Self-assembly Effect Of Angelica Sinensis Protein And Its Application

Angelica sinensis is one of the traditional Chinese medicines,the research on its pharmacological effects mainly focuses on the polysaccharides and small molecules of Angelica sinensis.However,there is rare study on Angelica sinensis protein.In this study,the Angelica sinensis protein(ASPR)was isolated from prepared slices of Angelica sinensis.Then,the self-assembly effect of ASPR and its ability to encapsulate insoluble drug Ferulic Acid(FA)were explored.Consequently,the biological activities of nanoparticles were studied to obtain a novel nanocarrier with the drug-encapsulate ability and biological safety.ASPR,glycoproteins with molecular weight of 18.33 kDa were purified from the prepared slices of Angelica sinensis by 80%ammonium sulfate precipitation and Sephadex G-50 gel filtration chromatography successively.ASPR mainly existed as-monomer in solution and formed part of dimer.The effects of pH,temperature and treatment time on the self-assembly of ASPR were investigated by dynamic light scattering.The results showed that ASPR(0.5mg/mL)can self-assemble to form uniform particle size particles(ASPR-NP)after heating at 100? for 15 min with a pH value of 8.0.We found some changes occured in heated ASPR including shifted ANS fluorescence wavelength,decreased ANS fluorescence intensity,decreased ?-helix content and increased ?-sheet content.These structural changes resulted in the unfolding of the ASPR structure,the exposure of hydrophobic groups,aggregation and self-assemble to the ASPR-NP nanoparticles.The ferulic acid(FA)encapsulated nanoparticles ASPR-FA-NP would be obtained when the methanol-dissolved FA(20 mg/mL)was added to the ASPR solution before thermal treatment.The ASPR-NP self-assembled by Angelica protein and the FA embedded nanoparticles ASPR-FA-NP were purified using 100 kDa ultrafiltration tubes.The average particle size of ASPR-NP was 129.76±3.28 nm and the Zeta potential was-11.64±2.66mV.The average particle size of ASPR-FA-NP was 216.3±18.3nm and the zeta potential was-14.65 ± 5.01 mV.Scanning electron microscopy showed that both ASPR-NP and ASPR-FA-NP particles were in irregularly spherical shape.The obtained nanoparticles are suitable for short-term preservation at 4? and long-term storage at-20?.Compared with ASPR with equal assembly content,the DPPH radical scavenging rate of ASPR-NP was significantly increased by 150%(P<0.05).The ABTS radical scavenging rate of ASPR-NP was significantly increased by 398%(P<0.01).ASPR-NP is capable of loading FA.In this study,the embedding rate of FA was approximately 30%,and the drug loading rate of ASPR-NP was about 65%.The radical scavenging activity of ASPR-FA-NP was significantly increased by 155%compared to the equivalent amount of ASPR involved in loading(P<0.05).Compared with the empty carriers ASPR-NP,the ABTS radical scavenging of ASPR-FA-NP was significantly increased by 79%(P<0.01).MTT assay showed that ASPR,ASPR-NP and ASPR-FA-NP all promoted the proliferation of human normal hepatic cells L-02.Compared with ASPR-NP,ASPR-FA-NP selectively protected human normal hepatic cells coincided with the inhibition proliferation of human hepatoma carcinoma cell Hep G2.With the FITC labeled ASPR and ASPR-NP and the confocal laser scanning microscopy,we found both ASPR and ASPR-NP can enter into the Hep G2 cells and reach mitochondria.Finally,the biocompatibility of ASPR,ASPR-NP and ASPR-FA-NP was tested by hemolysis experiments.It was found that none of them significantly induce the hemolysis of healthy red blood cells.In summary,ASPR has the capability of loading insoluble drugs FA and the preparation method is simple.The formed particles have various biological activities and high safety.Our research further broadened the application value of Angelica sinensis protein and provided a new direction for the design of nano drug carriers.