体外模拟消化对鸡爪槭叶多酚稳定性及其自由基清除和α-葡萄糖苷酶抑制活性的影响

本文旨在通过研究鸡爪槭叶多酚提取物在体外模拟口腔和胃肠道消化过程中总酚、总黄酮和缩合单宁含量,及其DPPH·、ATBS~+·清除能力和α-葡萄糖苷酶抑制活性的变化,阐述鸡爪槭叶中的多酚类化合物在人体消化过程中主要活性成分组成和活性的变化。研究结果表明,体外模拟口腔和胃消化过程会降低消化产物中总黄酮含量,但对酚类和缩合单宁类化合物的含量无显著性影响(P0.05);肠消化过程会大大降低样品中活性成分的含量,实验组样品中总黄酮、总酚和缩合单宁含量的损失率分别为99.26%、52.95%和100%。口腔和胃部消化会降低样品的自由基清除能力和α-葡萄糖苷酶活性抑制能力,但不显著。肠道消化会使样品的抗氧化和酶抑制活性急剧降低,在测试浓度范围内,DPPH·和α-葡萄糖苷酶活性抑制能力损失率达100%,清除ATBS~+·的IC50值增加了1.88倍,且实验组和p H组样品间不存在显著性差异(P0.05)。总之,胃肠道消化会降低鸡爪槭叶多酚的稳定性及其抗氧化和α-葡萄糖苷酶抑制活性,且主要发生在肠道消化阶段,p H环境的改变是其主要影响因素。     

余甘子活性成分含量与抗氧化性研究

研究了不同产地余甘子的总多酚、黄酮和多糖含量,同时测定了各产地余甘子提取物的抗氧化活性。结果表明,广东野生品种鲜果的总多酚含量、总黄酮含量和水溶性多糖含量最高,它们在每克干果中的含量分别为(204.15±6.85)mg没食子酸当量、(81.11±8.67)mg芦丁当量和(19.78±1.22)mg葡萄糖当量。广东野生品种提取物的抗氧化活性较高,其还原力为1.344±0.14,羟基自由基和超氧阴离子自由基的清除力分别为91.50%±3.53%,92.31%±1.30%。广东栽培种提取物的DPPH自由基清除力最高,达到92.09%±1.52%。     

额济纳绿洲胡杨（Populus euphratica）酚类物质含量和分布及其与土壤水分的关系

采用分光光度法测定了额济纳绿洲胡杨(Populus euphratica)的披针形叶、卵圆形叶、嫩枝、枝(D《5 mm)、枝(5～10 mm)、主干树皮、根(D《2 mm)、根(2～5 mm)和根(5～10 mm)9类器官中的总酚、黄酮和缩合单宁含量.结果表明,总酚含量较高的器官为皮(27.93 mg/s),叶、根、枝中总酚含量分别为17.64 mg/g(两类叶均值)、16.72mg/g(三类根均值)、12.19 mg/g(三类枝均值);黄酮含量较高的器官为皮(51.30 mg/g),叶、根、枝中黄酮含量分别为28.45 mg/g(两类叶均值)、39.99 mg/g(三类根均值)、23.67 mg/g(三类枝均值);根中缩合单宁含量较高,三类根均值为22.10 mg/g,皮、叶、枝中缩合单宁含量分别为8.41 mg/g、4.03 mg/g(两类叶均值)、4.47 mg/g(三类枝均值).披针形叶和卵圆形叶中酚类物质含量没有显著性差异(P》0.05);随着枝不断成熟,嫩枝、枝(D《5mm)、枝(5～10mm)中酚类物质逐渐减少;随着根直径减少,根中缩合单宁逐渐增加,细根(D《2mm)中的缩合单宁含量最高(25.95 mg/g).分析胡杨各器官中酚类物质含量与土壤水分的关系,结果表明卵圆形叶中酚类物质含量与土壤水分含量成显著负相关关系(P《0.05,总酚:r=-0.949;黄酮:r=-0.923;缩合单宁:r=-0.944).研究揭示了极端干旱地区胡杨各器官中酚类物质的变化规律,及其与环境因子的相互作用关系.     

双水相系统纯化山楂叶中黄酮类物质的研究

应用水、乙醇和硫酸铵三者形成双水相系统,并利用黄酮在乙醇中的溶解度要大于水中的溶解度,以脱除其中一些易溶于水和在高浓度乙醇中溶解度低的物质。尝试了不同盐、不同体积比的乙醇和水、以及盐加入量对分相的影响并测定了其在不同情况下的纯化效果。发现在硫酸铵中的分相情况最好,在加入量到2．5g时既可以达到分相完全。并测定：（1）在5mL乙醇＋5mL水＋5g硫酸铵＋0．15g粗体物（粗体物中黄酮的百分含量为25％）的双水相中,黄酮类物质的回收率为89．2％,而黄酮百分含量提升为47．6％;（2）在4mL乙醇＋5mL水＋5g硫酸铵＋0．15g粗体物（粗体物中黄酮的百分含量为25％）的双水相中,黄酮类物质的回收率为81．9％,而黄酮含量提升为50．9％。水、乙醇和硫酸铵三者形成双水相系统应用于山楂叶中黄酮类物质的纯化在文献中少有报道,而本文应用此法可以将黄酮含量从25％提升到将近47．6％,并且回收率为89％。     

野生地木耳中次生代谢产物含量测定与分析

测定了野生地木耳（Nostoc commune Vauch.）中次生代谢产物含量,并与4种常见食用菌藻黑木耳（Auricularia auricular-judae）、银耳（Tremella fuciformis）、紫菜（Porphyra）、海带（Laminaria japonica）进行比较分析,总酚、总黄酮和缩合单宁含量测定采用分光光度法,总生物碱含量测定采用高效液相色谱法（HPLC）。结果显示,野生地木耳中总酚含量为24.255 mg/g±1.631 mg/g,总黄酮含量为5.741 mg/g±0.239 mg/g,总生物碱含量为0.768 mg/g±0.073 mg/g,缩合单宁含量为0.069 mg/g±0.009 mg/g。4种食用菌藻中次生代谢产物含量范围为：总酚（5.520～62.326 mg/g）、总黄酮（0.847～7.010 mg/g）、总生物碱（0.408～4.132 mg/g）、缩合单宁（0.063～0.233 mg/g）;比较分析结果显示,野生地木耳次生代谢产物中的总酚和总黄酮物质含量较高,且总酚是主要次生代谢产物,缩合单宁与总生物碱含量较低;总酚含量约为黑木耳和银耳的2倍、紫菜的6倍;总黄酮含量约为黑木耳的7倍,银耳的3倍。     

老黑谷米中多酚化合物的提取及体外抗氧化活性研究

以老黑谷米面粉为原料,提取分离其中的可溶性多酚、游离多酚、酯化多酚、醚化多酚、键合多酚,利用比色法进行多酚和类黄酮含量测定,并采用DPPH自由基清除能力、铁离子还原/抗氧化能力测定（FRAP）、总抗氧化能力测定（TEAC）、还原能力测定（RP）和H2O2清除活性等5种不同的体外抗氧化测定体系进行抗氧化活性测定。结果表明：5种多酚中,可溶性多酚含量最高,酯化多酚含量次之,显著高于游离多酚、醚化多酚、键合多酚。多酚中类黄酮含量最高为键和多酚和可溶性多酚,显著高于其他3种多酚。5种多酚均有较高的体外抗氧化能力,键合多酚的DPPH自由基清除能力最高,可溶性多酚的H2O2清除能力、FRAP还原能力、RP还原能力和TEAC总抗氧化能力最高。本研究为老黑谷米的开发利用提供科学依据。     

黄连木叶提取物抗氧化活性及相关毒性、抗毒性研究

本文对黄连木叶进行提取,黄酮粗提物的得率达到6.05%。氯化铝法检测其黄酮含量为137.5±14.3mg/g芦丁当量。DPPH自由基清除能力的数据显示其IC50小于Vc,清除能力强于Vc。SOS反应圈提示黄连木叶粗提物在10 mg/mL浓度范围内没有诱导细菌的SOS反应,说明其没有毒性或者毒性微弱。对丝裂霉素C诱导的细菌SOS反应抑制实验和对细菌丝状化形态变化的抑制实验结果表明,黄连木叶粗提物有很强的抑制细菌SOS反应及抑制细菌丝状化形态改变的效应。     

沙棘叶多酚提取物抗氧化及体外降血糖活性研究

本实验以沙棘叶为原料,通过溶剂提取制得提取物A,将提取物A经大孔树脂纯化制得沙棘叶多酚提取物B,在研究提取物A及提取物B的抗氧化能力和自由基清除能力的基础上,探究了沙棘叶多酚提取物B对猪胰α-淀粉酶的抑制类型,并通过DNS法来评价提取物B的体外降糖能力。实验结果表明:提取物A、提取物B多酚含量分别为18.59%和30.25%;以VC当量计,总抗氧化能力分别为6.15 mgVC/g提取物A、9.79 mgVC/g提取物B,对应的DPPH自由基清除率IC_(50)值分别为20.19μg提取物A/mL、8.6μg提取物B/mL;当猪胰α-淀粉酶浓度为0.5 mg/mL以下,反应时间在1 min以内时,酶反应体系具有良好的线性趋势,在此条件下,沙棘叶多酚提取物B对猪胰α-淀粉酶具有一定的抑制作用,其半抑制浓度为0.25 mg/mL,属于可逆反竞争抑制。本研究可为沙棘叶的开发和利用提供数据支撑。     

构树叶性状分化的生态功能初步研究

叶性状分化在自然界中较为普遍,不同的叶性状特征与植物对资源获得及利用效率密切关联,反映了植物适应特定环境所形成的生存对策。叶性状分化的生态功能一直以来备受生态学家和进化生物学家的广泛关注。自然界构树(Broussonetia papyifera)在个体发育过程中出现全缘叶和裂缺叶的分化,但其生态功能尚不清楚,推测两者的叶型分化是构树对虫害规避的结果。为了探讨构树叶性状分化对应的可能生态功能,该研究采用野外监测和室内分析的方法,对构树全缘叶和裂缺叶的虫害发生率、叶面积、与抗虫有关的酚类物质(总酚、缩合单宁、黄酮)含量进行了比较。结果表明:(1)相对于裂缺叶,全缘叶虫害发生率显著增加,全缘叶虫害发生率是裂缺叶的两倍。(2)自然条件下,全缘叶叶面积显著高于裂缺叶,增加了约44个百分点。(3)自然条件下,裂缺叶中总酚、缩合单宁、黄酮含量均显著高于全缘叶,分别提高了6.0%、4.2%和16.2%。(4)除黄酮外,虫害处理下裂缺叶中总酚、缩合单宁含量显著高于全缘叶,均提高了约5.0%。(5)人为移除部分叶片,裂缺叶中总酚、缩合单宁、黄酮含量均显著高于全缘叶,分别提高8.0%、1.6%和25.4%。这说明构树全缘叶和裂缺叶中酚类物质含量对外来损伤响应不一致,裂缺叶虫害发生率较全缘叶低可能由于两种类型叶片中酚类物质含量存在差异所引起。     

黄花败酱黄酮类化合物的提取与分析

用70％乙醇提取黄花败酱黄酮类化合物,得到粗黄酮,经聚酰胺纯化,得到精制黄酮。结果表明：黄花败酱干粉中总黄酮含量为2．35％,粗黄酮、精制黄酮中黄酮含量分别为8．81％和21．39％。黄花败黄酮类化合物主要是黄酮和黄酮醇两类,其中含有芦丁成分。     

The involvement of polyphenols and peroxidase activities in heavy-metal accumulation by epidermal glands of the waterlily (Nymphaeaceae)

Co-localization of polyphenols and peroxidase activity was demonstrated in epidermal glands of the waterlily (Nymphaea) by histochemistry. Total phenols, tannins and peroxidase activity were determined quantitatively in plant extracts. Polyphenols were partially identified and were found to consist mainly of hydrolyzable tannins, gallic and tannic acid derivatives.Nymphaea polyphenols were shown to chelate Cr, Hg, and Pb in vitro, and Cd-binding by polymerized polyphenols was demonstrated in leaves exposed to Cd in vivo. Both polyphenols and peroxidases were found at very high constitutive levels, which were not induced or altered by external conditions, such as light and heavy-metal stress. It is suggested that the polymerization of polyphenols by peroxidases, enhanced after heavy-metal uptake and detoxification, is responsible for the binding of heavy metals in Nymphaea epidermal glands. Received: 29 April 2000 / Accepted: 8 June 2000     

水麻果多酚的提取纯化及其抗氧化、抗肿瘤活性作用

为提取分离水麻果多酚,探索水麻果多酚的抗氧化及抗肿瘤能力。本研究通过单因素试验和正交试验优化水麻果多酚超声提取工艺,使用大孔树脂纯化水麻果多酚,通过测定水麻果多酚的总还原力以及清除·OH、DPPH·、ABTS·的能力来表征其抗氧化活性,以宫颈癌Hela细胞和肺癌A549细胞为抗肿瘤研究对象,测定水麻果多酚的抗肿瘤作用。在最优超声提取工艺条件下,即乙醇浓度为60%,料液比为1∶30,超声功率200 W,超声温度为70℃,提取时间为40 min,水麻果多酚得率为3. 29%,纯化产物总酚含量为40. 47 mg/100 mg,水麻果多酚的总还原力与维生素C相当,对·OH、DPPH·、ABTS·均具有显著清除作用,可抑制Hela细胞和A549细胞的生长增殖,并导致癌细胞产生大量活性氧,出现凋亡形态特征。该提取工艺简单、高效且多酚得率高,提取物经大孔树脂纯化后总多酚含量显著提高,且具有显著的抗氧化和抗肿瘤活性。     

Antifungal chemical compounds identified using a C. elegans pathogenicity assay

There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (approximately 1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as "probe compounds" and may have antifungal activity against other fungi.     

Experiments on the biological activities of various fractions of the folic acid antagonist "X-methyl" folic acid

A crude synthetic preparation called crude "X-methyl" folate has previously been shown to function as a folate antagonist for rats and chicks. This product has been shown to contain two folate antagonists: 9-methyl folate, present as 6% by weight of the product and which has low activity as a folate antagonist for Streptococcus faecalis, and pyrrofolic acid, a compound present in small amounts (0.04%), but having high anti-folate biological activity for S. faecalis. These experiments deal with the antifolate activity of these two fractions for the rat as measured by their effects on histidine oxidation. Rats were fed a purified diet based on 20% vitamin-free casein and containing 1.0% sulfasuxidine. When this diet was supplemented with a marginal amount of folic acid (0.3 mg per kg diet), the addition of 4 g of crude antagonist decreased histidine oxidation and decreased liver folate levels. The addition of 240 mg of pure 9-methyl folic acid (amount of 9-methyl folic acid in 4 g of crude) produced similar decreases in histidine oxidation and liver folate levels. A concentrate of pyrrofolic acid (equivalent to 4 g of crude) free of 9-methyl folic acid produced no decrease in histidine oxidation and minimal changes in liver folate. This indicates that the folate antagonist activity observed previously with animals is probably due to the 9-methyl folic acid component rather than to the pyrrofolic acid activity.     

Purification of immunoprotective tumor antigens by preparative isotachophoresis

Summary Tumor-specific transplantation antigens (TSTA) were purified from 3 M KCl and butanol extracts of C3H/HeJ 3-methylcholanthrene-induced fibrosarcomas by preparative isotachophoresis (pITP). Fractions from pITP which reacted with antisera to TSTA preparations in an enzyme-linked immunospecific assay were tested in vivo for induction of resistance to the growth of transplanted tumor cells. Isotachophoresis of crude 3 M KCl extracts yielded TSTA that was immunogenic at doses between 17 and 124 g. Isotachophoresis of TSTA partially purified from crude 3 M KCl or butanol extracts by preparative isoelectric focusing (pIEF) of 3 M KCl and butanol extracts yielded TSTA that was immunogenic over a two-fold log dose range. As little as 10 ng purified TSTA reduced tumor growth by 50%. Tumor growth reduction was specific, and immunized animals survived longer than non-immunized controls. A purification of 10,000-fold over crude 3 M KCl extracts and 2,500-fold over crude butanol extracts was obtained. These results suggest that TSTA from murine tumor cells is preparatively purified by following extraction with pIEF and then pITP.     

植物多酚的防癌抗癌作用

植物多酚是植物中广泛存在的一大类多酚化合物的总称,包括多酚（如单宁）、黄酮、酚酸等。体外实验研究表明植物多酚对多种人癌细胞具有增殖抑制作用,抑制动物体内肿瘤生长。其作用机理与抗氧化、调控细胞周期、诱导肿瘤细胞凋亡和分化、影响血管生成和肿瘤细胞信号传导等有关。对植物多酚的防癌抗癌作用,值得进一步深入研究。     

取代短链脂肪酸对秀丽隐杆线虫的抗氧化作用

短链脂肪酸(short chain fatty acids, SCFAs)是碳原子数为1～6的有机脂肪酸,不但可以作为生物体内能源物质,而且还具有抗炎、影响肠道菌群代谢和预防早发1型糖尿病等重要作用。然而,目前多数是对传统短链脂肪酸的生物学作用进行研究,对取代短链脂肪酸(short branched-chain fatty acids, SBCFAs)的相关研究甚少。本研究以秀丽隐杆线虫为模式动物,系统探究SBCFAs的抗氧化生物活性作用。采用胡桃醌氧化应激模型,在体内评价SBCFAs对秀丽隐杆线虫生存能力的影响,并通过体外抗氧化及H_2DCFDA荧光染色实验,进一步评价SBCFAs清除活性氧自由基(reactive oxygen species,ROS)的能力。本研究证实,在氧化应激研究中,传统脂肪酸不能延长秀丽隐杆线虫的存活时间,而2′-甲基取代短链脂肪酸(n=4-6)具有显著的抗氧化作用。体外抗氧化实验表明,2′-甲基取代短链脂肪酸(n=4-6)不具有体外直接清除ROS的能力,但是H_2DCFDA荧光染色实验显示2′-甲基丁酸能够显著降低线虫体内的ROS水平,表明2′-甲基丁酸通过降低体内ROS水平,从而增强秀丽隐杆线虫的抗氧化应激能力。本研究提示,传统短链脂肪酸不具有抗氧化作用;2′-甲基取代短链脂肪酸(n=4-6)能显著增强秀丽隐杆线虫的抗氧化能力;甲基取代位置和取代烷基长度对短支链脂肪酸的抗氧化作用至关重要,其作用机制需进一步研究。     

Actin and myosin-linked calcium regulation in the nematode Caenorhabditis elegans. Biochemical and structural properties of native filaments and purified proteins.

Calcium regulation of actomyosin activity in the nematode, Caenorhabditis elegans, has been studied with purified proteins and crude thin filaments. Actin and tropomyosin have been purified from C. elegans and shown to be similar in most respects to actin and tropomyosin from rabbit skeletal muscle. The actin comigrates with rabbit actin on polyacrylamide-sodium dodecyl sulfate gel electrophoresis, forms similar filaments and paracrystals, and activates the Mg2+-ATPase of rabbit myosin heads as efficiently as rabbit actin. Nematode tropomyosin has a greater apparent molecular weight (estimated by mobility on polyacrylamide-sodium dodecyl sulfate gels) than the rabbit protein, yet it forms Mg2+-paracrystals with a slightly shorter periodicity. Native thin filaments extracted from nematodes activate rabbit myosin subfragment 1 Mg2+-ATPase in a calcium sensitive manner; the extent of activation is threefold greater in 0.2 mM CaCl2 than in the absence of calcium. This observation suggests that the thin filaments contain components which are functionally equivalent to vertebrate troponins. Calcium is also required for maximal activation of the Mg2+-ATPase of purified nematode myosin by pure rabbit F-actin. C. elegans therefore has both myosin and thin filament-linked calcium regulatory systems. The origin of the actin, tropomyosin, and myosin from different tissues and the use of genetic analysis to answer questions about assembly and function in vivo are discussed.     

LYCAT, a homologue of C. elegans acl-8, acl-9, and acl-10, determines the fatty acid composition of phosphatidylinositol in mice

Mammalian phosphatidylinositol (PI) has a unique fatty acid composition in that 1-stearoyl-2-arachidonoyl species is predominant. This fatty acid composition is formed through fatty acid remodeling by sequential deacylation and reacylation. We recently identified three Caenorhabditis elegans acyltransferases (ACL-8, ACL-9, and ACL-10) that incorporate stearic acid into the sn-1 position of PI. Mammalian LYCAT, which is the closest homolog of ACL-8, ACL-9, and ACL-10, was originally identified as a lysocardiolipin acyltransferase by an in vitro assay and was subsequently reported to possess acyltransferase activity toward various anionic lysophospholipids. However, the in vivo role of mammalian LYCAT in phospholipid fatty acid metabolism has not been well elucidated. In this study, we generated LYCAT-deficient mice and demonstrated that LYCAT determined the fatty acid composition of PI in vivo. LYCAT-deficient mice were outwardly healthy and fertile. In the mice, stearoyl-CoA acyltransferase activity toward the sn-1 position of PI was reduced, and the fatty acid composition of PI, but not those of other major phospholipids, was altered. Furthermore, expression of mouse LYCAT rescued the phenotype of C. elegans acl-8 acl-9 acl-10 triple mutants. Our data indicate that LYCAT is a determinant of PI molecular species and its function is conserved in C. elegans and mammals.