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Hydroxycinnamic acids, such as p-coumaric acid and ferulic acid, are a major class of compounds derived from the phenylpropanoid pathway. These compounds are widely conserved in plants and primarily accumulate in the secondary cell wall. They serve as important structural components that contribute to the overall strength and rigidity of plant cell walls and are also potent antioxidants valued for nutritional consumption. This protocol describes a method for analyzing hydroxycinnamic acids that are released after incubation under alkaline conditions.

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Cell Wall-bound p-Coumaric and Ferulic Acid Analysis
细胞壁结合的p-香豆和阿魏酸的分析

生物化学 > 其它化合物 >
作者: Nickolas Anderson
Nickolas AndersonAffiliation: Heartland Plant Innovations, Manhattan, USA
For correspondence: nanderson@heartlandinnovations.com
Bio-protocol author page: a3575
Vol 6, Iss 19, 10/5/2016, 1057 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1955

[Abstract] Hydroxycinnamic acids, such as p-coumaric acid and ferulic acid, are a major class of compounds derived from the phenylpropanoid pathway. These compounds are widely conserved in plants and primarily accumulate in the secondary cell wall. They serve as important structural components that contribute to the overall strength and rigidity of plant cell walls and are also potent antioxidants valued for nutritional consumption. This protocol describes a method for analyzing hydroxycinnamic acids that are released after incubation under alkaline conditions.
Keywords: Cell wall(细胞壁), P-coumaric acid(香豆酸), Ferulic acid(阿魏酸)

[Abstract] 羟基肉桂酸例如对 - 香豆酸和阿魏酸是衍生自苯丙素途径的一类主要化合物。 这些化合物在植物中广泛保守,并且主要累积在继发性细胞壁中。 它们作为重要的结构组分,有助于植物细胞壁的整体强度和刚性,并且是有价值的营养消耗的有效抗氧化剂。 该方案描述了用于分析在碱性条件下孵育后释放的羟基肉桂酸的方法。

Materials and Reagents

  1. 50 ml conical tubes (MIDSCI, catalog number: C50B )
  2. 2 ml microfuge tubes (MIDSCI, catalog number: AVX-T-20-C )
  3. Arabidopsis inflorescence tissue
  4. Liquid nitrogen
  5. Ethanol, pure (Sigma-Aldrich, catalog number: E7023 )
  6. Acetone (Sigma-Aldrich, catalog number: 320110 )
  7. 1 N NaOH (Sigma-Aldrich, catalog number: S8045 )
  8. 3,4,5-trimethoxycinnamic acid (Sigma-Aldrich, catalog number: T70408 )
  9. HCl (concentrated hydrochloric acid) (36-38%) (Sigma-Aldrich, catalog number: H1758 )
  10. Ethyl acetate (Sigma-Aldrich, catalog number: 270989 )
  11. Acetonitrile (Sigma-Aldrich, catalog number: 271004 )
  12. 0.1% formic acid (Sigma-Aldrich, catalog number: 94318 )
  13. p-coumaric acid (Sigma-Aldrich, catalog number: C9008 )
  14. Ferulic acid (Sigma-Aldrich, catalog number: 128708 )
  15. 50% methanol (Sigma-Aldrich, catalog number: 34860 )

Equipment

  1. Mortar and pestle
  2. Plastic spoon (appropriate for handling small volumes)
  3. Shaking incubator, 37 °C
  4. Centrifuge with a rotor that can accommodate 50 ml conical tubes and can reach 10,000 x g
  5. Microcentrifuge
  6. Vortex
  7. Drying oven, 70 °C
  8. Water bath, 65 °C
  9. Chemical scoop (appropriate for handling small volumes)
  10. Micropipette and tips (Mettler-Toledo, catalog number: L-STARTXLS+ )
  11. Repeating pipettor with adaptor syringe for dispensing 200 µl (Mettler-Toledo, model: AR-E1 )
  12. Speed Vac
  13. Analytical balance
  14. HPLC system with UV detector and autosampler (Shimadzu prominence modular HPLC: system controller CBM-20A, solvent delivery unit LC-20A, auto-sampler SIL-20A, column oven CTO-20A, UV-VIS detector SPD-20A)
  15. HPLC glass vials (VWR, catalog number: 89220-128 )
  16. Shim-pack XR-ODS column (column dimensions 3.0 x 75 mm, bead size 2.2 µm) (Shimadzu, model: Shim-pack XR-ODS)

Procedure

  1. Triplicate samples from three independent biological populations should first be washed to remove soluble metabolites. About 1 g of fresh Arabidopsis inflorescence tissue, stripped of leaves and seeds, was used for the analysis described in Anderson et al. (2015); however, this method can be used to analyze all plant tissues. Briefly, this process includes:
    1. Grind the tissue to powder form in liquid nitrogen using a mortar and pestle.
    2. Transfer the ground tissue to 50 ml conical tubes using a plastic spoon, a larger volume container may be needed depending on the amount of tissue used. The transfer of tissue becomes more cumbersome if the tissue is allowed to thaw. Immediate transfer of tissue is recommended.
    3. Wash the ground tissue using 40 ml 80% ethanol, allowing the mixture to incubate at 65 °C for 1 h with thorough mixing by vortex every 15 min. After 1 h, centrifuge the mixture for 10 min at 10,000 x g. Decant the supernatant being careful to not disturb the pelleted tissue.
    4. Repeat the washing, in 1 h intervals, 4 to 6 times until the solvent is clear (please see Figure 1).
      Note: Other plant tissues, such as leaves or roots, may require less or more washes.
    5. Wash once using 20 ml acetone. Do not incubate at 65 °C. Mix well by vortex and then centrifuge directly for 10 min at 10,000 x g. Decant the supernatant being careful to not disturb the pelleted tissue.
      Place in a drying oven (70 °C) until dried. For the sample described, sufficient drying occurred overnight. More tissue will increase the drying time. Proper drying is important for storage and handling. This washing step has previously been published in Meyer et al. (1998).
  2. Add 20 mg of the dried, washed cell wall tissue using a chemical scoop into 2 ml microfuge tubes.
  3. Add 600 µl of 1 N NaOH, containing a final concentration of 50 µM 3,4,5-trimethoxycinnamic acid as an internal standard, and incubate for 24 h at 37 °C with constant agitation.
  4. Acidify the solution by using 200 µl concentrated HCl using a repeating pipette. The final solution should be acidic for the proper extraction of hydroxicinnamic acids into ethyl acetate.
  5. Mix the samples well by vortex and centrifuged for 20 min at 14,000 x g.
  6. Carefully transfer 500 µl of the supernatant to a new tube and add 1 ml ethyl acetate.
  7. Mix samples well by vortex and centrifuge for 5 min at 14,000 x g.
  8. Transfer 900 µl of the organic phase into a new tube and dry to completion using a speed Vac.
  9. Redissolve the dried extracts in 50% MeOH and analyze by HPLC. Extracts were separated on a Shim-pack XR-ODS column using a gradient of increasing acetonitrile from 2.0% to 25% for 29.5 min in 0.1% formic acid at a flow rate of 0.9 ml min-1 (Figure 2).
  10. Quantify p-coumaric and ferulic acid content in samples. Authentic standards (purchased p-coumaric acid and ferulic acid in their purified form) were run separately using the same method for compound identification by retention time. Standard curves of these compounds of known concentrations were used to generate a linear formula that compares peak area to concentration. This formula was then used to quantify the concentration of the compounds in the sample. Concentrations for the standard curve should be selected so that they are similar to the amount of compound present in the sample. Peak area for these compounds should be adjusted to the peak area of the internal standard to account for transfer and pipetting error.

Representative data



Figure 1. A representative sampling of the extract from washed tissues. A progression showing the reduction of color present in the solvent during the washing process. The tube on the left shows what the solvent might look like after the first washing step. The tube in the middle shows what the solvent might look like after the third washing step and the tube on the right shows what it might look like after five washes where it is ready for the final wash with acetone.


Figure 2. A representative chromatogram from an Arabidopsis sample at 330 nm. Peaks corresponding to p-coumaric acid, ferulic acid, and 3,4,5-trimethoxycinnamic acid have been identified on this chromatogram; however, authentic standards should always be run separately for peak identification.

Acknowledgments

This protocol was originally modified from Meyer et al. (1998) and was published in Anderson et al. (2015).

References

  1. Anderson, N. A., Bonawitz, N. D., Nyffeler, K. and Chapple, C. (2015). Loss of FERULATE 5-HYDROXYLASE leads to mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis. Plant Physiol 169(3): 1557-1567.
  2. Meyer, K., Shirley, A. M., Cusumano, J. C., Bell-Lelong, D. A. and Chapple, C. (1998). Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis. Proc Natl Acad Sci U S A 95(12): 6619-6623.

材料和试剂

  1. 50ml锥形管(MIDSCI,目录号:C50B)
  2. 2ml微量离心管(MIDSCI,目录号:AVX-T-20-C)
  3. 拟南芥花序组织
  4. 液氮
  5. 纯乙醇(Sigma-Aldrich,目录号:E7023)
  6. 丙酮(Sigma-Aldrich,目录号:320110)
  7. 1N NaOH(Sigma-Aldrich,目录号:S8045)
  8. 3,4,5-三甲氧基肉桂酸(Sigma-Aldrich,目录号:T70408)
  9. HCl(浓盐酸)(36-38%)(Sigma-Aldrich,目录号:H1758)
  10. 乙酸乙酯(Sigma-Aldrich,目录号:270989)
  11. 乙腈(Sigma-Aldrich,目录号:271004)
  12. 0.1%甲酸(Sigma-Aldrich,目录号:94318)
  13. - 富马酸(Sigma-Aldrich,目录号:C9008)
  14. 阿魏酸(Sigma-Aldrich,目录号:128708)
  15. 50%甲醇(Sigma-Aldrich,目录号:34860)

设备

  1. 砂浆和杵
  2. 塑料勺(适合小容量处理)
  3. 摇匀培养箱,37°C
  4. 用可容纳50ml锥形管并可达到10,000×g 的转子离心机
  5. 微量离心机
  6. 涡流
  7. 干燥炉,70°C
  8. 水浴,65℃
  9. 化学勺(适用于处理小体积)
  10. 微量移液器和提示(Mettler-Toledo,目录号:L-STARTXLS +)
  11. 重复移液器与分配用适配器注射器200μl(Mettler-Toledo,型号:AR-E1)
  12. 速度飞越
  13. 分析天平
  14. 具有UV检测器和自动进样器的HPLC系统(Shimadzu prominence modular HPLC:系统控制器CBM-20A,溶剂输送单元LC-20A,自动进样器SIL-20A,柱温箱CTO-20A,UV-VIS检测器SPD-20A) >
  15. HPLC玻璃小瓶(VWR,目录号:89220-128)
  16. Shim-pack XR-ODS柱(柱尺寸3.0×75mm,珠尺寸2.2μm)(Shimadzu,型号:Shim-pack XR-ODS)

程序

  1. 来自三个独立生物群体的三份样品应首先洗涤以除去可溶性代谢物。将约1g新鲜的拟南芥花序组织,剥离的叶和种子用于Anderson等人所述的分析。 (2015);然而,该方法可用于分析所有植物组织。简而言之,该过程包括:
    1. 使用研钵和杵将组织在液氮中研磨成粉末形式
    2. 使用塑料勺将地面组织转移到50 ml锥形管,根据所使用的组织量,可能需要更大体积的容器。如果允许组织解冻,组织的转移变得更加麻烦。建议立即转移组织。
    3. 使用40毫升80%乙醇洗涤研磨的组织,允许混合物在65°C孵育1小时,通过每15分钟涡旋充分混合。 1小时后,将混合物在10,000×g离心10分钟。倾析上清液,小心不要打扰沉淀的组织。
    4. 重复洗涤,在1小时的间隔,4至6次,直到溶剂清除(请参见图1)。
      注意:其他植物组织,如叶或根,可能需要更少或更多的洗涤。
    5. 用20毫升丙酮洗一次。不要在65°C孵育。通过涡旋充分混合,然后在10,000×g下直接离心10分钟。倾析上清液小心不要打扰沉淀的组织。
      置于干燥炉(70℃)中直至干燥。对于所述样品,充分干燥过夜。更多的组织将增加干燥时间。适当的干燥对于储存和处理是重要的。该洗涤步骤先前已在Meyer等人中公布。 (1998)。
  2. 使用化学勺加入20毫克干燥,洗涤的细胞壁组织到2ml微量离心管中
  3. 加入600μl的1N NaOH,含有终浓度为50μM的3,4,5-三甲氧基肉桂酸作为内标,并在37℃下恒定搅拌孵育24小时。
  4. 使用重复移液器通过使用200μl浓HCl酸化溶液。最终的溶液应该是酸性的,以便将羟基肉桂酸适当地萃取到乙酸乙酯中
  5. 通过涡旋混合样品,并以14,000×g离心20分钟。
  6. 小心地将500μl上清液转移到新试管中,并加入1ml乙酸乙酯
  7. 通过涡旋混合样品,并以14,000×g离心5分钟
  8. 转移900微升的有机相到一个新的管,干燥完成使用速度Vac。
  9. 将干燥的提取物再溶解于50%MeOH中,并通过HPLC分析。在Shim-pack XR-ODS柱上分离提取物,使用在0.1%甲酸中从2.0%至25%的渐增29.5分钟的乙腈梯度,流速为0.9ml min -1 -1图2)。
  10. 定量样品中的p - 香豆酸和阿魏酸含量。使用相同的用于通过保留时间鉴定化合物的方法,分别运行真实标准(购买的p - 富马酸和其纯化形式的阿魏酸)。使用已知浓度的这些化合物的标准曲线来产生比较峰面积与浓度的线性公式。然后将该配方用于定量样品中化合物的浓度。应选择标准曲线的浓度,以使它们与样品中存在的化合物的量相似。这些化合物的峰面积应调整到内标的峰面积,以计及转移和移液误差。

代表数据



图1.来自洗涤组织的提取物的代表性取样。显示在洗涤过程中溶剂中存在的颜色减少的进程。左侧的管示出了在第一洗涤步骤之后溶剂可能看起来像什么。中间的管显示在第三次洗涤步骤后溶剂可能看起来像,右侧的管显示五次洗涤后可能看起来像什么,它准备用丙酮最后洗涤。


图2.来自拟南芥样品在330nm处的代表性色谱图。对应于p - 香豆酸,阿魏酸和3,4 ,5-三甲氧基肉桂酸已在该色谱图上鉴定;然而,真正的标准应该总是单独运行峰识别。

致谢

此协议最初是从Meyer 修改的。 (1998),并且在Anderson等人中公开。 (2015)。

参考文献

  1. Anderson,NA,Bonawitz,ND,Nyffeler,K.和Chapple,C。(2015)。  FERULATE 5-羟化酶的缺失导致拟南芥中可溶性苯丙素类生物合成的介体依赖性抑制。 植物生理学169 3):1557-1567
  2. Meyer,K.,Shirley,AM,Cusumano,JC,Bell-Lelong,DA和Chapple,C。(1998)。 
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Anderson, N. (2016). Cell Wall-bound p-Coumaric and Ferulic Acid Analysis. Bio-protocol 6(19): e1955. DOI: 10.21769/BioProtoc.1955; Full Text
  2. Anderson, N. A., Bonawitz, N. D., Nyffeler, K. and Chapple, C. (2015). Loss of FERULATE 5-HYDROXYLASE leads to mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis. Plant Physiol 169(3): 1557-1567.




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