HPLC法同时测定不同采收期木薯叶中6种类黄酮的含量

Simultaneous Determination of 6 Flavonoids in Cassava Leaves from Different Harvest Time by HPLC

建立同时测定木薯叶中6种类黄酮的高效液相色谱(high performance liquid chromatography)法,测定不同采收期下木薯叶中类黄酮含量,探讨木薯叶作为黄酮资源利用的最佳采收期。色谱柱为CNW Athena C18-WP(4.6 mm×250 mm,5μm),流动相为乙腈-乙酸-水,梯度洗脱,流速为1 m L/min,柱温室温,进样量5μL,检测波长儿茶素与二氢黄酮甙为281 nm,芦丁与山奈酚为360 nm,槲皮素367 nm、穗花杉双黄酮338 nm。儿茶素、芦丁、二氢黄酮甙、槲皮素、山奈酚、穗花杉双黄酮的浓度分别为1.5 mg/L^194.6 mg/L(R^2=0.999 9)、7.8 mg/L^992.6 mg/L(R^2=1.000 0)、0.7 mg/L^84 mg/L(R^2=0.999 9)、0.7 mg/L^93.8 mg/L(R^2=0.999 9)、0.8 mg/L^108 mg/L(R^2=0.995 8)、0.5 mg/L^67.2 mg/L(R^2=0.999 5)时与各自峰面积线性关系良好,回收率95.50%~100.01%,相对标准偏差(RSD)小于3%。该法适用于木薯叶中以上6种类黄酮成分的精确定量。检测结果表明,不同品种、不同采收期的木薯叶中类黄酮的组成和含量差异较大,芦丁与穗花杉双黄酮为主要类黄酮。儿茶素、芦丁、二氢黄酮甙、槲皮素含量最高采收期为种植后180 d,穗花杉双黄酮为种植后120 d,山奈酚因品种而异。

A high performance liquid chromatography （HPLC）method for the determination of six kinds of flavonoids was established and employed to the analysis of flavonoids in cassava leaves havested at different time. The chromatographic separation was performed on a CNW Athena C18-WP column kept at room temper-atur e and acetonitrile/ acetonitrile acid/ wat er mixture were used as the mobile phase with a flow rate of 1 mL/min through gradient elution. The injection volume of the sample was 5μL. The detection wavelengths were set at 281 nm for catechin and hesperidin, 338 nm for amentoflavone, 360 nm for rutin and kaempferol and 367 nm for quercetin. The linear ranges of catechin, rutin, hesperidin, quercetin, kaempferol and amentoflavone were 1.5 mg/L-194.6 mg/L（R2=0.9999）,7.8 mg/L-992.6 mg/L（R2=1.0000）,0.7 mg/L-84 mg/L（R2=0.9999）, 0.7 mg/L-93.8 mg/L（R2=0.9999）, 0.8 mg/L-108 mg/L（R2=0.9958）, 0.5 mg/L-67.2 mg/L（R2=0.9995）, respectively. This method was precise and accurate（RSD〈3%） and the average recoveries ranged from 95.50%to 100.01%, suitable for the precise quantification of the six flavoinds in cassava leaves. The results indicat-ed that rutin and amentoflavone were the major components with the total amounts over 6 times of other 4 flavoinds. The optimal harvest time to obtain the highest amouts for catechin, rutin, hesperidin, and quercetin was 180 d after planting, 120 d for amentoflavone, and it depend on cassava varieties for kaempferol.