响应面法优化超声辅助提取紫色小白菜花青苷的工艺研究

为探讨从紫色小白菜(Brassica campestris L.ssp.chinensis(L.)Makino var.communis Tsen et Lee)叶片中提取花青苷的最佳工艺条件,在超声功率为420 W、40%乙醇和两次超声提取条件下,采用Box-Behnken的中心组合试验设计原理,设计4因素3水平试验,研究了pH、料液比、超声温度、超声时间对花青苷得率的影响。结果表明,紫色小白菜叶片花青苷提取的优化工艺参数为料液比1∶16(pH 2.76),在超声功率为420 W、温度为58℃下超声12 min,花青苷的提取得率可达4.11 mg g–1 DW。     

反相高效液相色谱法测定厌氧菌有机酸代谢产物

建立反相高效液相色谱测定厌氧菌代谢发酵有机酸产物(乙酸、乳酸)的方法并用于测定乳酸菌代谢发酵产物中的含量。反相高效液相方法是一种简单、准确、灵敏的方法,可用于同时定量测定厌氧菌的有机酸代谢产物。     

干旱对紫色小白菜光合特性及营养品质的影响

以‘紫薇’不结球紫色小白菜为试验材料,采用盆栽控水试验,设置正常水分供应、轻度干旱、中度干旱和重度干旱4个干旱处理,测定了紫色小白菜植株营养品质和光合色素含量、光合与叶绿素荧光参数等指标,研究不同干旱胁迫处理对紫色小白菜光合特性及营养品质的影响。结果表明:(1)紫色小白菜叶片花青苷、可溶性糖、可溶性蛋白、维生素C含量均随干旱处理程度的加重呈先升高后下降的趋势,并均在轻度干旱下达到最大值。(2)随着干旱处理程度的加重,紫色小白菜叶片叶绿素a、叶绿素b、叶绿素(a+b)和类胡萝卜素含量均呈先缓慢上升后急剧下降的变化规律,且均于轻度干旱下达到最大值。(3)随着干旱处理程度的加重,紫色小白菜叶片净光合速率(P_n)、气孔导度(G_s)和蒸腾速率(T_r)均呈逐渐下降的趋势,而瞬时水分利用效率(WUE)表现出先升后降的趋势。(4)随着干旱处理程度的加重,紫色小白菜叶片以吸收光能为基础的性能指数(PI_(ABS))逐渐降低,而单位反应中心吸收的光能(ABS/RC)、单位反应中心捕获的光能(TR_O/RC)、单位反应中心传递的能量(ET_O/RC)、单位反应中心热耗散的能量(DI_O/RC)以及单位面积捕获的光能(TR_O/CS)、单位面积的热耗散(DI_O/CS)均呈现逐渐上升的趋势;叶片中PSⅡ最大光化学效率(ψ_(PO))、将电子传递到电子传递链中QA-下游的其他电子受体的概率(ψ_O)、反应中心吸收的光能用于电子传递的量子产额(ψ_(EO))值均随着干旱处理的加重呈先缓慢下降后急剧下降的趋势,受体侧J相的相对可变荧光(V_J)增幅比供体K相可变荧光占J相可变荧光的比例(W_K)大,PSⅡ受体侧受到的胁迫伤害大于供体侧。研究表明,干旱处理迫使紫色小白菜光合特性及品质都出现一定的变化,轻度干旱胁迫处理能增加紫色小白菜花青苷及其光合色素的含量,并提高净光合速率,进而改善植株的营养品质,值得在紫色小白菜耐旱生产中推广应用。     

紫叶李叶黄酮提取工艺的响应面优化及最佳采收月份的确定

使用高效液相色谱仪测定不同条件下超声辅助提取紫叶李叶中矢车菊素-3-O-葡萄糖苷花青素、芦丁、槲皮素、山奈酚4种黄酮类物质的得率,并根据4种物质的活性,建立紫叶李叶中黄酮类物质得率权重计算公式。在单因素试验的基础上,采用Box-Behnken设计-响应面法优化紫叶李叶黄酮类物质提取工艺。应用最佳提取工艺处理不同月份紫叶李叶,并根据4种成分权重计算黄酮类物质得率,确定最佳采收月份。结果表明,紫叶李叶黄酮类物质最佳提取工艺为乙醇体积分数63.93%、液料比25∶1(mL/g)、提取温度44.08℃、提取时间41.62 min;最佳采收月份为8月份。此结果重复性好,可信度高,其中矢车菊素-3-O-葡萄糖苷花青素得率随时间变化最明显,且与日照条件显著相关,该试验对后期相关具体成分的提取具有借鉴意义。     

杜仲叶中绿原酸提取工艺优化及分离制备

目的：优化绿原酸提取工艺,并进行单体制备。方法：在单因素试验基础上,以料液比、乙醇浓度和pH为影响因素,绿原酸提取收率为评价指标,依据中心组合法则,运用3因素3水平的响应曲面分析法优化提取工艺。以绿原酸提取液为原料,采用高压制备液相色谱法分离绿原酸。结果：最优提取工艺为料液比1∶10(g/mL)、乙醇浓度20%、pH 3.5、提取时间90 min、提取温度75℃,绿原酸的平均提取率为2.78%,模型预测值为2.94%。高压制备液相分离获得的绿原酸单体纯度大于98.75%,收率大于95.0%。结论：建立了一种简便高效的绿原酸提取分离工艺。     

火麻叶中大麻二酚提取工艺的响应面法优化及不同部位含量分布

本文研究一种超声波辅助提取火麻叶中大麻二酚(cannabidiol, CBD)的方法,同时分析火麻不同部位的CBD含量。首先建立CBD的HPLC检测方法,采用单因素实验确定超声提取功率、提取温度、提取时间、料液比4个因素对火麻叶中CBD提取率的影响;利用Design-expert软件进行Box-Benhnken实验设计确定并验证CBD的超声辅助提取的最优工艺条件,并检测火麻不同部位CBD含量。实验表明：采用Agilent HC-C₁₈柱(250 mm×4.6 mm, 5μm),检测波长：220 nm,流速：1.0 mL/min,柱温：28℃,流动相：甲醇-水=90∶10,等度洗脱,在此条件下的分离效果和峰型较好,并且结果准确可靠,稳定性好。以甲醇为提取溶剂,最佳提取工艺条件为：提取功率320 W,提取温度81℃,提取时间26 min,料液比1∶22 g/mL,在此条件下得到CBD含量的平均值为79.53±0.26 mg/g。火麻嫩叶中CBD含量最高,其余依次为老叶、种子和茎,根中含量最低。结果表明,超声波提取是一种高效的大麻二酚提取方法,且作为一种天然产物资源,大...     

蒜辣素UPLC检测体系优化及其在大蒜资源评价中的应用

大蒜是重要的调味蔬菜,大蒜辣素的含量是评价大蒜品质的最重要的一项指标。本研究通过对样品处理、提取方法和检测方法的研究,建立了完善的大蒜辣素超高效液相色谱(UPLC)检测方法:使用UPLC BEH C18色谱柱,流动相为甲醇∶水=1∶1,检测波长为254nm,进样体积为1μl,流速为0.3ml/min。大蒜辣素在2.04～510mg/L范围内呈良好线性关系(R2=0.9991)。利用建立的方法对212份大蒜鳞茎的大蒜辣素含量进行检测,发现供试212份大蒜资源的大蒜辣素含量差异显著,含量分布在0.82%～3.01%之间,最高含量与最低含量之间相差近4倍。     

大蒜辣素UPLC检测体系优化及其在大蒜资源评价中的应用

大蒜是重要的调味蔬菜,大蒜辣素的含量是评价大蒜品质的最重要的一项指标.本研究通过对样品处理、提取方法和检测方法的研究,建立了完善的大蒜辣素超高效液相色谱(UPLC)检测方法:使用UPLC BEH C18色谱柱,流动相为甲醇∶水=1∶1,检测波长为254nm,进样体积为1μl,流速为0.3ml/min.大蒜辣素在2.04 ～ 510mg/L范围内呈良好线性关系(R2 =0.9991).利用建立的方法对212份大蒜鳞茎的大蒜辣素含量进行检测,发现供试212份大蒜资源的大蒜辣素含量差异显著,含量分布在0.82％ ～3.01％之间,最高含量与最低含量之间相差近4倍.     

酢浆草的UPLC指纹图谱研究

酢浆草(Oxalis corniculata)为酢浆草科植物,资源分布广泛,全草均可入药。该研究为建立酢浆草超高效液相色谱指纹图谱,采用乙腈-0.05%醋酸水溶液梯度洗脱,以Agilent ZORBAX RRHD Eclipse Plus C18(2.1 mm×100 mm,1.8μm)色谱柱,流速0.3 mL·min~(-1),进样量为10μL,检测波长280 nm,柱温25℃等色谱条件,研究了15个不同产地30批酢浆草药材的指纹图谱。结果表明:确立了24个共有峰,相似度评价软件计算30批样品的相似度为0.839~0.987;应用聚类分析对各样品指纹图谱的共有峰峰面积统计分析得出,除贵州剑河、贵州印江及贵州天柱8月3批样品外,其余27批样品共有化学成分的含量较为接近。该研究结果表明建立的酢浆草UPLC指纹图谱方法简便、迅速、可靠,较其HPLC指纹图谱有更高的灵敏度和分辨率,极大地缩短了检测时间,可用于酢浆草质量评价的快速分析。     

Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS

To evaluate bile acid (BA) metabolism in detail, we established a method for analyzing BA composition in various tissues and intestinal contents using ultra performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). Twenty-two individual BAs were determined simultaneously from extracts. We applied this method to define the differences in BA metabolism between two rat strains, WKAH and DA. The amount of total bile acids (TBAs) in the liver was significantly higher in WKAH than in DA rats. In contrast, TBA concentration in jejunal content, cecal content, colorectal content, and feces was higher in DA rats than in WKAH rats. Nearly all BAs in the liver were in the taurine- or glycine-conjugated form in DA rats, and the proportion of conjugated liver BAs was up to 75% in WKAH rats. Similar trends were observed for the conjugation rates in bile. The most abundant secondary BA in cecal content, colorectal content, and feces was hyodeoxycholic acid in WKAH rats and omega-muricholic acid in DA rats. Analyzing detailed BA profiles, including conjugation status, in a single run is possible using UPLC/ESI-MS. This method will be useful for investigating the roles of BA metabolism under physiological and pathological conditions.     

高效液相色谱法测定必特螺旋霉素发酵液中有机酸

必特螺旋霉素是运用基因工程技术获得的工程茵产生的一组以异戊酰螺旋霉素为主要成分的多组分新型抗生素,其前体为乙酸、丙酸、丁酸和异戊酸等有机酸。本研究利用高效液相色谱法以0．01mol／L磷酸缓冲液（pH2．3）和甲醇为流动相,分别在反相C8（α-酮戊二酸、乙酸、柠檬酸、琥珀酸、丙酸）和C18（丁酸、异戊酸）柱上定量测定了必特螺旋霉素前体酸和三羧酸循环相关有机酸。所建立的测定方法的相对标准偏差为0．10％～0．42％,回收率为93．19％～102．08％。     

一株高抗氧化活性内生真菌的分子鉴定及产酚酸类物质研究

【目的】以疏花水柏枝(Myricaria laxiflora)淹水前根部分离到的内生真菌SG17为研究对象,为明确其抗氧化能力及可能的物质基础,对其进行生物学鉴定、抗氧化能力分析及产酚酸类物质研究。【方法】对该菌nr DNA的内转录间隔区进行测序;应用总抗氧化试剂盒,以维生素C为阳性对照,测定其抗氧化能力;采用高效液相色谱(HPLC)和薄层色谱(TLC)探讨其发酵液中的酚酸类化合物。【结果】SG17具有很高的抗氧化活性,其发酵液粗提物的抗氧化活性达到同浓度抗坏血酸的31.86%,分子和形态鉴定表明SG17是一株烟曲霉(Aspergillus fumigatus)。其发酵产物中酚酸类物质含量丰富,HPLC能检测到含有没食子酸,且通过HPLC和TLC分析还发现一种光学性质与绿原酸极为相似的类似物。【结论】内生真菌烟曲霉SG17发酵液的抗氧化能力远高于国际报道水平,是一种具有潜在开发价值的抗氧化剂资源。     

Case study and application of process analytical technology (PAT) towards bioprocessing: II. Use of ultra-performance liquid chromatography (UPLC) for making real-time pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. Two of the key goals that have been outlined for PAT are "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". Recently, we have been examining the feasibility of applying different analytical tools for designing PAT applications for bioprocessing. We have previously shown that a commercially available online high performance liquid chromatography (HPLC) system can be used for analysis that can facilitate real-time decisions for column pooling based on product quality attributes (Rathore et al., 2008). In this article we test the feasibility of using a commercially available ultra- performance liquid chromatography (UPLC) system for real-time pooling of process chromatography columns. It is demonstrated that the UPLC system offers a feasible approach and meets the requirements of a PAT application. While the application presented here is of a reversed phase assay, the approach and the hardware can be easily applied to other modes of liquid chromatography.     

Analysis of major tomato xylem organic acids and PITC-derivatives of amino acids by RP-HPLC and UV detection

Major amino acids and organic acids in xylem exudates of tomato plants were separated by reversed phase high performance liquid chromatography (RP-HPLC) and quantified by UV detection. Before separation, amino acids were converted into their phenylisothiocyanate (PITC) derivatives. In a single run, Asp, Glu, Ser, Gln, His, Thr, Ala, Tyr, Val, Met, Cys, Ile, Leu, Phe, and Lys could be separated and detected down to the pmol level. Unresolved peaks were obtained for Asn and Gly and for Arg and Pro. For organic acid analysis, exudates were pre-treated by perfusion over a prepacked Adsorbex SCX cation exchange column, to eliminate exudate amino acids. Elution recoveries for organic acids were close to 100%. The exudate organic acids were separated by ion suppression RP-HPLC chromatography, and peaks could be resolved for L-malic acid, malonic acid, maleic acid, citric acid and fumaric acid, down to the pmol level. UV signals for exudate ascorbic acid, and succinic acid were below the limits of detection. Determination of oxalic acid and tartaric acid was impossible, due to the presence of the exudate salt peak in the chromatogram. The results indicate the potential of the methods applied, and show the applicability of RP-HPLC analysis for the determination of both amino acids and organic acids in xylem exudates.     

Chromatographic techniques for the separation of Al and associated organic ligands present in soil solution

Organic acids including humic, fulvic, aliphatic and aromatic acids comprise part of the dissolved organic carbon (DOC) present in soil solution. They act as ligands for trace metals and are effective detoxifiers of monomeric aluminium (Al). Solid phase extraction (SPE) techniques permit fractionation of the DOC into organic classes but yield no information on the pre-existing Al/organic acid complexes. Aliphatic and aromatic acids may be separated and determined by High Performance Liquid Chromatography (HPLC); however, the conditions used dissociate the organic acid Al complexes. Humic and fulvic acids are of a variable and ill-defined nature and only limited information exists regarding their binding of Al. This paper reports on fractionation studies of soil solutions, using both SPE and molecular weight cut-off filters, to characterise the DOC components and on the subsequent development of a size exclusion chromatography (SEC) system for the separation of organically complexed Al into different species using a low ionic strength mobile phase at pH 4.2. Model complexes of Al and Cu citrate were used to evaluate chromatographic performance of a Fractogel TSK HW-40(S) column (1×30 cm). For soil solution samples, the column eluate, after passing through a UV detector, was directly coupled to an Inductively Coupled Plasma Atomic Emission Spectrometer (ICPAES) for on-line multi-element detection to characterise DOC and trace metal distribution.Fractionation studies revealed that polysaccharides constituted the major proportion of the DOC which passed the 10000 dalton molecular weight cut-off filter. Analysis of soil solutions from an organically amended soil by the SEC-ICPAES system showed that Al, Fe and Mn eluted as multiple peaks prior to the bed volume, indicating their presence as complexes with organic ligands.     

减磷配施有机肥对水旱轮作紫色水稻土磷素淋失的消减效应

为探索长江流域稻油轮作系统水稻季减少农田磷素流失的最佳施肥模式和有效耕作措施,降低其对长江水质的威胁。采用渗漏池长期田间原位定点试验并结合室内实验分析,研究了化肥配施猪粪有机肥和水稻秸秆还田对土壤磷素淋溶迁移的影响。结果表明在水稻生长期内土壤淋溶水中磷素浓度随时间延长呈逐渐下降的趋势,前期波动幅度大且下降迅速,到55d之后逐步稳定达到平衡。总可溶性磷(TDP)是渗漏水磷素的主要形态。土壤淋溶水中总磷(TP)和总可溶性磷(TDP)含量均表现为优化施肥+猪粪有机肥(MP)优化施肥+秸秆还田(SP)优化施肥(P)优化施肥量磷减20%+猪粪有机肥(MDP)优化施肥量磷减20%+秸秆还田(SDP)不施磷肥(P0)。土壤总磷(TP)淋失负荷在0.295—0.493 kg/hm2之间。施用有机肥提高了淋溶水中的磷素含量,促进了土壤中磷素的淋失,同时显著提高了土壤中有效磷的含量,猪粪有机肥的促进作用比水稻秸秆大。减少化肥施用量有利于降低土壤磷素淋失。在综合考虑农业生产省本增效和控制农田面源污染的情况下,可以采取减量化肥配施有机肥的施肥模式。     

有机酸和维生素对小麦某些生理生化特性的影响

生物试验研究表明,５０μｇ／ｇ的维生素Ｂ１、Ｂ２和Ｂ６混合液及２０μｇ／ｇ鸟囊素、阿魏酸、香草酸、水杨酸和苯甲酸单独或混合浸种可促进小麦早分蘖、多分蘖,提高小麦成穗率和产量,扬花期叶面喷施,可提高旗叶的过氧化物酶活性,降低丙二醛的积累,延缓叶片衰老,提高光合效率。     

超临界下有机酸对稻秆水解糖化的影响

采用间歇式反应器在超临界条件下,以有机酸（甲酸、乙酸和丙酸）为催化剂对稻秆进行水解糖化研究,重点考察反应温度、反应时间、固液比对还原糖产率的影响。实验表明：有机酸的加入有利于稻秆的水解糖化,稻秆水解速率和还原糖产量都有所提高,这种趋势在加入甲酸时最为明显;随着反应时间的延长,还原糖产量会逐渐减少;适当提高固液比有助于增加还原糖产量。稻秆超临界水解糖化的最佳条件：甲酸体积分数3％、固液比4：60（g／mL）、反应温度410℃、反应时间5min,在此条件下,还原糖产量最高,达6．65g／L。     

耕作方式对紫色水稻土有机碳和微生物生物量碳的影响

以位于西南大学的农业部紫色土生态环境重点野外科学观测试验站始于1990年的长期定位试验田为对象,研究了冬水田平作(DP)、水旱轮作(SH)、垄作免耕(LM)及垄作翻耕(LF)等4种耕作方式对紫色水稻土有机碳(SOC)和微生物生物量碳(SMBC)的影响。结果表明,4种耕作方式下SOC和SMBC均呈现出在土壤剖面垂直递减趋势,翻耕栽培下其降低较均匀,而免耕栽培下其富集在表层土壤中。同一土层不同耕作方式间SOC和SMBC的差异在表层最大,随着土壤深度的增加,各处理之间的差异逐渐减小。在0—60 cm剖面中,SOC含量依次为:LM(17.6 g/kg)>DP(13.9 g/kg)>LF(12.5 g/kg)>SH(11.3 g/kg),SOC储量也依次为:LM(158.52 Mg C/hm2)>DP(106.74 Mg C/hm2)>LF(93.11 Mg C/hm2)>SH(88.59 Mg C/hm2),而SMBC含量则依次为:LM(259 mg/kg)>SH(213 mg/kg)>LF(160 mg/kg)>DP(144 mg/kg)。与其它3种耕作方式比较,LM处理显著提高SOC含量和储量以及SMBC含量。对土壤微生物商(SMBC/SOC)进行分析发现,耕作方式对SOC和SMBC的影响程度并不一致。SMBC与SOC、全氮、全磷、全硫、碱解氮、有效磷均呈现极显著正相关(P<0.01),与有效硫呈显著正相关(P<0.05);表明SMBC可以作为表征紫色水稻土土壤肥力的敏感因子。