CO₂ Expanded Ethanol Extraction And Molecular Distillation Of Root Oil From Vetiveria Zizanioides?L.? Nash

Essential oil extracted from vetiver root,Vetiveria zizanioides?L.?Nash,is frequently used as a main odor contributor in the perfumery industry,and as a flavoring agent in the food industry.Besides its uses in various industries,vetiver root oil owns biological activities,such as antioxidant,antibacterial,anti-inflammatory,and so on.On account of its contributions,many researchers have focused on the development of extraction techniques to satisfy the market demand,but also to reduce the actual production costs,as well as environmental pollution.In this study,CO₂ expanded ethanol,a novel eco-friendly solvent,was first applied to isolate vetiver oil with the purpose of exceeding the traditional method and supercritical fluid extraction,and proving that extraction efficiency was superior to conventional methods.During CXE extraction,the major operating factors were optimized,and the chemical composition of vetiver oil was studied,along with bioactivities.Because of the complex chemical composition of vetiver oil,it was difficult to separate and identify using traditional methods.Furthermore,vetiver oil was fractionate by two-stage molecular distillation to remove most of the non-volatile components,significantly enhancing the chromatographic peak isolation,and combined with GC-MS analysis to identify the specific vetiver components and bioactive components.CO₂-expanded ethanol?CXE?is representing a mixed fluid formed by a typical polar solvent,ethanol,with the addition of compressed CO₂,which may effectively promote the inherent properties,including the specific dissolving ability,as well as the mass-transfer efficiency,being able to extract relatively complex oil from root matrix.CXE fluid has an enhanced solubility for various solutes,which can be used for the extraction of both polar and non-polar components.The investigation of chemical composition,antimicrobial and antioxidant activities of vetiver oil obtained from the novel method,CXE,was compared with traditional methods,including hydrodistillation?HD?,indirect vapor distillation?IVD?and supercritical fluid extraction?SFE?.Since the chemical composition of the oil extracted from the root material is more complex than the extracts from other parts of the plant,a further refining process was necessary.Molecular distillation is a separation and enrichment technique under vacuum and low temperature conditions,which is more suitable for heat-sensitive essential oil components.Two-stage molecular distillation is an effective pretreatment method for vetiver oil,which can effectively separate co-eluted components,and provide a more reliable MS identification.The primary research contents are as follows:?1?The vetiver oil was isolated by conventional extractions,including hydrodistillation,indirect vapor distillation,and supercritical fluid extraction.Based on the experimental results,the effect of extraction time,the particle size,the pressure,and temperature were significant for the isolation of vetiver oil yield.The distillation time of hydrodistillation and indirect vapor distillation was 12 to 18 hours,with supercritical fluid extraction being performed in 2 hours.The oil yield isolated by HD and IVD was 0.6%,and SFE oil yield was approximately 0.5%.?2?The novel green extraction technique,CXE,was firstly employed for obtaining vetiver oil,using polar solvent,such as ethanol,with the addition of compressed CO₂,known as CO₂expanded ethanol.The extraction process was optimized successfully relying on the Response surface methodology?RSM?,based on central composite design?CCD?,presenting the primary operating conditions,including temperature of 50oC,the pressure of 8.4 MPa,the CO₂ mole fraction of 0.22 and ethanol flow rate at 5 m L/min.The CXE extraction exhibited an oil yield,from 5.12%to 7.42%,higher than conventional methods.The Gas chromatography-Mass spectrometry?GC-MS?was employed to analyse the vetiver oil samples from four extraction methods.The results showed that four classes of components were identified,hydrocarbons,alcohols,carbonyl compounds,and carboxylic acids,with 22 components representative for CXE oil,and being similar to the number of components found in traditional oil.CXE oil presented three major components,valerenol?18.48%?,valerenal?10.21%?,and?-cadinene?6.23%?.This study revealed the necessity of an alternative for the extraction process of vetiver root oil,aiming to obtain a higher yield of vetiver oil,which is important for satisfying the production requirements.?3?Vetiver root oil is refined by molecular distillation performed in two-stages combined with GC-MS analysis.The first stage of molecular distillation was carried out through the adjustments of pressure,feeding temperature,evaporator temperature,feed rate,and rotor speed.Under low systemic pressure?0-2 Pa?and feeding temperature set at 80oC,the oil sample was flowable enough to inject into the evaporator,with its density of 0.878 g/mL,and its viscosity of 4.764 mPa·s,respectively.The evaporator temperature set at 80oC results in a relatively high total recovery of 79.81%,as well as proper distribution for three distinct fractions,including 1R_D of 28.61%,1D_M of 51.01%and 1D_L 0.19%.The rotor speed of 300rpm with the feed rate of 2 mL/min contributed jointly to the formation of an intact film of approximately 5 mm with a residence time around 15 sec.To obtain concentrated fractions and quality oil,the second stage of molecular distillation was conducted with similar conditions found in the first stage,but with a different value of the first condenser temperature,5oC.The GC-MS analysis showed that during the first stage of MD the high molecular weight components are partially removed,and whereas in the second stage,the exceptionally light fraction?2D_L?,presented an enrichment of components,such as khusimene?1.79%?,beta-vatirenene?5.86%?and humulene-1,6-dien-3ol?10.74%?.Molecular distillation could be considered a preprocess for vetiver oil through the fractionation of crude oil and beneficial to GC-MS analysis because of the effective isolation of components,ultimately resulting in dispersion of the peaks of the components.?4?The antioxidant activity of vetiver oils extracted by conventional and green extraction,CXE,were detected by the DPPH and ABTS free radical scavenging assay,demonstrated that the antioxidant abilities of root oils were sorted in the descending order:IVD>HD>CXE>SFE.Furthermore,the DPPH free radical scavenging activity revealed that the antioxidant activity of six distillate vetiver fractions were similar to the crude oil?5?The antimicrobial abilities of vetiver root oils were evaluated by the microdilution method,against Gram-negative bacteria Escherichia coli?ATCC NO.8739?and Pseudomonas aeruginosa?ATCC NO.27853?,as well as Gram-positive bacteria Bacillus subtilis?ATCC NO.14579?and Staphylococcus aureus?ATCC NO.6538?.The results showed that CXE oil exhibited an ability against Gram-positive bacteria,especially Staphylococcus aureus,approximately equivalent to traditional samples.In summary,the environmentally friendly technology,CO₂ expanded ethanol extraction,was first applied to isolate the oil from the root of Vetiveria zizanioides.The extraction yield of vetiver oil obtained by CXE extraction is significantly higher than that of traditional methods,such as distillation and supercritical fluid extraction,which greatly contribute to the reduction of production costs as well as environmental pollution.The chemical analysis showed a possible improvement of quality for vetiver oil extracted by CXE,such as the decline of the unpleasant smoky odor,enhancement of the bioactivity,and so on.Due to the complicated chemical composition of vetiver oil,the two-stage molecular distillation?MD?might be considered an alternative to process root oil for reducing the complexity of chemical analysis,as well as isolating or enriching the significant components.The first stage of MD effectively removed most of the high molecular components,while the second stage notably improved chromatographic peak isolation contributing to the identification of specific components.During the refining process,the bioactivity of distillate fractions remains active in contrast to original vetiver oil.The CXE technique and two-stage molecular distillation may be promising green manufacturing patterns for the production of quality vetiver oil.