Extraction, Separation And Pharmacological Activity Assay Of Garlic’s Functional Components Via Molecular Imprinting Technique

Garlic (Allium sativum L.), the underground bulbs of a kind of liliaceous alliumplants, is very popular for its edible and medicinal properties. It contains variousmedicinal components such as organic sulphides, flavonoids, saponins andpolysaccharides, etc. According to recent medical studies, garlic’s functionalcomponents possess many beneficial effects on antibacteria, lowering blood,antitumor, anti aging, enhancing immunity and so on, and consequently have manypotential applications in health care products. In this thesis, we extract alliin, allicin,flavonoid compounds, etc. functional components from garlic via molecularimprinting technique. Subsequently, we investigate their pharmacological activity.The main results are outlined as follows:1. Design and pre-assembly of template molecule and functional monomersBecause it is a complicated, hard process to separate the garlic’s functionalcomponents from aqueous system, we seek to find a kind of functional material whichcan effectively separate alliin, allicin and flavonoid compounds from aqueous system.With the combination of the principle of molecular imprinting and the theory ofmodern coordination chemistry, we established two molecular imprinting recognitionmodels, in which the structure of template molecule and functional monomers hasbeen optimized via PM3groups of GAMESS software packages. In addition, thebinding energy (ΔE) of the monomer template conjugates has been calculated withthe binding modes of noncovalent and coordinate bonds, respectively. The stabilitydiversities of the conjugates in different binding ways also were discussed bycomparing the binding energy of the conjugates. According to the results ofquantitative calculation, we designed the molecularly imprinted separation system ofgarlic’s functional components based on the metallic coordinate recognition model.Finally, In order to optimize the metallic coordinate molecular imprinting recognitionmodel for the separation of garlic’s functional components, we prescreened thetemplate molecule and functional monomers by the spectroscopy experiments toguide the synthesis of functional ligands and polymers.2. Synthesis and property of coordinate molecularly imprinted polymers for alliin A new type of functional ligand N (4styryl) oxamide has been designed andsynthesized. Replacing alliin with S propyl L cysteine sulfoxide as a“pseudo template molecule”, the coordinate monomer named S propyl L cysteinesulfoxide Zn oxamide has been synthesized and considered as a functionalcoordinate monomer to establish the bimetallic coordinate molecularly imprintedpolymer model which can selectively recognize the target alliin separated from garlic.With suspension polymerization technique, the directed synthesis of coordinatemolecularly imprinted polymer microspheres which can recognize alliin effectivelyhas been achieved. Through evaluating the recognition and separation effect of alliincoordinate molecularly imprinted polymers on the separated target, the coordinatemolecularly imprinted polymers with satisfying separation ability and largeadsorption capacity have been selected and applied to separate alliin in aqueoussolution. Compared with common cationic resin used presently, the purity of alliinseparated by alliin coordinate molecularly imprinted polymer microspheres can reach73.6%. This method will open a new way to develop the extraction and separation ofamino acid in aqueous solution.3. Synthesis and property of coordinate molecularly imprinted polymers for allicinA new type of functional ligand N (3propylamino) N’(4styryl) oxamide hasbeen designed and synthesized. Replacing allicin with dipropyl thioether as a“pseudo template molecule”, the bimetallic coordinate molecularly imprintedpolymer model which can selectively recognize the target allicin separated from garlichas been established. With suspension polymerization technique, the directedsynthesis of coordinate molecularly imprinted polymer microspheres which canrecognize allicin effectively has been achieved. Through evaluating the recognitionand separation effect of allicin coordinate molecularly imprinted polymers on theseparated target, the coordinate molecularly imprinted polymers with satisfyingseparation ability and large adsorption capacity have been selected and applied toseparate allicin in aqueous solution. The purity of allicin separated by allicincoordinate molecularly imprinted polymer microspheres can reach89.5%. Thismethod will provide a new methodological support for the separation andconcentration of trace thioethers compounds in aqueous solution.4. Synthesis and property of coordinate molecularly imprinted polymers for garlic’s flavonoidWith the major component myricetin in garlic flavonoids as template molecular, 4vinylpyridine as functional monomer and Zn(II) mediated，the garlic flavonoidcoordinate molecularly imprinted polymer microspheres, which can selectively adsorbthe template molecule analogues have been synthesized with the suspensionpolymerization technique. Through evaluating the separation effect of thesecoordinate molecularly imprinted polymers on several flavonoid compounds likemyricetin, quercetin, apigenin and kaempferol, the garlic’s flavonoids coordinatemolecularly imprinted polymers with proper separation ability and large adsorptioncapacity have been selected and applied to separate and purify the flavonoids crudeextract obtained by macroporous resin. And after the purification, the content ofgarlic’s flavonoids can reach81.2%. This method will lay a foundation for thepurification and large-scale preparation of garlic’s flavonoid.5. The pharmacological activity studies of garlic’s functional componentsThe inhibition activities of the four garlic’s functional components on H1N1influenza virus neuraminidase and the influenza A virus have been studied at themolecular and cellular level with H1N1influenza virus neuraminidase inhibitorscreening model and MDCK cellular model. The results indicated that theantioxidative activity IC50of the garlic’s flavonoids is2.064±0.026μg/mL, which issuperior to the positive control vitamin C (3.876±0.203μg/mL). The IC50values ofgarlic’s flavonoids in H1N1influenza virus neuraminidase inhibitor screening modeland MDCK cellular model are10.922±2.651μg/mL and3.15μg/mL, respectively,which are at the same level with the positive drug zanamivir in evaluating theanti influenza activities at the cellular level, while lower than the positive drug withselectivity and the largest non toxic concentration. Consequently garlic’s flavonoidshave good in vitro anti-influenza activities and potential application prospect, whichwill provide a new idea for the discovery of novel anti-influenza activecompounds.The oxidative stability and inhibition activities of the four garlic’sfunctional components (alliin, allicin, garlic’s flavonoids and saponins) onacetylcholinesterase and butyrocholinesterase have been studied with the DPPHanti oxidation model and the acetylcholinesterase and butyrocholinesterase inhibitormodel. The interaction between garlic’s flavonoids (myricetin, quercetin andKaempferol) and some biological macromolecules, such as DNA, has beeninvestigated at the molecular level with ultraviolet and fluorescence spectroscopy.These pharmacological studies have provided a substantial basis for the pharmaceutical development of garlic’s functional components.In conclusion, our researches built a new approach to separate and extractgarlic’s functional components. The results indicated that garlic’s flavonoidcompounds exhibited several pharmacological activities, such as oxidative stabilityand anti influenza virus, etc, which will not only provide important scientific data forthe research of China’s garlic medicinal functional components, but also are beneficialto the sustained and efficient development and utilization of China’s garlic resources.