| Honey is a nutritious food and health products, mainly collecting by bees from nectar secreted and plant sap. Flavonoids—secondary metabolites contained in honey which is to identify the geography attribute, plant sources and authenticity of other honeys has attracted wide attention and research. The rape honey was used to study extraction, determination and separation of total flavonoids, qualitative and quantitative analysis of flavonoids, setting up flavonoids Fingerprinting and the intrinsic link between bee pollen with the honey about flavonoids. The main contents and results are as follows:1. Compared AICl3 method with Al(NO3)3 method, the total extraction flavonoids of honey content were determined by the AICl3 color method, and the Standard Deviation was only 0.063 which comparing determined results with the HPLC method results. AICl3 method was better than Al(NO3)3 that the standard deviation between Al(NO3)3 and HPLC was 1.006.2. Researching six different polarity resins of NKA-9, AB-8, D3520, DM-130, Amberlite XAD-2 and Polyamide through static adsorption, desorption experiments and adsorption rate, and combining with pseudo-first-order empirical formula, intraparticle diffusion model and adsorption isotherms, the results were ultimately obtained that the best resin was Amberlite XAD-2 on flavonoids from honey. The equilibrium adsorption amount of XAD-2 was 39.70±0.55mg/g and desorption rate was 98.87±0.52%. The mainly rate-limiting step for XAD-2 resin adsorbed flavonoids was intraparticle diffusion. The best adsorptive temperature was 25℃.3. To study column chromatography conditions of XAD-2 resin about extracting and separating flavonoids from honey, the optimalizing conditions were:the ratio between sample quantity of honey and resin quantity,1.2:1; pH of solution,2; eluting solution, 90% methanol, and the amount of volume of methanol,3 times column bed, and the recovery was 82.8%. The condition could be better separation and extraction of flavonoids in honey.4. The optimal separation conditions of HPLC for the honey flavonoids were:The 0.3% formic acid solution was used as mobile phase A, methanol was used as mobile phase B. The column was Waters Symmetry C18, with the column temperature was set at 30℃, and flow at 1mL/min. Using the gradient elution,7 kinds of flavonoids were baseline separation and 15 flavonoids of honey sample were isolated. The relative standard deviations of the precision, stability and repeatability were controlled within 5%. 5. To using HPLC-MS/MS analysis of the fragment ions from flavonoids in rape honey, and combing with standard products of 7 flavonoids 23 species were identified, which including five kinds of phenolic acids and 17 flavonoids; Chrysin (267nm,314nm; UV),5,7-Dihydroxy flavanone(243nm,295nm; UV), Pinobanksin(290nm; UV), apigenin(267nm,339nm; UV),5,7-dimethoxy flavanone(243nm,328nm; UV), 3’,4’,5,7-tetrahydroxy-6-methoxyflavone(256nm,355nm; UV) were the first time identified in Rape honey, and those flavonoids provided basis for markers of rape honey.6.54 different origin and batches number of the rape honey were researched for quantitative analysis and fingerprints in this study. Significant different in flavonoid content among the 54 batches of rape honey samples has been observed and the content of which is vary from 0.989-6.477mg/100g, and the content of kaempferol and quercetin contribute to the total flavonoids is lager.29 fingerprint peaks were obtained with peak 25 as the reference peak (kaempferol). Cluster analysis and similarity analysis were performed, and the honey samples can be divided into 4 categories. Sample No.27 G049 from Suizhou in Hubei province, not only had the lowest total flavonoid content, but also was divided into a separate category. The sample may adulterated with other substances. By principal component analysis, sample No.7 and No.12 were divided into a separate category, separately. Although the result by PCA was difference with cluster analysis and similarity analysis, matching between cluster analysis and PCA could be reached to 81.48%. Followed by HPLC analysis using standard honey, commercial honey and Hybrid honey (25% Standard honey+75% commercial honey products) was found only 10 peaks, and the commercial and Hybrid honey were divided into a separate category compared with the different batch of 54 honey samples with Cluster and Similarity analysis.7. According to quadratic regression orthogonal rotation experiment, the results showed that the optimum extraction conditions with ultrasonic wave method were as follows:ethanol-water solvent concentration,76%; liquid ratio,1:30; ultrasonic time, 33min; ultrasonic temperature,51℃. Under these conditions, the theory yield of flavonoids was 2.12%, and the actual yield was 1.89% which reached to 89.15% of the theoretically predicted value.8. The result by HPLC-DAD and HPLC/MS showed that nine kinds of flavonoids aglycone were identified in the rape bee pollen. Which identified flavonoids were quercetin, kaempferol, isorhamnetin and naringenin. Compared rape bee pollen and honey flavonoids, differences were found. Such as:the flavonoids of quercetin, kaempferol and isorhamnetin were existed both bee pollen and honey, and honey have pinobanksin and pinocembrin, but no in rape bee pollen, and bee pollen contains naringenin, honey was not found. The reason may be a series of enzymatic function and process when bee pollen changed to honey, and flavonoids had derivatization to generate the other substances. This study was the basis of for identification of plant sources of rape honey traceability of geography and its authenticity. |
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