The Preparation Of Akebia Trifoliata Seed Oil And Its Effect On Wound Healing

Akebia trifoliate(Thunb.)koidz is a deciduous woody vine belonging to Akebia of Lardizabalaceae,which is widely distributed in Hunan and Sichuan.It has a large number of seeds,accounting for about 15%of the fresh fruit weight,and the highest oil content in the seeds can reach 43%.In the folk,there are examples of using Akebia trifoliata seed oil to promote wound healing and prevent scar formation,while the effect is remarkable,but the efficacy has not been scientifically certified,and it is mostly treated as waste at present.In this paper,the wound repair effect of Akebia trifoliata seed oil as the breakthrough point was taken,the extraction technology and deep processing were studied.Futhermore,the physicochemical properties,fatty acid composition and oxidation stability of seed oil treated by different methods were determined.At the same time,the wound repair was studied from the aspects of free radical scavenging,bacterial inhibition and pharmacodynamics in vitro.The main conclusions are as follows:(1)Determine the optimum extraction process and the best refining process of Akebia trifoliata seed oil.After dry heat pretreatment of Akebia trifoliata seeds,the effects of different extraction methods,such as squeezing,ultrasonic extraction method,Soxhlet extraction,water enzymatic extraction,three-phase extraction and supercritical CO₂ extraction,on the yield of Akebia trifoliata seed oil were compared.According to yield and appearance evaluation,supercritical CO₂ extraction was adopted to extract the seed oil.The optimal extraction process was determined by response surface analysis as extraction time 100 min,extraction kettle pressure 28 MPa and extraction kettle temperature 34?.The obtained seed oil is light yellow transparent clear liquid,which has the inherent smell of Akebia trifoliata seed oil and no peculiar smell.The best refining process is as follows:(1)Degumming:phosphoric acid 0.3%,water 5%,degumming time 30 min,degumming temperature 60?,the dephosphorization can rate up to 84.37%;(2)Deacidification:alkali liquor concentration 10%,super alkali content 0.25%,alkali refining time 40 min and temperature 60?,and the deacidification rate can reach 98.91%;(3)Decolorization:adding 5%activated clay,decolorizing at 70?for 35min,the rate can reach 83.46%.After refining,the phospholipid content and the acid value decreased obviously,the color became lighter,and the oil was clear and transparent without peculiar smell.The study on the stability of seed oil by Schaal oven method showed that the addition of antioxidants had an important influence on the oxidation stability of seed oil,while TBHQ had the best effect,which laid a foundation for solving the problem of easy oxidation during storage.(2)Compare the physicochemical properties,fatty acid composition and oxidation stability of untreated seed oil,dry heat treated seed oil and refined seed oil according to the national standard.The results showed that although there were some differences in Akebia trifoliata seed oil under different treatments,the overall indexes were outstanding and higher than the standard(QB/T 4079-2010,GB2716-2018).Meanwhile,the seed oil after dry heat treatment and refining has better physical and chemical properties,at the same time,it is more suitable for the application of seed oil.The fatty acid composition of seed oil was analyzed by gas chromatography.Compared with other plant seed oils,it was found that the content of unsaturated fatty acids in Akebia trifoliata seed oil was higher,and palmitic acid was the most abundant among saturated fatty acids,reaching more than 20%.At the same time,the oxidation stability of the seed oil was studied by using Schaal oven method.(3)Explain the role of Akebia trifoliata seed oil in wound repair from scavenging free radicals,inhibiting bacterial growth and pharmacodynamics in vitro.The antioxidant activity of untreated seed oil,dry heat-treated seed oil and refined seed oil was tested in vitro.V_C and BHT were used as positive controls to investigate the effect of seed oil on DPPH·,·OH and ABTS⁺·.In the test concentration range,with the increase of Akebia trifoliata seed oil concentration,its scavenging ability on different free radicals gradually increased,with obvious dose-effect relationship.According to the IC₅₀ calculated by fitting curve,it can be concluded that the antioxidant activity of dry heat treated seed oil is the best,while that of refined seed oil is the worst,but the scavenging effect is worse than Vc and BHT.The results showed that untreated,dry heat treated,refined and pressed seed oil all had good broad-spectrum antibacterial activity,and also had certain bactericidal effect.Among them,pressed seed oil had the best effect,refined seed oil had poor effect,and the antibacterial activity of the four treated seed oils was worse than that of gentamicin sulfate.S.aureus is more sensitive to seed oil than E.coli and P.aeruginosa.CCK-8 test,migration test,tube formation test and macrophage polarization test were used to evaluate the effects of untreated seed oil,dry heat-treated seed oil,refined seed oil and pressed seed oil on cytotoxicity,proliferation activity,migration activity,angiogenesis activity and cell inflammation.The results showed that Akebia trifoliata seed oil in a certain concentration range had no toxic effect on HaCaT and HUVEC cells,and had good cell compatibility.0.01,0.1,1?L/m L seed oil solution has significant proliferation effect on HaCaT,moreover,0.001,0.01,0.1?L/m L seed oil solution has significant proliferation effect on HUVEC.Futhermore,it has a positive effect on cell migration and is dose-dependent;it also has a certain positive effect on angiogenesis,while the pressing treatment of seed oil and dry heat treatment of seed oil have the most obvious effect on increasing the branch point and the tube length,and the concentration of seed oil in the middle is more suitable in general.Besides,seed oil can promote the polarization of macrophages into M2 type,inhibit the excessive inflammatory reaction of macrophages,and protect the wound from slowing down healing due to inflammatory reaction.