Pharmacologically significant constituents collectively responsible for anti-sepsis action of XueBiJing, a Chinese herb-based intravenous formulation.

Sepsis, a life-threatening health issue, lacks effective medicine targeting the septic response. In China, treatment combining the intravenous herbal medicine XueBiJing with conventional procedures reduces the 28-day mortality of critically ill patients by modulating septic response. In this study, we identified the combined active constituents that are responsible for the XueBiJing's anti-sepsis action. Sepsis was induced in rats by cecal ligation and puncture (CLP). The compounds were identified based on their systemic exposure levels and anti-sepsis activities in CLP rats that were given an intravenous bolus dose of XueBiJing. Furthermore, the identified compounds in combination were assessed, by comparing with XueBiJing, for levels of primary therapeutic outcome, pharmacokinetic equivalence, and pharmacokinetic compatibility. We showed that a total of 12 XueBiJing compounds, unchanged or metabolized, circulated with significant systemic exposure in CLP rats that received XueBiJing. Among these compounds, hydroxysafflor yellow A, paeoniflorin, oxypaeoniflorin, albiflorin, senkyunolide I, and tanshinol displayed significant anti-sepsis activities, which involved regulating immune responses, inhibiting excessive inflammation, modulating hemostasis, and improving organ function. A combination of the six compounds, with the same respective doses as in XueBiJing, displayed percentage survival and systemic exposure in CLP rats similar to those by XueBiJing. Both the combination and XueBiJing showed high degrees of pharmacokinetic compatibility regarding interactions among the six active compounds and influences of other circulating XueBiJing compounds. The identification of XueBiJing's pharmacologically significant constituents supports the medicine's anti-sepsis use and provides insights into a polypharmacology-based approach to develop medicines for effective sepsis management.

Novel assays for quality evaluation of XueBiJing: Quality variability of a Chinese herbal injection for sepsis management.

XueBiJing is an intravenous five-herb injection used to treat sepsis in China. The study aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS)- or liquid chromatography-ultraviolet (LC-UV)-based assay for quality evaluation of XueBiJing. Assay development involved identifying marker constituents to make the assay therapeutically relevant and building a reliable one-point calibrator for monitoring the various analytes in parallel. Nine marker constituents from the five herbs were selected based on XueBiJing's chemical composition, pharmacokinetics, and pharmacodynamics. A selectivity test (for "similarity of response") was developed to identify and minimize interference by non-target constituents. Then, an intercept test was developed to fulfill "linearity through zero" for each analyte (absolute ratio of intercept to C response, <2%). Using the newly developed assays, we analyzed samples from 33 batches of XueBiJing, manufactured over three years, and found small batch-to-batch variability in contents of the marker constituents (4.1%-14.8%), except for senkyunolide I (26.5%).

Novel assays for quality evaluation of XueBiJing:Quality variability of a Chinese herbal injection for sepsis management / 药物分析学报

XueBiJing is an intravenous five-herb injection used to treat sepsis in China.The study aimed to develop a liquid chromatography-tandem mass spectrometry(LC-MS/MS)-or liquid chromatography-ultraviolet(LC-UV)-based assay for quality evaluation of XueBiJing.Assay development involved identifying marker constituents to make the assay therapeutically relevant and building a reliable one-point cali-brator for monitoring the various analytes in parallel.Nine marker constituents from the five herbs were selected based on XueBiJing's chemical composition,pharmacokinetics,and pharmacodynamics.A selectivity test(for"similarity of response")was developed to identify and minimize interference by non-target constituents.Then,an intercept test was developed to fulfill"linearity through zero"for each analyte(absolute ratio of intercept to C response,＜2％).Using the newly developed assays,we analyzed samples from 33 batches of XueBiJing,manufactured over three years,and found small batch-to-batch variability in contents of the marker constituents(4.1％-14.8％),except for senkyunolide I(26.5％).

Paeoniflorin inhibits MRGPRX2-mediated pseudo-allergic reaction via calcium signaling pathway.

Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) has been shown to be a pivotal target for pseudo-allergic diseases. Therefore, MRGPRX2 might be a therapeutic target for allergic contact dermatitis, atopic dermatitis, and red man syndrome. Paeoniflorin (PF) was reported to have an antiinflammatory effect in neuroinflammation, enteritis, and so forth. In this study, we investigated the anti-pseudo-allergic effect of PF and the underlying molecular mechanisms. Our results showed that PF can suppress compound 48/80 (C48/80)-induced PCA and MCs degranulation in vivo, in a dose-dependent manner. Moreover, PF can reduce C48/80-induced calcium influx and suppress MC degranulation and chemokines release in vitro. PF can downregulate the phosphorylation levels of key kinases in PLCγ-regulated calcium influx and subsequent cytokine synthesis pathways. Our study revealed that PF could inhibit C48/80-induced allergic responses both in vivo and in vitro. As such, it may be regarded as a novel inhibitor for preventing MRGPRX2-mediated allergic diseases.

Nobiletin attenuates adverse cardiac remodeling after acute myocardial infarction in rats via restoring autophagy flux.

BACKGROUND: Our previous study showed that autophagy flux was impaired with sustained heart ischemia, which exacerbated adverse cardiac remodeling after acute myocardial infarction (AMI). Here we investigated whether Nobiletin, a citrus polymethoxylated flavonoids, could restore the autophagy flux and improve cardiac prognosis after AMI. AMI was induced by ligating left anterior descending (LAD) coronary artery in rats. Nobiletin improved the post-infarct cardiac dysfunction significantly and attenuated adverse cardiac remodeling. Meanwhile, Nobiletin protected H9C2 cells against oxygen glucose deprivation (OGD) in vitro. The impaired autophagy flux due to ischemia was ameliorated after Nobiletin treatment by testing the autophagy substrate, LC3BⅡ and P62 protein level both in vivo and in vitro. GFP-mRFP-LC3 adenovirus transfection also supported that Nobiletin restored the impaired autophagy flux. Specifically, the autophagy flux inhibitor, chloroquine, but not 3 MA, alleviated Nobiletin-mediated protection against OGD. Notably, Nobiletin does not affect the activation of classical upstream autophagy signaling pathways. However, Nobiletin increased the lysosome acidation which also supported that Nobiletin accelerated autophagy flux. Taken together, our findings suggested that Nobiletin restored impaired autophagy flux and protected against acute myocardial infarction, suggesting a potential role of autophagy flux in Nobiletin-mediated myocardial protection.

Pharmacokinetics and disposition of monoterpene glycosides derived from Paeonia lactiflora roots (Chishao) after intravenous dosing of antiseptic XueBiJing injection in human subjects and rats.

AIM: Monoterpene glycosides derived from Paeonia lactiflora roots (Chishao) are believed to be pharmacologically important for the antiseptic herbal injection XueBiJing. This study was designed to characterize the pharmacokinetics and disposition of monoterpene glycosides. METHODS: Systemic exposure to Chishao monoterpene glycosides was assessed in human subjects receiving an intravenous infusion and multiple infusions of XueBiJing injection, followed by assessment of the pharmacokinetics of the major circulating compounds. Supportive rat studies were also performed. Membrane permeability and plasma-protein binding were assessed in vitro. RESULTS: A total of 18 monoterpene glycosides were detected in XueBiJing injection (content levels, 0.001-2.47 mmol/L), and paeoniflorin accounted for 85.5% of the total dose of monoterpene glycosides detected. In human subjects, unchanged paeoniflorin exhibited considerable levels of systemic exposure with elimination half-lives of 1.2-1.3 h; no significant metabolite was detected. Oxypaeoniflorin and albiflorin exhibited low exposure levels, and the remaining minor monoterpene glycosides were negligible or undetected. Glomerular-filtration-based renal excretion was the major elimination pathway of paeoniflorin, which was poorly bound to plasma protein. In rats, the systemic exposure level of paeoniflorin increased proportionally as the dose was increased. Rat lung, heart, and liver exposure levels of paeoniflorin were lower than the plasma level, with the exception of the kidney level, which was 4.3-fold greater than the plasma level; brain penetration was limited by the poor membrane permeability. CONCLUSION: Due to its significant systemic exposure and appropriate pharmacokinetic profile, as well as previously reported antiseptic properties, paeoniflorin is a promising XueBiJing constituent of therapeutic importance.

Profiling of miRNA expression in immune thrombocytopenia patients before and after Qishunbaolier (QSBLE) treatment.

Immune thrombocytopenia (ITP), also known as idiopathic thrombocytopenic purpura, is an autoimmune disease characterized by low platelet count and increased bleeding tendency. Currently, glucocorticoid and splenectomy are the main therapies for ITP but with obvious side effects including tendency of relapse and risk of internal bleeding. In this study, we report the Mongolian medicine Qishunbaolier (QSBLE) can significantly and efficiently increase platelet count with a low recurrent rate and unnoticeable side effect. We profiled the microRNA (miRNA) expression in the blood sample of ITP patients and identified 44 miRNAs that are differentially expressed in ITP patients before and after QSBLE treatment. Out of these 44 miRNAs, 25 are expressed in control subjects and are downregulated in ITP patients, whereas the treatment with QSBLE restores their expressions to the level of control subjects. This result suggests that abnormal expression of these 25 miRNAs might be connected to the pathogenesis of ITP. Interestingly, 14 of those 44 miNRAs are predicted to target at least once on 31 known IPT associated genes, indicating the possible mechanism of QSBLE on ITP therapy.

Effects of chronic treatment with honokiol in spontaneously hypertensive rats.

The present study was performed to evaluate the antihypertensive effects of honokiol in vivo in spontaneously hypertensive rats (SHR). The effects of honokiol were investigated by determination of the blood pressure, vascular reactivity, oxidative parameters, and histologic change in the aorta. Long-term administration of honokiol (400 mg/kg/d) to SHR decreased systolic blood pressure significantly. Honokiol (200, 400 mg/kg/d) enhanced the aortic relaxation in response to acetylcholine after 49-d treatment, but had no significant effects on the relaxation to sodium nitroprusside. The oral administration of honokiol significantly increased the plasma level of NO(2(-))/NO(3(-)), but decreased the level of malondialdehyde in liver of SHR compared with the control vehicle. In addition, SHR administered honokiol showed significant reductions in the elastin bands and media thickness in the aorta. These results suggest that chronic treatment with honokiol exerts an antihypertensive effect in SHR, and its vasorelaxant action and antioxidant properties may contribute to reducing the elevated blood pressure.

Antiplasmodial isoflavanones from the roots of Sophora mollis.

Six new prenylated isoflavanones named sophoronols A-F (1-6), together with eight phenolic constituents, were isolated from the roots of Sophora mollis. Their structures and stereochemistry were established by 1D and 2D NMR techniques, especially HMBC and NOESY as well as CD results. Componds 3 and 5 exhibited moderate anitplasmodial activity against the CQS D10 strain of Plasmodium falciparum, with IC(50) values of 12.9 and 12.8 microM, respectively.

[Effect of ursolic acid on caspase-3 and PARP expression of human MCF-7 cells].

OBJECTIVE: To study the effect of ursolic acid (UA), apentacyclic triterpene acid, on MCF-7 cell apoptosis, and probable mechanism involved by detecting the expressions of caspase-3 and poly ADP-ribose polymerase(PARP) at protein level. METHOD: MCF-7 cells were cultured with different concentrations of UA. Growth inhibition of UA on MCF-7 cells was evaluated by MTT assay. Cell cycle and sub-G1 peak were performed by FCM. Morphologic changes of UA-treated cells were observed by light microscope. Apoptotic cells with condensed or fragmented nuclei were visualized by Ho 33258 staining by a fluorescence microscope (EX: U. V.). The protein expression of caspase-3 and PARP was analyzed by immunofluorescence cell staining (SABC-Cy3). RESULT: 24 hours after UA treatment, inhibition of MCF-7 cell growth was concentration-dependent. The IC50 value for UA was (22.6 +/- 3.0) micromo x L(-1). Cell cycle anaysis by FCM showed that 50 micromol x L(-1) of UA arrested MCF-7 cell cycle at G0 - G1 phase. Morphological changes of MCF-7 Cells exhibited many of the hallmark features of apoptosis, including chromatin clumps and aggregation and DNA fragmentation. UA increased caspase-3 protein expression. CONCLUSION: The results suggest that UA evokes MCF-7 cell apoptosis is correlation with the up-regulation of caspase-3. Our study indicated that UA might be a potential Chinese medical component for breast neoplasm.

[Experimental study on MCF-7 cell apoptosis induced by ursolic acid].

OBJECTIVE: To study the effects of ursolic acid (UA), apentacyclic triterpene acid, on MCF-7 cell apoptosis. METHODS: MCF-7 cells were cultured with different concentrations of UA. Viability of UA-induced MCF-7 cells was evaluated by MTT assay. Cell cycle and sub-G1 peak were performed by FCM. Morphologic changes of UA-treated cells were observed by light microscope. Apoptotic cells with condensed or fragmented nuclei were visualized by Ho33258 and PI staining by a fluorescence microscope (EX: U.V., Green light). p 53 protein expression was analyzed by fluorescence immunohistochemical method (SABC-Cy3). RESULT: 24 hours after UA treatment, inhibition of MCF-7 cell proliferation was concentration-dependent. The IC50 value for UA was 22.6 +/- 3.0 micromol/L. Cell cycle analysis by FCM showed that 50 micromol/L of the drug arrested MCF-7 cell cycle at G0-G1 phase. Morphological changes of MCF-7 cells exhibited many of the hallmark features of apoptosis, including condensation of chromatin and DNA fragmentation. UA increased p 53 protein expression. CONCLUSION: The results suggest that UA evokes MCF-7 cell apoptosis is correlation with the up-regulation of p 53. The study indicated that UA might be a potential Chinese medical component for breast neoplasm.

Effects of simvastatin on activities of endogenous antioxidant enzymes and angiotensin-converting enzyme in rat myocardium with pressure-overload cardiac hypertrophy.

AIM: To investigate effects of simvastatin (Sim) on the activities of antioxidant enzymes and angiotensin-converting enzyme in rat myocardium with pressure-overload cardiac hypertrophy. METHODS: Left ventricular hypertrophy (LVH) was induced by partly constricting rat abdominal aorta between the left and right renal artery. Rats were given ig Sim 1.8 and 3.6 mg . kg-1 . d-1 for 8 weeks following 6 weeks aortic constriction. Activities of antioxidant enzymes and angiotensin-converting enzyme, and lipid peroxidation of left ventricular (LV) tissue were determined. RESULTS: Contents of angiotensin II and thiobarbituric acid reactive substances (TBARS), and activity of ACE in LVH group (n = 8) were increased by 163 %, 90 %, and 130 %, respectively (P < 0.01)compared with sham-operated group (n = 7), and were decreased by 30 %, 37 %, and 51 %, respectively (P < 0.01) in high dosage Sim treatment group (n = 9) compared with LVH group. Activities of catalase and glutathione peroxidase of LV tissue in LVH group were decreased by 29 % and 23 % (P < 0.01) compared with sham-operated group, and were increased by 32 % and 22 % (P < 0.01) in high dosage Sim treatment group compared with LVH group. Activity of Cu, Zn-superoxide dismutase (SOD) of LV tissue was increased by 33 % in LVH group compared with sham-operated group. Sim treatment did not significantly affect activity of Cu,Zn-SOD. CONCLUSION: Alteration of redox status in myocardium is associated with cardiac hypertrophy and inhibitory effects of Sim on cardiac hypertrophy in rats model might be linked to its antioxidant effects.