AU2021103054A4 - Preparation method and extraction method of magnetic molecular imprinted polymer of larch flavonoid active ingredient - Google Patents

Abstract

The invention discloses a preparation method and an extraction method of magnetic molecularly imprinted polymer of larch flavonoid active ingredients, which comprises the following steps: mixing template raw materials with organic solvents and functional monomers, then adding crosslinking agent and initiator, introducing nitrogen to obtain a polymer, magnetically separating and drying under vacuum to obtain a reaction material, eluting and removing the template through Soxhlet extraction method, then removing residual organic matters, and drying, wherein the functional monomer is 4-vinyl pyridine, he template raw material is dihydroquercetin. The molar ratio of template raw material, functional monomer and crosslinking agent is 1: (2-6): (20-30). The preparation method stated is simple and low in cost.

Description

Preparation method and extraction method of magnetic molecular imprinted

polymer of larch flavonoid active ingredient

TECHNICAL FIELD

The invention relates to the technical field of separation and purification of flavonoids, in

particular to a preparation method and extraction method of magnetic molecular

imprinted polymer of larch flavonoid active ingredient.

BACKGROUND

At present, with the gradual improvement of people's awareness on the safety of animal

products, the food safety problems caused by the pollution of chemical drugs and

antibiotics and the severe challenges aroused by the "reduce and replace antibiotics" in

livestock and poultry breeding have become the focus of attention of the whole society.

Due to that wide variety of natural materia medica and the complexity and diversity of

active components with similar structure, higher requirements were put forward for the

efficient extraction and purification of natural active components for anti respiratory

diseases. Therefore, the construction of a convenient and efficient screening and

extraction technology of natural active products with high analysis efficiency, strong

specificity, and high flux is still the key technology for the development of new

veterinary drugs in China and the rest of the world.

Larix Griffithii (belongs to Pinaceae) is an important source of medicine for plateau

medicine - Tibetan medicine compounds because of its medicinal active components.

This kind of materia medica mainly distributed in the alpine region of Tibet and has rich

and unique active components. Nowadays, antibiotic poisoning and residues are often

found in high altitude animal husbandry. The extraction and separation of active ingredients from natural plants is essential for the development of new alternative antibiotics. As an integral part of Tibetan medicine, Larix Griffithii has not only various amazing drug effects, but also the effects like low toxicity, easy absorption and utilization, and is often used as the main component of Tibetan ethnic medicine.

Dihydroquercetin is an important flavonoid compound in many plants and is often used

as an important evaluation standard for Tibetan medicine. The good pharmacological

activity of this compound leads to its greatly increased use, especially in the treatment of

difficult and complex diseases that affecting people, livestock and poultry. In the aspects

of free radical scavenging, antioxidant, antitumor, antiviral, sterilization, etc,

dihydroquercetin is playing an increasingly important role.

In previous studies, dihydroquercetin was mainly extracted from Pinus massoniana,

Larix Olgensis Henry and Pinus Sylvestris Var, Mongolica. However, due to the complex

structure of Chinese medicine and the variety of active components as well as its low and

unstable content, it has brought certain difficulties to the separation and purification of

the active components in Chinese medicine. At present, the separation and purification

mainly rely on solid phase extraction with silica gel column, microporous adsorption

resin column chromatography, polyamide column chromatography and high performance

counter-current chromatography, but the recovery ratio remain low. Molecular imprinting

technology is based on bionic science, zymolyte simulation and natural receptor and

antibody. The basic principle is to introduce the molecular imprinted recognition site into

the polymer material by simulating the natural molecular recognition function to prepare

a polymer that is completely matched with the target molecule in the spatial structure and

binding site. The molecular imprinted polymer can realize specific selection on a target molecule. Along with the deepening of research and the continuous expansion of application fields, molecular imprinting technology has been widely used in the fields of chiral reparation, solid phase extraction, sensors, catalysis and organic synthesis. But there are still many problems and shortcomings. For example, traditional adsorbents have poor selective recognition ability, resulting in unavoidable matrix interference. In addition, the solid phase adsorption could not meet the extraction and detection requirements of dihydroquercetin. In a recent study, the content of curcumin was determined by using the magnetic molecularly imprinted adsorbent prepared on the surface of Fe304 nanocrystals combined with the ultraviolet-visible spectrophotometry.

Kolaei et al. also utilized Fe304 magnetic multi-layer carbon nanotubes with Fe304

nanoparticles and coated their surfaces with ethylene end groups. A novel molecularly

imprinted polymer was prepared using magnetic multi-layer carbon nanotubes as the

carrier and successfully applied to the extraction and determination of morphine.

Magnetic molecularly imprinted polymer nanoparticles with core-shell structure were

synthesized by enzyme-assisted sol-gel method. They showed good recognition and

selectivity for quercetin and catechin. By preparing an aqueous magnetic molecularly

imprinted polymer, the results showed that the molecularly imprinted polymer was

uniformly synthesized on the surface of Fe304. The adsorption experiments showed that

the extraction capacity and selectivity of morphine and its analogues were superior to

those of non-imprinted polymers. The molecularly imprinted solid phase extraction

column magnetic separation system is usually used for detecting pesticides and veterinary

drugs in foods and fruits. These magnetic molecularly imprinted polymers can overcome

the problems of long elution time, template leakage, and solid phase extraction, but are rarely used for quick extraction, separation, and determination of structures and activities of biological compounds in Tibetan medicine.

The structural types of flavonoid active components in Larix Griffithii are complex and

diverse. The trace and uncertain active components of some species and genera as well as

their inherently unstable side-chain structure, lead to difficulties in the separation and

purification of active molecules, and the use of drugs due to the complex components. At

present, the extraction and analysis methods of the flavonoid active components mainly

include solvent extraction, ultra-high pressure and low temperature extraction, ultrasonic

extraction, microwave-assisted extraction, supercritical C02 extraction, macroporous

adsorption resin extraction technology, and the like. But the extract components extracted

and prepared by the method above are still relatively complex, and the active components

can not be effectively enriched, so that the pharmacodynamic functions of the active

components are not fully exerted. Therefore, a new isolation method is urgently needed to

fully reveal the biological activity of multi-component Chinese herbal medicines. Multi

component knockout of medicinal Larix Griffithii and biological activity evaluation

would be helpful to clarify the pharmacological activity of Chinese medicine. Supposing

a specific selective isolation strategy (MIPs) was designed to comprehensively construct

a new isolation and extraction method and clarify the pharmacological effects of the

active components of Larix Griffithii, it would have important application value.

SUMMARY

The invention aims to provide a preparation method of larch flavonoid active ingredient

magnetic molecular imprinted polymer, which is simple and low in cost.

Another objective of the present invention is to provide a magnetic molecularly imprinted

polymer that maintains good morphology, good selective recognition, and physical and

chemical stability without affecting the adsorption efficiency of the polymer under the

dynamic conditions and high repeatability of the material.

The third objective of the invention is to provide a method for extracting

dihydroquercetin by using a magnetic molecular imprinted polymer, which can realize

the separation and detection of the active ingredient - dihydroquercetin of the Larix

Griffithii and other natural medicinal plants.

In order to achieve the purposes stated above, the invention provides the following

scheme:

The invention provides a preparation method of magnetic molecular imprinted polymer

of larch flavonoid active ingredient, which comprises the following steps:

Mix template raw materials with an organic solvent and a functional monomer, adding

crosslinking agent and initiator, introducing nitrogen to obtain a polymer, magnetically

separating and dry under vacuum to obtain a reaction material, eluting by using a soxhlet

extraction method to remove that template, removing residual organic substances, and

drying;

The functional monomer is 4-vinyl pyridine (4-vp);

The template raw material is larch flavonoid active ingredient.

The specific method comprises the following step of: (1) preparing magnetic Fe304

nanoparticles, and preparing Fe304@SiO2 nanoparticles by sol-gel method: mixing

ferrous salt (FeSO4-7H20) and trivalent iron salt (FeCl3-6H20) solution, wherein the total

concentration of the iron salt is 0.5mol/L, adding distilled water into a three-necked flask, controlling the temperature to be (30±1)°C, Then slowly drop ammonia water with concentration of 0.25mol/L into a three-necked flask until the pH value is equal to 10, vigorously stir, crystallization in high and constant temperature water bath for a certain period of time, gradually the colour of mix solution change from orange red to black, the continue to stir for 15min, and finishing the reaction, centrifuging the obtained Fe304 particles, repeatedly washing with distilled water until the pH value is equal to 7, removing the supernatant, keeping the temperature of the product at 80°C for 30min, aging, and grinding to obtain the nano magnetic Fe304 nanoparticles. Adding tetraethoxysilane into the 10% ethanol water solution of the magnetic Fe304 nanoparticles, stirring for 12 hours under the condition of 60°C, carrying out centrifugal precipitation on the obtained polymer particles, washing, and vacuum drying to obtain the

Fe304@SiO2 nanoparticles; wherein the mixing volume ratio of ethanol, pure water and

ammonia solution is 10: 4: 1. The molar ratio of FeSO4-7H20 to FeC3-6H20 is 1: 4.56;

(2) Preparation of Fe304@SiO2-NH2 particles: Fe304@SiO2 nanoparticles were dispersed

in toluene and aminopropyltriethoxysilane (APTES) (the mixing volume ratio of toluene

to APTES was 100mL:12mL), and then the mixtures (ammonia, APTES and

Fe304@SiO2) in a three-necked flask were stirred at 30°C for 5 hours under the

protection of nitrogen. Finally, Fe304@SiO2-NH2 microspheres were alternately washed

three times with water and ethanol, and dried under vacuum for 12 hours for later use;

(3) Preparation of Fe304@SiO2@MIPs: dispersing Fe304@SiO2-NH2 particles in a

template solution, adding 3-trihydroxymethylsilyl-propylmethyl phosphate (THPMP) and

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), adding functional monomer and

cross-linking agent, adding initiator, introducing nitrogen, sealing and stirring, adopting

Soxhlet extraction method, remove templates completely from the methanol-acetic acid

mixture until they could not be detected from the eluent by HPLC. Residual acetic acid

was removed 3 times with methanol and Fe304@SiO2@MIPs was obtained by magnetic

separation and drying under vacuum.

As a further improvement of the invention, the template material is dihydroquercetin.

As a further refinement of the invention, the crosslinking agent is ethylene glycol

dimethacrylate (EGDMA).

As a further refinement of the invention, the initiator is azobisisobutyronitrile (AIBN).

As a further improvement of the invention, the molar ratio of the template raw material,

the functional monomer, the crosslinking agent is 1: (2-6): (20-30).

As a further refinement of the present invention, the molar ratio of template material,

functional monomer, crosslinking agent is 1: 4: 20, 1: 6: 20, 1: 2: 25, 1: 4: 25, 1: 6: 25, 1:

2: 30 or 1: 4: 30.

The invention provides a magnetic molecular imprinted polymer which is prepared

according to the preparation method of the magnetic molecular imprinted polymer of the

Larix Griffithii flavonoid active ingredient.

The invention provides a method for extracting dihydroquercetin according to the

magnetic molecular imprinted polymer, which is extracted by a solid phase extraction

method.

As a further improvement of the invention, Fe304@SiO2@MIPs is used as adsorbent in

the solid phase extraction method.

As a further improvement of the present invention, in the solid phase extraction method,

elution is performed using a mixture of toluene and methanol as elution solution, and the volume ratio of the toluene to the methanol is 1-2:3-4. Preferably the volume ratio of toluene to methanol is 2:3.

In the invention, Fe304@SiO2@MIPs/NIPs is used as solid phase extraction adsorbent,

extraction conditions such as loading solution, washing, elution solvent, extraction time

and the like are optimized, high performance liquid chromatography (HPLC) is adopted

in combination with a solid phase extraction (MISPE) technology to determine

dihydroquercetin in Chinese herbal medicine and plants (especially Larix Griffithii) so

as to obtain the maximum adsorption amount of target molecules as objective. The results

showed that the optimal adsorbent for solid phase extraction is Fe304@SiO2@MIPs, 5mL

methanol is the optimal loading solvent, n-hexane is the optimal washing solvent, and the

mixture of toluene and methanol (mixed volume ratio of 2:3) is the optimal elution

solution.

The invention discloses the following technical effects:

In the invention, the magnetic molecular imprinted polymer material is synthesized by a

precipitation polymerization method, and the magnetic molecular imprinted polymer can

be used for extracting and selectively identifying dihydroquercetin. Under the dynamic

conditions and high repeatability of the materials, the magnetic molecularly imprinted

polymer maintained good morphology, good selective recognition and physical and

chemical stability, and did not affect the adsorption efficiency of the polymer. The

synthesis of magnetic surface molecularly imprinted material is a simple and effective

method to improve the binding performance. The dihydroquercetin in the Larix Griffithii

was separated and detected by the magnetic molecular imprinted polymer, and excellent

separation and detection performance was shown. In the invention, the dihydroquercetin magnetic molecular imprinted polymer is used as a solid phase extraction adsorbent, and the solid phase extraction method is suitable for practical application. The method had been successfully applied to the detection of samples of Larix Griffithii. By this method, it was found that the content of dihydroquercetin in Larix Griffithii was higher. The established magnetic molecular imprinted solid phase extraction and HPLC methods can be used for quick extraction, and to meet the requirements of separation and detection of the active ingredient dihydroquercetin by other natural medicinal plants at the same time.

BRIEF DESCRIPTION OF THE FIGURES

In order to explain more clearly the embodiments of the present invention or the technical

scheme in the currently available technology, the figures needed to be used in the

embodiments were briefly introduced below, and it is obvious that the figures in the

following description are only some embodiments of the present invention, and other

figures can be obtained according to the figures by a skilled person in the art without

paying creative labor.

Fig.1 is a flowchart of preparation process and magnetic SPE of Fe304@SiO2@MIPs

according to the present invention;

Fig.2 is a photograph of a Larix Griffithii sample and a chemical structural formula of

dihydroquercetin;

Fig.3 is a graph showing the binding and desorption amounts of Fe304@SiO2@MIPs and

Fe304@SiO2@NIPsversus the ratio of templating agent, monomer, and crosslinking

agent;

Fig.4 is an infrared spectrum analysis diagram of Fe304, SiO2@Fe34, Fe304@SiO2-NH2,

Fe304@SiO2@MIPs, and Fe304@SiO2@NIPs;

Fig.5 is a scanning and transmission electron microscopic view of Fe304(a),

Fe304@SiO2@MIPs(b and C), and Fe304@SiO2@NIPs(d);

Fig.6 is the hysteresis loops of Fe304@SiO2@MIPs and Fe304@SiO2@NIPs;

Fig.7 is the adsorption capacity of dihydroquercetin at different concentrations on

Fe304@SiO2@MIPs/NIPs;

Fig.8 is the kinetic curve of dihydroquercetin on Fe304@SiO2@MIPs/NIPs;

Fig.9 is a qualitative comparison of the selection (n=3) of different extraction solutions

(a), elution time (b) and elution solvent (c) in the magnetic solid phase extraction process.

Fig.10 is the chromatograms of dihydroquercetin and matrine standard solution, where a

is the chromatogram of the dihydroquercetin solution at a concentration of 0.24g/ml, b is

the total chromatogram of Larix Griffithii, c is the chromatogram of matrine in the

supernatant after magnetic solid phase extraction, d is the chromatogram of the n-hexane

eluent, e is the chromatogram of the dichloromethane eluent, and f is the chromatogram

of the eluent.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention are now described in detail,

which should not be construed as limiting the invention, but rather as a more detailed

description of certain aspects, features, and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing

particular embodiments only and is not intended to be limiting of the invention. In

addition, for numerical ranges in the present invention, it is understood that each

intermediate value between the upper and lower limits of the range is also specifically

disclosed. Each smaller range between any stated value or stated range of intermediate values and any other stated value or intermediate value within the stated range is also included within the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless otherwise indicated, all technical and scientific terms used herein have the same

meaning as commonly understood by one of ordinary skill in the art to which this

invention relates. While the present invention describes only the preferred methods and

materials, any methods and materials similar or equivalent to those described herein may

be used in the practice or testing of the present invention. All documents mentioned in

this specification are incorporated by reference to disclose and describe methods and/or

materials related to stated documents. In case of conflict with any incorporated literature,

the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations can

be made to the specific embodiments of the present specification without departing from

the scope or spirit of the invention. Other embodiments will be apparent to those skilled

in the art from the description of the invention. The specification and embodiment of that

present application are exemplary only.

As used herein, the terms "comprise", "include", "have", "contain", and the like are open

ended terms that mean including, but not limited to.

Example 1

(1) Preparation of Fe304@SiO2

Prepare magnetic Fe304nanoparticles, Fe304@SiO2nanoparticles were prepared by sol

gel method: 0.6g of a solution of ferrous salt (FeSO4-7H20) and 1.6g of a solution of

trivalent iron salt (FeC13-6H2O) at a total concentration of 0.5mol/L were mixed into a three-necked flask, dispersed in a mixture of ethanol (100mL), pure water (40mL) and an aqueous ammonia solution (lOmL, 15wt%) and N2 was used to remove oxygen. After ultrasonic mixing for 15 minutes, tetraethoxysilane (TEOS)(0.6ml, 2.2mmol) was added to the solution. After stirring for 12 hours at 60°C, the obtained polymer particles were subjected to centrifugal precipitation, washing three times, and vacuum drying for 10 hours to obtain Fe304@SiO2 nanoparticles. Fe304@SiO2(0.lg) activated microspheres were dispersed in 100mL of toluene and l2mL of APTES aminopropyltriethoxysilane.

The mixture in the three-necked flask was then stirred under nitrogen at 30°C for 5 hours.

Finally, Fe304@SiO2-NH2microspheres were alternately washed three times with water

and ethanol and dried under vacuum for 12 hours to obtain Fe304@SiO2-NH2particles.

(2) Preparation of Fe304@SiO2@MIPSandFe3O4@SiO2@NIPs

Approximately 0.Ig of Fe304@SiO2-NH2 particles were dispersed in 30ml of the

template solution (dihydroquercetin, 5mg/L) and the structure is shown in Figure 2

. Then, 0.05g of THPMP and 0.01mg of EDC were added, and the functional monomer (4

VP) and the crosslinking agent (EGDMA ethylene glycol dimethacrylate) were added to

the template (dihydroquercetin) in different molar ratios of template molecule, functional

monomer, crosslinking agent (molar ratios are 1: 2: 20, 1: 4: 20, 1: 6: 20, 1: 2: 25, 1:4:25,

1: 6: 25, 1: 2: 30, 1: 4: 30). Subsequently, an initiator AIBN azobisisobutyronitrile

(25mg) was added to the polymerization system, and nitrogen was supplied to remove air

for 15 minutes, the particles were sealed and stirred at 60°C for 24 hours, and after

polymerization, the template was removed by a Soxhlet extraction method using a

methanol-acetic acid mixed solvent, and the template was completely removed until they

could not be detected from the eluant by HPL. Residual acetic acid was removed 3 times by methanol, and Fe304@SiO2@MIPs was obtained by magnetic separation and drying under vacuum.

Fe304@SiO2@NIPs without template was similarly obtained by dispersing Fe304@SiO2

NH2 particles in methanol, adding THPMP and EDC, adding functional monomer and

crosslinking agent, adding initiator, introducing nitrogen, sealing and stirring, and

obtaining Fe304@SiO2@NIPswithout template by magnetic separation and drying under

vacuum. The preparation process and the magnetic SPE program of Fe304@SiO2@MIPs

in the invention are shown in Fig.1.

The adsorption properties of Fe304@SiO2@MIPs/NIPs obtained were evaluated:

Adsorption property

Prepare a stock solution of acetonitrile (1mg/mL) containing dihydroquercetin and

matrine. A series of standard solutions were diluted as needed for the experiment:

Approximately 5mg of polymer was added to lOmL of dihydroquercetin and matrine

solution (10Og/mL). The static binding abilities of the four compounds adsorbed on

Fe304@SiO2 were determined by the following equation: Qe=(Co-Ce)V/m(1), where Co

and Ce( g/L) were the initial and equilibrium concentrations of the compound in the

matrix solution respectively; V(L) is the volume of the standard solution; m(g) is the

weight of Fe304@SiO2@MIPs; Qe is the static binding capacity. The adsorption

isotherms were calculated by Scatchard (Equation 2), Langmuir (Equation 3) and

Freundlich model (Equation 4): Q/Ce=(Qmax-Q)/Ks (2), 1/Qe=l/(KCeQmax)+1/Qmax (3),

LnOe=LnKf+(1/n)LnCe(4), t/Qt=l/k2Q2cal 2+t/Q2cai(5), where Qmax is the maximum

saturated adsorption capacity(mg/g), Ks is the dissociation constant, K is the

Langmuir(L/mg) adsorption rate constant, Kf is the Freundlich adsorption rate constant(mg/g), n is the heterogeneity parameter, t(min) is the adsorption time, Qt is the binding capacity during static binding(mg/g), K2 is the rate constant [g/(mg min)], and

Q2ca is the theoretical adsorption capacity of pseudo-second-order kinetics(mg/g).

Sample analysis

Accurately weighed 100mg of the Larix Griffithii sample, labeled (0.1, 1, 5, and

mg/kg), immersed in a 1OmL centrifuge tube containing ethyl acetate for 1 hour and

sonicated for 30 minutes. The process was repeated three time. The extract solution was

then centrifuged at 5000 rpm for 5 minutes. The collected supernatant was defatted,

concentrated in vacuum at 4°C and the crude extract was stored in flasks for the

following magnetic solid phase extraction procedure. Then added 1OmL of the solution

and 5mg of Fe304@SiO2@MIPsto the tube and mix for 30 minutes at room temperature.

The extract was obtained by external magnetic field. Eluted the bound compound on

Fe304@SiO2@MIPsto a constant volume of 10mL and analyzed by HPLC.

In the invention, the functional 4-VP was used as monomer and the EGDMA was used as

crosslinking agent, different proportions of a template, the monomer and the crosslinking

agent were investigated, and optimal preparation conditions were obtained. When the

template (dihydroquercetin), the functional monomer 4-vinylpyridine (4-VP) and the

crosslinking agent ethylene glycol dimethacrylate (EGDMA) were added in the ratio of

1:4:25, the prepared Fe304@SiO2@MIPs exhibited the optimal adsorption and desorption

properties. The maximum binding and desorption capacity of dihydroquercetin was

7.56mg/g (see Figure 3).

The infrared spectrum analysis results of Fe304@SiO2@MIPs/NIPs are shown in Fig.4.

The characteristic absorption peak of -Fe-O- was observed at 580cm- 1, and the bending vibration absorption peak of Si-O-Si was observed at 747.94 and 475.85cm-1 . Before amination, the peak at 1676cm-' was attributed to the amino group because the absorption peak of the amino group moved to the low direction. An absorption peak appeared at

2813cm- 1. Band corresponding to -OH tensile vibration at 3450cm-1 . By introducing the

NH2 group, the tensile vibration of 3420cm- 1 and the N-H bending vibration at 1540cm-'

were recorded. The peaks of 1735cm-'(C=O), 3100.58cm-1 (N-H) and 1169cm-'(-C-O-C-)

in the polymer indicated that the polymerization of Fe304@SiO2@MIPs was successful.

The surface morphology of Fe304@SiO2@MIPs was studied by TEM and SEM (see

Figure 5). The structure of Fe304 is a regular sphere. The agglomeration of Fe304 is due

to the high specific surface area of Fe304 nanoparticles and dipolar interaction of particle

anisotropy (Fig.5a). Figures 5b and 5c show that the MIP surface is covered with holes,

and the surface is rough and fluffy. The core-shell structure of Fe304@SiO2 is clear.

Therefore, after elution, the template molecular dihydroquercetin was washed out from

Fe304@SiO2@MIPs and regular pores were formed, which was beneficial to the internal

adsorption of dihydroquercetin through the pores. Figure 5d shows a magnified SEM

image of Fe304@SiO2@NIPs at the same time. The pore size was very small and the

surface became stiff, indicating that Fe304@SiO2@NIPs has no specific sites and pores

for dihydroquercetin molecules. In the invention, Fe304 nanoparticle were wrapped by

the MIPs lay, and Fe304@SiO2@MIPs was successfully prepared. The magnetic

properties of Fe304@SiO2@MIPs were characterized by a particle size of approximately

250nm and were characterized by a vibrating sample magnetometer (figure 6). The

hysteresis loops show that the magnetic saturation strength of the magnetic particles is

67.6emu/g, the residual magnetic field strength is 0.64emu/g, and the coercive force is e. Fe304@SiO2@MIPs exhibited good magnetic properties under an external magnetic field. The polymer dispersion and agglomeration characteristics enable rapid magnetic extraction and separation.

Adsorption performance study

The invention evaluates the static adsorption performance based on the adsorption

capacity of dihydroquercetin with different concentrations. As the concentration

increased, the adsorption of dihydroquercetin increased (Figure 7). When the

dihydroquercetin concentration reached 10mg/L, the binding capacity almost reached

saturation. Fe304@SiO2@MIPs showed a specific selectivity for dihydroquercetin

compared to Fe304@SiO2@NIPs. The static adsorption process was studied using

different models including Scatchard, Langmuir and Freundlich models. The correlation

coefficient (R) of the Langmuir isothermal model was higher than that of other models

(Table 1). The adsorption process was in good agreement with the Langmuir monolayer

adsorption. The kinetic curve is shown in the figure. With the increase of time,

dihydroquercetin was more and more adsorbed on Fe304@SiO2@MIPs. Compared with

Fe304@SiO2@NIPs, Fe304@SiO2@MIPs had a significant adsorption effect on

dihydroquercetin. The adsorption process maintained equilibrium within 16 hours.

According to the pseudo-second-order kinetic model based on the data in Table 1, there

was a good linear relationship between T/Q1 and T, and R2 was 0.9865, proving that the

adsorption kinetics conformed to the pseudo-second-order model.

Table 1

Compound Model analysis Calibration R equation . Scatchard Y=1.0021X+7.6606 0.2463 Dihydroquercetin Langmuir Y=0.3565X+1.0145 0.9440

Freundlich Y=0.2332X+1.3466 0.6631 Pseudo-second-order Y=0.3456X+9.4000 0.9865 Magnetic SPE program research

The loading solutions were studied with different solvents to obtain maximum adsorption

of the target molecules. Considering that the optimal solution can enhance the template

binding site complex, the recognition ability was affected by the loading scheme; In

addition, template leakage usually occurs before saturated adsorption. In the present

invention, 5mL of methanol resulted in a maximum adsorption of 77.72±3.56 mg/g

during loading (Figure 9a). Therefore, methanol was selected as the optimal loading

solvent for Fe304@SiO2 on MIPs. The washing step may weaken or even destroy the

interaction between the impurity molecules and the polymer. The washing process with

different solvents was studied and the best solution was selected. In the present invention,

mL of n-hexane can remove a large amount of impurities and also some target

molecules (fig.9b). Examining the elution solvent by using different ratios of toluene and

methanol may disrupt the binding interaction between the target molecule and the

polymer. Through the elution process, 3mL of a mixture of toluene and methanol(2:3,

v/v) eluted the maximum dihydroquercetin with a recovery of 68.56%1.74%(Figure 9c).

Therefore, n-hexane was selected as the wash solvent with toluene and methanol (2:3,

v/v) as the eluents.

Method validation

Calibration curves were good in the range of 1-1000 g/g for the dihydroquercetin of

Larix Griffithii according to the IUPAC recommendations and previously reported

method validation methods. The correlation coefficient is 0.9999 for the standard solution

determined by using a magnetic SPE protocol. The detection and quantitation limits were

1.23-4.0 and 2.50[tg/g respectively. To evaluate the precision of the magnetic solid phase

extraction protocol, different concentrations of dihydroquercetin (50 and 100[g/g) were

analyzed and relative standard deviations of 2.16% and 3.45% were obtained

respectively. In addition, inter-day precision was evaluated by five dihydroquercetin

concentration determinations at the same level, which calculated RSD to be 3.72% to

2.04%.

Application in actual samples

Approximately 0.lg of dried Larix Griffithii leaves were extracted and analyzed by

magnetic molecular imprinted solid phase conjugates using HPLC (Figure 10). A

chromatogram of a 0.2g/ml dihydroquercetin standard is shown in Figure.10a. The

crude sample is shown in Fig.10b. After loading, the dihydroquercetin molecules were

fully bound to Fe304@SiO2@MIPs (Figure 10c). As shown in Fig.10d, that purity can be

washed. After elution, the dihydroquercetin was completely eluted (Figure 10f). In this

protocol, the accuracy of the method was evaluated by adding dihydroquercetin at four

concentrations (0.1, 1, 5, and 10 g/g). The results are shown in Table 2. The recovery is

74.64%~101.80%, and RSD is 2.00%~3.07%. The successful application of Fe304@SiO2

in the extraction and determination of the active compounds in Larix Griffithii has proved

the feasibility of the magnetic molecularly imprinted polymer.

Table 2

Compound Actual Actual Average Addition Measured Recovery Average RSD sample (g) content content (Vg • g- 1) content rate(%) recovery (%) (pg • g- 1 (pg • g- ) (pg • g-1) (%) 1) Dihydroquercetin 0.10 5.81 5.54 0.1 4.02 71.28 74.64 3.07 4.25 75.35 4.36 77.30 5.62 1 6.22 95.11 93.37 2.37 5.93 90.67 6.17 94.34 5.33 5 10.02 95.07 97.47 2.57

10.24 97.15 10.56 100.19 5.40 10 16.12 103.73 101.80 2.00 15.50 99.74 15.84 101.93 The invention characterized the amino functionalized core-shell magnetic nanoparticles

by using scanning electron microscope, transmission electron microscope, vibrating

sample magnetometer and infrared spectrometer. The average particle size of the polymer

was 250+2.56nm. Fe304@SiO2@MIPs showed good stability and adsorption capacity for

the template molecules, and they were enriched through hydrogen bond interaction. A

comprehensive evaluation of the extraction and enrichment ability of dihydroquercetin

was conducted using the developed material as the adsorbent for solid phase extraction.

The extraction conditions such as drug loading procedure, washing, elution solvent, and

extraction time were optimized. Dihydroquercetin in Larix Griffithii was determined by

high performance liquid chromatography (HPLC) with the developed

Fe304@SiO2@MIPssolid phase extraction (MISPE) technology. The detection limit of

the method was 1.23-4.0jg/g. The precision of the method was determined by using

three concentration levels of dihydroquercetin in the actual samples. The results showed

that the recovery rate of the samples by this method could reach 74.64%-101.80%, and

the relative standard deviation was less than 2.04%, indicating that the extraction

efficiency was high. Therefore, trace dihydroquercetin in Larix Griffithii can be

selectively extracted and analyzed for effective separation, purification and determination

of dihydroquercetin in other complex Chinese medicinal samples.

In conclusion, that present invention synthesized a magnetic molecularly imprinted

polymer material by precipitation polymerization, which can be use for extraction and

selective recognition of dihydroquercetin. Under the dynamic conditions and high repeatability of the materials, the magnetic molecularly imprinted polymers maintained good morphology, good selective recognition and physical and chemical stability, and did not affect the adsorption efficiency of the polymers. The synthesis of magnetic surface molecularly imprinted material is a simple and effective method to improve the binding performance. The excellent performance of separation and detection of dihydroquercetin in Larix Griffithii was studied. Dihydroquercetin magnetic molecularly imprinted polymer was used as the adsorbent for solid phase extraction, and the solid phase extraction method was suitable for practical application. The method has been successfully applied to the detection of samples of Larix Griffithii. By this method, it was found that the content of dihydroquercetin in Larix Griffithii was higher. The established magnetic molecular imprinted solid-phase extraction and HPLC methods can be used for quick extraction, and to meet the requirements of separation and detection of the active ingredient of other natural medicinal plants at the same time.

The above-mentioned embodiments are only for describing the preferred embodiments of

the present invention and are not intended to limit the scope of the present invention. On

the premise of not departing from the design spirit of the present invention, various

modifications and improvements made to the technical scheme of the present invention

by those of ordinary skill in the art should fall within the protection scope determined by

the claims of the present invention.

Claims (10)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. The preparation method of the magnetic molecular imprinted polymer of the larch

flavonoid active ingredient is characterized by comprising the following steps of:

Mix template raw materials with an organic solvent and a functional monomer, adding

crosslinking agent and initiator, introducing nitrogen to obtain a polymer, magnetically

separating and drying under vacuum to obtain a reaction material, eluting by using a

Soxhlet extraction method to remove that template, removing residual organic

substances, and drying;

The functional monomer is 4-vinyl pyridine;

The template raw material is flavonoid active ingredient of Larix Griffithii.

2. The preparation method of the larch flavonoid active ingredient magnetic molecular

imprinted polymer according to claim 1 is characterized in that the template raw material

is dihydroquercetin.

3. The preparation method of the larch flavonoid active ingredient magnetic molecular

imprinted polymer according to claim 1 is characterized in that the crosslinking agent is

ethylene glycol dimethacrylate.

4. The preparation method of the larch flavonoid active ingredient magnetic molecular

imprinted polymer according to claim 1 is characterized in that the initiator is

azobisisobutyronitrile.

5. The preparation method of the larch flavonoid active ingredient magnetic molecular

imprinted polymer according to claim 1 is characterized in that the molar ratio of the

template raw material, the functional monomer, the crosslinking agent is 1: (2-6): (20

).

6. The preparation method of the larch flavonoid active ingredient magnetic molecular

imprinted polymer according to claim 5 is characterized in that the molar ratio of the

template raw material, the functional monomer, the crosslinking agent is 1: 4: 20, 1: 6:

,1:2:25,1:4:25,1:6:25,1:2:30or1:4:30.

7. The invention relates to a magnetic molecular imprinted polymer, which is

characterized in that the magnetic molecular imprinted polymer is prepared by a

preparation method of a larch flavonoid active ingredient according to any one of claims

1 to 6 and its composition is Fe304@SiO2@MIPs.

8. A method for extracting dihydroquercetin according to claim 7, characterized in that it

is extracted through a solid phase extraction method.

9. The method for extracting dihydroquercetin according to claim 8, wherein

Fe304@SiO2@MIPsis used as adsorbent in the solid phase extraction method.

10. The method for extracting dihydroquercetin according to claim 8, wherein during

elution through the solid phase extraction method, a mixture of toluene and methanol is

used as elution solution, and the volume ratio of the toluene to the methanol is 1-2:3-4.