【作者】 李晓娟；

【导师】 付玉杰；

【作者基本信息】 东北林业大学 ， 植物学， 2012， 博士

【摘要】 香鳞毛蕨是一种具有特殊香气的多年生草本植物,其特点是老叶宿存,系鳞毛蕨科鳞毛蕨属。香鳞毛蕨主要分布于亚洲温带、欧洲和北美洲,通常生长在岩屑坡和岩石的裂缝中。特别是在五大连池分布的香鳞毛蕨,其只生长于火山喷发后所形成的熔岩环境中,当地居民用它来治疗多种皮肤病(牛皮癣、痤疮、皮疹和皮炎),民间验方利用香鳞毛蕨已有几十年的历史。此外,香鳞毛蕨还具有抗菌、镇痛、抗氧化、抗肿瘤和治疗类风湿性关节炎的作用。本文对香鳞毛蕨的精油和间苯三酚类成分进行了分离提取,并对纯化出的成分进行了抗菌活性考察。研究结果如下：1.建立了香鳞毛蕨精油以及4种间苯三酚类成分的分析方法。(1) GC-MS结合保留指数对香鳞毛蕨精油成分进行了鉴定,共鉴定了18个成分,占精油总量的89.65%,结果表明香鳞毛蕨精油富含倍半萜类化合物。(2)确定了RP-HPLC同时柃测香鳞毛蕨中4种间苯三酚类成分的色谱条件。色谱柱为ODS C18色谱柱(100mm×4.6mm.i.d.,2.6μm),流动相为乙腈-水-甲酸,柱温为25℃。本方法重复性好、准确度高、稳定性强,适用于香鳞毛蕨及其他植物中香鳞毛蕨素等间苯三酚类成分的分析及检测。2.采用无溶剂微波萃取技术对香鳞毛蕨中的精油进行了提取,中心组合设计结合响应而分析优化提取工艺,考察了提取时间、辐射功率和含水率对提取率的影响,确定了最佳的提取工艺参数：提取时间：34min辐射功率：500w含水率：50%在上述条件下对香鳞毛蕨精油进行提取,精油的平均提取率为0.33%,其R.S.D.为5.19%。响应面分析结果表明,在各影响因素中,提取时间对香鳞毛蕨精油提取率的影响最大,其次是辐射功率,最后是含水率。提取率与试验因素之间并不是简单的线性关系,对香鳞毛蕨精油提取率有较强交互作用的是提取时间和辐射功率。无溶剂微波提取法实现了香鳞毛蕨精油的高效提取,精油提取后的材料可继续用于其它目标成分的提取。3.采用负压空化提取法对香鳞毛蕨中间苯三酚类成分进行提取。考察了液固比、提取时间、空气流量、压力、提取次数等因素对负压空化提取过程中间苯三酚类成分提取率的影响,确定了最佳工艺参数：液固比：10：1(mL/g)提取时间：20min空气流量：30mL/min压力：-0.04～-0.05MPa提取次数：3次在上述条件下,香鳞毛蕨素、绵马酚、绵马素PB和绵马素BB的提取率均达到94%以上。由于负压空化提取技术以负压为动力,提取过程在密闭体系和常温操作系统下进行,所以这一方法具有节能效果好,无有机溶剂释放,有效成分无高温降解发生等优点,适用于香鳞毛蕨以及其他植物中活性成分的高效提取。4.对香鳞毛蕨中绵马酚、绵马素PB、香鳞毛蕨素和绵马素BB的分离富集方法进行了研究。(1)采用树脂柱吸附技术对香鳞毛蕨中的绵马酚和绵马素PB的分离富集进行了研究,洗脱流程为：5BV30%乙醇溶剂用于移除杂质,10BV50%乙醇溶剂被用于收集绵马酚,15BV70%乙醇溶液用来富集绵马素PB,沈脱流速为2BV/h。(2)采用树脂柱色谱技术对香鳞毛蕨中的香鳞毛蕨素和绵马素BB的分离富集进行了研究,对HPD-826,AB-8和HPD-600三种树脂的分离性能进行了考察,结果表明AB-8树脂对香鳞毛蕨素和绵马素BB的分离性能较好。确定了最佳洗脱流程为：10BV70%乙醇溶剂用于移除杂质,30BV80%乙醇溶剂被用于收集香鳞毛蕨素,20BV95%乙醇溶液用来富集绵马素BB,洗脱流速为6BV/h。(3)经过树脂富集后,绵马酚的含量从1.40mg/g增加到了10.4mg/g,提高了7.4倍；绵马素PB在产品中的含量从4.9mg/g增加到了41.8mg/g,增长了8.52倍。他们的回收率分别为85.73%和78.41%。采用AB-8树脂柱色谱动态分离后,香鳞毛蕨素的含量从4.10mg/g增加到了34.4mg/g,提高了8.39倍：绵马素BB在产品中的含量从9.5mg/g增加到了56.9mg/g,增长了5.99倍。他们的回收率分别为91.22%和75.64%。5.采用中压硅胶柱层析及重结晶技术对香鳞毛蕨中绵马酚、绵马素PB、香鳞毛蕨素和绵马素BB进行了纯化,并采用一系列方法对纯化所得化合物结构进行了确认。(1)确定了香鳞毛蕨样品中绵马酚、绵马素PB、香鳞毛蕨素和绵马素BB的中压硅胶柱层析分离方法以及重结晶条件。经HPLC检测表明绵马酚、绵马素PB、香鳞毛蕨素和绵马素BB的纯度依次为96.2%,95.1%,97.7%和95.3%。回收率依次为78.26%,63.27%,73.24%和65.63%。(2)经纯化得到具有一定纯度的化合物,通过理化性质、紫外(UV)、质谱(ESI-MS-MS)与核磁共振谱(’H-NMR和13C-NMR)技术,结合文献报道信息确定所得产品为目标化合物。6对香鳞毛蕨不同溶剂萃取物、纯化得到的4种间苯三酚类成分以及香鳞毛蕨精油的抗菌活性进行了初步研究,研究结果如下：(1)考察了不同极性溶剂萃取物的抗菌活性,受试菌种包括痤疮杆菌、金黄色葡萄球菌、表皮葡萄球菌、枯草芽孢杆菌、大肠杆菌、变形杆菌、绿脓杆菌和白色念珠菌。不同极性部位对细菌和真菌都具有较好的抑制作用,其中对石油醚、乙酸乙酯、正丁醇和水这四种极性溶剂萃取物最为敏感的菌株是大肠杆菌,其最小抑菌浓度分别为1.95,1.95,3.9和3.9mg/mL。(2)香鳞毛厥精油对痤疮杆菌的最小抑菌浓度为0.0391%(v／v),香鳞毛蕨精油的最小杀菌浓度为0.0782%(v/v)。香鳞毛蕨精油对痤疮杆菌的抑菌活性优于文献报道的迷迭香精油,与J‘香精油抑制痤疮杆菌的活性相当。(3)对香鳞毛蕨中4种间苯三酚类成分较为敏感的菌株是表皮葡萄球菌,金黄色葡萄球菌、枯草芽孢杆菌、变形杆菌、白色念珠菌和痤疮杆菌。香鳞毛蕨素对白色念珠菌的最小杀菌浓度为125μg/mL,杀菌效果与氯霉素相当,优于红霉索和青霉素。绵马酚、绵马素PB和绵马素BB对表皮葡萄球菌、金黄色葡萄球菌、枯草芽孢杆菌、变形杆菌和白色念珠菌的杀菌效果与阳性对照相当或优于阳性对照。香鳞毛蕨素、绵马酚、绵马素BB和绵马素PB对痤疮杆菌的最小抑菌浓度分别为15.63,15.63,31.25和125μg/mL。(4)绵马素BB的动态杀菌曲线表明浓度为MBC(2MIC)的绵马素BB对瘁疮杆菌在5小时内可以将细菌全部杀灭。通过聚丙烯酰胺凝胶电泳(SDS-PAGE)研究了绵马素BB对痤疮杆菌的蛋白表达量的变化,结果表明绵马素BB可能通过抑制26kDa和33kDa两种特异蛋白而起作用。采用琼脂糖凝胶电泳研究了绵马素BB对痤疮杆菌DNA的裂解作用,结果表明浓度MBC的绵马素BB作用痤疮杆菌能导致其DNA出现碎裂。综上所述,无溶剂微波萃取技术和负压空化提取技术适用于香鳞毛蕨中精油及间苯三酚类成分的提取。树脂柱吸附和树脂柱色谱可以有效地分离富集香鳞毛蕨中四种主要的间苯三酚类成分,纯化后,绵马酚、绵马素PB、香鳞毛蕨素和绵马素BB的纯度分别达到96.2%,95.1%,97.7%和95.3%,总回收率为46.07%,36.63%,45.80%和38.66%。香鳞毛蕨不同极性溶剂萃取物和纯化得到的间苯三酚类成分均具有抑菌效果。对绵马素BB抑制痤疮杆菌的分子机制进行了初步的探索,为进一步开发利用植物药提供了良好的基础。更多还原

【Abstract】 Dryopleris fragrans (L.) schott, a member of the Dryopteris genus, is a deciduous perennial herb characterized by distinctive fragrance and persistent old fronds. This aromatic medicinal plant normally occurs on talus slopes and cliff crevices, and mainly distributed in Asia-temperate, Europe and North America. Especially in Wu Da Lian Chi (Heilongjiang province, China), a place of interest with volcanic geological landforms, it possessed a strong affinity for lava and molten rocks formed after volcanic eruption. The indigenous plant has been used in traditional folk medicine for decades in treating various dermatosis (such as psoriasis, acne, erythra and dermatitis) and using as an effective agent of antibacterial, analgesic, antioxidant, anti-tumour as well as antirheumatic arthritis. In the present study, extraction and separation of essential oil and phloroglucinols from D. frangrans were investigated. The antibacterial activities of purified constituents were studied.The results were as follows:1. The analysis method of essential oil and four phloroglucinols from D. fragrans were developed.(1) GC-MS combined with retain index were used to identify the constituents of the essential oil of D. fragrans. Eighteen compounds, representing89.65%of the oil, were identified. The essential oil was found to be rich in sesquiterpenes.(2) A RP-HPLC method was developed for the simultaneous separation and determination of four phloroglucinols from D. fragrans.Chromatography column:ODS C18reversed phase column (100mm×4.6mm. i.d.,2.6μm); mobile phase:acetonitrile-water-Formic acid; column temperature:25℃. This method was repeatable, accurate and stable, which was suitable for the determination of phloroglucinols in D. fragrans as well as in other plants.2The solvent-free microwave extraction was developed for extraction of the essential oil from D. fragrans. A central composite design combined with response surface methodology was applied to optimize the technique. The influence parameters of extraction time, irradiation power and humidity were investigated. The optimal operating condition was as follows: extraction time34min, irradiation power500W, humidity50%.Under the opimized condition, the extraction yield of essential oil was0.33%, and R.S.D. was5.19%. In the results of response surface analysis, extraction time is the main factor which affects the microwave extraction process followed by irradiation power and humidity. The relationship between the extraction yield and the influence factor is not simple linearity. There is strong interaction between extraction time and irradiation power. The solvent-free microwave extraction was an efficient technique for extraction of the essential oil from D. fragrans. After extracted by solvent-free microwave extraction, the residua can be used as raw material to extract other target components.3. The negative-pressure cavitation extraction (NPCE) was developed for extraction of four main phloroglucinols from D. fragrans. The effects of liquid/solid ratio, extraction time, air flow, pressure and number of extration cycles on the recovery of flavonoids were optimized. The optimum parameters were as follows:liquid/solid ratio10:1(mL/g), extraction time20min, air flow30mL/min, pressure-0.04～-0.05Mpa,3extraction cycles.Under the above opimized conditions, the relative recoveries of dryofragin, aspidin BB, aspidin PB and aspidinol were all higher than94%. Negative pressure is the impetus of NPCE; and the extraction process was performed under the obturation condition and normal temperature. So the method was energy saving, solvent free and avoiding the thermal decomposition, which was suitable for the extraction of active components in D. fragrans as well as in other plants.4. The method of separation and enrichment of aspidinol, aspidin PB, dryofragin and aspidin BB from D. fragrans was studied.(1) Resin column adsorption was applied for separation and enrichment of aspidinol and aspidin PB. Considering both contents and recoveries, the optimal enrichment and separation conditions were confirmed as follows:30%ethanol5BV was used for removing impurities,50%ethanol10BV was selected to wash aspidinol and70%ethanol15BV to elute aspidin PB, the flow rate was3BV/h.(2) Resin column chromatography was applied for separation and enrichment of dryofragin and aspidin BB. Considering both contents and recoveries, the optimal enrichment and separation conditions were confirmed as follows:70%ethanol10BV was used for removing impurities,80%ethanol30BV was selected to wash dryofragin and95%ethanol20BV to elute aspidin BB, the flow rate was6BV/h.(3)After AB-8resin column adsorption, the content of aspidinol increased from1.40mg/g to10.4mg/g, increased7.4times; the content of aspidinol increased from4.9mg/g to41.8mg/g, increased8.52times.The recoveries of aspidinol and aspidin PB were85.73%and78.41%, separately. After AB-8resin column chromatography, the content of dryofragin and aspidin BB increased to34.4mg/g and56.9mg/g, separately, increased8.39and5.99times. The recoveries of dryofragin and aspidin BB were91.22and75.64%, respectively.5. Medium-pressure silica gel column chromatography and recrystallization were applied for purification of aspidinol, aspidin PB, dryofragin and aspidin BB from D. fragrans were studied. A series of schemes were employed for identification the structure of the purified compounds. (1)1he condition of medium-pressure silica gel column chromatography and recrystallization were determined. After determined by HPLC, the purities of aspidinol, aspidin PB, dryofragin and aspidin BB were96.2%,95.1%,97.7%and95.3%, separately, with the recoveries of78.26%,63.27%,73.24%and65.63%.(2)The products were identified as the target compounds by the reaction of physical and chemical property combined with UV spectrum, mass spectrum (ESI-MS-MS) and nuclear magnetic resonance (1H-NMR和13C-NMR).6. Antibacterial activities of different fractions, four main phloroglucinols and essential oil from D. fragrans were studied. The results were as follows:(1)The antibacterial activities of different fractions were tested, and the bacterium include Propionibacterium Acnes, Staphylococcus aureus, Staphylococcus epidermidis, Bacilus subtillis, Escherichia coli, Proteus vuLgaris, Pseudomonas aeruginos and Candida albicans. All fractions showed antimicrobial activity towards both gram-positive bacteria and gram-negative bacteria. The most sensitive stain towards petroleum ether, ethyl acetate, n-butanol and water fractions was Escherichia coli with the MIC values of1.95,1.95,3.9and3.9mg/mL(2)The MIC and MBC values of D. fragrans essential oil towards P. Acnes were0.0391%and0.0782%(v/v). The antibacterial activity of D. fragrans essential oil towards P. Acnes was better than that of reported Rosmarinus officinalis essential oil and nearly to that of Syzygium aromaticum essential oil.(3)The most sensitive stains towards four phloroglucinols were Staphylococcus epidermidis, Staphylococcus aureus, Bacilus subtillis, Proteus vμLgaris, Candida albicans and Propionibacterium Acnes.The MIC values of dryofragin towards Candida albicans were125μg/mL. The bactericidal effect of dryofragin approach to that of positive control penicillium and better than that of chloromycetin and erythromycin. The bactericidal effect of aspidinol, aspidin PB and aspidin BB towards Staphylococcus epidermidis, Staphylococcus aureus, Bacilus subtillis, Proteus vμLgaris and Candida albicans are nearly to or better than that of positive control (penicillium, chloromycetin and erythromycin). The MIC values of dryofragin, aspidinol, aspidin BB and aspidin PB towards P. Acnes were15.63,15.63,31.25and125μg/mL.(4) According to time-kill curves of aspidin BB (MBC), all the P. Acnes were killed in5hours. The results of SDS-PAGE showed the molecular mechanisms of aspidin BB against P. acnes, aspidin BB could inhibit the two special proteins (26kDa and33kDa). The DNA (?)leavage activity of aspidin BB towards P. Acnes showed that aspidin BB inhibits the growth of P. Acnes by DNA cleaving.In conclusion, the method of solvent-free microwave extraction (SFME) and negative- pressure cavitation extraction (NPCE) was suit for extraction of essential oil and four main phloroglucinols from D.fragrans. Resin column adsorption and Resin column chromatography could enrich the four main phloroglucinols effectively. After extraction, separation and purification, the purities of aspidinol, aspidin PB, dryofragin and aspidin BB were96.2%,95.1%,97.7%and95.3%, separately, with the recoveries of46.07%,36.63%,45.80%and38.66%. Different fractions, purified constituents and essential oil from D.fragrans possessed antibacterial activities. Aspidin BB showed great antimicrobial activity. The results benefit of develop and make full use of plant medicinal.更多还原