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Carotenoid Extraction and Quantification from Capsicum annuum
甜椒中的类胡萝卜素提取和定量测定   

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Abstract

Carotenoids are ubiquitous pigments that play key roles in photosynthesis and also accumulate to high levels in fruit and flowers. Specific carotenoids play essential roles in human health as these compounds are precursors for Vitamin A; other specific carotenoids are important sources of macular pigments and all carotenoids are important anti-oxidants. Accurate determination of the composition and concentration of this complex set of natural products is therefore important in many different scientific areas. One of the richest sources of these compounds is the fruit of Capsicum; these red, yellow and orange fruit accumulate multiple carotenes and xanthophylls. This report describes the detailed method for the extraction and quantification of specific carotenes and xanthophylls.

Materials and Reagents

  1. Fresh Capsicum fruit (dissected into pericarp tissue, frozen at -80 °C or lyophilized pericarp tissue)
  2. N2 gas
  3. CHCl3 (HPLC grade) (Sigma-Aldrich, catalog number: 472476 )
  4. 2-propanol (HPLC grade) (Sigma-Aldrich, catalog number: 34863 )
  5. Methanol (HPLC grade) (Sigma-Aldrich, catalog number: 179337 )
  6. KOH (Sigma-Aldrich, catalog number: P-1767 )
  7. Methyl-t-butyl ether (MTBE) (HPLC grade) (Sigma-Aldrich, catalog number: 34875 )
  8. β-carotene (Sigma-Aldrich, catalog number: C4582 )
  9. Lutein (Sigma-Aldrich, catalog number: 07168 )
  10. Lycopene (Sigma-Aldrich, catalog number: 75051 )
  11. Antheraxanthin (CaroteNature, catalog number: 0231 )
  12. Capsanthin (CaroteNature, catalog number: 0335 )
  13. Capsorubin (CaroteNature, catalog number: 0413 )
  14. Zeaxanthin (CaroteNature, catalog number: 0119 )
  15. Violaxanthin (CaroteNature, catalog number: 0259 )
  16. β-cryptoxanthin (CaroteNature, catalog number: 0055 )
  17. Methanolic KOH (add solid KOH crystals to methanol until the solution is saturated)

Equipment

  1. Farberware soft grips (Food Chopper, model: 83427-93 )
  2. Polytron generator (Polytron Technologies, model: PT 10-35 )
  3. Temperature block (with 1.8 ml microfuge tube rack) (Thermolyne Dri-Bath, model: DB-17615 )
  4. HPLC system equipped with a photodiode array detector and YMC carotenoid column (4.6 x 250 mm) (Waters)
  5. Bath sonicator (Branson Ultrasonic Cleaner, model: 2510 )
  6. Centrifuge (capable of 1,000 x g, 5 min at 4 °C)
  7. Microcentrifuge
  8. UV/Vis spectrophotometer
  9. Vortex mixer
  10. Fume hood
  11. Nitrogen evaporator (Organomation Associates, model: N-EVAP-112 )

Procedure

  1. Extraction
    1. If using dried (lyophilized) pericarp samples use ~0.25 to 0.5 g for each extraction, grind samples to a powder in a mortar and pestle. If using fresh frozen pericarp samples, use ~2.5 to 5 g for each extraction. Chop the frozen pericarp into small pieces measuring ~4 mm2 using a manual food chopper. See Figure 1.


      Figure 1. Processing of fresh Capsicum pericarp. A. Pericarp samples are placed in lower chamber of food chopper. B. Samples are chopped to very small pieces C.

    2. Place samples in a 50 ml plastic tube, add ~25 ml CHCl3 and use the polytron to thoroughly homogenize the sample, ~20 to 30 sec. See Figure 2.


      Figure 2. Homogenization of fresh Capsicum pericarp. A. Chopped pericarp in a 40 or 50 ml tube with 25 ml CHCl3. B. Sample is homogenized with polytron for 20 to 30 sec. C. Fresh fruit sample is completely dispersed in CHCl3.

    3. Allow the homogenized tissue to sit in the CHCl3 for 30 min with occasional mixing by either a vortex mixer or by a bath sonicator.
    4. If using frozen fresh samples, centrifuge the sample to separate the CHCl3 phase from the aqueous phase (upper layer); 1,000 x g, 5 min at 4 °C. Keep the CHCl3 phase. If using dried samples, this step is not performed.
    5. Filter the CHCl3 extract using Whatman 1 (or equivalent) filter paper and a vacuum filter.
    6. Transfer filtrate to a ~40 ml glass amber vial and evaporate the solvent using a stream of N2 gas and gentle heating (50 °C). A nitrogen evaporator works well for this step.
    7. Resuspend the dried sample in 1.1 ml of 2-propanol. Bath sonication is advised to help resuspend the material.
    8. Material can be stored at -20 or -80 °C for several weeks.
    9. We keep the extracted sample out of bright light as much as possible. During the time the sample is on the nitrogen evaporator in the fume hood, we keep the lights off in the hood; we store the samples in amber vials and we keep the vials in the dark as much as possible.

  2. Saponification
    1. In a standard microfuge tube, combine 0.5 ml of the 2-propanol extract with 0.1 ml of methanolic KOH, in a 1.8 ml microfuge tube.
    2. Mix well by pipetting, then incubate for 30 min at 50 °C.
    3. Cool the sample by immersion in an ice bath for ~2 min, then add 0.5 ml of water.
    4. Add 0.4 ml of CHCl3. Vortex vigorously.
    5. Centrifuge for 1-2 min at top speed (10,000 to 12,000 x g) in a microcentrifuge to resolve the phases. Lower centrifugal forces will also resolve the CHCl3 and aqueous phases.
    6. Remove (discard) the upper phase; recover the lower phase.

  3. HPLC Analysis
    1. Sample volume for the HPLC is typically 10 to 30 µl. The volume used depends on the concentration of the carotenoid preparation. Determine the OD 450 nm for a 1:100 dilution of the carotenoid sample. If the A450 of the diluted sample is 0.5 to 1.0, then inject 10 µl of concentrated sample onto the HPLC. If the A450 is 0.25 to 0.5, then inject 20 µl. If the A450 is 0.1 to 0.25, then inject 30 µl. If the absorbance is above 1.0, then dilute the sample appropriately. If the absorbance is below 0.1, then concentrate the sample appropriately (e.g. evaporate some of the solvent under a stream of N2 gas).
    2. Chromatography is conducted on an HPLC system with a photodiode array detector using the full visible spectrum (400 - 700 nm) with monitoring employed at 450 nm. Column is a YMC carotenoid column, 250 mm x 4.6 mm, S = 5 µm.
    3. Flow rate, 2 ml/min, with a linear gradient for 0 to 30 min as below:
      1. Solvent A: methanol: MTBE: water:: 81:15:4 (vol: vol: vol)
      2. Solvent B: MTBE: methanol: water:: 88:8:4 (vol: vol: vol)

        Time (min)         
        A           
        B
        0      
        100%            
        0%
        30        
        33%       
        67%

        At 31 min we start washing/reconditioning the column:    
        31         0%        100        
        34         0%        100        
        35        100%         0        
        39        100%         0   

  4. Calibration curve
    1. Calibration curves were generated at 450 nm with the reference standards: β-carotene, lutein, lycopene, capsanthin, capsorubin, zeaxanthin, antheraxanthin, violaxanthin and β-cryptoxanthin.
    2. Stock solutions at 1 mg/ml are prepared in CHCl3 (hexane can also be used).
    3. Samples representing the range of 10 ng to 3 µg are prepared by dilution of the stock solution and independently analyzed by HPLC as described above. The retention time of the single peak and the area under the curve is recorded and plotted against the amount of the carotenoid injected.
    4. In our lab most carotenoids have a linear response at 450 nm for detection between 10 ng to 3 µg, with R2 values of 0.999.
    5. The formulas we used to convert HPLC peak area for the specific carotenoids into concentrations expressed as ng/g tissue are:
      Antheraxanthin (ng/g tissue): (peak area - 1316.8)/5,816.9/ injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      β-carotene (ng/g tissue): (peak area - 55,271)/4,253.5/ injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      β-cryptoxanthin (ng/g tissue): (41,437 + peak area)/6,758.4/injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      Capsanthin (ng/g tissue): (28,266 + peak area)/4,489.5 injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      Capsorubin (ng/g tissue): (26,864 + peak area)/5,541.4/injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      Lutein (ng/g tissue): (peak area - 1,428.9)/6312/injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      Lycopene (ng/g tissue): (28,266 + peak area)/4,489.5/injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      Violaxanthin (ng/g tissue): (28,616 + peak area)/7,453.4/injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      Zeaxanthin (ng/g tissue): (92,586 + peak area)/7,220.6/injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
      Total carotenoids (ng/g tissue): (peak area of entire chromatogram - 55,271)/4,253.5/ injected volume (μl) x sample dilution x undiluted sample volume (μl)/tissue mass (grams)
    6. For plant sample characterization, triplicate independent extractions are performed on each genotype and the average concentration for each carotenoid is calculated following independent HPLC analyses using the specific calibration curve to determine the abundance of each respective peak (e.g. peaks 1-7 in Figure 3). We use the calibration curve for β-carotene to estimate the abundance of unassigned carotenoid peaks, those that do not elute at the retention time of a reference standard (e.g. peaks a-g in Figure 3).


      Figure 3. HPLC separation of Capsicum annum pericarp extracts. Saponifed carotenoid extracts from mature fruit pericarp from three Capsicum varieties, Costeño Amarillo, Costeño Red and NuMex Garnet. Known peaks are: 1, violaxanthin; 2, lutein; 3, capsanthin; 4, zeaxanthin; 5, β -cryptoxanthin; 6, α-carotene; 7, β-carotene. Carotenoid peaks that are not identified are labeled with letters, a-g. Absorption spectra from the photodiode array for the unknown peaks are provided on the right. HPLC conditions as described.

Representative data

  1. Examples of typical chromatograms are shown in Figure 3. The top sample (green line) is from an orange colored Capsicum fruit pericarp sample, while the two lower traces (red and blue lines) represent the carotenoids in red colored Capsicum fruit samples.
  2. The coefficient of variation in our lab for this protocol is <5.0% when we are re-running samples from the same saponified fruit extract. The biological variability for carotenoid abundance in Capsicum fruit can be much higher, ~20% variation. We routinely sample three to five independent samples of a particular fruit stage and/or genotype to capture the biological variability in these samples.

Notes

  1. To help reduce the biological variability in the samples we prefer to use dried or lyophilized plant tissue. This reduces the variability in water weight that occurs when you allocate fruit samples for extraction. Depending on the fruit type, there can be quite a bit of difference in sample weight depending on fruit maturity stage and the thinness of the exocarp.
  2. It is important to run calibration standards regularly, to ensure that the chromatography conditions are kept constant. The less expensive carotenoid standards, β-carotene and lutein, can be used to monitor the chromatography conditions. The frequency of these calibration runs depends on the work load of the research program, but checking that peaks are eluting at their appropriate retention times should be checked at least bi-monthly or at the start of any new analysis project.
  3. We have not tested a wide range of HPLC columns for the separation of the carotenoids, we do know that the C30 YMC carotenoid column works very well, while a C18 column will not resolve the carotenoids at least with our solvent system.

Recipes

All of the solutions are made up as described above using standard methods. There are no unusual aspects to the preparation of these solutions. All solvents are HPLC grade; the water is deionized water. We have not found it necessary to degas or to vacuum filter the solvents prior to use on the HPLC.
Solvent A: methanol: MTBE: water:: 81:15:4 (vol: vol: vol)
Solvent B: MTBE: methanol: water:: 88:8:4 (vol: vol: vol)

Acknowledgments

This protocol was adapted from previous work in our laboratory; see Rodriguez-Uribe et al. (2012). This work was supported in part by the NM Agricultural Experiment Station, USDA CSREES grant 2009-34604-19939 and USDA NIFA 2010-34604-20886.

References

  1. Kilcrease, J., Collins, A. M., Richins, R. D., Timlin, J. A., O’Connell, M. A. (2013). Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit. Plant J 76: 1074-1083.
  2. Rodriguez-Uribe, L., Guzman, I., Rajapakse, W., Richins, R. D. and O'Connell, M. A. (2012). Carotenoid accumulation in orange-pigmented Capsicum annuum fruit, regulated at multiple levels. J Exp Bot 63(1): 517-526.

简介

类胡萝卜素是在光合作用中起关键作用的普遍存在的颜料,并且在果实和花中积累到高水平。 特定类胡萝卜素在人类健康中起着重要作用,因为这些化合物是维生素A的前体; 其他特定类胡萝卜素是黄斑色素的重要来源,并且所有类胡萝卜素是重要的抗氧化剂。 因此,在许多不同的科学领域中,精确测定天然产物的复杂集合的组成和浓度是重要的。 这些化合物最丰富的来源之一是辣椒的果实。 这些红色,黄色和橙色果实积累多个胡萝卜素和叶黄素。 本报告描述了特定胡萝卜素和叶黄素的提取和定量的详细方法。

材料和试剂

  1. 新鲜的辣椒水果(解剖成果皮组织,在-80℃冷冻或冻干的果皮组织)
  2. N <2>气体
  3. CHCl 3(HPLC级)(Sigma-Aldrich,目录号:472476)
  4. 2-丙醇(HPLC级)(Sigma-Aldrich,目录号:34863)
  5. 甲醇(HPLC级)(Sigma-Aldrich,目录号:179337)
  6. KOH(Sigma-Aldrich,目录号:P-1767)
  7. 甲基叔丁基醚(MTBE)(HPLC级)(Sigma-Aldrich,目录号:34875)
  8. β-胡萝卜素(Sigma-Aldrich,目录号:C4582)
  9. 叶黄素(Sigma-Aldrich,目录号:07168)
  10. 番茄红素(Sigma-Aldrich,目录号:75051)
  11. 花青素(CaroteNature,目录号:0231)
  12. 辣椒红(CaroteNature,目录号:0335)
  13. Capsorubin(CaroteNature,目录号:0413)
  14. 玉米黄质(CaroteNature,目录号:0119)
  15. 紫黄质(CaroteNature,目录号:0259)
  16. β-隐黄素(CaroteNature,目录号:0055)
  17. 甲醇KOH(将固体KOH晶体加入到甲醇中,直到溶液饱和)

设备

  1. Farberware软握把(Food Chopper,型号:83427-93)
  2. Polytron发生器(Polytron Technologies,型号:PT 10-35)
  3. 温度块(具有1.8ml微量离心管架)(Thermolyne Dri-Bath,型号:DB-17615)
  4. 装备有光电二极管阵列检测器和YMC类胡萝卜素柱(4.6×250mm)(Waters)的HPLC系统
  5. 浴超声波仪(Branson超声波清洗机,型号:2510)
  6. 离心机(能够1,000×g,在4℃下5分钟)
  7. 微量离心机
  8. UV/Vis分光光度计
  9. 涡流搅拌器
  10. 通风橱
  11. 氮气蒸发器(Organomation Associates,型号:N-EVAP-112)

程序

  1. 萃取
    1. 如果使用干燥(冻干)果皮样品,使用〜0.25至0.5g 每次提取,用研钵和研杵将样品研磨成粉末。 如果 使用新鲜冷冻的果皮样品,每个使用约2.5至5g 萃取。 使用手动食品剁碎机将冷冻的果皮切成测量〜4mm 2的小块。 见图1.


      图1.处理 新鲜的辣椒。 A.将果皮样品置于下室中   的食物斩波器。 B.样品切成非常小的块C.
    2. 将样品置于50ml塑料管中,加入〜25ml CHCl 3并使用 polytron彻底匀化样品,〜20〜30秒。 参见图 2.


      图2.新鲜辣椒的均质化。 A。 切碎的 果皮在具有25ml CHCl 3的40或50ml管中。 B.样品是 用polytron均质化20至30秒。 C.新鲜水果样品 完全分散在CHCl 3中。

    3. 允许均质组织   置于CHCl 3中30分钟,偶尔混合 涡流混合器或浴槽超声波仪
    4. 如果使用冷冻新鲜 样品,离心样品以从CHCl 3相分离CHCl 3 水相(上层); 1,000×g/g,在4℃下5分钟。 保持CHCl <3>相。 如果使用干燥样品,则不执行此步骤。
    5. 使用Whatman 1(或等效的)滤纸和真空过滤器过滤CHCl 3萃取物。
    6. 转移滤液到〜40 ml玻璃琥珀色小瓶,蒸发 溶剂,使用N 2气体流并温和加热(50℃)。 氮 蒸发器在此步骤中工作良好
    7. 将干燥的样品重悬在1.1ml 2-丙醇中。 浴超声处理建议帮助重悬材料。
    8. 材料可以在-20或-80°C下储存数周
    9. 我们尽可能保持提取的样品在明亮的光线之外。 在样品在烟气中的氮气蒸发器上的时间期间 罩,我们保持灯罩在敞篷; 我们将样品存储在琥珀色中 小瓶,我们尽可能保持在黑暗中的小瓶。

  2. 皂化
    1. 在标准微量离心管中,合并0.5毫升2-丙醇提取物 用0.1ml甲醇KOH,在1.8ml微量离心管中
    2. 用移液器充分混合,然后在50℃下孵育30分钟
    3. 通过在冰浴中浸泡约2分钟冷却样品,然后加入0.5ml水
    4. 加入0.4ml CHCl 3。 大力涡旋
    5. 以最高速度(10,000至12,000×g/min)离心1-2分钟 微量离心机以分离相。 较低的离心力将 也分解CHCl 3和水相
    6. 移除(丢弃)上层相; 恢复下相。

  3. HPLC分析
    1. HPLC的样品体积通常为10至30μl。 使用的体积 取决于类胡萝卜素制剂的浓度。确定 对于类胡萝卜素样品的1:100稀释的OD 450nm。如果 稀释样品的a <450>为0.5至1.0,然后注入10μl 浓缩样品到HPLC上。如果A 450是0.25到0.5,则 注射20μl。如果A <450> 为0.1至0.25,则注射30μl。如果 吸光度高于1.0,然后适当稀释样品。如果 吸光度低于0.1,然后适当地浓缩样品(例如在N 2气体流下蒸发一些溶剂)。
    2. 在具有光电二极管阵列的HPLC系统上进行色谱法 检测器使用完全可见光谱(400 - 700 nm)监测 。柱是YMC类胡萝卜素柱,250mm×4.6mm,S  =5μm。
    3. 流速,2ml/min,用线性梯度洗脱0至30分钟,如下:
      1. 溶剂A:甲醇:MTBE:水= 81:15:4(体积:体积:体积)
      2. 溶剂B:MTBE:甲醇:水= 88:8:4(体积:体积:体积)
        时间(分钟)        
        A           
        B
        0      
        100%            
        0%
        30        
        33%       
        67%

        31分钟后,我们开始清洗/翻新色谱柱:    
        31         0%         100        
        34         0%         100        
        35         100%         0        
        39         100%         0   

  4. 校准曲线
    1. 使用参考在450nm处产生校准曲线 标准:β-胡萝卜素,叶黄素,番茄红素,辣椒红,辣椒红素, 玉米黄质,花青素,紫黄质和β-隐黄质。
    2. 在CHCl 3中制备1mg/ml的储备溶液(也可以使用己烷)。
    3. 通过制备代表10ng至3μg范围的样品 稀释储备溶液并通过HPLC独立分析 如上所述。 单峰的保留时间和面积 记录并绘制曲线下的量 类胡萝卜素注射。
    4. 在我们的实验室中,大多数类胡萝卜素具有线性   在450nm处的反应在10ng至3μg之间检测,具有R 2 2值 为0.999。
    5. 我们用于转换HPLC峰面积的公式 将特定的类胡萝卜素浓度表示为ng/g组织 是:
      花青素(ng/g组织):(峰面积-1316.8)/5,816.9/ 注射体积(μl)x样品稀释x未稀释的样品体积 (μl)/组织质量(克)
      β-胡萝卜素(ng/g组织):(峰面积 - 55,271)/4,253.5/注射体积(μl)x样品稀释度x未稀释 样品体积(μl)/组织质量(克)
      β-隐黄质(ng/g组织):   (41,437 +峰面积)/6,758.4/注射体积(μl)x样品稀释x 未稀释的样品体积(μl)/组织质量(克)
      辣椒红(ng/g 组织):(28,266+峰面积)/4,489.5注射体积(μl)×样品 稀释x未稀释的样品体积(μl)/组织质量(克)
      Capsorubin   (ng/g组织):(26,864 +峰面积)/5,541.4/注射体积(μl)x 样品稀释x未稀释的样品体积(μl)/组织质量(克)
      叶黄素   (ng/g组织):(峰面积-1,428.9)/6312 /注射体积(μl)×样品   稀释x未稀释的样品体积(μl)/组织质量(克)
      番茄红素   (ng/g组织):(28,266+峰面积)/4,489.5/注射体积(μl)x 样品稀释x未稀释的样品体积(μl)/组织质量(克)
      紫草黄   (ng/g组织):(28,616 +峰面积)/7,453.4/注射体积(μl)x 样品稀释x未稀释的样品体积(μl)/组织质量(克)
      玉米黄质   (ng/g组织):(92,586 +峰面积)/7,220.6/注射体积(μl)x 样品稀释x未稀释的样品体积(μl)/组织质量(克)
      总   类胡萝卜素(ng/g组织):(整个色谱峰面积 - 55,271)/4,253.5/注射体积(μl)x样品稀释度x未稀释 样品体积(μl)/组织质量(克)
    6. 植物样品 表征,一式三份独立提取 每个基因型和每个类胡萝卜素的平均浓度 使用具体的独立HPLC分析计算 校准曲线以确定每个相应峰的丰度 (例如图3中的峰1-7)。我们使用的校准曲线 β-胡萝卜素来估计未分配的类胡萝卜素峰的丰度, 在参考标准的保留时间不洗脱的那些 (例如图3中的峰a-g)。


      图3. 辣椒的果皮提取物的HPLC分离。皂化的类胡萝卜素提取物 来自三个辣椒品种的成熟果皮,CosteñoAmarillo, Costeño红和NuMex石榴石。已知的峰是:1,紫黄质;如图2所示, 叶黄素3,辣椒红; 4,玉米黄质; 5,β-隐黄质;如图6所示, α-胡萝卜素7,β-胡萝卜素。未鉴定的类胡萝卜素峰 标有字母,a-g。光电二极管阵列的吸收光谱 为未知峰提供在右边。 HPLC条件为 描述。

代表数据

  1. 典型的色谱图的实例显示在图3中。顶部样品(绿色线)来自橙色的辣椒果皮果皮样品,而两个下部迹线(红色和蓝色线)代表红色的类胡萝卜素 着色的辣椒水果样品。
  2. 当我们从相同的皂化的水果提取物重新运行样品时,本方案的实验室的变异系数为<5.0%。 辣椒果实中类胡萝卜素丰度的生物变异性可以高得多,约20%变异。 我们常规地对特定水果阶段和/或基因型的3至5个独立样本进行采样,以捕获这些样本中的生物变异性。

笔记

  1. 为了帮助减少样品中的生物变异性,我们优选使用干燥或冻干的植物组织。这减少了当您分配水果样品进行提取时发生的水重量的变异性。根据果实类型,样品重量可能有相当多的差异,取决于果实成熟阶段和外果皮的厚度。
  2. 定期运行校准标样是重要的,以确保色谱条件保持恒定。较便宜的类胡萝卜素标准品,β-胡萝卜素和叶黄素可用于监测色谱条件。这些校准运行的频率取决于研究项目的工作负荷,但是应该至少每两个月或在任何新的分析项目开始时检查峰值是否在适当的保留时间内洗脱。
  3. 我们没有测试大量的HPLC色谱柱用于分离类胡萝卜素,我们知道C30 YMC类胡萝卜素柱效果非常好,而C18色谱柱至少在我们的溶剂系统中不能分离出类胡萝卜素。

食谱

所有溶液如上所述使用标准方法制备。这些解决方案的准备没有异常的方面。所有溶剂均为HPLC级;水是去离子水。在HPLC上使用前,我们没有发现有必要对溶剂进行脱气或真空过滤 溶剂A:甲醇:MTBE:水= 81:15:4(体积:体积:体积) 溶剂B:MTBE:甲醇:水= 88:8:4(体积:体积:体积)

致谢

该协议改编自我们实验室以前的工作;参见Rodriguez-Uribe等人(2012)。这项工作部分由NM农业实验站,USDA CSREES批准2009-34604-19939和USDA NIFA 2010-34604-20886支持。

参考文献

  1. Kilcrease,J.,Collins,A.M.,Richins,R.D.,Timlin,J.A.,O'Connell,M.A。(2013)。 多种显微镜方法证明了在不同的辣椒中的色质体结构和类胡萝卜素组成之间的联系 fruit。 Plant J 76:1074-1083。
  2. Rodriguez-Uribe,L.,Guzman,I.,Rajapakse,W.,Richins,R.D.and O'Connell,M.A。(2012)。 橙色色素的辣椒果实中的类胡萝卜素积累,受多个水平调节 。 J Exp Bot 63(1):517-526。
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引用:Richins, R. D., Kilcrease, J., Rodgriguez-Uribe, L. and O’Connell, M. A. (2014). Carotenoid Extraction and Quantification from Capsicum annuum. Bio-protocol 4(19): e1256. DOI: 10.21769/BioProtoc.1256.
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