CJI (Traditional Chinese Medicine)
基于UPLC指纹图谱及多指标成分含量的罗汉果质量分析
张芳平,刘晓霞,位翠杰,冯涌微,何民友,夏天睿,陈向东,孙冬梅,李振雨广东一方制药有限公司,广东省中药配方颗粒企业重点实验室,广东 佛山 528244
摘要:目的 建立罗汉果超高效液相色谱(UPLC)指纹图谱及多指标成分含量测定方法,为完善罗汉果药材的质量评价提供依据。方法 采用Waters Cortecs T3 C18色谱柱(2.1 mm×100 mm,1.6 μm),流动相为乙腈-0.1%磷酸水,梯度洗脱,流速0.30 mL/min,检测波长0~25 min为300 nm、25.1~40 min为203 nm,柱温43 ℃,进样量1 μL;利用《中药色谱指纹图谱相似度评价系统》(2012版)对15批罗汉果药材UPLC指纹图谱进行相似度分析;采用SPSS25.0和SIMCA14.1软件对罗汉果药材进行化学计量学分析;测定罗汉果药材中多指标成分的含量。结果 15批罗汉果药材指纹图谱有16个共有峰,指认其中4种成分;聚类分析和主成分分析均将15批罗汉果药材分为两类,其中广西柳州样品为一类,广西桂林样品为一类;以变量重要性投影(VIP)值>1.0为条件,共筛选出6个差异性标志物,按VIP值排序,峰13>峰9>峰5>峰7>峰15(罗汉果皂苷Ⅴ)>峰2(5-羟甲基糠醛);广西柳州样品5-羟甲基糠醛含量高于广西桂林样品,罗汉果皂苷Ⅴ含量则略低。结论 本研究建立的UPLC指纹图谱方法和含量测定方法可行,可为罗汉果质量评价提供依据。关键词:罗汉果;超高效液相色谱法;指纹图谱;化学计量学分析;多指标成分
中图分类号:R284.1 文献标识码:A 文章编号:1005-5304(2023)10-0139-06 DOI:10.19879/j.cnki.1005-5304.202304005 开放科学(资源服务)标识码(OSID): Quality Analysis on Siraitiae Fructus Based on UPLC Fingerprint and Multi-index Component Content
ZHANG Fangping, LIU Xiaoxia, WEI Cuijie, FENG Yongwei, HE Minyou, XIA Tianrui,
CHEN Xiangdong, SUN Dongmei, LI Zhenyu
Guangdong Yifang Pharmaceutical Co. Ltd., Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Formula, Foshan 528244, China
Abstract: Objective To establish the UPLC fingerprint and multi-index component content determination method of Siraitiae Fructus; To provide a basis for optimize the quality evaluation of Siraitiae Fructus. Methods Waters Cortecs T3 C18 column (2.1 mm × 100 mm, 1.6 μm) was used. The mobile phase was acetonitrile-0.1% phosphoric acid water with gradient elution. The flow rate was 0.30 mL per minute. The detection wavelength was 300 nm in 0–25 min and 203 nm in 25.1–40 min. The column temperature was 43 °C and the injection volume was 1 μL. The UPLC fingerprints of 15 batches of Siraitiae Fructus were analyzed by the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition); the Chemometrics analysis of Siraitiae Fructus was carried out by SPSS 25.0 and SIMCA 14.1 software; multi index components in Siraitiae Fructus were determined. Results The results showed that there were 16 common peaks in the fingerprints of 15 batches of Siraitiae Fructus, and 4 components were identified. The 15 batches of Siraitiae Fructus were divided into two categories by clustering analysis and PCA, among which Liuzhou in Guangxi was classified into one category and Guilin in Guangxi was classified into one category. The VIP value > 1.0 was used as the screening condition to screen the differential markers. A total of 6 differential markers were found. The VIP value was ranked as peak 13 > peak 9 > peak 5 > peak 7 > peak 15 (mogroside V) > peak 2 (5-hydroxymethylfurfural); the results of基金项目:中华人民共和国工业和信息化部2022年产业技术基础公共服务平台项目(2022-230-221)通讯作者:李振雨,E-mail:lzy1083656123@126.com
content determination showed that the content of 5-hydroxymethylfurfural in Siraitiae Fructus from Liuzhou, Guangxi was higher than that from Guilin, while the content of mogroside V was slightly lower than that from Guilin. Conclusion The UPLC fingerprint method and content determination method established in this study are feasible and can provide a basis for the quality evaluation of Siraitiae Fructus.
Keywords: Siraitiae Fructus; UPLC; fingerprint; chemometric analysis; multi-index components罗汉果为葫芦科植物罗汉果Siraitia grosvenorii (Swingle)C. Jeffrey ex A. M. Lu et Z. Y. Zhang的干燥果实,有清热润肺、利咽开音、滑肠通便功效[1],是原
卫生部于2002年公布的我国首批药食同源中药(卫法监发〔2002〕51号)。罗汉果是广西道地药材之一,多分布于广西壮族自治区桂林市、柳州市等地[2]。研究表明,罗汉果含有黄酮类、皂苷类、多糖类、多酚类等活性成分,有抗菌、抗炎、抗氧化等作用[3-4],可用于治疗肿瘤、肝脏疾病,具有丰富的营养价值与药用价值[5-7]。目前,罗汉果应用广泛,2020年版《中华人民共和国药典》仅以单一成分罗汉果苷Ⅴ作为罗汉果的质量标志物(Q-Marker),无法全面评价罗汉果的内在质量,因此,亟需建立完整的罗汉果质量评价体系。同时,相关研究表明,除罗汉果皂苷Ⅴ外,葫芦烷型四环三萜类罗汉果苷Ⅱe、罗汉果苷Ⅲe、11-O-罗汉果皂苷Ⅴ等,黄酮苷类山柰苷等,小分子醛类
5-羟甲基糠醛等是罗汉果的重要化学成分[8],具有抗炎、抗氧化、抗肿瘤等药理作用。因此,本研究通过建立罗汉果药材UPLC指纹图谱并对其多指标成分进行含量测定,结合化学计量学分析,全面评价罗汉果药材的质量,为完善罗汉果药材质量标准提供依据。
1 仪器与试药
U3000超高效液相色谱仪,赛默飞世尔科技(中国)有限公司;ME204E万分之一天平、XP26百万分之一天平,梅特勒-托利多公司;HWS28电热恒温水浴锅,上海一恒科技有限公司。
15批罗汉果药材,经广东一方制药有限公司孙冬梅主任中药师鉴定,均为葫芦科植物罗汉果Siraitia grosvenorii (Swingle) C. Jeffrey ex A. M. Lu et Z. Y. Zhang的干燥果实,批号LHG01~LHG05样品(编号L1~L5)产地为广西壮族自治区柳州市(广西柳州),批号LHG06~LHG15样品(编号L6~L15)产地为广西壮族自治区桂林市(广西桂林)。对照品罗汉果皂苷Ⅴ(批号
111754-201703,含量94.0%,中国食品药品检定研究院),山柰苷(批号 20110503 ,含量99.63%,成都普菲德生物有限公司),11-O-罗汉果皂苷Ⅴ(批号
DST200903-032,含量99.39%,成都乐美天生物技术有限公司),5-羟甲基糠醛(批号111626202215,含量
99.5%,中国食品药品检定研究院)。甲
醇(分析纯,西陇科学股份有限公司),乙腈(色谱纯,默克股份有限公司),磷酸(色谱纯,天津市科密欧化学试剂有限公司),超纯水(实验室自制)。
2 方法与结果
2.1 指纹图谱建立
2.1.1 色谱条件
采用 Waters Cortecs T3 C18 色谱柱( 2.1 mm×
μm),流动相为乙腈(A)-0.1%
100 mm,1.6 磷酸溶液(B),梯度洗脱(0~6 min,2%~7%A;6~17 min, 7%~15%A;17~19 min,15%~19%A;19~28 min, 19%~22%A;28~35 min,22%~27%A;35~40 min, 27%~90%A),流速0.30 mL/min,检测波长0~25 min为300 nm、25.1~40 min为203 nm,柱温43 ℃,进样
μL。
量1
2.1.2 对照品溶液制备
5-羟甲基糠醛、山柰苷、11-O-罗汉果皂苷Ⅴ、取罗汉果皂苷Ⅴ对照品适量,精密称定,置
25 mL量瓶
μg、山中,加甲醇制成每1 mL含5-羟甲基糠醛51.614
μg、11-O-罗汉果皂苷Ⅴ μg、罗汉柰苷22.914 31.656
果皂苷Ⅴ μg
52.230 的溶液,即得混合对照品溶液。2.1.3 供试品溶液制备
取罗汉果药材粉末(过四号筛)约1.0 g,精密称定,置具塞锥形瓶中,精密加入甲醇50 mL,称定质量,加热回流2 h,放冷,再称定质量,用甲醇补足减失的质量,摇匀,过滤,取续滤液,即得。
2.1.4 方法学考察
2.1.4.1 精密度试验取罗汉果药材(编号L2)供试品溶液,按“2.1.1”
罗汉果皂苷Ⅴ为项下色谱条件连续进样6次,以峰15
参照峰S,计算各共有指纹峰与S峰的相对保留时间RSD为0.32%~1.53%,相对峰面积RSD为 0.65%~ 1.59%,均小于3.00%,表明仪器精密度良好。2.1.4.2 重复性试验
取罗汉果药材粉末(过四号筛,编号L2)约1.0 g,平行6份,按“2.1.3”项下方法制备供试品溶液6份,按“2.1.1”项下色谱条件进样分析,以峰15罗汉果皂苷Ⅴ为参照峰
S,计算共有指纹峰与S峰的相对保留时