目的: 利用超高效液相色谱-质谱联用(UPLC-MS/MS)法比较款冬花碱制前后毒性成分克氏千里光碱(SK)含量的变化,确定炮制减毒工艺条件,并根据测定结果进行初步的风险评估。方法: 在前期研究基础上优化UPLC-MS/MS分析方法,并建立款冬花炮制减毒工艺,通过单因素实验结合正交实验设计,以SK含量为评价指标,分别对碱水用量、加热温度及加热时间进行考察,筛选出最佳炮制工艺,并与传统蜜炙法对比。结果: SK浓度在1.00~503.04 ng·mL-1范围内与峰面积呈现良好的线性关系(r>0.999);平均加样回收率为93.8%,RSD为3.0%;碱处理工艺为:水浴加热80 ℃,反应时长120 min、加5%碳酸钠溶液体积比(反应液与碱液)为2∶1时,16批款冬花SK水解率均达到90%以上,4批蜜款冬花SK含量下降率为40%左右。结论: 与生品相比,该方法处理后的16批款冬花SK含量呈显著降低趋势,且炮制前的16批款冬花药材均为风险等级,通过此炮制方法,5批转为无风险等级,11批转为低风险等级。虽然蜜炙法也能降低SK的含量,但与该碱制法相比,4批蜜款冬花仍属于风险等级药材,这为款冬花的安全用药提供了可行性指导依据。
Objective: To compare the changes of the contents of toxic component Senkirkine in crude and alkali processing Farfarae Flos using UPLC-MS/MS method. The preliminary risk assessment was carried out based on the results. Methods: Based on the previous research, UPLC-MS/MS analysis method was optimized. The alkaline treatment process of Farfarae Flos was established. Through single factor experiment and orthogonal experimental design, the amount of alkaline water, heating temperature and heating time were investigated with SK content as the evaluation index, and the best process was selected. The optimized method was compared with traditional honey roasting method. Results: The linear ranges of SK were 1.00-500 ng·mL-1 (r>0.999); the average sample recovery rate was 93.8%, and the RSD was 3.0%. The alkali treatment process was as follows: the water bath was heated at 80 ℃, the reaction time was 120 min, and the volume ratio of 5% Na2CO3(reaction solution-alkali volume)was 2∶1, the hydrolysis rate of SK in 16 batches of Farfarae Flos reached more than 90%. The decreasing rate of SK content in 4 batches of honey-processing Farfarae Flosis about 40%. Conclusion: Compared with the raw product, the content of toxic alkaloids in 16 batches of Farfarae Flos treated by this method showed a significant decreasing trend, and the 16 batches of Farfarae Flos before processing were all at risk level. Through this concocting method, 5 batches were transferred to the no-risk level, and 11 were transferred to the low-risk level. Although the honey roasting method can also reduce the content of SK, the 4 batches of honey-processing Farfarae Flos still belong to the risk grade medicine compared with the alkali preparation method, which provided possible guidance for the safe medication of Farfarae Flos.
[1] 中华人民共和国药典2020年版. 一部[S].2020
ChP 2020.VolⅠ[S].2020
[2] 曹娟, 王福刚, 刘克. 蜜炙款冬花HPLC指纹图谱研究 [J].中药材, 2012, 35(1):33
CAO J, WANG FG, LIU K, et al. Study on the fingerprint of honey-fried Farfarae Flos by HPLC [J].J Chin Med Mater, 2012, 35(1):33
[3] PARK HR, YOO MY, SEO JH, et al. Sesquiterpenoids isolated from the flower buds of Tussilago farfara L. inhibit diacylglycerol acyltransferase [J].J Agric Food Chem, 2008, 56 (22): 10493
[4] LEE J, SONG K, HUH E, et al. Neuroprotection against 6-OHDA toxicity in PC12 cells and mice through the Nrf2 pathway by a sesquiterpenoid from Tussilago farfara [J].Redox Biol, 2018, 18:6
[5] CHEN S, DONG L, QUAN H, et al. A review of the ethnobotanical value, phytochemistry, pharmacology, toxicity and quality control of Tussilago farfara L. (coltsfoot) [J].J Ethnopharmacol, 2021, 267:113478
[6] ANELY N, KOSTOVA N, SIDJIMOV A, et al. Pyrrolizidine alkaloids in Tussilago farfara from Bulgaria [J].Biotechnol Biotechnol Equip, A, 2015, 29(Suppl.1):S1
[7] LIN G, WANG J Y, LI N, et al. Hepatic sinusoidal obstruction syndrome associated with consumption of Gynura segetum [J].J Hepatol, 2011, 54 (4): 666
[8] RUAN J, YANG M, FU P, et al. Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis [J].Chem Res Toxicol, 2014, 27 (6): 1030.
[9] GEBUREK I, PREISS-WEIGERT A, LAHRSSEN-WIEDERHOLT M, et al. In vitro metabolism of pyrrolizidine alkaloids-metabolic degradation and GSH conjugate formation of different structure types [J].Food Chem Toxicol, 2020, 135 110868
[10] DAI N, YU YC, REN TH, et al. Gynura root induces hepatic veno-occlusive disease: a case report and review of the literature [J].World journal of gastroenterology, 2007, 13 (10): 1628
[11] KA AS, MICHEL G, IMBERT P, et al. Hepatic veno-occlusive disease due to dietary toxins in a five-year-old child in Senegal [J].Med Trop, 2006, 66 (5): 514
[12] 张瑞环, 安薇薇, 王慧.浅析中药炮制对药物毒性的影响 [J].齐鲁药事, 2006, (6): 355
ZHANG RH, AN WW, WANG H. Analysis of the effect of processing of traditional Chinese medicine on drug toxicity [J].Qilu Pharm Aff, 2006, (6): 355
[13] 李红军, 王增绘, 李文涛等. UPLC-Q-TOF/MS法分析款冬花蜜炙前后的化学成分变化 [J].中国药房, 2015, 26 (6): 792
LI HJ, WANG ZH, LI WT, et al. Analysis of the variation of chemical composition of Tussilago farfara by UPLC-Q-TOF/MS before and after fried with honey[J].China Pharm, 2015, 26 (6): 792
[14] 吴笛, 雷昌, 唐林. HPLC-ESI-ITMS法分析蜜炙前后款冬花中毒性成分克氏千里光碱的变化 [J].中成药, 2019, 41 (3): 694
WU D, LEI C, TANG L. The change of senkirkine in Tussilago farfara before and after honey roasting by HPLC-ESI-ITMS [J].Chin Tradit Pat Med, 2019, 41 (3): 694
[15] ROEDER E. Medicinal plants in China containing pyrrolizidine alkaloids [J].Pharmazie, 2000, 55 (10): 711-726
[16] 李宁, 姚令文, 刘丽娜,等. UPLC-MS/MS法测定款冬花炮制前后3个吡咯里西啶生物碱的含量 [J].药物分析杂志, 2020, 40 (7): 1262
LING N, YAO LW, LIU LN, et al. Determination of three main pyrrolizidine alkaloids in crude and prepared Farfarae Flos by UPLC-MS/MS [J].Chin J Pharm Anal 2020, 40 (7):1262
[17] EFSA. Scientific opinion on Pyrrolizidine alkaloids in food and feed [J].EFSA Jl, 2011, 9 (11): 2406.
[18] CHAN P. NTP technical report on the toxicity studies of Riddelliine (CAS No. 23246-96-0) Administered by Gavage to F344 Rats and B6C3F1 Mice [J/OL].US, 1993[2022-02-07].https://ntp.niehs.nih.gov/go/tox027
[19] EFSA. Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data [J/OL].EFSA Scientific Committee: EFSA Journal, 2012[2022-03-18].http://www.efsa.europa.eu
[20] COT. COT statement on pyrrolizidine alkaloids in food. COT statement 2008-06 [2022-03-01].https://cot.food.gov.uk/sites/default/files/cot/
[21] BfR. Updated risk assessment on levels of 1, -unsaturated pyrrolizidine alkaloids (PAs) in foods [J/OL].Berlin, Germany, 2020[2022-03-11].https://www.bfr.bund.de/en
[22] HMPC. Public statement on contamination of herbal medicinal products, traditional herbal medicinal products with pyrrolizidine alkaloids transitional recommendations for risk management and quality control. London. European Medicines Agency[BD/OL]. 2016 [2021-03-01]. https://www.ema.europa.eu/en/documents/public-statement/
[23] DOULL J, ROZMAN KK. Using Haber's law to define the margin of exposure [J].Toxicology, 2000, 149 (1): 1
[24] ROEDER E, WIEDENFELD H. Pyrrolizidine alkaloids in plants used in the traditional medicine of Madagascar and the Mascarene islands [J].Pharmazie, 2011, 43(15): 637
