Preparation method of natural eutectic solvent modified attapulgite and application of natural eutectic solvent modified attapulgite in specific recognition and separation of quercetin

Abstract

The application belongs to the technical field of material preparation and natural medicine separation, and relates to a preparation method of natural eutectic solvent modified attapulgite, mainly including acidification of attapulgite, amino modification of attapulgite, preparation of natural eutectic solvent, and preparation of natural eutectic solvent / attapulgite composite nanomaterial. The application selects NADES containing organic acid, modifies natural clay ATP through amidation reaction, increases surface functional groups of the natural clay adsorbent, and further improves adsorption selectivity. The prepared composite material is low in cost, good in biocompatibility, and can be recycled for multiple times, and meets the green development concept. The application covalently modifies the natural eutectic solvent containing organic acid on the surface of attapulgite through amidation reaction, solves the problems of single attapulgite functional group and poor adsorption selectivity, and can be effectively applied to the sample pretreatment process of quercetin in complex components.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of material preparation and natural medicine separation, and particularly relates to a preparation method of natural eutectic solvent modified attapulgite and application of the attapulgite in specific recognition and separation of quercetin. BACKGROUND

[0002] Quercetin (QC) is a kind of polyphenolic flavonoids, which is widely present in fruits, vegetables, tea and other plants. It is concerned due to its high biological activity. It includes very strong antioxidant, anticancer, anti-allergic, anti-inflammatory, antiviral and heart protection activity, and is widely used in medical and pharmaceutical fields. However, due to its low solubility and weak stability, it is very difficult to extract and separate from plant extracts and to detect. At present, the relatively mature methods for separating and enriching QC include macroporous adsorption resin, organic solvent extraction and reverse phase silica gel column chromatography. These methods are complicated in process, and a large amount of organic solvent is consumed, which not only inhibits the biological activity, but also does not meet the green development concept. More importantly, the separation process is easily disturbed by other flavonoids with similar molecular structures, greatly reducing the selectivity. Therefore, it is necessary to establish an efficient, environmentally friendly and highly selective separation method.

[0003] Solid phase extraction (SPE) as a common sample pretreatment technology is to extract and enrich target compounds by using the interaction between solid adsorbent and target substances. On this basis, dispersive solid phase extraction (DSPE) directly disperses the adsorbent in the sample solution, so that the adsorbent and the target substances are in more sufficient contact, reducing unnecessary consumption of organic solvents, and being more environmentally friendly and efficient than traditional SPE technology. The performance of the adsorbent directly restricts the extraction efficiency of the technology, so the development of new adsorbents with high efficiency and stability has become a research hotspot in the field of DSPE technology.

[0004] Attapulgite (ATP) is a kind of hydrous magnesium silicate clay mineral with layer chain structure. Influenced by many factors such as crystallization characteristics, crystallization morphology, deposition method and internal pore, ATP has a very high specific surface area, and the unbalanced charge on its surface makes ATP a natural inorganic adsorbent with good mechanical properties, low price and excellent performance. However, due to the single type of functional groups of ATP, it is difficult for ATP to play an advantage in selective separation of natural medicines with complex composition.

[0005] Nature deep eutectic solvents (NADES) is a new green solvent, which is composed of natural components such as sugars, organic acids, organic bases and the like existing in various cells or tissue organs. These components have better health safety and lower environmental hazard compared with traditional organic solvents. Hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) are combined by hydrogen bonds to produce eutectic mixtures that are stable at low temperatures. In addition, NADES also has some ideal characteristics similar to ionic liquids, such as very low volatility and high solubility. The strong designability of NADES enables it to change its composition for different target objects, thereby changing the physicochemical properties such as functional group type, viscosity and polarity. NADES has been used as an excellent solvent for liquid-liquid extraction of natural pharmaceutical ingredients. Therefore, the present application modifies ATP by NADES, which can retain the high adsorption performance of ATP while significantly improving the specific recognition effect of QC. SUMMARY

[0006] The first object of the present application is to provide a kind of NADES covalently modified ATP-based adsorbent material (ATP-NADES) with HBD as organic acid and a preparation method thereof, to solve the problem of single functional group and poor selectivity when separating and identifying target object quercetin from natural mineral attapulgite (ATP).

[0007] To achieve the above-mentioned object of the application, the specific technical scheme is as follows:

[0008] A preparation method of attapulgite modified by natural deep eutectic solvent, comprising the following steps:

[0009] S1: Preparation of ATP: add attapulgite (ATP) raw soil to hydrochloric acid solution and stir thoroughly, wash with deionized water until no Cl is detected in the supernatant of AgNO3 solution - , centrifugal separation, take the ATP with lighter color in the upper layer after centrifugation, dry to constant weight, crush and sieve to obtain pretreated ATP;

[0010] S2: Preparation of ATP-NH2: take the pretreated ATP and place it in toluene for ultrasonic treatment for 10 min, add 3-aminopropyltriethoxysilane (APTES) dropwise under stirring, reflux at 40-50℃ under N2 protection for 2 h, wash with toluene and anhydrous ethanol to remove impurities, vacuum dry the obtained ATP-NH2 to constant weight, crush and sieve;

[0011] S3: Preparation of three kinds of NADES with HBD as organic acid: Choline chloride and levulinic acid (ChCl / Lev), glycerol and L-proline (Gly / Pro), glucose and lactic acid (Glu / Lac) were mixed in proportion as HBA and HBD, respectively, heated to a transparent and uniform liquid by stirring, and placed at room temperature until no crystals were precipitated, to prepare three kinds of natural deep eutectic solvents (NADES);

[0012] S4: Preparation of three kinds of ATP-NADES: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in a morpholine ethanesulfonic acid buffer solution, the pH was adjusted to 5-6, the natural deep eutectic solvents (NADES) prepared in S3 were added, stirred uniformly, a phosphate buffer solution was added, the pH was adjusted to 7-8, ATP-NH2 prepared in S2 was added, stirred for 12 h, unreacted substances were removed by deionized water washing, and freeze-drying was performed to obtain the product.

[0013] In the preferred disclosure of the present application, the concentration of the hydrochloric acid solution in S1 is 12-15 wt%.

[0014] In the preferred disclosure of the present application, the solid-liquid ratio of the raw attapulgite and the hydrochloric acid solution in S1 is 1 g: 9-10 mL.

[0015] In the preferred disclosure of the present application, the solid-liquid ratio of ATP and APTES in S2 is 1 g: 1-1.2 mL.

[0016] In the preferred disclosure of the present application, the molar ratio of HBA and HBD in S3 is 1:2-1:5.

[0017] In the preferred disclosure of the present application, the heating temperature in S3 is 50-80°C, preferably 50-60°C, and the heating temperature should not be too high to avoid the generation of ester substances from part of the alcohol-containing NADES.

[0018] In the preferred disclosure of the present application, the concentration of the morpholine ethanesulfonic acid buffer solution and the phosphate buffer solution in S4 is 0.1-0.2 M.

[0019] In the preferred disclosure of the present application, the molar ratio of NHS, EDC and HBD in NADES in S4 is 6 mmol: 30 mmol: 10-60 mmol, and the molar ratio is preferably 6 mmol: 30 mmol: 20 mmol.

[0020] In the preferred disclosure of the present application, the solid-liquid ratio of ATP-NH2 and HBD in NADES in S4 is 375 mg: 20 mmol.

[0021] The natural low eutectic solvent modified attapulgite (ATP-NADES) prepared by the method has a rod needle-like fiber bundle diameter of ATP obviously increased, and presents more aggregate and cluster morphologies.

[0022] The second object of the application is to disclose the application of the prepared natural low eutectic solvent modified attapulgite (ATP-NADES) in specific recognition and separation of quercetin.

[0023] The NADES type specific to quercetin is screened according to the homoeopathic dihydroxy, carbonyl, benzene ring structure and surface charge contained in the target quercetin.

[0024] A separation method of quercetin in a solution, comprising the following steps:

[0025] The prepared quercetin sample solution and the ATP-NADES prepared by the application are mixed for adsorption, and three specific adsorption materials adsorbing quercetin are obtained by centrifugation; the content of residual quercetin in the supernatant is tested; the three specific adsorption materials are the ATP-NADES in the above technical solution or the ATP-NADES prepared by the preparation method in the above technical solution; the three ATP-NADES adsorbents adsorbing quercetin are eluted, and the recyclable three ATP-NADES adsorbents are obtained.

[0026] Preferably, the quercetin sample is prepared by using a solution of methanol: phosphate buffer solution (0.2M) at a ratio of 1:4 (v / v) as a solvent.

[0027] Preferably, the elution solvent for elution is a mixed solvent of methanol and acetic acid, and the ratio of methanol to acetic acid is 1:1 (v / v).

[0028] A method for selectively separating quercetin and its analogues, comprising the following steps:

[0029] A sample solution of quercetin and its analogues with consistent concentration is prepared, and mixed with three specific adsorption materials for adsorption, and then three specific adsorption materials adsorbing quercetin or its analogues are obtained by centrifugation; the content of residual quercetin or its analogues in the supernatant is tested; the three specific adsorption materials are the ATP-NADES in the above technical solution or the ATP-NADES prepared by the preparation method in the above technical solution.

[0030] Preferably, the quercetin analogue is Rutin hydrate or Luteolin.

[0031] The ATP-NADES prepared by the application forms a large number of amide groups on the basis of ATP-NH2 after NADES modification, and builds a basic framework for specifically adsorbing quercetin. Different components of NADES form various active groups on the surface of ATP, such as carbonyl, hydroxyl, benzene ring, secondary amine group and quaternary ammonium group. These groups can participate in the completion of hydrogen bond, π-π stacking, electrostatic interaction and other non-covalent interactions for the cis-dihydroxyl, carbonyl, benzene ring and surface charge contained in quercetin, so as to further improve the selectivity of the adsorbent when adsorbing quercetin and its analogues.

[0032] Advantages

[0033] The application selects NADES containing organic acid, modifies natural clay ATP through amidation reaction, increases the surface functional groups of the natural clay adsorbent, and further improves the adsorption selectivity. The ATP-NADES adsorbent prepared by natural clay and NADES composed of natural components not only has good quercetin adsorption effect, but also has excellent biocompatibility, low cost and simple preparation process, meets the green development concept, and can be effectively applied to the sample pretreatment process of active ingredient quercetin. Different compositions of NADES are used to further explore the interaction between the adsorbent and the target, and find the NADES that specifically binds to quercetin. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 .ATP and infrared spectra of three kinds of ATP-NADES.

[0035] Figure 2 .ATP and scanning electron micrographs of three kinds of ATP-NADES.

[0036] Figure 3 .ATP and XRD graphs of three kinds of ATP-NADES.

[0037] Figure 4 . Adsorption isotherm graph of three kinds of ATP-NADES.

[0038] Figure 5 .ATP-NH2 and three kinds of ATP-NADES adsorption capacity comparison graph of quercetin and its analogues. DETAILED DESCRIPTION

[0039] The application will be described in detail below in conjunction with examples, so that those skilled in the art can better understand the application, but the application is not limited to the following examples.

[0040] Example 1

[0041] S1: Preparation of ATP

[0042] The raw soil was added to 15wt% hydrochloric acid solution at a solid-liquid ratio of 1:10 (g / mL), stirred for 3 h, and then washed with deionized water until no Cl was detected in the supernatant using AgNO3 solution - , centrifuged, and the upper layer of ATP after centrifugation was dried to constant weight. The dried sample was crushed through a 200-mesh sieve.

[0043] S2: Preparation of ATP-NH2

[0044] 6 g of ATP in S1 was placed in 100 mL of toluene and ultrasonicated for 10 min. 6 mL of 3-aminopropyltriethoxysilane (APTES) was added dropwise to the above mixture under stirring, and then refluxed at 45°C for 2 h under N2 protection. The unreacted substances were removed by washing with toluene and anhydrous ethanol three times, respectively. The product ATP-NH2 was vacuum dried to constant weight, and the dried sample was crushed through a 200-mesh sieve.

[0045] S3: Preparation of NADES with components ChCl / Lev

[0046] Choline chloride (ChCl, 10 mmol) and levulinic acid (Lev, 20 mmol) were mixed in a ratio of 1:2, and stirred and heated at 60°C until a transparent and uniform liquid was obtained. No crystals were precipitated at room temperature, and thus [ChCl][Lev] was prepared.

[0047] S4: Preparation of ATP-[ChCl][Lev]

[0048] 30 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6 mmol of N-hydroxysuccinimide (NHS) were dissolved in a morpholine ethanesulfonic acid buffer solution (0.1 M), and the pH was adjusted to 6. [ChCl][Lev] in S3 was added, and stirred at room temperature for 30 min. Phosphate buffer solution (0.2 M) was added, and the pH was adjusted to 8. 375 mg of ATP-NH2 in S2 was added, and stirred at room temperature for 12 h. The unreacted substances were removed by washing with deionized water three times, and collected after freeze-drying.

[0049] The samples were characterized by scanning electron microscopy, infrared spectroscopy, and XRD. The results are shown in the graphs or curves labeled as b) in FIGS. Figure 1 、 2 and 3.

[0050] Figure 1 In FIG. 3, b) retained the significant 1033 cm -1 (V Si-O-Si ) peak of ATP, and produced new peaks at 3253 cm -1 (V N-H ), 2973 cm -1 (VC-H ), 2924 cm -1 (V C-H ), 1654 cm -1 (V C＝O ) and 1533 cm -1 (δ C-H ), indicating that [ChCl][Lev] was successfully modified on the surface of ATP by covalent reaction.

[0051] Figure 2 In the middle, the ATP rod needle-like significantly thickened after surface modification of NADES.

[0052] Example 2

[0053] S1 and S2 steps are the same as example 1;

[0054] Preparation of NADES with Gly / Pro as components in S3;

[0055] According to the ratio of 1:2.5, glycerol (Gly, 8 mmol) and proline (Pro, 20 mmol) were mixed uniformly, heated to a transparent uniform liquid at 60°C, and placed at room temperature without crystal precipitation, that is, [Gly][Pro] can be prepared.

[0056] S4: Preparation of ATP-[Gly][Pro]

[0057] 30 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6 mmol N-hydroxysuccinimide (NHS) were dissolved in morpholine ethanesulfonic acid buffer solution (0.1 M), the pH was adjusted to 6, [Gly][Pro] in S3 was added, stirred at room temperature for 30 min, then phosphate buffer solution (0.2 M) was added, the pH was adjusted to 8, 375 mg ATP-NH2 in S2 was added, stirred at room temperature for 12 h, washed with deionized water 3 times to remove unreacted substances, and then collected after freeze-drying.

[0058] The samples were characterized by scanning electron microscopy, infrared and XRD. The results are shown in Figure 1 , 2 and 3, the figure or curve marked as c) or the results of characterization are basically similar to those of example 1, indicating the successful preparation of ATP-[Gly][Pro].

[0059] Example 3

[0060] S1 and S2 steps are the same as example 1,

[0061] Preparation of NADES with Glu / Lac as components in S3

[0062] According to the ratio of 1:5, glucose (Glu, 4 mmol) and lactic acid (Lac, 20 mmol) were mixed uniformly, heated to a transparent uniform liquid at 60°C, and placed at room temperature without crystal precipitation, to prepare [Glu][Lac].

[0063] S4: Preparation of ATP-[Glu][Lac]

[0064] 30 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6 mmol of N-hydroxysuccinimide (NHS) were dissolved in a morpholine ethanesulfonic acid buffer solution (0.1 M), and the pH was adjusted to 6. [Glu][Lac] in S3 was added, stirred at room temperature for 30 min, then phosphate buffer solution (0.2 M) was added, the pH was adjusted to 8, 375 mg of ATP-NH2 in S2 was added, stirred at room temperature for 12 h, washed with deionized water 3 times to remove unreacted substances, and then collected after freeze-drying.

[0065] The sample was characterized by scanning electron microscopy, infrared, and XRD, and the results are shown in the figures or curves labeled d) in Figs. Figure 1 , 2 and 3, which are basically similar to those of Example 1, indicating the successful preparation of ATP-[Glu][Lac].

[0066] Application Example 1

[0067] The ATP-[ChCl][Lev] material was used for adsorbing quercetin in a solution, which specifically included the following steps:

[0068] I. Sample preparation: The quercetin standard was prepared into adsorbate solutions with a mass concentration of 0.004, 0.006, 0.008, 0.01, 0.02, 0.04, and 0.06 mg / mL using methanol: phosphate buffer solution (1:4 v / v) as the solvent.

[0069] II. Adsorption: 8 mL of each concentration of the adsorbate solution prepared in step I above was taken, 2 mg of ATP-[ChCl][Lev] was added, and adsorption was carried out in a water bath shaker at 25°C for 120 min. After adsorption, centrifugal separation was performed, the supernatant was measured for the concentration of residual quercetin using a UV-visible spectrophotometer, and the adsorption capacity of ATP-[ChCl][Lev] was calculated. The test results are shown in the a) curve in Fig. Figure 4 , and the adsorption capacity Q was 30.46 mg / mL.

[0070] The adsorption rate Q (mg / g) was defined as:

[0071]

[0072] Where C0 (mg / mL) represents the concentration of the target analyte before adsorption, C e (mg / mL) represents the concentration of the target analyte after adsorption, V(mL) represents the volume of the adsorbate solution added, and m(g) represents the mass of the adsorbent used.

[0073] Application Example 2

[0074] The ATP-[Gly][Pro] material was used to adsorb quercetin from the solution. The specific implementation process was the same as in Application Example 1, and the test results are as follows. Figure 4 As shown in curve b), the adsorption capacity Q is 26.61 mg / mL.

[0075] Application Example 3

[0076] The ATP-[Glu][Lac] material was used to adsorb quercetin from the solution. The specific implementation process was the same as in Application Example 1, and the test results are as follows. Figure 4 Curve c) shows an adsorption capacity Q of 25.52 mg / mL.

[0077] Application Example 4

[0078] Three ATP-NADES materials were used for the selective adsorption of quercetin and its analogues rutin and luteolin in solution, specifically including the following steps:

[0079] I. Sample preparation: Quercetin, rutin, and luteolin standards were prepared into adsorbate solutions with a mass concentration of 0.02 mg / mL using methanol:phosphate buffer solution (1:4 v / v) as the solvent.

[0080] II. Adsorption: Take 8 mL of the adsorbate solutions of different target compounds prepared in step I above, and add 5 mg of ATP-[ChCl][Lev] to each. Then, repeat the above steps with ATP-[Gly][Pro] and ATP-[Glu][Lac], and adsorb on a water bath shaker at 25℃ for 120 min. After adsorption, centrifuge and take the supernatant to measure the concentration of the remaining target compounds using a UV-Vis spectrophotometer, and calculate the adsorption capacity of ATP-[ChCl][Lev], ATP-[Gly][Pro], and ATP-[Glu][Lac] for different target compounds.

[0081] Test results are as follows Figure 5 As shown, compared with ATP-NH2, all three ATP-NADES exhibited excellent selectivity for quercetin.

[0082] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing attapulgite modified with a natural eutectic solvent for quercetin-specific recognition and separation, characterized in that, Includes the following steps: S1: Add the ATP from the original attapulgite soil to a 12-15 wt% hydrochloric acid solution and stir thoroughly. The solid-liquid ratio of the original attapulgite soil to the hydrochloric acid solution is 1 g: 9-10 mL. Wash with deionized water until Cl in the supernatant is undetectable by AgNO3 solution. - Centrifuge the ATP, take the lighter-colored upper layer after centrifugation, dry it to constant weight, crush and sieve it to obtain pretreated ATP; S2: Take the pretreated ATP and place it in toluene and sonicate for 10 min. Add 3-aminopropyltriethoxysilane APTES dropwise while stirring. The solid-liquid ratio of pretreated ATP to APTES is 1 g: 1~1.2 mL. Reflux at 40~50℃ for 2 h under N2 protection. Wash with toluene and anhydrous ethanol to remove impurities. Dry the obtained ATP-NH2 under vacuum to constant weight, pulverize and sieve. S3: Choline chloride and levulinic acid, glycerol and L-proline, glucose and lactate as hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) were mixed evenly in a molar ratio of 1:2 to 1:5, stirred and heated at 50 to 80°C until a transparent and homogeneous liquid was obtained, and left at room temperature until no crystals precipitated, thus obtaining three natural eutectic solvents, NADES. S4: Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in morpholine ethanesulfonic acid buffer solution, adjust the pH to 5-6, and add the three natural eutectic solvents NADES prepared in S3, wherein the molar ratio of NHS, EDC, and HBD in NADES is 6 mmol:30 mmol:10-60 mmol. Stir thoroughly, then add phosphate buffer solution, adjust the pH to 7-8, and add ATP-NH2 prepared in S2, wherein the solid-liquid ratio of ATP-NH2 to HBD in NADES is 375 mg:20 mmol. Stir for 12 h, wash with deionized water to remove unreacted substances, and freeze-dry to obtain natural eutectic solvent modified attapulgite. The concentrations of morpholine ethanesulfonic acid buffer solution and phosphate buffer solution are both 0.1-0.2 M.

2. The method for preparing natural eutectic solvent-modified attapulgite for quercetin-specific recognition and separation according to claim 1, characterized in that: In S3, the heating temperature is 50~60℃.

3. The method for preparing natural eutectic solvent-modified attapulgite for quercetin-specific recognition and separation according to claim 1, characterized in that: In S4, the molar ratio of HBD in the NHS, EDC, and NADES is 6 mmol: 30 mmol: 20 mmol.

4. Natural eutectic solvent-modified attapulgite prepared by any one of the methods described in claims 1-3.

5. An application of attapulgite modified with a natural eutectic solvent as described in claim 4, characterized in that: It was applied to the specific recognition and separation of quercetin.

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