羊栖菜中α-淀粉酶抑制剂的提取及研究

研究羊栖菜浸提液中α-淀粉酶抑制剂对猪胰α-淀粉酶的抑制作用。实验用乙醇溶液对羊栖菜进行浸泡得到羊栖菜浸提液,将浸提液用乙酸乙酯萃取后,采用透析法及动力学方法研究其对猪胰α-淀粉酶的抑制机制。结果表明羊栖菜对猪胰α-淀粉酶的抑制类型为非竞争性可逆抑制,抑制常数为10.07 mg/m L。     

苦荞提取物成分分析及不同极性提取物对α-淀粉酶的抑制作用

采用石油醚、乙酸乙酯与正丁醇萃取获得不同极性的苦荞提取物,采用超高效液相色谱-串联四极杆飞行时间质谱法分析提取物的成分。结果表明苦荞提取物中含有大量的黄酮类物质,主要包括芦丁、槲皮素、异槲皮素及其糖苷等,还含有羟基苯甲酸等酚酸类物质。再以抑制率、抑制类型、荧光猝灭效应实验分析提取物对α-淀粉酶的抑制作用。结果表明,苦荞提取物对α-淀粉酶的抑制作用表现为乙酸乙酯相提取物、正丁醇相提取物与粗提物的结果类似,都有较为显著的抑制作用,而石油醚相提取物则没有明显且规律的抑制效果,可推断出苦荞提取物中各成分共同发挥了对α-淀粉酶活性的抑制作用,且抑制类型为非竞争性抑制。通过荧光猝灭实验得知抑制的机理为苦荞提取物中的成分与α-淀粉酶结合,从而使α-淀粉酶的荧光特性发生静态猝灭。     

绿茶和红茶浸提液对α-淀粉酶的抑制作用研究

研究绿茶和红茶浸提液对猪胰α-淀粉酶的抑制作用。对绿茶和红茶进行浸泡提取得到茶浸提液,采用非连续测定和Dixon作图法研究其对猪胰α-淀粉酶的半抑制浓度和抑制机理。发现绿茶和红茶对猪胰α-淀粉酶的半抑制浓度分别为8.99mg/mL和10.01mg/mL(以干茶叶含量计),二者的抑制类型都为可逆非竞争型抑制,绿茶和红茶的抑制常数分别为9.42mg/mL和11.51mg/mL(以干茶叶含量计)。茶浸提液与猪胰α-淀粉酶相互作用后的紫外差谱显示茶浸提物导致了猪胰α-淀粉酶紫外吸收的增强和峰的移动。     

大豆多肽中α-葡萄糖苷酶和α-淀粉酶抑制剂的虚拟筛选及体外活性验证

目的:通过抑制碳水化合物消化酶的活性延缓碳水化合物的消化,来预防和控制糖尿病的作用已经成为多领域的研究热点。β-伴大豆球蛋白是大豆蛋白中含量最高的功能性蛋白,但其胃肠道消化后产生的多肽是否具有抑制碳水化合物消化酶的活性还不得而知。方法:将β-伴大豆球蛋白进行胃肠道模拟消化,通过虚拟筛选和ADMET预测选出α-葡萄糖苷酶抑制肽和α-淀粉酶抑制肽,检测多肽对α-葡萄糖苷酶和α-淀粉酶的抑制作用。结果:通过胃酶、胰酶和糜蛋白酶将β-伴大豆球蛋白虚拟酶解成95个小分子多肽;筛选出8个α-葡萄糖苷酶抑制肽和α-淀粉酶抑制肽;发现氢键和静电相互作用在多肽与α-葡萄糖苷酶和α-淀粉酶结合中起重要作用;体外验证发现四肽EASY具有较强α-葡萄糖苷酶抑制效果,其IC50值为208.6 μg/mL,未发现明显α-淀粉酶抑制效果。结论:α-葡萄糖苷酶抑制肽EASY具有抑制碳水化合物消化酶的活性,可能成为控制糖尿病的潜在抑制剂。     

亚麻籽饼粕蛋白提取工艺优化及其水解物抑制α-淀粉酶活性研究

该研究以亚麻籽加工副产物-亚麻籽饼粕为原料制备α-淀粉酶抑制活性肽。采用响应面优化法对亚麻籽蛋白提取工艺进行优化,利用碱性蛋白酶、中性蛋白酶、胰蛋白酶对所提取亚麻籽蛋白进行酶解,采用3,5-二硝基水杨酸法（3,5-dinitro salicylic acid,DNS）来测定α-淀粉酶活性,比较不同蛋白酶酶解产物的α-淀粉酶抑制活性。根据优化结果与实际条件调整,亚麻籽蛋白的提取条件为pH 9.5,料液比为1∶20（g/mL）,温度为50℃,一次浸提时间为120 min以及二次浸提时间为60 min。测得最佳试验条件下亚麻籽饼粕蛋白的提取率为83.27%。α-淀粉酶抑制活性表明,经碱性蛋白酶酶解后得到的酶解产物具有较高的活性,其α-淀粉酶的抑制活性为27%。     

多穗柯三叶苷的抑制糖尿病关键酶活性和抗氧化性

从多穗柯中提取分离得到三叶苷,以抗糖尿病药物阿卡波糖为阳性对照,检测多穗柯三叶苷对小肠α-葡萄糖苷酶和胰脏α-淀粉酶活性的抑制作用和对α-葡萄糖苷酶活性抑制类型,并以芦丁为阳性对照检测其清除DPPH自由基能力。结果显示：多穗柯三叶苷对α-葡萄糖苷酶有明显抑制作用,为非竞争性抑制,抑制效果与阿卡波糖无明显差别,对α-淀粉酶活性的抑制率低于阿卡波糖;清除DPPH自由基能力强于芦丁。表明多穗柯三叶苷是一种有应用潜力的抗糖尿病活性物质。     

燕麦β-葡聚糖的提取和初步纯化

为了提高燕麦β-葡聚糖的纯度和功能特性,用α-淀粉酶去除燕麦粗提液中的淀粉,分别用Sevag法、胰蛋白酶法、等电点法、胰蛋白酶结合Sevag法和胰蛋白酶结合等电点法去除燕麦β-葡聚糖粗提液中蛋白质。结果表明:提取之前,加入20 U/(170 mL)α-淀粉酶,反应20 min,提取完成后,加入50 U/(100 mL)α-淀粉酶,反应时间25 min,即可完全去除淀粉;用Sevag法脱蛋白时,需要重复5～6次,胰蛋白酶结合Sevag法可以减少重复次数,胰蛋白酶去除蛋白质的效果最差,等电点法去除蛋白质时,操作简单,成本低,而胰蛋白酶结合等电点法去除蛋白质的效果最好,其蛋白质去除率94.8%,β-葡聚糖保留率85%。     

芦丁、槲皮素对α-淀粉酶抑制活性研究

以α-淀粉酶为评价指标,探讨了芦丁和槲皮素的辅助降血糖功能。研究结果芦丁和槲皮素对α-淀粉酶抑制均呈阳性,表明芦丁和槲皮素都具有辅助降血糖的功能。芦丁溶液、槲皮素溶液和阿卡波糖对α-淀粉酶的半抑制浓度分别为0.258mg/mL、0.233mg/mL、0.095mg/mL。芦丁、槲皮素、阿卡波糖对α-淀粉酶的抑制作用大小顺序为:阿卡波糖>槲皮素>芦丁。     

葛根、桑叶、山药3种天然产物对α-淀粉酶抑制作用研究

研究了葛根、桑叶、山药3种天然产物的提取溶液对α-淀粉酶活性的抑制作用。结果显示除了山药对α-淀粉酶无明显抑制作用,葛根、桑叶对α-淀粉酶均有显著抑制作用,对α-淀粉酶半抑制浓度(IC50)分别为56.72,46.57 mg/mL,抑制效果可分别达到现有α-淀粉酶抑制剂拜糖平的1.12,1.36倍。     

3种黄酮类化合物对α-淀粉酶的抑制机制

黄酮类化合物可以通过抑制α-淀粉酶的活性来调节血糖水平,然而,黄酮类化合物对α-淀粉酶的抑制机制和二者构效关系的研究十分有限,特别是B环上的羟基化和C环上的糖基化对黄酮类化合物抑制α-淀粉酶的影响尚不清楚。为探究黄酮类化合物对α-淀粉酶的抑制机制和构效关系,分析了芦丁、槲皮素、山奈酚3种黄酮类化合物对α-淀粉酶的抑制类型、荧光淬灭特性、构象变化、结合力和结合位点。结果表明,山奈酚对α-淀粉酶的抑制能力最强,芦丁最弱。C环C3处的糖基化和B环C3’处的羟基化均削弱了黄酮类化合物对α-淀粉酶的抑制作用。淬灭亲和力被C环C3处的糖基增强,被B环C3’处的羟基削弱。3种黄酮类化合物主要通过氢键和疏水作用与α-淀粉酶自发结合,导致酶的结构和疏水微环境被改变,进而有效抑制α-淀粉酶的活性。研究旨在为富含黄酮类化合物的降血糖食品的开发和应用提供理论参考。     

Screening for cysteine proteinase inhibitor activity in legume seeds

Extracts of 44 legume seeds were assayed for cysteine proteinase inhibitory (CPI) activity, using a papain/azocasein assay. Extracts with high papain inhibitory activity were further assayed with a crude gut extract (CGE) of larval Acanthoscelides obtectus (Say) (common bean weevil) against ³H-methemoglobin. Soybeans (Glycine max L Merrill), lima beans (Phaseolus lunatus L), tepary beans (Phaseolus acutifolius A Gray), and lupine seeds (Lupinus albus L) were found to be highest in CPI activity. Soybean hypocotyl extracts were found to have much greater CPI activity, relative to protein concentration, than cotyledon extracts. Papain was much more inhibited by soybean hypocotyl and cotyledon extracts than was A obtectus CGE. Water extracts of soybean hypocotyl flour were slightly higher in CPI activity than extracts with 0.2 M sodium phosphate buffer, pH 6.2.     

太湖蓝藻水华胰蛋白酶抑制剂和凝血酶抑制剂的测定

为配合太湖蓝藻的收集处理 ,研究了蓝藻水华的资源化可能 ,在前期工作的基础上 ,通过层析分离并结合活性测定 ,发现太湖蓝藻水华中有多种胰蛋白酶和凝血酶抑制剂     

Multiple molecular forms of finger millet subtilisin and amylase inhibitors

Nine varieties of finger millet (Eleusine coracana Gaertn) including a wild form were screened for proteinase and α-amylase inhibitory activities. Subtilisin inhibitory activity was present in all the varieties examined and was highest in the wild form, while the cultivated varieties had more trypsin inhibitory activity. Isoelectric focusing of a 60% ammonium sulphate fraction of the wild form, before and after complexing with subtilisin, showed multiple forms of subtilisin inhibitors one of which was also active against trypsin. Isoforms of finger millet subtilisin inhibitors were isolated from the ammonium sulphate fraction by ion exchange chromatography on DEAE-Sephadex followed by SP-Sephadex chromatography. Copurification of trypsin and α-amylase inhibitory activities with subtilisin inhibitory activity was observed during cation exchange chromatography. PAGE analysis revealed them to be charge isomers with pI in the range 5·5–6·0. SDS–PAGE gave an estimate of 12 000 mol wt for each of them. Finger millet subtilisin inhibitors were more active on bacterial proteinases than on bovine trypsin and chymotrypsin.     

植物中酪氨酸酶的筛选及不同抑制剂对其活性的影响

以新鲜马铃薯、红富士苹果、红蛇果、秀珍菇和紫茄子为研究对象,从中分别提取酪氨酸酶并测定其酶活力,发现紫茄子中提取的酪氨酸酶具有最大酶活力。因此选紫茄子中提取的酪氨酸酶做进一步研究。选邻苯二酚作为茄子酪氨酸酶底物,进一步研究了有机类、无机类和天然类三大类抑制剂对酪氨酸酶催化活性的影响,并测定了这些抑制剂的IC50值。      

Rice bean (Vigna umbellata Thunb. Ohwi and Ohashi) protection against Callosobruchus maculatus F.” by presence of protein profile

To identify defence responses of rice bean against Callosobruchus maculatus, 10 genotypes of rice bean and a mungbean variety, PAU 911 as susceptible check were evaluated. The rice bean genotypes were found to restrain the growth and development of C. maculatus at different larval stages. To justify these results, seeds of different test genotypes were examined for the presence of plant protease inhibitors which inhibit insect proteases causing reduction in the availability of amino acids necessary for their growth and development. Trypsin, chymotrypsin and cysteine protease inhibitor content was significantly higher in rice bean genotypes which ranged from 299.99 to 342.97 TIU g⁻¹ seeds, 235.07 to 251.45 CIU/g seeds and 1060.70 to 1574.99 CPIU/g seeds, respectively as compared to mungbean variety, PAU 911 (235.52 TIU/g seeds, 217.91 CIU/g seeds and 499.99 CPIU/g seeds, respectively). The protein profile showed a polypeptide band of 25–30 kDa corresponding to presence of protease inhibitors in rice bean genotypes except PAU 911. The results indicate that protease inhibitors in different genotypes of rice bean are very effective against C. maculatus.     

Preparation and optimisation of liposome‐in‐alginate beads containing oyster hydrolysate for sustained release

Oyster (Crassostrea gigas) hydrolysate shows antihypertensive effect in our previous study. Oral administration of oyster hydrolysate can loss bioactive peptides due to enzymatic degradation in vivo. To maximise its bioavailability, liposome‐in‐alginate (LA) beads were used to encapsulate the oyster hydrolysates to protect from degradation and obtain sustained release. The preparation conditions of the LA beads were optimised by response surface method using a model peptide of tyrosylalanine (YA). Their characterisation, swelling and release properties were investigated. The optimised conditions for the concentration of calcium chloride, sodium alginate and the amount of ethanol‐dissolved lecithin (EDL) were 0.5 m , 3% and 95.4 mg, respectively. The encapsulation efficiencies of YA and the oyster hydrolysate in the optimised condition were 74.9% and 84.3%, respectively. The release time of the oyster hydrolysate in the simulated gastrointestinal fluid was up to 16 h. The LA beads can be recommended to encapsulate oyster hydrolysate for bioavailability improvement.     

Proteinaceous Inhibitors of Trypsin and of Amylases in Developing and Germinating Seeds of Pigeon Pea ( Cajanus cajan(L) Millsp)

Inhibitors of trypsin and amylase in the extracts of developing seeds of 12 pigeon pea cultivars were analysed using a gel-X-ray film contact print technique and an enzyme-inhibitor assay, respectively. The inhibitors of amylase and trypsin in the extracts of germinating seeds of a pigeon pea cultivar (BDN2) were also studied. Nine trypsin inhibitor bands were detected in mature seeds of all the 12 cultivars. Inhibitory activities against amylase and trypsin were not detected in the extracts of seeds collected 11 and 27 days after flowering (DAF) by the enzyme-inhibitor assay. However, up to three trypsin inhibitor bands could be detected in the extracts of seeds collected 27 DAF by the gel-X-ray film contact technique. Two new slow-moving trypsin inhibitor bands were detected in the extracts of germinating seeds of BDN2 cultivar. These bands were prominent in extracts of seeds 10 days after germination (DAG). The amylase inhibitors and trypsin inhibitors in pigeon pea seeds are late synthesised proteins, their highest levels were observed in mature seeds and they were found to be slowly degraded during germination. Significant inhibitor activities were observed even 15 DAG. The amylases in developing seeds are insensitive to endogenous inhibitors.     

Subtilisin inhibitors from legume seeds: A purification procedure

Subtilisin inhibitors were prepared from ethanol-acetone precipitated seed extracts of chick peas, jack beans and broad beans, heated to destroy endogenous proteases. Trypsin inhibitors were complexed with trypsin and separated from subtilisin inhibitors by molecular sieve chromatography. The eluates were again heated to avoid tryptic degradation. The procedure was easy to handle and gave satisfactory yields. In a final step two subtilisin isoinhibitors from chick peas were purified to electrophoretic homogeneity. They constitute 0.01% of the defatted dry weight.     

The nutraceutical value of grain legumes: characterisation of bioactives and antinutritionals related to diabesity management

Beyond the nutritional value, legumes and particularly common beans are found in several dietary supplements used to treat diabesity (diabetes and obesity). These products contain not only inhibitors of carbohydrate-hydrolising enzymes (α-amylase, α-glucosidase), but also antinutritional factors that can cause adverse effects on human health. In the present research, twenty-two accessions of grain legumes were screened for bioactive (α-amylase and α-glucosidase inhibitors) and antinutritional (lectins, flatulence-producing sugars, trypsin inhibitors) phytochemicals. Results showed that four accessions had high α-amylase inhibiting activities (AI > 30%), and particularly the common bean ‘Great Northern’ resulted of interest for its high carbo-blocker activity (AI = 42.6 ± 0.5%), absence of lectins, low amounts of flatulence-producing oligosaccharides (2.5 ± 0.2 g/100 g DW) and low anti-trypsin activity (22.5 ± 4.3 trypsin inhibiting unit/mg DW). The knowledge offered from this work provides leads to the ultimate goal of developing new, more effective and safer dietary supplements for diabesity management.     

太湖蓝藻水华胰蛋白酶抑制剂的含量测定

采用紫外分光光度法对太湖蓝藻提取液中的胰蛋白酶抑制剂进行了含量测定,测定的线性范围在4～40μg/mL.此法简单方便,精密度和回收率均较理想.