Purification of Botanical Medicine by Hydrophobic Interaction Membrane Chromatography

Author:Peng Rong

Supervisor:chen xiao nong


Degree Year:2016





Botanical medicine is playing an increasingly significant role in the treatment of diseases with its unique merits,such as rich source,less side effect and low cost.Currently,column chromatography technology is the main purification technology for the purification of active constituents in botanical medicine.However,there are some limitations related to column chromatography,such as high back pressure,time-consuming,and large consumption of organic solvents.These limits the further development of botanical medicine.Membrane chromatography is a combination of membrane separation technology and liquid chromatography.Its separation mechanism is mainly dependent on interaction difference between membrane and solutes.Membrane chromatography has attracted increasing attention due to its advantages,such as low back pressure,high flow-rate,easy to scale up and no organic solvent involved.Membrane chromatography technology has been extensively applied in the purification of bio-macromolecules such as protein,DNA and antibody isolation.However,study on the separation of botanical medicine by membrane chromatography is rarely reported.Membrane chromatography technology was applied to the isolation of active components from botanical medicine in this study.Compared with biological macromolecules,botanical drugs have lower molecular weight,smaller molecular size and weaker electrostatic and hydrophobic interaction between membrane and drugs.Hence,drug molecules are difficult to bind to membrane,which impedes the application of chromatography technology in botanical medicine purification.Therefore,it will be a key issue to enhance and meanwhile maintain the difference of interaction strength between membrane and drug molecules to extend the application of membrane chromatography in botanical medicine purification.Graphene oxide(GO)is an ideal adsorbent material for its significant advantages,e.g.,two-dimensional planar structure,large surface area and large delocalized π-structure.So far functionalized GO materials are widely used to load anti-cancer drugs with low hydrophilicity,which provides a new way to resolve the problem of drug delivery.When GO carries anticancer-drugs,a strong physical bonding forms between GO and drugs by hydrophobic interaction and π-π conjugation.These GO properties lead to GO high drug loading and slow drug release.Inspired by the idea,in this study,GO was selected as a hydrophobic interaction ligand to enhance membrane adsorption capacity for relevant components in extract.The work would create a prerequisite for chromatographic separation of botanical medicine.GO modified cotton fiber(GO-CF)was prepared to fabricate GO-CF membrane.GO-CF membrane performance was studied and the membrane was then utilized as hydrophobic interaction membrane to purify botanical medicine,e.g.,polyphenols,ellagic acid(EA)and danshensu(DSS).A new environment-friendly and safe separation method based on hydrophobic interaction membrane chromatography(lHIMC)with low operating pressure,rapid separation and no organic solvents involved was developed for botanical extract purification.Main contents are as follows:1.Preparation and characterization of graphene oxide modified cotton fiber membraneCotton fiber(CF)was selected as membrane substrate.GO was covalently grafted onto CF to provide a strongly hydrophobic adsorption site.And the modified fiber was made into a sheet-like GO-CF membrane.SEM images,Raman spectra,FT-IR spectra and XPS spectra clearly proved that GO was grafted to CF by epoxy ring-opening reaction.Basic properties of the membrane were investigated by separating polyphenols from crude extract of pomegranate peel.The effect of flow rate on transmembrane pressure drop was studied and results indicated that transmembrane pressure drop would increase with increasing flowrate.However,the increased transmembrane pressure drop was still significantly lower than that of commercial column,which indicates that GO-CF membrane is more energy efficient.The transmembrane pressure drop did not significantly change after several adsorption-desorption cycles,which indicated that membrane had good reusability.Pure water and 0.04 M NaOH aqueous solution were the optimum binding solution and eluting solution for crude polyphenols purification,respectively.Polyphenols were adsorbed to the membrane mainly by hydrophobic interaction.Desorption process can be achieved by enhancing the alkalinity of mobile phase.The increasing buffer alkalinity will enhance the dissociation of phenolic hydroxyl groups in the buffer,which reduces the hydrophobic interaction between polyphenols and membrane.Based on peak ratio(Flow-through peak/elution peak),the binding capacity of(0.40%-GO)-CF membrane improved significantly which is 140 times higher than that of unmodified CF membrane.The result exhibits the significant effect of GO on improving cotton fiber based membrane performance.After single binding-elution cycle,about 72.5%of sugar and almost all of proteins in the feed solution were removed.Polyphenols recovery was~73.0%and its content was enriched from approximately 48.0%to~76.1%.2.Estimation of relative retention of pharmaceutical molecules in hydrophobic interaction membrane chromatographyCurrent hydrophobic interaction theory cannot be applied to properly predict drug separation behavior due to the significant differences between bio-macromolecules and small molecule drugs,such as molecular weight,molecular charge and molecular structure.In this study,a series of drugs with different hydrophobicity were selected as model compounds to study the relationship between relative retention time(tRR)of the model compounds in the reversed-phase high performance liquid chromatography(HPLC)and its hydrophilic group ratio(HGR).Then,an empirical model is established(tRR = 1.06-0.52HGR-1.16(HGR)2,R2=0.96,water/acetonitrile as mobile phase).The effects of gradient slope and organic solvent on tRR were studied.Results showed that sample tRR was independent on gradient slope but would increase with increasing polarity of organic solvent.Based on target drug structure,HGR can be calculated and it can be used to compare the relative hydrophilicity of different drug molecules.Comparilg calculatedt tRR of sample with the experimental tRR of model compounds,the relative retention order of sample was estimated.The estimation can facilitate the rapid determination of appropriate time to collect target component and provide a guidance for experiment design.3.Purification of ellagic acid and danshensu by hydrophobic interaction membrane chromatographySince column chromatography used for EA and DSS purification has some shortcomings such as high back pressure,time-consuming and large consumption of organic solvent,HIMC technology was applied to purify botanical medicine by using(0.40%-GO)-CF membrane as HIMC membrane in the study.Suitable HIMC operating mode and operating conditions were investigated.EA is highly hydrophobic based on the estimatied tRR;therefore,its HIMC purification mode is binding-elution mode,i.e.,impurities directly flow through membrane and target drugs are collected in elution stage.While flow-through operation mode,i.e.,target drugs are collected in the adsorption stage and impurities are desorbed from membrane,would be a suitable mode for hydrophilic DSS.The effects of mobile phase composition,pH,flow rate and other factors on purification efficiency were systematically investigated.Results indicated that the optimum binding solution and eluting solution were 5 mM Na2CO3 aqueous solution and 0.04 M-0.06 M NaOH aqueous solution for EA purification,respectively.Under optimized conditions,EA purity increased from 7.5%(Crude extraction)to 75.0%~80.0%.The binding capacity of(0.40%-GO)-CF membrane to EA was~397.8 μg·ml-1,equaling to 27.5%mass fraction of GO(EA,mg/GO,mg)when converted into the binding capacity of GO on GO-CF membrane.The value,27.5%,was in the similar range with the reported loading rate of functional GO for drug medicine.Considering stability and economy,5mM sodium phosphate buffer with pH 6 and 0.04 M NaOH were suitable as binding solution and elution solution for DSS purification,respectively.After single binding-elution cycle,DSS purity increased from 24.8%(Crude extraction)to 70.0%~75.0%which was higher than that obtained from adsorbent resin(57.3%)or ion exchange resin(71.1%).DSS recovery was high,~96.0%.Crude DSS was successfully purified by adsorbing impurities,which facilitate the application of HDMC technology in hydrophilic drugs purification.