| China is the world's largest country of Camellia oleifera oil production. Over one-hundred-thousand kilograms of C. oleifera oil are produced annually in China. But a big amount of by-product, the seed cake of C. oleifera is produced after the oil refining process. The defatted seed cakes are always used as detergent or organic fertilizer with low economic value. Therefore, the comprehensive utilization improvement of C. oleifera seed cake and the exploration of active compounds in it are urgent. The flavonoids in seed cake have been researched in system in this paper, which will be expected to provide a useful guideline for utilization of the flavonoids in seed cake, and enhance the commercial value of seed cake in all-round way. The main investigations and results in this paper were listed as following:1. Different techniques of extracting flavonoid compounds from the seed cake of C. oleifera were analyzed and compared. They were solvent extraction, heat reflux (HRE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE). The results showed that in rapid extraction of 15 min, the extraction efficiency of MAE was 1.17%, which bringed higher yield of flavonoids than conventional solvent extraction and HRE in 2 h. The flavonoid yield of MAE in 15 min was slightly lower than that of UAE in 2 h, but MAE took only one by eighth time of UAE and saved a lot of time. Therefore, considering the extraction efficiency, time-consuming and energy consumption, microwave-assisted rapid extraction was choosed to extract the flavonoids from the seed cake.2. The preparative separation and purification of flavonoid glycosides from seed cake was studied by high-speed counter-current chromatography (HSCCC). A rapid and efficient HSCCC method was developed:the two-phase solvent system composed of ethyl acetate/butanol/3%acetate (1:4:4, V/V), the flow-rate of mobile phase 1.5 mL/min, the rotation speed 800 rpm, the sample volume 120 mg in 3 mL lower phase. The results showed that two flavonoid compounds (11.2 mg and 15.4 mg) were obtained within 350 min, with the purity of 99.1% and 94.5%, respectively.3. The chemical structures of the two compounds isolated in HSCCC were identified by UV, IR, LC-MS and 1H NMR,13C NMR. According to spectrum analyses, the structures were elucidated and characterized as kaempferol 3-O-{α-L-rhamnopyranosyl-(1â†'6)-β-D-glucopyranoside}-7-O-β-D-glucopyranosid e and kaempferol 3-O-{β-D-glucopyranosyl-(1â†'4)-a-L-rhamnopyranoside}-7-O-α-L-rhamnopyranoside, which are identified as novel compounds in the C. oleifera seed cake.4. Study on DPPH free radical scavenging efficiency and the protective effect on injured human umbilical vein endothelial cells (HUVECs) of two flavonoid monomers from the seed cake. The results showed that in concentration range of 5-25μg/mL, the clearance of two flavonoids were 17.3%～81.3% and 19.1%～83.9%, respectively, which showed favourable DPPH scavenging efficiency. Otherwise, the protective effect on injured HUVECs of flavonoids were analysed by MTT method. The experiments showed that the OD490nm of the first monomer in 10,100μg/mL were increased significantly (P<0.05), indicating that it had protective effect on HUVECs. When the concentration was 1000μg/mL, the first monomer had no protective effect. Besides, the OD490nm of the second monomer in 10,100,1000μg/mL were all increased significantly (P<0.05), suggesting that the second monomer had protective effect on HUVECs. These results show that the flavonoids in seed cake exhibit strong biological functions, which have great value to exploit. |
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