Study on HPLC method to determine contents of Schisandrin A and Schisandrin B in Schisandra chinensis extraction

The determination method of Schisandrin A and Schisandrin B in Schisandra chinensis was improved with the high performance liquid chromagraphy （HPLC）. The sample was extracted exceedingly in the critical limit of CO2. The retention time of Schisandrin A and Schisandrin B was reduced, with methano/water （75 ： 25） as mobile phase. The wavelength for detection was 254 nm. The R^2 of standard curve was 0.9998 and the relative standard deviation was 2.31% and 3.17% with the recovery of 96.45% and 97.37%, respectively. The result shows that the rate of veracity of this method is higher and it proves that the determination method of Sehisandrin A and Schisandrin B in Schisandra chinensis is a feasible method.

Novel discovery of schisandrin A regulating the interplay of autophagy and apoptosis in oligoasthenospermia by targeting SCF/c-kit and TRPV1 via biosensors

Oligoasthenospermia is the primary cause of infertility.However,there are still enormous challenges in the screening of critical candidates and targets of oligoasthenospermia owing to its complex mechanism.In this study,stem cell factor(SCF),c-kit,and transient receptor potential vanilloid 1(TRPV1)biosensors were successfully established and applied to studying apoptosis and autophagy mechanisms.Interestingly,the detection limit reached 2.787×10^(-15)g/L,and the quantitative limit reached 1.0×10^(-13)g/L.Furthermore,biosensors were used to investigate the interplay between autophagy and apoptosis.Schisandrin A is an excellent candidate to form a system with c-kit similar to SCF/c-kit with a detection constant(K_(D))of 5.701×10^(-11)mol/L,whereas it had no affinity for SCF.In addition,it also inhibited autophagy in oligoasthenospermia through antagonizing TRPV1 with a K_(D) of up to 4.181×10^(-10)mol/L.In addition,in vivo and in vitro experiments were highly consistent with the biosensor.In summary,high-potency schisandrin A and two potential targets were identified,through which schisandrin A could reverse the apoptosis caused by excessive autophagy during oligoasthenospermia.Our study provides promising insights into the discovery of effective compounds and potential targets via a well-established in vitro-in vivo strategy.

Chemoproteomics identifies Ykt6 as the direct target of schisandrin A for neuroprotection

Schisandrin A is a natural dibenzocyclooctene lignan with potent neuroprotective activity.However,the specific mechanisms or direct target proteins have not been clarified up to now.In this study,we designed and synthesized the probes of schisandrin A with photoreactive diazirine and clickable alkyne to identify its direct target in SH-SY5Y cells by employing activity-based protein profiling(ABPP)technique.Ykt6 was prominent among the 13 proteins obtained with high confidence and we confirmed Ykt6 as the direct target of schisandrin A by CETSA,IF,SPR and knockdown assay.Functionally,schisandrin A protected the cells against the injury induced by glutamate by regulating autophagy via Ykt6.This discovery may provide a novel therapeutic option for various neuronal cell damage-mediated diseases.

Schisandrin B exerts anticancer effects on human gastric cancer cells through ROS-mediated MAPK,STAT3,and NF-κB pathways

Schisandrin B(Sch B)is a monomer with anti-cancer and anti-inflammatory effects,which are isolated from the plant Schisandra chinensis(Turcz)Baillon.We investigated the anti-gastric cancer(GC)effects of Sch B and its underlying molecular mechanisms.The Cell Counting Kit-8 assay was used to determine the effects of Sch B on the viability of GC and normal cell lines.Hoechst/propidium iodide staining and flow cytometry were used to assess the apoptosis induction of Sch B.Western blotting was used to evaluate the effects of Sch B on downstream apoptotic proteins.The DCFH-DA fluorescent probe was used to assess the regulatory effects of Sch B on reactive oxygen species(ROS)levels and related signaling pathways in GC cells.The results showed that Sch B could regulate the phosphorylation level of mitogen-activated protein kinase(MAPK)by upregulating ROS accumulation in gastric cancer cells,and then reduce the expression of nuclear factor kappa B(NF-κB)and phosphorylated transcription 3(p-STAT3).In addition,Sch B downregulated the cell cycle proteins cyclin-dependent kinase 2/4/6 and cyclin D1/E,and arrested cells in the G0/G1 phase.Moreover,it also inhibited cell migration,which was reversed with Nacetylcysteine pretreatment.In summary,Sch B has killing effects on GC cells by upregulating the production of intracellular ROS and regulating the MAPK/STAT3/NF-κB signaling pathway,leading to the migration arrest and apoptosis of GC cells.

Network pharmacology-based prediction and verification of the molecular targets and pathways for schisandrin against cerebrovascular disease

AIM: To illuminate the molecular targets for schisandrin against cerebrovascular disease based on the combined methods of network pharmacology prediction and experimental verification. METHOD: A protein database was established through constructing the drug-protein network from literature mining data. The protein-protein network was built through an in-depth exploration of the relationships between the proteins. The computational platform was implemented to predict and extract the sensitive sub-network with significant P-values from the protein-protein network. Then the key targets and pathways were identified from the sensitive sub-network. The most related targets and pathways were also confirmed in hydrogen peroxide(H2O2)-induced PC12 cells by Western blotting. RESULTS: Twelve differentially expressed proteins(gene names: NFKB1, RELA, TNFSF10, MAPK1, CHUK, CASP8, PIGS2, MAPK14, CREB1, IFNG, APP, and BCL2) were confirmed as the central nodes of the interaction network(45 nodes, 93 edges). The NF-κB signaling pathway was suggested as the most related pathway of schisandrin for cerebrovascular disease. Furthermore, schisandrin was found to suppress the expression and phosphorylation of IKKα, as well as p50 and p65 induced by H2O2 in PC12 cells by Western blotting. CONCLUSION: The computational platform that integrates literature mining data, protein-protein interactions, sensitive sub-network, and pathway results in identification of the NF-κB signaling pathway as the key targets and pathways for schisandrin.

Investigation of in Vitro and in Vivo Metabolism of Schisandrin B from Schisandrae Fructus by Liquid Chromatography Coupled Electrospray Ionization Tandem Mass Spectrometry

Schisandrin B (Sch B) is one of the active dibenzocyclooctadiene lignans found in the Schisandrae Fructus. Experimental studies have shown that Sch B possesses various pharmacological properties, including anti-cancer, neuroprotective and nephroprotective activities. However, no detailed information on its biotransformation was reported in the literature. Here, we investigated the in vitro and in vivo metabolism of Sch B by using ultra-performance liquid chromatography coupled with tandem mass spectrometry. In vitro study detected and identified one oxygenated metabolite. Four metabolites were detected and identified from the in vivo study. The results indicated that the metabolism of Sch B mainly involved the demethylation of methoxy groups, the opening of five-member ring and the glucuronidation of metabolites in rats. The metabolites were identified for the first time by MS/MS analyses.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.

Scavenger receptor A-mediated nanoparticles target M1 macrophages for acute liver injury

Acute liver injury(ALI)has an elevated fatality rate due to untimely and ineffective treatment.Although,schisandrin B(SchB)has been extensively used to treat diverse liver diseases,its therapeutic efficacy on ALI was limited due to its high hydrophobicity.Palmitic acid-modified serum albumin(PSA)is not only an effective carrier for hydrophobic drugs,but also has a superb targeting effect via scavenger receptor-A(SR-A)on the M1 macrophages,which are potential therapeutic targets for ALI.Compared with the common macrophage-targeted delivery systems,PSA enables site-specific drug delivery to reduce off-target toxicity.Herein,we prepared SchB-PSA nanoparticles and further assessed their therapeutic effect on ALI.In vitro,compared with human serum albumin encapsulated SchB nanoparticles(SchB-HSA NPs),the SchB-PSA NPs exhibited more potent cytotoxicity on lipopolysaccharide(LPS)stimulated Raw264.7(LAR)cells,and LAR cells took up PSA NPs 8.79 times more than HSA NPs.As expected,the PSA NPs also accumulated more in the liver.Moreover,SchB-PSA NPs dramatically reduced the activation of NF-κB signaling,and significantly relieved inflammatory response and hepatic necrosis.Notably,the high dose of SchB-PSA NPs improved the survival rate in 72 h of ALI mice to 75%.Hence,SchB-PSA NPs are promising to treat ALI.

五味子乙素对人卵巢癌Skov3细胞株的增殖、凋亡及Wnt／β-catenin信号通路的影响

目的探讨五味子乙素(Sch B)对人卵巢癌Skov3细胞株的增殖、凋亡及Wnt/β-catenin信号通路的影响。方法采用0、1.0、10.0、20.0、50.0μmol/L Sch B处理Skov3细胞,采用四甲基偶氮唑盐(MTT)法检测各浓度处理24、48、72和96h的增殖抑制率,Annexin-FITC/PI双染法检测不同浓度处理48h后的细胞凋亡情况,流式细胞仪检测各浓度处理48h后的细胞周期分布情况,Western blotting检测各浓度处理48h后细胞核Wnt/β-catenin信号通路中β-连接素(β-catenin)及下游靶分子C-myc、细胞周期素D1(Cyclin D1)的蛋白水平,同时于各浓度处理48h后检测细胞质和细胞核的糖原合成酶激酶-3β(GSK-3β)活性。结果 Sch B可呈剂量和时间依赖方式增加细胞增殖抑制率(P<0.05),作用48h后可呈剂量依赖方式升高早、晚期凋亡率及细胞质/胞核GSK-3β活性(P<0.05);除1.0μmol/L外,其余浓度作用48h的G0/G1期细胞比例均高于0μmol/L,S期、G2/M期细胞比例及β-catenin、C-myc和Cyclin D1蛋白水平均低于0μmol/L(P<0.05),且10.0、20.0、50.0μmol/L Sch B间的差异有统计学意义(P<0.05)。结论 Sch B可以抑制Skov3细胞株的增殖并促进其凋亡和细胞周期阻滞,以及抑制Wnt/β-catenin通路的激活。

Effects of Schisandra B on neurotransmitter contents, inflammation and oxidative stress in hippocampus of depression model rats

Objective:To study the effects of Schisandra B on neurotransmitter content,inflammation and oxidative stress in the hippocampus of depression model rats.Methods:A total of 30 male SD rats were randomly divided into the control group,the model group,and the Schisandra B group.The latter two groups established depression model according to chronic mild unpredictable stress.The Schisandrin B group was given Schisandrin B gavage for 6 weeks.The differences in depressive behavior and hippocampal neurotransmitter,inflammatory index,oxidative stress index were compared among the three groups.Results:Compared with the control group,the sugar preference score was significantly lower;the immobility time was significantly prolonged;the contents of 5-hydroxytryptamine(5-HT),norepinephrine(NE),dopamine(DA),superoxide dismutase(SOD),glutathione peroxidase(GPx)in hippocampus were significantly decreased;the contents of malondialdehyde(MDA),the expression of nuclear factor-kappa B(NF-κB),interleukin-1beta(IL-1β)and tumor necrosis factor-α(TNF-α)in hippocampus were significantly increased in the model group(P<0.05).Compared with the model group,the sugar preference score was significantly increased;immobility time significantly shortened;the contents of 5-HT,NE,DA,SOD and GPx in hippocampus were significantly increased;the content of MDA and the expression of NF-κB,IL-1βand TNF-αin hippocampus were significantly decreased in ketamine group(P<0.05).Conclusion:Low dose of Schisandra B can improve depressive behavior,increase monoamine neurotransmitter secretion,and inhibit inflammation and oxidative stress in depression model rats.

Schisandrin B Protects against Ischemic Brain Damage by Regulating PI3K/AKT Signaling in Rats

Objective To explore the effect and mechanism of schisandrin B(Sch B)in the treatment of cerebral ischemia in rats.Methods The cerebral ischemia models were induced by middle cerebral artery occlusion(MCAO)and reperfusion.Sprague-Dawley rats were divided into 6 groups using a random number table,including sham,MCAO,MCAO+Sch B(50 mg/kg),MCAO+Sch B(100 mg/kg),MCAO+Sch B(100 mg/kg)+LY294002,and MCAO+Sch B(100 mg/kg)+wortmannin groups.The effects of Sch B on pathological indicators,including neurological deficit scores,cerebral infarct volume,and brain edema,were subsequently studied.Tissue apoptosis was identified by terminal transferase-mediated dUTP nick end-labeling(TUNEL)staining.The protein expressions involved in apoptosis,inflammation response and oxidative stress were examined by immunofluorescent staining,biochemical analysis and Western blot analysis,respectively.The effect of Sch B on phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling was also explored.Results Sch B treatment decreased neurological deficit scores,cerebral water content,and infarct volume in MCAO rats(P<0.05 or P<0.01).Neuronal nuclei and TUNEL staining indicated that Sch B also reduced apoptosis in brain tissues,as well as the Bax/Bcl-2 ratio and caspase-3 expression(P<0.01).Sch B regulated the production of myeloperoxidase,malondialdehyde,nitric oxide and superoxide dismutase,as well as the release of cytokine interleukin(IL)-1βand IL-18,in MCAO rats(P<0.05 or P<0.01).Sch B promoted the phosphorylation of PI3K and AKT.Blocking the PI3K/AKT signaling pathway with LY294002 or wortmannin reduced the protective effect of Sch B against cerebral ischemia(P<0.05 or P<0.01).Conclusions Sch B reduced apoptosis,inflammatory response,and oxidative stress of MCAO rats by modulating the PI3K/AKT pathway.Sch B had a potential for treating cerebral ischemia.

Effectiveness of the analogue of natural Schisandrin C (HpPro) in treatment of liver diseases : an experience in Indonesian patients

Abstract Objective To determine the effect of dimethyl 4, 4' dimethoxy 5, 6, 5', 6 dimethylene dioxybiphenyl 2, 2' dicarboxylate (HpPro) on patients with acute and chronic liver diseases. Methods An open trial and a prospective randomized and controlled study were performed. The open trial consisted of 56 cases (16 cases of acute hepatitis, 20 cases of chronic hepatitis, 14 cases of liver cirrhosis and 6 cases of fatty liver). Controlled study consisted of 20 cases of Child A chronic hepatitis which were randomly treated with either HpPro or a mixture of known drugs which used as a liver protective agent in Indonesia as control for one week. The patients were then crossed over those two drugs in the next week. Results In the open trial, after 4 weeks' treatment with HpPro 7.5 mg orally three times daily, acute hepatitis, chronic hepatitis and fatty liver cases showed rapid decrease of SGOT and SGPT. In the liver cirrhosis cases, SGOT and SGPT were decreased slowly. In the controlled trial, nine patients received HpPro 7.5 mg three times daily orally and eleven were treated with a mixture of known drugs as the controls. After one week treatment, HpPro group clinically showed significant decrease of SGPT and SGOT levels compared to control group (P=0.035). At the second week, HpPro group showed significant decrease of SGOT compared to control group (P=0.038) but the decrease of SGPT was not significant (P=0.096). Conclusion Treatment with HpPro is effective to reduce liver impairment in acute and chronic liver diseases on Indonesian patients. No side effect of HpPro was observed.

Schisandrin B Inhibits NLRP3 Inflammasome Pathway and Attenuates Early Brain Injury in Rats of Subarachnoid Hemorrhage

Objective: To determine whether Schisandrin B(Sch B) attenuates early brain injury(EBI) in rats with subarachnoid hemorrhage(SAH). Methods: Sprague-Dawley rats were divided into sham(sham operation), SAH, SAH+vehicle, and SAH+Sch B groups using a random number table. Rats underwent SAH by endovascular perforation and received Sch B(100 mg/kg) or normal saline after 2 and 12 h of SAH. SAH grading, neurological scores, brain water content, Evan’s blue extravasation, and terminal transferase-mediated dUTP nick end-labeling(TUNEL) staining were carried out 24 h after SAH. Immunofluorescent staining was performed to detect the expressions of ionized calcium binding adapter molecule 1(Iba-1) and myeloperoxidase(MPO) in the rat brain, while the expressions of B-cell lymphoma 2(Bcl-2), Bax, Caspase-3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3), apoptosis-associated specklike protein containing the caspase-1 activator domain(ASC), Caspase-1, interleukin(IL)-1β, and IL-18 in the rat brains were detected by Western blot. Results: Compared with the SAH group, Sch B significantly improved the neurological function, reduced brain water content, Evan’s blue content, and apoptotic cells number in the brain of rats(P<0.05 or P<0.01). Moreover, Sch B decreased SAH-induced expressions of Iba-1 and MPO(P<0.01). SAH caused the elevated expressions of Bax, Caspase-3, NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the rat brain(P<0.01), all of which were inhibited by Sch B(P<0.01). In addition, Sch B increased the Bcl-2 expression(P<0.01). Conclusion: Sch B attenuated SAH-induced EBI, which might be associated with the inhibition of neuroinflammation, neuronal apoptosis, and the NLRP3 inflammatory signaling pathway.