CN114213382A

CN114213382A - Method for synthesizing micro-nano-grade EGCG based on supercritical anti-solvent process

Info

Publication number: CN114213382A
Application number: CN202111506644.4A
Authority: CN
Inventors: 孙冰冰; 王森森; 薛长颖; 葛丹; 刘洋
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2021-12-10
Filing date: 2021-12-10
Publication date: 2022-03-22

Abstract

The invention discloses a method for synthesizing micro-nano EGCG based on a supercritical anti-solvent process, which takes ethyl acetate as a solvent, fully dissolves initial EGCG through magnetic stirring and ultrasound, ensures that the requirements of equipment are met through filter filtration, and can regulate and control the particle size of a product by controlling parameters of experimental equipment. The method disclosed by the invention is simple to operate, environment-friendly and good in repeatability of reaction products, and the prepared micro-nano EGCG is uniform in appearance and has a good prospect in preparation and application of medicines.

Description

Method for synthesizing micro-nano-grade EGCG based on supercritical anti-solvent process

Technical Field

The invention relates to a preparation method of micro-nano-scale epigallocatechin gallate (EGCG), in particular to a green and environment-friendly method with controllable particle size based on a supercritical anti-solvent process.

Background

Oxidative stress is defined as a pathophysiological response due to an imbalance between the production of oxidative species and endogenous antioxidants. The oxidizing species (ROS and RNS) may react with and cause damage to cell membranes, subcellular organelle membranes, proteins, lipids and DNA, thereby impairing the normal function of the cell, subsequently leading to mutations, apoptosis and failure within these systems. Green tea is a beverage widely cited worldwide, and its component is catechin. EGCG is the highest antioxidant activity of catechin, has antioxidant activity at least 100 times that of vitamin C and 25 times that of vitamin E, and can protect cell and DNA from oxygen free radical damage. However, natural antioxidants are susceptible to degradation, and their bioavailability is limited by low absorption and degradation during administration. Therefore, the invention improves the bioavailability of EGCG by micronizing the EGCG through a supercritical anti-solvent process. In the preparation process, the EGCG micro-nano particles with controllable particle size are prepared by adjusting experimental parameters of a supercritical anti-solvent process, such as temperature, pressure, initial sample injection concentration, sample injection rate and the like.

Disclosure of Invention

The invention aims to provide a method for synthesizing micro-nano-scale epigallocatechin gallate (EGCG) based on a supercritical anti-solvent process, and the aim of controlling the particle size of the EGCG is achieved by regulating and controlling parameters in an experimental process.

A method for synthesizing micro-nano-grade EGCG based on a supercritical anti-solvent process comprises the following steps:

1) adding a certain mass of EGCG into a proper glass container, adding one or more organic solvents with proper volume into the container, dissolving the EGCG, and preparing an EGCG solution; the EGCG is dissolved in the organic solvent by shaking at room temperature.

2) Adopting magnetic stirring and ultrasound to fully dissolve the EGCG; the process of using magnetic stirring and ultrasound to fully dissolve EGCG is stirring and dissolving at room temperature.

3) Sucking the solution by using a syringe, filtering by using a filter of an organic phase, and collecting filtrate to obtain the EGCG solution.

4) And (3) setting experiment parameters by using supercritical anti-solvent equipment, and preparing the EGCG solution into micro-nano EGCG.

In the above-mentioned technical solutions of the preparation methods, the preferred cases areIn the present invention, the organic solvent is mainly but not limited to ethyl acetate C₄H₈O₂Ethyl acetate C may be used₄H₈O₂With ethanol C₂H₆O or methanol CH₄And O, a mixed solvent.

In the technical scheme of the preparation method, the EGCG in the step 1) has the purity of 95% or more in a preferable condition.

In the technical scheme of the preparation method, the concentration of the EGCG solution in the step 1) is preferably 10-15 mg/mL.

In the above-described embodiment of the preparation method, it is preferable that the dissolution of EGCG is performed under a dark condition.

In the above technical solution of the preparation method, preferably, the time of the magnetic stirring in step 2) is 15-25min, and the stirring speed is 300-400 rpm; the ultrasonic time is 20-30min, and the ultrasonic power is 99W.

In the technical scheme of the preparation method, the diameter of the filter in the step 3) is 13mm, and the pore diameter is 0.22-0.45 μm.

In the above technical solution of the preparation method, preferably, the experimental parameters set during the operation of the supercritical anti-solvent equipment in step 4) include: at 35-37 deg.C and 10-12MPa of pressure, CO₂The flow rate of the EGCG solution is 33-42 mL/min, and the liquid inlet speed of the EGCG solution is 1-2 mL/min.

Another aspect of the present invention is to protect the micro-nano grade EGCG prepared by the above method.

According to the invention, ethyl acetate or a mixed organic solvent thereof is selected as a solvent, an EGCG solution with a proper concentration is prepared, the EGCG solution is fully dissolved by magnetic stirring and ultrasonic, the filter of an organic phase is utilized for filtering to ensure that the requirements of equipment are met, and filtrate is collected as feed liquid; and then, the micro-nano EGCG is prepared by a supercritical anti-solvent process and setting appropriate experimental parameters. The method disclosed by the invention is simple in process, environment-friendly and low in synthesis temperature, is suitable for synthesizing temperature-sensitive drugs, and can be used for synthesizing micro-nano EGCG with controllable particle size. The reaction product has good repeatability, and the prepared micro-nano EGCG has uniform appearance, thereby having good application prospect in the medical fields of preparation and use of medicines and the like.

Drawings

FIG. 1 is SEM characterization results before and after supercritical in example 1: (a) is an SEM image of the initial EGCG and (b) is an SEM result of the EGCG after the supercritical state, and the scale is 1 μm.

FIG. 2 is the X-ray diffraction characterization results of example 1: (a) is the XRD pattern of the original EGCG and (b) is the XRD pattern of EGCG after supercritical.

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

Example 1

1200mg of EGCG with the purity of 95% is accurately weighed and added into a conical flask with magnetons (the flask body is wrapped by aluminum foil and is protected from light), then 100mL of ethyl acetate is weighed and added into the conical flask, and the mixture is uniformly mixed by shaking to prepare a solution with the concentration of 12 mg/mL. At normal temperature, the conical flask is placed on a magnetic stirrer to be stirred for 15-25min at the stirring speed of 300-400rpm, so that EGCG is fully dissolved; then the solution was further dissolved by sonication for 20-30min using a sonicator (99W). After sufficient dissolution, the solution was filtered using a syringe and a filter for the organic phase (diameter 13mm, pore size 0.22 μm) to prevent clogging of the nozzle during the preparation. The preparation of the feed liquid is finished through the steps. Setting the parameters of supercritical anti-solvent equipment at 35 deg.C and 12Mpa in CO₂The flow rate of (2) was 37.5mL/min, and the flow rate of the feed solution was 1 mL/min. And when the parameters reach a set value, adding the feed liquid into the crystallization kettle through a pump, performing a supercritical anti-solvent process, and collecting the product of the EGCG in the micro-nano level.

The above materials were characterized and found to have an average particle size of EGCG after supercritical processing of 2.47 μm and a Zeta potential of-32.43 mV (as shown in Table 1). By comparing the SEM images of the EGCG before and after the supercritical process, the appearance of the sample after the supercritical process is changed into a more uniform sheet structure (see figure 1). Through the XED detection of the EGCG before and after the supercritical, the position of the main peak of the EGCG after the supercritical is found to be shifted, which indicates that the crystal form of the EGCG after the supercritical is changed (see figure 2).

TABLE 1 characterization results of hydrated particle size distribution and Zeta potential of EGCG after supercritical extraction in example 1

It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.

Claims

1. A preparation method for synthesizing micro-nano EGCG based on a supercritical anti-solvent process comprises the following steps:

1) adding EGCG into a glass container, adding an organic solvent, and fully dissolving the EGCG by adopting magnetic stirring and ultrasound to obtain an EGCG solution;

2) sucking EGCG solution with syringe, filtering with organic phase filter, and collecting filtrate;

3) setting experiment parameters by using supercritical anti-solvent equipment, and preparing filtrate into micro-nano EGCG;

the organic solvent in the step 1) is ethyl acetate or mixed liquid of ethyl acetate and ethanol or methanol;

the experimental parameters of the supercritical anti-solvent equipment in the step 3) during operation comprise: the temperature is 35-37 ℃, the pressure is 10-12MPa, and CO is₂The flow rate of the filter is 33mL/min to 42mL/min, and the liquid inlet speed of the filter liquid is 1mL/min to 2 mL/min.

2. The preparation method for synthesizing micro-nano grade EGCG based on the supercritical anti-solvent process according to claim 1, characterized in that: the purity of the EGCG in the step 1) is 95% or more.

3. The preparation method for synthesizing micro-nano grade EGCG based on the supercritical anti-solvent process according to claim 1, characterized in that: the concentration of the EGCG solution in the step 1) is 10-15 mg/mL.

4. The preparation method for synthesizing micro-nano grade EGCG based on the supercritical anti-solvent process according to claim 1, characterized in that: the glass container in the step 1) is a conical flask or a round-bottom flask.

5. The preparation method for synthesizing micro-nano grade EGCG based on the supercritical anti-solvent process according to claim 1, characterized in that: the rotating speed of the magnetic stirring in the step 1) is 300-400rpm, and the time is 15-25 min.

6. The preparation method for synthesizing micro-nano grade EGCG based on the supercritical anti-solvent process according to claim 1, characterized in that: the power of the ultrasound in the step 1) is 99W, and the time of the ultrasound is 20-30 min.

7. The preparation method for synthesizing micro-nano grade EGCG based on the supercritical anti-solvent process according to claim 1, characterized in that: the diameter of the filter in the step 2) is 13mm, and the pore diameter is 0.22-0.45 μm.

8. The micro-nano grade EGCG prepared according to the method of claim 1.