甜叶菊中9种甜菊醇糖苷积累与其生物合成关键基因表达量的相关性

为了研究甜叶菊中主要甜菊醇糖苷积累特点与其生物合成关键基因表达量的关系,本研究选取3个品种甜叶菊(普赛科3号、中山2号、同心)为试验材料,定期在幼苗期、营养生长期、花蕾期采收植株的上位3对叶片,采用液相色谱-质谱法(LC-MS)检测9种甜菊醇糖苷,即莱宝迪苷A、B、C、D、E、F(RA、RB、RC、RD、RE、RF)、...     

不同基因型甜叶菊光合生理特性与产量品质比较

为了筛选适宜甘肃河西绿洲灌区种植的甜叶菊及高光效育种材料,对引进的14个不同基因型甜叶菊种质资源光合生理特性、产量和甜菊醇糖苷含量进行差异性分析。结果表明,不同基因型甜叶菊整个生育期光合速率(photosynthetic rate,Pn)、蒸腾速率(transpiration rate,Tr)、气孔导度(stomata conductance,Gs)变化均呈"低-高-低"的单峰曲线,且峰值都出现在现蕾期。各次测定表明各基因型间Pn、Tr和Gs与干叶产量、甜菊醇糖苷含量具有一定的相关性。HX-2和ZY-0911两个基因型光合速率和干叶产量均较高,分别较平均值提高9.18%、8.66%和6.41%、9.68%。基因型间甜菊醇糖苷含量差异达到极显著水平(P0.01),BY-0915-2和ZY-0911甜菊醇糖苷含量最高,分别为15.33%和14.30%,较平均增加31.31%和22.49%。综合分析得出,甘肃河西绿洲灌区要引种高光效甜叶菊进行栽培或作为育种亲本时,应首选引自安徽的8(ZY-0911)。     

高效液相色谱法测定栀子苷的含量

建立了一种快速测定栀子及栀子黄中栀子苷含量的高效液相色谱分析方法。采用超声波法提取栀子苷,使用Shimpack HRC-ODS色谱柱(4.6mm×250mm,5μm),以甲醇-水(30:70,V/V)为流动相,采用二极管阵列检测器(检测波长240nm)对栀子苷进行测定。结果表明,采用超声波法提取1.0h可将栀子苷提取完全,在2~24μg/ml范围内栀子苷含量与峰面积呈良好的线性关系,相关系数r=0.9986,精密度和稳定性试验相对标准偏差均小于5%,加样回收率达到98.13%。该测定方法简单、准确、精密度高、重现性好。     

SPE-UPLC-MS/MS测定葡萄酒中的高氯酸盐

为建立前处理简单、定量准确、灵敏度高的葡萄酒中高氯酸盐的快速测定和分析方法,本研究使用酸化甲醇提取葡萄酒样品,最终选择WAX固相萃取柱前处理净化,亲水性聚丙烯滤膜(GHP)过滤,经Phenyl-Hexyl色谱柱分离,以电喷雾离子源(ESI)在多反应监测(MRM)负离子模式下测定葡萄酒中高氯酸盐。结果表明,该方法的检测时间为12 min,检出限和定量限分别为0.15 μg·L^-1和0.5 μg·L^-1,在1、2、5、10和20 μg·L^-1水平的加标回收率介于94.5%~105%之间,相对标准偏差(RSD)小于3.17%。该方法的建立可为葡萄酒生产企业和相关监管部门提供一定的技术支撑。     

60Co-γ辐照结合1-MCP处理对蓝莓贮藏品质的影响

为研究⁶⁰Co-γ辐照结合1-甲基环丙烯(1-MCP)处理对蓝莓贮藏品质的影响,以粉蓝蓝莓为试验材料,对采后蓝莓生理指标、营养指标及相关酶活性进行测定,研究0±0.5℃条件下6种处理(1.5 kGy辐照处理记为A、2.5 kGy辐照处理记为B、1.5 kGy辐照+1 μL·L^-1 1-MCP 处理记为C、2.5 kGy辐照+1 μL·L^-1 1-MCP处理记为D、1 μL·L^-1 1-MCP处理记为E,不进行任何处理记为F)对蓝莓贮藏品质的影响。结果表明,与对照(F)比较,4种处理(A、C、D、E)均能够抑制果实腐烂率的上升和风味指数的下降,延缓果实的生理代谢,更好地保持果实的营养品质和酶活性,而2.5 kGy辐照处理(B)降低了果实的硬度、L^*值、可溶性固形物含量和花色苷含量,加快了果实多聚半乳糖醛酸酶活性的上升。其中,在贮藏80 d时,A、B、C、D、E、F组蓝莓的腐烂率分别为24.94%、38.36%、13.87%、30.78%、22.96%和48.38%。因此,1.5 kGy ⁶⁰Co-γ辐照结合1 μL·L^-1 1-MCP处理蓝莓对果实的贮藏效果最好。本研究结果为蓝莓的贮藏保鲜提供了新思路。     

刺葡萄皮中花色苷的分离纯化与结构鉴定

为研究刺葡萄花色苷的结构及其纯化分离的柱层析法,将刺葡萄色素粗提液依次经大孔树脂HP-20、聚酰胺树脂、葡聚糖凝胶Sephadex LH-20吸附纯化,利用超高效液相色谱三重四级杆飞行时间质谱联用技术对分离所得花色苷进行结构鉴定,并运用荧光光度法探索荧光图谱与花色苷结构的关系。研究发现,聚酰胺树脂对部分花色苷产生了吸附作用,而Sephadex LH-20凝胶能起到较好的分离作用,最终得到3种色素,经鉴定,确定色素I为锦葵素-3,5-O-双葡萄糖苷,通过质谱信息初步确定色素III可能为锦葵素-3,5-O-双葡萄糖苷-香豆酰,色素IV可能为飞燕草素-3-O-芸香糖苷和锦葵素-3-O-芸香糖苷的混合物,经高效液相色谱以归一法计算峰面积,色素I和色素III的纯度分别达到了98.64%、98.33%,得率分别为0.114%和0.076%。研究结果为花色苷的分离及鉴定提供参考。     

大孔吸附树脂一次性分离杜仲叶中杜仲总苷和杜仲黄酮的研究

本文就9种大孔树脂吸附分离杜仲总苷和杜仲黄酮的工艺进行了研究，结果表明：一次性分离杜仲总苷(富含绿原酸、桃叶珊瑚苷等活性物质)和杜仲黄酮的适宜的大孔树脂为XDA-1和X-5。适宜的吸附解吸条件分别为：上柱液pH 6，吸附时间应大于10 h；XDA-1树脂用40%乙醇洗脱杜仲总苷，用80%的乙醇洗脱黄酮；X-5树脂用30%的乙醇洗脱杜仲总苷，用70%的乙醇洗脱黄酮。两种树脂的分离效果分别为：XDA-1分离杜仲总苷(其中绿原酸含量为14.23%，桃叶珊瑚苷含量为7.69%)粗品得率为8.01%，杜仲总黄酮粗品得率为4.76%，含量为15.82%；X-5分离杜仲总苷(其中绿原酸含量为15.39%，桃叶珊瑚苷含量为9.07%)粗品得率为7.35%，杜仲总黄酮粗品得率为5.11%，含量为16.15%。     

体外消化对油茶蒲提取物抑制α-葡萄糖苷酶活性的影响

为探索体外消化对油茶蒲活性成分的影响,本试验以油茶蒲为原料,分析消化前后油茶蒲提取物、水相、正丁醇相、乙酸乙酯相对α-葡萄糖苷酶抑制活性的变化,采用高效液相色谱(HPLC)、分子对接进一步探索油茶蒲中抑制α-葡萄糖苷酶的主要活性物质。结果表明,油茶蒲提取物、乙酸乙酯相、正丁醇相、水相对α-葡萄糖苷酶的IC₅₀为0.27~1.17 μg·mL^-1,经体外模拟消化后,IC₅₀上升至0.34~2.36 μg·mL^-1,其中乙酸乙酯相的活性最强。油茶蒲提取物成分分析结果显示,体外消化能影响各相萃取物的总酚含量,其中乙酸乙酯相的总酚含量最高(44.49 μg·mL ^-1)且在体外消化中具有良好的稳定性。相关性分析结果表明,油茶蒲提取物中有5个化合物与α-葡萄糖苷酶的IC₅₀呈显著相关,其中包括3-O-甲基鞣花酸-4'-O-β-D-吡喃葡萄糖苷(MEAG)。AutoDock分子对接结果也表明,MEAG具有良好的α-葡萄糖苷酶抑制活性,以及最佳的结合能(-7.99 kcal·mol^-1)和估计抑制常数(1.40 μmol·L ^-1), 其主要通过氢键和疏水作用与α-葡萄糖苷酶的氨基酸残基相互作用。本研究结果为开发新型α-葡萄糖苷酶抑制剂以及油茶蒲资源利用提供了数据参考。     

施钾对甜叶菊糖苷跃变期生理指标和产量品质的影响

为提高河西绿洲灌区甜叶菊产量和品质,提升该区甜叶菊市场竞争力。以谱星6号为试验材料,比较了不同施钾水平(K2O 0、60、90、120、150 kg/hm2)对甜叶菊糖苷跃变期生理指标以及产量品质的影响。结果表明,不同施钾处理下,甜叶菊整个糖苷跃变期叶片各生理指标含量达到峰值的时间有所不同,叶片SOD活力、含钾量均在现蕾前5 d达到最高,可溶性蛋白、SPAD值、含氮磷量、总苷含量以及叶片干物质量在现蕾初期达到最高,POD活力、可溶性糖含量在现蕾后5 d达到最高,以上各指标均以施用K2O 120 kg/hm2处理表现最佳。其中总苷含量最高,达128.4 g/kg,较不施钾肥增加14.19%;产量也最高,达7 007.64 kg/hm2,较不施钾肥增加57.24%;净收益达5.26万元/hm2,比不施钾多3.40万元/hm2。通过甜叶菊叶片理化指标与产量品质的相关性分析,以上各生理指标与产量品质均呈现显著或者极显著的正相关关系。在当地甜叶菊生产中施用K2O 120 kg/hm2,且在现蕾初期采摘叶片可获得较好收益。     

外源褪黑素对镉胁迫下豌豆种子萌发、幼苗抗性生理及镉含量的影响

为探究外源褪黑素(MT)对镉胁迫下豌豆种子萌发和幼苗生长影响的生理机制,以豌豆品种中豌6号为试验材料,采用培养皿滤纸法,研究不同浓度外源褪黑素(0、50、100、200、400 μmol·L^-1)对镉胁迫(0、10、100 μmol·L^-1)下豌豆种子萌发、幼苗生长及生理指标的影响。结果表明,镉胁迫显著抑制了豌豆种子萌发及幼苗生长;施用外源褪黑素能够显著提高镉胁迫下豌豆种子的发芽势和发芽率,促进豌豆幼芽和幼根的生长,提高豌豆幼芽的超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,并降低丙二醛(MDA)和镉含量。当Cd²⁺浓度为10 μmol·L^-1时,施用100 μmol·L^-1 MT较未添加MT豌豆的发芽势、发芽率、芽长、根长、芽鲜重和根鲜重分别增加90.47%、82.94%、127.27%、129.47%、131.71%和83.33%,SOD、POD和CAT活性分别提高24.17%、40.50%和76.91%,MDA含量下降57.64%,镉含量下降57.62%。结果表明,适宜浓度的外源褪黑素能够通过提高抗氧化酶活性,降低活性氧的积累,抑制豌豆幼苗对镉的吸收,从而缓解镉胁迫对豌豆幼苗的毒害作用,促进种子萌发及幼苗生长。本研究结果为进一步探索外源褪黑素缓解豌豆镉胁迫的机理提供了一定的理论依据。     

Productivity of two stevia varieties under reduced irrigation and fertilization inputs

A 3-year field study was conducted in central Greece to determine the productivity of two stevia [(Stevia rebaudiana (Bertoni) Bertoni] varieties (‘Morita’ and ‘Candy-stevia’) under normal and reduced irrigation (100% and 75% of the evapotranspiration) and fertilization [1:0.8:1.1 or 1:0.4:0.8 N:P:K ratio in the first year and only N fertilization (100% or 74% of the recommended rate) in the second and third years] inputs. Averaged across years, stevia cv. Morita achieved greater dry leaf yield (3.48 t ha^?1) than the cv. Candy-stevia (2.85 t ha^?1). Irrigation and fertilization inputs did not significantly affect stevia cv. Morita dry leaf and steviol glycosides (stevioside plus rebaudioside-A) yields; however, decreasing irrigation and fertilization caused slight reduction of cv. Candy-stevia yields. In cv. Morita leaves, the concentrations of stevioside and rebaudioside-A ranged from 5.97% to 7.78% and 3.73% to 4.79%, respectively, while the corresponding concentrations in cv. Candy-stevia leaves were 8.21–9.36% and 3.89–6.33%. Conclusively, both stevia varieties could achieve satisfactory dry leaf biomass and steviol glycosides yield, even when grown under reduced irrigation (at 75% of evapotranspiration) and reduced N fertilization (74% of the recommended rate). Thus, stevia could represent an alternative crop to tobacco in the Mediterranean conditions.     

The response of Stevia (Stevia rebaudiana Bertoni) to nitrogen supply under greenhouse condition

Stevia (Stevia rebaudiana Bertoni) is a perennial plant producing some natural sweeteners. Stevia is considered as a new crop in some countries. This study was conducted to find the stevia response to nitrogen fertilizer supply. Different levels of nitrogen (0, 30, 60, 90, 120, and 150 kg/ha from urea source) were used in a greenhouse condition and then the stevia growth and metabolites were assessed under different availability of nitrogen. Results showed that the optimum growth of stevia was obtained by 60 kg/ha nitrogen and more nitrogen supply did not enhance the stevia growth. It was observed that the total steviol glycosides (SVglys) content of Stevia was significantly increased by nitrogen fertilizer application just up to 30 kg/ha, while it decreased by more rates of nitrogen fertilizer. Our result clearly showed that SVglys yield reached to maximum value by application of 60 kg/ha of nitrogen fertilizer. Since the variation of SVglys content and shoot growth of the stevia were compromised by 60 kg/ha nitrogen, it can be concluded that 60 kg/ha of nitrogen fertilizer could be considered as an optimum rate of nitrogen for stevia and could also be recommended for greenhouse conditions.     

Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method

An improved analytical method was developed which may be applied to quality control of stevioside and rebaudioside A contents in dried leaves of Stevia rebaudiana before processing; in a selective sampling program searching for plants of higher yield in diterpene glycosides content; or when a large number of samples are sent to the laboratory for analysis. The procedure developed involves two steps: solvent extraction followed by an isocratic HPLC analysis. The sample, 1 g of dried leaves of S. rebaudiana, is ground and solvent-extracted with EtOH 70% (w/w) in Erlenmeyer flasks by shaking for 30 min in a 70 degrees C water bath. After the extract was cooled, it was filtered and analyzed by HPLC using an NH(2) column (250 x 4.6 mm) and a mixture of acetonitrile/water (80:20, v/v) as mobile phase, pH 5 adjusted with acetic acid. The detection was in the UV range at 210 nm (0.04 AUFS). Quantitation was performed by means of an external standard calibration curve for each analyte which had been obtained from standard solutions of pure stevioside and rebaudioside A. Working under these conditions there were no observed interference effects. The method saves time in sample preparation, and reduces sample handling and chromatographic analysis time, while having little loss of precision [coefficient of variation (CV%) between 1.8% and 3.0%] and recovery [between 98.5% and 100.5%]. The method was applied to 30 samples of S. rebaudiana from Misiones (Northeastern Argentina), and the stevioside content found ranged between 3.78 and 9.75% (weight) whereas Rebaudioside A content ranged between 1.62 and 7.27% (weight).     

The Effect of the Plant Age and Growth Period on the Nutritional Substance,Chlorophyll and Steviol Glycoside Rates in Stevia (Stevia Rebaudiana Bertoni) Leaves

Stevia (Stevia rebaudiana Bertoni) is a perennial and subshrub herb of the Asteraceae family, which originates from South America. Stevia has long been used as a sweetener and herbal medicine throughout the world. The most important feature of stevia is its sweetness with zero calories. This natural feature of Stevia is of utmost importance to everyone, especially those with diabetes and those who diet. In this study, samples were taken from Stevia rebaudiana plants of different ages (2, 3 and 4 years) in different growth periods (H1: 1 July, H2: 1 August, H3: 1 September and H4: 1 October) and an evaluation was made on the nutritional content values, the chlorophyll and sugar rates found in these samples according to the age and growth period of the plant. It was determined that the age of the plant and the harvesting period had an effect on the macro-micro nutritional elements, and chlorophyll and steviol glycoside rates contained in the stevia plant. In addition, as a result of correlation analysis, significant correlations were determined between the parameters measured in the plant. The results of the study demonstrated that in the cultivation of stevia, the best values were obtained from plants that were 3 years old and harvested in July.     

The effect of different nitrogen levels on yield and quality of stevia (Stevia rebaudiana bert.)

In the present work, the efficiency of different nitrogen doses (0, 50, 100, 150, and 200 kg ha^?1) on growth, yield, and quality of stevia (Stevia rebaudiana Bert.) was investigated in 2011–2013. The study was conducted in Antalya located in the Mediterranean Region of Turkey. Terra rossa type soil (LVx, FAO) characteristics of the experimental field were clay loam, with high amounts of lime (33,9%) and slightly alkaline (pH 7.7). The experiment was carried out in randomized block design with four replications. All the results were summarized as mean of three years. The highest fresh and dry biomass yields (26.75 t ha^?1 and 7.5 ha^?1, respectively) were obtained from 150 kg ha^?1 N dose and followed by 100 kg ha^?1 N dose (26.29 t ha^?1 and 7.24 ha^?1, respectively). Whereas the highest fresh and dry leaf yields (13.27 t ha^?1 and 3.82 t ha^?1, respectively) were realized in 100 kg ha^?1 N dose. Actually, all nitrogen doses gave higher biomass and leaf yields compared to the control. On the hand, major steviol glycosides (stevioside and rebaudioside A) in the leaf were not influenced by nitrogen levels. In conclusion, 100 kg ha^?1 N dose was found to be suitable for cultivation of stevia under field conditions.     

Photostability of rebaudioside A and stevioside in beverages

The Coca-Cola Company and Cargill, Inc. have initiated the development and commercialization of the Stevia rebaudiana (Bertoni) derived sweetener rebaudioside A. Efforts were focused on high purity rebaudioside A (>97% by HPLC), commonly known as rebiana. In the course of the development program, extensive stability studies were carried out on rebiana, all supporting good stability for use in all food and beverage applications, including conditions where rebiana-sweetened beverages were exposed to light. Our findings on rebiana light stability refute those of an earlier study that suggested rebaudioside A to be unstable to sunlight exposure, while the structurally homologous stevioside is stable. We replicated the earlier study and found no significant photodegradation for either rebaudioside A or stevioside.     

Preparative separation and purification of rebaudioside a from steviol glycosides using mixed-mode macroporous adsorption resins

Preparative separation and purification of rebaudioside A from steviol glycosides using mixed-mode macroporous adsorption resins (MARs) were systematically investigated. Mixed-mode MARs were prepared by a physical blending method. By evaluation of the adsorption/desorption ratio and adsorption/desorption capacity of mixed-mode MARs with different proportions toward RA and ST, the mixed-mode MAR 18 was chosen as the optimum strategy. On the basis of the static tests, it was found that the experimental data fitted best to the pseudosecond-order kinetics and Temkin-Pyzhev isotherm. Furthermore, the dynamic adsorption/desorption experiments were performed on the mini column packed with mixed-mode MAR 18. After one run treatment, the purity of rebaudioside A in purified product increased from 40.77 to 60.53%, with a yield rate of 38.73% (W/W), and that in residual product decreased from 40.77 to 36.17%, with a recovery yield of 57.61% (W/W). The total recovery yield reached 96.34% (W/W). The results showed that this method could be utilized in large-scale production of rebaudioside A from steviol glycosides in industry.     

Metabolism of stevioside and rebaudioside A from Stevia rebaudiana extracts by human microflora

Stevia rebaudiana standardized extracts (SSEs) are used as natural sweeteners or dietary supplements in different countries for their content of stevioside or rebaudioside A. These compounds possess up to 250 times the sweetness intensity of sucrose, and they are noncaloric and noncariogenic sweeteners. The aim of this study was to investigate the in vitro transformation of stevioside and rebaudioside A after incubation with human microflora, the influence of these sweeteners on human microbial fecal community and which specific groups metabolize preferentially stevioside and rebaudioside A. The experiments were carried out under strict anaerobic conditions in batch cultures inoculated with mixed fecal bacteria from volunteers. The hydrolysis was monitored by HPLC coupled to photodiode array and mass spectrometric detectors. Isolated bacterial strains from fecal materials incubated in selective broths were added to stevioside and rebaudioside A. These sweeteners were completely hydrolyzed to their aglycon steviol in 10 and 24 h, respectively. Interestingly, the human intestinal microflora was not able to degrade steviol. Furthermore, stevioside and rebaudioside A did not significantly influence the composition of fecal cultures; among the selected intestinal groups, bacteroides were the most efficient in hydrolyzing Stevia sweeteners to steviol.