Study On The Repair Effect Of Anthocyanin Extract Of Aronia Melanocarpa On Acute Lung Injury

Acute lung injury(ALI)is a kind of lung pathological injury that leads to the damage of lung cells and uncontrolled response due to direct or indirect injury factors.In severe cases,it leads to acute respiratory distress syndrome(ARDS),which is also one of the main lethal factors of COVID-19.In addition,ALI is a major disease that endangers human health and has a mortality rate of up to 40%.It has attracted people’s attention because of its complex pathogenesis.At present,many studies have shown that some anti-inflammatory,antioxidant and anti apoptotic drugs have potential therapeutic effects on ALI,but there are still some adverse effects.Therefore,it is of great significance to find more effective prevention and control methods.Aronia melanocarpa is a very special small berry.Its fruit is rich in a variety of nutrients,especially polyphenols.As the main polyphenols,anthocyanins have a wide range of effects,such as anti-inflammatory effect.However,there are few reports on whether the anti-inflammatory effect of anthocyanins can inhibit ALI and the related mechanism.Therefore,this paper studies the extraction and purification,species identification,in vitro antioxidant capacity of anthocyanins from Aronia melanocarpa,and the anti-inflammatory repair effect of LPS induced acute lung injury in mice,in order to provide new ideas for the adjuvant treatment and drug processing of acute lung injury.The main results are as follows:The process conditions of conventional acidified ethanol extraction(Et OH),natural eutectic solvent(NADES),ultrasonic microwave synergistic ethanol extraction(UMAE-Et OH)and ultrasonic microwave synergistic natural eutectic solvent extraction(UMAE-NADES)were optimized by single factor test and response surface methodology.Taking the anthocyanin extraction content as the index,the four extraction methods were compared from the aspects of test cycle,energy consumption and consumables.The optimum technological conditions of Et OH were as follows:ethanol volume fraction 60%,solid-liquid ratio 1:36g/m L,extraction time 72 min and extraction temperature 60℃.The anthocyanin content was 2.996±0.013mg/g and the extraction rate was 82.25±0.80%.The optimum technological conditions of NADES were choline chloride glycerol(Ch Gly),molar ratio of 1:2,water content of 30%,solid-liquid ratio of 1:26g/m L,extraction time of 51 min and extraction temperature of 51°C.The anthocyanin content was 3.311±0.018 mg/g and the extraction rate was 87.66±0.67%.The optimum technological conditions of UMAE-Et OH were as follows:solid-liquid ratio 1:25 g/m L,extraction time 485 s,extraction temperature 45°C and microwave power 226W.The anthocyanin content was3.599±0.093 mg/g and the extraction rate was 93.09±0.75%.The optimum technological conditions of UMAE-NADES were as follows:solid-liquid ratio 1:15 g/m L,extraction time 367 s,extraction temperature 52°C and microwave power 230W.The anthocyanin content was4.456±0.032 mg/g and the extraction rate was 97.05±0.85%.Compared with the other threemethods,UMAE-NADES has the advantages of high anthocyanin content and short time.AB-8 macroporous resin was used to purify and identify the anthocyanin crude extrac ts extracted by Et OH,NADES,UMAE-Et OH and UMAE-NADES.After purification,the a nthocyanins extracted from Et OH,NADES,UMAE-Et OH and UMAE-NADES were 29.99%,32.88%,36.51%and 44.89%respectively.The anthocyanins extracted by four method s were identified by high performance liquid chromatography-mass spectrometry(HPLC-M S).Six anthocyanins were identified:the dimer of cyanidin-hexoside,cyanidin-3-O-galactosi de,cyanidin-3-O-glucoside,cyanidin-3-O-arabinoside,cyanidin-3-O-xyloside,and cyanidin-3,5-O-dihexoside.Compared with the other three methods,the anthocyanins extracted by U MAE-NADES improved the purity in the purification process.ABTS~+·,FRAP and T-AOC were used to study the antioxidant activity of anthocyanins obtained by four extraction methods.The results showed that the anthocyanins of Aronia melanocarpa had the ability to scavenge ABTS~+·free radicals and reduce iron ions,and the antioxidant abilities of FRAP and T-AOC of anthocyanins were higher than those of V_Cat the same concentration(p<0.05).The antioxidant capacity of anthocyanins obtained by UMAE-NADES is 1-2 times that of the other three extraction methods,in which the antioxidant capacity of FRAP and T-AOC is 2 times and 4.2 times that of V_C,respectively.Therefore,the anthocyanins extracted and purified by UMAE-NADES have the strongest antioxidant activity.Lipopolysaccharide(LPS)-induced acute lung injury in mice was used for in vivo study to investigate the anti pneumonia effect of rowan anthocyanin on ALI mice.The results showed that anthocyanins could significantly alleviate the symptoms of pulmonary edema in ALI mice,basically restore the pulmonary septum and alveolar cavity in the lung tissue of mice,and the repair effect was equivalent to that of positive control dexamethasone(DXM).Its main mechanism is to reduce the proinflammatory factor(TNF-α,IL-1βand IL-6)in mouse serum,lung tissue and bronchoalveolar lavage fluid(BALF),reduce the number of monocyte macrophages,neutrophils and the total number of cells in BALF,reduce the release of MDA in the lung,reduce MPO enzyme activity and increase SOD enzyme activity,so as to reduce the oxidative stress response in mice and inhibit NF-κB related p65 signal pathway and regulation IκBα/p-IκBαprotein expression of genes.TUNEL staining showed that anthocyanins could significantly reduce the apoptosis rate of AIL mice.Conclusion the anthocyanins of Aronia melanocarpa can significantly inhibit the inflammatory response of ALI mice.