| In China,Eucommia ulmoide has the advantage of abundance resource,but most raw materials are idle because of the low utilization efficiency,which led to a great waste of its medicinal value,food value and plant resources.To obtain high-valued E.ulmoide components including gutta-percha,seed oil,chlorogenic acid and genipin and to promote forestry economics and social development,in this paper,we present a novel concept for multistage and efficient utilization of plant resources.The contents of the concept are as follows:using renewable resources of plants as the main utilization parts;realizing multistage and continuous extraction for active ingredients in plants;paying attention to innovate extraction and separation technology;consummating ecological separation and conversion processes.In this research,we have carried out an innovative design about green separation process for active ingredients in different parts of E.ulmoide,providing a scientific basis for the rational and effective usage of E.ulmoide resources.1.HPLC condition was identified to simultaneous separation of four target ingredients from E.ulmoide,including aucubin,chlorogenic acid,genipin and geniposide.HPLC condition:HiQ sil-C18 reversed-phase column(4.6mm×250mm×5?m);column temperature:30?;detection wavelength:205 nm,240 nm;mobile phase:0.04%aqueous solution of phosphoric acid(A)-methanol(B);gradient elution:0-10 min,5%(B);10-20 min,5-20%(B);20-35 min,20-25%(B);35-40 min,25-5%(B);injection volume:10 ?L;flow rate:1 mL/min.The detection method has high precision,good reproducibility and stability,and is suitable for the analysis and detection target components in the E.ulmoide.The HPLC results indicated that high contents ingredients bark,leaves and seeds E.ulmoide are geniposide,chlorogenic acid and aucubinin,respectively.2.Ionic liquids-enzyme assisted extraction technology was firstly developed and applied for extraction of chlorogenic acid from E.ulmoide leaves.0.5M[C6mim]Br cellulase was used as extraction solution.The optimum process conditions were as follows:incubation and extraction time 2 h,extraction temperature 50 ?,pH value 3,enzyme concentration 2 mg/mL.Comparing extraction dynamics for chlorogenic acid in four solvents system,[C6mim]Br solution has obvious advantages as extraction solvent.Addition of cellulase improved the penetrable ability of the extraction solvent,which also effectively improve the ionic liquid solution's bad penetrability against plant matrixes.The results of methodology study demonstrate ionic liquids-enzyme assisted extraction has good reproducibility and stability.The difficulty that separate target analyte from ionic liquids solution has been solved by the macroporous resin technology.The chosen HPD750 resin has ability to achieve effective separation the adsorbed target substance from ionic liquid aqueous solution.So an effective method was found to reduce ionic liquid residue for plant extracts.Moreover,this method also provides a reference to improve safety for application of ionic liquids in extracting other botanical drugs.Furthermore,we investigated the macroporous resin regeneration and recycling of the ionic liquid,providing a theoretical basis to reduce the cost of the proposed extraction method.3.We have develoed continuous multistage s extraction for three representative components of E.ulmoide seed,seed oil,gutta-percha and aucubin glycosides,and obtained high efficient utilization process routes of E.ulmoide seed.Firstly,E.ulmoide seed oil was extracted by supercritical CO2 extraction technology,then gutta-percha was separated using petroleum ether heating extraction,lastly moderately polar components including aucubin were separated by ionic liquids based ultrasound-assisted method,in which we can achieve the objective of maximize usage of E.ulmoide seed resources.By single factor experiment and response surface optimization experiments,we obtained optimal process condition of E.ulmoide seed oil supercritical CO2 extraction:extraction pressure 41 MPa,extraction temperature 43 ?,CO2 flow rate of 11.5 kg/h.Under optimal conditions,extraction yield of E.ulmoide seed oil achieved 41%.And supercritical CO2 extraction has superiority on the quality and chemical composition comparing with traditional organic solvents heating extraction method.We extracted aucubin from E.ulmoide seeds using ionic liquids based ultrasound-assisted method.Single factor experiments were carried out to get optimum conditions as follows:soaking time 2 h,the solid-liquid ratio of 1:20(w/v),ultrasonic power 225W,ultrasonic time 20min,[C2mim]Br concentration 1.0 M,the extraction times 2 times,aucubin yield 16.74 mg/g.4.Ionic liquid-enzyme in situ extraction hydrolysis method was applied to extract genipin from the E.ulmoide.The released geniposide from cells was hydrolysed in the extraction process,conveting to genipin,in which takes advantage of characteristic of cellulase complex containing ?-glucosidase,avoiding some complex preparation process of geniposide and ?-glucosidase.And both the extraction and hydrolysis were completed in the same system,which provides a simple and feasible preparation method for genipin.Optimum Ionic liquid-enzyme in situ extraction hydrolysis process:0.5 mol/L[C6mim]Cl used as the reaction medium,hydrolysis and extraction time 2 h,the solid-liquid ratio 1:20(g/mL),pH = 5,enzyme concentration of 2 mg/mL.Under optimum conditions,genipin increment was 0.55 mg/mL.Meanwhile we have established dynamic model of hydrolysis reaction of geniposide catalyzed by ?-glycosidase under different reaction mediums.In the three chosen reaction medium,the ranking of reaction rate of geniposide hydrolysis reaction catalyzed ?-glucosidase is as follows:H2O>0.5 mol/L[C6mim]Cl>50%enthanol.The ?-Glucosidase within 0.5 mol/L[C6mim]Cl solution exhibited better catalytic activity than 50%ethanol,indicating that inhibition degree of enzyme in a certain concentration ionic liquid aqueous solution is weaker than the organic solvent.Ionic liquid-enzyme in situ extraction hydrolysis method has feasibility and superiority on genipin preparation.5.The preparation,physical and chemical properties and immunocompetence of E.ulmoide polysaccharide were investigated.Microwave assisted extraction was applied to extract polysaccharide and obtain optimum process condition by single factor test:solid-liquid ratio of 1:25 g/mL,microwave power of 540 W,irradiation time of 20 min.Under this condition,the polysaccharides yield of E.ulmoide reached 27.07 mg/g.As for specific immune activity,when polysaccharides group was in 0.05?1.6 mg/mL concentration range,various concentration E.ulmoide polysaccharide have significant enhancement on the proliferation rate of mouse spleen lymphocytes(P<0.01,P<0.05)comparing to the blank control group.This result implies E.ulmoide polysaccharides have obvious immunocompetence,and itself has the role of mitogen.At the same time,E.ulmoide polysaccharide also can promote T or B lymphocyte proliferation actived by Con A or LPS(P<0.05 or 0.01),the results show that the E.ulmoide polysaccharides can enhance proliferation of T lymphocytes and B lymphocytes,and its effect on B lymphocyte proliferation was stronger than the T lymphocytes.6.The remaining solids of E.ulmoide after active substances extracting were used as raw materials,with phosphoric acid as an activator,we prepared powdered activated carbon using microwave heating in order to achieve multistage usage of E.ulmoide plant resources and reduce the environment pollution.Through orthogonal experiments,we determine the optimum conditions to prepare activated carbon by microwave irradiation phosphate method:microwave power 540 W,the fraction of phosphoric acid 60%,microwave irradiation time 15 min.Under this condition,the activated carbon yield was 38.41%,iodine sorption value 699 mg/g,methylene blue adsorption value 69.5 mL/g.This process provides a feasible way to deal with solid residue after E.ulmoide active substances extracting,and open up a new approach for the comprehensive utilization of E.ulmoide,implement efficient multistage use of E.ulmoide resources. |
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