葛根先煎时间对升麻葛根汤中的葛根素含量及药效学的影响

目的：通过研究不同时间煎煮葛根对升麻葛根汤中的葛根素含量及药效学的影响，探讨葛根的最佳煎煮时间，为葛根的特殊煎煮方法的临床应用提供理论依据。方法：采用高效液相色谱（HPLC）法测定不同时间煎煮葛根对升麻葛根汤中的葛根素含量影响。色谱条件：Agilent 5 TC-C₁₈柱（250 mm×4.6 mm，5 μm）；流动相为甲醇-水（25：75），流速为1 mL·min^-1，检测波长为250 nm，柱温30℃。采用氨水诱咳法和热板法观察不同时间煎煮葛根的升麻葛根汤对小鼠的镇咳作用和镇痛作用的影响。将ICR小鼠（雌性180只，雄性60只）随机分为12组，即空白对照组、感冒疏风胶囊组、阿司匹林肠溶片组、不同煎煮时间的升麻葛根汤9组（葛根先煎0，5，10，15，20，25，30，35，40 min），按照0.02 mL·g^-1剂量分别灌胃给药，连续7 d。镇咳实验各组小鼠（雌雄各半）末次给药1 h后，将小鼠放入雾化器中，观察和记录各组小鼠的咳嗽潜伏期（首次开始咳嗽的时间）和2 min内咳嗽次数。镇痛实验各组小鼠（雌性）末次给药1 h后，将小鼠放置于（55±0.5）℃的智能热板仪上，记录其足底接触热板到出现舔足或跳跃的时间（热板反应潜伏期）。结果：在0.02~0.12 mg范围内与峰面积呈良好的线性关系（r=0.999 6）；葛根先煎15 min时葛根素含量为43.6 mg·g^-1，含量最高。与空白对照组比较，葛根先煎10，15，20，25 min组可使小鼠咳嗽潜伏期明显延长（P<0.01，P<0.05），葛根先煎15、20 min组可使小鼠咳嗽次数减少（P<0.05）；葛根先煎10，15，20 min时，小鼠痛阈时间明显延长（P<0.05）。结论：葛根素含量测定结果表明，葛根在先煎15 min时，葛根素的含量最高，煎煮时间过短，葛根素不能充分溶出，煎煮时间过长，葛根素含量下降；药效学实验结果表明，葛根在先煎15 min时，升麻葛根汤对风寒表证的镇咳、镇痛作用显著。提示，葛根入汤剂应先煎15 min为宜。

Effect of Puerariae lobata decoction time on puerarin content and pharmacodynamics of Shengma Gegen Decoction

OBJECTIVE To investigate the effects of different Pueraria lobata decocting times on the content of puerarin and the pharmacodynamics of Shengma Gegen Decoction, the optimal decoction time of Pueraria lobata, and provide the theoretical basis for the clinical application of special decoction method of Pueraria lobata. METHODS The HPLC was adopted to determine the effects of decoction times of Pueraria lobata on the content of puerarin in Shengma Gegen Decoction with the following conditions:mobile phase of methanol-water (25:75), the flow rate at 1 mL· min^-1, the detection wavelength of 250 nm, and the column temperature at 30℃. The influence of decoction times of Shengma Gegen Decoction on the antitussive effect and analgesic effect was observed by ammonia induced coughing and hot plate method in mice. ICR mice (female 180, male 60) were randomly divided into 12 groups:blank control group, Ganmao Shufeng capsule group, aspirin enteric-coated tablet group, and 9 groups of Shengma Gegen Decoction with different decoction times (10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min). Animals were intragastrically administered at 0.02 ml·g^-1 for 7 days consecutively. One hour after last medication in antitussive groups, mice (half male and half female) were placed in a nebulizer to observe and record the coughing latency (the time to first cough) and the number of coughs in 2 min. And 1 hour after the last medication (female) in different analgesic groups, mice were placed on a (55±0.5)℃ intelligent hot plate instrument to record the times of licking or jumping since the animal feet firstly touched the hot plate (hot plate reaction latency). RESULTS In the range of 0.02-0.12 mg, it showed good linear relationship with peak area (r=0.999 6). The content of puerarin was 43.6 mg·g^-1, the highest after decoction for 15 min. Compared with the control group, Pueraria lobata after decoction for 10 min, 15 min, 20 min, 25 min prolonged cough latency (P<0.01,P<0.05). Pueraria lobata after decoction for 15 min and 20 min reduced the count of cough (P<0.05). Pueraria lobata after decoction for 10 min, 15 min and 20 min obviously prolonged the pain threshold (P<0.05). CONCLUSION The results of puerarin content show that, the content of puerarin is the highest after decoction for 15 min. Shorter decoction cannot sufficiently dissolute puerarin, while longer decoction will reduce the puerarin content. Pharmacodynamics results show that, after Pueraria lobata was decocted for 15 min, Shengma Gegen Decoction shows obvious antitussive and analgesic effects against exterior syndrome due to wind-cold, suggesting that decoction for 15 min is appropriate for decoction with Pueraria lobata.