Study On The Extraction And Separation Of Fat-soluble And Alcohol-soluble Components From Alpinia Officinarum

Galangal is the dried rhizome of Alpinia officinarum Hance,a plant belonging to the ginger family.It is a commonly used Traditional Chinese Medicine（TCM）.It is widely used in the treatment for abdominal pain,cold stomach,vomiting,belching and so on.Galangal is rich in fat-soluble components such as diphenylheptanes,volatile oils,as well as alcohol-soluble components like flavonoids.They are the main components for its anti-tumor,anti-oxidant,anti-bacterial and other pharmacological effects.So far,the research on galangal has focused on the volatile oil or flavonoids,and less on the overall extraction,development and utilization of fat-soluble parts rich in diphenylheptane and alcohol-soluble parts rich in flavonoids.It is difficult to reflect the overall components and efficacy of galangal,which also leads to the waste of galangal resources.In terms of the extraction of fat-soluble components,high-temperature steam distillation is mainly used.Although there were reports on supercritical CO₂ extraction,the research mainly focused on aroma ingredients,while traditional extraction methods such as solvothermal reflux are mainly used for the alcohol-soluble components such as flavonoids.And in further separation and purification of the target monomer components,the traditional extraction methods such as silica gel column chromatography are mainly used.In general,the extraction and separation methods are backward and the enrichment effect of active components is poor.In this study,technology combination of the advanced supercritical CO₂ extraction with the low-temperature and high-pressure crushing extraction were developed.Galangal fat-soluble and overall alcohol soluble parts were extracted,and the main component composition,the key components and their synergistic antitumor effect were explored.It can provide reference for the overall extraction,enrichment,development and utilization of the active ingredients of galangal,as well as improve the utilization of galangal medicinal resources.The main research contents and results are as follows:1.A HPLC method for the determination of diphenylheptane A,B,C,galangin and kaempferin was established,and the method was verified.This method is stable and feasible and can be used for the content determination of samples.At the same time,GC-MS was used to establish an analysis method for other volatile components in the fat-soluble part;LC-MS was used to establish a qualitative analysis method for the constituents of the alcohol-soluble part,and UV method was used to establish a determination method for the total flavonoids content of the fractions.2.The supercritical CO₂ extraction technology was used to study the effects of extraction factors on the extraction efficiency of fat soluble components by taking the yield of extraction and the yield and content of main diphenylheptane components as evaluation indexes.The optimized process conditions were as follows:particle size of 60 mesh,xtraction time of 1.5 h,CO₂ flow rate of 40 Kg/h,extraction pressure of 30 MPa,extraction temperature of 50℃,pressure in separation vessel I of 15 MPa with temperature of 55℃.Under these conditions,the yield of fat-soluble extracts was 5.48%,and the total yield of diphenylheptanes was 2.56%.The content determination showed that the total content of diphenylheptanes in the fat-soluble extract was up to 45.22%,and there was no diphenylheptanes in the dregs after extraction,indicating that the diphenylheptanes have been completely extracted.At the same time,GC-MS analysis proved that the fat-soluble extract also contains a variety of other volatile components including low and high boiling points.The traditional steam distillation extract contains only low-boiling volatile components,but no diphenylheptanes and other high-boiling volatile components.3.Using ethanol as the solvent,low-temperature and high-pressure crushing extraction technology was used to extract the dregs.The yield of galangin and kaempferin in the extract was taken as the evaluation indexes and the optimal extraction conditions were as follows:Extraction solvent was 78%ethanol solution,material-to-liquid ratio of 1:37,extraction pressure of 75 MPa,material particle size of 100 mesh,one extraction,extraction temperature of 4℃.Under these conditions,the yield of alcohol-soluble extracts was 8.84%,the yields of galangin and kaempferol were 0.64%and 0.22%,and the contents were 7.20%and 2.53%,respectively.LC-MS and HPLC analysis proved that the extract mainly contained flavonoids,followed by organic acids,phenylpropanoids and other polar components.The total flavonoids content was 17.11%,of which the main representative components were galangin and kaempferol and their combined content was 9.53%,and the transfer rate reaches95.85%.The traditional hot reflux extraction method had a longer extraction time and higher extraction temperature,and the yields of galangin and kaempferin were 0.627%and 0.219%,respectively,and the contents were 5.58%and 1.59%,respectively,which were lower than the low-temperature and high-pressure crushing extraction method.4.It was easy to separate and prepare the main diphenylheptane components from the fat-soluble extracts by industrial chromatography.Polyamide column chromatography and industrial chromatography were used to separate and prepare flavonoids from alcohol-soluble extracts easily.¹H-NMR,¹³C-NMR and HRMS spectroscopy methods were used to confirm the structures of diphenylheptane A,B,C,D,galangin and galangin flavanone.5.MTT method was used to screen the anti-tumor activity of SH-SY5Y cells from the fat-soluble and alcohol-soluble extracts of and isolated monomers of galangin.The results showed that the fat-soluble and alcohol-soluble extracts as well as the monomers isolated had good anti-tumor activity,and the combination of the fat-soluble and alcohol-soluble extracts of galangal showed synergistic anti-tumor effect at the combination ratio of 1:1 and 1:2.