In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors.

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC50: 7.54 ± 1.10 µM), 5e (IC50: 9.00 ± 0.97 µM), and 5 h (IC50: 9.57 ± 0.62 µM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC50: 32.18 ± 1.66 µM), 5 h (IC50: 31.47 ± 1.42 µM), and 5 s (IC50: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.HighlightsA series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.Compound 5g exhibited promising activity (IC50 = 7.54 ± 1.10 µM) against α-glucosidase.Compound 5s exhibited promising activity (IC50 = 30.91 ± 0.86 µM) against α-amylase.In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

The synthesis of cyanoformamides via a CsF-promoted decyanation/oxidation cascade of 2-dialkylamino-malononitriles.

A mild and efficient method for the synthesis of cyanoformamides from N,N-disubstituted aminomalononitriles with CsF as the promoter has been developed. This method features a wide substrate scope and high reaction efficiency, and will facilitate corresponding cyanoformamide-based biological studies and synthetic methodology development.

The synthesis of cycloalka[b]furans via an Au(i)-catalyzed tandem reaction of 3-yne-1,2-diols.

An Au(i)-catalyzed cyclization/1,2-rearrangement/aromatization cascade of 3-yne-1,2-diols has been successfully realized. This reaction not only provides a new and efficient strategy for the synthesis of substituted cycloalka[b]furan compounds as well as their derivatives, but might also facilitate related biological studies.

Cyclovirobuxinum D suppresses lipopolysaccharide-induced inflammatory responses in murine macrophages in vitro by blocking JAK-STAT signaling pathway.

AIM: Cyclovirobuxinum D (CVB-D), an alkaloid isolated from the Chinese medicinal plant Buxus microphylla, has been found to be effective to treat cardiac insufficiency, arrhythmias and coronary heart disease. In the present study, we investigated the effects of CVB-D on the inflammatory responses in lipopolysaccharide (LPS)-stimulated murine macrophages in vitro and the underlying mechanisms. METHODS: Murine macrophage cell line RAW264.7 cells were incubated in the presence of LPS (0.1 µg/mL) for 24 h. The cell viability was measured using MTT assay. The release of NO and cytokines were detected using the Griess test and ELISA, respectively. The mRNA and protein levels were determined using RT-PCR and Western blot, respectively. Reporter gene assays were used to analyze the transcriptional activity of NF-κB. RESULTS: Treatment of RAW264.7 cells with CVB-D (25-300 µmol/L) did not affect the cell viability. Pretreatment with CVB-D (50, 100 and 200 µmol/L) concentration-dependently decreased NO release and iNOS expression in LPS-treated RAW264.7 cells (its IC50 value in inhibition of NO production was 144 µmol/L). CVB-D also concentration-dependently inhibited the secretion and mRNA expression of IL-1ß and IL-6 in LPS-treated RAW264.7 cells. Furthermore, CVB-D remarkably inhibited the phosphorylation of STAT1 and STAT3, as well as JAK2 in LPS-treated RAW264.7 cells, but did not affect the activation of NF-κB and MAPKs pathways. Pretreatment with the JAK2 specific inhibitor AG490 (30 µmol/L) produced similar effects on NO release and iNOS expression in LPS-treated RAW264.7 cells. CONCLUSION: CVB-D exerts anti-inflammatory effects in LPS-stimulated murine macrophages in vitro at least in part by blocking the JAK-STAT signaling pathway. The anti-inflammatory actions of CVB-D may contribute to its cardioprotection.

Sinomenine induces apoptosis in RAW 264.7 cell-derived osteoclasts in vitro via caspase-3 activation.

AIM: Sinomenine (SIN) is an alkaloid found in the roots and stems of Sinomenium acutum, which has been used to treat rheumatic arthritis in China and Japan. In this study we investigated the effects of SIN on osteoclast survival in vitro and the mechanisms of the actions. METHODS: Mature osteoclasts were differentiated from murine monocyte/macrophage cell line RAW264.7 through incubation in the presence of receptor activator of NF-κB ligand (RANKL, 100 ng/mL) for 4 d. The cell viability was detected using the CCK-8 method. The survival and actin ring construction of the osteoclasts were scored using TRACP staining and phalloidin-FITC staining, respectively. The apoptosis of the osteoclasts was detected by DNA fragmentation and Hoechst 33258 staining, and the cell necrosis was indicated by LDH activity. The activation of caspase-3 in osteoclasts was measured using Western blotting and the caspase-3 activity colorimetric method. RESULTS: SIN (0.25-2 mmol/L) inhibited the viability of mature osteoclasts in dose-dependent and time-dependent manners, but did not affect that of RAW264.7 cells. Consistently, SIN dose-dependently suppressed the survival of mature osteoclasts. The formation of actin ring, a marker associated with actively resorbing osteoclasts, was also impaired by the alkaloid. SIN (0.5 mmol/L) induced the apoptosis of mature osteoclasts, which was significantly attenuated in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. SIN increased the cleavage of caspase-3 in mature osteoclasts in dose-dependent and time-dependent manners. Furthermore, SIN dose-dependently enhanced caspase-3 activity, which was blocked in the presence of Ac-DEVD-CHO. CONCLUSION: Sinomenine inhibits osteoclast survival in vitro through caspase-3-mediated apoptosis, thus it is a potential agent for treating excessive bone resorption diseases.

[Study on the anti-endotoxin effect of saponin from Tupistra chinensis].

OBJECTIVE: To investigate the effect of saponin from Tupistra chinensis Baker (STCB) on lethal toxicity of endotoxin in mice and explore the underlying mechanism. METHODS: Mouse models of endotoxin-induced death and endotoximia were established by intraperitoneal administration of KM mice with lipopolysaccharides (LPS) from Pseudomonas aeruginosa in doses of 60 mg/kg and 10 mg/kg respectively. Mouse survival rate and survival time were recorded and the serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in endotoximia mice were measured by enzyme-linked immunosorbent assay (ELISA). Mouse peritoneal exudate cells induced by LPS were used as an in vitro inflammatory model,which was then intervened with STCB and the levels of IL-1beta and TNF-alpha in the culture supernatants were measured by ELISA. RESULTS: The survival rates of mice prophylactically treated with STCB (200 and 400 mg/kg, in 5 consecutive days) were slightly higher compared with that in model group,but no statistical difference was observed (P>0.05). The survival time was much longer in the treated group (P<0.05). The serum levels of IL-1beta and TNF-alpha in STCB-treated mice (200 and 400 mg/kg, in 5 consecutive days) were significantly lower compared with those in model group (P<0.05). STCB (20 and 40 microg/mL) remarkably inhibited LPS-induced IL-1beta and TNF-alpha production by peritoneal exudate cells in vitro (P<0.05). CONCLUSION: Saponin from Tupistra chinensis showed beneficial effect on the prevention of mice from lipopolysaccharides-induced death, in which down regulation of IL-1beta and TNF-alpha expression might be involved.

[Process optimization for extraction of immune active polysaccharides from Fomes fomentarius by introduction of ultrasonication].

OBJECTIVE: To study the optimal extraction process of immune active polysaccharides from Fomes fomentarius by introduction of ultrasonication. METHODS: An orthogonal experimental design of L9 (3(4)) was used to investigate the effects of ultrasonication time, extraction temperature and extraction time on the extraction ratio, sugar content and immune stimulating activity (mouse splenocyte metabolic activity measured with MTT colorimetry) of the polysaccharides and the optimal extraction process was evaluated. RESULTS: Ultrasonication treatment had the most remarkable effect on the immune stimulating activity of the polysaccharides. The optimal extraction process for extraction of immune active polysaccharides was as follows: ultrasonication for 30min, extraction temperature at 80 degrees C and water extraction time for 2h. CONCLUSION: Ultrasonication can be used as a useful technique for extraction of immune active polysaccharides from Fomes fomentarius.

[Effect of Flammulina velutipes polysaccharides on production of cytokines by murine immunocytes and serum levels of cytokines in tumor-bearing mice].

OBJECTIVE: To study the effect of Flammulina velutipes polysaccharides (FVP) on the production of tumor necrosis factor alpha (TNF-alpha), interferon-gamma (INF-gamma) and interleukin 2 (IL-2) by murine immunocytes. METHODS: The cell's metabolic activity was determined with methylthiazolyl tetrazolium (MTT) colorimetry assay and the amounts of TNF-alpha, INF-gamma and IL-2 were measured by ELISA. RESULTS: FVP (200, 100, 50 microg/mL) could promote the metabolic activity of murine splenocytes and peritoneal exudate cells (PEC) and increase the amounts of TNF-alpha, INF-gamma and IL-2 in the supernatants of splenocyte cultures, and the amount of TNF-alpha in PEC cultures, with the most marked increase on TNF-alpha level. FVP (100, 50, 25 mg/kg) could raise the serum levels of TNF-alpha and INF-gamma in S180 tumor-bearing mice. CONCLUSION: FVP may regulate murine immune function through promoting the production of TNF-alpha, INF-gamma and IL-2.

A catalytic asymmetric one-pot [3+2] cyclization/semipinacol rearrangement sequence: an efficient construction of a multi-substituted 3H-spiro[benzofuran-2,1'-cyclopentane] skeleton.

A facile and efficient method to form a chiral multi-substituted 3H-spiro[benzofuran-2,1'-cyclopentane] structural unit has been developed via a one-pot [3+2] cyclization/semipinacol rearrangement cascade. A catalysis system of Cu(ii)/BOX has been used to efficiently construct a key stereogenic center via a cyclization between substituted benzoquinones and allylic alcohols affording the desired products in good yields and with excellent enantioselectivities and diastereoselectivities (21 examples; up to 67% yields; up to 92% ee and up to >20 : 1 dr). This method provides an alternative strategy for the synthesis of the corresponding bioactive molecules containing spiro[benzofurancyclopentane] skeleton units.

Copper-catalyzed cyclization of 2-cyanobenzaldehydes and 2-isocyanoacetates: an efficient strategy for the synthesis of substituted 1-aminoisoquinolines.

A Cu(acac)2-catalyzed cyclization reaction of 2-cyanobenzaldehydes with 2-isocyanoacetates has been successfully developed providing an efficient strategy for the synthesis of substituted 1-aminoisoquinolines. The reaction proceeds smoothly under mild conditions with high efficiency, and might provide an alternative strategy for the synthesis of 1-aminoisoquinoline containing molecules.

[Study of anticoagulant activity of ethanol extracts from leech in vitro].

OBJECTIVE: To study the anticoagulant activity of different portions of leech ethanol extracts (LEEs). METHODS: Anticoagulant activity was determined by measuring prothrombin time(PT), thrombin time (TT), activated partial throboplstin time (APTT) and fibrinogen coagulation time (FCT). RESULTS: PT, TT, APTT and FCT were remarkably prolonged by ethyl acetate portion of LEEs. Portions of petroleum ethrer, n-butanol and water extracted from LEEs was much weaker on anticoagulant activity. CONCLUSION: The anticoagulant effect of ethyl acetate extract portion of LEEs is the strongest among the four portions, and this results may be from its inhibitory effect on thrombin-catalyzed fibrinogen hydrolysis.

Zinc-promoted cyclization of tosylhydrazones and 2-(dimethylamino)malononitrile: an efficient strategy for the synthesis of substituted 1-tosyl-1H-pyrazoles.

A Zn(OTf)2-promoted cyclization reaction of tosylhydrazones with 2-(dimethylamino)malononitrile has been successfully developed providing an efficient strategy for the synthesis of substituted 1-tosyl-1H-pyrazoles.

[Isolation and structural analysis of a new polysaccharid, Streptomyces polysaccharide].

OBJECTIVE: To isolate and purify a new polysaccharid Streptomyces polysaccharid polysaccharide (SMP ), from cultured broth of a Streptomyces sp.strain and perform its structural analysis. METHODS: Ethanol was used to precipitate the polysaccharides and macromolecules from the broth. The proteins in the precipitate were removed by Sevage method and purification of SMP was carried out by DEAE-celluloseion-exchange chromatography and Sephadex G-25 gel filtration. The chemical structure of the SMP was determined by combined application of high performance liquid chromatography (HPLC), UV, IR and 1H-NMR spectroscopy, supplemented by periodate oxidation and Smith degradation. RESULTS: The purified SMP was neutral by a relative molecular mass of approximately 4 855. Sugar analysis showed that SMP contained glucose and fructose residues in an approximate molar ratio of 22:1 (10.96 to 0.48). The glycosidic linkages were estimated to be (1-6)- alpha-D- pyranoside form. CONCLUSION: SMP is characterized as a (1-6)- alpha-D- pyranose.

[Effects of cyclosporin A on gene expression profiles of NIT-1 pancreatic beta cell line].

OBJECTIVE: To observe the effects of cyclosporin A (CsA) on gene expression profiles of NIT-1 pancreatic beta cells cell line using microarray technique. METHODS: NIT-1 cells were exposed to cyclosporin A treatment (10 micromol/L) for 24 h and the differential gene expressions were assessed using microarray technique. RESULTS: After 24h of CsA treatment, 38 of the 4096 genes tested were up-regulated, including 13 genes with known functions involving stress response, cell growth and protein synthesis. Meanwhile 46 genes were down-regulated, including 25 genes with known functions involving cell growth and maturation, oxidative phosphorylation and protein synthesis. Changes of gene expression of Zfr,Tpi and Pax6 were confirmed by semi-quantitative reverse transcription polymerase chain reaction. CONCLUSIONS: CsA treatment for 24 h induces changes in the gene expression profiles of NIT-1 pancreatic beta cells. Down-regulation of the genes related to cell growth and maturation, oxidative phosphorylation and protein synthesis may partly explain the mechanisms that CsA inhibits the release of insulin from pancreatic beta cells.

[Effects of cyclosporine A on NIT-1 beta cell proliferation and pol alpha1 gene expression in vitro].

OBJECTIVE: To investigate the effects of cyclosporine A on the proliferation and pol alpha1 mRNA expression of cultured NIT-1 beta cells. METHODS: After exposure to cyclosporine A at various concentrations (0.05 to 10 micromol/L) for 48 h and 72 h, NIT-1 cell proliferation was analyzed by MTT assay and the gene expression determined by reverse transcriptional PCR (RT-PCR). RESULTS: Forty-eight-hour and 72-hour cyclosporine A exposure inhibited the cell proliferation in a concentration- dependent manner, and at the concentration of 10 micromol/L, cyclosporine A also decreased pol alpha1 mRNA expression after a 48-hour exposure. CONCLUSION: Cyclosporine A can effectively inhibit the proliferation of NIT-1 cells possibly through down-regulating the expression of pol alpha1 mRNA.

[Cyclosporin A inhibits insulin release and down-regulates gene expressions of mitochondrial oxidative phosphorylation enzymes in NIT-cells].

OBJECTIVE: To investigate the effects of cyclosporin A on insulin release and the gene expression profiles of mitochondrial oxidative phosphorylation in NIT-1 cells. METHODS: NIT-1 cells were exposed to cyclosporin A (10 micromol/L) for 24 and 48 h respectively, after which the amount of insulin release was determined by means of radioimmunoassay (RIA), and the differential expressions of Nuox23, Cox7c and Atp5K genes assessed by semi-quantitative reverse transcription polymerase chain reaction. RESULTS: Cyclosporin A reduced insulin release in the cell culture after 24 and 48 h exposure and decreased Nuox23, Cox7c and Atp5K mRNA expressions. CONCLUSION: Cyclosporin A induces inhibition of insulin release in NIT-1 cells, possibly due to the reduction of ATP synthesis involving the down-regulation of the gene expression of mitochondrial oxidative phosphorylation enzymes.

[Changes in GAT-3 and GABA-T mRNA expression of C6 glioma cells in response to a 2-week treatment with sodium valproate and withdrawal].

OBJECTIVE: To examine the effects of sodium valproate(VPA) treatment and withdrawal on the expression of GABA transporter-3 (GAT-3) and GABA transaminase (GABA-T) mRNA in C6 glioma cells, and to explore the role of GAT-3 and GABA-T in the rebound mechanism of VPA withdrawal. METHODS: C6 glioma cells were maintained for 2 weeks in DMEM medium containing VPA (50 mg/L) to establish the cell model of chronic exposure to VPA. Semi-quantitative RT-PCR was used to examine the changes of GAT-3 and GABA-T mRNA expression in response to VPA treatment and withdrawal. RESULTS: Chronic exposure to VPA down-regulated GAT-3 mRNA expression to 39.1% +/-0.5% from 46% +/-1.3% in the control group; After VPA withdrawal, GAT-3 mRNA expression level kept decreasing, reaching the minimum (11.7% +/-1.6%) 24 h after the withdrawal and with an increase to the level of 33.5%+/-1.1% after another 24 hr. GABA-T mRNA expression was up-regulated to 71.31% +/-8.39% from 34.77% +/-2.36% of the control level after VPA treatment, the withdrawal of which resulted in decreased GABA-T mRNA expression. Till 12 h after the withdrawal, the GABA-T mRNA expression level decreased to the minimum, 25.36% +/-7.68%. CONCLUSIONS: Chronic treatment with VPA can down-regulate GAT-3 mRNA expression and up-regulate GABA-T mRNA expression in C6 glioma cells, and this undulation may involve VPA withdrawal rebound.

[Effects of chronic valproic acid sodium treatment and withdrawal on glutamate and glutamine release of C6 glioma cells].

OBJECTIVE: To determine the effects of chronic valproic acid sodium (VPA) treatment and subsequent withdrawal on the release of glutamate (Glu) and glutamine (Gln) by C6 glioma cells, so as to understand the role of Glu and Gln released by astrocytes in the antiepileptic mechanism of VPA and the rebound mechanism of VPA withdrawal. METHODS: C6 glioma cells were maintained for 2 weeks in DMEM medium containing 50 mg/L VPA to establish the cell model of chronic VPA treatment. High-performance liquid chromatography (HPLC) was performed to detect the levels of Glu and Gln released by C6 glioma cells after chronic VPA treatment and subsequent withdrawal. RESULTS: Chronic VPA treatment increased Glu release of C6 glioma cells, and subsequent VPA withdrawal resulted in sustained decrease in the Glu level, reaching the lowest level 12 h after VPA withdrawal, which was obviously lower than the control level. Gradual but significant increase of Glu level was then observed to approach the control level till 48 h after VPA withdrawal. For Gln release, chronic VPA treatment resulted in its decrease while after VPA withdrawal, gradual increase occurred to recover the control level. CONCLUSIONS: Chronic VPA treatment can increase Glu and decrease Gln release by C6 glioma cells. Glu level undergoes a rebound in response to VPA withdrawal, while the relatively even changes of Gln level in the same setting may not involve VPA withdrawal rebound mechanism.

[General pharmacology of koumine parenteral solution].

OBJECTIVE: To determine the effect of koumine parenteral solution on the nervous, respiratory and cardiovascular systems of experimental animals. METHODS: Mouse spontaneous activities under the influence of the koumine injection were recorded with a photoelectric counter, and canine femoral artery pressure was determined by CYS-0.5 pressure transducer, respiratory curve described with TB-611 tension transducer and electrocardiogram (ECG) recorded with subcutaneous electrodes in the extremities after the injection. The above indices were automatically sampled and processed by multifunctional signal processor after being inputt to a computer. In this experiment, we observed the changes in the general behavior of the mice and their spontaneous activities within 15 min, along with the heart rate, maximum, minimum, and mean value of cardiac electric voltage, mean arterial pressure, respiratory rate and respiratory depth of the dogs before and at 10, 20, 30, 60, 90, 120 min after koumine injection. RESULTS: Koumine injection significantly decreased mouse spontaneous activities in moderate and high-dose groups, but did not produce obvious effect on the respiratory system, mean arterial pressure, and maximum, minimum, and mean values of cardiac electric voltage in dogs. The heart rate of the dogs did not undergo obvious changes in response to the injection at a low dose, but median and high doses of the injection produced obvious effects. CONCLUSION: Koumine injection has definite sedative effect in mice, and does not affect the respiratory and cardiovascular systems of dogs with the exception of the heart rate.

[Study of koumine-induced apoptosis of human colon adenocarcinoma LoVo cells in vitro].

OBJECTIVE: To study the apoptosis-inducing effect of koumine on human colon adenocarcinoma LoVo cells in vitro. METHODS: After koumine (50 mmol/L) treatment in vitro, the LoVo cells were examined under light microscope, transmission electron microscope and fluoroscope respectively for apoptosis, and the cell cycle distribution was analyzed using flow cytometry. RESULTS: The percentage of apoptotic cells increased in a time-dependent manner after the cells were treated with koumine, whose action exhibited remarkable cell cycle specificity. The percentage of LoVo cells in G(0)/G(1) phase rose from 31.3% to 42.3% and the percentage of cells in S phase fells from 62.0% to 38.7%. CONCLUSION: Koumine can induce apoptosis of LoVo cells in a time-dependent manner and inhibit the DNA synthesis in LoVo cells, thereby blocking the cell cycle from G1 to S phase.