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.

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.

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.

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.

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.

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.

[Prostaglandin E2 promotes hepatocellular carcinoma cell proliferation through EP2 prostanoid receptor].

OBJECTIVE: To investigate the effect of prostaglandin E2 (PGE(2)) on the proliferation of cultured hepatocellular carcinoma cells and explore which subtypes of EP prostanoid receptor mediate the action. METHODS: RT-PCR was used to determine COX-2 and EP receptor mRNA expression levels in human hepatocellular carcinoma cell line Hep3B and human normal hepatocyte line QSG7701. Cell counting kit-8 (CCK-8) assay was employed to investigate the effect of PGE(2), selective EP2 receptor agonist butaprost and EP3/EP4 receptor agonist PGE1 alcohol on the proliferation of the cells. RESULTS: COX-2 mRNA was highly expressed in Hep3B cells but scarcely in QSG7701 cells. Hep3B cells expressed the mRNAs for all the EP receptor subtypes, but EP2 and EP4 receptors were much more strongly expressed than EP1 and EP3 receptors. PGE(2) significantly promoted Hep3B cell proliferation in a time- and dose-dependent manner, and 10 µmol/L PGE(2) increased the cell proliferation by 22.57% (P<0.001) after a 48-h incubation; treatment with 0.1, 1.0, and 10 µmol/L PGE(2) for 72 h resulted in significantly increased cell proliferation by 12.13% (P<0.01), 17.58% (P<0.01) and 33.07% (P<0.001), respectively. EP2 receptor agonist butaprost (20 µmol/L) increased Hep3B cell proliferation by 21.96% (P<0.001), but the EP3/EP4 receptor agonist PGE(1) alcohol (2-20 µmol/L) exhibited no significant mitogenic effect in Hep3B cells, and 200 µmol/L PGE(1) alcohol decreased the cell viability. CONCLUSION: Selective activation of EP2 receptor promotes Hep3B cell proliferation, indicating the predominant role of EP2 receptor in mediating the mitogenic effect of PGE2.

[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.

[Establishment of mouse model of humoral immune response using rabbit red blood cells as the antigen].

OBJECTIVE: To establish a mouse model of humoral immune response by immunization with rabbit red blood cells (RRBCs). METHODS: The mice were immunized with RRBCs and the serum hemolysin level was measured by micro-hemolysis spectrophotometry. RESULTS: The peak time needed for hemolysin production against RRBCs was 6 days after the immunization, and 20% RRBCs in a total volume of 0.2 ml was optimal for intraperitoneal injection. Hydrocortisone (25 mg/kg) and cyclophosphamide (20 mg/kg) inhibited hemolysin production. Mannatide (4 mg/kg) produced no significant effect on serum hemolysin level in normal mice, but significantly potentiated hemolysin production in immunosuppressed mice induced by cyclophosphamide (20 mg/kg). CONCLUSION: Intraperitoneal RRBC injection is feasible for establishing mouse models of humoral immune response.

[Effects of lipopolysaccharides of Bacterium prodigiosum on tumor growth and immunosuppression in mice].

OBJECTIVE: To observe the effects of lipopolysaccharides of Bacterium prodigiosum (BP-LPS) in inhibiting tumor growth and improving immunosuppression in mice. METHODS: In mice bearing S180 tumor and a mouse model of immunosuppression induced by cyclophosphamide (CTX), the tumor growth, indexes of the immune organs and peripheral white blood cell count were measured after intraperitoneal injection of BP-LPS. RESULTS: Injections of BP-LPS (40 U/kg) for 8 consecutive days resulted in a significant inhibition of the tumor growth in mice bearing S180 tumor (P<0.01), with a dose-dependent increase of the spleen indexes but no obvious changes in the thymus indexes. Intraperitoneal injections of BP-LPS for 7 days inhibited the reduction of peripheral white blood cells and spleen indexes in immunosuppressive mice, but did not produce any significant changes in normal mice. CONCLUSION: BP-LPS can inhibit the tumor growth in tumor-bearing mice and enhance the immune functions of immunosuppressive mice.

[Antineoplastic effect of koumine in mice bearing H22 solid tumor].

OBJECTIVE: To investigate the antitumor effects of koumine in mice bearing H22 solid tumor and its effect on the immune system of the mice. METHODS: The changes in spleen and tumor weights and blood cell count were observed after koumine treatment in BALB/c athymic mice bearing H22 solid tumor, using normal saline solution and 5-Fu as the controls. RESULTS: Koumine significantly inhibited the tumor growth in a dose-dependent manner. The spleen index and blood cell counts in koumine group showed no significant differences from those in the saline control group, but higher than those in 5-Fu group. CONCLUSION: Koumine can significantly inhibit the growth of H22 solid tumor without obvious inhibitory effect on the immune system in mice.

[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.

[Immunomodulatory effects of Fomes fomentarius polysaccharides: an experimental study in mice].

OBJECTIVE: To investigate the immunomodulatory effects of Fomes fomentarius polysaccharides (FFP) in mice. METHODS: MTT assay was employed to evaluate the in vitro metabolic activity of the mouse splenocytes treated with FFP at different concentrations, and the secretion of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma) and interleukin 2 (IL-2) from the cells were measured by enzyme-linked immunosorbent assay. The changes in the phagocytotic activity of mouse macrophage in response to FFP treatment were evaluated by phagocytosis percentage of chicken red blood cells (CRBCs). The effect of FFP on the humoral immunity was assessed in mice immunized with sheep red blood cells (SRBCs) by measuring the serum levels of specific antibody (hemolysin) against SRBCs. RESULTS: FFP at the concentrations of 25, 50, and 100 microg/ml all significantly enhanced the metabolic activity of mouse splenocytes in vitro and increased the production of TNF-alpha, IFN-gamma and IL-2. FFP treatment also markedly enhanced the metabolic activity of mouse peritoneal exudate cells and TNF-alpha production by the cells. At the doses of 25, 50, and 100 mg/kg, FFP significantly increased serum hemolysin level in mice immunized with SRBCs, and FFP at 50 and 100 mg/kg obviously increased the capacity of mouse peritoneal macrophages in vivo for CRBC phagocytosis. CONCLUSION: FFP can promote the secretion of TNF-alpha, IFN-gamma and IL-2 by mouse immunocytes and enhance mouse humoral immune response and the phagocytotic activity of the macrophages.

[Changes of serum interferon-gamma levels in mice bearing S-180 tumor and the interventional effect of immunomodulators].

OBJECTIVE: To investigate the changes of serum inteferon-gamma (IFN-gamma) in mice bearing S-180 tumor and explore the role of the endogenous IFN-gamma in confining the transplanted tumor by intervention with immunomodulators. METHODS: Mouse models bearing S-180 solid tumor were established and subjected to intragastric administration of Ganoderma lucidum polysaccharides (GLP) or cyclosporine A (CsA) at different daily doses for 9 consecutive days. Serum IFN-gamma levels were measured in untreated tumor-bearing mice and in those after completion of GLP or CsA treatments by enzyme-linked immunosorbent assay (ELISA), and the changes of the tumor weight in the treated mice were evaluated. RESULTS: It was found for the first time that serum IFN-gamma levels in the tumor-bearing mice increased progressively within the initial 20 days after tumor implantation. The serum IFN-gamma levels in the 3 GLP-treated groups (at daily doses of 400, 200, and 100 mg/kg) all increased, which was the most obvious in 400 mg/kg GLP-treated group, and the tumor weight decreased significantly in response to GLP treatment, but the most conspicuous effect occurred with the daily dose of 200 mg/kg, and no significant statistical correlation was found between the two parameters. CsA treatment (at 20, 10, and 5 mg/kg, respectively) resulted in reduced serum IFN-gamma levels but produced virtually no effect on the tumor weight, and no obvious correlation was found between serum IFN-gamma level and the tumor weight. CONCLUSION: Increased serum IFN-gamma levels following GLP treatment are not significantly correlated to tumor growth inhibition in mice, and CsA reduces serum IFN-gamma levels without affecting the tumor weight, suggesting that endogenous IFN-gamma is not a major immunomodulating factor in growth inhibition of transplanted S-180 tumor.

[Study of dynamic changes of blood sugar and body signs in streptozotocin-induced diabetic animal models].

OBJECTIVE: To explore the dynamic changes of blood sugar and body's signs in streptozotocin diabetic animal models. METHODS: Rat and mouse diabetic models were established by a single intraperitoneal (ip) injection and 5-day successive ip injections of streptozotocin respectively. Blood sugar levels were measured. The food consumption index (consumption of food/body weight) and the water consumption index (consumption of water/body weight) were calculated. RESULTS: Sixty five point zero percent male rats received streptozotocin, 60 mg/kg ip, developed diabetes mellitus. The blood sugar remained in high level between the 15th day and the 25th day after injection, and it began to decline afterwards. By 5-day ip injections of streptozotocin, 40 mg/kg daily, 90.0% male mice developed diabetes mellitus. Dynamic changes of blood sugar of diabetic mouse were similar to those of rats, except that the blood sugar of mice did not decline as obvious as that of rats. The changes of water consumption index were in best fit with the changes of blood sugar in both models, with correlation index r>0.970. CONCLUSION: The blood sugar of diabetic animal model stayed in high level from the 15th day to the 25th day after the beginning of injection. And the period is suitable for observing effect of anti-diabetic drugs. The water consumption index can reflect the blood sugar levels of diabetes animals.

[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.

[Effect of Ganoderma lucidum polysaccharides on tumor cell nucleotide content and cell cycle in S180 ascitic tumor-bearing mice].

OBJECTIVE: To investigate the effect of Ganoderma lucidum polysaccharides (GLP) on the nucleotide contents and cell cycle distribution of the tumor cells in S180 ascitic tumor-bearing mice and explore the possible mechanism of the antitumor effect of GLP. METHODS: Mice bearing S180 ascitic tumor were subjected to intragastric administration of GLP (100, 200, and 400 mg/kg), normal saline or subcutaneous injection of cyclophosphamide (CTX) at 25 mg/kg, respectively. The treatment was given once daily for 9 consecutive days, after which the ascitic tumor cells were harvested for determination of the RNA and DNA contents and their ratio as well as the cell cycle alterations. Laser scanning confocal microscopy and acridine orange staining was performed to evaluate the DNA and RNA fluorescence intensity, and flow cytometry with propidium iodide (PI) staining was utilized for cell cycle analysis of the tumor cells. RESULTS: Compared with normal saline group, the tumor cells in the 3 GLP groups all showed reduced RNA and DNA contents, and this reduction was statistically significant in 200 mg/kg GLP group (P=0.000). Significantly reduced RNA/DNA ratio was noted in all the 3 GLP groups (P=0.003, 0.000, 0.008 corresponding to 400, 200, and 100 mg/kg groups), suggesting that ganoderma polysaccharides more effectively reduced RNA content than DNA content. CTX also resulted in reduced RNA and DNA contents but not the RNA/DNA ratio. At the doses of 400, 200, and 100 mg/kg, GLP increased the percentage of G2/G2 phase cells (P=0.003, 0.000, and 0.000) whereas CTX showed the contrary effect (P=0.000). GLP produced no obvious effect on S-phage cells but CTX significantly reduced their percentage (P=0.000). GLP at the 3 doses all decreased the percentage of G2/M phase tumor cells (P=0.014, 0.049, 0.016) and CTX again induced contrary effect (P=0.000). CONCLUSION: With different effects from CTX on DNA and RNA contents and cell cycle, GLP inhibits DNA and RNA synthesis in the tumor cells by mobilizing the host immune function to interfere with the normal cell cycles, which might be one of the mechanisms for the antitumor effect of GLP.