【作者】 马艳艳；

【导师】 陈康；

【作者基本信息】 广州中医药大学 ， 中药学， 2008， 硕士

【摘要】 【目的】通过采用现代分析技术及药效对比,对化橘红的主要有效部位——挥发油、总黄酮、总多糖等进行组合规律研究,综合分析,阐述化橘红镇痛抗炎、化痰和抗氧化作用的科学内涵,为揭示其药效物质基础,探明主要有效部位或成分的协同作用提供实验依据。【方法】采用水蒸汽蒸馏法提取化橘红的挥发油、采用溶剂法提取分离总黄酮、多糖类等成分;采用分光光度法测定总黄酮含量、总多糖含量,RP—HPLC法测定柚皮苷含量,并用HPLC指纹图谱法对总黄酮进行质量监控;采用用小鼠氨水引咳法、小鼠气管酚红法、小鼠扭体法、热板法及小鼠耳肿胀法等进行药效研究,寻找与化橘红功效相关的有效部位或成分及其组合;采用均匀设计方法,优化化橘红有效部位或成分的组合,寻找其组合规律。【结果】1化橘红有效部位的研究对化橘红化学成分中的挥发油、总黄酮、总多糖及其他成分(如脂肪酸、香豆素等),进行提取分离。对总黄酮、总多糖部位进行的含量测定。总黄酮中以柚皮苷计,不得少于80.0%;总多糖用苯酚-硫酸法测定,以葡萄糖计,不得少于75.0%,确定为主要有效部位。2化橘红有效部位及组合的化痰、镇咳的药效研究化橘红各有效部位的化痰、镇咳药效研究表明,与空白组相比较,各有效部位组药效均较优,其差异均有统计学意义,呈现一定的量效关系。在化痰方面,以挥发油高剂量组效果较为显著;在镇咳方面,以挥发油高剂量组效果较为显著。利用均匀设计将各有效部位进行组合药效研究,结果表明:在化痰方面,通过方程理论选出较佳组合为挥发油高剂量组,与单因素考察结果相符合。由统计过程分析,挥发油与总黄酮、总多糖配伍可起到增效作用。与传统水煎液相比,挥发油组药效较优,其差异有显著性意义。从数据分析选出较佳组合11(组合11为各有效部位的高剂量组,各部位之间比例为挥发油:总黄酮:总多糖:其它成分=0.8:40:80:20),与传统水煎液比,组合11效果较优,差异有显著性意义;与挥发油高剂量组比较,组合11没有显著性差异。在镇咳方面,以挥发油与总黄酮起主要作用,根据数据优选出组合11,与传统水煎液比,组合11药效值与水煎液接近,差异没有显著性意义。3化橘红有效部位及组合的镇痛抗炎的药效研究通过热板法、扭体法测定化橘红各有效部位的镇痛作用,结果表明,与空白组比较,各实验组均有一定的镇痛的作用,以总黄酮高剂量组较为显著。利用均匀设计对各有效部位进行组合研究,优选的较佳组合为组合11。热板法中,与传统水煎液相比,总黄酮高剂量组在给药后30分钟药效较好,差异有统计学意义;其它时间段差异没有显著性意义;从组合中优选出的组合11,与传统水煎液比较,组合没有显著性差异。扭体法中,与传统水煎液比较,总黄酮高剂量组及组合11药效值接近,差异没有显著性意义。通过二甲苯致小鼠耳肿胀法、蛋清致小鼠足趾肿胀法测定化橘红各有效部位的抗炎作用,结果表明,与空白组比较,各有效部位药效较优,差异有显著性意义,以总黄酮高剂量组效果较为显著。利用均匀设计对各有效部位进行组合研究,初步优选的较佳组合为组合4、6;与传统水煎液相比,各组药效相当,差异均无统计学意义。验证组合11在致炎后30分钟时药效较为显著,与传统水煎液相比,差异有统计学意义。其它时间段差异无显著性意义。4化橘红有效部位及组合的体外抗氧化的药效研究通过邻苯三酚法和邻二氮菲法体外比较化橘红各有效部位的抗氧化作用,通过抑制率比较表明,各试样均有一定的抗氧化性。邻苯三酚法中单因素考察以总黄酮高剂量组效果显著。邻二氮菲法中单因素考察以其它成分高剂量组效果显著。利用均匀设计对各有效部位进行组合研究,邻苯三酚法中,优选出总黄酮高剂量组,从对邻苯三酚自氧化抑制率来分析,组合中以实验组11效果显著,均高于传统水煎液组;邻二氮菲法中,优选出总黄酮高剂量组,从对羟自由基清除ΔA来分析,组合中以实验组4(组合4各有部部位之间比例为挥发油:总黄酮:总多糖:其它成分=0.2:40:80:10)效果显著,但以传统水煎液的抗氧化作用最为突出。5传统水煎液药效学的研究中药主要是以水煎液的形式应用于临床,起到防病治病的目的。本课题通过传统水煎液与空白对照组比较表明,有一定的化痰平喘及镇咳作用;有一定的镇痛抗炎作用:有较强的抗氧化作用。【结论】1.化橘红有效部位的研究通过制订含量限度标准来控制所用有效部位的质量,可保证药效考察时药物质量与药效的均一性。2.化橘红有效部位及组合的化痰、镇咳的药效研究在镇咳化痰方面,化橘红各有效部位考察中均以挥发油高剂量组效果较为显著。组合中以组合11较为显著,化痰药效中,与传统水煎液比较,组合11及挥发油高剂量组药效较好,差异均有显著性意义;镇咳实验中,与传统水煎液比较,药效接近,差异没有统计学意义。3.化橘红有效部位及组合的镇痛抗炎的药效研究在镇痛抗炎方面,化橘红各有效部位均有一定的药效作用。以总黄酮高剂量组效果较为显著。各有效部位组合研究表明,在镇痛实验中,组合11效果较为显著,与传统水煎液比较,组合11药效接近,差异没有显著性意义;在抗炎实验中,优选组合为总黄酮高剂量组、总多糖高剂量组,与传统水煎液比较,药效接近,差异没有显著性意义。组合11在致炎后30分钟药效较为显著,与传统水煎液相比,差异有统计学意义。其它时间段差异无显著性意义。4.化橘红有效部位及组合的体外抗氧化的药效研究通过邻苯三酚法和邻二氮菲法两种方法体外比较化橘红各有效部位的抗氧化作用结果表明,各试样均有一定的抗氧化性。邻苯三酚法中以总黄酮高剂量组效果显著;邻二氮菲法中以其它成分高剂量组效果显著。组合结果表明,邻苯三酚法中,优选出总黄酮高剂量组,从对邻苯三酚自氧化的抑制率来分析,组合中以实验组11效果显著,均高于传统水煎液组;邻二氮菲法中,优选出总黄酮高剂量组,从对羟自由其清除ΔA分析,组合中以实验组4效果显著,但以传统水煎液的抗氧化作用最为突出。5.传统水煎液药效学的研究传统水煎液有一定的合理性,但由于其中挥发油含量损失比较多,对化痰的疗效不如其它一些实验组;有一定的镇咳、镇痛抗炎作用;但在抗氧化作用方面效果相对比较显著。更多还原

【Abstract】 Objective:By adopting modern analyzing techniques and drug effect comparision, studying combination rules of the efficiency parts such as volatile oil、total flavone、total polysaccharides, etc. By analyzing integrately, we expound the scientific meaning of exocarpium’s effect on easing pain and resisting inflammation、turing phlegrn and stoping asthma, anti-oxygenation, which can afford experimental gists for revealing material bases of efficacy and proving synergism the main effective parts and components.Mothod:Using steam distillation method to extract the volatile oil and solvent mothod to extract total flavone and polysaccharide components; Using Spectrophotometry to determine the content of totle flavone and amylose, the RP-HPLC method to mensurate content of naringin , using ammonia causing rat cough method、injecting hydroxybenzene red to rats’ trachea、making rats wresting mothed、heating broad method, making rats’ ear swelling method to study the drug effect and search the feasible effect parts and their combination of exocarpium;we also introduce the equality designing method to optimize the most effective part or their combination and look for the combination rules.Result:1. research on exocarpium’s effect partsSeparating the volatile oil, total flavone and amylose and other components (such as fatty acid and coumarin, etc)from chemical components of exocarpium. Determine the content of total flavone and amylose , the total flavone is calculateed by naringin, not fewer than 80%; use the phenol-vitriol method to calculate the glucose, the part which contains more than 75% is the determined as the main effective part.2. Studies on eliminating phlegm and relieving asthma , antitussive effect of effective parts and combination of exocarpium Compared with the blank team, studies on eliminating phlegm and relieving asthma、antitussive effect of each effective part show that every effective part has statistic significance and it presents certain dose-effect relationship. As to eliminating phlegm effect, the high dose group of volatile oil is the best;and it is also the best team in relieving cough. Using uniform design to study effects of effective parts combination, the results shows:As to the eliminating phlegm and relieving asthma effect, using the equation theory to choose the best combination, the high dose of volatile oil team, which is accord with the single-dose investigation result. By analyzing the statistic process, compatibility of volatile oil、total flavone and total polysaccharides can enhance effect. Compared with traditional water decoction, there is significant distinction with volatile oil team;We choose the better combinatiom group 11(combination 11 is composed of all high-dose of exocarpium, the equare is 0.8:40:80:20) by analyzing statistics and compare with traditional water decoction, there is significant distinction of combination ll;Compared with high-dose team of volatile oil team, combination 11 has no significant distinction.. As to the antitussive effect, volatile oil and total flavone play main roles. Based on the data, we choose combinatiom 11, compared with traditional water decoction, there is no significant distinction with group 11.3. Studies on analgesic and anti-inflammatory effect of effective parts and combination of exocarpiumUsing hot plate test and twisting method to determine analgesic effect of each effective part of exocarpium, the result is that compared with blank group, each experiment team have certain analgesic effect, among them . The combinatiom 11 has the significant effect. Using uniform design to study combination of every effective part, the best combination is high-dose of total flavone. In the hot plate test, compared with traditional water decoction, high-dose of total flavone group have statistic significance 30 minites after taken drugs;other periods have no significant distinction;The better combinatiom 11, compared with water decoction, has no significant difference. In the twising method, compared with water decoction, total flavone and combination 11 of high-dose team have no significant difference. With xylene inducing rats’ ears swelling method, and albumen inducing rats’ toes swelling method, we determine anti-inflammatory effect of each effective part of exocarpium,the result shows that compared with blank group, each team has significant distinction, and the most significant team is combination 4 and 6;compared with traditional water decoction, both have no significant difference.4. Studies on antioxidation of effective parts and combination of exocarpiumComparing antioxidation of every effective parts of exocarpium by pyrogallol atoxidation method and o-Phenanthroline method vitroly, comparesion of inhibition rate shows that every sample has certain oxidation resistance. Single factor investigation in pyrogallol atoxidation method,the high-dose of total flavone team has the most significant effect. Single factor investigation in o-Phenanthroline method, high-dose of other teams have better effect. Studying effective parts combination by uniform design , with pyrogallol atoxidation method, we choose high-dose of total flavone team, by analyzing inhibition rate, group 11 has the most significant effect, all are higher than the traditional water decoction team;with o-Phenanthroline method, we choose the high-dose of total flavone team, analyzingΔA, group 4 (combination 4 is composed of low-dose of volatile oil、tolal flavone of high-dose、high-dose of total polysaccharide、middle-dose of other contents) have the significant effect, but antioxidation of the regular water decoction is the best.5. Studies on pharmacodynamics of traditional water decoctionDrugs are used in clinic mostly by water decoction form, and make the effect of preventing and treating diseases. The research compare traditional water decoction group with other experimental teams, it shows that it has certain eliminating phlegm and relieving asthma、antitussive effect; it has certain analgesic and anti-inflammatory effect; it has strong antioxidation.Conclusion:1. Studies on effective parts of exocarpiumBy content determination controlling quality of drugs, which ensure homogeneity of quality and efficacy.2. Studies on eliminating phlegm and relieving asthma、antitussive effect of effective parts and combination of exocarpiumAs to eliminating phlegm and antitussive effect areconcerned, the high-dose of volatile oil group has the most instinctive effect in every investigation. Of the combination, the group 11 is the most instinctive,Compared with traditional water decoction, combination 11 and high-dose of volatile oil team both have instinctive distinction in eliminating phlegm efficacy experiment; in antitussive experiment, Compared with traditional water decoction, there is no statistic signification.3. Studies on analgesic and anti-inflammatory effect of effective parts and combination of exocarpiumAs to the analgesic and anti-inflammatory effect, each effective part has certain efficacy, but the high-dose total flavone group is the most effective group. the study of combination shows that in the analgesic effect experiment, combination 11 are most effective, but compared with water decoction, it has no significant difference; in the anti-inflammatory effect experiment, the super combination is the high-dose of total flavone group and total polysaccharide, compared with water decoction, the two teams have no significant distinction.4. Studies on antioxidation of effective parts and combination of exocarpiumComparing the antioxidation effect of each effective part exocarpium by pyrogallol method and phenanthroline method show that each sample have certain antioxidation. In pyrogallol method, high-dose of total flavone group have the most instinctive effct;in phenanthroline method, high-dose of other team are most instinctive. Combination study shows that in pyrogallol method, we choose the high-dose of total flavone team, by analyzing inhibition rate, group 11 is the most instinctive, and it is better than water decoction; by phenanthroline method, we choose the high-dose of total flavone team, analyzingΔA, group 4 (the proportion of combination 4 is, volatile oil、tolal flavone、total polysaccharide other contents, equare0.2:40:80:10) is most distinctive, but as to the antioxidation, the water decoction is most effective.5. Studies on pharmacodynamics of traditional water decoctionTraditional water decoction may have some rationality,but because of morevolatile oil is lost than others, the on eliminating phlegm and relieving asthmaeffect is not so good as other groups; it has certain antitussive effect andanalgesic and anti-inflammatory effect; the most instinctive effect is inantioxidation.更多还原