Inhibition of Cyanogenesis by tannins

During isolation of two biosynthetic types of cyanogenic glycosides fromCarica papaya, weak cyanide tests were obtained from initial fractions. Upon final purification, strongly positive cyanide tests were obtained. Pretreatment of extracts to remove polyphenolics alleviated inhibition of cyanogenesis, which led us to suspect that tannins were inhibitory agents. Qualitative and quantitative measures of inhibition were made using standard cyanogenic glycosides and polyphenolics. Cyanogenesis was inhibited quantitatively when condensed tannins (quebracho, wattle, and chestnut), or hydrolyzable tannin (tannic acid) were added. When tannins were precipitated from the reaction mixture, cyanide tests proceeded optimally. These results stress the need to interpret negative cyanide tests with care and indicate possible ecological synergisms between plant defensive chemicals.     

HPLC测定长柄扁桃仁及饼粕中苦杏仁苷与野黑樱苷

为确保长柄扁桃仁在加工与其副产物利用过程中,更快更有效地明确降解产物中有害物质含量,本文建立了一种同时测定样品中苦杏仁苷与其降解产物野黑樱苷的高效液相色谱方法。在采用甲醇提取长柄扁桃仁、饼粕中苦杏仁苷和野黑樱苷,色谱柱Aglient ZORBAX SB-C18(5μm,4.6 mm×250 mm),流动相为20%甲醇和80%水,流速1.00 mL/min,检测波长210 nm,柱温30℃的条件下,苦杏仁苷和野黑樱苷色谱峰分离良好;苦杏仁苷浓度在0.50～300μg/m L间线性关系良好,R2=0.9999,野黑樱苷浓度在0.30～100μg/m L间线性关系良好,R2=0.9999;长柄扁桃仁提取苦杏仁苷和野黑樱苷平均加标回收率分别为87.10%～96.19%(RSD为4.64%～7.83%)和88.65%～103.10%(RSD为3.03%～7.55%);而在长柄扁桃饼粕中分别为98.32%～107.99%(RSD为1.44%～3.36%)和107.58%～117.60%(RSD为1.45%～2.26%)。本方法准确、可靠,适用于长柄扁桃仁、长柄扁桃饼粕中苦杏仁苷与野黑樱苷的测定。     

长柄扁桃加工产品中苦杏仁苷及其降解产物野黑樱苷质谱定性和定量分析

建立了长柄扁桃加工产品中苦杏仁苷及其降解产物野黑樱苷的定性和定量分析方法。长柄扁桃饼(粕)、长柄扁桃油样品经甲醇超声萃取后,采用HPLC-Q-TOF,经C18柱(100 mm×2. 1mm,2. 7μm)分离进行定性分析;采用UPLC-TSQ,在C18柱(100 mm×2. 1 mm,1. 7μm),电喷雾ESI源,正离子模式条件下进行定量分析。结果表明:建立的定性方法,经相对保留时间、精确相对分子质量和二级碎片比对,鉴定出苦杏仁苷及其降解产物野黑樱苷;建立的定量方法对于饼粕、油脂样品中苦杏仁苷和野黑樱苷线性范围分别为50～1 000 ng/m L,50～800 ng/m L;对于饼粕样品苦杏仁苷、野黑樱苷平均加标回收率分别为99. 2%～100. 5%和102. 0%～105. 8%,精密度分别为0. 1%～3. 3%和1. 6%～3. 6%,检出限分别为2. 5、5 mg/kg;对于油脂样品苦杏仁苷、野黑樱苷平均加标回收率分别为99. 1%～108. 3%和102. 9%～108. 9%,精密度分别为1. 5%～2. 2%和1. 0%～3. 5%,检出限分别为0. 06、0. 12 mg/kg。该定量方法快速简便,为保障长柄扁桃加工产品的安全提供了技术支撑。     

Quantification of amygdalin,prunasin, total cyanide and free cyanide in powdered loquat seeds

ABSTRACT

Amygdalin, prunasin, total cyanide and free cyanide concentrations in 12 powdered loquat seeds were investigated. Loquat (Eriobotrya japonica) is a species of flowering plant in the family Rosaceae, and its fruit is quite popular in Japan. Amygdalin and prunasin were measured using LC–MS/MS. Total cyanide was measured by enzymatic treatment, steam distillation and colorimetric quantification using the pyridine–pyrazolone method. Free cyanide was measured without enzymatic treatment. The mean concentrations of amygdalin, prunasin, total cyanide and free cyanide in powdered loquat seeds were 5900, 760, 410 and 44 mg/kg, respectively. The range of each quantitative value was extensive. Seven out of twelve samples were at risk for exceeding the acute reference dose (ARfD) of cyanide.     

甜杏仁和苦杏仁野黑樱苷水解酶基因的克隆

以轮台小白杏（甜杏仁品种）和甲麦黄杏（苦杏仁品种）为实验材料,采用同源基因克隆方法克隆野黑樱苷水解酶（prunasin hydrolase,PH）基因。轮台小白杏PH基因命名为PaLTPh（GenBank登录号：KF888615）,序列长度3 686 bp;甲麦黄杏PH基因命名为PaMJPh（KF888616）,序列长度3 690 bp。PaLTPh和PaMJPh与扁桃Ph691基因相似性为94%。根据PaLTPh和PaMJPh保守区序列设计引物,克隆编码区（coding sequence,CDS）序列。PaLTPh CDS（KF888617）和PaMJPh CDS（KF888618）序列长度均为1 635 bp,与扁桃Ph691全CDS区相似性为97%。PaLTPh CDS和PaMJPh CDS序列均为全长的开放阅读框,编码蛋白质长度为544 个氨基酸。PaLTPH蛋白和PaMJPH蛋白均包含长度为26 个氨基酸的信号肽和糖基水解酶家族Ⅰ结构域,可催化野黑樱苷水解为扁桃腈和葡萄糖。杏PH基因与扁桃Ph691具有高度的同源性。