Action of the natural compound gomisin a on Ca2+ movement in human prostate cancer cells.

Gomisin A is a dietary lignan compound isolated from the fruit of Schisandra chinensis and has many pharmacological properties, including hepato-protective, anti-diabetic, and anti-oxidative activities. However, the benefit of gomisin A is still not well understood. The action of gomisin A is diverse. However, the effect of gomisin A on Ca2+ signaling in prostate cancer cells is unknown. Ca2+ is a pivotal second envoy that triggers and regulates cellular processes such as apoptosis, fertilization, energy transduction, secretion, and protein activation. The goal of this study was to explore the action of gomisin A on [Ca2+]i and cytotoxicity in PC3 prostate cancer cells. Gomisin A at 100-200 µM provoked [Ca2+]i raises. 20% of the response was reduced by removing external Ca2+. The Ca2+ influx provoked by gomisin A was suppressed by 20% by store-caused Ca2+ entry suppressors: econazole, SKF96365, nifedipine; also by phorbol 12-myristate 13 acetate and GF109203X. Without external Ca2+, gomisin A-caused [Ca2+]i raises were abolished by thapsigargin. In contrast, gomisin A suppressed the [Ca2+]i raises caused by thapsigargin. U73122 fell short to change gomisin A-caused [Ca2+]i responses. Gomisin A (20-100 µM) elicited cytotoxicity in a dose-associated fashion. Blockade of [Ca2+] elevations with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl failed to inhibit cytotoxicity of gomisin A. Collectively, gomisin A evoked [Ca2+]i raises and provoked cytotoxicity in a Ca2+-dissociated fashion in prostate cancer cells.

Gomisin A Alleviates Obesity by Regulating the Phenotypic Switch between White and Brown Adipocytes.

Although gomisin A (GA) alleviates cancer and inflammation, its anti-obesity effect and the underlying mechanism have not yet been elucidated. Therefore, in this study, we aimed to elucidate the anti-obesity effects of GA by investigating the phenotypic changes involved in the browning and whitening of adipocytes. Here, obesity was induced to C57BL/6J mice using a high-fat diet (HFD). We administrated GA and checked weight changes for 12 weeks. We found that GA decreased the weight of weight gain, epididymal white adipose tissue (eWAT), and liver in the mice. In addition, the administration of GA elevated the levels of high-density lipoprotein (HDL)-cholesterol in the mice serum. Moreover, even after 12 weeks of treatment with GA, it did not cause any hepatic and renal toxicity. However, we found that GA induced the browning of eWAT and inhibited the whitening of brown adipose tissue. We further confirmed the anti-obesity mechanism of GA using 3T3-L1 cells, the human adipose mesenchymal stem cells (hAMSCs), and primary brown adipocytes (BAs) in vitroexperiments. We found that GA suppressed adipogenesis via the activation of AMP-activated protein kinase (AMPK). Furthermore, GA-induced browning by increasing the expression levels of uncoupling protein 1 (UCP1) in hAMSCs. The results of our study indicate that GA can inhibit weight gain by regulating the phenotypic changes involved in the browning and whitening of adipose tissues, which makes it a potential therapeutic agent for the treatment of obesity.

Inhibitory Effectiveness of Gomisin A, a Dibenzocyclooctadiene Lignan Isolated from Schizandra chinensis, on the Amplitude and Gating of Voltage-Gated Na+ Current.

Gomisin A (Gom A), a lignan isolated from Schisandra chinensis, has been reported produce numerous biological activities. However, its action on the ionic mechanisms remains largely unanswered. The present experiments were undertaken to investigate the possible perturbations of Gom A or other related compounds on different types of membrane ionic currents in electrically excitable cells (i.e., pituitary GH3 and pancreatic INS-1 cells). The exposure to Gom A led to the differential inhibition of peak and end-pulse components of voltage-gated Na+ current (INa) in GH3 cells with effective IC50 of 6.2 and 0.73 µM, respectively. The steady-state inactivation curve of INa in the presence of Gom A was shifted towards a more hyperpolarized potential. However, neither changes in the overall current-voltage relationship nor those for the gating charge of the current were demonstrated. The application of neither morin (10 µM) nor hesperidin (10 µM) perturbed the strength of INa, while sesamine could suppress it. However, in the continued presence of Gom A, the addition of sesamine failed to suppress INa further. Gom A also effectively suppressed the strength of persistent INa activated by long ramp voltage command, and further application of tefluthrin effectively attenuated Gom A-mediated inhibition of the current. The presence of Gom A mildly inhibited erg-mediated K+ current, while a lack of change in the amplitude of hyperpolarization-activated cation current was observed in its presence. Under cell-attached current recordings, the exposure to Gom A resulted in the decreased firing of spontaneous action currents with a minimal change in AC amplitude. In pancreatic INS-1 cells, the presence of Gom A was also noticed to inhibit peak and end-pulse components of INa differentially with the IC50 of 5.9 and 0.84 µM, respectively. Taken together, the emerging results presented herein provide the evidence that Gom A can differentially inhibit peak and sustained INa in endocrine cells (e.g., GH3 and INS-1 cells).

Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes.

BACKGROUND: Gomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods. PURPOSE: The objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma. STUDY DESIGN: The anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo. METHODS: WST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays. RESULTS: G.A (25-100 µM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5-20 µM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2-50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells. CONCLUSION: These results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.

Effects of crushed Schisandra Fruit on the content of lignans in Kampo decoction.

Arboreous fruit of Schisandra chinensis Baillon, Schisandra Fruit (SF), is a crude drug used in Japanese traditional Kampo medicine. The marker compounds of SF for quality control are lignans, such as schizandrin (Sz) and gomisin A (GmA). Kampo formulation containing SF is usually prepared as decoctions in the dosage form of whole crude drug (W), as its size is small enough to measure using a spoon. However, in some traditional books, it has been described that SF must be used in the dosage form of crushed or cut pieces (Cr). In this study, we evaluated the transferring ratio of lignans from SF to the decoction, and the stability and taste of the decoctions of shoseiryuto (SST) and ninjin'yoeito (NYT) using each dosage form, i.e., Cr and W, of SF. The transferring ratio of Sz and GmA was significantly higher in the decoction prepared with the Cr form than that prepared using the W form in both SST and NYT. The concentration of Sz and GmA in the decoctions was stable when maintained at 4 °C for 35 days. The taste of SST decoction prepared using the Cr form was more acidic, harsher, and bitterer than SST decoction prepared using the W form, and the taste of NYT decoction prepared using the Cr form was harsher than NYT decoction prepared using the W form. In conclusion, when SF is used in Kampo prescription, crushing the fruits and seeds can increase its effectiveness.

Gomisin A Suppresses Colorectal Lung Metastasis by Inducing AMPK/p38-Mediated Apoptosis and Decreasing Metastatic Abilities of Colorectal Cancer Cells.

Gomisin A (G.A) is a dietary lignan compound from Schisandra chinensis. In this study, the effect of G.A on the proliferation and metastasis of colorectal cancer (CRC) cells was investigated using several CRC cell lines and a lung metastasis mouse model. Both oral and intraperitoneal administration of G.A (50 mg/kg) inhibited lung metastasis of CT26 cells. Various concentrations of G.A were incubated with CRC cell lines and their viability was determined using a cell counting kit-8 assay. G.A significantly decreased the viability of various CRC cell lines, whereas it did not change the proliferation of normal colon cells. G.A induced G0/G1 phase arrest and apoptosis of CT26 and HT29 cells by regulating cyclin D1/cyclin-dependent kinase 4 (CDK4) expression and apoptotic proteins such as caspases and B-cell lymphoma-2 (Bcl-2) family proteins, respectively. G.A-induced apoptosis was mediated by AMPK/p38 activation in CRC cells. A non-cytotoxic concentration of G.A inhibited epithelial-mesenchymal transition of CRC cells by modulating E-cadherin and N-cadherin expression levels. Moreover, the migration and invasion of CRC cells were reduced by G.A treatment. Especially, G.A decreased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities. G.A ameliorated lung metastasis of CRC cells by decreasing cell survival and metastatic abilities of CRC cells. Thus, G.A might be a potential novel therapeutic agent for metastatic CRC.

Enhancement of gomisin A in pro-apoptotic effect of carboplatin on human ovarian cancer Skov3 cells and its mechanism / 吉林大学学报(医学版)

Objective: To explore the effect of gomisin A (GA) in combination with carboplatin (CBP) on the apoptosis of ovarian cancer Skov3 cells, and to illustrate the synergistic antitumor effect of GA. Methods: The ovarian cancer Skov3 cells were treated with GA combined with CBP. MTT assay was used to detect the inhibitory rates of proliferation and the appropriate concentrations of GA and CBP were selected according to the results. The Skov3 cells were divided into control group, GA (0. 04 μmol · L-1) group, CBP (16 mg · L-1) group and GA (0.04 μmol · L-1) + CBP (16 mg · L-1) group. After 48 h treatment, the cell morphology was observed by microscope, the apoptotic rate was measured by flow cytometry, the apoptotic index (AI) was observed by TUNEL staining, the mitochondrial membrane potential was detected by JC-1 staining, and the mRNA and protein expression levels of apoptosis-related genes (Bax, caspase-3, Stat3) were determined by RT-PCR and Western blotting method. Results: The inhibitory rate of proliferation of Skov3 cells in GA + CBP group (52. 1%) was significantly higher than those in GA and CBP groups (P<0. 01). The concentrations of GA and CBP for the best dose ratio were 0. 04 μmol · L-1 and 16 mg · L-1, respectively. Compared with control group, the cell refractivities were decreased, the cells retracted, and part of cells were suspended in GA group and CBP groups; there were more suspended cells and cell retraction phenomenon was more prominent in GA + CBP group. The results of TUNEL staining and flow cytometry showed that the AI and apoptotic rate of cells in GA + CBP group were significantly increased compared with control group, GA and CBP group (P<0. 05 or P<0. 01); the JC-1 staining results suggested that the mitochondrial membrane potential of Skov3 cells in GA + CBP group was decreased (P<0. 05 or P<0. 01). The RT-PCR and Western blotting results showed that the expression levels of Bax and caspase-3 mRNA and protein were up-regulated (P<0. 05) and the expression levels of Bcl-2 and Stat3 mRNA and protein in GA + CBP group were down-regulated compared with control, GA and CBP groups (P< 0. 05). Conclusion: GA can enhance the ability of CBP to induce the apoptosis of human ovarian cancer Skov3 cells, and its mechanisms may be related to the up-regulation of the Bax and Caspase-3 expressions and the down-reguation of the Bcl-2 expression; GA can synergize the induction of CBP on apoptosis.

Inhibitory effects of gomisin A combined with carboplatin on proliferation, invasion and metastasis of human ovarian cancer Skov3 cells and their mechanisms / 吉林大学学报(医学版)

To explore the inhibitory effects of gomisin A (GA) in combination with carboplatin (CBP) on the proliferation, invasion and metastasis of human ovarian cancer Skov3 cells and their mechanisms, and to illustrate the synergistic antitumor effect of GA. Methods: The ovarian cancer Skov3 cells were treated with different concentrations of GA combined with different concentrations of CBP. MTT assay was used to detect the inhibitory rates of proliferation. According to the results, the appropriate concentrations of GA and CBP were conformed. The Skov3 cells were treated with PBS, GA 0.04 μmol · L-1), CBP 16 mg · L-1) and GA 0.04 jumol · L-1) in combination with CBP 16 mg · L-1), respectively, and used as control group, GA group, CBP group and GA+CBP group. After 48 h of treatment, the cell morphology was observed by optical microscope, the cell reproductive ability was determined by clonogenic cell survival assay, and the cell migration ability was measured by cell wound healing test; Transwell experiment was used to detect the cell invasion ability. The expression levels of MMP-2 and MMP-9 mRNA were determined by RT-PCR and the protein expression levels of MMP-2, MMP-9, AKT1 and pAKT1 were determined by Western blotting method. Results: Compared with GA and CBP groups, the inhibitory rate of proliferation of Skov3 cells in GA + CBP group was significantly increased (P<0.01). The concentrations of GA and CBP for the best dose ratio were 0.04 μmol · L-1 and 16 mg · L-1, respectively. The results of clone formation assay showed that the colony formation rates in CBP and GA+CBP groups were decreased compared with control group (P<0.05 or P<0.01); the colony formation rate of cells in GA + CBP group was significantly decreased compared with GA and CBP groups (P<0.01); the wound scratch assay results suggested that the number of metastic Skov3 cells in GA and CBP groups were decreased compared with control group; and the number of metastic Skov3 cells in GA + CBP group was decreased compared with GA and CBP groups. The results of Transwell expriment showed that the capacities of cells passing the ECM in GA and CBP groups were decreased compared with control group; the capacity of cells passing the ECM was decreased compared with GA and CBP groups. The RT-PCR and Western blotting results showed that the expression levels of MMP-2 and MMP-9 mRNA in GA+CBP group were down-regulated compared with GA and CBP groups (P< 0.01), and the expression levels of MMP-2, MMP-9, AKT1 and pAKTl protein were down-regulated (P<0.01). Conclusion: GA can enhance the ability of CBP to inhibit the proliferation, invasion and metastasis of human ovarian cancer Skov3 cells, and thus play a role in decreasing the toxicity and increasing the efficacy of chemotherapy.

Inhibitory effects of gomisin A combined with carboplatin on proliferation,invasion and metastasis of human ovarian cancer Skov3 cells and their mechanisms / 吉林大学学报(医学版)

Objective:To explore the inhibitory effects of gomisin A(GA)in combination with carboplatin (CBP)on the proliferation,invasion and metastasis of human ovarian cancer Skov3 cells and their mechanisms,and to illustrate the synergistic antitumor effect of GA.Methods:The ovarian cancer Skov3 cells were treated with different concentrations of GA combined with different concentrations of CBP.MTT assay was used to detect the inhibitory rates of proliferation.According to the results,the appropriate concentrations of GA and CBP were conformed.The Skov3 cells were treated with PBS,GA(0.04 μmol·L-1),CBP(16 mg·L-1)and GA (0.04 μmol·L-1)in combination with CBP(16 mg·L-1),respectively,and used as control group,GA group, CBP group and GA+CBP group.After 48 h of treatment,the cell morphology was observed by optical microscope, the cell reproductive ability was determined by clonogenic cell survival assay,and the cell migration ability was measured by cell wound healing test;Transwell experiment was used to detect the cell invasion ability.The expression levels of MMP-2 and MMP-9 mRNA were determined by RT-PCR and the protein expression levels of MMP-2,MMP-9,AKT1 and pAKT1 were determined by Western blotting method.Results:Compared with GA and CBP groups,the inhibitory rate of proliferation of Skov3 cells in GA + CBP group was significantly increased (P<0.01).The concentrations of GA and CBP for the best dose ratio were 0.04 μmol·L-1and 16 mg·L-1, respectively.The results of clone formation assay showed that the colony formation rates in CBP and GA+CBP groups were decreased compared with control group(P<0.05 or P<0.01);the colony formation rate of cells in GA + CBP group was significantly decreased compared with GA and CBP groups(P<0.01);the wound scratch assay results suggested that the number of metastic Skov3 cells in GA and CBP groups were decreased compared with control group;and the number of metastic Skov3 cells in GA+ CBP group was decreased compared with GA and CBP groups.The results of Transwell expriment showed that the capacities of cells passing the ECM in GA and CBP groups were decreased compared with control group;the capacity of cells passing the ECM was decreased compared with GA and CBP groups.The RT-PCR and Western blotting results showed that the expression levels of MMP-2 and MMP-9 mRNA in GA+CBP group were down-regulated compared with GA and CBP groups(P<0.01),and the expression levels of MMP-2,MMP-9,AKT1 and pAKT1 protein were down-regulated(P<0.01).Conclusion:GA can enhance the ability of CBP to inhibit the proliferation,invasion and metastasis of human ovarian cancer Skov3 cells,and thus play a role in decreasing the toxicity and increasing the efficacy of chemotherapy.

Enhancement of gomisin A in pro-apoptotic effect of carboplatin on human ovarian cancer Skov3 cells and its mechanism / 吉林大学学报(医学版)

Objective:To explore the effect of gomisin A (GA) in combination with carboplatin (CBP) on the apoptosis of ovarian cancer Skov3 cells,and to illustrate the synergistic antitumor effect of GA.Methods:The ovarian cancer Skov3 cells were treated with GA combined with CBP.MTT assay was used to detect the inhibitory rates of proliferation and the appropriate concentrations of GA and CBP were selected according to the results.The Skov3 cells were divided into control group,GA (0.04 μmol · L-1) group,CBP (16 mg · L 1) group and GA (0.04 μmol· L-1)+ CBP (16 mg · L-1) group.After 48 h treatment,the cell morphology was observed by microscope,the apoptotic rate was measured by flow eytometry,the apoptotic index (AI) was observed by TUNEL staining,the mitochondrial membrane potential was detected by JC-1 staining,and the mRNA and protein expression levels of apoptosis-related genes (Bax,caspase-3,Stat3) were determined by RT PCR and Western blotting method.Results:The inhibitory rate of proliferation of Skov3 cells in GA + CBP group (52.1％) was significantly higher than those in GA and CBP groups (P＜0.01).The concentrations of GA and CBP for the best dose ratio were 0.04 μmol· L-1 and 16 mg · L-1,respectively.Compared with control group,the cell refractivities were decreased,the cells retracted,and part of cells were suspended in GA group and CBP groups;there were more suspended cells and cell retraction phenomenon was more prominent in GA + CBP group.The results of TUNEL staining and flow cytometry showed that the AI and apoptotic rate of cells in GA + CBP group were significantly increased compared with control group,GA and CBP group (P＜0.05 or P＜0.01);the JC-1staining results suggested that the mitochondrial membrane potential of Skov3 cells in GA + CBP group was decreased (P＜0.05 or P＜0.01).The RT-PCR and Western blotting results showed that the expression levels of Bax and caspase-3 mRNA and protein were up-regulated (P＜0.05) and the expression levels of Bcl-2 and Stat3mRNA and protein in GA + CBP group were down-regulated compared with control,GA and CBP groups (P＜0.05).Conclusion:GA can enhance the ability of CBP to induce the apoptosis of human ovarian cancer Skov3 cells,and its mechanisms may be related to the up-regulation of the Bax and Caspase-3 expressions and the down-reguation of the Bcl-2 expression;GA can synergize the induction of CBP on apoptosis.

Time- and NADPH-Dependent Inhibition on CYP3A by Gomisin A and the Pharmacokinetic Interactions between Gomisin A and Cyclophosphamide in Rats.

The traditional Chinese medicine Schisandra chinensis has remarkable protective effects against chemical-induced toxicity. Cyclophosphamide (CTX), in spite advances in chemotherapy and immunosuppressive regimes, is prone to cause severe toxicity due to its chloroacetaldehyde (CAA) metabolite produced by CYP3A. Our previous study identified that S. chinensis extract (SCE) co-administration potently decreased CAA production and attenuated liver, kidney and brain injuries in CTX-treated rats. Gomisin A (Gom A) is proved to be one of the most abundant bioactive lignans in S. chinensis with a significant CYP3A inhibitory effect. To find out whether and how Gom A participated in the chemoprevention of SCE against CTX toxicity, the Gom A-caused CYP3A inhibition in vitro as well as the pharmacokinetic interactions between Gom A and CTX in vivo were examined in this study. Using human liver microsomes, a reversible inhibition assay revealed that Gom A was a competitive inhibitor with a KI value of 1.10 µM, and the time- and NADPH-dependent CYP3A inhibition of Gom A was observed in a time-dependent inhibition assay (KI = 0.35 µM, kinact = 1.96 min-1). Hepatic CYP3A mRNA expression experienced a significant increase in our rat model with Gom A administration. This explained why CAA production decreased in the 0.5 h- and 6 h-pretreatment rat groups while it increased in the 24 h- and 72 h-pretreatment groups, indicating a bidirectional effect of Gom A on CYP3A-mediated CTX metabolism. The present study suggested that Gom A participates like SCE in the pharmacokinetic intervention of CTX by blocking CYP3A-mediated metabolism and reducing CAA production, and thus plays an important role in the chemopreventive activity of S. chinensis against CTX toxicity, in addition to the previously recognized protective effects. Also, the combined use of S. chinensis preparation or other drugs containing Gom A as the main component with CTX needed to be addressed for better clinical intervention.

Current knowledge of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) as a medicinal plant species: a review on the bioactive components, pharmacological properties, analytical and biotechnological studies.

Schisandra chinensis Turcz. (Baill.) is a plant species whose fruits have been well known in Far Eastern medicine for a long time. However, schisandra seems to be a plant still underestimated in contemporary therapy still in the countries of East Asia. The article presents latest available information on the chemical composition of this plant species. Special attention is given to dibenzo cyclooctadiene lignans. In addition, recent studies of the biological activity of dibenzocyclooctadiene lignans and schisandra fruit extracts are recapitulated. The paper gives a short resume of their beneficial effects in biological systems in vitro, in animals, and in humans, thus underlining their medicinal potential. The cosmetic properties are depicted, too. The analytical methods used for assaying schisandra lignans in the scientific studies and also in industry are also presented. Moreover, special attention is given to the information on the latest biotechnological studies of this plant species. The intention of this review is to contribute to a better understanding of the huge potential of the pharmacological relevance of S. chinensis.

Schisandra lignans production regulated by different bioreactor type.

Schisandra chinensis (Chinese magnolia vine) is a rich source of therapeutically relevant dibenzocyclooctadiene lignans with anticancer, immunostimulant and hepatoprotective activities. In this work, shoot cultures of S. chinensis were grown in different types of bioreactors with the aim to select a system suitable for the large scale in vitro production of schisandra lignans. The cultures were maintained in Murashige-Skoog (MS) medium supplemented with 3mg/l 6-benzylaminopurine (BA) and 1mg/l 1-naphthaleneacetic acid (NAA). Five bioreactors differing with respect to cultivation mode were tested: two liquid-phase systems (baloon-type bioreactor and bubble-column bioreactor with biomass immobilization), the gas-phase spray bioreactor and two commercially available temporary immersion systems: RITA® and Plantform. The experiments were run for 30 and 60 days in batch mode. The harvested shoots were evaluated for growth and lignan content determined by LC-DAD and LC-DAD-ESI-MS. Of the tested bioreactors, temporary immersion systems provided the best results with respect to biomass production and lignan accumulation: RITA® bioreactor yielded 17.86g/l (dry weight) during 60 day growth period whereas shoots grown for 30 days in Plantform bioreactor contained the highest amount of lignans (546.98mg/100g dry weight), with schisandrin, deoxyschisandrin and gomisin A as the major constituents (118.59, 77.66 and 67.86mg/100g dry weight, respectively).

Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells.

Nearly half of prescription medicines are metabolized by human cytochrome P450 (CYP) 3A. CYP3A4 and 3A5 are two major isoforms of human CYP3A and share most of the substrate spectrum. A very limited previous study distinguished the activity of CYP3A4 and CYP3A5, identifying the challenge in predicting CYP3A-mediated drug clearance and drug-drug interaction. In the present study, we introduced gomisin A (GA) with a dibenzocyclooctadiene skeleton as a novel selective probe of CYP3A4. The major metabolite of GA was fully characterized as 8-hydroxylated GA by LC-MS and NMR. CYP3A4 was assigned as the predominant isozyme involved in GA 8-hydroxylation by reaction phenotyping assays, chemical inhibition assays, and correlation studies. GA 8-hydroxylation in both recombinant human CYP3A4 and human liver microsomes followed classic Michaelis-Menten kinetics. The intrinsic clearance values indicated that CYP3A4 contributed 12.8-fold more than CYP3A5 to GA 8-hydroxylation. Molecular docking studies indicated different hydrogen bonds and π-π interactions between CYP3A4 and CYP3A5, which might result in the different catalytic activity for GA 8-hydroxylation. Furthermore, GA exhibited a stronger inhibitory activity towards CYP3A4 than CYP3A5, which further suggested a preferred selectivity of CYP3A4 for the transformation of GA. More importantly, GA has been successfully applied to selectively monitor the modulation of CYP3A4 activities by the inducer rifampin in hepG2 cells, which is consistent with the level change of CYP3A4 mRNA expression. In summary, our results suggested that GA could be used as a novel probe for the selective sensing of CYP3A4 in tissue and cell preparations.

Analysis on resource material and discussion on reutilization methods of Schizandrae Fructus residues in process of Shengmai Injection production / 中草药

Objective: Multi-type resource chemical constituents in Schizandrae Fructus residues were analyzed in the process of Shengmai Injection production, in order to provide the scientific basis for Schizandrae Fructus in the further process of industrialization. Methods: The lignan components were analysed and evaluated by HPLC-UV method. After using NaOH to extract sample, the BCA method was adopted to measure the mass fraction of total protein and take bovine serum albumin as the reference. Using UV-Vis spectrophotometer to measure the mass fraction and constitutes of neutral polysaccharide and acidic polysaccharide, respectively. The equipment for raw fiber determination was taken to gauge the crude fiber content of Schizandrae Fructus residues. Results: The mass fractions of schisandrin A, schizandrin B, schizandrin C, gomisin A, gomisin B, and schisantherin A were 1.442 4, 3.788 0, 1.350 9, 4.399 3, 3.231 3, and 0.505 3 mg/g, respectively. Compared with the original medicinal materials, the technology utilization rate of gomisin A was 20.84% during the process of Shengmai Injection production. But the gomisin B virtually was unused and remained in the residues. The mass fractions of schisandrin A, schizandrin B, schizandrin C, and schisantherin A were higher than that of the original medicinal material. The available macromolecular substances are proteins, polysaccharide, and crude fiber. The content of total proteins was 14.69%. The mass fractions of neutral polysaccharide and acidic polysaccharide were 3.82% and 1.31%, respectively. The analysis on crude fiber showed that the mass fraction of crude fiber in Schizandrae Fructus residues was 43.80%. Conclusion: The analysis shows that Schizandrae Fructus residues contain many resource chemical components in the process of Shengmai Injection production, such as lignins, protein, saccharides, and crude fiber. The strategy for recycling and possible way is proposed exploringly based on the available resource chemical components of Schizandrae Fructus residues and water-extracting technology. It provides the reference for the utilization of waste resource in further process of industralization, promoting the resource conservation, and development of environmental protection, realizing the harmonious coexistence between the economy and ecology.

Gomisin A decreases the LPS-induced expression of iNOS and COX-2 and activation of RIP2/NF-κB in mouse peritoneal macrophages.

Gomisin A (GA), a lignan component contained in the fruit of Schisandra chinensis Baillon, improves hepatic cell degeneration, vasodilatory activity and insulin sensitivity. These effects also impact the immune system, including various inflammatory mediators and cytokines. In this study, the anti-inflammatory effect of GA on lipopolysaccharide-stimulated mouse peritoneal macrophages was studied. Pretreatment with GA attenuated the expression of receptor-interacting protein 2 (RIP2) and IκB kinase-ß (IKK-ß) as well as IKK-ß phosphorylation. The activation of nuclear factor-kappa B (NF-κB) in the nucleus, the phosphorylation of IκBα and degradation of IκBα in the cytosol were suppressed by GA. GA decreased the production and mRNA expression of the inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. In addition, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and production of nitric oxide were decreased by pretreatment with GA. In conclusion, these results show that the anti-inflammatory properties of GA potentially result from the inhibition of COX-2, iNOS, IL-6, TNF-α and NO through the down-regulation of RIP2 and NF-κB activation. These results impact the development of potential health products for preventing and treating inflammatory diseases.

Gomisin A inhibits lipopolysaccharide-induced inflammatory responses in N9 microglia via blocking the NF-κB/MAPKs pathway.

Gomisin A, one of the major dibenzocyclooctadiene lignans isolated from Schisandra chinensis Baill., has proved to possess a variety of pharmacological effects. The aim of the present study was to investigate the anti-inflammatory and neuroprotective effects of gomisin A as well as its potential molecular mechanisms. It was found that gomisin A not only inhibited the production of NO and PGE2 in a concentration-dependent manner but also suppressed the expressions of iNOS and COX-2 in LPS-stimulated N9 microglia without observable cytotoxicity. Gomisin A was also able to attenuate the mRNA expression and the production of pro-inflammatory factors TNF-α, IL-1ß and IL-6. Moreover, LPS induced reactive oxygen species (ROS) production, NADPH oxidase activation, and gp91phox expression, which were markedly inhibited by gomisin A in microglia. Furthermore, the data showed that gomisin A significantly down-regulated the TLR4 protein expression, and inhibited nuclear transcription factor (NF)-κB and mitogen-activated protein kinases (MAPKs) signaling pathways. Additionally, gomisin A alleviated the cell death of SH-SY5Y neuroblastoma, rat primary cortical and hippocampal neurons induced by the conditioned-media from activated microglia. In summary, gomisin A may exert neuroprotective effects by attenuating the microglia-mediated neuroinflammatory response via inhibiting the TLR4-mediated NF-κB and MAPKs signaling pathways.

Simultaneous analysis on 13 components in Yiqi Fumai Injection (freeze-dried) by UPLC-MS/MS method / 中草药

Objective: To establish a UPLC-MS/MS analytical method for the simultaneous analysis of ginsenosides (ginsenosides Rb1, Re, Rg1, Rc, Rd, Rf, Rg3, F2, and notoginsenoside R1) and lignans (gomisin A, schisandrol B, deoxyschizandrin, and schisandrin B) in Yiqi Fumai Injection (freeze-dried) (YFI), and measure the contents of these constituents in YFI. Methods: Quantitative research of 13 components in YFI was done by reversed-phase liquid chromatography on a C18 column using a gradient elution (0.1% formic acid in water and 0.1% formic acid in methanol). A triple quadrupole mass spectrometer operating in positive electrospray ionization mode with multiple reaction monitoring was used. Results: Thirteen components in YFI have good linear relationship, precision, stability, and repeatability according to the requirements of the methodology determination. The recoveries were 98.28%-101.08%. The 13 components in three batches of YFI were determined by UPLC-MS/MS method. Conclusion: The developed UPLC-MS/MS method is simple, sensitive, and accurate, and has good repeatability. The 13 components in YFI could be rapidly and accurately quantified by UPLC-MS/MS, which provides the helpful information for the comprehensive quality evaluation of YFI.

Optimization of Smashing Tissue Extraction Technology of Schisandra chinensis Fruits by Orthogonal Test / 中草药·英文版

Objective To optimize the extract technology of active lignins from the fruits of Schisandra chinensis.Methods The content of schizandrin,gomisin A,and deoxyschizandrin were selected as standards to evaluate the efficiency of smashing tissue extraction (STE).Solid-liquid ratio,extracting times,ethanol concentration,and extracting time were investigated through orthogonal test.Results The optimized conditions for STE were ten times amount of 80％ EtOH,extracting for three times,and 2 min for each time.Conclusion STE could obtain relatively higher yield,simplicity of operation,and benefit for environment protection.It could be better choice for the extraction ofS.chinensis.

Transcriptome analysis of the effects of gomisin a on the recovery of carbon tetrachloride-induced damage in rat liver.

Gomisin A possesses a hepatic function-facilitating property in liver-injured rats. Its preventive action on carbon tetrachloride-induced cholestasis is due to maintenance of the function of the bile acids-independent fraction. To investigate alterations in gene expression after gomisin A treatment on injured rat liver, DNA microarray analyses were performed on a Rat 44K 4-Plex Gene Expression platform with duplicated reactions after gomisin A treatment. We identified 255 up-regulated and 230 down-regulated genes due to the effects of gomisin A on recovery of carbon tetrachloride-induced rat liver damage. For functional characterization of these genes, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes biochemical pathways analyses were performed. Many up-regulated or down-regulated genes were related to cell cycle or focal adhesion and cell death genes, respectively. Our microarray experiment indicated that the liver repair mechanism induced by gomisin A was strongly associated with increased gene expressions related to cell cycle and suppression of the gene expression related in cell death.