Shay Philip-Edouard, Trofymow J A, Constabel C Peter
Department of Biology & Centre for Forest Biology, University of Victoria, P.O. Box 3020, STN CSC, Victoria, BC V8W 3N5 Canada.
Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, Victoria, BC Canada.
Plant Methods. 2017 Aug 2;13:63. doi: 10.1186/s13007-017-0213-3. eCollection 2017.
Condensed tannins (CT) are the most abundant secondary metabolite of land plants and can vary in abundance and structure according to tissue type, species, genotype, age, and environmental conditions. Recent improvements to the butanol-HCl assay have separately helped quantification of soluble and insoluble CTs, but have not yet been applied jointly. Our objectives were to combine previous assay improvements to allow for quantitative comparisons of different condensed tannin forms and to test protocols for analyses of condensed tannins in vegetative plant tissues. We also tested if the improved butanol-HCl assay can be used to quantify water-soluble forms of condensed tannins.
Including ~50% acetone in both extraction solvents and final assay reagents greatly improved the extraction and quantification of soluble, insoluble and total condensed tannins. The acetone-based method also extended the linear portion of standard integration curves allowing for more accurate quantification of samples with a broader range of condensed tannin concentrations. Estimates of tannin concentrations determined using the protocol without acetone were lower, but correlated with values from acetone-based methods. With the improved assay, quantification of condensed tannins in water-soluble forms was highly replicable. The relative abundance of condensed tannins in soluble and insoluble forms differed substantially between tissue types.
The quantification of condensed tannins using the butanol-HCl assay was improved by adding acetone to both extraction and reagent solutions. These improvements will facilitate the quantification of total condensed tannin in tissues containing a range of concentrations, as well as to determine the amount in water-soluble, acetone:MeOH-soluble and insoluble forms. Accurate determination of these three condensed tannin forms is essential for careful investigations of their potentially different physiological and ecological functions.
缩合单宁（CT）是陆地植物中含量最丰富的次生代谢产物，其含量和结构会因组织类型、物种、基因型、年龄和环境条件而有所不同。最近对丁醇 - 盐酸法的改进分别有助于定量分析可溶性和不溶性CT，但尚未联合应用。我们的目标是结合以前的分析改进方法，以便对不同缩合单宁形式进行定量比较，并测试用于分析植物营养组织中缩合单宁的方案。我们还测试了改进后的丁醇 - 盐酸法是否可用于定量缩合单宁的水溶性形式。
在提取溶剂和最终分析试剂中加入约50%的丙酮，极大地改善了可溶性、不溶性和总缩合单宁的提取和定量。基于丙酮的方法还扩展了标准积分曲线的线性部分，从而能够更准确地定量分析缩合单宁浓度范围更广的样品。使用不含丙酮的方案测定的单宁浓度估计值较低，但与基于丙酮方法的值相关。通过改进的分析方法，水溶性缩合单宁的定量具有高度可重复性。不同组织类型中可溶性和不溶性缩合单宁的相对丰度差异很大。
通过在提取液和试剂溶液中添加丙酮，改进了使用丁醇 - 盐酸法对缩合单宁的定量。这些改进将有助于定量分析含有不同浓度范围的组织中的总缩合单宁，以及确定水溶性、丙酮：甲醇可溶性和不溶性形式的含量。准确测定这三种缩合单宁形式对于仔细研究它们潜在的不同生理和生态功能至关重要。
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