Ultrasonic-assisted Extraction Of Sea Buckthorn Leaf Flavonoids And Their Biological Activities

Sea buckthorn（Hippophae rhamnoides L.）,as one of the herbs of medicinal food,is an excellent plant species for windproof and soil-fixing,and sea buckthorn leaves,as one of the medicinal parts of sea buckthorn,is now a new resource food in China.Sea buckthorn leaves are rich in flavonoids,polysaccharides,volatile oil and other active compounds,which have pharmacological effects such as hypoglycemic and hypotensive,antioxidant,antibacterial and antitumor.Especially flavonoids are widely used as an important active substance in the treatment of hypoglycemia,antitumor drugs and the development of related health products,and its content in sea buckthorn leaves is higher than that of fruits,but the current research mainly focuses on fruits,and the exploitation of leaves is less,resulting in a large waste of our by-products.There are more methods about the extraction and purification of sea buckthorn leaf flavonoids,but the extraction rate and purification rate are not high.In order to improve the utilization value of sea buckthorn leaf flavonoids,ultrasound-assisted extraction of sea buckthorn leaf flavonoids was used and the best extraction conditions were obtained to obtain sea buckthorn leaf crude flavonoids（FSL）;the first purification of sea buckthorn leaf flavonoids（FSL-1）was obtained by the secondary separation of AB-8macroporous resin and polyamide resin,and then analyzed by LC MS/MS was used to analyze the composition of FSL-1,and then the antioxidant and antibacterial biological activities of FSL-1 were evaluated by chemical methods;finally,the effect of different concentrations of sea buckthorn leaf flavonoids on the quality of fresh-cut lettuce was investigated by making a composite preservative with chitosan.The specific studies were as follows:（1）The best extraction conditions of sea buckthorn leaf flavonoids by ultrasonic-assisted extraction were 19:1 material-to-liquid ratio,47%ethanol ratio,65°C,34 min,and 360 W ultrasonic power,and the validation experiment FSL was extracted to 66.006mg/m L;（2）The purification was carried out by AB-8 macroporous adsorption resin and polyamide resin,and the purified product FSL-1 was obtained,and the flavonoid content was increased from 12.43%to 50.18%before purification,and the composition analysis of FSL-1 was carried out by LC-MS/MS,and the main components of FSL-1were rutin,isorhamnetin and hesperidin;（3）Chemical antioxidant experiments were conducted using FSL-1,and it was found that when the concentration of FSL-1 was higher than 0.125 mg/m L,the highest scavenging rate of other free radicals except O₂^-radicals and good total reducing ability were obtained,which was close to the positive control V_C,indicating that FSL-1 has good antioxidant activity.（4）The antibacterial activity of FSL-1 against four foodborne pathogens,namely Staphylococcus aureus,Escherichia coli,Salmonella typhimurium and Listeria monocytogenes,was determined by punching method,twofold dilution method and absorbance method.The results showed that FSL-1 inhibited the growth of all the four bacteria,and FSL-1 had the best inhibitory effect on S.aureus,and the higher the concentration,the more significant the inhibitory effect.（5）The inhibition stability of FSL-1 on S.aureus was explored by UV irradiation time,disinfection temperature,p H,Na₂SO₃,metal ions,food flavoring and food preservatives,and it was found that the inhibition activity of FSL-1 decreased after the influence of these factors,indicating that FSL-1 is not a stable inhibition substance;（6）A new composite preservative（FCCP）was made by wrapping FSL-1 with chitosan,and the results of Fourier transform infrared spectroscopy showed that the viscosity and contact angle of the composite preservative increased when FSL-1 was combined with CS substrate,and its film-forming performance was superior;the scavenging ability and total reducing ability of FCCP for DPPH and ABTS radicals also increased with the increase of FSL-1 concentration The scavenging ability and total reduction capacity of FCCP for DPPH and ABTS radicals also increased with the increase of FSL-1 concentration,which was close to the positive control VC;the bacterial inhibitory performance of FCCP also improved.（7）FCCP was used as an active packaging material to coat fresh-cut lettuce,and it was found that FCCP could effectively prevent the oxidation and deterioration of fresh-cut lettuce to extend its shelf life,and the compound preservative of FCCP-2 with the addition of 1.0 mg/m L of FSL-1 could more effectively maintain the quality of fresh-cut lettuce.In conclusion,the response surface optimized ultrasonic-assisted extraction process extracted sea buckthorn leaf flavonoids with high extraction rate and short extraction time.the secondary separation and purification of sea buckthorn leaf flavonoids by AB-8 and polyamide resin showed high purification rate,and FCCP had better antioxidant activity and antibacterial activity than chitosan or FSL-1 alone,and more low concentration and high biological activity.