Separation And Preparation, Structural Analysis And In Vitro Activity Of Anthocyanins From Mulberry Fruits

Mature mulberry fruits are rich in anthocyanin pigments, which may be used as apromising source of such pigments. Mulberry anthocyanins show good water solubility,non-toxic and varieties of biological functions, which make these pigments a natural colorant,antioxidant, and nutraceuticals in the food industry. The fresh Morus alba L. mulberry fruitsfrom Dingtao county, Heze city of Shandong province were used as raw material, and thepaper carried out a systematic study of the extraction and purification process, structurecharacterization, antioxidant and anticancer activity of mulberry anthocyanins. The mainconclusions were summarized as follows:Anthocyanins in mulberry were extracted by water bath shaking, ultrasonic-assisted andultrasonic-microwave-assisted extraction methods, and the process conditions of the threeextraction methods were optimized. The optimal conditions of water bath shaking methodwere: liquid-to-solid ratio10, the extraction was kept at50°C for1.5h. The optimalextraction conditions of ultrasonic-assisted extraction were: ultrasonic power400W,liquid-to-solid ratio10, the extraction was stayed at30°C for5min. The best extractionprocess of ultrasound-microwave-assisted extraction was: microwave power, liquid-to-solidratio and extraction time were90W,3and180s, respectively. Comparing the three extractionmethods at the optimal extraction conditions respectively, it was found thatultrasound-microwave-assisted extraction was superior to ultrasound-assisted extraction, andultrasound-assisted extraction was better than water bath shaking extraction.The static adsorption and desorption capacities of12kinds of macroporous adsorptionresins and6kinds of cation exchange resins to mulberry anthocyanins were compared, and itwas found that the best macroporous adsorption resin was LX-68and the optimal cationexchange resin was D001on the basis of static experiment results. Through the static anddynamic conditions optimization, the best purification conditions of mulberry red pigmentused by LX-68resin were sample with concentration of0.991Abs, pH3, absorption velocity8BV/h and with pH2,80%of acidic ethanol as elution agents, elution velocity1BV/h. Thepigment purified by LX-68resin was purplish dark powder, the chromosphere was114, theanthocyanins content was39.9%, and the recovery of anthocyanins was91.5%. D001resin’soptimum purification conditions were that the concentration, pH, feeding rate of solution were1.411Abs, pH2and6BV/h respectively, and elution solvent was pH1,60%of acidic ethanolwith3BV/h elution velocity. The pigment purified by D001resin was atropurpureus powderwith color-value of65, anthocyanins content of24.1%, and the recovery of anthocyanins was 67.6%. Both LX-68resin and D001resin presented good adsorption separation properties tomulberry anthocyanins. However, the separation effect of LX-68resin was better than D001resin.The analysis of the mulberry anthocyanins employed UV-Visible, HPLC-DAD,HPAEC-PAD, UPLC-MS and1H NMR. Four anthocyanins were identified as cyaniding3-O-glucoside (68.61%), cyanidin3-O-rutinoside (30.13%), pelargonidin3-O-glucoside(0.87%) and pelargonidin3-O-rutinoside (0.34%). Moreover, the non-anthocyanin polyphenoland flavonoid compounds of pyrogallic acid, chlorogenic acid, gentisic acid, caffeic acid,vanillic acid, syringic acid, rutin and quercetin were found in MAR.In the antioxidant activity assays, it was found that the antioxidant activities of thesamples were increased dose-dependently. The MAHP, C3G and C3RG purified bysemi-preparative HPLC have strong antioxidant activity, which could be proved by the factthat their quenching˙OH capacities were even higher than Vitamin C and the activity ofMAHP’s scavenging˙DPPH had no significant difference with Vitamin C. While theantioxidant activities of other samples were all lower than Vitamin C. In the˙DPPH,˙OH, and˙O2ˉsystem, the radical scavenging activities of the five samples decreased in the order ofMAHP> C3G> C3RG> MAR> CME. In the reducing power assay, the reducing power ofthe samples decreased in the order of MAHP> MAR> C3G> C3RG> CME. However, theantioxidant activity of MAR was less than the MAHP, the process of MAR was simple,lower-costing, and considering its antioxidant activity was much higher than CME, MAR wasan ideal natural antioxidant, which could be used as an industrial production.In the inhibition of cancer cell proliferation system, CME, MAR and MAHP were allshowed inbihition activities of liver (Hepa1-6, HepG-2), breast (MDA-MB-231, MCF-7) andlung (A549) cancer cells, which were dose-dependently. For the proliferation inhibition ofHepa1-6, HepG-2, MDA-MB-23, MCF-7and A549, the IC50of CME were10.30mg/mL,17.75mg/mL,17.13mg/mL,18.90mg/mL and21.56mg/mL, the MAR’s were0.51mg/mL,1.38mg/mL,1.48mg/mL,1.49mg/mL and1.75mg/mL and the MAHP’s were0.54mg/mL,1.20mg/mL,0.94mg/mL,1.24mg/mL and1.79mg/mL, respectivily. Comparision betweenthe five cancer cells, Hepa1-6was the most vulnerable and A549was the least sensitive tomulberry anthocyanins. The anticancer activities of MAHP and MAR were approximate,which were significantly greater than CME’s. With simple processing and significantanticancer activity, MAR could be used as potential anticancer drugs.