| Sea buckthorn（Hippophae rhamnoides L.）is a kind of perennial deciduous shrub or tree belonging to the genus Hippophae,the family of Elaeagnaceae,not only has good edible and medicinal value,but also has environmental protection capability.The berry is mainly be utilized part of sea buckthorn,which is rich in nutrients and bioactive substance.At present,the berry of sea buckthorn is widely exploited to produce sea buckthorn juice,sea buckthorn seed oil,sea buckthorn oil and pomace and seed dregs are the main byproduct during its processing.Many studies have demonstrated that these residues remain a large amount of natural polyphenolic compounds,including proanthocyanidins,flavonoids and polyphenols,which are closely related to the antioxidant,anti-inflammatory,antibacterial and protection of cardiovascular activity.Consequently,in order to promote the comprehensive utilization of sea buckthorn and extend industrial chain,making full use of the byproduct and forming green pattern of the resource utilization for industrial solid waste are of considerable interest.For the purpose of sufficient utilization of these resources,this present study,sea buckthorn dregs that are the residues after supercritical CO2 extracting sea buckthorn oil were used as experimental materials.Based on optimization of the extraction and purification condition of the antioxidative substances,sea buckthorn seed dregs extract with high antioxidant activity and proanthocyanidins content were preparing prepared.Furthermore,the structure of concentrates were preliminary analyzed,and evaluated its antioxidant activity and free radical scavenging capacity in vitro.In addition,its protective effect on human hepatocellular carcinoma cells HepG2 damaged by H2O2 was also investigated.The results of this research as follows:1.Conditions for DPPH scavenging capacity and FRAP value were studied under ultrasonic assisted deep eutectic solvent extraction.The optimal extract conditions for the antioxidant substances were determined by single factor and response surface experiment.The results showed that optimal conditions for the extraction can be used 30%water in choline chloride-oxalic acid（molar rate 1:1）as the suitable solvent and liquid to material ratio of14:1（mL/g）,ultrasonic power 420 W,ul trasonic time 52 min,ultrasonic temperature 44oC.Under these conditions,the elimination ration of DPPH radicals and FRAP value of extractions was the best of value in 60.06±0.08%,63.86±0.13 mmol/L,respectively.2.The main active constituents of the extract were detected by UV method and the results showed that the content of proanthocyanidins was（22.71 mg/gDW）,which was the highest,follow by phenolic（11.61 mg/gDW）and it also had a small amount of flavonoids（3.08mg/gDW）.And the correlation between antioxidant capacity and total contents of phenolic compounds、flavonoids and proanthocyanidins was also studied.The correlation analysis showed that the antioxidant capacity was a significant positive correlation with the contents of total phenolic compounds、total flavonoids and total proanthocyanidins.3.Among the seven kinds of resin,HPD400 resin performed good adsorption-desorption properties,which were a suitable material to enrichment and purification the proanthocyanidins from sea buckthorn seed dregs.Optimum absorption-desorption conditions were as follows:initial extract solution dilution about 4 times,flow rate,1.0 mL/min,the adsorption time about5 hours;elution solvent,70%ethanol,elution volume,2.5 BV.Under the above optimal conditions,the content of sea buckthorn seed proanthocyanidins in the brown powder was60.17±1.14%.4.Sea buckthorn seed dregs deep eutectic solvent extracts were purified and identified preliminarily by using the method of UV、IR and HPLC.The UV wavelength scanning analysis showed that sea buckthorn seed proanthocyanidins has the characteristic absorption peak at wavelengths 270 nm and displayed the properties of phenolic compounds.And IR spectra confirmed the basic skeleton structure of proanthocyanidins from sea buckthorn seed dregs.In addition,the monomers,（+）-catechin and（-）-epicatechin were also identified by reverse phase HPLC.5.The antioxidant activity of sea buckthorn seed proanthocyanidins was determined by DPPH assay,ABTS assay and FRAP assay,respectively.At the same time,the H2O2 damage HepG2 cell model was established to further study the biological activity of sea buckthorn seed proanthocyanidins.The results showed that sea buckthorn seed proanthocyanidins had high free radical scavenging ability and Fe2+reduction ability at low concentration.The IC50 values of DPPH radical and ABTS radical scavenging rate were 0.83 mg/mL,0.12mg/mL,respectively.When the concentration of sea buckthorn seed proanthocyanidins was 1.5mg/mL,the FRAP values was 7.42 mmol/L.And it could protect human hepatocellular carcinoma cells HepG2 from H2O2 induced cytotoxicity.At the concentration of 50μg/mL-400μg/mL,sea buckthorn seed proanthocyanidins could significantly improved the survival rate rate of cells.It should be noted that the cell survival rate reached 82.76%when the pretreatment concentration was 200μg/mL.At the same time,the morphologic observations confirmed the reduction of apoptosis cells.In conclusion,sea buckthorn seed dregs could be used as a new source of natural antioxidants. |
