| This work was supported by the National Natural Science Foundation of China (No. 81373889)-Study on the mechanisms and integral regulation of multi-components of mulberry leaves for diabetes and its complications based on metabolomics.This dissertation was divided into four chapters.In Chapter 1, chemical composition, pharmacological effects, resource chemistry and its applications of mulberry leaves was summarized. Phytochemical studies have revealed that mulberry leaf contains various constituents, including flavonoids, alkaloids, polysaccharides, steroids, organic acids, nucleosides, amino acids, and so on. It has been becoming a hotspot for its efficacies of hypoglycemic, hypolipidemic, anti-platelet aggregation, anti-inflammatory, antioxidant, and so on. The functional products of mulberry leaves have caught people’s eye, such as, mulberry tea. Besides, Ramulus Mori, Fructus Mori, Cortex Mori, is also widely used, there is culture medium made from Ramulus Mori, mulberry wine, drinks, vinegar made from Fructus Mori and Cortex Mori. However, resource chemistry of Folium Mori is so plain that this paper focus on the dynamic analysis of chemical constituents in Genus Morus in China during different harvesting time and the optimum drying process to provide a scientific evidence for rational harvest and comprehensive utilization of mulberry leaves.In Chapter 2,6 type of chemical compositions were carried out on 88 different samples of mulberry leaves. Flavonoids, alkaloids, nucleosides, amino acids, polysaccharides and protein was tested based on the ultraperformance liquid chromatography coupled with a triple quadrupole mass detector, method of phenol-concentrated sulphuric acid and kjeldahl method. As a result, the chromatographic conditions in the determination method of Mori Folium in Chinese Pharmacopoeia was optimized, and an ultraperformance liquid chromatography coupled with a triple quadrupole mass detector and electro spray ionization-mass spectrometer method (UPLC-TQ/MS) has been established for the simultaneous determination of the 14 nucleosides and nucleobases,24 amino acids and 2 main alkaloids in mulberry leaf. The analysis of flavonoids were performed on Acquity UPLC BEH C18 column (100 mm×2.1 mm,1.7 μm), with the gradient elution (0～12.5 min,95%Aâ†'5%A; 12.5～14 min,5%) of 0.1% formic acidaqueous (A)-acetonitrile (B) at a flow rate of 0.4 mL·min-1. The injection volume was 1μL and the column temperature was controlled at 35℃. It was found that the difference between varies varieties were huge, the range of rutin is 0.01-2.34 mg/g, for isoquercitrin is 0.02-2.35 mg/g, for astragalin is 0.01～1.49 mg/g, for resveratrol is 0.07～1.37 mg/g. The analysis of alkaloids, amino acids and nucleosides were performed on an Acquity UPLC BEH Amide (100 mm×2.1 mm,1.7 μm) column was applied for all analyses. The mobile phase was composed of A (5 mM ammonium formate and ammonium acetate solution,0.2% formic acid) and B (Acetonitrile with 1 mM ammonium formate, ammonium acetate solution and 0.2% formic acid) with a gradient elution:0～3 min,10% A; 3～9 min,10â†'18%A; 9～15 min,18â†'20% A,15～16 min,20â†'46% A, 16～18 min,46% A. The flow rate of the mobile phase was 0.4 mL·min-1, the column temperature was maintained at 35℃. It turns out that the content of different samples is abundant but unequal. The range of DNJ is 0.24～8.49 mg/g, the content of fagomine is low. The highest content of polysaccharides is 3.49% in Morus alba, whereas the lowest content is 0.79% in Morus multicaulis. The highest content of protein is 22.14% in Morus australis, whereas the lowest content is 9.54% in Morus mongolica. This part of our research reveals the difference in various chemicals between 88 samples, which provides an important basis of rational utilization of mulberry leaves.In Chapter 3, The optimum harvesting time and utilization of mulberry leaves during different growth periods was investigated based on the content of alkaloids and flavonoids,88 samples of 11 species of mulberry leaves. UPLC-TQ/MS method was applied and the results showed that the ingredients of alkaloids and flavonoids in mulberry leaves are quite different in different growth periods and different species. There was a sharp decline of the average content of alkaloids in all samples from October, while the content of flavonoids dropped either from October but with less volatile. The content of flavonoids in M. atropurpurea was much higher than alkaloids, while M. australis was opposite completely. There was a sharp decline of alkaloids in M. cathayana and M. mongolica from July to August, however, the content of alkaloids and flavonoids in M. alba is neither too high nor too low. In summary, it is more suitable to harvest tender mulberry leaves harvested from the end of September to beginning of October. Leaves of Lun Ha No.2, Yu 711 and He Sang were collected every three days from Oct. 13～Nov.30,2014, in which the flavonoids and alkaloids were tested to discuss the dynamic change of mulberry leaves before and after the frost. It was found that the content of all compounds was with a slight fluctuation but no obvious increasing trend. DNJ in all samples is high, whereas fagomine is very low. There is a trend of declining of DNJ in Lun Ha No.2 and He Sang, whereas the changing curve of DNJ in Yu 711 is ’U’, that is, there is a increase trend of DNJ in Yu 711 after the frost. The research in this part provides a scientific evidence for rational harvest and comprehensive utilization of mulberry leaves, but the real connotation of’Shuang Sang Ye’needs further elucidation.In Chapter 4, five drying technology were compared in Yu 711 including drying in sun or shade, hot air drying, MWIR drying and controlled temperature and humidity drying, in which fixing time and temperature were controlled. Suitable drying method was confirmed based on the color of dried mulberry leaf and the content of flavonoids and alkaloids. It was found that content of all compounds in fresh leaves, dried in sun or shade was lower than others. Finally,hot air drying at 50℃,60℃, and 60℃ after 60 s fixing at 100℃ was better than others which provided standards for drying supporting the quality of mulberry leaves. Meanwhile, samples were collected during hot air drying at 60℃ after 60 s fixing at 100℃ every half an hour, all tested analytes were determined to probe into dynamic change of the tested compounds during the drying process. |
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