Technical Optimization Of Ultrasound Assisted Extraction Of Active Substances Of Iberis Amara Seeds And Their Chemical Component Analysis

Iberis amara belongs to the family of Cruciferae,which is native to Spain.is a bloomer cultivated as an ornamental and medicinal plant.Iberis amara has pharmacological effects such as antioxidant,anti-insect,anti-inflammatory,anti-microbial,antiallergic and anti-cancer effects.This plant is rich in amines,glucosinolates,cucurbitacins and flavonoids.Now studies of Iberis amara mainly focus on cultivation techniques and activity of crude extract instead of extraction of active substances and chemical component analysis,which limits the applications of this plant in the pharmaceutical industry to a great extent.For another,ultrasound as a clean and efficient modern extraction technology,which plays an important role in the field of natural medicine.Ultrasonic extraction has many advantages,such as high efficiency,environmental protection,low temperature and low energy consumption,when comparing it with the traditional techniques.Therefore,different ultrasonic assisted extraction methods are used in this study,it is the first time to systematically study on chemical components analysis and optimization of extraction process of active substances about Iberis amara seeds.The chemical components and the efficient ultrasound-assisted extraction routes of various characteristic active substances from Iberis amara were studied and compared with the traditional techniques.The effects of ultrasonic assisted extraction on the yield,chemical composition and biological activity of volatile oil from Iberis amara were investigated;The correlations between ultrasonic pretreatment parameters and the yield,physicochemical properties,fatty acid profile,contents of phytochemical components and antioxidant activity of Iberis amara seed oil were explored;The unique relationship between the ultrasonic power and the dynamic model of cucurbitacin extraction from Iberis amara was analyzed.Through this research,trying to provide the sufficient reference information for in-depth development of Iberis amara,and provide the theoretical basis for further development of the efficient industrial production of the active ingredients.The main study contents and conclusions are as follows:(1)Firstly,Ultrasound-assisted hydro-distillation(UAHD)extraction of essential oil from Iberis amara seeds was adopted for the first time.The impacts of extraction time,ultrasound power and solvent-to-sample ratio on essential oil yield were studied.Based on the results of one-factor trials,the extraction parameters was optimized via response surface methodology.The maximum recovery of essential oil was 0.42±0.05%(v/w,fresh weight)using the optimal extraction parameters(extraction time of240 min,ultrasound power of 75 W and solvent-to-sample ratio of 6.7 m L/g).UAHD showed a significantly higher amount of EO when compared with the traditional methods hydro-distillation(HD)and steam distillation(SD).Subsequently,28 kinds of compounds were identified by GC–MS for the EOs extracted by UAHD,HD and SD and with similar quantitative characters.The principal constituents were4-Carvomenthenol,Menthone,Neodihydrocarveol and?-Curcumene.Further study on bioactivity of EO samples was carried out.The oils obtained with the three techniques exerted slightly different but good antioxidant and anti-inflammatory effects,cytotoxicity on human colon cancer cells,as well as antibacterial and anti-feedant effects against Pieris rapae larvea.In conclusion,this study suggests that UAHD is an efficient,convenient and green technology for essential oil extraction,the introduction of ultrasound can significantly improve the extraction efficiency of traditional distillation technique without damaging the original chemical composition and biological activity of the product.Therefore,ultrasound can be considered as an auxiliary extraction method for industrial production of aromatic essential oil.In addition,the essential oil from Iberis amara seeds shows extensive biological activities,which has an excellent application prospects in food,pharmaceutical and agricultural industries.(2)Secondly,ultrasound pretreatment(USP)combined with supercritical CO2extraction(SCE)was carried out to obtain Iberis amara seed oil(IASO)for the first time.Effects of different parameters of ultrasonic pretreatment(power and duration)on yield,physicochemical properties,fatty acid profile,contents of various phytocompounds and antioxidant activity of IASO were explored.The maximal oil yield(25.28±0.39%,w/w,dry basis)acquired by SCE from ultrasound-treated with ultrasonic power of 100 W during 10 min,which was about 28%higher than that obtained from the untreated seeds.Physicochemical and antioxidant properties of IASO are comparable to that of edible oil,IASO contains abundant Monounsaturated fatty acids and nutrients.USP+SCE resulted in a better oil quality,enhanced slightly the selectivity of monounsaturated fatty acids in obtained oil,as well as improved the contents of diverse phytocompounds and antioxidant activity of IASO samples,when compared with traditional techniques.The above results indicated that USP+SCE is an efficient and environmentally-friendly extraction technique,Ultrasonic pretreatment of seed samples can significantly enhance the IASO yield and improve the oil quality,which has potential application value in the industrial extraction of natural vegetable oil.Furthermore,IASO has high nutritional value,thus Iberis amara seeds could be used as easily available sources of natural plant oil in the pharmaceutical,biodiesel and dietary industries.(3)Subsequently,an unique ultrasound-assisted supercritical CO2 extraction(UASCE)was exploited to extract Cucurbitacin E(Cu E)from the Iberis amara seeds in this study.This natural compound is a characteristic active substance with heterogeneous biological activity and is abundant in the Iberis amara seeds.The Taguchi design method was adopted for optimizing the experimental conditions of UASCE using L₂₅(5⁶)orthogonal array.The highest Cu E yield(8.61±0.06 mg/g,dry mass)for UASCE was achieved at 60 min dynamic extraction time,25 MPa pressure,50^oC,2m L/min CO₂ flow rate,12%co-solvent concentration,and 200 W ultrasonic power.The obtained results revealed that UASCE improved the Cu E yield by 26.1%and decreased extraction duration by 34.8%as well as reduced the usage amount of CO₂ by 52%compared with orthodox supercritical CO₂ extraction.Furthermore,the kinetics study demonstrated that the adopted the models of Broken and Intact Cells fitted well with the experimental extraction curves.The convective mechanism plays a prominent role in UASCE process and mass transfer coefficients increased with the amplification of ultrasonic power.The above results showed that UASCE is a efficient,green and feasible extraction technique,the introduction of ultrasound can improve the extraction efficiency and save the operation costs of traditional supercritical CO₂ extraction,it can provide some reference value for the implementation of efficient Cu E production in industrial scale.(4)Finally,The extract of Iberis amara seeds was obtained by ultrasound-assisted solvent extraction,then petroleum ether and ethyl acetate as well as n-butanol were used orderly for extraction.Silica gel column,Sephadex LH-20 gel column,MCI column,preparative thin layer chromatography,semi-preparative HPLC and recrystallization were adopted to separation and purification from ethyl acetate part of the Iberis amara seeds extract.By using chromatography techniques such as MS and NMR,26monomeric compounds were identified.Including 5 lignins,8 cucurbitacins and their derivatives,7 flavones and flavonoid glycosides,2 sesquiterpenoids,as well as 4 other compounds.They were Ethyl 4-methoxycinnamate(1),p-Coumaric acid ethyl ester(2),?-sitosterol(3),Tiliroside(4),Neolignan(5),Corchoionol C(6),3-methoxy-4',7-epoxy-8,3'-neolignane-4,9,9'-triol(7),Phlorizin(8),Cucurbitacin D(9),3-epi-isocucurbitacin D(10),Cucurbitacin B(11),Cucurbitacin I(12),25-acetoxy-2?,16?,20(R)-trihydroxy-cucurbita-5,23-dien-3,11,22-trione(13),25-acetoxy-3?,16?,20(R)-trihydroxy-cucurbita-5,23-dien-2,11,22-trione(14),Cucurbitacin E(15),Arvenins I(16),(-)-Pinoresinol(17),(±)-Syringaresinol(18),(±)-Lariciresinol(19),Blumenol B(20),Afzelin(21),(-)-Catechin(22),Taxifolin(23),Kaempferol(24),Sinensin(25),p-Hydroxybenzonic acid(26),respectively.22 of these compounds were obtained from Iberis amara for the first report.The above results indicated that the Iberis amara seeds contain abundant active ingredients.Further expanding the knowledge on the phytochemical compositions of this plant,it has positive effects on promoting the development of Iberis amara industry and utilization of related crops products.