Advances in the pharmacological activities and mechanisms of diosgenin

Diosgenin, a well-known steroid sapogenin derived from plants, has been used as a starting material for production of steroidal hormones. The present review will summarize published literature concerning pharmacological potential of diosgenin, and the underlying mechanisms of actions. Diosgenin has shown a vast range of pharmacological activities in preclinical studies. It exhibits anticancer, cardiovascular protective, anti-diabetes, neuroprotective, immunomodulatory, estrogenic, and skin protective effects, mainly by inducing apoptosis, suppressing malignant transformation, decreasing oxidative stress, preventing inflammatory events, promoting cellular differentiation/proliferation, and regulating T-cell immune response, etc. It interferes with cell death pathways and their regulators to induce apoptosis. Diosgenin antagonizes tumor metastasis by modulating epithelial-mesenchymal transition and actin cytoskeleton to change cellular motility, suppressing degradation of matrix barrier, and inhibiting angiogenesis. Additionally, diosgenin improves antioxidant status and inhibits lipid peroxidation. Its anti-inflammatory activity is through inhibiting production of pro-inflammatory cytokines, enzymes and adhesion molecules. Furthermore, diosgenin drives cellular growth/differentiation through the estrogen receptor(ER) cascade and transcriptional factor PPARγ. In summary, these mechanistic studies provide a basis for further development of this compound for pharmacotherapy of various diseases.

Structural characterization and identification of major constituents in Radix Scrophulariae by HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

AIM: To analyze the major constituents in Radix Scrophulariae(Scrophularia ningpoensis). METHOD: Radix Scrophulariae was analyzed by HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry(ESI-Q-TOF MS/MS). Compounds were separated by HPLC using a C18 column and gradient elution of acetonitrile and 0.1 %(V/V) acetic acid-water. Negative ion mode was employed. RESULTS: A total of thirty-six compounds, including fourteen iridoid glycosides, nineteen phenylpropanoid glycosides, and three organic acids, were identified from Radix Scrophulariae based on the accurate mass measurement of precursor and product ions. Twenty-one of the constituents were identified by comparing their retention times(tR) and ESI-MS/MS data with those of reference standards and/or previous publications, while another fifteen compounds were tentatively identified or deduced according to their Q-TOF MS/MS data which afforded sufficient structural information. CONCLUSION: It is believed that this study is useful for the identification of constituents in Radix Scrophulariae, as well as related plants and complex prescriptions.

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.

A combination of four effective components derived from Sheng-mai san attenuates hydrogen peroxide-induced injury in PC12 cells through inhibiting Akt and MAPK signaling pathways

The present study was designed to investigate whether a combination of four effective components derived from Sheng-mai san(SMXZF; ginsenoside Rb1: ginsenoside Rg1: DT^(–1)3: Schizandrol A as 6 : 9 : 4 : 5) could attenuate hydrogen peroxide(H_2O_2)-induced injury in PC12 cells, focusing on the Akt and MAPK pathways. The PC12 cells were exposed to H_2O_2(400 mmol·L^(–1)) for 1 h in the presence or absence of SMXZF pre-treatment for 24 h. Cell viability was measured by MTT assay. The efflux of lactate dehydrogenase(LDH), the intracellular content of malondialdehyde(MDA), the activities of superoxide dismutase(SOD), and caspase-3 were also determined. Cell apoptosis was measured by Hoechst 33342 staining and Annexin V-FITC/PI staining method. The expression of Bcl-2, Bax, cleaved caspase-3, Akt, and MAPKs were detected by Western blotting analyses. SMXZF pretreatment significantly increased the cell viability and SOD activity and improved the cell morphological changes, while reduced the levels of LDH and MDA at the concentrations of 0.1, 1 and 10 μg·m L^(–1). SMXZF also inhibited H_2O_2-induced apoptosis in PC12 cells. Moreover, SMXZF reduced the activity of caspase-3, up-regulated the protein ratio of Bcl-2 and Bax and inhibited the expression of cleaved caspase-3, p-Akt, p-p38, p-JNK and p-ERK1/2 in H_2O_2-induced PC12 cells. Co-incubation of Akt inhibitor or p38 inhibitor partly attenuated the protection of SMXZF against H_2O_2-injured PC12 cells. In conclusion, our findings suggested that SMXZF attenuated H_2O_2-induced injury in PC12 cells by inhibiting Akt and MAPKs signaling pathways, which might shed insights on its neuroprotective mechanism.

Novel cytotoxic steroidal glycosides from the roots of Liriope muscari

The present study was designed to investigate the chemical constituents and bioactivities of the roots of Liriope muscari(Decne.) L.H. Bailey. The compounds were isolated through various chromatography techniques, including silica gel, Sephadex LH-20, and semi-preparative HPLC. The structures were elucidated by infrared(IR), mass spectrometric(MS), 1D- and 2D-NMR analyses in comparison with reference data. In addition, the cytotoxicity of these compounds against human breast cancer MDA-MB-435 cells was evaluated by the MTT assay. Two new steroidal glycosides, 25(R, S)-ruscogenin-1-O-[β-D-fucopyranosyl(1→2)]-[ β-D-xylopyranosyl(1→3)]-β-D-glucopyranoside(Liriopem I, 1) and 25(R, S)- ruscogenin-1-O-[β-D-fucopyranosyl(1→2)]-[β-D-xylopyranosyl(1→4)]-β-D-fucopyranoside(Liriopem II, 2) and two known compounds LM-S6(3) and DT-13(4) were isolated and identified. Liriopem I(1), liriopem II(2) and DT-13(4) showed remarkable cytotoxicity with IC50 values being(0.58 ± 0.08),(0.05 ± 0.10), and(0.15 ± 0.09) μg·m L-1, respectively. In summary, compounds 1 and 2 identified in the present study exerted cytotoxicity against breast cancer cells, providing a basis for future development of these compounds as novel anticancer agents.

Laminaria japonica increases plasma exposure of glycyrrhetinic acid following oral administration of Liquorice extract in rats

The present study was designed to investigate the effects of Laminaria japonica(Laminaria) on pharmacokinetics of glycyrrhetinic acid(GA) following oral administration of Liquorice extract in rats.Following oral administrations of single-dose and multi-dose Liquorice extract and Liquorice-Laminaria extract,respectively,plasma samples were obtained at various times and the concentrations of GA,liquiritigenin,and isoliquiritigenin were measured by LC-MS.The effects of Laminaria extract on pharmacokinetics of GA were also investigated,following single-dose and multidose of glycyrrhizic acid(GL).The effects of Laminaria extract on intestinal absorption of GA and GL were studied using the in situ single-pass intestinal perfusion model.The metabolism of GL to GA in the contents of small and large intestines was also studied.The results showed Liquorice-Laminaria extract markedly increased the plasma concentration of GA,accompanied by a shorter Tmax.Similar alteration was observed following multidose administration.However,pharmacokinetics of neither liquiritigenin nor isoliquiritigenin was affected by Laminaria.Similarly,Laminaria markedly increased concentration and decreased Tmax of GA following oral GL were observed.The data from the intestinal perfusion model showed that Laminaria markedly increased GL absorption in duodenum and jejunum,but did not affect the intestinal absorption of GA.It was found that Laminaria enhanced the metabolism of GL to GA in large intestine.In conclusion,Laminaria increased plasma exposures of GA following oral administration of liquorice or GL,which partly resulted from increased intestinal absorption of GL and metabolism of GL to GA in large intestine.

The saponin DT-13 inhibits gastric cancer cell migration through down-regulation of CCR5-CCL5 axis

AIM: To investigate the effect of DT-13 on gastric cancer cell migration, and to explore the possible mechanisms underlying the anti-metastasis activity of DT-13. METHODS: Growth inhibition of DT-13 was analyzed by the MTT assay. Cell migration was measured by the scratch-wound assay and transwell double chamber assay. To investigate the possible mechanisms underlying the anti-metastasis activity of DT-13, chemokine receptors that are involved in cancer metastasis(CCR2, CCR5, CCR7, CXCR4, and CXCR6) were detected by conventional PCR. The effect of DT-13 on CCR5 and CXCR4 expression was further evaluated by quantitative PCR and Western blot, respectively. The secretion of CCL5(ligand of CCR5) and SDF-1(ligand of CXCR4) were detected by enzyme-linked immunosorbent assay(ELISA). RESULTS: DT-13 inhibited BGC-823 and HGC-27 cell growth in a dose dependent manner, and the estimated IC50 value for 24 h treatment was 23.5 ± 5.1 μmol·L-1 for BGC-823 cells and 35.6 ± 7.6 μmol·L-1 for HGC-27 cells. DT-13 also significantly decreased gastric cancer cell migration. DT-13 significantly decreased the gene expression of CCR5 in both BGC-823 and HGC-27 gastric cancer cells, and moderately reduced the expression of CXCR4. Similar to the results of gene expression, significant down-regulation of CCR5 protein was observed, but CXCR4 protein levels were much less affected. CCL5 secretion, but not SDF-1 production, was inhibited by DT-13. CONCLUSION: DT-13 inhibited gastric cancer cell migration by down-regulation of the CCR5-CCL5 axis.

Simultaneous Determination of Saponins in Dripping Pills Made from Astragali Radix and Panax notoginseng by UPLC-ELSD

Objective To develop a simple and fast method for removing polyethylene glycol （PEG） and simultaneous determination of fives saponins, i.e. astragaloside IV, noto- ginsenoside R1, ginsenoside Rgl, ginsenoside Rbl, and ginsenoside Rd in dripping pills made from Astragali Radix and Panax notoginseng. Methods The extraction method was based on a liquid-liquid extraction using water-saturated n-butanol and the quantitative determination was based on ultra-performance liquid chromatography coupled with evaporative light scattering detection （UPLC-ELSD）. The chromatographic analysis was performed on an Acquity UPLC HSS T3 column （100 mm ~ 2.1 mm, 1.8 IJm） with a gradient elution of acetonitrile-0.1% formic acid aqueous solution within a runtime of 15 min. Results Compared to different methods, the proposed method could remove the interference of PEG in formulation. And the calibration curves showed good linearity during the test ranges. The method was validated for limits of detection and quantification, precision, and reproducibility. The recoveries were within the range of 96.87% - 99.97%. In addition, the verified method was firstly applied to determination of the five active ingredients in Qishen Yiqi Dripping Pills （QYDP） simultaneously. Conclusion The contents of five active ingredients are stable and homogeneous in QYDP, which indicates that the method could be readily utilized as a quality evaluation method for this traditional Chinese medicine dripping pills made from AstragaliRadix and Panax notoginseng.

Glycosylation and sulfation of emodin by Gliocladium deliquescens NRRL 1086

The present study was designed to explore the substrate scope and biocatalytic capability of Gliocladium deliquescens NRRL 1086 on phenolic natural products. Emodin was subjected to the fermentation culture of Gliocladium deliquescens NRRL 1086 according to the standard two-stage protocol. The biotransformation process was monitored by HPLC-DAD-MS, the main product was isolated by column chromatography, and the structure was elucidated on the basis of NMR spectroscopy. Emodin could be fully metabolized by Gliocladium deliquescens NRRL 1086, resulting in high yield of emodin 6-O-β-D-glucopyranoside and small amount of sulfated product. In conclusion, our results may provide a convenient method to prepare emodin 6-O-β-D-glucopyranoside and the microbe catalyzed glucosylation/sulfation will give an inspiration to pharmacokinetic model studies in vitro.

An optimized micro-assay of myosin Ⅱ ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors

Myosin Ⅱ plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin Ⅱ ATPase activity based on molybdenum blue method, using a known myosin Ⅱ ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate(ATP) and calcium chloride, p H, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin Ⅱ ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L^(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin Ⅱ ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin Ⅱ ATPase inhibitors in vitro.

Two new steroidal glycosides from Ophiopogon japonicus

二新 C27-steroidal glycosides 从 Ophiopogon japonicus 的含纤维的根被孤立。光谱分析和化学证据揭示了他们的化学结构是(25R )-spirost-5,14-dien-3 尾 -yl-O- 伪 -l-rhamnopyranosyl-(1 鈫 ? 2 )- 尾 -d-xylopyranosyl-(1 鈫 ? 4 )- 尾 -d-glucopyranoside (1 ) 和 ophiogenin 3-O- 尾 - d-glucopyranoside (2 ) 。

Ge-Gen Decoction attenuates oxytocin-induced uterine contraction and writhing response: potential application in primary dysmenorrhea therapy

The uterine tetanic contraction and uterine artery blood flow reduction are possible reasons for primary dysmenorrhea(PD). In the present study, we aimed to evaluate the uterine relaxant effect and the influence on uterine artery blood velocity of Ge-Gen Decoction(GGD), a well-known Chinese herbal formula. In female ICR mice, uterine contraction was induced by oxytocin exposure following estradiol benzoate pretreatment, and the uterine artery blood velocity was detected by Doppler ultrasound. Histopathological examination of the uterine tissue samples were performed by H&E staining. Ex vivo studies demonstrated that oxytocin, posterior pituitary, or acetylcholine induced contractions in isolated mouse uterus. GGD inhibited both spontaneous and stimulated contractions. In vivo study demonstrated that GGD significantly reduced oxytocin-induced writhing responses with a maximal inhibition of 87%. Further study demonstrated that GGD normalized oxytocin-induced abnormalities of prostaglandins F_2 alpha(PGF_(2α)) and Ca^(2+) in mice. In addition, injection of oxytocin induced a decrease in uterine artery blood flow velocity. Pretreatment with GGD reversed the oxytocin response on blood flow velocity. Histopathological examination showed pretreatment with GGD alleviated inflammation and edema in the uterus when compared with the model group. Both ex vivo and in vivo results indicated that GGD possessed a significant spasmolytic effect on uterine tetanic contraction as well as improvement on uterine artery blood velocity which may involve PGF_(2α) and Ca^(2+) signaling, suggesting that GGD may have a clinic potential in PD therapy.

Sheng-Mai-San attenuates contractile dysfunction and structural damage induced by chronic intermittent hypoxia in mice

Sheng-Mai-San(SMS), a well-known Chinese medicinal plant formula, is widely used for the treatment of cardiac diseases characterized by deficiency of Qi and Yin syndrome. A mouse chronic intermittent hypoxia(CIH) model was established to mimic the primary clinical features of deficiency of Qi and Yin syndrome. Mice experienced CIH for 28 days(nadir 7% to peak 8% oxygen, 20 min per day), resulting in left ventricle(LV) dysfunction and structure abnormalities. After administration of SMS(0.55, 1.1, and 5.5 g·kg-1·d-1) for four weeks, improved cardiac function was observed, as indicated by the increase in the ejection fraction from the LV on echocardiography. SMS also preserved the structural integrity of the LV against eccentric hypotrophy, tissue vacuolization, and mitochondrial injury as measured by histology, electron microscopy, and ultrasound assessments. Mechanistically, the antioxidant effects of SMS were demonstrated; SMS was able to suppress mitochondrial apoptosis as indicated by the reduction of several pro-apoptotic factors(Bax, cytochrome c, and cleaved caspase-3) and up-regulation of the anti-apoptosis factor Bcl-2. In conclusion, these results demonstrate that SMS treatment can protect the structure and function of the LV and that the protective effects of this formula are associated with the regulation of the mitochondrial apoptosis pathway.