| In recent years,marine algae have served as important sources of bioactive natural substances.These natural pigments exhibit various beneficial biological activities,such as antioxidant,anticancer,anti-inflammatory,anti-obesity,anti-angiogenic and neuroprotective activities.Due to the NP has the advantages of being non-toxic,high safety,and so on.The application of NP is more and more widespread,and the application of synthetic pigments in food,medicine,cosmetics and other fields is strictly limited.Therefore,the application of natural pigments instead of synthetic pigments in foods,cosmetics and pharmaceuticals has become an inevitable trend.In this study,seaweeds were used as raw materials to study the extraction,separation and structural identification of its high antioxidant pigments.There are the main findings: 1?Screening high antioxidant activity pigments in seaweedIn order to screen algae with high antioxidant activity pigments,the contents of chlorophyll,carotenoid and total pigment in Laminaria.japonica,Undaria pinnatrfida(Harv.)Suringar,Sargassumm horneri,Enteromorpha,Ulva pertusa,Porphyra,Gracilaria and Nannochloropsis oculata were comparatively analyzed.The in vitro antioxidative activity of their total pigments was compared.In terms of total pigment S content,the experimental results show that Undaria pinnatrfida(Harv.)Suringar > Ulva pertusa>Porphyra>Enteromorpha>Laminaria.japonica>Nannochloropsis oculata>Sargassumm horneri>Gracilaria,and the difference in chlorophylls content and carotenoids content is large,contrasting the chlorophylls content and the carotenoids content shows that the content of chlorophylls is greater than the content of carotenoids;In terms of the antioxidant of the total pigments in seaweed,the experimental results showed that the DPPH radical scavenging rate of the Laminaria.japonica pigments was up to 50% at the final concentration of 0.8 mg/m L.The hydroxyl radical scavenging rates of the pigments of Laminaria.japonica,Undaria pinnatrfida(Harv.)Suringar,Sargassumm horneri,Ulva pertusa were all reached above 50%,the order of their order was Undaria pinnatrfida(Harv.)Suringar>Sargassumm horneri>Laminaria.japonica>Ulva pertusa;andthe Scavenging of superoxide anion free radicals by the pigments of Laminaria.japonica,Enteromorpha and Ulva pertusa allreached 50%,and the order of their size was Enteromorpha>Laminaria.japonica>Ulva pertusa.In summary,the antioxidative activity of Laminaria.japonica pigments is the best,so Laminaria.japonica is selected as a source of active pigments.2?Optimization of total pigment extraction conditions in Laminaria.japonicaThe single-factor test and orthogonal test were used to optimize the extraction of total pigment from Laminaria.japonica,and the optimal conditions and total pigment content values were obtained: Fresh Laminaria.japonica was used as raw material(water content was approximately 85%),After vacuum freeze-drying,ethanol was used as extraction solvent.The optimum extraction conditions were as follows:cellulase addition 3%,extraction temperature 40°C,extraction time 1.1 h,and solid-liquid ratio 1:26.Under this condition,a verification test was conducted and the yield of the pigment was 1.655 mg/g(calculated as dried Laminaria.japonica).3.Comparing the effects of different processing methods on the antioxidant of total pigment in Laminaria.japonicaBy comparing the effects of three different processing methods fresh,salted,and dried on the antioxidant of Laminaria.japonica pigments,the following experimental results can be obtained: fresh Laminaria.japonica has the highest pigments content,followed by salted Laminaria.japonica,and dried Laminaria.japonica has the lowest pigment contents;The antioxidant activity of the crude extracts of the three kinds of Laminaria.japonica was as follows: fresh Laminaria.japonica>salted Laminaria.japonica>dried Laminaria.japonica.The correlations between the Scavenging rates of three free radicals,chlorophyllcontents and carotenoid contents were analyzed by SPSS,and the following results were obtained: The correlations between the three free radical scavenging rates and the two types of pigment content were good,and both were extremely significant.Kelp pigment is a major antioxidant substance.Among them,the relationship between the three free radical scavenging rates and the carotenoid contents was higher,indicating that carotenoids play a greater role.4?Separation and purification of Laminaria.japonica pigments,screening of pigments with high antioxidant and identificate its structureAccording to the different solubility of various pigments in Laminaria.japonica in different solvents and similar dissolving principle,the pigments were first separated by silica gel column chromatography.The eluent was petroleum ether/acetone(90/10),petroleum ether/ acetone(70/30)and pure acetone respectively,the three components of pigments S1,S2,and S3 were isolated by silica gel column chromatography.Among them,S3 had the highest antioxidant in vitro.The S3 was further purified by high-speed countercurrent chromatography.High-speed countercurrent chromatography conditions as follows: solvent system/n-hexane/ methanol/water(4/3/1),the upper phase was the stationary phase,the lower phase was the mobile phase,and the column volume was 230 m L;Flow rate:1.0 m L/min;injection volume:200 mg;rotation speed:800 rpm;retention rate of the stationary phase:58%;detection wavelength:450 nm.After separation and purification by high-speed countercurrent separation,S3 was separated into two components named S3-1 and S3-2.The antioxidant of S3-1 was highest in vitro.The structure of S3-1 was identified by HPLC,LC-MS and NMR,and the molecular weight of S3-1 was 658.91,both LC-MS and NMR spectra showed distinctly characteristic peaks of fucoxanthin.According to the separation and purification method of the kelp pigment used in this experiment,the purity of S3-1 was 93.3% as determined by HPLC. |
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