Enzyme-assisted Salting-out Extraction Of Lutein And Polyphenols From Marigold Flowers

Marigold?Tagetes erecta L.?,also known as scented hibiscus,golden chrysanthemum,etc.,belongs to the genus Marigold.Marigold has anti-oxidant,anti-bacterial,anti-diabetic,anti-obesity,anti-inflammatory,and reduces the risk of chronic diseases such as heart disease,cancer and age-related eye diseases,and it has been widely used in the food,cosmetic and pharmaceutical industries.At present,there are many reports on lutein in marigold flowers,but there are few reports on other active ingredients such as phenolic substances,polysaccharides and pectin.In this study,enzyme-assisted salting-out extraction technique was used to extract lutein and phenolic substances from marigold flower,and the crude extract of marigold flower was extracted by enzyme-assisted salting-out extraction to achieve one-step extraction and crude separation of various active ingredients.First,enzyme-assisted aqueous two-phase extraction of lutein and polyphenols from marigold flowers was studied.The effects of two-phase salting-out extraction under microwave and the influencing factors of enzymatic hydrolysis conditions were investigated.On this basis,the response surface method was used to optimize the extraction process of lutein and polyphenols from marigold flower by enzyme-assisted two-phase salting-out extraction.The results showed that when the aqueous two-phase system consisted of 30%?w/w?ethanol/19%?w/w?ammonium sulfate,the meshing number was 120 mesh,and the ratio of material to liquid was 1:45 g/mL.The amount of enzyme was 4.2 U/g,and the yields of lutein and polyphenols were the highest at pH 4.00 and 37 ^oC for 117 min,which were 5.59±0.13 mg/g and 83.56±0.69 mg/g,respectively.It is 99.81%and 95.45%.In addition,the IC₅₀ of the enzyme-assisted aqueous two-phase extracts measured by DPPH and ABTS were 49.16?g/mL and 45.97?g/mL,respectively,and the antioxidant activity was better than the two-phase extraction and Soxhlet extraction.Secondly,microwave-enzyme-assisted two-phase salting-out extraction of lutein and polyphenols from marigold flowers was studied.The influencing factors of two-phase salting-out extraction under microwave and the influencing factors of enzymatic hydrolysis conditions were investigated.The results showed that the optimal process for the extraction of lutein and polyphenols from marigold by microwave-enzyme-assisted aqueous two-phase extraction was:28%?w/w?ethanol/20%?w/w?ammonium sulfate,and the mesh was 160 mesh.The ratio of material to liquid is 1:45 g/mL,the amount of pectinase is4.5 U/g,pH 5.00,enzymatic hydrolysis at 45 ^oC for 150 min,microwave power 270 w,microwave time120 s;the yields of lutein and polyphenol by this method are high than microwave-assisted aqueous two-phase,enzyme-assisted aqueous two-phase and soxhlet extraction.In addition,the surface morphology of marigold flower powder before and after extraction by different extraction methods was observed by scanning electron microscopy.Thirdly,enzyme-assisted three-liquid extraction of lutein and polyphenols from marigold flowers was studied.The factors affecting the three-liquid extraction and the effects of enzymatic hydrolysis conditions on the extraction were investigated.The results showed that when the system composition was 12%?w/w?n-hexane/26%?w/w?ethanol/14%?w/w?ammonium sulfate,the meshing number was 160 mesh,and the ratio of material to liquid was 1:45 g/mL,7.5 U/g pectinase,pH 4.50,enzymatic hydrolysis for 150 min at35 ^oC,the maximum yields of lutein and polyphenols were 16.67±0.09 mg/g and 87.71±0.37 mg/g;DPPH The test showed that the polyphenols(IC₅₀ is 45.36?g/mL)enriched in the middle phase had higher antioxidant activity than the lutein(IC₅₀ is 54.28?g/mL)enriched in the upper phase;the intermediate extract was solidified by HLB solid phase extraction cartridges.After purification of the phase extraction cartridge,the main components were gallic acid,syringic acid,rutin and quercetin.Finally,the macroporous resin was used to purify the intermediate phase crude extract of the enzyme-assisted three-liquid phase.Through adsorption and analysis experiments,the adsorption and analytical ability of four macroporous adsorption resins AB-8,D101,NKA-9 and X-5 on crude phenolic extracts were investigated.The effect of elution flow rate and eluent concentration on the adsorption and resolution effects.The results showed that the D101 macroporous resin had the best adsorption and analytical properties for phenolic substances,and ethanol was selected as the adsorption solvent for polyphenols.The optimum parameters for separation and purification of phenolic materials are:6 mg/mL polyphenol loading concentration,1.3 mL/min elution flow rate and 80%methanol as elution solvent.Under the purification conditions,marigold flowers are obtained.Polyphenols are mainly gallic acid,syringic acid and quercetin.