A method for preparing dihydroflavonoid glycosides compounds using industrial chromatography technology

Abstract

The invention discloses a method for preparing dihydroflavonoid glycoside compounds by industrial chromatography technology, comprising taking a sample solution of a total flavonoid glycoside extract as a raw material, purifying by a dynamic axial compression column system of industrial chromatography technology, and detecting separation by a dual-wavelength ultraviolet online detector; collecting the eluate of the prepared dihydroflavonoid glycoside compounds according to the peak time and chromatographic peak height of a naringin component, wherein the collection start and end time is 155-235min, and the eluate is concentrated and dried. The prepared dihydroflavonoid glycoside compounds have high purity; and have the characteristics of good process reproducibility, simple operation, short preparation time, small amount of organic solvent, economic and environmental protection, and large-scale industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of active ingredients of traditional Chinese medicines, and particularly relates to a method for preparing dihydroflavonoid glycoside compounds by utilizing industrial chromatography technology. Background Art

[0002] Naringin is a dihydroflavonoid glycoside compound. Literature reports show that it is widely present in Chinese medicinal materials such as Citrus aurantium, Citrus aurantium, Changshan Huyou, Huazhou Pomelo, and Huajuhong. It is also used as an active ingredient or representative ingredient for quality control in dozens of Chinese medicine varieties such as Aolanti Weikang Tablets, Chenxiang Huaqi Tablets, Chenxianglu Bailu Tablets, Xiaoer Fupi Granules, Weisu Granules, Zhishi Xiaopi Pills, and Huoxiang Zhengqi Oral Liquid. However, research on low-content ingredients such as naringin is still insufficient, and due to the low content of this ingredient and the cumbersome and complicated separation process, naringin components cannot be prepared in large quantities at present, which seriously limits the quality control of medicinal materials containing naringin and related products. Therefore, the development of a simple and rapid method for separating and purifying high-purity naringin components is the focus of current research.

[0003] Chen Jianwei et al. reported that Citrus aurantium contains naringenin (determination of four total flavonoid components in Citrus aurantium by a one-test, multiple-evaluation method. Chinese Herbal Medicine, 2015, 46(9): 1374-1377); Yang Lili et al. also reported that Citrus aurantium contains naringenin (research on flavonoid control extracts from Citrus aurantium and its application in quality control of Citrus aurantium medicinal materials, but no report on the preparation method of this component, Chinese Journal of Traditional Chinese Medicine Information, 2019, 26(9): 73-79); CN101062165A discloses a total flavonoid extract extracted from the Chinese herbal medicine Citrus aurantium and its preparation method, but no prior art for preparing naringenin monomer compounds was found. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention provides a method for preparing a dihydroflavonoid glycoside compound naringin by using industrial chromatography technology. The method has the advantages of small amount of organic solvent, simple preparation process, short production cycle and easy industrialization.

[0005] The objective of the present invention is to be achieved through the following technical solutions:

[0006] A method for preparing dihydroflavonoid glycoside compound naringin by industrial chromatography technology comprises the following steps:

[0007] The total flavonoid glycoside extract is taken, solvent 1 is added to dissolve it and prepare a solution of a certain concentration, and the solution is filtered to obtain a sample solution; the sample solution is injected into a dynamic axial compression column system according to a certain injection volume, and the solvent 2 is used for elution, and the separation is detected by a dual-wavelength ultraviolet online detector. According to the peak time and chromatographic peak height of the naringin component, the eluate of the prepared naringin compound is collected, and the collection start and end time is 155-235 minutes, and the eluate is concentrated and dried.

[0008] Preferably, the total flavonoid glycoside extract is a total flavonoid glycoside extract of Citrus aurantium and / or Citrus aurantium; and / or the content of naringenin in the total flavonoid glycoside extract is 2.0%-5.0% (W / W), preferably 2.5%-3.5% (W / W).

[0009] Preferably, the solvent 1 is a 1.0-5.0% (V / V) acetonitrile aqueous solution or water, preferably a 3.0%-3.5% (V / V) acetonitrile aqueous solution.

[0010] Preferably, the sample solution concentration is 85-125 mg / mL; preferably 95-110 mg / mL.

[0011] Preferably, the dynamic axial compression column system filler is a reverse phase polymer filler, preferably UniPS 40-300, UniPSN40-300 or CG161M.

[0012] Preferably, the solvent 2 is one or more of acetonitrile, water, and phosphoric acid; preferably a mixed solution of acetonitrile, water, and phosphoric acid.

[0013] Preferably, the volume ratio of acetonitrile-water-phosphoric acid in the mixed solution is 15-25:85-75:0.1; such as: 15:85:0.1, 25:75:0.1, 18:82:0.1.

[0014] Preferably, the elution flow rate is 1250-2000 mL / min; preferably 1500-1750 mL / min; and / or the injection volume is 90-115 L.

[0015] Preferably, the detection wavelength of the ultraviolet online detector is 283 nm and the reference wavelength is 210 nm.

[0016] Preferably, the concentration is reduced-pressure concentration; and / or the concentration temperature is 40-50°C, preferably 45°C.

[0017] Preferably, the drying is freeze vacuum drying; and / or the drying temperature is -25 to -45°C, preferably -35°C.

[0018] Beneficial technical effects of the present invention:

[0019] 1. The present invention adopts industrial chromatography technology to separate and purify the dihydroflavonoid glycoside compound naringin from the total flavonoid extract. For example, the total flavonoid glycoside extract of Citrus aurantium or Citrus aurantium is directly separated and purified by a dynamic axial compression column system to obtain naringin, and the purity thereof can reach more than 99.0%, which can solve the problem of lack of naringin reference material.

[0020] 2. Freeze vacuum drying can effectively solve the problem of sample stability and ensure the high purity of the sample.

[0021] 3. The present invention utilizes an industrial chromatography technology dynamic axial compression column system to prepare naringin, which can realize real-time online monitoring of the separation process, has the advantages of good repeatability, simple operation, short preparation time, small amount of organic solvent, high purity of separated naringin, etc., and can be industrially produced in large quantities. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :Process flow chart for preparing dihydroflavonoid glycoside compound naringin using industrial chromatography technology;

[0023] Figure 2 : HPLC chromatogram of total flavonoid glycosides extract from Citrus aurantium;

[0024] Figure 3 : HPLC chromatogram of naringenin. DETAILED DESCRIPTION

[0025] In order to better understand the present invention, the technical scheme of the present invention is further described and illustrated by the following examples.

[0026] Example 1

[0027] A method for preparing dihydroflavonoid glycoside compound naringin using industrial chromatography technology, the process flow chart is as follows Figure 1 As shown, the specific steps are as follows:

[0028] Take the total flavonoid glycosides extract of Citrus aurantium (its HPLC chromatogram is shown in Figure 2), wherein the content of naringin is about 3.5% (W / W), 4.0% (V / V) acetonitrile aqueous solution is added to dissolve it and prepare a 125 mg / ml solution, filter it and set aside to obtain a sample solution; 90 L of the sample solution is injected into a dynamic axial compression column system, the industrial chromatography filler is UniPSN 40-300 (reverse phase polymer filler), acetonitrile: water: phosphoric acid solution (15:85:0.1, V / V / V) is used as the eluent, the elution flow rate is 2000 mL / min, the ultraviolet detection wavelength is 283 nn, and the reference wavelength is 210 nm. According to the peak time of the naringin component and the height of the chromatographic peak, the eluate of the prepared naringin was collected, and the collection start and end time was 185-235min. The eluate containing high purity naringin was concentrated under reduced pressure at 45°C, and then freeze-dried in a vacuum at -35°C for 12h to obtain 354.38g of naringin with a purity of 99.41% (its HPLC chromatogram is shown in Figure 3 ).

[0029] Example 2

[0030] A method for preparing dihydroflavonoid glycoside compound naringin using industrial chromatography technology, the process flow chart is as follows Figure 1 As shown, the specific steps are as follows:

[0031] Take the total flavonoid glycoside extract of Citrus aurantium immaturus, in which the content of naringin is about 2.5% (W / W), add 3.0% (V / V) acetonitrile aqueous solution to dissolve it and prepare a solution of 85 mg / ml, filter to obtain a sample solution; inject 115L of the sample solution into a dynamic axial compression column system, the industrial chromatography filler is UniPS 40-300 (reverse phase polymer filler), acetonitrile: water: phosphoric acid solution (25:75:0.1, V / V / V) is used as the eluent, the elution flow rate is 1500mL / min, the ultraviolet detection wavelength is 283nn, and the reference wavelength is 210nm. According to the elution time of the naringin component and the height of the chromatographic peak, the eluate of the prepared naringin was collected, and the collection start and end time was 155-205 min. The eluate containing high purity naringin was concentrated under reduced pressure at 45° C., and then freeze-dried in a vacuum at -35° C. for 12 h to obtain 232.13 g of naringin with a purity of 99.63%.

[0032] Example 3

[0033] A method for preparing dihydroflavonoid glycoside compound naringin using industrial chromatography technology, the process flow chart is as follows Figure 1 As shown, the specific steps are as follows:

[0034] Take the total flavonoid glycoside extract of Citrus aurantium, wherein the content of naringin is about 2.0% (W / W), add water to dissolve it and prepare a solution of 100 mg / ml, filter it to obtain a sample solution; inject 100L of the sample solution into a dynamic axial compression column system, the industrial chromatographic filler is or CG161M (reverse phase polymer filler), acetonitrile: water: phosphoric acid solution (18:82:0.1, V / V / V) is the eluent, the elution flow rate is 1750mL / min, the ultraviolet detection wavelength is 283nn, and the reference wavelength is 210nm. According to the peak time of the naringin component and the height of the chromatographic peak, collect the eluent of the prepared naringin, the collection start and end time is 170-225min, and the eluent containing high purity of naringin is concentrated under reduced pressure at 45°C, and then freeze-dried in a vacuum at -35°C for 12h to obtain 285.45g of naringin with a purity of 99.91%.

[0035] The above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Those skilled in the art should understand that any modification or equivalent replacement of the technical solutions of the present invention without departing from the purpose and scope of the technical solutions of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing naringin, a dihydroflavonoid glycoside compound, by industrial chromatography technology, characterized in that The method comprises the following steps: Take a total flavonoid glycoside extract, wherein the content of naringenin in the total flavonoid glycoside extract is 2.0%-3.5% W / W, add 1.0-5.0% V / V acetonitrile aqueous solution or water to dissolve it and prepare a solution with a concentration of 85-125 mg / mL, filter, and obtain a sample solution; inject the sample solution into a dynamic axial compression column system according to a certain injection volume, wherein the dynamic axial compression column system filler is a reverse phase polymer filler UniPS 40-300, UniPSN 40-300 or CG161M, eluted with a mixed solution of acetonitrile, water and phosphoric acid, wherein the volume ratio of acetonitrile, water and phosphoric acid is 15-25:85-75:0.1, the elution flow rate is 1250-2000mL / min, the injection volume is 90-115L, and the separation is detected by a dual-wavelength ultraviolet online detector. According to the peak time and chromatographic peak height of the naringin component, the eluate of the prepared naringin compound is collected, and the collection start and end time is 155-235min, and the eluate is concentrated and dried.

2. The method according to claim 1, wherein the total flavonoid glycoside extract is a total flavonoid glycoside extract of Fructus Aurantii Immaturus and / or Fructus Aurantii Immaturus.

3. The method according to claim 1, wherein the acetonitrile aqueous solution is a 3.0%-4.0% V / V acetonitrile aqueous solution.

4. The method according to claim 1, wherein the volume ratio of acetonitrile, water and phosphoric acid in the mixed solution is 15:85:0.1, 25:75:0.1 or 18:82:0.

1.

5. The method according to claim 1, wherein the elution flow rate is 1500-2000 mL / min.

6. The method according to claim 1, wherein the detection wavelength of the ultraviolet online detector is 283 nm and the reference wavelength is 210 nm. The method according to claim 1 , wherein the concentration is concentration under reduced pressure.

8. The method according to claim 7, wherein the concentration temperature is 40-50°C.

9. The method according to claim 1, wherein the drying is freeze vacuum drying.

10. The method according to claim 9, wherein the drying temperature is -25 to -45°C.

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