Studies On Flavonoids Effective Components In Vaccinium Bracteatum Thunb. Leaves And Function Of Anti-tumor

Vaccinium bracteatum Thunb. is medicinal plant, which is of Vaccinium(Erioaoeae). Effective components of its leaves have been applied in food addition and eye-disease medicine abroad, TCM holds that, black rice, which is made of the juice of Vaccinium bracteatum Thunb. leaves and glutinous rice, has several functions, including light-body, bright-eyes, black-hair, good-face, and long-life. This research focused on analysis of flavonoids and other effective components in Vaccinium bracteatum Thunb. leaves and its function of anti-tumor. The effects of different drying methods on appearance quality, effective and nutritional components were contrasted, the conditions based on RSM for extracting flavonoids from Vaccinium bracteatum Thunb. leaves were optimized, extraction conditions of flavonoids from leaves with SC-CO₂ were sought, acute toxicity test and function of anti-tumor of ethanol extract were studied, and the anti-tumor active component——quercetin by combining HSCCC and HPLC was separated. The main results were as follows:1. Fresh materials were treated respectively with hot blast drying, natural drying, vacuum low-pressure drying, and vacuum freeze-drying, the effects of different drying methods on appearance quality, effective components(including flavonoids, flavonoid glycosides, polyphenol, and tannin) and nutritional components(including soluble protein, soluble sugar, raw fat) were contrasted. Besides, change tendency of effective and nutritional components in different growth periods, distribution law of flavonoid glycosides in different growing zones, and flavonoid glycosides regional differences were analyzed. The results showed that, the effects of vacuum freeze-drying on appearance quality, effective and nutritional components were least, the effective and nutritional components content of the leaves in fall were more than that in spring and summer except polyphenol. There was a significant difference of flavonoid glycosides content in different origins and pick times (p<0.05), the experimental materials were with branchlets, and moisture content of experimental material was 0.1～0.5%, pick time was from June to September, the origin was Guling in Fuzhou.2. Considering saving and safety, extraction solvent was ethanol. Single - factor experiment, fraction factorial design experiment and response surface methodology (RSM) were applied to optimize the solvent extraction conditions of flavonoids from Vaccinium bracteatum Thunb. leaves. Analysis of main components in ethanol extract and the temperature effect on ethanol extract active component by HPLC were made. The results suggested that: ethanol concentration and extraction temperature should be two statistically significant factors. The optimized conditions were as follows: soaking time (60 min), the ratio (V:W) of solvent to material (50:1), ethanol concentration (86.7%), extraction temperature (61℃) and extraction time (2 h). Under the optimized operating conditions, the once extraction rate of flavonoids was 68.12%, flavonoids had large content (20.76%) in ethanol extract, tannin had a certain content(8.82%), the temperature near 60℃had little effect on flavonoids in ethanol extract. High temperature(70℃) had a bad effect on ethanol extract.3. Single-factor experiment and orthogonal test were applied to optimize SC-CO₂ extraction conditions of flavonoids from Vaccinium bracteatum Thunb. leaves. It was the first time to discuss the effect of co-solvent, co-solvent concentration, and dosage on extraction amount of flavonoids. Extraction rate of flavonoids and components in extract with SC-CO₂ extraction were compared with that of solvent extraction. The results suggested the optimized conditions were extraction pressure 16MPa, extraction time 1.5h, extraction temperature 50℃, co-solvent ethanol concentration 75%, flow of CO₂ 20 kg/h, addition of co-solvent 5mL/g. Co-solvent can increase the extraction amount to a certain extent, but most of flavonoids can not be extracted. Analysis of SC-CO₂ extract by HPLC showed SC-CO₂ extraction can extract more chemical components compared with solvent extraction, but the extraction rate with SC-CO₂ extraction was less 11 % than that with solvent extraction.4. On the basis of earlier stage work, the acute toxicity test was done with ethanol-extract from Vaccinium bracteatum Thunb. leaves. The result showed that, LD₅₀ of ethanol extract from leaves calculated by DAS ver1.0 Bliss method was 39.1g/kg, 95% confidence limit was 36.8～41.6g/kg, and the mice had no acute toxicity side effect, so this ethanol extract is safe in clinical application.5. Model groups(transplanted U14 and S180 model in mice), control groups(CTX) and treated groups of ethanol extract (5,2.5,1.25 g/kg for U14, 8,7,6 g/kg for S180) to observe the inhibition by calculating tumor inhibitory rates. The results showed that high dose of ethanol extract had significant inhibition to U14 (p<0.05), tumor inhibitory rate reached 28.76%, middle and low dose had no inhibition to U14.High, middle, and low dose of ethanol extract had no inhibition to S180.6. This part focused on separating the anti-tumor active component——quercetin from ethanol extract by combining HSCCC and HPLC. The results showed that, there were two solvent systems obtained from the primary-selecting test by TLC, the system of chloroform-methanol-water(4∶3∶2)had good separating effect, the fraction A obtained has the similar UV-Vis absorption spectral characteristics with control substance of quercetin, the purity of fraction A caculated by area normalization method was 98.3%. While the fraction A obtained by the system of chloroform-methanol-butanol-water(5∶1∶3∶1l)was mixed materials, and was separated two main components by HPLC, the component 1 with large content was proved the flavonoids——quercetin, compared with standard chromatogram.