| Flavonoids, such as naringin, are widely used in pharmaceutical, food and cosmetic industry. The bioavailability and bioactivity of natural flavonoids are improved after proper modification. Naringin, one of flavonoids, was employed in this study, and modified by esterification to improve its lipophilicity and bioavailability. Magnetic field was introduced to strengthen the enzymatically synthesis of naringin palmitat in organic solvent with immobilized lipase. Besides, the effect of magnetic field on the lipase was discussed. Furthermore, the interaction between naringin palmitate and protein molecules was investigated so as to obtain the information about drug actions of the esterified naringin. The main contents and results are as follows:1. Enzymatically synthesis of naringin palmitate in organic solvent.Effect of the chain length of the fatty acids, the origin and concentration of the enzyme, the type of organic solvent, the initial water activity, the reaction temperature, the shaking speed or the molar ratio between the substrates on the the enzymatically reaction of naringin with fatty acids were studied. The proper operation conditions were: immobilized lipase Novozym 435 in t-amyl alchohol at 60℃, with the orbitally shaking speed of 150 r/min. Molecular sieves were used to removed the water produced during the the esterifiation. At the fixed conditions, naringin(50 mmol/L) was esterified with palmitic acid(250 mmol/L), the enzyme concentration of 10 g/L. The conversion rate of naringin reached 34.80% after 48 h. A kinetic study showed that the reaction followed the Michaelis-Menton kinetic and a Ping-Pong Bi-Bi mechanism.2. Synthesis of naringin palmitate catalyzed by enzyme under magnetic field.Novozym 435, and Novozym 435 and the organic solvent were separately pretreated by magnetic field. The effects of magnetic field intensity and magnetic pretreatment time on the enzyme activity and conversion rate were studied. The results showed that when with proper magnetic field intensity and pretreatment time magnetical pretreatment would improve the enzyme activity as well as the conversion rate. When the enzyme and the organic solvent were pretreated with magnetic field intensity of 100, 300 or 500 mT and pretreatment time of 1, 2 or 3 h, the enzyme activity was raised. The conversion rate was reached 45.31% from 34.80% after 48 h at the ratio of 37.4%. When only Novozym 435 was pretreated by magnetic field, at 500 mT for 2 h, the enzyme activity was greatly increased and the conversion rate reached 24.20% from 18.66% after 24 h, increased by 29.2%. The secondary structure of Candida antarctica lipse B(CALB), which is the free form of Novozym 435, was investigated with FT-IR after magnetic pretreatment. The results showed that the secondary structure of CALB after magnetically pretreated was almost indistinguishable from the origin. It seemed that the change of enzyme activity was not induced by the secondary structure change of the molecule, but by the subtle change at the active site of the enzyme, that is, the change of the flexibility of the protein.3. Purification and structrual identification of enzymatically synthesized naringin palmitate.Naringin palmitate was purification by a silica column using a solvent mixture system of acetic acetate: ethnol: formic acid(15:1:1) as the eluent or by preparative HPLC with a C18 column(21.5×250 mm) and a UV detector(283 nm) using methanol as the eluent. The injection volume of samples was 3 mL at the concentration of 30 mg/mL, and the flow rare of 20 mL/min.The product prepared by magnetically assisted enzyme catalysis was purified. The structure of the purified product was identified as mono-ester of naringin and palmitic acid by IR spectra and mass spectra. 1H NMR and 13C NMR data showed the regioselective acylation preferred to the 6′′-hydroxyl group of the naringin glucose. It indicated that the regioselectivity of the enzyme did not change after magnetic pretreatment.4. Interaction between naringin palmitate and protein.Fluorescence quenching technique was used to investigate the interaction of naringin or its esterified derivative naringin palmitate with bovine serum albumin(BSA) or lysozyme. The results showed that the interaction between naringin palmitate and protein followed the regular principles as other flvonoids. Naringin palmitate caused the flurescence quenching of BSA or lysozyme through a static quenching procedure. The binding of naringin palmitate with BSA or lysozyme was spontaneous and the energy transfer from the protein to naringin palmitate occurred with high probability. The number of binding sites was calculated as 0.7 and 0.6 at 37℃with BSA and lysozyme respectively. It was more likely that electrostatic interaction was involved in the binding process with BSA, while the binding with lysozyme might mainly account for hydrogen bonding force and Van der Waals force. The decomposing constant of naringin palmitate binding with BSA or lysozyme was lower than that of naringin, which indicated that naringin palmitate might perform longer retention when transported by protein and exert pharmacological effect persistly due to slow release.
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