长形肉豆蔻药用相关研究

【作者】 王远志；

【导师】 贾天柱；

【作者基本信息】 辽宁中医药大学 ， 中药学， 2009， 博士

【摘要】 目的:肉豆蔻（Mystica fragrans Houtt.）为《中国药典》2005版收载的中药,一般称圆形肉豆蔻。具有温中、下气、消食、固肠等作用,用于心腹胀痛、虚泻冷痢、呕吐、宿食不消等症。而圆形肉豆蔻肉豆蔻醚含量较高,有一定毒性,因此寻找毒性更小,作用相近的类似品显得格外重要。所以本研究在中医药理论的指导下,应用现代科学技术手段,首次对长形肉豆蔻（Myristica argentea Warb.）进行全面深入的研究,制定炮制工艺,对其化学成分与圆形肉豆蔻进行比较研究,制定质量标准。进一步考察长形肉豆蔻挥发油的制剂和长形肉豆蔻挥发油对人癌细胞体外增殖的影响等药理作用,以期扩大药源,为长形肉豆蔻的研究开发和临床应用提供可靠的依据,为中药现代化研究做出有益的尝试。材料与方法:1.药材:圆形肉豆蔻药材购于沈阳市药材公司,经辽宁中医药大学翟延君教授鉴定为肉豆蔻科植物肉豆蔻（Mystica fragrans Houtt.）的干燥成熟种仁。长形肉豆蔻药材购于安徽亳州,经辽宁中医药大学翟延君教授鉴定为肉豆蔻科植物长形肉豆蔻（Mysticaargontea.Warb.）的干燥成熟种仁。2.方法:通过常规观察确定其外观性状。通过薄层色谱法及高效液相色谱法对两种肉豆蔻挥发油含量及成分进行比较分析。利用现代层析技术和分离方法对长形肉豆蔻的挥发油和乙醇提取物进行了分离。采用GC/MS方法分析长形肉豆蔻和圆形肉豆蔻挥发油。采用电感耦合等离子体质谱（ICP-MS）法测定样品中多种元素的含量。通过对两种肉豆蔻的面裹煨、滑石粉煨、麸煨、泡后麸煨四种炮制方法研究确认长形肉豆蔻的炮制方法及采用综合加权评分的方法确定其工艺。参考中国药典2005年版一部肉豆蔻标准,并结合三个市售地长形肉豆蔻及麸煨长形肉豆蔻的质量研究结果提出长形肉豆蔻及麸煨长形肉豆蔻的标准。为使长形肉豆蔻挥发油发挥效用,根据药物本身的性质、剂型和工艺的要求,以自乳化系统为载体,将其制成软胶囊,用总体液平衡反向透析法对长形肉豆蔻挥发油自乳化软胶囊的体外溶出过程进行考察。为了比较长形肉豆蔻及圆形肉豆蔻的药用价值,本文通过两种肉豆蔻挥发油直接作用于肿瘤细胞,进行体外抗肿瘤试验。结果:1.长形肉豆蔻和圆形肉豆蔻药材外观明显不同,因为长形肉豆蔻的长度为3～4 cm,而圆形肉豆蔻长度为2～3cm。2.两者挥发油含量及其油中成分的含量有较大差异,长形肉豆蔻挥发油含量低于圆形肉豆蔻,其挥发油中止泻成分甲基丁香酚和异甲基丁香酚的含量也略微低于圆形肉豆蔻,毒性成分黄樟醚高于圆形肉豆蔻,而肉豆蔻醚在长形肉豆蔻中不易被液相检测出,含量甚少。经提取分离共得到13个化合物,通过经典的化学方法和波谱学技术鉴定了结构,为木脂素类化合物。其中,6个化合物为种内首次分离,1个为科内首次分离。采用GC/MS方法分析长形肉豆蔻和圆形肉豆蔻挥发油,鉴定出长形肉豆蔻生品32个化合物,其炮制品29个化合物,减少了α-蒎烯、α,α,4-三甲基苯甲醇、3-甲基-6-异丙基-2环己烯-1-醇3种化合物。圆形肉豆蔻生品35个化合物,其炮制品36个化合物,增加的化合物为乙酸龙脑酯。两种肉豆蔻在炮制前后无机元素的含量情况:Mg、Ca、Fe、Na的含量在炮制后有所升高;K有所降低。而且圆形肉豆蔻中的必需元素含量比长形肉豆蔻中的含量多。两种肉豆蔻炮制前后的有毒有害重金属元素含量基本没有变化。3.通过对两种肉豆蔻的四种炮制方法研究,可以看出两种肉豆蔻的泡后麸煨法优于其他方法,而长形肉豆蔻含的肉豆蔻醚很少,所以初步认为长形肉豆蔻的毒性小于圆形肉豆蔻。进一步对长形肉豆蔻的泡后麸煨工艺研究,在长形肉豆蔻炮制工艺中,选用L9（3⁴）正交表设计实验,以止泻有效成分甲基丁香酚和异甲基丁香酚的含量为主要指标,以黄樟醚为次要指标,进行综合评分,优选出长形肉豆蔻最佳炮制工艺为加水浸泡6小时,麸煨温度160～170℃,麸煨时间25min,药麸比100:40。4.本研究完成了长形肉豆蔻质量标准及麸煨长形肉豆蔻质量标准及起草说明。建立了长形肉豆蔻中甲基丁香酚、异甲基丁香酚、黄樟醚的含量测定方法。并进行了显微鉴别、薄层鉴别、挥发油、水分、总灰分、酸不溶性灰分等测定。挥发油照挥发油测定法（中国药典2005附录XD）测定,不得少于3.0%。水分照水分测定法（中国药典2005附录ⅨH第二法）测定,不得过6.0%。总灰分照总灰分测定法（中国药典2005附录ⅨK）测定,不得过5.0%。酸不溶性灰分照酸溶性灰分测定法（中国药典2005附录ⅨK）测定,不得过1.0%5.长形肉豆蔻挥发油,25℃在油酸乙酯中平衡溶解度较大;在Tween80等亲水性表面活性剂和Transcutol P等助溶剂中的平衡溶解度较大。亲脂性较好。确定了一个最优的油、表面活性剂和助溶剂配伍,即油相油酸乙酯、非离子表面活性剂吐温-80、助溶剂Transcutol P。温度、溶液的流动形态、pH和体积等外在因素对自乳化效率的影响实验结果显示,溶液体积对自乳化性能无显著性影响;在一定范围内升高温度、加大搅拌速度可以缩短体系的自乳化时间;在水、0.1 mol·L^-1 HC1、pH 7.4 PBS三种介质中,0.1 mol·L^-1 HCI使自乳化时间稍微延长。SEDDS（自乳化药物传递系统）中,药物的加入、非离子表面活性剂和助溶剂的比例都对SEDDS的乳化性能有影响。以油酸乙酯、吐温-80、Transcutol P作为油相、乳化剂、助溶剂绘制的假三元相图,同时根据乳化后的粒径大小及自乳化效率,确定了一个最优处方比例:长形肉豆蔻挥发油:油酸乙酯:吐温-80:Transcutol P（5:45:50:5）（w）。用总体液平衡反向透析法对长形肉豆蔻挥发油自乳化软胶囊的体外溶出过程进行了初步考察,其溶出曲线最符合Hixson-Crowell经验方程。空白SEDDS和长形肉豆蔻挥发油SEDDS的动电电位均为0 mV,但长形肉豆蔻挥发油SEDDS的粘度大于空白SEDDS。同时,药物的加入使得体系的自乳化区域明显减小,而乳化后所得乳剂的油水间界面张力降低,自乳化区内各组成的乳化效率增强,所得乳剂粒径减小。长形肉豆蔻挥发油SEDDS的稳定性考察试验表明,SEDDS遇强光照射会导致长形肉豆蔻挥发油含量降低,故须避光保存;在避光留样试验中,长形肉豆蔻挥发油SEDDS的外观、药物含量、乳化速度、乳化后所得乳剂的平均粒径等各项指标基本无变化,稳定性良好。长形肉豆蔻挥发油SEDDS经溶液以不同比例稀释后,4 h内溶液稳定,未见药物析出,保证了药物在动物体内的生物利用度。6.主要研究考察了两种肉豆蔻挥发油对人癌细胞HepG-2、SGC-7901、KB和KB的体外增值抑制作用,结果显示:圆型和长型肉豆蔻挥发油对HepG-2、SGC-7901、KB细胞的体外增殖均具有一定的抑制作用,抑制强度呈一定的剂量依赖性。结论:根据长形肉豆蔻和圆形肉豆蔻的化学成分、炮制工艺、质量标准、药理和制剂等全面研究,长形肉豆蔻可以作为肉豆蔻的一种使用。更多还原

【Abstract】 Purpose:Nutmeg that carried in"Chinese Pharmacopoeia" 2005 versions as a Chinese herbal medicine with warming middle,lower qi functions was generally called the round nutmeg.It is used for the bellyache,dysentery,vomit symptoms.Myristicin and safrol which had certain toxicity in the nutmeg were higher.So the search of another Chinese herbal medicine with similar functions seemed to be particularly important.We wanted to Proceed the research under the Chinese medicine theories by the Modern science techniques.For the first time,we proceed the in-depth research of Myristica argentea(Myristica argentea Warb.),established optimal processing method and quantity standard,compared with chemistry compositions of round nutmeg,further investigated the volatile oil from processed products and pharmacological functions that influenced the cancer cell multiplication outside the body.The subject Provided the dependable basis for the developing research and clinical application of the Myristica argentea,gived a useful attempt for Chinese herbal medicine modernization.Material and method:1.medicinal materials:The round nutmeg(Mystica fragrans Houtt.) bought in Shenyang Medicine Material Company,identified by prof ZhaiYanJun from Liaoning University of Chinese Medicine.It was Dry mature kernel of the nutmeg from the nutmeg section.The Myristica argentea(Mystica argente Warb.) bought in Bozhou City Anhui Province,identified by prof ZhaiYanJun from Liaoning University of Chinese Medicine.It was Dry mature kernel of the Myristica argentea from the nutmeg section. 2.method:Confirmed the external appearance through general observations.Analysed the compositions of volatile oil from two kinds of nutmegs by TLC and HPLC methods. Separated the compositions of volatile oil from Myristica argentea and alcohol extract with the modern layer-analysis technique and method.Analysed the compositions of volatile oil from Myristica argentea by GC/MS methods.Determined Contents of various chemical elements by ICP-MS.Confirmed optimal processing method and technic of Myristica argentea by analyzing different processed products of Myristica argentea.Established quantity standard of Myristica argentea and its processed product according to "Chinese Pharmacopoeia" 2005 versions.Examine preparation evaluation and pharmacokinetic study of simvastatin self-emulsifying capsules.Compared The anti- tumor functions in tumor cells directly with two kinds of volatile oil.Results:1.The Myristica argentea was very different from the round nutmeg in length.2.The contents and compositions of two kinds of nutmegs were different.Myristica argentea had less volatile oil,less active compositions(methyleugenol,methylisoeugenol),more toxic composition(safrol),minimum amount of myristicin.3.The results showed that the optimal processing condition was that:Nutmeg was soaked with water for 6hr,then bran at 160-170℃for 25min.every 100kg nutmeg with 40kg wheat.4.This subject established quantity standard of Myristica argentea and the product roasted with wheat,and gived drafting explanation.5.According to SEDDS system,optimal prescription of self-emulsifying capsule was that: Volatile oil:Ethyl Oleate:Tween 80:Transcutol P(5:45:50:5)(w).And the dissolving curves matched up to the "Hixson-Crowell" formula.Self-emulsifying capsules were steady with good appearance,appropriate emulsifying speed,appropriate particle diameter,favorable bioavailability.6.The volatile oil from two kinds of Nutmegs had the inhibiting function to the multiplication of cancer cells(HepG-2,SGC-7901,KB) outside the body.And the inhibiting intensity depended on the dosage.Conclusion:Myristica argentea Can be used as medicinal materials of nutmeg herb with the research of Chemistry composition,processing method,quantity standard,pharmacological function, preparation studies.更多还原