CN110681183A

CN110681183A - Research on process for purifying liquorice total flavonoids by NKA-9 type macroporous resin and effect of liquorice total flavonoids on nitrite elimination

Info

Publication number: CN110681183A
Application number: CN201910870342.1A
Authority: CN
Inventors: 宋吉明; 朱泽华; 董庄庄; 方晶
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2019-09-16
Filing date: 2019-09-16
Publication date: 2020-01-14

Abstract

The invention discloses a process for purifying a crude extract of total flavonoids of liquorice by using NKA-9 type macroporous resin, researches the effect of the crude extract on removing nitrite, and belongs to the technical field of separation and purification of flavonoids in Chinese herbal medicines. And obtaining process parameters such as sample loading concentration, sample loading crude extract, sample loading volume, sample loading speed, elution volume, elution speed and the like through a single-factor experiment. The content of the licorice total flavonoids in the crude extract purified by NKA-9 type macroporous resin is increased from 44.5% to 57.8%, and the purified licorice total flavonoids have a certain effect of removing nitrite. The method is easy to operate, the process conditions are simple, the used solvent is non-toxic and harmless, and the purified product has a function of removing nitrite and has a good practical application value.

Description

Research on process for purifying liquorice total flavonoids by NKA-9 type macroporous resin and effect of liquorice total flavonoids on nitrite elimination

Technical Field

The invention belongs to the technical field of separation and purification of flavonoid compounds in Chinese herbal medicines, and particularly relates to a process for separating and purifying a licorice flavone crude extract by using NKA-9 type macroporous resin, and research on nitrite removal.

Background

In recent years, a great deal of research shows that the flavonoid compounds in the liquorice have important physiological activity and have important pharmacological effects in the aspects of resisting HIV, tumors, inflammations, bacteria, sedation, analgesia, melanoma and the like. Because the types of natural components contained in the liquorice are complex, a series of impurities are inevitably introduced in the extraction process, so that the content of total flavonoids in the liquorice flavone crude extract is low, the liquorice flavone crude extract cannot be directly applied to actual production, and the development of the liquorice flavone crude extract is greatly limited. In recent years, macroporous resin purification technology is developed rapidly, and the principle is to separate by utilizing the difference of adsorption force between compounds and macroporous resin and the difference of molecular weights of different substances, so that the macroporous resin is widely applied to separation and purification of various natural products. There are many reports of using macroporous resin to purify licorice total flavone, and the related patents are: (1) patent numbers: CN 103623041B, name: the macroporous resin is used for separating and purifying flavone in the liquorice extract, and the patent is in the validity period; (2) patent numbers: CN 108261438A, name: a method for extracting and purifying licoflavone component is disclosed, which is in validity period. Different from the related patents, the NKA-9 type macroporous resin is selected for purifying the liquorice total flavonoids for the first time, and the purity of the liquorice total flavonoids is successfully improved to 57.8 percent. The scavenging effect on nitrite is researched, and the result shows that the licorice total flavonoids have good scavenging effect on nitrite, and the scavenging effect is better than that of the control L-ascorbic acid even at low concentration.

Disclosure of Invention

The invention provides a method for purifying a licorice total flavone crude extract by using NKA-9 type macroporous resin, which obtains a purified licorice total flavone product by optimizing process parameters and researches the nitrite removing capability of the licorice total flavone product. The method comprises the following steps:

preparation of licorice total flavone crude extract

Accurately weighing a proper amount of liquorice, performing reflux extraction for 2-3 hours by using 15-25 times of 60-80% ethanol to obtain a coarse liquorice total flavone extract, adding a certain volume of ethyl acetate for extraction, collecting an upper oil phase, performing reduced pressure concentration, and drying and collecting the obtained crude extract.

Secondly, purifying the crude extract of the liquorice total flavone

Static experiment of NKA-9 type macroporous adsorption resin:

(1) dissolving Glycyrrhrizae radix total flavone crude product, diluting to constant volume, and collecting 5 parts of solutionAdjusting to different pH, adding certain amount of macroporous resin, reacting in constant temperature shaking table, and measuring absorbance of the solution (total flavone content adopts NaNO)₂-Al(NO₃)₃NaOH chromogenic assay, same as below).

(2) Taking a certain volume of licoflavone crude extract solution with known concentration, adding a certain amount of macroporous resin, placing in a constant temperature shaking table for reaction, and taking 1mL of solution at regular intervals to measure absorbance.

(3) Taking a plurality of the resin with saturated adsorption in the step (2), dividing into five parts, respectively adding ethanol with the same volume and different concentrations for desorption, and measuring the absorbance of the desorption solution.

(4) Preparing a crude extract solution of the licorice total flavonoids with a certain concentration, diluting the crude extract solution into a series of solutions to be detected with concentration gradients, adding a certain amount of macroporous resin respectively, placing the solutions in a constant-temperature shaking table for reaction for a certain time, and detecting the absorbance of the solution.

Dynamic experiment of NKA-9 type macroporous adsorption resin:

(1) treating with a certain amount of macroporous resin, and packing with wet method (chromatography column 30 mm × 16 mm) to obtain a resin bed column with volume of 1 BV. The sample loading speed is controlled by using an SK-500 III injection pump, one sample is collected every 0.5 BV, the absorbance is measured (1/10 of the concentration of the sample solution is taken as a leakage standard), and macroporous resin leakage curves at different sample loading speeds are drawn.

(2) A certain amount of macroporous resin is taken and loaded under the optimal process conditions obtained in the dynamic experiment (1), the SK-500 III injection pump is used for controlling the elution speed, one sample is collected every 0.5 BV, and the absorbance is measured.

Thirdly, research on nitrite scavenging capacity of liquorice total flavonoids

Accurately weighing a certain mass of purified liquorice total flavonoids to prepare a solution to be detected with a known concentration, and then diluting the solution to be detected with a certain concentration gradient. Taking the above 1mL solutions to be tested with different concentrations, adding 5 μ g/mL NaNO respectively₂1mL of the solution was placed in a water bath at 37 ℃ for 30 min, taken out, added with 1mL of 0.4% sulfanilic acid, left for 5 min, added with 0.5 mL of 0.2% naphthyl ethylenediamine hydrochloride, left for 15 min, and then added with distilled waterFor reference, the absorbance a of the solution was determined at a wavelength λ =545 nm₁. The control group uses distilled water instead of NaNO₂Solution determination of solution Absorbance A₂Measuring solution absorbance A by using distilled water instead of Glycyrrhrizae radix total flavone solution to be measured₀And calculating the clearance rate of the solution to be detected to the nitrite under different concentrations.

Nitrite clearance calculation:

nitrite clearance/% = (A)₀+A₂-A₁)/A₀×100%

In the formula A₀Representing the absorbance of the solution when the liquorice total flavone solution to be tested is replaced by distilled water; a. the₁Representing the absorbance of the solution when the liquorice total flavone solution to be tested with different concentrations is added; a. the₂Represents distilled water instead of NaNO₂Absorbance of the solution when in solution.

Description of the drawings:

FIG. 1 is a standard curve for measuring the content of total flavonoids in licorice;

FIG. 2 is the influence of different pH values of the solution on the adsorption amount of the resin in the static experiment (1);

FIG. 3 is the determination of the adsorption saturation time of NKA-9 macroporous resin in static experiment (2);

FIG. 4 is the influence of ethanol with different concentrations on the elution effect of adsorption saturated macroporous resin in the static experiment (3);

FIG. 5 is the determination of the adsorption isotherm curve of NKA-9 macroporous resin at room temperature in the static experiment (4);

FIG. 6 is a graph showing the effect of different loading speeds and loading volumes on macroporous resin column packing in dynamic test (1);

FIG. 7 is a graph showing the dynamic test (2) on the effect of different elution speeds and elution volumes on the purification of crude extract of licorice total flavonoids by macroporous resin;

FIG. 8 shows the nitrite scavenging ability of licorice total flavonoids at different concentrations in the third invention.

The specific implementation mode is as follows:

the invention is illustrated in detail below with reference to the examples:

example 1: change of licorice total flavone content in crude extract before and after NKA-9 type macroporous resin purification

(1) Precisely weighing a proper amount of liquorice, and then taking 75% ethanol as an extraction solvent according to a material-liquid ratio of 1: 20, extracting for 3 hours to obtain a coarse extract of licoflavone, adding a proper volume of ethyl acetate for extraction, collecting an upper oil phase, concentrating under reduced pressure, drying the obtained coarse extract at 80 ℃, and collecting.

(2) Dissolving a certain amount of the licoflavone crude extract in 15% ethanol solution, ultrasonically dissolving and filtering, and adjusting pH to 5.0 to obtain a sample solution with concentration of 2.8 mg/mL for later use;

(3) accurately weighing 5.0 g of pretreated NKA-9 macroporous resin, and filling the pretreated NKA-9 macroporous resin into a column (the specification of the chromatographic column is 300mm multiplied by 16 mm) by a wet method, wherein the height of the column is 8.5 cm;

(4) the sample loading speed is controlled to be 2.0 BV/h by using an SK-500 III injection pump, and the sample loading volume is 3.5 BV. Then 80% ethanol is used for eluting the eluent, the elution speed is 2.5 BV/h, and the elution volume is 8.0 BV. The content of the liquorice total flavonoids in the crude extract is changed from 44.5 percent to 57.8 percent after the crude extract is purified by NKA-9 macroporous resin.

Example 2: research on nitrite scavenging effect of purified licorice total flavonoids

NaNO₂Diazotizing with sulfanilic acid under weak acidity condition, coupling with naphthyl ethylenediamine hydrochloride to obtain red compound, measuring the absorbance of the red compound with ultraviolet spectrophotometer to obtain NaNO₂The content is small. Therefore, the method can be used for determining NaNO under the same conditions₂The change of the content reflects the strength of the nitrite eliminating capability of the licorice total flavone solution, and the nitrite eliminating capability of the licorice total flavone solution is measured according to the steps in the third experimental content. As can be seen from FIG. 8, licorice total flavonoids has a certain scavenging ability for nitrite, and its scavenging action is enhanced with increasing concentration, and the scavenging effect of licorice total flavonoids is better than that of its control L-ascorbic acid at low concentration.

Claims

1. The invention relates to a research on a NKA-9 type macroporous resin process for purifying liquorice total flavonoids and an effect of the research on nitrite removal, and the patent specification is as follows:

1. a process for purifying liquorice total flavonoids by NKA-9 type macroporous resin comprises the following process parameters: the sample loading concentration is 1.0-3.0 mg/mL, the pH of the crude sample loading extract is = 3.0-7.0, the sample loading volume is 1.0-9.0 BV, the sample loading speed is 1.5-2.5 BV/h, the elution volume is 1.0-9.0 BV, the elution speed is 1.5-2.5 BV/h, and the total flavone content of the liquorice after the crude extract is purified by NKA-9 macroporous resin is increased to 57.8%.

2. The research on the nitrite scavenging effect of the licorice total flavonoids prepared by the process of claim 1, wherein the nitrite scavenging effect of the licorice total flavonoids is measured in the range of 0.02-0.12 mg/mL, and the result shows that the nitrite scavenging effect is enhanced with the increase of the concentration, and the effect is better than that of L-ascorbic acid at low concentration.