不同采收期苗药土党参中党参炔苷、总多糖和总黄酮含量的测定以及土党参最佳采收期的确定

Determination of Lobetyolin,Total Polysaccharides and Total Flavonoids in Miao Medicine Campanumoea Javanica from Different Harvesting Periods and Determination of the Optimal Harvesting Period of Campanumoea Javanica

分别采用高效液相色谱法(HPLC)和紫外-可见分光光度法(UV-Vis)测定不同采收期(1~12月)土党参中党参炔苷、总多糖、总黄酮含量,并用总评归一化法确定其最佳采收期。在土党参样品中加入甲醇,超声提取40 min,过0.45μm滤膜,用HPLC测定滤液中的党参炔苷含量。在土党参样品中加入80%(体积分数)乙醇溶液,回流2次,每次1 h,过滤后,滤渣以水为介质在微波炉中消解2次,每次5 min,稀释后加入50 g·L^(-1)苯酚溶液和硫酸,于100℃水浴加热10 min,冰水浴中冷却20 min,用UV-Vis在490 nm下测定总多糖含量,用葡萄糖作为对照品绘制标准曲线。在土党参样品中加入60%(体积分数,下同)乙醇溶液,回流2次,每次1 h,过滤、浓缩并稀释,加入50 g·L^(-1)亚硝酸钠溶液、100 g·L^(-1)硝酸铝溶液和40 g·L^(-1)氢氧化钠溶液,用60%乙醇溶液稀释至10 mL,用UV-Vis在509 nm下测定总黄酮含量,用芦丁作为对照品绘制标准曲线。以Hassan法计算党参炔苷、总多糖和总黄酮的归一值,以总归一(OD)值确定最佳采收期。结果显示:党参炔苷、总多糖和总黄酮的质量浓度均在一定范围内与其对应的峰面积(党参炔苷)和吸光度(总多糖和总黄酮)呈线性关系,党参炔苷的检出限(3S/N)、总多糖的检出限(3.143s)、总黄酮的检出限(3.143s)分别为0.131,0.046,0.071 mg·L^(-1)。方法用于实际样品的分析,峰面积(党参炔苷)和吸光度(总多糖和总黄酮)的相对标准偏差(n=6)均不大于3.0%,平均加标回收率分别为98.8%,99.1%,98.5%;10月份的OD值较高,可将其确定为土党参最佳采收期,此时党参炔苷、总多糖和总黄酮的质量分数分别为3.08,185.15,5.85 mg·g^(-1)。

Lobetyolin,total polysaccharides and total flavonoids in Campanumoea javanica from different harvesting periods(January to December)were determined by HPLC and UV-Vis,and the optimal harvesting period was obtained by the comprehensive evaluation normalization method.The Campanumoea javanica sample were mixed with methanol for extraction with ultrasound for 40 min.Aftering passing through 0.45μm filter membrane,lobetyolin in the filtrate was determined by HPLC.The ethanol solution of 80%(volume fraction)was added into the Campanumoea javanica sample,and the mixture was refluxed twice for 1 h each time.After filtering,the filtrate was digested twice with water as media in the microwave oven for 5 min each time,diluted and mixed with 50 g·L^(-1) phenol solution and sulfuric acid.The above solution was heated in the water bath at 100℃for 10 min,cooled in an ice water bath for 20 min,and total polysaccharides was determined by UV-Vis at 490 nm,with glucose as reference substance for preparation of standard curve.The Campanumoea javanica sample together with 60%(volume fraction,the same below)ethanol solution was refluxed twice for 1 h each time.After filtration,concentration and dilution,50 g·L^(-1) sodium nitrite solution,100 g·L^(-1) aluminum nitrate solution and 40 g·L^(-1) sodium hydroxide solution were added.The solution obtained was diluted to 10 mL with 60% ethanol solution,and total flavonoids was determined by UV-Vis at 509 nm,with rutin as reference substance for preparation of standard curve.The normalized values of lobetyolin,total polysaccharide and total flavonoids were calculated by Hassan method,and the optimal harvesting period was obtained by the total normalized(OD)value.As found by results,linear relationships between mass concentrations of lobetyolin,total polysaccharides and total flavonoids and their corresponding peak areas(for lobetyolin)and absorbances(for total polysaccharides and total flavonoids)were kept in definite ranges,and detection limits of lobetyolin(3 S/N),total polysaccharides(3.143s)and total flavonoids(3.143s)were 0.131,0.046,0.071 mg·L^(-1),respectively.The proposed method was applied to the analysis of actual samples,RSDs(n=6)of the peak areas(for lobetyolin)and absorbances(for total polysaccharides and total flavonoids)were not more than 3.0%,and the average values of test for spiked recovery were 98.8%,99.1% and 98.5%,respectively.The OD values in October were higher,which could be identified as the optimal harvesting period for Campanumoea javanica,with mass fractions of lobetyolin,total polysaccharides and total flavonoids of 3.08,185.15,5.85 mg·g^(-1),respectively.