转化白藜芦醇苷虎杖内生真菌的分离和鉴定

为提高虎杖中白藜芦醇得率,采用虎杖内生真菌直接发酵虎杖转化白藜芦醇苷为白藜芦醇,通过从虎杖根、茎中初筛得到6株内生真菌,复筛以虎杖煮提液为底物发酵,采用高效液相色谱法定量检测发酵液中白藜芦醇苷和白藜芦醇含量。结果表明：6株内生真菌均具有转化白藜芦醇苷为白藜芦醇能力,其中分离于茎内的J1转化率最高为25.6%。J1经形态学观察及ITS-5.8S rDNA序列分析,鉴定为草酸青霉Penicillium oxalicum,Genbank登录号为HQ732137。     

转化虎杖苷虎杖内生真菌的分离及鉴定

为寻找可高效转化虎杖苷生成白藜芦醇的微生物新菌株,采用以虎杖苷为唯一碳源的选择性培养平板,从中药虎杖中分离筛选出4株生长良好的内生真菌,分别编号为HZ001、HZ002、HZ003和HZ004;考察了4菌株细胞和发酵液转化虎杖苷的性能,并对菌株进行了形态学和分子生物学鉴定.菌体细胞生物转化实验结果表明,HZ001和HZ...     

酶解法制备白藜芦醇的工艺优化

本文对虎杖提取物中白藜芦醇苷酶解制备白藜芦醇的工艺进行研究,以样品中白藜芦醇的含量为指标,对虎杖复合酶、纤维素酶、β-葡萄糖苷酶、虎杖苷专用酶进行了筛选,结果表明,以β-葡萄糖苷酶的转化率最高.采用单因素实验对影响β-葡萄糖苷酶酶解的因素:底物浓度、酶用量、酶解温度、pH和酶解时间进行考察;得出较佳的酶解工艺条件:酶用量3％,pH=5,40℃酶解10h.用该方法制备白藜芦醇的可以得到较高的产率,且苷水解酶可循环使用,重现性较好,适用于工业化大规模生产.     

白藜芦醇和白藜芦醇苷抗氧化作用的研究

采用亚油酸体系、H2O2诱导的红细胞溶血试验研究白藜芦醇、白藜芦醇苷的体外抗氧化作用。研究结果表明:白藜芦醇、白藜芦醇苷能抑制亚油酸的过氧化作用,其抑制率高于VC、BHT、虎杖粗提物。白藜芦醇、白藜芦醇苷均可降低H2O2诱导红细胞氧化性溶血率,其作用效果好于VC、VE和虎杖粗提物,抑制率具有剂量效应。     

虎杖中虎杖苷提取工艺的响应面优化方法

考察虎杖中虎杖苷的最佳提取工艺,采用无毒的乙醇作为提取溶剂,利用响应面法优化了虎杖中虎杖苷的提取条件,采用紫外分光光度法和HPLC法检测虎杖苷的含量。最佳提取条件为乙醇浓度78%,提取温度51℃,料液比10.70 mL/g,提取时间为2 h,得到虎杖苷理论吸光度值为0.445,与实际值0.439接近。经HPLC检测得到虎杖苷提取率为1.83%,与最高含量理论值2%接近。实验结果表明,采用响应曲面法优化得到的提取条件参数准确、可靠,具有实用价值。     

虎杖中白藜芦醇发酵和提取工艺

目的：对虎杖中白藜芦醇的发酵和提取工艺进行研究。方法：以汉中地区的虎杖为原料,通过正交试验研究虎杖中白藜芦醇自然发酵和提取条件。结果：最佳发酵条件为温度45℃、料液比1:3、发酵时间48h、白藜芦醇苷的转化率为96.1%;自然发酵后最佳提取条件为98% 甲醇、料液比1:6、提取3 次、每次1h,白藜芦醇的得率达1.912%;通过发酵和提取,虎杖中白藜芦醇的提取率达90%。结论：本实验可为虎杖中白藜芦醇的发酵和提取提供一定参考。     

湖北虎杖主要化学成分的含量分析及其与色差的相关性研究

目的分析湖北虎杖中主要成分的含量与粉末色差值,评价不同来源和产地药材质量,并探讨成分含量与颜色之间的相关性。方法采用高效液相色谱法测定58批药材中虎杖苷、白藜芦醇和大黄素含量,利用色差仪测定粉末色差值,分析主要成分与色差的相关性。结果野生品与栽培品的大黄素含量差异有统计学意义(P<0.01);总白藜芦醇的质量分数均达到了1.0%;虎杖苷的含量均达到了药典标准;大黄素与颜色指标值L*、a*存在极显著相关关系(P<0.01)。结论荆州公安县章庄铺、黄冈英山县红山镇、荆州松滋县杨林市镇等地药材质量较优,可作为优质种质资源进行更深入的研究,通过分析虎杖粉末色差值,可以预测大黄素的含量。     

虎杖内生真菌Aspergillus aculeatus HZ001产β-葡萄糖苷酶的酶学特性

中药虎杖含有丰富的虎杖苷,利用生物转化技术将其转化为白藜芦醇是制备天然白藜芦醇的有效方法。虎杖内生真菌Aspergillus aculeatus HZ001细胞可催化虎杖苷转化为白藜芦醇,该文对该菌株产胞内β-葡萄糖苷酶的酶学性质进行研究。结果表明:该酶的最适反应温度为60℃,在低于60℃时有较好的稳定性;最适反应pH为4.8,在pH 3.6~4.0时有较好的稳定性;Fe^2+对其活力有激活作用,相对酶活力可达到120%,Ca^2+、Co^2+、Mg^2+等离子对酶活力有明显的抑制作用,其中Co^2+的抑制作用最明显;十二烷基硫酸钠、巯基乙醇、二甲基亚砜和乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)4种抑制剂对该酶活力具有抑制作用,其中EDTA的抑制效果最为明显,相对酶活力仅有10%左右。在最适催化条件下,进行酶催化动力学分析,米氏常数K m值为2.571 mmol/L,最大反应速率V max值为0.594μmol/(mL·h)。     

虎杖内生菌转化虎杖苷和转化产物抗氧化活性研究

研究虎杖内生真菌对虎杖苷转化及转化产物抗氧化活性。采用薄层层析法筛选转化虎杖苷的内生真菌;通过形态学特征和ITS-5.8S r DNA基因序列分析对内生真菌进行鉴定;利用大孔吸附树脂、制备薄层色谱和高效液相色谱分离纯化生物转化产物;以VC、BHT、虎杖苷及5种虎杖苷衍生物为对照,对转化产物清除DPPH·自由基和OH·自由基的生物活性进行比较研究。经筛选发现其中l株内生真菌J3能转化虎杖苷形成转化产物。内生真菌J3 ITS-5.8S r DNA序列全长573bp,Gen Bank登录号KM 434883,确定为烟曲霉Aspergillus fumigatus。转化产物A-2在体外具有清除DPPH·自由基和OH·自由基的能力,且清除OH·自由基的能力高于底物虎杖苷,A-1在体外无清除DPPH·自由基和OH·自由基的能力。虎杖内生真菌Aspergillus fumigatus能对虎杖苷进行生物转化,形成具抗氧化活性的物质。     

虎杖中白藜芦醇生物转化菌的筛选及鉴定

白藜芦醇具有多种生物学活性,具有很高的应用价值,白藜芦醇的前体物质白藜芦醇苷在虎杖中含量较高,可通过微生物转化成白藜芦醇。论文采用添加有白藜芦醇苷的固体培养基,从虎杖内筛选能将白藜芦醇苷转化成白藜芦醇的内生菌,然后对筛选到的菌种进行鉴定。通过菌种形态学、显微镜观察和18srDNA序列分析等方法,鉴定结果显示该菌种为卡门培尔青霉（Penicilliumcamemberti）。     

Post-column on-line photochemical derivatization for the direct isocratic-LC-FLD analysis of resveratrol and piceid isomers in wine

Resveratrol is a stilbene produced by plants, e.g. grapes, in response to stress. Resveratrol is extracted during winemaking, being present in wine as 3-O-β-d-gluocoside (piceid) and as aglycone. Both, resveratrol and piceid exist in two isomeric forms, trans and cis. Resveratrol and piceid are weakly fluorescent in both of their isomeric forms, but highly fluorescent compounds are obtained when the original molecules are UV-irradiated. A chromatographic method with post-column on-line photoderivatization, has been developed for the analysis of resveratrol and piceid isomers. The four analytes are firstly separated in a C18 column (150 mm × 3.9 mm i.d., 4 μm) by isocratic elution, at 15 °C, with a mobile phase consisting of a mixture acetonitrile:o-phosphoric acid (0.04%), 18:82, v:v, at 0.9 mL min⁻¹, and secondly they are on-line phototransformed into their fluorescent photoproducts in a 3 m PTFE tube coiled around a 4 W xenon lamp. The elution conditions have been chemometrically optimized by means of the experimental design and the response surface methodology. Linearity ranges from 0.10 to 1.50 and from 0.10 to 1.00 μg mL⁻¹ and LOD around 0.001 and 0.01 μg mL⁻¹ have been calculated for trans- and cis-isomers, respectively. The method has been satisfactorily applied to red and white wine samples by standard addition and external calibration, respectively.     

Differential stilbene induction susceptibility of seven red wine grape varieties upon post-harvest UV-C irradiation

The stilbene induction capacity of the red wine grape varieties 'Tempranillo' (TEM), 'Cabernet Sauvignon' (CAS), 'Merlot' (MER), 'Syrah' (SYR), 'Monastrell' (MON), 'Garnacha' (GAR) and 'Cariñena' (CAR) exposed to post-harvest UV-C irradiation is reported for the first time. The induction of trans-piceatannol, trans-resveratrol and viniferins, among other stilbenes, was characterized using high-performance liquid chromatograph with diode-array and mass spectrometric detection (HPLC-DAD-MS-MS). All the wine grape varieties increased their resveratrol, piceatannol and viniferin content after UV-C treatment with the exception of the variety MON in which only piceatannol was induced. Other stilbenes such as astringin (piceatannol glucoside) or piceid (resveratrol glucoside) were induced to a lesser extent and only in some varieties. The stilbene induction capacity of grapes submitted to UV-C irradiation did not necessarily match the final corresponding maximum stilbene content reached in each variety. The total stilbene induction capacity of the grapes ranged from 2.4-fold (in MER) to 10.9-fold (in both CAS and CAR) with respect to untreated grapes (control). The total stilbene content (mg stilbene/100 g fresh weight, fw) after UV-C irradiation ranged from 1.16 in MON to 3.5 in MER. In general, the maximum induction of specific stilbenes in all the varieties followed the sequence: resveratrol >piceatannol >₁-viniferin >piceid. This maximum induction was 22.7-fold for resveratrol in CAS, 6.4-fold for piceatannol in CAR, 3.8-fold for piceid in TEM and 8.4-fold for ₁-viniferin in both CAS and CAR with respect to the control. The maximum concentration of the specific stilbenes reached after UV-C irradiation was 2.5 (mg stilbene/100 g fresh grape weight) for resveratrol in MER, 0.42 for piceatannol in MER, 0.16 for piceid in MON and 0.39 for ₁-viniferin in CAR. The screening of stilbene induction capacity in red wine grapes treated with UV-C irradiation could be useful for selecting the most suitable variety to produce stilbene-enriched red wines with potentially increased health-promoting properties.     

Supercritical CO2 extraction of resveratrol and its glycoside piceid from Chinese traditional medicinal herb Polygonum cuspidatum

Resveratrol (3,5,4′‐trihydroxystilbene) and its glycoside piceid are phenolic compounds found to be beneficial to health. In this paper, resveratrol and piceid were successfully extracted from the Chinese traditional medicinal herb Polygonum cuspidatum (Hu Zhang in Chinese), with supercritical CO₂ plus ethanol modifier. The effects of ethanol content, pressure and temperature on the yields of resveratrol and piceid were investigated. Results showed that the extraction pressure was the main factor for the effective extraction of the two substances in the presence of ethanol modifier, especially for piceid. The optimal extraction condition was obtained: 30 MPa, 50 °C, (100 ml l^?1), ethanol and 20 kg h^?1 of CO₂, at which the yields of resveratrol and piceid were 7.1 g kg^?1 and 16.0 g kg^?1, respectively. The yields obtained by supercritical fluid extraction (SFE) was comparable with those obtained by traditional organic solvent methods, indicating that SFE is an alternative method for extraction of the two compounds. Copyright © 2004 Society of Chemical Industry     

Pulsed electric field treatment enhanced stilbene content in Graciano,Tempranillo and Grenache grape varieties

The purpose of this paper was to study the effect of pulsed electric fields (PEF) on the stilbene content of three grape varieties. For this purpose, four different PEF treatments were applied using a continuous system over three varieties, Graciano, Tempranillo and Grenache, destemmed and crushed. In addition, the influence of PEF on their physicochemical composition was studied. PEF treatments did not affect the pH or total acidity of Graciano, however, musts from Tempranillo and Grenache had higher pH values and lower total acidity. In the three varieties, all treatments resulted in an increase of potassium content, deeper colour intensity and total polyphenol index and lower tonality, more pronounced in the treatments with higher time and energy. The stilbene content of the must significantly increased with respect to the control. This increase depended on the variety and the treatment applied. Tempranillo increased up 200% the total stilbene concentration, Grenache 60% and Graciano 50%. For the three varieties, the treatment with the highest time and energy was the most effective on the total stilbene extraction. These results indicate that PEF could be a suitable technology for obtaining musts with deeper colour and higher phenolic content, including resveratrol and piceid.     

Quantification of free and protein-bound trans-resveratrol metabolites and identification of trans-resveratrol-C/O-conjugated diglucuronides - two novel resveratrol metabolites in human plasma

The polyphenol trans-resveratrol (t-RES) is present as t-RES-3-O-beta-D-glycoside, termed piceid, in several plant-derived foods. Although data on the metabolism and on in vivo effects of t-RES have been reported, quantitative data on the metabolites formed after dietary intake of t-RES or piceid are still lacking. In this study, 85.5 mg of piceid per 70 kg of body weight (bw) were administered to healthy volunteers in a bolus dose. t-RES metabolites formed in plasma and urine were identified and quantified by LC-MS/MS, NMR, and HPLC-DAD analysis using chemically synthesized t-RES conjugate standards. In addition, the amount of t-RES metabolites bound noncovalently to plasma proteins was determined for the first time in humans. The metabolites identified and quantified were t-RES-3-sulfate, t-RES-3,4'-disulfate, t-RES-3,5-disulfate, t-RES-3-glucuronide and t-RES-4'-glucuronide, with t-RES-sulfates being the dominant conjugates in plasma and urine. Besides these metabolites, two novel t-RES-C/O-conjugated diglucuronides have been identified and quantified in plasma and urine. Moreover, it could be shown that up to 50% of the plasma t-RES-3-sulfate, t-RES-disulfates, and the novel t-RES-C/O-diglucuronides were bound to proteins. Total recovery of the dietary administered piceid in urine ranged between 13.6 and 35.7%.     

Maximising resveratrol and piceid contents in UV and ultrasound treated peanuts

The effects of varying doses of UV and ultrasound (US) treatments on the concentrations of resveratrol, piceid and total stilbenes, and sensory overall acceptance of peanuts were evaluated using response surface methodology. UV and US treatments resulted in increased (α < 0.05) resveratrol, piceid and total stilbenes but decreased overall acceptance compared to untreated controls. US produced higher concentrations of stilbenes compounds but lower overall acceptance compared to UV treatment. The optimum US process will produce 2.64–4.40 μg/g resveratrol and 4.50–6.50 μg/g total stilbenes which are more than that will be achieved by the optimum UV process with 2.00–2.06 and 2.10–2.27 μg/g, respectively. On a per serving basis, if a peanut bar contains 30 g peanuts, 3–5 bars of US- or 6 bars of UV-treated containing bars will have the same resveratrol content as a serving of 140 mL-glass of red wine.     

Profiling and accurate quantification of trans‐resveratrol,trans‐piceid,trans‐pterostilbene and 11 viniferins induced by Plasmopara viticola in partially resistant grapevine leaves

Background and Aims: In the Vitaceae, stilbenoid viniferins constitute a relatively restricted group of molecules based on a trans‐resveratrol structure and represent the main known phytoalexins. The aim of this work was the development of a new method for the chromatographic separation and accurate quantification of viniferins found in grapevine leaves following infection with Plasmopara viticola. Methods and Results: An interspecific F1 population was used, deriving from the cross between Merzling and Teroldego, respectively, partially resistant and susceptible to P. viticola. Analysis was carried on a liquid chromatography‐mass spectrometry (LC‐MS) system, using a reverse phase column. A method was validated for detailed metabolite profiling, with accurate identification and quantification of trans‐resveratrol, trans‐piceid, trans‐pterostilbene, a condensation product between (+)‐catechin and caffeic acid, and the whole class of stilbenoid viniferins in P. viticola‐infected grapevine leaves, using their respective standards. Synthesis was ongoing, and 6 days after infection, it was possible to see the highest concentration of stilbenoids for all genotypes. On the other hand, the intensity of stilbene induction did not show a clear and homogeneous correlation with the position of the leaves in the young shoot. Conclusions: The method described in this paper provides the basis for performing in a single run a targeted analysis of resveratrol, piceid, pterostilbene and the whole class of stilbenoids in P. viticola‐infected grapevine genotypes. Significance of the Study: To our knowledge, this is the first paper that has reported on detailed metabolite profiling with accurate identification and quantification of the main viniferins in grape leaves following infection with P. viticola, using the respective standards.     

Comparison of polyphenols and aroma in red wines from Portuguese mainland versus Azores Islands

trans-Resveratrol (trans-3,5,4′.trihydroxystilbene), piceid (resveratrol 3-O-β-D-glucopyranoside), together with their isomers and quercetin were measured in red wines from cultivars grown in Portuguese mainland and Azores Islands. Crude hydroxystilbenes and quercetin were extracted by solid-phase extraction-C₁₈ cartridges, and eluted with ethyl acetate. Recoveries of added trans-resveratrol and quercetin were between 90.2 to 101% (mean 96.8%) and between 95.2 and 97.6% (mean 96.2%), respectively. The precision measured by coefficients of variation (overall mean) ranged from 1.8 to 3.3%, and the calibration curves were linear with the mean correlation coefficients of 0.9997 and 0.9978 for trans-resveratrol and quercetin, respectively. The wine polyphenolic compounds were separated by reverse-phase C18 HPLC column and identified by UV fast scanning, retention times and HPLC/MS. The average content of total stilbenes was 4.75 and 6.33 mg/l, that of quercetin was 4.38 and 5.57 mg/l, and the trans/cis ratio was 8.32 and 10.89 for piceid and 5.2 and 4.09 for resveratrol in Portuguese mainland and Azorean wines, respectively. The Azorean wine (Basalto) was notable in having the highest trans-resveratrol concentration. The aroma profile of red wine samples from different regions were compared using SPME/GC-MS headspace methodology.     

Effect of yeast strain selection on the concentration of cis- and trans-resveratrol and resveratrol glucoside isomers in wine

The influence of four fermentative yeast strains belonging to the Saccharomyces cerevisiae species, on the content of cis‐ and trans‐resveratrol and resveratrol glucoside isomers in Vitis vinifera L. cv. Shiraz grape musts during alcoholic fermentation was studied by reverse phase high performance liquid chromatographic (RP‐HPLC) analysis. During vinification an increase in both the free and the glucosidic forms was observed up to day three/five (cis‐ resveratrol/piceid) or day four (trans‐ isomers) after which varible yeast, β‐glucosidase activity was noted until dryness where a general increase in free forms of resveratrol was accompanied by a decrease in the corresponding glucoside. In the finished wines ready for bottling or aging, the total amount of all resveratrol forms reflected (P < 0.05) the manufacturers polyphenol extraction ratings for the strains. Relative extraction of total phenolics in red wine amongst different yeast strains was demonstrated to mimic relative stilbene content.