| 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. |
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