Study On The Preparation And Physiological Activity Of Naringin Dihydrochalcone

Exocarpium Citri Grandis（ECG）belongs to Rutaceae and Citrus,it belongs to a mutant Huazhou pomelo.It is mainly produced in Huazhou City,Guangdong Province,its fruit is small and sour and bitter,which is not suitable for consumption.Its medicinal components are the dry exocarp of immature or near-mature fruit of Huazhou pomelo.Flavonoids are one of the main active components of ECG,in which naringin accounts for more than 80%of the total flavone content.the content of naringin in Chinese pharmacopoeia is the main evaluation index of ECG‘quality.As a kind of citrus fruit,amino acid is also one of the important nutritional components of citrus,studies have shown that amino acids in addition to the composition of proteins,there are some special pharmacological functions.Naringin of ECG is an effective monomer extracted from traditional Chinese medicine ECG,and studies have shown that the monomer and its metabolites have obvious anti-inflammatory,antitussive and expectorant effects.In this paper,the preparation technology of Naringin dihydrochalcone（Naringin DC）was optimized,and the antioxidant activity and inhibitory tyrosinase activity of Naringin DC were discussed in detail.Through the combination of experiment and calculation,the antioxidant activity and tyrosinase inhibition ability of Naringin DC were systematically explained in this paper,which provided a theoretical basis for expanding the efficacy and application of Naringin DC.The related research results of this paper are as follows:1.The amino acid contents of fruit,leaves and flowers of ECG in three different months were determined by amino acid analyzer,the results showed that:The more mature the ECG fruit,the more total and species of amino acids will be.The total amount of 22 kinds of amino acids and the total amount of effective amino acids were higher in flowers than in fruits and leaves,which indicates that different parts of ECG may be quite different in nutrition and medical application.Naringin from ECG was extracted by ethanol assisted by ultrasonic wave,and the optimum extraction parameters were obtained by response surface optimization.:The ultrasonic power was 180 W,the ultrasonic time was 50 min,the extraction temperature was 57.5℃,and the ratio of liquid to material was 60.9 m L/g,The volume fraction of ethanol was 50.8%.Under these conditions,the extraction rate of naringin from ECG was 7.87%.Naringin in alcohol extract of ECG was purified by water precipitation method designed by orthogonal experiment.The structure of naringin was characterized by IR,SEM and LC-Ms.the purity of naringin monomer was 95.1%by liquid chromatography.2.The prepared naringin monomer was added to the stainless steel autoclave for catalysis and hydrogen was added to change its properties.the single factor experiment was designed with the yield of Naringin DC as the index and the optimum conditions for hydrogenation to change the properties were obtained.The structure of Naringin DC prepared under these conditions was characterized by IR,SEM and LC-Ms.its purity was 95.5%-99.9999%by liquid chromatography,and its sweetness was 400 by sensory comparison,which indicates that Naringin DC has high purity and sweetness.Naringin DC was bound to human sweet receptors T1R2 and T1R3 by Discovery Studio software,the order of affinity between Naringin DC and sweet receptors was determined by the binding energy,it is:T1R3>T1R2.It is very likely that Naringin DC binds to human sweet receptor T1R3 first and then to T1R2,thus showing sweetness.The pharmacophore models of 10 kinds of dihydrochalcone were calculated by Discovery studio software.the sweetness of dihydrochalcone was shown by comparing Fit Value and the pharmacophore model can also be used to screen new or synthetic dihydrochalcone in order to find novel lead compound molecules.3.The optimum bond length,bond angle and vibration frequency of Naringin DC molecule were obtained by Materials Studio software,and the parameters of entropy,heat capacity,enthalpy,free energy and two orbital energy level differenceΔ_ELUMO-HOMO of Naringin DC molecule were also obtained.ItsΔ_ELUMO-HOMO value is small,so the energy level of electron transition is low and the molecular activity is high.The results of Mulliken charge distribution,Fukui index and frontier orbital calculation show that the oxygen atom on phenolic hydroxyl group in the molecule is the site of electrophilic reaction of Naringin DC.Naringin DC has high reactivity and strong antioxidant activity.The antioxidant experiment showed that naringin,Naringin DC,NHDC and quercetin had certain scavenging ability on DPPH free radical,ABTS free radical,hydroxyl free radical and oxygen free radical,and it can reduce iron ions.The results of the five antioxidant methods were in good agreement with the Fukui function diagram andΔ_ELUMO-HOMOcalculated by Material Studios 8.0.the results showed that the oxidation resistance of the hydrogenation product Naringin DC was improved due to the change of structure in the process of naringin hydrogenation modification.In order to confirm the correlation between the experimental results and the calculated results,the DPPH free radical scavenging ability andΔ_ELUMO-HOMO value of 18 flavonoids and sweeteners were determined,it was concluded that:The antioxidant activity of the compounds was negatively correlated with theirΔ_ELUMO-HOMO values.the stronger the antioxidant activity was,the smaller theΔ_ELUMO-HOMO was.The thermodynamic data of Naringin DC scavenging DPPH radical reaction transition state calculated by Gaussian09 software show that,Naringin DC scavenging DPPH radical reaction is thermodynamically favorable,thus explaining the antioxidant activity mechanism of Naringin DC from the quantum chemical level.The docking of Naringin DC with SOD showed that the terminal benzene ring of Naringin DC could form cation-πinteraction with amino acid residue B/Arg-115.Moreover,Naringin DC can interact with amino acid residues A/Leu-106,A/Gly-108and A/Cys-111 to form three hydrogen bonds with lengths of 2.3?,2.1?and 2.6?respectively.These interactions make Naringin DC form a stable complex with SOD.The results showed that,Naringin DC might be an inhibitor of SOD,and the mechanism of antioxidant activity of Naringin DC was expounded at the molecular level.4.The inhibitory effect of Naringin DC on TYR from Pleurotus ostreatus showed that:Naringin DC could effectively inhibit the activity of TYR from Pleurotus ostreatus.In vitro cell experiments showed that,Naringin DC inhibited TYR in mouse melanoma cell line B16 in a safe concentration range,as well as proteins related to melanin production,such as MITF,TRP1 and TRP2,and the m RNA expression values of protein TYR,MITF,TRP1 and TRP2 were lower than those of blank group and naringin group,which were consistent with the experimental results of related protein expression values,which proves that Naringin DC can inhibit the expression of melanin in B16 cells at both protein and gene levels.The molecular docking of Naringin DC with TYR of Agaricus bisporus,Naringin DC can form double hydrogen bonds with amino acid residues His-244 and Met-280 with the lengths of 2.6?and 2.0?respectively.These interactions make Naringin DC form a stable complex with TYR.The results showed that,Naringin DC might be an inhibitor of TYR,so it was speculated that Naringin DC could inhibit TYR at the molecular level.