A method for preparing dihydroflavonoid glycosides compounds using industrial chromatography technology

Abstract

The present invention discloses a method for preparing dihydroflavonoid glycoside compounds by industrial chromatography technology, the method comprising preparing a total flavonoid glycoside extract sample solution, adopting dynamic axial compression industrial chromatography purification, and detecting the separation condition by a dual-wavelength ultraviolet online detector; determining the start and end time of collecting the eluent of the dihydroflavonoid glycoside compounds according to the peak time and the height of the chromatographic peak, and concentrating the eluent under reduced pressure and vacuum drying. The purity of the prepared dihydroflavonoid glycoside compounds is above 98.5%; the separation process can be detected in real time online, the process has good reproducibility, simple operation, short preparation time, less organic solvent usage, economic and environmental protection, and can be mass-produced industrially.

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] Neospermum officinale is a dihydroflavonoid glycoside compound, which is widely found in Chinese medicinal materials such as Citrus aurantium, Citrus aurantium, sweet orange, Huazhou pomelo, Huajuhong, Changshan Huyou, and Drynaria, and is used as an active ingredient or representative ingredient in Sanqi Shangyao Tablets, Guci Tablets, Qianggu Capsules, Hongzhu Capsules, Shudan Granules, Shangshi Pills and other varieties for content control. However, the research on low-content ingredients such as neospermum officinale is still insufficient, and due to the low content of this ingredient and the complicated separation process, the neospermum officinale component cannot be prepared in large quantities at present, which seriously limits the quality control of medicinal materials containing neospermum officinale components and related products. Therefore, the development of a simple and rapid method for separating and purifying high-purity neospermum officinale components is the focus of current research.

[0003] After reviewing the literature, it was found that Wei Jining et al. reported that the total flavonoid glycoside extract of Citrus aurantium contains elaiocarpon (Study on the Characteristic Spectrum of the Total Flavonoid Glycoside Extract of Citrus aurantium. Journal of Jiangxi University of Traditional Chinese Medicine, 2016, 28(5):72-74,87), but the preparation method of this ingredient was not reported; Yue Chunhua et al. reported a method for preparing elaiocarpon and naringin reference substances from Drynaria fortunei (Study on the Preparation of Laiocarpon and Naringin Reference Substances from Drynaria fortunei. Chinese Herbal Medicine, 2008, (4):529-531), but the preparation method has a relatively small batch size, a complicated preparation process, a long production cycle, and cannot achieve large-scale preparation. After consulting the patents, it was found that Chinese patents CN102397374B, CN102397373B, CN102406734B, and CN102048874B disclosed a total flavonoid extract of Citrus aurantium or Citrus aurantium extracted by decoction and its uses, but no prior art for preparing new erythroposide monomer compounds was found. Summary of the invention

[0004] The invention provides a method for preparing a dihydroflavonoid glycoside compound, a new eriocin monomer compound, from a flavonoid glycoside extract, which has the characteristics of small amount of organic solvent used, simple preparation process, short production cycle and easy industrialization.

[0005] A method for preparing a dihydroflavonoid glycoside compound, erythrocetoside, by using industrial chromatography technology comprises the following steps:

[0006] (1) Sample solution preparation: Take the total flavonoid glycoside extract, add a solvent to dissolve it and prepare a sample solution of a certain concentration, filter it and set aside;

[0007] (2) separation, purification and drying: injecting the sample solution of step (1) into a dynamic axial compression industrial chromatograph according to a certain injection volume, eluting with an organic solvent, detecting the separation condition with a dual-wavelength ultraviolet online detector, collecting the eluate of the prepared new north american erythroside monomer compound according to the peak elution time and chromatographic peak height of the new north american erythroside component, the collection start and end time is 105-165min, and the eluate is concentrated and dried.

[0008] Preferably, in step (1), the total flavonoid glycoside extract is the total flavonoid glycoside extract of Citrus aurantium and / or Citrus aurantium; and / or the content of eriocin in the total flavonoid glycoside extract is 0.5%-2.5% (W / W); preferably 1.0%-2.0% (W / W).

[0009] Preferably, in step (1), the solvent is 0-10% (V / V) methanol aqueous solution, such as 2%, 5%, 8% (V / V); preferably 2%-5% (V / V) methanol aqueous solution.

[0010] Preferably, in step (1), the sample solution concentration is 70-100 mg / mL; preferably 90-100 mg / mL.

[0011] Preferably, in step (2), the industrial chromatographic filler is a reverse phase C18 filler, preferably ODS-A or ODS-AQ.

[0012] Preferably, in step (2), the organic solvent is one or more of methanol, water, and trifluoroacetic acid; preferably a mixed solution of methanol, water, and trifluoroacetic acid; the volume ratio of the three is preferably 30-40:70-60:0.3, for example 40:60:0.3, 30:70:0.3, 35:65:0.3.

[0013] Preferably, in step (2), the elution flow rate is 2.5-10.0CV / h; preferably 5.0-7.5CV / h; and the injection volume is 100-110L.

[0014] Preferably, in step (2), the detection wavelength of the ultraviolet online detector is 283 nm and the reference wavelength is 210 nm.

[0015] Preferably, in step (2), the concentration is concentrated under reduced pressure; and / or the concentration temperature is 60-70°C, preferably 65°C.

[0016] Preferably, in step (2), the drying is vacuum drying; and / or the drying temperature is 40-50°C, preferably 45°C.

[0017] Beneficial technical effects of the present invention:

[0018] 1. The present invention dissolves the total flavonoid glycoside extract of Citrus aurantium or Citrus aurantium and directly separates and purifies it through industrial chromatography to obtain the dihydroflavonoid glycoside monomer compound neoeriocin with a purity of more than 98.5%.

[0019] 2. The present invention utilizes industrial chromatography to prepare the dihydroflavonoid glycoside monomer compound erythroposide, 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, and high purity of the separated erythroposide monomer, and can be mass-produced industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Process flow chart for preparing new eriocin monomer using industrial chromatography;

[0021] Figure 2 : HPLC chromatogram of total flavonoid glycosides extract from Citrus aurantium;

[0022] Figure 3 :HPLC chromatogram of new eriocin. DETAILED DESCRIPTION

[0023] In order to better understand the present invention, the technical scheme of the present invention is further described and illustrated by the following examples.

[0024] Example 1

[0025] A method for preparing a dihydroflavonoid glycoside compound, eriocin, from a total flavonoid glycoside extract of Citrus aurantium using industrial chromatography technology, comprising sample solution preparation and separation, purification and drying. The process flow chart is as follows: Figure 1 As shown, the specific steps are as follows:

[0026] (1) Sample solution preparation: Take the total flavonoid glycosides extract of Citrus aurantium (its HPLC chromatogram is shown in Figure 2 ), wherein the content of new erioglossin is about 2.0% (W / W), 5% (V / V) methanol aqueous solution is added to dissolve it and prepare a sample solution with a concentration of 100 mg / ml, and the sample solution is filtered and set aside.

[0027] (2) Separation, purification and drying: 100L of the sample solution in step (1) was injected into an industrial chromatograph, the industrial chromatograph filler was ODS-A (C18), methanol: water: trifluoroacetic acid solution (40:60:0.3, V / V / V) was used as the eluent, the elution flow rate was 5.0CV / h, the ultraviolet detection wavelength was 283nn, and the reference wavelength was 210nm. According to the peak time of the new erythroposide component and the height of the chromatographic peak, the eluate of the prepared new erythroposide monomer was collected, and the collection start and end time was 115-150min, and the eluate containing the purified target component was concentrated to dryness at 65°C under reduced pressure and then dried in vacuum at 45°C for 24h to obtain 185.12g of new erythroposide monomer compound with a purity of 98.65% (its HPLC chromatogram is shown in Figure 3 ).

[0028] Example 2

[0029] A method for preparing a dihydroflavonoid glycoside compound, eriocin, from a total flavonoid glycoside extract of Citrus aurantium using industrial chromatography, comprising sample solution preparation, separation, purification and drying. The process flow chart is as follows: Figure 1 As shown, the specific steps are as follows:

[0030] (1) Preparation of sample solution: Take the total flavonoid glycoside extract of Citrus aurantium immaturus, in which the content of eriocin is about 1.0% (W / W), add 2.0% methanol aqueous solution to dissolve it and prepare a sample solution with a concentration of 90 mg / ml, filter the sample solution and set aside.

[0031] (2) Separation, purification and drying: 110L of the sample solution in step (1) was injected into an industrial chromatograph, the industrial chromatograph filler was ODS-AQ (C18), methanol: water: trifluoroacetic acid solution (30:70:0.3, V / V / V) was the eluent, the elution flow rate was 7.5CV / h, the ultraviolet detection wavelength was 283nn, and the reference wavelength was 210nm. According to the peak time of the new eriocin component and the height of the chromatographic peak, the eluate of the prepared new eriocin monomer was collected, and the collection start and end time was 105-145min, and the eluate containing the purified target component was concentrated to dryness at 65°C under reduced pressure and then vacuum dried at 45°C for 24h to obtain 99.25g of the new eriocin monomer chemical component with a purity of 98.83%.

[0032] Example 3

[0033] A method for preparing a dihydroflavonoid glycoside compound, eriocin, from a total flavonoid glycoside extract of Citrus aurantium using industrial chromatography, comprising sample solution preparation, separation, purification and drying. The process flow chart is as follows: Figure 1 As shown, the specific steps are as follows:

[0034] (1) Preparation of sample solution: Take the total flavonoid glycosides extract of Citrus aurantium, in which the content of neoerioglossin is about 1.5% (W / W), add water to dissolve it and prepare a sample solution with a concentration of 95 mg / ml, filter the sample solution and set aside.

[0035] (2) Separation, purification and drying: 105L of the sample solution in step (1) was injected into an industrial chromatograph, the industrial chromatograph filler was ODS-AQ (C18), methanol: water: trifluoroacetic acid solution (35:65:0.3, V / V / V) was the eluent, the elution flow rate was 2.5CV / h, the ultraviolet detection wavelength was 283nn, and the reference wavelength was 210nm. According to the peak time of the new eriocin component and the height of the chromatographic peak, the eluate of the prepared new eriocin monomer was collected, and the collection start and end time was 120-165min, and the eluate containing the purified target component was concentrated to dryness at 65°C under reduced pressure and then dried at 45°C in vacuum for 24h to obtain 149.28g of the new eriocin monomer chemical component with a purity of 98.57%.

[0036] The above preferred embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Those skilled in the art should understand that any modification or equivalent replacement of the technical solution of the present invention without departing from the purpose and scope of the technical solution of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a dihydroflavonoid glycoside compound, erythroposide, by using industrial chromatography technology, characterized in that The method comprises the following steps: (1) Preparation of sample solution: taking a total flavonoid glycoside extract, adding a solvent to dissolve it and preparing a sample solution of a certain concentration, filtering it for later use; the total flavonoid glycoside extract is a total flavonoid glycoside extract of Citrus aurantium and / or Citrus aurantium, and the content of eriocin in the total flavonoid glycoside extract is 0.5%-2.5% W / W; the solvent is 0-5% V / V methanol aqueous solution; the concentration of the sample solution is 90-100 mg / mL; (2) separation, purification and drying: injecting the sample solution of step (1) into a dynamic axial compression industrial chromatography according to a certain injection volume, eluting with an organic solvent, detecting the separation situation with a dual-wavelength ultraviolet online detector, collecting the eluate of the prepared new north American eriocin monomer compound according to the peak time and chromatographic peak height of the new north American eriocin component, the collection start and end time is 105-165min, and concentrating and drying the eluate; the industrial chromatography filler is a reverse phase C18 filler; the organic solvent is a mixed solution of methanol, water and trifluoroacetic acid, wherein the volume ratio of methanol, water and trifluoroacetic acid is 30-40:70-60:0.3; the elution flow rate is 2.5-7.5CV / h; the injection volume is 100-110L; the detection wavelength of the ultraviolet online detector is 283nm, and the reference wavelength is 210nm; the concentration is reduced pressure concentration, and the concentration temperature is 60-70℃; the drying is vacuum drying, and the drying temperature is 40-50℃.

2. The method according to claim 1, wherein in step (1), the content of eriocin in the total flavonoid glycoside extract is 1%-2% W / W.

3. The method according to claim 1, wherein in step (1), the solvent is 2%-5% V / V methanol aqueous solution.

4. The method according to claim 1, wherein in step (2), the industrial chromatography filler is ODS-A or ODS-AQ.

5. The method according to claim 1, wherein in step (2), the volume ratio of methanol, water and trifluoroacetic acid is 40:60:0.3, 30:70:0.3 or 35:65:0.

3.

6. The method according to claim 1, wherein in step (2), the elution flow rate is 5.0-7.5 CV / h.

7. The method according to claim 1, wherein in step (2), the concentration temperature is 65°C.

8. The method according to claim 1, wherein in step (2), the drying temperature is 45°C.

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