Application of metal-organic framework materials in active ingredients of traditional Chinese medicine for selective fishing

Abstract

This application belongs to the technical field of new methods for extracting traditional Chinese medicine, and specifically relates to the application of metal organic framework materials in selective fishing of traditional Chinese medicine active ingredients. The metal organic framework material is zirconium-based MOFs; the specific surface area of ​​the zirconium-based MOFs is 800-950 m 2 / g: pore size range is 2-5 nm; the active ingredients include isoquinoline alkaloids. The metal organic framework material is directly prepared by a simple solvothermal method, the steps are simple, no other catalysts need to be added, and the prepared metal organic framework material has a large specific surface area and rich porosity, and can be directly used as a carrier material for selective fishing of active ingredients of traditional Chinese medicine components, so as to achieve efficient enrichment of trace active ingredients in complex matrices of traditional Chinese medicine.

Description

Technical Field

[0001] The present application belongs to the technical field of new methods for extracting traditional Chinese medicine, and specifically relates to the application of metal-organic framework materials in selectively fishing active ingredients of traditional Chinese medicine. Background Art

[0002] As the core material basis of the efficacy of traditional Chinese medicine, the active ingredients of traditional Chinese medicine are the key to the therapeutic effect of traditional Chinese medicine. These ingredients can regulate human physiological functions, enhance immunity, and improve pathological conditions through multi-pathway and multi-target mechanisms, thereby achieving the purpose of treating or assisting the treatment of diseases. However, the matrix of traditional Chinese medicine is very complex and the content of most active ingredients is very low, which has brought a lot of resistance to the screening of active ingredients of traditional Chinese medicine. The traditional screening method of active ingredients of traditional Chinese medicine requires continuous separation and purification, which leads to the problems of long time, large workload and low efficiency in the whole process. Among them, alkaloid active ingredients show significant biological activity in anti-tumor, antibacterial and anti-inflammatory, antiviral and regulating metabolism, and have great application potential in health and medicine. At present, the extraction methods of alkaloid active ingredients mainly include dilute acid extraction, alcohol solvent extraction and lipophilic organic solvent extraction, but the matrix is ​​complex and it is difficult to achieve targeted enrichment. In addition, the extraction efficiency is low, the amount of various solvents used is large, and it usually requires repeated extraction, which takes a long time. Therefore, the development of an efficient and simple method is of great significance for the research and application of active ingredients of traditional Chinese medicine.

[0003] In the exploration of efficient and rapid extraction strategies for active ingredients in traditional Chinese medicine, bioaffinity chromatography, ultrafiltration and ligand fishing, which integrate cutting-edge nanotechnology, are gradually becoming the focus of scientific research. Metal-organic frameworks (MOFs), with their outstanding structural characteristics, rich active sites, highly uniform pore structure, huge specific surface area and flexible and ordered framework structure, have shown extraordinary application potential in many key fields such as medicine, energy, environmental protection and food. In particular, the composite carrier system constructed based on MOFs, through the use of ligand fishing strategy, can not only achieve efficient capture and loading of target active ingredients in traditional Chinese medicine, but also give these ingredients the ability of controlled release and precise targeted delivery, opening up a new path for the modernization of traditional Chinese medicine and the development of precision medicine.

[0004] In our previously disclosed patents (CN117567533A, CN118162118A), organic acids in honeysuckle and mono / diester alkaloids in aconite were used as target components, and metal organic frameworks and MOFs-based core-shell materials were prepared for targeted extraction and separation. Compared with traditional analytical methods, the extraction and separation efficiency was significantly improved. However, it is difficult to achieve the selective enrichment and fishing of isoquinoline alkaloid active ingredients in Coptis chinensis, especially for berberine, the main active ingredient in Coptis chinensis, because the recovery rate is low and it is difficult to achieve the ideal effect. This is mainly because berberine is a quaternary ammonium alkaloid with strong polarity. It is often dissolved in highly polar solvents such as water or acid water. However, the stability and acid-base stability of the above materials in highly polar solvents need to be further improved. Therefore, it is an urgent problem to achieve the selective fishing of isoquinoline alkaloid active ingredients in Coptis chinensis efficiently and quickly. Summary of the invention

[0005] In view of the problems of long time consumption, heavy workload and low efficiency in the extraction of trace active ingredients of traditional Chinese medicine, especially isoquinoline alkaloid active ingredients, the present application provides a metal organic framework material with strong water stability and acid-base stability, such as Figure 1 As shown, its structural advantages and interactions are utilized as a carrier to selectively extract the isoquinoline alkaloid active ingredients in Coptis chinensis. This method is efficient, rapid, has a high recovery rate, and strong stability, providing technical support for the extraction of trace active ingredients in traditional Chinese medicine.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] Application of metal organic framework materials in selective fishing of traditional Chinese medicine active ingredients, wherein the metal organic framework materials are zirconium-based MOFs; the specific surface area of ​​the zirconium-based MOFs is: 800-950 m 2 / g, and the pore size range is 2-5 nm.

[0008] The active ingredients include isoquinoline alkaloids.

[0009] Preferably, the raw materials used for the zirconium-based MOFs include: zirconium oxychloride, trimesic acid (1,3,5-BTC); and also include solvents, and the solvents include N,N-dimethylformamide (DMF), formic acid (FA), hydrochloric acid (HCl), and trifluoroacetic acid (TFA).

[0010] Preferably, the volume ratio of DMF to FA in the solvent is (2-3):2;

[0011] And / or, the mass ratio of zirconium oxychloride to 1,3,5-BTC is (13-13.5):5;

[0012] And / or, the volume ratio of HCl to TFA is 1:(2-3).

[0013] Preferably, the method for preparing zirconium-based MOFs comprises the following steps:

[0014] (1) Add zirconium oxychloride and trimesic acid into N,N-dimethylformamide and formic acid solvent and dissolve;

[0015] (2) adding hydrochloric acid and trifluoroacetic acid to the solution of step (1) to react; after the reaction is completed, collecting the solid product by centrifugation;

[0016] (3) The product obtained in step (2) is centrifuged and washed, then immersed and activated; centrifuged, and dried.

[0017] Preferably, the reaction conditions of step (2) are: reacting at 120-130° C. for 48-72 h;

[0018] And / or, the centrifugal washing solution in step (3) is: DMF and / or ethanol;

[0019] And / or, the soaking activation conditions of step (3) are: time 2 to 3 days, during which the DMF and / or ethanol solvent is replaced 3 to 5 times.

[0020] Preferably, the traditional Chinese medicine is Coptis chinensis.

[0021] Furthermore, the active ingredient is one or more of jatrorrhizine hydrochloride, epiberberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride and berberine hydrochloride.

[0022] Preferably, the method of selective fishing comprises the following steps:

[0023] S1 Preparation of crude extract of Coptis chinensis: Add acid-alcohol aqueous solution to the rhizome of Coptis chinensis for extraction;

[0024] S2 Selective fishing of alkaloid active ingredients: add the above-mentioned metal organic framework material to the crude extract of step S1, incubate, centrifuge, desorb and filter to obtain the active ingredients.

[0025] Preferably, the relationship between the amount of the dried rhizome of Coptis chinensis and the metal organic framework material added is: 10-30 mg of the metal organic framework material is added to the crude freeze-dried powder obtained from 5 g of the dried rhizome of Coptis chinensis;

[0026] And / or, the specific process of step S2 is: incubating on a shaker at room temperature for 1 to 2 hours, removing the supernatant by centrifugation, adding a formic acid-acetonitrile mixed solution to the precipitate for ultrasonic desorption, obtaining the desorbed liquid by centrifugation, and filtering it through a liquid phase filter for later use.

[0027] Preferably, the volume ratio of the formic acid to acetonitrile solution is 3%-5% formic acid.

[0028] The present invention directly prepares the metal organic framework material by a simple solvent thermal method. The preparation steps are simple, no other catalysts need to be added, and the prepared metal organic framework material has a large specific surface area ( Figure 2 Figure A) and rich pore structure ( Figure 2 (Figure B) can be directly used as a carrier material for the selective extraction of active ingredients from traditional Chinese medicine components, thereby achieving efficient enrichment of trace active ingredients in the complex matrix of traditional Chinese medicine.

[0029] like Figure 1 As shown, the metal organic framework material prepared by the present invention is used as a carrier, and its structural advantages and interactions are used to achieve targeted fishing and enrichment of active ingredients of traditional Chinese medicine. The crude extract of Coptis chinensis is incubated with the prepared metal organic framework material, and the incubation conditions are optimized to selectively fish for one or more active ingredients in Coptis chinensis, including jatrorrhizine hydrochloride, epiberberine hydrochloride, coptisine hydrochloride, bamipine hydrochloride and berberine hydrochloride. Zirconium-based MOFs can use their huge surface area, molecular sieve effect and interaction to achieve selective fishing of isoquinoline alkaloids in Coptis chinensis. In addition, by adjusting the volume ratio of hydrochloric acid to trifluoroacetic acid, optimizing its water stability and acid-base stability, it can be reused many times.

[0030] Beneficial Effects of the Invention

[0031] 1. The metal organic framework material of the present invention retains the excellent properties of the metal organic framework material such as huge specific surface area, rich porosity and adjustable pore size, and at the same time has satisfactory hydrophilicity and stability, so that it maintains structural stability during the incubation process in polar solvents.

[0032] 2. The preparation method of the present application is simple and efficient, and the preparation cost is relatively cheap, and the subsequent preparation process is simple and efficient.

[0033] 3. The efficiency of enrichment of alkaloid active ingredients is significantly improved. The zirconium-based MOFs used in the present invention have a specific surface area of ​​up to 900 m² / g, which can significantly improve the enrichment efficiency of active ingredients and has a high selective fishing efficiency for alkaloid active ingredients, with a recovery rate of more than 85%.

[0034] 4. It effectively improves the problem of difficult targeted enrichment of alkaloid active ingredients. On the one hand, the strong adsorption properties of the metal organic framework itself are utilized, and on the other hand, the Zr group clusters and van der Waals interactions of the zirconium-based MOFs with hydrophilicity and stability can achieve multiple selective fishing, thereby improving efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a fishing diagram;

[0036] Figure 2 N2 adsorption-desorption curve (A) and pore size distribution result (B) of MOF-808 prepared in Example 1;

[0037] Figure 3 The results of direct liquid phase analysis of the lyophilized crude extract of Coptis chinensis prepared in Example 1 (A) and the liquid phase analysis results after selective enrichment with MOF-808 (B). DETAILED DESCRIPTION

[0038] In order to better understand the content of the present invention, a method of selectively fishing for alkaloid active ingredients in Coptis chinensis based on metal-organic framework interactions is described through specific examples.

[0039] Instruments: CPA225D 1 / 100,000 electronic balance (Sartorius Scientific Instruments, Germany); F7JA36578 clear ultrapure water system (Merck Millipore Laboratory Equipment Co., Ltd.); vacuum drying oven, DZF-6020, Shanghai Yiheng Technology Co., Ltd., China; DD-5M medical centrifuge (Hunan Pingfan Technology Co., Ltd.); 90-1 constant temperature magnetic stirrer (Shanghai Huxi Analytical Instrument Factory); high performance liquid chromatograph, Agilent 1206 series, USA; low temperature circulation (vacuum) pump (DLSB-ZC) Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.; ultrasonic cleaner KQ-500DE Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.; hydrothermal reactor, Shanghai Shenfu Instrument Co., Ltd.

[0040] Reagent: Zirconium oxychloride (ZrOCl 2· 8H2O) and trimesic acid (1,3,5-BTC) were from Bailingwei Technology Co., Ltd.; N,N-dimethylformamide (DMF) was from Foshan Xilong Chemical Co., Ltd.; formic acid (FA) and trifluoroacetic acid (TFA) were from Maclean Technology Co., Ltd.; anhydrous ethanol, methanol, and hydrochloric acid were from Tianjin Damao Chemical Reagent Factory; berberine hydrochloride reference substance, coptisine hydrochloride reference substance, epiberberine hydrochloride reference substance, bamipine hydrochloride reference substance, and jatrorrhizine hydrochloride reference substance were all from Chengdu Pusi Biotechnology Co., Ltd.

[0041] Example 1

[0042] (1) Preparation of MOF-808 mixed solution 1: Accurately weigh 1.08 g ZrOCl 2·8H2O and 0.41 g of 1,3,5-BTC were dissolved in a mixture of DMF (75 mL) and FA (75 mL), and sonicated at room temperature for 5 min to form a uniform transparent solution. Subsequently, HCl (1 mL) and TFA (3 mL) were added and sonicated for 1 min to obtain a uniform transparent mixed solution 1.

[0043] (2) Add the mixed solution 1 into the reactor and react at 130 °C for 72 h. After cooling to room temperature, centrifuge at 8000 r / min. -1 The mixture was centrifuged, washed three times with DMF and ethanol respectively, and dried at 80 °C to obtain solid white powder MOF-808.

[0044] (3) Preparation of crude extract of Coptis chinensis: Weigh 10 g of dried rhizome of Coptis chinensis, crush it and grind it into fine powder through No. 5 or 6 sieves. Place it in a beaker and add it to 400 mL of 0.5% HCl + 80% ethanol aqueous solution for ultrasonic treatment at 40 °C for 30 min. Filter and retain the supernatant and precipitate respectively.

[0045] (4) The precipitate from step (3) was added to 400 mL of 0.5% HCl + 80% ethanol aqueous solution, and the extraction and filtration were repeated according to the conditions in step (3). The filtrates obtained twice were combined and concentrated to dryness on a 55°C rotary evaporator. The filtrates were freeze-dried and set aside for use. Accurately weigh 10 mg of the lyophilized powder and dissolve it in 100 mL of methanol solution to prepare 0.1 mg mL -1 Coptis chinensis extract.

[0046] (5) Preparation of reference solution: Weigh berberine hydrochloride reference, coptisine hydrochloride reference, epiberberine hydrochloride reference, palmatine hydrochloride reference and jatrorrhizine hydrochloride reference respectively and place them in volumetric flasks. Add methanol to make a reference solution containing 0.394 mg berberine hydrochloride, 0.412 mg coptisine hydrochloride, 0.422 mg epiberberine hydrochloride, 0.413 mg palmatine hydrochloride and 0.401 mg jatrorrhizine hydrochloride per 1 mL.

[0047] (6) Take 10 mg of the lyophilized crude extract of Coptis chinensis obtained in step (4) and dissolve it in 100 mL of methanol solution. Use the methanol solution as the solvent to prepare 0.05 mg / mL -1 , 0.1mg·mL -1 , 0.4mg·mL -1 The concentration of the standard mixed solution in step (5) using the crude extract as solvent.

[0048] (7) Selective enrichment of alkaloid active ingredients: Accurately weigh 80 mg of dried MOF-808 and add it to the crude extract of Coptis chinensis (0.1 mg / mL) obtained in step (2) and the mixed solution obtained in step (6). After ultrasonication at 40 °C for 10 min, incubate on a shaker at 37 °C for 15 min, and centrifuge at 2000 rpm for 5 min to obtain a precipitate. Add 3% formic acid acetonitrile solution to the precipitate and ultrasonicate at 40 °C for 50 min. Repeat the centrifugation step and take the supernatant and filter it through a liquid filter membrane for later use.

[0049] (8) Chromatographic conditions: Stationary phase: C18 column; Mobile phase: acetonitrile -0.05 mol·L -1 Potassium dihydrogen phosphate solution (50:50) (add 0.015 mol·L -1 Sodium dodecyl sulfate, and then adjusted to pH 4.0 with phosphoric acid); flow rate: 0.6 mL min -1 ; Column temperature: 30 ℃; Detection wavelength: 345 nm; Injection volume: 10 μL. Figure 3 As shown in Figure B, the metal organic framework material has a high selective enrichment efficiency for jatrorrhizine hydrochloride, epiberberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride and berberine hydrochloride in Coptis chinensis, with a recovery rate of more than 80%. Compared with the traditional method of extracting active ingredients of traditional Chinese medicine, this method saves a lot of organic solvents and is very convenient for routine operation. Figure 3 A picture and Figure 3 From the comparison of Figure B, it can be seen that in addition to significantly and effectively improving the enrichment efficiency, the matrix interference effect is also reduced, making the chromatographic separation efficiency higher. The material prepared by Example 1 efficiently achieves the targeted enrichment and fishing of the alkaloid active ingredients in Coptis chinensis, and the RSD% after repeated use is less than 5%. This shows that this type of metal organic framework material can be used for the efficient enrichment of trace alkaloid active ingredients in traditional Chinese medicine components, and has good application prospects and potential in the field of traditional Chinese medicine analysis.

[0050] Example 2

[0051] Steps (1) to (6) are the same as in Example 1;

[0052] (7) Selective enrichment of alkaloid active ingredients: Accurately weigh 80 mg of dried MOF-808 and add it to the crude extract of Coptis chinensis (0.1 mg / mL) obtained in step (2) and the mixed solution obtained in step (6). After ultrasonication at 40°C for 10 min, incubate on a shaker for 15 min under hydrochloric acid conditions with a pH of about 3-4. Centrifuge at 2000 rpm for 5 min to obtain a precipitate. Add 3% formic acid acetonitrile solution to the precipitate and ultrasonicate at 40°C for 50 min. Repeat the centrifugation step and take the supernatant and filter it through a liquid filter membrane for later use.

[0053] (8) The chromatographic conditions were the same as those in Example 1.

[0054] Example 3

[0055] Steps (1) to (6) are the same as in Example 1;

[0056] (7) Selective enrichment of alkaloid active ingredients: The MOF-808 used three times in Example 1 was dried, 80 mg was accurately weighed, and added to the crude extract of Coptis chinensis (0.1 mg / mL) in step (2) and the mixed solution obtained in step (6), respectively. After ultrasonication at 40°C for 10 min, the mixture was incubated on a shaker at 60°C for 15 min, and centrifuged at 2000 rpm for 5 min to obtain a precipitate. A 3% formic acid acetonitrile solution was added to the precipitate, and the mixture was ultrasonicated at 40°C for 50 min. The centrifugation step was repeated, and the supernatant was taken and filtered through a liquid filter for later use.

[0057] (8) The chromatographic conditions were the same as those in Example 1.

[0058] Comparative Example 1

[0059] (1) Preparation of crude extract of Coptis chinensis: Weigh 10 g of dried rhizome of Coptis chinensis, crush it and pass it through a No. 5 or 6 sieve to make fine powder, place it in a beaker, add it to 400 mL of 0.5% HCl + 80% ethanol aqueous solution and ultrasonically treat it at 40 °C for 30 min. After filtering, retain the supernatant and precipitate respectively.

[0060] (2) Add the precipitate from step (1) to 400 mL of 0.5% HCl + 80% ethanol aqueous solution, repeat the extraction and filtration under the conditions in step (1), combine the filtrates obtained twice, concentrate to dryness on a 55°C rotary evaporator, and freeze-dry for later use. Accurately weigh 10 mg of the lyophilized powder and dissolve it in 100 mL of methanol solution to prepare 0.1 mg mL -1 Coptis chinensis extract.

[0061] (3) The chromatographic conditions were the same as those in Example 1.

[0062] Comparative Example 2

[0063] Steps (1) to (6) are the same as in Example 1;

[0064] (7) Selective enrichment of alkaloid active ingredients: According to the previously disclosed patent (CN118162118A), 80 mg of the core-shell material was accurately weighed and added to the crude extract of Coptis chinensis (0.1 mg / mL) in step (2) and the mixed solution obtained in step (6). After ultrasonication at 40°C for 10 min, the mixture was incubated on a shaker at 37°C for 15 min, and centrifuged at 2000 rpm for 5 min to obtain a precipitate. 3% formic acid acetonitrile solution was added to the precipitate, and ultrasonication was performed at 40°C for 50 min. The centrifugation step was repeated, and the supernatant was taken and filtered through a liquid filter for later use.

[0065] (8) The chromatographic conditions were the same as those in Example 1.

[0066] Comparative Example 3

[0067] Steps (1) to (6) are the same as in Example 1;

[0068] (7) Selective enrichment of alkaloid active ingredients: Metal-organic framework materials were prepared according to the previously disclosed patent (CN117567533A). Accurately weigh 80 mg and add them to the crude extract of Coptis chinensis with a concentration of 0.1 mg / mL in step (2) and the mixed solution obtained in step (6). After ultrasonication at 40°C for 10 min, the mixture was incubated on a shaker at 37°C for 15 min, and centrifuged at 2000 rpm for 5 min to obtain a precipitate. A 3% formic acid-acetonitrile solution was added to the precipitate and ultrasonicated at 40°C for 50 min. The centrifugation step was repeated, and the supernatant was taken and filtered through a liquid filter for later use.

[0069] Implementation Effect

[0070] The selective enrichment of active ingredients for fishing in Examples 1-3 and Comparative Examples 1-3 was compared, and the results are shown in Table 1. By comparison, it can be seen that the MOF-808 prepared by this method has a highly efficient and selective fishing effect on the isoquinoline alkaloid active ingredients in Coptis chinensis, proving that the MOF-808 prepared by simply adjusting the ratio of hydrochloric acid to trifluoroacetic acid has good acid stability, as well as good stability and reusability.

[0071]

Claims

1. Application of metal organic framework materials in selective fishing of active ingredients of traditional Chinese medicine, characterized in that: The metal organic framework material is a zirconium-based MOFs; the specific surface area of ​​the zirconium-based MOFs is 800-950 m 2 / g; pore size range is 2-5 nm; The active ingredients include isoquinoline alkaloids; The raw materials used in the zirconium-based MOFs include: zirconium oxychloride, trimesic acid 1,3,5-BTC; and also include solvents, and the solvents include N,N-dimethylformamide DMF, formic acid FA, hydrochloric acid HCl, and trifluoroacetic acid TFA; The volume ratio of DMF and FA in the solvent is (2-3):2; And / or, the mass ratio of zirconium oxychloride to 1,3,5-BTC is (13-13.5):5; And / or, the volume ratio of HCl to TFA is 1:(2-3); The method for preparing zirconium-based MOFs comprises the following steps: (1) Add zirconium oxychloride and 1,3,5-BTC into DMF and FA solvents and dissolve them; (2) adding hydrochloric acid and trifluoroacetic acid to the solution of step (1) to react; after the reaction is completed, collecting the solid product by centrifugation; (3) The product obtained in step (2) is centrifuged and washed, then immersed and activated; centrifuged, and dried to obtain; The reaction conditions of step (2) are: 120-130°C for 48-72 h; And / or, the centrifugal washing solution in step (3) is: DMF and / or ethanol; And / or, the soaking activation conditions of step (3) are: time 2 to 3 days, during which the DMF and / or ethanol solvent is replaced 3 to 5 times.

2. The use according to claim 1, characterized in that: The traditional Chinese medicine is Coptis chinensis.

3. The use according to claim 1, characterized in that: The active ingredient is one or more of jatrorrhizine hydrochloride, epiberberine hydrochloride, coptisine hydrochloride, palmatine hydrochloride and berberine hydrochloride.

4. The use according to claim 2, characterized in that: The method of selective fishing includes the following steps: S1 Preparation of crude extract of Coptis chinensis: Add acid-alcohol aqueous solution to the rhizome of Coptis chinensis for extraction; S2 Selective fishing of alkaloid active ingredients: add metal organic framework material to the crude extract of step S1, incubate, centrifuge, desorb and filter to obtain the active ingredients.

5. The use according to claim 4, characterized in that The relationship between the amount of the dried rhizome of Coptis chinensis and the metal organic framework material added is as follows: 10-30 mg of the metal organic framework material is added to the crude freeze-dried powder obtained from 5 g of the dried rhizome of Coptis chinensis; And / or, the specific process of step S2 is: incubating on a shaker at room temperature for 1 to 2 hours, removing the supernatant by centrifugation, adding a formic acid-acetonitrile mixed solution to the precipitate for ultrasonic desorption, obtaining the desorbed liquid by centrifugation, and filtering it through a liquid phase filter for later use.

Images