBD24 on osteosarcoma and to determine the molecular mechanism. Our results showed that BBD24 inhibited the growth of the human osteosarcoma cell lines HOS and MG63 in a time- and dose-dependent manner. BBD24 also exhibited significant inhibitory effects on primary osteosarcoma cells. In contrast, BBD24 did not affect normal blood cells under the same conditions. Treatment with BBD24 induced apoptosis, necrosis and autophagy in osteosarcoma cells. Western blot analysis revealed that BBD24 activated the caspase-dependent pathway and downregulated the NF-kB, AKT, and ERK pathways. Finally, BBD24 treatment induced a significant inhibitory effect on the growth of osteosarcoma in nude mice. Our findings indicate that BBD24 is a multitarget inhibitor and may represent a new type of anticancer agent for osteosarcoma treatment.

[Simultaneous determination of four components of Shuganning Injection in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study].

This study aims to reveal the pharmacokinetics of Shuganning Injection in normal rats. In this experiment,ultra-high performance liquid chromatography-electrospray-tandem mass spectrometry( UPLC-ESI-MS/MS) was used to establish an analytical method for simultaneous determination of chlorogenic acid,gardenioside,oroxylin A and baicalin in rat plasma. Then,the non-compartmental model( NCA) in Phoenix WinN onL in 6. 4 software was used to fit pharmacokinetic parameters. The methodological validation showed that the linear relationship of the components in rat plasma samples were good( r>0. 995). The recovery rate and matrix effect of plasma samples with low,middle and high concentration were 79. 14%-101. 4%. The intra-day and inter-day precision,accuracy and stability meet the requirements of biological sample analysis. The half-life( t1/2) of chlorogenic acid,gardenioside,oroxylin A did not change significantly and the area under blood concentration-time curve( AUC0-t) is proportional to the dose,which suggested that three components showed a linear kinetic characteristics,but baicalin showed nonlinear kinetic characteristics. Moreover,the retention time of each component in rats was short. The established UPLC-MS/MS quantitative analysis method is rapid,sensitive and accurate,which can be used for the determination of chlorogenic acid,gardenioside,oroxylin A and baicalin in rat plasma and pharmacokinetic study of Shuganning Injection.

Tissue Distribution Comparison of Six Active Ingredients from an Eucommiae Cortex Extract between Normal and Spontaneously Hypertensive Rats.

Eucommiae Cortex (EC), a rare, nourishing medicinal herb that is native in China, has good effect in the treatment of hypertension. In this study, we compared tissue distribution of six representative active components of EC extract-genipinic acid (GA), protocatechuic acid (PCA), neochlorogenic acid (NCA), chlorogenic acid (CA), (+)-pinoresinol di-O-ß-D-glucopyranoside (PDG), and (+)-pinoresinol 4'-O-ß-D-glucopyranoside (PG)-between normal rats and spontaneously hypertensive rats (SHRs). Each rat was intragastrically given EC extract at a dose of 5.4 g/kg. Rats were sacrificed at 10 min, 30 min, 2 h, and 8 h after administration; six rats were sacrificed at each time point. Then, we quickly harvested their major organs, including heart, liver, spleen, lungs, kidneys, and brain. Using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), we determined the levels of the above mentioned six components in the organs of both types of rats and then analyzed differences in the tissue distribution. The results showed that levels of each component differed between SHRs and the normal group at each time point. As time progressed, the number of organs in which GA distribution in each tissue of SHRs differed from that of the normal group gradually increased: SHRs showed lower GA levels than normal rats. Levels of PG and PDG in both groups at 10 and 30 min were similar. NCA and CA in the SHR group and the normal group at 10 min, 30 min, and 2 h were also similar to some extent. The results indicated that the pathological state of spontaneous hypertension could affect tissue distribution of EC active components in rats.

Loss of TIMP3 expression induces inflammation, matrix degradation, and vascular ingrowth in nucleus pulposus: A new mechanism of intervertebral disc degeneration.

Low back pain (LBP) is one of the most common complains in orthopedic outpatient department and intervertebral disc degeneration (IDD) is one of the most important reasons of LBP. The mechanisms of IDD contain a complex biochemical cascade which includes inflammation, vascular ingrowth, and results in degradation of matrix. In our study, we used both in vitro and in vivo models to investigate the relation between tissue inhibitor of metalloproteinase-3 (TIMP3) expression and IDD. Loss of TIMP3 expression was found in degenerative intervertebral disc (IVD), this change of expression was closely related with the dephosphorylation of smad2/3. Overexpression of TIMP3 significantly inhibited the release of TNF-α and matrix degradation induced by Lipopolysaccharide. Vascular ingrowth was also suppressed by TIMP3 in the in vitro and in vivo models. Further, animal experiments confirmed that the degeneration of IVD was reduced after overexpression of TIMP3 in nucleus pulposus. Taken together, our results indicated TIMP-3 might play an important role in the pathogenesis of IDD and therefore be a potential therapeutic target in the future.

Comparative absorption kinetics of seven active ingredients of Eucommia ulmoides extracts by intestinal in situ circulatory perfusion in normal and spontaneous hypertensive rats.

Eucommia ulmoides Oliv. (E. ulmoides) is a valuable and nourishing medicinal herb in China that has been used in the treatment of hypertension. Given the fact that most traditional Chinese medicine is mainly used to treat disease, investigating the pharmacokinetics of traditional Chinese medicines in the pathological state is more useful than that in the normal state. However, the differences in the absorption kinetics of active ingredients of E. ulmoides extract between pathological and physiological conditions have not been reported. Therefore, in this study, the rat intestinal in situ circulatory perfusion model was used to investigate the differences in absorption kinetics of seven active ingredients of E. ulmoides extract in normal and spontaneously hypertensive rats, namely, genipinic acid, protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, (+)-pinoresinol di-O-ß-D-glucopyranoside and (+)-pinoresinol 4'-O-ß-D-glucopyranoside. Our results indicate that the pathological state of spontaneous hypertension may change the absorption of active components of E. ulmoides extracts, and these findings may provide a reference for improving the rational use of E. ulmoides in the clinic.

[Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis].

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

A UPLC-ESI-MS/MS Method for Simultaneous Quantitation of Chlorogenic Acid, Scutellarin, and Scutellarein in Rat Plasma: Application to a Comparative Pharmacokinetic Study in Sham-Operated and MCAO Rats after Oral Administration of Erigeron breviscapus Extract.

Erigeron breviscapus, a traditional Chinese medicine, is clinically used for the treatment of occlusive cerebral vascular diseases. We developed a sensitive and reliable ultra-performance liquid chromatography-electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS) method for simultaneous quantitation of chlorogenic acid, scutellarin, and scutellarein, the main active constituents in Erigeron breviscapus, and compared the pharmacokinetics of these active ingredients in sham-operated and middle cerebral artery occlusion (MCAO) rats orally administrated with Erigeron breviscapus extract. Plasma samples were collected at 15 time points after oral administration of the Erigeron breviscapus extract. The levels of chlorogenic acid, scutellarin, and scutellarein in rat plasma at various time points were determined by a UPLC-ESI-MS/MS method, and the drug concentration versus time plots were constructed to estimate pharmacokinetic parameters. The concentration of chlorogenic acid in the plasma reached the maximum plasma drug concentration in about 15 min and was below the limit of detection after 4 h. Scutellarin and scutellarein showed the phenomenon of multiple absorption peaks in sham-operated and MCAO rats, respectively. Compared with the sham-operated rats, the terminal elimination half-life of scutellarein in the MCAO rats was prolonged by more than two times and the area under the curve of each component in the MCAO rats was significantly increased. The results showed chlorogenic acid, scutellarin, and scutellarein in MCAO rats had higher drug exposure than that in sham-operated rats, which provided a reference for the development of innovative drugs, optimal dosing regimens, and clinical rational drug use.

Ginkgolide B exerts anti-inflammatory and chondroprotective activity in LPS-induced chondrocytes.

BACKGROUND: Osteoarthritis (OA) is one of degenerative and chronic diseases of articular joints. Articular cartilage is an avascular tissue, and its primary cellular component is chondrocytes. The main characteristic of OA is non-classic inflammation and cartilage degeneration. Ginkgolide B (GB) is a component of Ginkgo biloba which has diverse bioactivities. OBJECTIVES: The present study uses an in vitro experimental model to detect the underlying anti-inflammatory and chondroprotective effects of GB and provides a new way for future clinical therapy of OA. MATERIAL AND METHODS: Rat chondrocytes were isolated, cultured and treated with 1 µg/mL lipopolysaccharide (LPS) and/or different concentrations of GB. Cell Counting Kit-8 (CCK-8) was used to test the cell viability of chondrocytes, and chondrocytes apoptosis was detected using a cell apoptosis kit. Collagen-II and aggrecan expression were detected by immunohistochemistry. Relative expression of genes was detected by real-time PCR and western blot. RESULTS: Ginkolide B did not inhibit chondrocyte proliferation, and ginkgolide B inhibited LPS induced matrixdegradation in chondrocytes. Ginkgolide B also reversed LPS-induced collagen-II and aggrecan decreased in chondrocytes via upregulated synthesis-related gene expression and downregulated matrix-degrading enzyme gene expression. Furthermore, we found that ginkgolide B significantly inhibited LPS-induced MAPK pathway activation. CONCLUSIONS: The results of our study suggest that ginkgolide B exerted anti-inflammatory and chondroprotective effects in LPS-induced chondrocytes, and might be an underlying therapy for OA afterwards.

[Effect of leptin on inducible NO and MMP-13 in rabbit articular chondrocytes in vitro].

OBJECTIVE: To observe the in vitro effect of leptin, alone or in combination with tumor necrosis factor-alpha (TNF-alpha) on inducible nitric oxide(NO)and on inducible matrix metalloproteinase-13 (MMP-13) in rabbit articular chondrocytes. METHODS: The chondrocytes from the articular cartilage of 2-month-old rabbits were cultivated and identified, and the second filial generation chondrocytes were cocultured on plates with different concentrations of leptin alone or in combination with TNF-alpha for 48 h or 96 h after 12 h starvation. The concentration of NO and MMP-13 was measured in the chondrocytes culture supernatant fluid. The results were statistically analyzed. RESULTS: There was no significant difference in the concentrations of NO between the different concentrations of leptin alone groups and the blank control group (P>0.05). In combination with the same concentration of TNF-alpha (10 ng/mL), leptin could dose-dependently increase the concentration of NO in the chondrocytes culture supernatant fluid in vitro. There was significant value in average concentration of MMP-13 on the main effect of both time and dose (P<0.05). No MMP-13 was detected in the blank control group. CONCLUSION: Leptin can induce MMP-13 and have synergistic induction effect on NO with TNF-alpha in rabbit articular chondrocytes in vitro.