| Toona sinensis(T. sinensis) leaves are usually used to treat enteritis, dysentery in clinic practice for functions of clearing heat and eczema, anti-inflammatory as well as detoxification, and they are also edible vegetables preferred by common populations. Recent research has demonstrated that leaves extraction have some bioactivities including antioxidant, anti-cancer and anti-hyperlipidemia, etc. T. sinensis leaves are an edible Chinese medicine with broad prospects for containing many types of compounds. In this study, the chemistry constitutions in T. sinensis leaves were explored and flavonoids were analyzed as the following four parts:Part 1 Isolation and identification of phenolic compounds in T. sinensis leaves Liquid-liquid partition, and column chromatographic technologies were applied to isolate and purify chemical compounds, and then UV-Vis, ESI--MS, 1H-NMR and 13C-NMR were used to identify their chemical structures. 8 phenolic compounds were obtained from T. sinensis leaves, namely, dibutyl phthalate(1), 1,2,3,6-tetra-O-galloyl-?-D-glucopyranose(2), 1,2,3,4,6-penta-O-galloyl-?-D-glucopyranose(3),(-)-epigallocatechin gallate(4), quercetin-3-O-?-D-glucopyranoside(5), quercetin-3-O-?-L-rhamopyranoside(6), kaempferol-3-O-?-D-glucopyranoside(7) and kaempferol-3-O-?-L-rhamopyranoside(8). Compound 1 and compound 4 were separated for the first time from T. sinensis, and compound 4 was separated for the first time from Toona genus.Part 2 Content determination of total flavonoids in T. sinensis leaves Rutin was used as reference substance and UV method was established to determine the total flavonoids. Results showed linear relationships between concentrations and peak areas were good when the concentration ranged from 4.05?g/m L to 50.6 ?g/m L. Moreover, stability, repeatability and reproducibility were good, and recovery was 97.27%, which suggested that the established method can be used for total flavonoids determination. Furthermore, flavonoids in leaves harvested from various areas and times were analyzed, and results showed apparent difference among them. Namely, the amount of total flavonoids is higher from April to June than other time within the year. And the amount of total flavonoids in leaves harvested in Shandong Yantai is the highest, while the lowest was harvested from Shangdong Yuncheng.Part 3 Extraction of total flavonoids from T. sinensis leaves Ethyl alcohol was used to extract total flavonoids from T. sinensis leaves by use of refluxing in hot water bath, and extraction conditions were optimized using orthogonal design. The best condition was obtained after optimization, namely, solid-70% ethyl alcohol(1:30), temperature at 80 oC, extraction duration at 1.5 hrs, reflux twice. Under the best condition, the extraction rate was 3.356%, and the yield of the total flavonols after purification using macroporous resin was 90.866%. The method after optimization in this study was sample, economy, safe, clean and efficient, and it is promising to be applied in industry application.Part 4 Determination of four flavonol glycosides and hierarchy cluster analysis of T. sinensis leaves from different areas Four flavonol glycosides including Rutin(I), quercetin-3-O-?-D-glucopyranose(II), quercetin-3-O-?-L-rhamopyranoside(III), kaempferol-3-O-?-L-rhamopyranoside(IV) were chosen to be analyzed. And then the extraction and separation condition were optimized to establish a simple, stable, accurate and well separated analysis method. After optimization, the samples were loaded on an Waters RP18(2.1 mm × 150 mm, 1.7 ?m) column, eluted using mobile phases consisting of 0.3% TFA H2O(A) and 0.15% TFA ACN(B), and the percentage of A was decreased from 85% to80% from 0 to 20 min, then continued to decrease from 80% to 70% from 20 min to 31 min. A flow rate of 1.0 m L/min and temperature at 30°C were kept constant during analysis. A detection wavelength of 254 nm and a sample injection volume of 10 ?L were used for solution analysis. Under the HPLC condition, compound I, compound II, compound III and compound IV showed good linear relationships in the range of 1.038~66.4, 1.147~73.4, 2.566~164.2 and 1.691~108.2 ?g/m L, and correlation coefficient(r2) of the method is 0.9994, 0.9996, 0.9995 and 0.9990, respectively. Furthermore, the established method has a good precision(0.13%~1.76%, n=6) and repeatability(RSD<5%, n=6) and sample solution is stable within 48 hrs. Average recovery of this method is from 96.01% to 108.53%(n=3), RSD<5%. The contents of four flavonoid glycosides in T. sinensis leaves from 11 areas were determined. As a result, the contents of compound I, II, III and IV were 0.007%~0.200%, 0.026%~0.453%, 0.085%~1.507% and 0.026%~0.833%, respectively. Consequently, the contents of flavonoid glycosides in the leaves of T. sinensis from different areas were various. Among those, the leaves harvested from Shandong Yantai contained the highest of the four flavonol glycosides, while Zhejiang Wengxian contained the lowest. Then, hierarchy cluster analysis was carried out to distinguish leaves from 11 areas, the results showed A, B, D, E, G, I, J and K belong to same cluster, while C, F and H belong to the other cluster. |
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