Extraction, Purification, Antioxidation Activity And Stability Of Red Pigment From Mulberries

Mulberry, named Sang Zao, Sang Guo, Sang Shi, Sang Zi, is mature collective fruit for mulberry branch mulberry plant (Morus alba L.). It includes the massive variety glucoside pigment which is one kind of very good anthocyanines pigment resources. Mulberry anthocyanidin is water-soluble, which shows blue color under the alkalinity conditions and purple in the acid solution conditions. It has many kinds of pharmacology activeness, such as enriching blood, moistening brain, benefiting liver, diuresis, anti-oxidation, anti-mutation, anti-tumor. For these reasons, Mulberry anthocyanidin draw more and more attentions when it is taken as a natural pigment. At present there are few related reports to the mulberry overseas, the home mainly focus on extraction purification crafts, however, the high-purity mulberry anthocyanidin's preparation research has not been seen yet, therefore the separation purification and oxidation resistance activeness were studied emphatically in this study, provides the rationale for its application.Frozen mulberry was used as the raw material, the qualitative quantitative analysis methods, the purification craft, anti-oxidation activeness and the stability were conducted systemly, the main contents and the results are as follows:Investigation on the optimum extraction crafts: The mulberry red pigment was extracted by the solvent-extraction method from fresh fruit. The primary factors which affected the pigment yield was temperature, time, fluid material and extracting density. The absorption value under biggest absorption wave length 513nm was taken as index to carry on the single factor experiment for investigating the effect of single factor on pigment yield. Based on this study, soak temperature, soak time, leaching solution density and fluid material ratio were taken as factors as well as absorption value as index for orthogonal test. Results showed extraction's best technological conditions of mulberry anthocyanidin including: the temperature was at 50℃, extraction time was 90min, the fluid material ratio was 15:1, the leaching solution density is 40% (80% ethyl alcohol: 0.1%HCl=1:1), and extraction yield was 102.6mg/g in these conditions. The verification tests indicated that this craft was stable and suitable for its extraction.Analysis methods on investigating mulberry glucoside: Four qualitative methods, colors to respond tests, the paper chromatographic analysis, the thin layer chromatographic analysis and the ultraviolet - obvious spectral analysis were used to analyze qualitatively mulberry glucoside which was the main component. After these methods, the component was preliminary judged as cornflower-3-glucose glucoside.Quantitative analysis methods of mulberry glucoside mainly includes colored value, the extinction method of correlates, pH show the method of difference and NaNO₂-Al (NO₃)₃ colorimetric method. These four methods were used respectively for quantitative analysis of mulberry glucoside.The results indicated that the value after the purification was 14.8 times before purification, and the anthocyanidin content was 0.015% according to the extinction method, 0.017% according to pH show difference method and 0.0076% according to NaNO₂-Al (NO₃)₃ colorimetric method. In these methods, pH show difference method was the maturer method of predecessor, which is relatively more accurate, easily, compared with other methods.Optimum purification conditions of macroporous resin: four macroporous resins—NKA, D101, AB-8 and D1300 were screened by static adsorption and resolution experiment, its adsorption rate and resolution rate were calculated, respectively. Results showed that adsorption rate of D101 was the best one, 72.1% and resolution rate was 68.5%, so D101 was considered as the best resin.The main fators which effect the adsorption of macroporous resin were eluent concentration, eluent volume and elution speed.Do the dynamic adsorption and resolution mono-factor experiments,and then do the orthogonal test on the basis of preceding tests.The optimum purification condition of mulberry red pigment used by D101 macroporous adsorptive resin were:the concentration 1.071Abs, feed solution pH3, feeding rate 1mL/min;eluent concentrate were 80%, eluent rate 1mL/min, and eluent volume 2BV, and resolve rate 86.1%.14 anthocyanidins were obtained by HPLC-DAD-ESI-MS, they were pelargon idin-3-glucoside,pelargonidin-3-rahamnose,pelargonidin-3-rutionside,cyandin-3-gluco side, cyandin-3-rutinsambubioside,peonid-3-arabside,cyandin-3-rutionside, cyandin -3-sophonoside,petunidin-3-glurutinoside,delphindin-3,5-arabside,petunidin-3-glucosi de,cyandin-3-xylosylrutinoside,malvidin-3-glucoside, cyandin-3-arabside.Four acidity methanol solution:20%,40%,60%,80% were prepared. The fraction collection was carried out by sephadex LH-20,and three compositions were obtained. The content of main substance in composition 1 was 85.8% cyandin-3-glucoside(m/z 449.5), and 78.5% cyandin-3-sambubioside (m/z 581.58) in composition 2 by HPLC.The antioxidative test of mulberry red pigment shows: mulberry red pigment had good scavenge effect on superoxide anion, hydroxide radical and hydrogen peroxide radical. The clearance rate on DPPH·was 93.5% when pigment density was 40μg/mL; the clearance rate onμOH was 88.21% when pigment density was 600μg/mL, IC50 were 123.25,132.25μg/mL of Vc and mulberry pigment.It also had some lipid antioxygen effect, but was worse than BHT.The stability test of mulberry red pigment:the main factors which influence the stability of mulberry was pH value,and it was stable in acidity condition,bright colored.Heating can cause the degradation and descending the stability; the pigment was more sensitive to oxidant, even a low concentration can cause degradation; Na⁺,Mg²⁺ had less effect on the pigment stability, Fe³⁺,Cu²⁺ had larger effect on the pigment,it can make the red change into amethyst.