| Pearl is a medicine of channel tropism of heart and liver in traditional Chinese medicine.It has effects of promoting granulation and resisting skins senility.At present,conventional solid-liquid extraction(SLE),acidolysis and enzymolysis are common methods for the extraction of pearl powder.However,SLE is ineffective,enzymolysis and acidolysis can destroy the original structure and naturality of the protein in pearl powder.It is still unclear whether the biological activities of pearl powder are affected by different extraction methods or not.Therefore,it is necessary to seek a more effective and safety method for improving the extraction efficiency and utilization of pearl powder.The main contents of this study:(1)The extraction process of total protein from pearl powder by ultrasonic-assisted extraction(UAE)was studied in this experiment.Based on single-factor experiments,the optimum parameters were set up by the response surface.The optimum parameters about extraction were concluded as follows:solid-liquid ratio 0.350:10 g/mL,ultrasonic power 200 W and extraction time 70.6 min,then the extraction rate could reach to 0.126 ± 0.002 mg/g.(2)The extraction process of total protein from pearl powder by microwave-assisted extraction(MAE)was studied in this experiment.On the basis of single-factor experiments,the optimum parameters were set up by the response surface.The optimum parameters about extraction were concluded as follows:solid-liquid ratio 0.280:10 g/mL,reaction temperature 21 ℃ and extraction time 84 s,then the extraction rate could reach to 0.117 ± 0.002 mg/g.(3)In this study,the total protein content and molecular weight distribution of pearl powder extracts produced by different extraction methods were investigated,including SLE,UAE,MAE,acidolysis and enzymolysis.And the results showed that the total protein contents of pearl powder extracts were 0.051,0.126,0.119,0.021 and 0.551 mg/g respectively.The macromolecules which the molecular weight(MW)were greater than 130 kDa and small molecules(MW was less than 14 kDa)existed in the extracts by SLE,UAE and MAE,but only small molecules were found in the extract by acidolysis(MW was between 5 and 50 kDa)and extract by enzymolysis(MW was less than 14 kDa).Strong acidolysis lead to an excess degree of hydrolysis and the lost of protein.Protein could also be hydrolyzed into small molecular by enzymolysis which was moderate,so that,the total protein yield was much higher and MW was less than 14 kDa.In a short,the protein in extract produced by SLE,UAE or MAE was original,but was non-original after acidolysis and enzymolysis.What’s more,UAE and MAE could enhance the yield without destroying the protein structure.(4)The total antioxidant activity(the effect on scavenging DPPH ·radicals,ABTS+ radicals and TRAP value)of the pearl powder extracts by different methods were investigated.And the results showed that the antioxidant activity of extract by enzymolysis was extremely strong.The antioxidant activity of the extract by enzymolysis was much stronger than other extracts,IC50 for DPPH· scavenged activity was 0.028 pearl powder(g)/mL,IC50 for ABTS+ scavenged activity was 0.023 g/mL and TRAP value was 152.13 ± 1.66 μg/mL at 2 g/mL.However,the antioxidant activity of extract by acidolysis was weak.And other extracts don’t have scavenging effect on DPPH· radicals,but all of them have good scavenging effect on ABTS+ radicals and small TRAP value.The antioxidant activities of the extracts produced by SLE,UAE and MAE were basically the same.And protein could be hydrolyzed into small molecular by enzymolysis to enhance the antioxidant activities.However,small molecules produced by acidolysis were lost during desalting,and there were no obvious antioxidant activities.(5)The cytotoxicities of the pearl powder extracts by different methods for B16F10 were investigated.And the results showed that the pearl powder extracts by SLE,UAE and MAE had no cytotoxicity for B16F10,but extracts by acidolysis and enzymolysis had cytotoxicity,and the IC50 were 0.639,0.323 g/mL respectively.Therefore,the cytotoxicity of pearl powder extracts produced by acidrolysis and enzymalysis were worthy of attention.The dose of them should be controlled during use,and their safety were still needf further investigation.(6)The effects of the pearl powder extracts by different methods on B16F10’s tyrosinase activity were investigated.And the result showed that the pearl powder extracts could inhibit tyrosinase activity,the tyrosinase inhibition activity of the extracts prepared by SLE,UAE,MAE,acidolysis and enzymolysis were 21.85 ± 3.42%,21.53 ± 4.24%,26.28 ±3.01%at 1 g/mL and 6.10 ± 2.8%,26.41 ± 3.04%at 0.1 g/mL respectively.In cell experiment of extract produced by enzymolysis,the high concentration could not be used because of its obvious cytotoxicity.However,its activity was much lower than extract produced by MAE at the safe concentration.Therefore,there were other small molecules besides protein could play the whitening effect.(7)The effects of the pearl powder extracts by different methods on production of melanin of B16F10 were investigated.And the results showed that the pearl powder extracts could prevent the production of melanin which was similar to the tyrosinase inhibition activity.The antimelanogenesis activity of the extracts prepared by SLE,UAE,MAE,acidolysis and enzymolysis were 33.81 ± 2.68%,33.23 ± 1.94%,38.16 ±2.53%at 1 g/mL and 16.80 ± 2.14%,30.57 ± 2.45%at 0.1 g/mL respectively.This study showed that pearl powder extracts could inhibit the activity of tyrosinase and the production of melanin.And it also suggested that MAE was the best method to produce nontoxic extract with better effect.Furthermore,the safety of the extract produced byenzymolysis should be in attention which was wildly used in the application of pearl powder. |
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