通痹方药效部位提取工艺优化及抗炎镇痛作用研究

Study on the Optimization of Extraction Technology and Analgesic Effect of Tongbi Prescription

目的:优化通痹方中挥发油和醇溶性部位的提取工艺,研究通痹方对疼痛模型小鼠的镇痛、抗炎作用。方法:(1)采用L9(33)正交试验法,考察加水倍数、提取时间、浸泡时间对挥发油得率的影响;考察乙醇浓度、乙醇用量、提取时间对醇溶性部位得率的影响,筛选得到挥发油及醇部位最优提取工艺。(2)热板实验:将70只小鼠随机分为空白对照组、通痹方挥发油组(3mL/kg)、醇部位组(3g/kg)、挥发油+醇部位组(1.5mL/kg+1.5g/kg)、阳性对照组1(双氯芬酸钠,0.04g/kg)、阳性对照组2(对乙酰氨基酚0.3g/kg)、中药对照组(1.2g/kg)。每组每天灌胃给药1次,连续给药7天,采用智能热板仪测定末次给药0、15、30、60min后小鼠的痛阈值;(3)抗炎实验:取70只SPF级小鼠,分组及给药同上,末次给药30min后于右耳涂抹二甲苯致炎,比较耳肿胀度,观察通痹方的抗炎镇痛效果。结果:(1)最佳挥发油提取工艺为:加入10倍量水浸泡1h,提取时间10h;最佳醇部位提取工艺为:加入6倍量85%乙醇,提取时间3h;(2)在镇痛实验中,与空白对照组比较,醇部位组和挥发油+醇部位组末次给药0、15、30min后小鼠痛阈值均显著提高(P<0.05),挥发油组在给药0、30min后小鼠痛阈值明显提高(P<0.05);(3)在抗炎实验中,与空白对照组比较,通痹方醇部位组和阳性对照组小鼠致炎后耳肿胀度明显降低(P<0.05),其中醇部位组肿胀抑制率达到了33.4%。结论:筛选的提取工艺对通痹方挥发油和醇溶性部位有效成分提取率高、工艺稳定、方法简便可行。该方中挥发油与醇部位具有明显的镇痛效果并存在一定的抗炎作用,可作为其物质基础进行进一步研究,为通痹方制剂开发等方面提供理论支持。

Objective:To optimize the extraction process of volatile oil and alcohol-soluble parts in Tongbi Prescription;To observe the anti-inflammatory and analgesic effects of Tongbi Prescription.Methods:(1)Taking the water multiple,extraction time and soaking time as factors,L9(33)orthogonal test was designed to investigate the effects of on the yield of volatile oil.Taking the amount and concentration of ethanol,ethanol dosage and extraction time as factors,orthogonal test was adopted to investigate the effects of on the yield of alcohol-soluble fractions;(2)Analgesic experiment.Totally 70 mice were randomly divided into blank control group,the essential oil group(3 mL/kg),alcohol parts group(3 g/kg),the essential oil and alcohol parts group(1.5 mL/kg+1.5 g/kg),positive control group 1(diclofenac sodium,0.04 g/kg),positive control group 2(acetaminophen,0.3 g/kg),Chinese medicine control group(1.2 g/kg).They were given relevant medicine once a day intragastrically for consecutive 7 d.The response pain threshold(Tr)of mice was determined by intelligent hot plate instrument 0,15,30,60 min after last administration;(3)Anti-inflammatory experiment.Other 70 mice were collected,they were grouped and given medicine as above.60 min after last medication,Application of xylene to the right ear causes inflammation.To observe the anti-inflammatory and analgesic effect of Tongbi prescription.Results:(1)The best volatile oil extraction technology for Tongbi Prescription was reflux extracting for 10 heach time with 10 times the amount of water for 1 hour;The best ethanol extraction technology for Tongbi Prescription was reflux extracting for 3 hwith six times of 85%ethanol;(2)In analgesic experiment,compared with blank control group,Trwere increased significantly0,15,30 min after last medication(P<0.05);(3)In anti-inflammatory experiment,compared with the control group,the degree of paw swelling were decreased significantly(P<0.05),the inhibition rate of alcohol swelling reached 33.4%.Conclusion:The extraction method of volatile oil and alcohol fractionin Tongbi Prescription is simple and feasible.Tongbi Prescription has good analgesic effect and certain anti-inflammatory effect.The volatile oil and alcohol fraction can be further studied as its material basis,and provide theoretical support for the development of Tongbi Prescriptions.