龙胆和秦艽钴60辐照前后指纹图谱变化的研究

目的:建立龙胆和秦艽的高效液相色谱指纹图谱分析方法,用于分析钴60辐照前后龙胆和秦艽的成分含量变化情况。方法:以C₁₈柱(250 mm×4.6 mm,5 μm)为色谱柱,0.1%醋酸溶液-乙腈为流动相,梯度洗脱,在240 nm波长处检测。以未辐照样品为对照,按龙胆15个特征峰、秦艽13个特征峰,采用夹角余弦法计算经系列剂量辐照后样品的相似度。以各特征峰的峰面积/称样量为参数,比较辐照后样品对未辐照样品的变化百分率。并对4个已知成分马钱苷酸、獐芽菜苦苷、龙胆苦苷和当药苷按外标法进行定量。结果:龙胆与秦艽中龙胆苦苷的含量随辐照剂量的增加呈明显下降的趋势,龙胆中峰1(未知峰)辐照后含量有所增加,秦艽中獐芽菜苦苷含量有较明显下降。由于没有峰个数的增加或缺失,辐照前后指纹图谱的相似度很高,但呈现出随辐照剂量的增加相似度减小的趋势。采用外标法对已知4个成分定量,测得未经辐照的3批龙胆样品中4个成分的含量范围为:马钱苷酸0.37%~0.39%,獐芽菜苦苷0.12%~0.14%,龙胆苦苷2.7%~3.1%,当药苷0.035%~0.036%;经辐照25 kGy后,4个成分的含量范围为:马前苷酸0.35%~0.36%,獐芽菜苦苷均为0.11%,龙胆苦苷1.1%~1.3%,当药苷0.032%~0.035%。未经辐照的3批秦艽样品中4个成分的含量范围为:马钱苷酸0.64%~0.98%,獐芽菜苦苷0.06%~0.25%,龙胆苦苷1.4%~2.2%,当药苷0.043%~0.054%;经辐照25 kGy后,4个成分含量范围为:马前苷酸0.60%~0.84%,獐芽菜苦苷0.047%~0.18%,龙胆苦苷0.65%~1.3%,当药苷0.038%~0.048%。结论:所建立的指纹图谱方法验证结果良好,能够满足龙胆、秦艽辐照前后成分含量变化研究的要求。本文得到的龙胆、秦艽整体成分含量变化情况为中药合理利用辐照灭菌手段提供了参考。

Objective:To establish fingerprints and quantitative analysis of Gentianae Radix et Rhizoma and Gentianae Macrophyllae Radix by HPLC,and study the change of the ingredients before and after irradiation by ⁶⁰Co.Methods: The C₁₈ column(250 mm×4.6 mm,5 μm)was adopted and the wavelength of UV detection was 240 nm.The mobile phase consisted of 0.1% acetic acid(A)and acetonitrile(B)with a gradient elution.15 common chromatographic peaks in Gentianae Radix et Rhizoma and 13 common chromatographic peaks in Gentianae Macrophyllae Radix were observed.The reference fingerprints were established on non-irradiated samples, and a similarity evaluation was carried out based on included angle cosine.The changes were compared using the parameter which is the radio of peak area to the sample weight.The content of loganic acid, swertiamarin, gentiopicroside and sweroside were also simultaneously determined.Results: Gentiopicroside in Gentianae Radix et Rhizoma and Gentianae Macrophyllae Radix was significantly decreased after irradiation.Peak 1(unknown substance)in Gentianae Radix et Rhizoma was increased, and swertiamarin in Gentianae Macrophyllae Radix exhibited a downward trend.The amounts of the four components were determined using the external standard method, and the content ranges of three batches non-irradiated Gentianae Radix et Rhizoma were: loganic acid 0.37%-0.39%, swertiamarin 0.12%-0.14%, gentiopicroside 2.7%-3.1%, sweroside 0.035%-0.036%;the content ranges of the samples irradiated by 25 kGy were: loganic acid 0.35%-0.36%, swertiamarin 0.11%, gentiopicroside 1.1%-1.3%, and sweroside 0.032%-0.035%.The content ranges of three batches non-irradiated Gentianae Macrophyllae Radix were: loganic acid 0.64%-0.98%, swertiamarin 0.06%-0.25%, gentiopicroside 1.4%-2.2%, sweroside 0.043%-0.054%;the content ranges of the samples irradiated by 25 kGy were: loganic acid 0.60%-0.84%, swertiamarin 0.047%-0.18%, gentiopicroside 0.65%-1.3%, and sweroside 0.038%-0.048%.Conclusion: The established method is repeatable and accurate, which can meet the requirements of our study.The results can provide reference to the rational utilization of irradiation on traditional Chinese medicine.