【作者】 赵锐；

【导师】 马伟；

【作者基本信息】 黑龙江中医药大学 ， 生药学， 2019， 硕士

【摘要】 柳属植物作为中国许多省市的绿色植被作物之一,在我国分布广泛、资源丰富,不仅可以作为建筑材料、做家具,还可以制成保健品、入药用。但柳属植物的分类及其药用成分及疗效的探究却是我国乃至世界的难题。本文对文献综述、柳属植物的鉴定及其中有效成分的含量进行研究。文献综述:通过查阅文献,对柳属植物的化学成分及药理作用进行总结及综述,为柳属植物的鉴定及成分测定提供理论支持。本研究以哈尔滨植物园及黑龙江中医药大学校园内所采集的部分柳属植物叶片为材料,分别对柳属植物的分类及总黄酮、原花青素质量浓度测定进行了研究,并运用HPLC法对选定样品的木犀草苷的质量浓度进行测定,观测其变化并进行分析。本实验先对所选取的柳属植物样本进行宏观的考察,观测其外观性状,再对其叶片进行显微观测及扫描电镜的检测,结合以上三点来鉴定柳属植物的类别。为了使采集的柳属植物叶片中的有效成分能够提取的更加充分和均匀,我们对采集的叶片进行了烘干及粉碎,并过30目筛网。本实验用乙醇溶液为提取溶剂,提取了柳属植物树叶样本中的总黄酮及原花青素,然后利用紫外-可见分光光度法分别检测了样品树叶中总黄酮及原花青素的质量浓度。样品中木犀草苷的提取我们选择乙醇为提取溶剂,以黑龙江中医药大学校园内采集的旱柳树叶为原料,采用L₉（3⁴）的正交实验优化木犀草苷的提取工艺,并采用优化的提取方法对哈尔滨植物园所采集的样品进行了提取,以木犀草苷为已知质量浓度的成分对样品进行指纹图谱的分析。实验结果如下:1.通过对采集的柳属植物的外观性状以及显微观察和扫描电镜检测的结果可以鉴定所采集的样品为沙柳、旱柳、三蕊柳、垂柳及旱垂柳。2.用乙醇溶液回流提取法对粉碎过筛后的树叶粉末进行提取,精密量取样品4g,加入浓度为50%的乙醇120m L,回流提取1h,通过紫外-可见分光光度法分别测定样品的总黄酮和原花青素质量浓度。测定结果发现:（1）采集的样品中沙柳叶中的总黄酮含量最高,三蕊柳叶片中的总黄酮含量最低;各品种的柳属植物总黄酮质量浓度在6、7、8、9月份变化较大,变化趋势基本都是先降低、再增高、再增高,7月份的总黄酮质量浓度最低,6、9月份总黄酮质量浓度相较于各月较高;（2）各品种的柳属植物原花青素质量浓度的变化趋势基本都是随着月份的增加而增加,故9月份各品种的原花青素含量达到最高,其中旱柳的最高,垂柳的最低。3.取黑龙江中医药大学校园内采集已确认种属的旱柳叶片为原料,对柳属植物叶片中木犀草苷成分的高效液相条件进行研究,最后确定紫外检测波长为350nm,流动相为乙腈-0.1%乙酸水溶液,梯度洗脱程序为:0～15min,乙腈10%～20%;15～40min,乙腈20%;40～50min,乙腈20%～30%;进样量10μL,柱温25℃。按照上述条件对样品进行洗脱,得到木犀草苷的相对含量,通过对木犀草苷的提取条件进行单因素考察,选择乙醇浓度、提取时间、料液比、提取次数作为考察的影响因素,进行正交实验,对柳树叶中木犀草苷的提取方法进行优化,从所得数据可知:各因素中,提取次数为影响旱柳叶中木犀草苷含量提取的主要因素,提取时间与乙醇浓度之。优化后的提取工艺为:提取液乙醇浓度为80%,提取时间4h,料液比1:25,提取次数为3次。4.按照最佳提取工艺对旱垂柳及垂柳叶片进行提取,利用软件中药色谱指纹图谱相似性评价系统（2012版本）对样品进行分析,结果表明:旱垂柳及垂柳的树叶提取液指纹图谱相似性均大于0.9,相似性较高,其中旱垂柳中有4个共有峰,确定一个成分,即木犀草苷,且面积最大;垂柳中有7个共有峰,确定一个成分即峰面积最大的木犀草苷。更多还原

【Abstract】 As one of the green vegetation crops in many provinces and cities in China,Salix is widely distributed and rich in resources.It can be used not only as building materials,furniture,but also as health products and medicine.However,the classification of Salix and its medicinal ingredients and therapeutic effects are difficult problems in China and the world.This paper reviews the literature review,the identification of Salix and the content of active ingredients in it.Literature review:Through reviewing the literature,the chemical composition and pharmacological effects of Salix are summarized and reviewed,which provides theoretical support for the identification and composition determination of Salix plants.In this study,some of the leaves of Salix plant collected from the Harbin Botanical Garden and Heilongjiang University of Traditional Chinese Medicine were used as materials to classify the willow plants and determine the total concentration of flavonoids and proanthocy anidins,and select samples by HPLC.The mass concentration of luteolin was measured,and the change was observed and analyzed.In this experiment,the selected specimens of Salix were firstly examined,and their appearance traits were observed.Then the microscopic observation of the leaves and the scanning electron microscopy were carried out.The above three points were combined to identify the species of Salix.In order to make the active ingredients in the leaves of the collected Salix plants more fully and evenly extracted,the collected leaves were dried and pulverized and passed through a 30 mesh screen.In this experiment,the total flavonoids and proanthocyanidins in the leaves samples of Salix were extracted with ethanol solution as extracting solvent.Then the mass concentration of total flavonoids and proanthocyanidins in the leaves of the samples were detected by UV-vi sible spectrophotometry.Extraction of luteolin in the sample we chose ethanol as the extraction solvent,and used the L9(34)orthogonal experiment to optimize the extraction process of luteolin,and optimized it.The extraction method was used to extract the samples collected by Harbin Botanical Garden,and the fingerprints of the samples were analyzed with luteolin as a component of known mass concentration.The experimental results are as follows:1.Through the appearance traits of the collected Salix plants and the results of microscopic observation and scanning electron microscopy,the collected samples can be identified as Salix cheilophila,Salix matsudana Koidz,Salix triandra L,Salix babylonica and Salix matsudana.2.Using the ethanol solution reflux extraction method to extract the pulverized leaf powder,accurately measure the sample 4g,add 120mL of ethanol with a concentration of 50%,reflux for 1 h,and determine the total sample by UV-vi sible spectrophotometry.Flavor and proanthocyanidin mass concentration.The results showed that:(1)The total flavonoids content in the leaves of Salix psammophila was the highest,and the total flavonoids content in the leaves of S.chinensis was the lowest.The total flavonoids concentration of Salix in each variety was 6,7,8,and 9.The monthly changes are relatively large,and the trend of change is basically reduced first,then increased,and then increased.The total flavonoids concentration in July is the lowest,and the total flavonoids concentration in June and September is higher than that in each month;(2)The variation trend of the original anthocyanin concentration of Salix is basically increased with the increase of the month.Therefore,the proanthocyanidin content of each variety reached the highest in September,with the highest of the Salix matsudana Koidz and the lowest of the Salix babylonica.3.The high-efficiency liquid phase conditions of the luteolin in the leaves of Salix were studied on the campus of Heilongjiang University of Traditional Chinese Medicine.The UV detection wavelength was 3 50 nm and the mobile phase was determined.Acetonitrile-0.1%acetic acid aqueous solution,gradient elution procedure:0～15min,acetonitrile 10%～20%;15～40min,acetonitrile 20%;40～50min,acetonitrile 20%～30%;injection volume 10μL,column temperature 25℃.The samples were eluted according to the above conditions to obtain the relative content of luteolin.The factors of ethanol concentration,extraction time,ratio of material to liquid and extraction times were selected as factors influencing the extraction conditions of luteolin.Orthogonal experiment was carried out to optimize the extraction method of luteolin in willow leaves.From the data obtained,the extraction times were the main factors affecting the extraction of luteolin in the leaves of the willow,the extraction time and the ethanol concentration.The optimized extraction process was as follows:the ethanol concentration of the extract was 80%,the extraction time was 4 h,the ratio of material to liquid was 1:25,and the number of extractions was 3 times.4.The leaves of Salix matsudana Koidz and Salix babylonica were extracted according to the best extraction process.The samples were analyzed by the software traditional Chinese medicine chromatographic fingerprint similarity evaluation system(2012 version).The results showed that the similarity of the fingerprints of the leaves of the Salix matsudana Koidz and Salix babylonica,all of them are greater than 0.9,and the similarity is higher.Among them,there are 4 common peaks in the Salix matsudana Koidz,and one component is determined,that is,luteolin,and the area is the largest;there are 7 common peaks in the Salix babylonica,and one component is the largest rhinoceros--Glycosides.更多还原