| Eucommia ulmoides is a unique treasure tree species of our country,and is one of the rare medicinal herbs,which is used for a long history.It contains a variety of natural active ingredients so that it has extensive pharmacological actions.Therefore,elaborate processing of active ingredient from E.ulmoides is of great significance to improve its economic value.It is not only can effectively utilize our unique and valuable resources,developed a series of medical and health drugs,food,but also can promote E.ulmoides industry transfered from resources superiority to economic advantages,and speed up the internationalization of Chinese herbal medicine,and enhance its competitiveness in the international market,thus bring us great social and economic benefits.In this dissertation,the bark of E.ulmoides were taken as raw materials.The ultrasonic circulation technique was used for extracting geniposide from the bark of E.ulmoides,and the response surface method was used for the optimization of the extraction process.The geniposide was separated and purified by macroporous resin.Several methods including electronic scanning microscope,Fourier transform infrared,thermogravimetric,element analysis and X-ray diffraction were used to analyze the magnetic silica gel immobilized ionic liquids and magnetic silica gel immobilized enzyme.Geniposide was catalyzed by immobilized ionic liquids and immobilized enzyme to prepare geniposidic acid and genipin,respectively.The hydrolysis processes were optimized by single factor tests.Using macroporous resin to preliminary separate and purify the hydrolysate.The hydrolysate was analyzed by FTIR and LC-MS/MS.This paper systematically studied the extraction of geniposide from the bark of E.ulmoides,and build a high efficiency extraction and separation technology,as well as build a novel catalytic conversion technology of geniposide by magnetic silica gel immobilized catalyst.The main content is as follows:HPLC conditions for analysis of geniposide,geniposidic acid and genipin were as follow:detection wavelength was 240 nm;mobile phase is methanol-0.04%phosphoric acid solution(22:78,v:v).Three components had good linear relationship(R2?0.9994).HPLC conditions for analysis of geniposide and chlorogenic acid were as follow:detection wavelength was 328 nm;mobile phase is methanol-0.1%formic acid solution(32:68,v:v).Two components had good linear relationship(RA>0.9992).HPLC conditions for analysis of geniposide and pinoresinol diglucoside were as follow:detection wavelength was 256 im;mobile phase is acetonitrile-water-glacial acetic acid(15:85:1.5,v:v:v).Two components had good linear relationship(R2? 0.9996).Methodology validation experiments showed that this method was of high accuracy,good stability.It is suitable for determination of geniposide,geniposidic acid,genipin,chlorogenic acid and pinoresinol diglucoside.Ultrasonic circulation technique is used to extract geniposide from bark of E.ulmoides.The single variable method was used to evaluate the effects of different concentration of ethanol,extraction temperature,liquid ratio,ultrasonic power and time,rotational speed impact on the yield of geniposide.The optimal conditions were as follow:40%ethanol solution used as solvent,extraction temperature was 40 ?,liquid to solid ratio was 40 mL/g,ultrasonic power was 600 W,ultrasonic time was 40 min,and speed was 1500 rpm.Under these optimum conditions,the actual yield of geniposide can be up to 4.33±0.21 mg/g,the concentration of geniposide was 215.82 ?10.15 ?g/mL.The optimal extraction conditions of geniposide and chlorogenic acid from leaf of E.ulmoides were optimized by single factor experiments.The result were as follow:concentration of ethanol was 40%;liquid-solid ratio was 50 mL/g;temperature was 40 ?;revolution was 1500 rpm;ultrasound power and time was 400 W and 20 min.At these conditions,the yield of geniposide and chlorogenic acid wer 0.48 ± 0.02 mg/g and 4.81 ± 0.24 mg/g,respectively.The performance of macroporous resins for the enrichment and seperation of chlorogenic acid and geniposide had been evaluated.The optimum seperation parameters were established:HPD600 was optimum macroporous resin;adsorption time was 4 hours;20%ethonal was used as eluant;elution time was 30 min.At these conditions,a good separation behavious was observed for chlorogenic acid and geniposide.Based on the differences between bark and leaf,ultrasonic circulation technique is used to extract geniposide efficiently.Compared with taditional ultasound extraction,this new extraction technology is more efficient and easier to be used for industrial expanded experiment.The performance of macroporous resins for the enrichment and purification of geniposide had been evaluated,including D1011 DM130,HPD400A,HPD80,HPD600,ADS-7,ADS-17 and HPD100B.The optimum purification parameters were established:HPD600 was optimum macroporous resin to purify geniposide.The best purification conditions:adsorption temperature was 25 ?;saturated adsorption time was 3 hours;10%ethonal was used as eluant;desorption time was 20 min;desorption temperature was 45 ?;the concentration of crude extract was 42.35±2.12?g/mL,the saturated adsorption capacity was 402.37 ?g/g;desorption ratio was 94.38%.Dynamic feeding speed was 3 BV/h;saturated adsorption capacity was about 8 BV.The recovery of geniposide eluted by 24 BV of 10%ethonal was 95.73%.The purity of geniposide was increased from 1.79%to 41.25%.After extracted by ethyl acetate and acetone,the purity of geniposide was 98.51%.The results of FTIR and LC-MS/MS improved that the purification product was geniposide.SEM analysis results show that the magnetic SiO2 immobilized ionic liquids was spherical sample,and the average diameter was of 98.26 nm.FTIR analysis results show that the infrared spectra of immobilized ionic liquids had the characteristic absorption peak of Fe3O4.The infrared spectra of immobilized ionic liquids had a imidazole ring characteristic peak.XRD results improved that the immobilized process of ionic liquids had no damage to the crystalline structure of the magnetic silica gel carrier.Based on the result infrared spectra and elemental analysis,the immobilized catalyst contained the elements of the ionic liquid.Therefore,the ionic liquids were immobilized on the magnetic silica particles.Loading capacity of[VHim]Im was 292.87 ± 11.05 mg/g.The magnetic silica gel immobilized ionic liquids was developed and applied for hydrolysis of geniposide.The ratio of different raw material for synthesis of magnetic silica gel inmmobilized ionic liquids and different conditions of hydrolysis were optimized.The optimal synthesis of magnetic silica gel immobilized ionic liquids was as follow.The mixture of 3.00 g of Fe3O4 and 800 mL of ethanol was ultrasonic for 10 min.Then,120 mL of water,60 mL of hartshorn,and 10 mL tetraethoxysilane were added into above mixture for reaction for 5 h at room temperature to prepare Fe3O4@SiO2.3.00 g of Fe3O4@SiO2 was mixed with 10 mmoL vinyl triethoxy silane.The mixture was refluxed at 110 ?C for 24 h to prepare Fe304@SiO2@VTES.3.00 g of Fe304@SiO2@VTES was mixed with 1.50 g 1-vinyl-3-hexylimidazolium imidazole,0.04 g of azodiisobutyronitrile,and 30 mL of trichloromethane.The mixture was reacted at at room temperature for 1 h.Then,the mixture was heated to 100 ? and lasted for 2 h.The optimal hydrolysis conditions of geniposide were as follow:imidazole was selecte as the best anion for synthesis of ionic liquid,catalyst was 40 mg;middle power was used;hydrolysis time was 3.0 h.Under these conditions,the conversion rate of geniposide was 96.53%.The same batches immobilized ionic liquids were repeated used for five times,the conversion rate of geniposide were over 50%.The purity of geniposidic acid was changed from 7.95%to 35.68%after purification by DM130 macroporous resin.After extracted by ethyl acetate and acetone,the purity of geniposidic acid was 95.67%.Infrared spectrum analysis of purification products illustrated that the purified product had the characteristic absoption peaks of functional group of geniposidic acid reference.LC-MS/MS analysis further demonstrated that the purified product was geniposidic acid.The magnetic silica gel immobilized ionic liquids is efficiency and easy to separate from sample solution,and is also could be reused for at least four times.This method was not been reported.SEM analysis results show that the magnetic SiO2 immobilized enzyme was spherical sample,and the average diameter was of 99.21 nm.FTIR analysis results show that the infrared spectra of immobilized enzyme had the characteristic absorption peak of Fe3O4.The infrared spectra of immobilized enzyme had a characteristic peak at 1094 cm-1,which demonstrated that there was dentate bettwen histidine and iron.XRD results improved that the immobilized process of enzyme had no damage to the crystalline structure of the magnetic silica gel carrier.Based on the result infrared spectra and elemental analysis,the immobilized catalyst contained the elements of the enzyme.Therefore,the enzyme was immobilized on the magnetic silica particles.Loading capacity of cellulase was 135.91 ± 6.81 mg/g.The optimum hydrolysis conditions of geniposide by immobilized enzyme catalytic were as follows:cellulose was selected for the preparation of immobilized catalyst;catalyst was 60 mg;hydrolysis temperature was 50 ?;pH was 3.6;the hydrolysis time was 8 h.Under thess conditions,the concentration of genipin was 22.64 ug/mL.The conversion rate of geniposide was 97.58%.Catalyst for repeated use five times,the conversion rate of geniposide was greater than 55%.The HPD700 macroporous resin was used for purification of hydrolyzate.After purification,tihe purity of genipin was 97.38%.Infrared spctrum analysis of purification product indicated that the hydrolyzate had the characteristic absorption peak of functional groups of genipin reference.Further the LC-MS/MS analysis fturther demonstrated that the purified product was genipin.The magnetic silica gel immobilized enzyme is efficiency,low cost and easy to separate from sample solution,and is also could be reused for at least four times.This method had not been reported. |
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