| Walnut (Juglans regia L.) green husk is one of the major by-products of walnut fruit, which is produced in a large quantity. During the mature period, most of the walnut green husk are piled up as wast, causing environmental pollution and waste of resources. Only a few walnut green husks are used for medice. Research shows that walnut green husk contains a large amount of polyphenols, and polyphenols have anti-cancer, anti-oxidation, anti-aging, fall blood sugar and other health care function. The comprehensive utilization of effective substances in walnut green husk can not only protect the environment, making the poison forest waste into a kind of important beneficial human resources, but also can enhance the economic income of fruit growers. In this study, walnut green husk was used as raw material. Its nutrient chemical components and nutritional value were determined. At the same time, walnut green husk polyphenols were extracted by 75% ethanol, and its antioxidant activity, a-amylase inhibitory activity and its stability were studied. The components of polyphenols of the extract were identified by LC-MS. Besides, we analyzed the interaction between the different polyphenol components for the inhibition of a-amylase and the interaction mechanism between gallic acid and a-amylase. Also, we preparaed walnut green husk ferment using walnut green husk together with compound fruits and vegetables under normal conditions. The main contents and results were as follows:1. The results of conventional nutrients analysis showed that walnut green husk has good nutrients and the contents of each index were very high. Walnut green husk is rich in potassium, calcium, phosphorus, magnesium, iron and other mineral elements, and it has the characteristic of high potassium and low sodium. Excepted tryptophan, the content of amino acid in walnut green husk was 33.309%, theratio of essential amino acids and total amino acid ratio was 34.56%, the ratio of essential amino acids and essential amino acid was 52.81%, which were close to the ideal proportion by WHO/FAO (40% and 60%).2. Extract the walnut green husk polyphenols by 75% ethanol. Polyphenol contents of the extracts were determined using Folin-Ciocalteu assays. The antioxidant activity of extracts was evaluated using the ability of the scavenge DPPH radical, hydroxyl radical and superoxide radical. The anti-diabetic activity was evaluated by the ability of a-amylase inhibition. The results showed that the content of total polyphenols in the juce and residue extracts were 125.58mg/g and 112.42mg/g, respectively, and the general flavone content were 92.60mg/g and 61.72mg/g, respectively. The IC50 values of the extracts on DPPH radical, hydroxyl radical and superoxide radical were 3.41μg/mL,33.94μg/mL and 0.47mg/mL respectively. The IC50 value on the a-amylase inhibitory was 20.70mg/mL Walnut green husk has strong antioxidant activity and amylase inhibitory activity, besides, they were doed-dependent.3. To dicuss the effect of different environmental conditions including temperature, humidity and illumination on the stability of the polyphenols from walnut green husk. Treated the polyphenol extract by temperature of 30±2℃, relative humidity of 60±5% and illumination conditions and then determined the stability of polyphenols and the functional stability, including antioxidant activity and amylase inhibition for anti-diabetic activity. Results indicated that with the extension of processing time under the high temperature, high humidity and light conditions, the total polyphenol content of flavonoids reduced, and the IC50 for DPPH and hydroxyl free radical clearance and for a-amylase inhibition increased, noticing that the antioxidant activity and amylase inhibition for hypoglycemic activity decreased after the treatment. LC-MS analysis showed that there were 16 kinds of polyphenols in the extract before treatment, in which the content of monogalloyl-glucose, gallic acid were high. However, after oxidation treatment, polyphenols composition and composition changed, vanillic acid, gallic acid and other five kinds of polyphenols absent, HHDP-glucose and digalloyl-glucose only existed in the treated extract. Besides, the content of some polyphenols changed.4. Isobologram analysis found synergy interaction among several polyphenols on the inhibition of a-amylase, including neochlorogenic acid and trigalloyl-glucose isomer, ellagic acid and pyrogallic acid. Antagonistic interaction among several polyphenols, including neochlorogenic acid and pyrogallic acid, gallic acid and ellagic acid. The antagonistic interaction of neochlorogenic acid and pyrogallic acid was more obvious.5. Interaction of gallic acid and a-amylase were analyzed by ultraviolet spectrum, fluorescence spectroscopy, synchronous fluorescence spectrum and the three dimensional fluorescence spectra. The results revealed that there was obvious quenching effect between gallic acid and α-amylase and quenching mechanism was static quenching, a complex of gallic acid-α-amylase was formed. The association between gallic acid and α-amylase changed the molecular conformation of a-amylase. The number of alpha helix, energy levels of π-π* conjugate system in α-amylase decreased. Gallic acid altered the microenvironment around aromatic amino acids, making it more polarity hydrophilic.6. Making full use of walnut resources, the study preparaed walnut green husk ferments using walnut green husk together with compound fruits and vegetables under normal conditions. The ferments had good taste and clear color, it was orange/red brown. |
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