昆仑雪菊提取物对α- 葡萄糖苷酶的抑制作用

目的:探讨昆仑雪菊提取物对α-葡萄糖苷酶的抑制活性。方法:将昆仑雪菊干燥花序粉碎,分别用水提法和乙醇法制备5种提取物。采用α-葡萄糖苷酶体外活性抑制模型,测定昆仑雪菊的5种提取物对α-葡萄糖苷酶的抑制活性。结果:这5种提取物对α-葡萄糖苷酶活性有较强的抑制作用,抑制活性均高于阿卡波糖。其中提取物Ⅰ的抑制活性最强,IC50=28.2 mg/L。结论:昆仑雪菊提取物具有较高的α-葡萄糖苷酶抑制活性,提示昆仑雪菊在抗糖尿病产品开发方面具有很好的应用前景。     

昆仑雪菊提取物对α-葡萄糖苷酶的抑制作用

目的：探讨昆仑雪菊提取物对α-葡萄糖苷酶的抑制活性。方法：将昆仑雪菊干燥花序粉碎,分别用水提法和乙醇法制备5种提取物。采用α-葡萄糖苷酶体外活性抑制模型,测定昆仑雪菊的5种提取物对α-葡萄糖苷酶的抑制活性。结果：这5种提取物对α-葡萄糖苷酶活性有较强的抑制作用,抑制活性均高于阿卡波糖。其中提取物Ⅰ的抑制活性最强,IC50=28.2 mg/L。结论：昆仑雪菊提取物具有较高的α-葡萄糖苷酶抑制活性,提示昆仑雪菊在抗糖尿病产品开发方面具有很好的应用前景。     

河南鼠尾草抑制体内外α-糖苷酶活性研究

本文采用96微孔板法,首次对河南鼠尾草抑制酵母和大鼠小肠α-葡萄糖苷酶活性进行研究。河南鼠尾草乙酸乙酯提取物(IC50=28.73μg/mL)和正丁醇提取物(IC50=73.90μg/mL)抑制酵母α-葡萄糖苷酶活性远高于阳性对照Acarbose(IC50=1081.27μg/mL),但只有乙酸乙酯提取物(IC50=366.79μg/mL)具有抑制大鼠小肠α-葡萄糖苷酶活性,阳性对照Acarbose未检测出其IC50。结果表明,河南鼠尾草乙酸乙酯提取物和正丁醇提取物均具有较好的酵母α-葡萄糖苷酶抑制活性,但只有乙酸乙酯提取物具有良好的大鼠小肠α-葡萄糖苷酶抑制活性。     

球毛壳菌H6次级代谢产物及其α-葡萄糖苷酶抑制活性

采用体外α-葡萄糖苷酶抑制模型对一株球毛壳菌H6的发酵液和菌丝体两种乙酸乙酯提取物进行活性评价,结果表明,发酵液乙酸乙酯提取物对α-葡萄糖苷酶具有较强的抑制活性,其IC₅₀值为(510.76±23.46)μg/mL。采用硅胶、Sephadex LH-20、半制备高效液相等色谱技术从其发酵液乙酸乙酯提取物中分离得到12个化合物。通过各种光谱分析,依次鉴定为chaetoviridins A-B(1-2),chaetoglobosins A-D(3-6),chaetoglobosin J(7),chaetoglobosin Q(8),prochaetogobosinsⅠ-Ⅲ(9-11),vibratilicin(12),其中化合物12为首次从毛壳属中分离得到。对化合物进行α-葡萄糖苷酶抑制活性测定发现,化合物12显示弱的抑制活性,其IC₅₀为(1 182.75±19.14)μg/mL。     

内蒙古产白刺、小果白刺和霸王α-葡萄糖苷酶抑制活性

首次利用体外α-葡萄糖苷酶抑制模型对内蒙古产3种蒺藜科植物的9个提取物进行活性评价,并与阳性对照Acarbose比较,发现3种植物均有抑制α-葡萄糖苷酶活性。其中白刺石油醚提取物对α-葡萄糖苷酶的抑制活性(IC50=81.80 mg/L)最高,其余依次为小果白刺乙酸乙酯提取物(IC50=610.29 mg/L),霸王石油醚(IC50=627.22 mg/L)和乙酸乙酯提取物(IC50=838.40 mg/L),它们的抑制活性远大于阳性对照Acarbose(IC50=1103.01 mg/L)。结果发现,不同植物不同溶剂提取物的α-葡萄糖苷酶抑制活性不同。同一植物不同溶剂提取物相比较,甲醇提取物的α-葡萄糖苷酶抑制活性不及乙酸乙酯和石油醚提取物。     

五种苦苣苔科植物α-葡萄糖苷酶抑制活性研究

利用体外α-葡萄糖苷酶抑制模型对5种苦苣苔科植物进行活性评价,并与阳性对照Acarbose进行比较,发现5种植物不同部位均有一定的α-葡萄糖苷酶抑制活性。其中,牛耳岩白菜石油醚部位的抑制活性最高(IC50=26.19μg/mL,活性均远大于阳性对照Acarbose(IC50=1081.27μg/mL)。不同植物比较,牛耳岩白菜的α-葡萄糖苷酶抑制活性最好,其3种不同溶剂提取物与Acarbose相比均有很高抑制活性;对牛耳岩白菜提取物的α-葡萄糖苷酶抑制动力学研究结果表明,石油醚和乙酸乙酯提取物对α-葡萄糖苷酶抑制作用属于非竞争性抑制类型,Ki值分别为4.24和40.04μg/mL。正丁醇提取物则属于竞争性抑制类型(Ki=205.48μg/mL)     

新疆盐生植物对α-葡萄糖苷酶抑制活性的研究

对10种新疆盐生植物提取物的α-葡萄糖苷酶抑制活性进行初步研究.在10种盐生植物的70%乙醇提取物中,大叶补血草的α-葡萄糖苷酶抑制活性较高,IC_(50)值为1.77 mg/mL;水提取物中,琵琶柴、多枝柽柳、花花柴的α-葡萄糖苷酶抑制活性较高,IC_(50)值分别为2.53、1.21、1.52 mg/mL.本文选取的10种新疆盐生植物中部分植物提取物具有一定的抑制α-葡萄糖苷酶活性.     

黄连提取物对α-葡萄糖苷酶抑制作用研究

利用体外α-葡萄糖苷酶抑制模型对黄连不同部位提取物进行活性评价,并与阳性对照Acarbose比较,发现黄连不同部位均有一定的α-葡萄糖苷酶抑制活性.其中,黄连根茎乙酸乙酯提取物的抑制活性最高(IC_(50)=20.72 μg/mL),黄连种子石油醚部位(IC_(50)=40.86 μg/mL)和黄连叶石油醚部位(IC_(50)=62.85 μg/mL)的活性次之.3个部位的提取物活性均远大于阳性对照Acarbose(IC_(50)=1081,27 μg/mL).不同部位比较,根茎对α-葡萄糖苷酶抑制活性最好,这3种提取物抑制活性均比阳性对照高;同一部位不同提取物比较,石油醚和甲醇提取物α-葡萄糖苷酶抑制活性一般要高于乙酸乙酯提取物.     

基于网络药理学对蒲公英抑制α-葡萄糖苷酶活性成分及作用机制的研究

为阐明蒲公英对α-葡萄糖苷酶抑制的活性成分。实验对蒲公英进行活性筛选和分子对接,结果表明,其乙醇提取物的石油醚萃取成分能良好抑制α-葡萄糖苷酶的作用(IC_(50)=0.9μg/mL),其抑制效果强于阳性药阿卡波糖(IC_(50)=174.3μg/mL)。故采用GC-MS联用技术从上述成分中分离鉴定了37个化合物,由虚拟筛选后,共得到5个成药性强的小分子化合物;通过与人源性α-葡萄糖苷酶进行分子对接后,证实其中2个小分子化合物能与α-葡萄糖苷酶存在亲和性。基于网络药理学构建蒲公英"降糖成分—靶点"图,阐述蒲公英的降糖机理。     

血竭超临界提取物的降血糖作用及其机制研究

为了研究血竭超临界提取物的降血糖作用及其机制,用正常小鼠和四氧嘧啶糖尿病小鼠模型,以血糖、糖耐量为指标研究血竭超临界提取物的降糖作用。通过测定体外α-葡萄糖苷酶的活力,观察血竭超临界提取物对α-葡萄糖苷酶的抑制作用。结果发现血竭超临界提取物对正常小鼠空腹血糖无明显降低作用,能改善小鼠对蔗糖的耐受力,能降低四氧嘧啶诱导的糖尿病小鼠的空腹血糖水平,能抑制α-葡萄糖苷酶的活性。表明血竭超临界提取物对糖尿病小鼠有较好的降糖作用,对α-葡萄糖苷酶的抑制作用可能是其降血糖的机制之一。     

Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.     

Regulation of protein tyrosine phosphatase 1B by sumoylation

Protein-tyrosine phosphatase 1B (PTP1B) is an ubiquitously expressed enzyme that negatively regulates growth-factor signalling and cell proliferation by binding to and dephosphorylating key receptor tyrosine kinases, such as the insulin receptor. It is unclear how the activity of PTP1B is regulated. Using a yeast two-hybrid assay, a protein inhibitor of activated STAT1 (PIAS1) was isolated as a PTP1B-interacting protein. Here, we show that PIAS1, which functions as a small ubiquitin-like modifier (SUMO) E3 ligase, associates with PTP1B in mammalian fibroblasts and catalyses sumoylation of PTP1B. Sumoylation of PTP1B reduces its catalytic activity and inhibits the negative effect of PTP1B on insulin receptor signalling and on transformation by the oncogene v-crk. Insulin-stimulated sumoylation of endogenous PTP1B results in a transient downregulation of the enzyme; this event does not occur when the endogenous enzyme is replaced with a sumoylation-resistant mutant of PTP1B. These results suggest that sumoylation, which has been implicated primarily in processes in the nucleus and nuclear pore, also modulates a key enzyme-substrate signalling complex that regulates metabolism and cell proliferation.     

Protein tyrosine phosphatase 1B and α-glucosidase inhibitory Phlorotannins from edible brown algae, Ecklonia stolonifera and Eisenia bicyclis

The present work investigates protein tyrosine phosphatase 1B (PTP1B) and the α-glucosidase inhibitory activities of two edible brown algae, Ecklonia stolonifera and Eisenia bicyclis, as well as in their isolated phlorotannins. Since the individual extracts and fractions showed significant inhibitory activities, column chromatography was performed to isolate six phlorotannins, phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofurofucoeckol-A (4), dieckol (5), and 7-phloroeckol (6). Phlorotannins 3-6 were potent and noncompetitive PTP1B inhibitors with IC(50) values ranging from 0.56 to 2.64 μM; 4-6 exhibited the most potent α-glucosidase inhibition with IC(50) values ranging from 1.37 to 6.13 μM. Interestingly, 4 and 6 were noncompetitive, while 5 exhibited competitive inhibition in an α-glucosidase assay. E. stolonifera and E. bicyclis as well as their isolated phlorotannins therefore possessed marked PTP1B and α-glucosidase inhibitory activities; this could lead to opportunities in the development of therapeutic agents to control the postprandial blood glucose level and thereby prevent diabetic complications.     

Protein-tyrosine phosphatase 1B (PTP1B) deficiency confers resistance to transforming growth factor-β (TGF-β)-induced suppressor effects in hepatocytes

Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, mediating both tumor suppressor and promoter effects. The suppressor effects of the cytokine can be negatively regulated by activation of survival signals, mostly dependent on tyrosine kinase activity. The aim of our work was to study the role of the protein-tyrosine phosphatase 1B (PTP1B) on the cellular responses to TGF-β, using for this purpose immortalized neonatal hepatocytes isolated from both PTP1B(+/+) and PTP1B(-/-) mice. We have found that PTP1B deficiency conferred resistance to TGF-β suppressor effects, such as apoptosis and growth inhibition, correlating with lower Smad2/Smad3 activation. Both responses were recovered in the presence of the general tyrosine kinase inhibitor genistein. PTP1B(-/-) cells showed elevated NF-κB activation in response to TGF-β. Knockdown of the NF-κB p65 subunit increased cell response in terms of Smads phosphorylation and apoptosis. Interestingly, these effects were accompanied by inhibition of Smad7 up-regulation. In addition, lack of PTP1B promoted an altered NADPH oxidase (NOX) expression pattern in response to TGF-β, strongly increasing the NOX1/NOX4 ratio, which was reverted by genistein and p65 knockdown. Importantly, NOX1 knockdown inhibited nuclear translocation of p65, promoted Smad phosphorylation, and decreased Smad7 levels. In summary, our results suggest that PTP1B deficiency confers resistance to TGF-β through Smad inhibition, an effect that is mediated by NOX1-dependent NF-κB activation, which in turn, increases the level of the Smad inhibitor Smad7 and participates in a positive feedback loop on NOX1 up-regulation.     

Protein-tyrosine Phosphatase 1B Antagonized Signaling by Insulin-like Growth Factor-1 Receptor and Kinase BRK/PTK6 in Ovarian Cancer Cells

Ovarian cancer, which is the leading cause of death from gynecological malignancies, is a heterogeneous disease known to be associated with disruption of multiple signaling pathways. Nevertheless, little is known regarding the role of protein phosphatases in the signaling events that underlie the disease; such knowledge will be essential to gain a complete understanding of the etiology of the disease and how to treat it. We have demonstrated that protein-tyrosine phosphatase 1B (PTP1B) was underexpressed in a panel of ovarian carcinoma-derived cell lines, compared with immortalized human ovarian surface epithelial cell lines. Stable restoration of PTP1B in those cancer cell lines substantially decreased cell migration and invasion, as well as proliferation and anchorage-independent survival. Mechanistically, the pro-survival IGF-1R signaling pathway was attenuated upon ectopic expression of PTP1B. This was due to dephosphorylation by PTP1B of IGF-1R β-subunit and BRK/PTK6, an SRC-like protein-tyrosine kinase that physically and functionally interacts with the IGF-1R β-subunit. Restoration of PTP1B expression led to enhanced activation of BAD, one of the major pro-death members of the BCL-2 family, which triggered cell death through apoptosis. Conversely, inhibition of PTP1B with a small molecular inhibitor, MSI-1436, increased proliferation and migration of immortalized HOSE cell lines. These data reveal an important role for PTP1B as a negative regulator of BRK and IGF-1Rβ signaling in ovarian cancer cells.     

Protein Tyrosine Phosphatase 1B and α-Glucosidase Inhibitory Phlorotannins from Edible Brown Algae,Ecklonia stolonifera and Eisenia bicyclis

The present work investigates protein tyrosine phosphatase 1B (PTP1B) and the α-glucosidase inhibitory activities of two edible brown algae, Ecklonia stolonifera and Eisenia bicyclis, as well as in their isolated phlorotannins. Since the individual extracts and fractions showed significant inhibitory activities, column chromatography was performed to isolate six phlorotannins, phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofurofucoeckol-A (4), dieckol (5), and 7-phloroeckol (6). Phlorotannins 3–6 were potent and noncompetitive PTP1B inhibitors with IC₅₀ values ranging from 0.56 to 2.64 μM; 4–6 exhibited the most potent α-glucosidase inhibition with IC₅₀ values ranging from 1.37 to 6.13 μM. Interestingly, 4 and 6 were noncompetitive, while 5 exhibited competitive inhibition in an α-glucosidase assay. E. stolonifera and E. bicyclis as well as their isolated phlorotannins therefore possessed marked PTP1B and α-glucosidase inhibitory activities; this could lead to opportunities in the development of therapeutic agents to control the postprandial blood glucose level and thereby prevent diabetic complications.     

Acylsulfonamide-containing PTP1B inhibitors designed to mimic an enzyme-bound water of hydration

Previously, it had been reported that 6-(phosphonodifluoromethyl)-2-naphthoic acid binds to the protein-tyrosine phosphatase PTP1B with its 2-carboxyl group interacting only indirectly through a bridging water molecule. Reported herein is a family of new analogues that utilize acylsulfonamido functionality both to mimic this water of hydration and to provide an additional new site for elaboration not found in the parent carboxyl-containing analogue. Target acylsulfonamides were prepared in two steps from commercially available primary sulfonamides, which were selected based on in silico screening for their potential ability to interact with one of three binding surfaces proximal to the PTP1B catalytic site. In general, modest potency enhancements were observed. Arylacylsulfonamides represent a structure-based extension of inhibitor design that may have broader utility in the development of PTP1B inhibitors.     

Benzothiazole benzimidazole (S)-isothiazolidinone derivatives as protein tyrosine phosphatase-1B inhibitors

Benzothiazole benzimidazole (S)-isothiazolidinone ((S)-IZD) derivatives 5 were discovered through a peptidomimetic modification of the tripeptide (S)-IZD protein tyrosine phosphatase 1B (PTP1B) inhibitor 1. These derivatives are potent, competitive, and reversible inhibitors of PTP1B with improved caco-2 permeability. An X-ray co-crystal structure of inhibitor 5/PTP1B at 2.2A resolution demonstrated that the benzothiazole benzimidazole forms bi-dentate H-bonds to Asp48, and the benzothiazole interacts with the surface of the protein in a solvent exposed region towards the C-site. The design, synthesis, and SAR of this novel series of benzothiazole benzimidazole containing (S)-IZD inhibitors of PTP1B are presented herein.     

Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.     

Small molecule approach to studying protein tyrosine phosphatase

Understanding the function of protein tyrosine phosphatases (PTPs) is crucial to deciphering cellular signaling in higher organisms. Of the 100 putative PTPs in human genome, only a little is known about their precise biological functions. Thus establishing novel ways to study PTP function remains a top priority among researchers. Classical genetics and more recently the use of RNA interference (RNAi) for gene silencing remains a popular choice to study function. However, the one gene-one function hypothesis is now recognized as an oversimplified scenario, especially among the signaling proteins such as PTPs. Therefore, there is a need to understand gene function in an appropriate cellular context. Since proteins are the work horses of the cell, alteration of protein function by various means is a particularly attractive strategy. In this context, the chemical approach, where a small molecule is used to affect the function of the desired protein is increasingly being recognized as a method of choice. In this review, we describe how small molecules can be used to study the function of a prototypical PTP, PTP1B, which is a negative regulator in insulin signaling. This includes our initial strategies for finding the most potent and specific PTP1B inhibitor to date, synthesizing cell permeable analogues suitable for cellular studies, and using them to dissect the role of PTP1B in the insulin signaling pathway. This approach is potentially general and thus could be utilized to study the function of other PTPs.