Research Progress of Citri Reticulatae Semen / 中国实验方剂学杂志

Citri Reticulatae Semen is a traditional Chinese medicine in our country. By referring to the relevant literature and materials such as the works of ancient and modern materia medica， this paper summarizes the historical evolution of processing， quality analysis， pharmacological effects and other aspects of Citri Reticulatae Semen. The results showed that the processing methods of Citri Reticulatae Semen in the past dynasties mainly included stir-frying， processing with wine， baking with wine， etc. The modern stir-baking with salt solution was developed on the basis of the ancient stir-frying with green salt， most of the national and provincial processing standards contained crude and salt-processed products of Citri Reticulatae Semen. The properties and flavors of Citri Reticulatae Semen in the past dynasties were basically consistent with those in modern times. It is bitter in taste and neutral in nature. It belongs to the liver and kidney meridians and has the effects of regulating Qi， dispersing knots and relieving pain. Citri Reticulatae Semen contains limonins， fatty acids， proteins and mineral elements， etc. High performance liquid chromatography （HPLC）， gas chromatography-mass spectrometry （GC-MS） and thin layer chromatography （TLC） were commonly used to detect and analyze Citri Reticulatae Semen. Citri Reticulatae Semen has anti-tumor， anti-inflammatory， analgesic and antibacterial activities， and processing can change the content of its chemical components to a certain extent. In a word， the current research about Citri Reticulatae Semen is not enough in-depth， it is necessary to further analyze the material basis of efficacy of Citri Reticulatae Semen and its processed products， explore the change law of chemical components before and after processing and the relationship between chemical composition and activity， and clarify the processing mechanism for providing scientific basis for its standardized processing， quality control and clinical rational use.

Analysis of Equivalence Between Traditional and Nontraditional Medicinal Varieties of Citri Reticulatae Semen from Sichuan Province / 中国实验方剂学杂志

Objective:To study the differences in genetic relationship， shape， size， and flavonoid content between traditional and nontraditional medicinal varieties of Citri Reticulatae Semen produced in Sichuan province as well as their equivalence. Method:Six batches of traditional medicinal Citri Reticulatae Semen （<italic>Citrus reticulata</italic> 'Dahongpao'） and 23 batches of nontraditional medicinal varieties were collected， and their genetic relationship was explored using sequence-related amplified polymorphism （SRAP） markers. Following the observation of their shapes and sizes under a stereomicroscope， the contents of naringin， hesperidin， and neohesperidin were measured by high performance liquid chromatography （HPLC）. SIMCA 14.1 software was used for cluster analysis of their shapes， sizes， and flavonoid contents， thus figuring out the similarities between the traditional and nontraditional medicinal varieties in character， size， and chemical components. Result:SRAP markers-based genetic relationship analysis effectively distinguished different Citri Reticulatae Semen varieties from each other. Some samples collected from the same or adjacent places exhibited a close genetic relationship and they shared high similarities in shape， size， and flavonoid content. However， the traditional medicinal Citri Reticulatae Semen was still quite different from most nontraditional medicinal varieties. Conclusion:The analysis of differences in genetic materials， appearance， character， and active ingredient content between the traditional and nontraditional medicinal varieties revealed that the equivalence<italic> </italic>of <italic>C.</italic> <italic>reticulata</italic> 'Ponkan' samples from some regions with the traditional medicinal variety was the largest， enabling them to be considered as the emerging medicinal variety.

Correlation analysis between genetic diversity and limonin compounds content of Citri reticulatae Semen / 中草药

Objective To study the correlation between the genetic diversity and limonin compounds content of Citri reticulatae Semen germplasm resources in Sichuan to provide a reference for screening the main source varieties of C. reticulatae Semen. Methods Firstly, NTSYSpc2.10e software was used to analyze the ISSR markers results of 35 C. reticulatae Semen samples from different habitats and different varieties in Sichuan. Then, UPLC was used to determine the contents of limonin compounds (limonin, nomilin, and obacunone). Finally, all the results were analyzed by SPSS 21.0 software. Results A total of 240 clear and detectable DNA bands were generated from 33 ISSR primers, and the variation range of genetic similarity coefficient were 0.72-0.92. Citrus reticulate ‘Ponkan’ and C. reticulate ‘Dahongpao’ have the closest relationship and their Limonin compounds contents were similar, but the relationship between other varieties and Citrus reticulate ‘Dahongpao’ was far and limonin compounds contents had great difference. Conclusion The relationship of variety is closer to the C. reticulata ‘Dahongpao’, the content of limonin compounds is higher, and Citrus reticulate ‘Ponkan’ could be considered as the main source variety of Citri reticulatae Semen.

Grey relational analysis of relationship between limonin compounds accumulation and functional genes expression of Citri Reticulatae Semen / 中草药

Objective: To study the biosynthesis mechanism and transshipment law of limonin compounds in Citri Reticulatae Semen, traditional medicinal variety Citrus reticulata "Dahongpao". Methods: In the process of seed growth and development, the contents of limonin, nomilin, and obacunone in stem, vane, peel, and seeds were determined by UPLC tangerine; Using grey relational analysis method, the correlation analysis between the contents and the cloning of squalene synthase gene (ss), squalene epoxidase gene (se), and glucose transferase (lgt) expression was performed. Results: Limonin compounds in different organs had the largest accumulation in seeds; The accumulation of limonin compounds in different organs was significantly correlated with ss, se, and lgt gene expression, but in the seeds the accumulation of limonin has the most closely correlation with ss, se, and lgt gene expression; The ss gene expression had the largest contribution on the accumulation of limonin, nomilin, and obacunone. Conclusion: The study provides reliable and scientific envidendce for the synthesis mechanism and transshipment law of limonin compounds.

Cloning, analysis, and expression of limonoid UDP-glucosyl transferase gene in Citri Reticulatae Semen / 中草药

Objective: To cloning limonoid-UDP-glucosyl transferase (LGT) gene in Citrus reticulata, analyze the bioinformations of LGT, and compare the expression to provide the foundation for the composite and regulation mechanism of the active ingredient in Citri Reticulatae Semen. Methods: LGT gene was cloned the protein characteristics were analyzed using bioinformatics and constructing phylogenetic tree. The expression of LGT in the different parts was analyzed using real time-PCR. Results: A 1 530 bp LGT sequence in in C. reticulata, was obtained, which had a 1509 bp ORF and could encode 502 amino acids. And protein analysis and forecast of its secondary and tertiary structures were carried out. Gene expression analysis revealed the expression of citrus LGT was the lowest in semen, and was followed by skin, and the highest expression of fresh. Conclusion: LGT gene of C. reticulata, and is successfully cloned, analyzed, and expressed a basis for the study of matter in the synthesis and regulation of limonoids is provided.