【作者】 孙静贤；

【导师】 陆树刚；

【作者基本信息】 云南大学 ， 植物学， 2019， 博士

【摘要】 贯众(Cyrtomiumlike)为较常用传统中药,始载于《神农本草经》,位列下品。其性味苦、微寒、有小毒,归肝、脾经;有清热解毒、凉血止血、杀虫等功效,临床上用于治疗风热感冒,温毒斑疹,咳血,吐血,便血,衄血,血痢,崩漏,带下及肠道寄生虫病。《中国药典》2015年版收载粗茎鳞毛蕨Dryopteris crassirhizoma Nakai和紫萁Osmunda japonica Thunb.为贯众的基原植物。除药典收载品种外,多种药用蕨类植物因形态、药材的功效主治与贯众相似,加之全国各地又普遍存在同名异物、地区性习惯用药等原因,导致市场上作为贯众入药的来源植物很多,严重影响其质量和用药安全。本文针对贯众地方习用品种和混淆品种较多,在本草考证的基础上,对19种贯众类植物进行形态学、DNA分子鉴别和生物活性研究,进而对贯众类植物进行系统的鉴别研究和质量研究,其研究结果如下:1.对贯众类植物进行形态学鉴别研究。结果显示:贯众类植物形态学的鉴别点主要为根状茎、叶柄分体中柱、孢子囊群的形态,其次为孢子表面纹饰、鳞片形态等,而叶表皮对贯众类植物的鉴别支持不足。2.对贯众类植物进行分子鉴别研究。选取5个叶绿体基因或间隔区序列psb A-trn H、rbc L、mat K、rps4-trn S和atp B-rbc L以及2个核基因片段ITS和ITS2为候选DNA条形码,对贯众类植物进行分子鉴别研究,发现mat K、ITS2和atp B-rbc L片段在贯众类植物中通用性较差,不适合作为贯众类植物鉴别的DNA条形码。根据贯众类物种鉴定率结果,推荐psb A-trn H为鉴别贯众类植物的首选DNA条形码片段。然而,在本研究中psb A-trn H单片段虽然能区分贯众类植物的科属,但在鳞毛蕨属和贯众属的类群中区分度不高,而组合片段psb A-trn H+rps4-trn S既能区分贯众类植物的科属种,且物种分辨率最高,对贯众类植物的区分趋势最为明显,因此建议psb A-trn H+rps4-trn S片段作为贯众类植物DNA分子鉴别的首选组合序列。3.对贯众类植物进行抑菌活性研究。通过对贯众类植物乙醇提取物对大肠杆菌Escherichia coli、白色念珠菌Monilia albican等包括细菌和真菌在内的9种微生物抑菌活性的研究,发现粗茎鳞毛蕨D.crassirhizoma、金冠鳞毛蕨D.chrysocoma、黑鳞鳞毛蕨D.lepidopoda和半育耳蕨P.semifertile具有较好的抑制革兰氏阳性菌和革兰氏阴性菌的活性;乌毛蕨B.orientale、单芽狗脊蕨W.unigemmata、狗脊蕨W.japonica和桫椤A.spinulosa具有对真菌的抑制作用。4.对贯众类植物进行抗氧化活性研究。比较研究了贯众类植物乙醇提取物对DPPH自由基、ABTS~+自由基的清除能力,发现贯众C.fortunei、单芽狗脊蕨W.unigemmata、刺齿贯众C.caryotideum、大叶贯众C.macrophyllum、半育耳蕨P.semifertile有很强的清除DPPH的活性;而乌毛蕨B.orientale、苏铁蕨B.insignis、单芽狗脊蕨W.unigemmata、狗脊蕨W.japonica、刺齿贯众C.caryotideum、贯众C.fortunei、大叶贯众C.macrophyllum、粗茎鳞毛蕨D.crassirhizoma、对马耳蕨P.tsus-simense和半育耳蕨P.semifertile均表现出很高的清除ABTS~+自由基的活性。综合来看,贯众C.fortunei、单芽狗脊蕨W.unigemmata、刺齿贯众C.fortunei、大叶贯众C.macrophyllum和半育耳蕨P.semifertile对两种自由基均有很强的清除作用。5.对贯众类植物进行抗炎活性研究。利用脂多糖(LPS)诱导的RAW 264.7细胞体外炎症模型,采用MTT法检测细胞存活率;用Griess试剂法检测细胞上清液中一氧化氮(NO)含量,考察了贯众类植物乙醇提取物的抗炎活性。实验结果显示,除边果鳞毛蕨D.marginata外,其他贯众类植物提取物对RAW 264.7细胞均无毒性作用。在抑制LPS诱导的RAW 264.7细胞释放NO的作用方面,活性从强到若依次是桫椤A.spinulosa>对马耳蕨P.tsus-simense>边果鳞毛蕨D.marginata>毛柄短肠蕨A.dilatata>紫萁O.japonica,桫椤A.spinulosa和对马耳蕨P.tsus-simense抑制NO释放的能力接近L-NMMA,具有较好的抗炎活性。6.对贯众类植物进行降糖活性研究。采用α-葡萄糖苷酶活性抑制能力试验评价了贯众类植物乙醇提取物的降血糖潜力,发现除狗脊蕨W.japonica和大叶贯众C.macrophyllum外,其他样品都表现出较好的体外抑制α-葡萄糖苷酶活性,具有降糖活性。本论文系统地研究了19种贯众类植物的形态特征,筛选出适合对其进行鉴别的DNA条形码,为贯众类植物提供了形态学和分子鉴别依据;同时比较研究了贯众类植物乙醇提取物在抑菌、抗氧化、抗炎和降血糖等方面的生物活性,为贯众药材的质量控制、资源的合理开发利用提供了参考。更多还原

【Abstract】 Cyrtomiumlike is a common traditional Chinese medicine with the properties of bitter taste,slight cold and slight toxicity,which was first recorded in Shen Nong’s Herbal Classic and listed as inferior.Its rhizomes are clinically used for treating wind-heat common cold,toxic heat macula,hemoptysis,hematemesis,hematochezia,epistaxis,bloody dysentery,metrorrhagia,and intestinal parasitic diseases based on the effects of clearing away heat and toxic materials,cooling blood,stopping bleeding,and killing parasites,associated with the liver and spleen meridians.Dryopteris crassirhizoma Nakai and Osmunda japonica Thunb.are documented in the Chinese Pharmacopoeia(2015)as the original plants of Cyrtomiumlike.Except for these two plants,many species of the medicinal ferns are used as Cyrtomiumlike because of their similarities in morphology,efficacy and indications.In addition,homonymy and regional habitual drug used are common in all over the country.As a result,there are many plants for Cyrtomiumlike in the market,which caused serious problems in the quality control and drug safety.In this paper,the morphology,DNA molecular identification and biological activities of 19 species of Cyrtomiumlike plants were studied for the systematic authentication and quality research of herbal medicine.The results are as follows:1.Morphological identification of Cyrtomiumlike led to the key points of morphological identification for the plants,including the morphology of rhizomes,split steles of petioles and sporangia.Spore surface ornamentation and scale morphology were determined as the second identifications,while the leaf epidermis did not provide sufficient support for the identification of Cyrtomiumlike plants.2.Molecular identification of Cyrtomiumlike plants was studied.Five chloroplast genes or spacer sequences psb A-trn H,rbc L,mat K,rps4-trn S and atp B-rbc L,as well as two nuclear gene fragments ITS and ITS2,were selected as candidate DNA barcoding of molecular identification for 18 species.The results showed that mat K,ITS2 and atp B-rbc L fragments were not suitable as the DNA barcoding for identification the Cyrtomiumlike plants for poor universality.According to the identification rate of these plants,psb A-trn H is recommended as the preferred DNA barcoding fragment for identification of the Cyrtomiumlike plants.The single psb A-trn H fragment could distinguish the families and genera of the Cyrtomiumlike plants,but there was not highly distinction between Dryopteris and Cyrtomium by using psb A-trn H.The combined fragment psb A-trn H+rps4-trn S could distinguish the families and genera of these plants with the highest species resolution and the most obvious tendency to distinguish 16 species.Therefore,the fragment psb A-trn H+rps4-trn S was suggested as the first combined sequence for DNA molecular identification of Cyrtomiumlike plants.3.The antimicrobial activities of the ethanol extracts from Cyrtomiumlike were assayed on the inhibition to 9 kinds of microorganisms including bacteria and fungi.The plants,D.crassirhizoma,D.chrysocoma,D.lepidopod and P.semifertile showed moderate activity to inhibit Gram-positive bacteria and Gram-negative bacteria.However,B.orientale,W.unigemmata,W.japonica and A.spinulosa exhibited weak inhibitory activity against fungus.4.The DPPH radical and ABTS~+radical scavenging activities of the ethanol extracts from Cyrtomiumlike plants were evaluated.The plants,C.fortunei,W.unigemmata,C.caryotideum,C.macrophyllum and P.semifertile showed strong DPPH scavenging activities.B.orientale,B.insignis,W.unigemmata,W.japonica,C.caryotideum,C.fortunei,C.macrophyllum,D.crassirhizoma,P.tsus-simense and P.semifertile showed high activity of scavenging ABTS~+free radicals.On the whole,C.fortunei,W.unigemmata,C.fortunei,C.macrophyllum and P.semifertile showed strong scavenging effects on both two free radicals.5.The anti-inflammatory activities of the ethanol extracts from Cyrtomiumlike plants were studied by examining their inhibitory effects on lipopolysaccharide(LPS)-induced inducible nitric oxide synthase(i NOS)-dependent NO production in the murine macrophage cell line RAW264.7.No cytotoxicity against RAW264.7 cells were observed by MTT method for those plants at their effective concentrations except for D.marginata.In terms of inhibiting LPS-induced NO release from RAW 264.7 cells,the order of activity from strong to weak was A.spinulosa,P.tsus-simense,D.marginata,A.dilatata,and O.japonica.A.spinulosa and P.tsus-simense exhibited the good ability to inhibit NO release close to positive control L-NMMA,indicating the extracts of the two plants had good anti-inflammatory activities.6.The hypoglycemic potential of ethanol extracts from Cyrtomiumlike plants was evaluated byα-glucosidase activity inhibition test.The results indicated that all the samples showed good in vitroα-glucosidase inhibition activity and hypoglycemic activity except W.japonica and C.macrophyllum.In this paper,the morphological characteristics of 19 species of Cyrtomiumlike plants were systematically studied,and the suitable DNA barcoding for identification were selected,which provided morphological and molecular identification basis for Cyrtomiumlike plants.Moreover,the biological activities of ethanol extracts from Cyrtomiumlike plants in antibacterial,antioxidant,anti-inflammatory and hypoglycemic aspects were compared and studied,providing a reference for the quality control of Cyrtomiumlike plants medicinal materials and the rational development and utilization of resources.更多还原