【作者】 刘荣；

【导师】 刘友平； 陈鸿平；

【作者基本信息】 成都中医药大学 ， 药物化学， 2014， 硕士

【摘要】 中药陈皮来源于芸香科Rutaceae柑橘属Citrus L.植物橘Citrus reticulata Blanco及其栽培变种的干燥成熟果皮。橘的栽培品种较多,地域分布广泛,我国长江以南各省均有栽培,作为陈皮来源品种的橘类植物主要为宽皮柑橘类,栽培品种有茶枝柑C.reticulata’Chachi’、红橘C. tangerina Hort. et Tanaka、樟头红C. reticulata Blanco cv. zhangshuensi、南丰蜜橘C. reticulata Blanco cv. Kinokuni、福橘C.reticulata’Tangerina’等,主产于广东、四川、重庆、江西、福建等地。陈皮是中药多栽培品种入药的代表药物,近年来由于柑橘品种不断更新交替以及栽培更重视其食用价值,加之2010年版《中国药典》对陈皮来源橘的栽培变种未作进一步规定,药典收载的质量标准也无法客观评价各栽培变种的品质差异,造成陈皮传统来源品种大红袍等面临淘汰,而一些药用质量未经评价的柑橘品种以“杂陈皮”流通于市场,导致陈皮药材质量问题突出,临床疗效难以保证。因此,亟需对市场上流通的陈皮入药品种的遗传差异、化学成分、药理作用等进行系统评价研究,为进一步规范陈皮入药品种,确保陈皮药材质量稳定可控和临床用药安全有效提供科学依据。本文首次对川陈皮传统来源品种大红袍C.reticulata ’Dahongpao’(红橘品系)、赣陈皮来源品种南丰蜜橘和樟头红中主要药效成分一黄酮、总酚酸、生物碱、多糖含量在不同采收期内的动态变化进行研究,明确不同药效成分的动态变化规律,为选择陈皮入药品种的次生代谢产物的等同性研究奠定基础；并对大红袍果实发育期(7-12月份)内叶片和果皮中黄酮类成分的合成积累与3种次生代谢关键酶(PAL、C4H、4CL)活性的关系进行系统研究,首次初步阐释了大红袍果实发育过程中黄酮类成分生物合成代谢机制,揭示了陈皮成分积累与陈皮入药成熟度的科学内涵,为进一步规范陈皮传统入药品种的采收加工提供依据；对陈皮传统入药品种茶枝柑、大红袍、南丰蜜橘及其市场上混杂入药的柑橘品种天草C. reticulata hybrid等22个柑橘品种的遗传多样性及DNA条形码进行研究,以期为陈皮种质资源的开发和合理利用提供参考,也为陈皮入药品种的进一步筛选提供遗传学依据。具体研究内容及结果如下：1不同栽培品种陈皮药材主要药效成分动态变化研究本研究采用HPLC法测定不同采收期大红袍、樟头红、南丰蜜橘中4种主要黄酮类成分芸香柚皮苷、橙皮苷、川陈皮素、橘皮素和生物碱类成分辛弗林的含量,UV分光光度法测定总黄酮、总酚酸和多糖的含量。结果表明：3个栽培品种中黄酮类成分芸香柚皮苷、川陈皮素、橘皮素和辛弗林均随采收时间的推迟而逐渐降低至果实成熟时趋于稳定。橙皮苷、总黄酮和总酚酸、多糖在整个采收期内呈现起伏波动的变化趋势,橙皮苷、总黄酮和总酚酸在整个采收期内的总体变化呈下降趋势,橙皮苷和总黄酮在幼果期至果实成熟前含量均呈降低趋势,在果实成熟后含量有所回升；总酚酸在果实膨大期至着色期含量变化幅度较大,先迅速升高,后持续降至果实成熟时趋于稳定；多糖在果实着色期前均持续升高至着色期后急剧下降,于成熟期含量大幅上升至最高水平。从各药效成分在整个采收期内的积累量来看,3个品种中黄酮类成分芸香柚皮苷的含量存在明显差异,其积累量的相对高低为樟头红>南丰蜜橘>大红袍；橙皮苷、总黄酮及其生物碱类成分辛弗林的含量也存在明显差异,其含量高低为南丰蜜橘>大红袍>樟头红,而其它类成分的积累量差异不明显,原因可能与调控黄酮类成分合成积累的次生代谢关键酶的活性表达有关,具体的调控机制还有待继续深入研究。该研究初步明确了3个栽培品种陈皮中主要药效成分的动态变化规律，为进一步选择陈皮入药品种的次生代谢产物的等同性研究奠定基础。2大红袍叶片、果皮中黄酮类成分积累的初步研究对大红袍、南丰蜜橘、樟头红中主要药效成分的动态变化研究表明,3个栽培品种中黄酮类成分橙皮苷和总黄酮的含量随果实发育期的推迟存在明显差异。本研究首次以川陈皮来源品种大红袍叶片和果皮为对象,对其果实发育的幼果期至成熟期内(7-12月份)叶片中黄酮类成分积累与其次生合成代谢关键酶PAL、C4H、4CL活性的相关性及其同期果皮中黄酮类成分积累的内在关联性进行系统研究,共比较了40批大红袍叶片和果皮样品。2.1大红袍叶片中黄酮类成分积累与其次生代谢3种关键酶的相关性研究本研究在前期课题组研究基础上建立了UPLC测定大红袍叶片中橙皮苷、川陈皮素和橘皮素的含量方法,对不同采收期内大红袍叶片中3种主要黄酮类成分的含量进行测定,并运用UV分光光度法追踪测定了叶片中黄酮类成分合成代谢的3种关键酶PAL、C4H、4CL活性和总黄酮的含量,分析黄酮类成分积累与3种关键酶活性的关系。结果表明：大红袍叶片中橙皮苷和总黄酮的积累与PAL、 C4H活性从果实发育的幼果期到膨大期内呈现相似的变化趋势,橙皮苷和总黄酮的含量逐渐增加,PAL、C4H酶的活性也逐渐升高,从果实着色期至成熟期出现了分化,橙皮苷和总黄酮含量持续升高,而PAL、C4H活性持续降低,而在整个采收期内二者含量与4CL酶活性之间没有明显的关系；4CL酶活性的变化与川陈皮素和橘皮素含量之间也呈现相似的变化,从果实膨大期至着色前期呈现先降低后升高的同步变化趋势,之后从果实着色期至成熟期出现分化,川陈皮素和橘皮素含量升高,4CL酶活性降低,而在整个采收期内二者含量与PAL、C4H酶活性的关系不明显。大红袍叶片中黄酮类成分积累与3种次生代谢合成关键酶出现分化的具体原因可能与其栽培环境的变化及调控黄酮类成分合成的其他关键酶活性的调控有关,其中复杂的合成代谢调控机制还有待进一步深入研究。该研究初步揭示了大红袍叶片中黄酮类成分生物合成积累与PAL、C4H、4CL关键酶活性的关系,为研究大红袍叶片中黄酮类成分的合成代谢机制奠定基础。2.2大红袍叶片与果皮中黄酮类成分积累的内在关联性研究为进一步深入研究大红袍果皮中黄酮类成分合成积累的代谢机制,本研究在对大红袍叶片中黄酮类成分合成积累研究的基础上,对同期采集的大红袍果皮中黄酮类成分的积累进行系统研究,利用HPLC法测定了与叶片同期采收的大红袍果皮中3种主要黄酮类成分橙皮苷、川陈皮素和橘皮素的含量,并运用UV分光光度法测定其叶片中总黄酮的含量,分析大红袍果实发育期内大红袍果皮和叶片中黄酮类成分积累的内在关联性。结果表明：在大红袍果实发育的幼果期到成熟期内叶片和果皮中黄酮类成分的变化趋势不尽相同,在果实发育的膨大期到成熟期内二者黄酮类成分的积累变化较为明显,呈现出增减互补的变化趋势,叶片中橙皮苷和总黄酮的含量随成熟度的增加逐渐升高,而果皮中橙皮苷和总黄酮的含量逐渐降低,叶片中橘皮素和川陈皮素含量随成熟度的增加缓慢升高,而果皮中则逐渐降低。该研究初步表明了大红袍叶片和果皮中黄酮类成分积累的内在关联性,为阐释大红袍中黄酮类成分积累与陈皮入药成熟度的相关性研究奠定基础。3不同栽培品种柑橘的遗传多样性分析研究本研究对市场上流通的陈皮入药品种进行遗传多样性分析研究,为进一步筛选陈皮入药的柑橘品种提供遗传学依据。采用AFLP分子标记技术对茶枝柑、福橘、大红袍、南丰蜜橘、樟头红、天草等22个柑橘品种进行遗传多样性研究。结果表明：从12对EcoR Ⅰ/Mse Ⅰ引物组合中筛选出6对引物,对样本进行PCR批量扩增,经高效毛细管电泳检测得到的样本DNA多态性条带总数为472条,多态性比例达到80%以上,平均每对引物扩增出78条DNA多态性条带,能够较好地反映柑橘样本间的遗传信息。且基于遗传相似性系数构建的UPGMA聚类图能够将不同栽培品种的茶枝柑、大红袍、福橘、椪柑、脐橙、不知火、天草等各聚为一类,其中同一品种不同产地的茶枝柑、大红袍、椪柑、脐橙、天草等各聚为一类,表明品种间的遗传差异受地域的影响较小；化学成分相似的南丰蜜橘和樟头红也单独聚为一类,与正品茶枝柑比较椪柑与其亲缘关系最近,而毛气柑与其亲缘关系最远。以上研究结果显示AFLP分子标记能够较好地界定划分不同栽培品种的柑橘,从DNA水平上表征了柑橘各栽培品种的遗传多样性,建议在进行陈皮入药品种筛选时可以从与陈皮道地品种亲缘关系较近的柑橘品种入手。4不同栽培品种柑橘的DNA条形码初步研究本研究以陈皮主要栽培品种茶枝柑、大红袍、南丰蜜橘等13个柑橘品种为研究对象,以ITS2序列为基础对其进行DNA条形码分子鉴定分析研究。结果表明：基于K2P距离构建的NJ发育树不能较好地区分鉴定各陈皮栽培品种,研究结果与文献报道一致。原因在于陈皮来源植物宽皮柑橘的栽培品种之间普遍存在自然杂交和芽变现象,特别是经杂交育种的杂柑(天草、不知火等),其DNA片段上仅存在少数位点的单核苷酸变异,其ITS2序列存在高度相似性。因此,仅从具有特征片段的DNA条形码序列难以进行区分,但ITS2序列在研究柑橘属植物亲缘关系方面仍然具有很大潜力。更多还原

【Abstract】 Traditional Chinese medicine Pericarpium Citri Reticulatae derived from dry mature peel of Rutaceae Citrus L. Citrus reticulata Blanco and its cultivated variation.There are many cultivars of Citrus reticulata Blanco, whose cultivated region is widespread, cultivated in provinces to the south of Yangtze River. The main variety of Pericarpium Citri Reticulatae is C.reticulata. The cultivated variations include C.reticulata’Chachi’, C.tangerina Hort. et Tanaka, C. reticulata Blanco cv. zhangshuensis Hort, C. reticulata Blanco cv. Kinokuni, reticulata ’Tangerina’ ect, produced mainly in Guangdong, Sichuan, Chongqing, Jiangxi, Fujian and other places.Pericarpium Citri Reticulatae is the representative medicine of multiple cultivars used as Chinese medicine.In recent years, Citrus cultivated varieties have been continuously updated and more attention were paid to their food value, and there aren’t further provisions for Pericarpium Citri Reticulatae source Citrus cultivated variation in2010edition of China Pharmacopoeia. Quality standards in China Pharmacopoeia can not evaluate the quality differences among cultivars objectively, resulting traditional variety source C. reticulata’Dahongpao’of Pericarpium Citri Reticulatae is facing elimination.However some Citrus varieties without medicinal quality evaluation is circulating in the market as miscellaneous Pericarpium Citri Reticulatae, resulting in quality problems of Pericarpium Citri Reticulatae medicinal materials outstanding and their clinical efficacy is difficult to guarantee.Therefore,it desiderates systematic evaluation and studies on genetic differences, chemical composition, pharmacological effects of Citrus cultivars that circulating in the market, so that provide a scientific basis for specificating Pericarpium Citri Reticulatae cultivars.This paper firstly research the dynamic content change of flavonoids, total phenolic acids, alkaloids, polysaccharides,the main medicinal components of Pericarpium Citri Reticulatae in Sichuan traditional variety C. reticulata ’Dahongpao’(C. tangerina Hort. et Tanaka strains), and Pericarpium Citri Reticulatae in Jiangxi variety C. reticulata Blanco cv. Kinokuni, C. reticulata Blanco cv. zhangshuensis Hort in different harvest period. To clear the dynamic changes regular of different efficacy component, and provide a theoretical basis for screening Pericarpium Citri Reticulatae variety which used as medicine.Scientific studies relationship of flavonoids Synthesis and accumulation in the leaves and peel with three kinds of secondary metabolism key enzymes (PAL, C4H,4CL)activity in C. reticulata ’Dahongpao’during fruit developing period (July to December). We firstly preliminary interpret the flavonoids biosynthesis mechanisms in C. reticulata ’Dahongpao’during the fruit developing period, revealing the scientific connotation of relationship between harvesting Pericarpium Citri Reticulatae to use as medicine and its maturity, and provide the basis for further regulate the harvesting and processing of Pericarpium Citri Reticulatae traditional cultivars, and lay to foundation choose Pericarpium Citri Reticulatae cultivars secondary metabolites equivalence research. I have studied the genetic diversity and DNA barcodes of Pericarpium Citri Reticulatae traditional cultivars C.reticulata’Chachi’, C. reticulata ’Dahongpao’, C. reticulata Blanco cv. Kinokuniand, C. reticulata hybrid whose medicinal quality without evaluation arieties and other22Citrus cultivars, to provide a reference to Pericarpium Citri Reticulatae Germplasm resources development and rational use, and also provides genetic evidence for further screening Pericarpium Citri Reticulatae varieties. Specific studies as follows:1. Dynamic Variation Regularities of Effective Composition in C. reticulata ’Dahongpao’, C. reticulata Blanco cv. Kinokuni, C. reticulata Blanco cv. zhangshuensis Hort.This study determine content of four main flavonoids naringin, hesperidin, nobiletin, tangeretin and synephrine alkaloids in three Pericarpium Citri Reticulatae cultivars during different harvest period by HPLC,determine total flavonoids, total phenolic acids and polysaccharides by UV spectrophotometric. The results showed that:three cultivars flavonoids naringin, nobiletin, tangeretin and synephrine were delayed and decreased with harvest time to stabilize when the fruit maturation. hesperidin, total flavonoids and total phenolic acids, polysaccharides throughout the harvest period showed fluctuating trend of hesperidin, total flavonoids and phenolic acids in general, changes in the total harvest period showed a downward trend, hesperidin and flavonoids in young fruit to fruit maturation before content showed a decreasing trend in the levels rebounded after fruit maturation; total phenolic acids in fruit enlargement to big variations in the coloration of content, the first rise rapidly, the continued tends dropped fruit ripening steady; polysaccharide before coloring of the fruit continued to rise to a sharp decline in the coloration, surged to the highest level in mature content. According to each medicinal component accumulation throughout the harvest period, in perspective, there are obvious differences between three varieties of flavonoids naringin, its accumulation of the relative level is C. reticulata Blanco cv. zhangshuensis Hort> C. reticulata Blanco cv. Kinokuni> C. reticulata ’Dahongpao’. There are significant differences of flavonoids hesperidin, total flavonoid and synephrine content in the three varieties. Its accumulation relative high or low is C. reticulata Blanco cv. Kinokuni> C. reticulata ’Dahongpao’> C. reticulata Blanco cv. zhangshuensis Hort, and differences between other types components is not obvious, it may be related to the activity of secondary metabolism key enzymes that regulate synthesis and accumulation of flavonoids, but specific regulation mechanism remains to be continued depth study. This study primarily clear the dynamic changes of main medicinal components in three Pericarpium Citri Reticulatae cultivars, laying foundation for choosing Pericarpium Citri Reticulatae cultivars secondary metabolites equivalence research.2. Preliminary analysis of the accumulation of flavonoids in leaves and peel of C. reticulata ’Dahongpao’We focus on dynamic changes about effective compositio in C. reticulata ’Dahongpao’、C. Kinokuni Tanaka and C. reticulata Blanco cv. Studies have shown that the content of3cultivars of Citrus flavonoids and total flavonoids hesperidin significantly different with fruit development period extended. This study taken the source of C. reticulata ’Dahongpao’ of which’s leaves and peels as meterials at the first time. Targeting its young fruit to mature fruit development period (July to December) leaves flavonoids accumulation and metabolism of a key enzyme in the biosynthesis followed PAL, C4H,4CL activity and its relevance in the same period peel flavonoids that accumulated intrinsic relevance systematic study of comparative batch of40leaves and rind samples.2.1The study correlation of C. reticulata ’Dahongpao’about accumulation of flavonoids with second born of3key metabolic enzymes.The study on the basis of pre-established UPLC determination C. reticulata ’Dahongpao’leaves hesperidin, nobiletin and methods tangeretin. Leaves of different harvest levels during the period of three main flavonoids were measured about C. reticulata ’Dahongpao’, and we used of UV spectrophotometric determination to trace leaves flavonoids anabolic3key enzymes PAL, C4H,4CL activity and total flavonoids, analyzes the relationship between the accumulation of flavonoids3key enzyme activities. The results show:the accumulation of total flavonoid hesperidin in the leaves of C. reticulata ’Dahongpao’and PAL, C4H activity showed a similar trend from the young fruit to the enlargement of fruit development period, and total fla(?)id hesperidin content gradually increased, PAL, C4H enzyme activity gradually increased, from fruit to fruit ripening period coloration were divided, and total flavonoid hesperidin content continues to rise, while PAL, C4H continue to decrease, both in the content of the entire harvest period and4CL no significant correlation between enzyme activity; Also showed changes in activity between4CL and nobiletin and tangeretin content similar change from pre-enlargement period show synchronized to coloring trend lower trend after the first rise, followed by coloring from fruit to fruit ripening period of differentiation, nobiletin and tangeretin content increased,4CL activity decreased, while the entire harvest period in both content and PAL, C4H activity relationship is not obvious. Flavonoids accumulation in the leaves of C. reticulata’Dahongpao’differentiation occurs with3kinds of key enzymes in the synthesis of secondary metabolites of which may be due to the specific cultural environment C. reticulata’Dahongpao’about dramatic changes, including anabolic complex regulatory mechanisms need to be further studied. The preliminary study reveals the relathion between biosynthesis of flavonoids accumulation and the metabolic enzymes, which can lay a foundation for the study of anabolic mechanisms of flavonoids in C. reticulata ’Dahongpao’.2.2Studies on intrinsic relationship of flavonoids accumulation in C. reticulata ’Dahongpao’ of leaves and peelsTo further study the metabolic mechanisms C. reticulata ’Dahongpao’ peel flavonoids biosynthesis, the study of the C. reticulata ’Dahongpao’ leaves in flavonoids biosynthesis study, based on the same period of accumulation in the acquisition of C. reticulata ’Dahongpao’ peel flavonoids system research. Measured by HPLC different harvest C. reticulata ’Dahongpao’ peel content of the three main flavonoids hesperidin, nobiletin and tangeretin, and the use of UV spectrophotometric determination of total flavonoids in leaves, analysis C. reticulata ’Dahongpao’ peel fruit development period and the accumulation of flavonoids in leaves intrinsic relevance. The results show:C. reticulata ’Dahongpao’ in fruit development of young fruit to maturity leaves and peel flavonoids period varies. In the enlargement of fruit development to changes in both the accumulation of flavonoids maturity period is more obvious, showing a trend of increase or decrease in complementary, leaf content of total flavonoid and hesperidin gradually increased with increasing maturity of fruit and the content of the peel and total flavonoid and hesperidin decreased, leaves nobiletin and tangeretin content elevated with increasing fruit maturity, while the peel is decreased. The preliminary study showed that the C. reticulata ’Dahongpao’ leaves and peel flavonoids accumulated intrinsic relevance, as explained C. reticulata ’Dahongpao’ flavonoids accumulation and Pericarpium Citri Reticulatae medicine maturity correlation foundation.3. The study of genetic diversity about different Citrus cultivarsIn this study, the flow of the market as a peel of citrus cultivars studied medicine for analysis of genetic diversity; provide a basis step for further screening genetic medicine Citrus varieties. We analysised the genetic diversity among different cultivars C.reticulata ’Chachi’、C.reticulata ’Tangerina’、C. reticulata ’Dahongpao’、 C.reticulata Blanco cv. zhangshuensis Hort、C. reticulata Blanco cv. Kinokuni、 C.reticulata ’Unshiu’、C. reticulata hybrid etc.22different Citrus cultivars by the technology of AFLP. The results showed that:12pairs of EcoR Ⅰ/Mse Ⅰ primer combinations were screened from six pairs of primers; bulk samples for PCR amplification by high performance capillary electrophoresis to detect DNA polymorphism samples obtained from a total of472bands, the proportion reached more than80%,average per pair of primers amplified polymorphic DNA bands78. So we could conclude that it can better reflect the genetic information between samples of citrus. UPGMA dendrogram based on genetic similarity coefficient could objectively divided into different cultivars of citrus, such as C.reticulata’Chachi’、C. reticulata ’Dahongpao’、C. reticulata Blanco cv. zhangshuensis Hort、C. reticulata Blanco cv. Kinokuni、C.reticulata’Unshiu’、C. reticulata hybrid, meanwhile C.reticulata’Chachi’、C. reticulata ’Dahongpao’、C. poonensis Tanaka. C. reticulata hybrid etc.were clustered into one class. So we can say that genetic differences indicate less affected by geographical varieties. They clustered together because of similar chemical composition between C. Kinokuni Tanaka and C. reticulata Blanco cv. zhangshuensis Hort. If we compared C.reticulata’Chachi’with them, C. poonensis Tanaka has close kinship, while Maoqigan has farthest kinship. The results shown that AFLP marker can better define the division of different citrus cultivars, characterize the genetic diversity of cultivars from the DNA level. Pericarpium Citri Reticulatae varieties conducting medicine recommended screening can start from authentic varieties of citrus varieties which has close relationship.4. The preliminary study of different cultivars in citrus by DNA barcode.In this study, we took the main source of medicinal C.reticulata’Chachi’. C. reticulata ’Dahongpao’、C. reticulata Blanco cv. Kinokuni etc.13cultivated varieties of citrus as the research object, and we resersched ITS2sequence based on DNA barcode, of which it could provide Molecular identification of citrus. The results manifested that the distance of K2P based on NJ phylogenetic tree could not better distinguish each citrus cultivars, the results of this study were consistent with other reports. The reason lies in the existence of universal natural hybridization between cultivated varieties of C.reticulata and mutant phenomenon, especially hybrid citrus (Amakusa Tangor, Shiranuhi Tangor etc) that exited after natural hybridization, citrus varieties between DNA fragments present on only a few sites in a single nucleotide variation, and exists had a high degree of similarity in ITS2sequence of the DNA fragment. Therefore, only DNA barcode sequence fragments with characteristic differences are difficult to distinguish between them. But ITS2sequences still have a great potential in the study of genetic relationships among the citrus.更多还原