A method for simultaneously preparing two dihydroflavonoid glycoside monomer compounds using industrial chromatography technology

Abstract

The invention discloses a method for simultaneously preparing two dihydroflavonoid glycoside monomer compounds by using industrial chromatography technology, wherein the dihydroflavonoid glycoside monomer compounds are naringin and neohesperidin, and the purity is above 99.5%. The method of the invention can realize real-time online monitoring of the separation process, has good process reproducibility, simple operation, short preparation process, less organic solvent consumption, economic and environmental protection, high economic benefits, 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 simultaneously preparing two dihydroflavonoid glycoside monomers by utilizing industrial chromatography technology, and specifically to a method for simultaneously preparing naringin and neohesperidin by utilizing an industrial dynamic axial compression column system. Background Art

[0002] Citrus aurantium is the dried young fruit of Citrus aurantium L. and its cultivars or Citrussinensis Osbeck, which are Rutaceae plants. It has the effects of breaking up qi and eliminating stagnation, resolving phlegm and dispersing stagnation, and is used for stagnation of internal stagnation, fullness and pain, heaviness after diarrhea, constipation, phlegm stagnation and qi obstruction, chest pain, chest congestion, gastroptosis, rectal prolapse, uterine prolapse and other symptoms. Literature reports show that Citrus aurantium mainly contains flavonoids, alkaloids, volatile oils, coumarins and other ingredients. In terms of pharmacological effects, it includes effects on the gastrointestinal tract, antioxidant effects, anti-inflammatory effects, antibacterial effects, etc. For the effects on the gastrointestinal tract, the main active ingredients are flavonoids and volatile oils. Studies have shown that several dihydroflavonoids in Citrus aurantium (such as neohesperidin, naringin, etc.) can improve gastric emptying and small intestinal propulsion in rats with functional dyspepsia, and can improve the symptoms of gastric ulcers in rats induced by indomethacin, and are suitable for the development of drugs for the treatment of functional dyspepsia. However, there are problems such as cumbersome and complicated separation process and large amount of organic solvents for dihydroflavonoids, resulting in a small number of dihydroflavonoid standard products and high cost, which seriously limits the quality control of Citrus aurantium / Citrus aurantium and its related products, and affects the further research and development of Citrus aurantium / Citrus aurantium. Therefore, the development of a simple and rapid method for separating and purifying high-purity dihydroflavonoids is the focus of current research.

[0003] The separation and purification of monomer active ingredients is one of the most important contents of Chinese herbal medicine research. It is the preliminary basis for the research of herbal medicine and also the key link that affects the research speed. There are many traditional separation and purification methods and technologies, including organic solvent extraction, recrystallization, precipitation and chromatography. All of the above methods have the defects of large amount of organic solvent, cumbersome preparation process, long production cycle, low purity of prepared monomers and difficulty in industrial promotion. At present, neohesperidin and naringin components cannot be prepared in large quantities, which seriously limits the quality control of herbal medicines containing neohesperidin and naringin components and their related products. Therefore, the development of a simple and rapid method for separating and purifying high-purity neohesperidin and naringin components is the focus of current research.

[0004] CN1053997874A discloses a method for extracting hesperidin, neohesperidin and synephrine from Citrus aurantium immaturus, CN106220698A discloses a method for separating high-purity hesperidin, neohesperidin, naringin and synephrine from Citrus aurantium immaturus, CN102617673A discloses a method for separating and purifying naringin and neohesperidin from the white cortex of Citrus grandis, CN1594344A discloses a method for separating high-purity neohesperidin from Citrus aurantium fructus, CN101293906A discloses a method for extracting high-purity neohesperidin from Citrus aurantium fructus, CN1431216A discloses a method for extracting naringin from Citrus aurantium fructus, and CN109912666A discloses a method for simultaneously extracting high-purity naringin and neohesperidin from Citrus aurantium fructus. However, these processes either produce active ingredients of low purity, which are mixtures of multiple substances without thorough separation and purification; or the purity and yield of various components are low; or the separation and purification process is difficult to industrialize and can only be achieved in small batches, and the separation process cannot be monitored online in real time, and the separation process is blind and has low sensitivity. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a method for simultaneously preparing two dihydroflavonoid glycoside monomers by using industrial chromatography technology. The method comprises injecting a total flavonoid glycoside extract sample solution into a dynamic axial compression column system for purification and separation to obtain two monomer compounds, naringin and neohesperidin. The method has the characteristics of small amount of organic solvent used, simple preparation process, short production cycle, easy industrialization, environmental friendliness, etc.

[0006] The present invention provides a method for simultaneously preparing two dihydroflavonoid glycoside monomer compounds by using industrial chromatography technology, wherein the dihydroflavonoid glycoside monomer compounds are naringin and neohesperidin, comprising the following steps:

[0007] Take the total flavonoid glycoside extract, add an appropriate amount of solvent 1 to dissolve it and prepare a solution of a certain concentration, filter it, and prepare a sample solution; inject the sample solution into an industrial dynamic axial compression column system according to a certain injection volume, then elute it with solvent 2, use a dual-wavelength ultraviolet online detector to detect the separation of each component, collect the eluate of each component, and then concentrate and dry it respectively.

[0008] Preferably, the total flavonoid glycoside extract is a total flavonoid glycoside extract of Citrus aurantium and / or Citrus aurantium; and / or, the naringin content in the total flavonoid glycoside extract is 10.0%-40.0% (W / W), preferably 15.0%-40.0% (W / W); the neohesperidin content is 15.0%-50.0% (W / W), preferably 20.0%-45.0% (W / W).

[0009] Preferably, solvent 1 is a 2.0%-10.0% (V / V) ethanol aqueous solution; preferably a 5.0%-8.0% (V / V) ethanol aqueous solution.

[0010] Preferably, the concentration of the solution is 85-110 mg / mL; preferably 90-105 mg / mL.

[0011] Preferably, the filler of the industrial dynamic axial compression column system is a reverse phase filler, preferably UniSil 30-100 (C18) or CG161M filler.

[0012] Preferably, the solvent 2 is one or more of ethanol, water, and trifluoroacetic acid; preferably a mixed solution of ethanol, water, and trifluoroacetic acid; the volume ratio of the three is preferably 35-45:65-55:0.3, for example 35:65:0.3, 40:60:0.3, 45:55:0.3.

[0013] Preferably, the elution flow rate is 5.0-10.0CV / h; preferably 7.5-10.0CV / h; and / or, the injection volume is 100-120L; and / or, the collection start and end times of the eluates of the two monomer compounds of naringin and neohesperidin are 145-175min and 225-260min, respectively.

[0014] Preferably, the detection wavelength of the ultraviolet online detector is 283 nm and the reference wavelength is 210 nm.

[0015] Preferably, the concentration is reduced pressure concentration; and / or the concentration temperature is 60-70°C, preferably 65°C; and / or the drying is vacuum drying; and / or the drying temperature is 40-50°C, preferably 45°C.

[0016] Preferably, the preparation method of the total flavonoid glycoside extract is:

[0017] Take an appropriate amount of medicinal materials containing total flavonoid glycosides, decoct in water and filter, pass the filtrate through a non-polar macroporous resin, elute with water, ethanol and sodium hydroxide in sequence, collect the eluate, concentrate and dry.

[0018] More preferably, in the ethanol elution step, low concentration ethanol (such as below 20% (V / V)) can be used first, and then higher concentration ethanol (such as 25%-90% (V / V)) can be used; and / or, the concentration of sodium hydroxide is 0.5%-2% (W / W).

[0019] More preferably, the concentration is reduced-pressure concentration, more preferably reduced-pressure concentration to a fluid extract with a relative density of 1.20-1.40; and / or the concentration temperature is 50-70°C.

[0020] More preferably, the drying is reduced pressure drying; and / or the drying temperature is 60-80°C.

[0021] More preferably, the method further comprises a pulverizing step.

[0022] More preferably, the preparation method is: take Citrus aurantium or Citrus aurantium, cut into slices or crush, add 4-15 times the weight of water, decoct several times (such as 1, 2, 3 times, etc.), decoct for 1-3 hours each time, combine the decoctions, filter, pass the filtrate through a non-polar macroporous resin, elute with water and 20% (V / V) or less ethanol in turn to remove impurities, then elute with 25%-90% (V / V) ethanol and 0.5-2% sodium hydroxide, collect the 25%-90% (V / V) ethanol eluate, concentrate under reduced pressure at 50°C-70°C to a fluid extract with a relative density of about 1.20-1.40, dry under reduced pressure at 60-80°C, and crush.

[0023] Beneficial technical effects of the present invention:

[0024] 1. The present invention separates and purifies the total flavonoid glycoside extract (such as the total flavonoid glycoside extract of Citrus aurantium or Citrus aurantium) directly through an industrial dynamic axial compression column system to obtain two monomer compounds, naringin and neohesperidin, both of which have a purity of more than 99.5%, thereby solving the problems of difficult preparation and high price.

[0025] 2. The present invention utilizes industrial chromatography technology, that is, a method for simultaneously preparing naringin and neohesperidin monomer compounds using a dynamic axial compression column system, which can realize real-time online monitoring of the separation process, has good repeatability, simple operation, short preparation process, and low organic solvent usage. The method has high economic benefits and can be industrially prepared in large quantities.

[0026] 3. The present invention adopts industrial dynamic axial compression column system preparation technology to simultaneously prepare multiple monomer compounds from one medicinal material, thereby improving the utilization rate of the medicinal material, saving costs, and can be extended to other similar varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : HPLC chromatogram of total flavonoid glycosides extract from Citrus aurantium;

[0028] Figure 2 :Process flow chart for preparing neohesperidin and naringin;

[0029] Figure 3 : HPLC chromatogram of naringin;

[0030] Figure 4 : HPLC chromatogram of neohesperidin. DETAILED DESCRIPTION

[0031] In order to better understand the present invention, the technical scheme of the present invention is further described and illustrated by the following examples.

[0032] Example 1

[0033] Take the immature bitter orange, cut it into slices or crush it, add 4 times the amount of water, decoct it 3 times, decoct it for 3 hours each time, combine the decoctions, filter, pass the filtrate through the treated non-polar macroporous resin, and elute with water, 20% or less ethanol (19%) to remove impurities, then elute with 25% ethanol and 2% sodium hydroxide, collect the 25% ethanol eluate, concentrate it under reduced pressure at 50-70°C to a fluid extract with a relative density of about 1.40, dry it under reduced pressure at 80°C, and crush it to obtain the total flavonoid extract of the immature bitter orange (its HPLC chromatogram is shown in Figure 1 ).

[0034] Example 2

[0035] Take Citrus aurantium, cut into slices or crush, add 15 times the amount of water, decoct once, decoct and extract for 1 hour each time, combine the decoctions, filter, pass the filtrate through a treated non-polar macroporous resin, and elute with water and ethanol (15%) below 20% in sequence to remove impurities, then elute with 90% ethanol and 0.5% sodium hydroxide, collect the 90% ethanol eluate, concentrate under reduced pressure at 50-70°C to a fluid extract with a relative density of about 1.20, dry under reduced pressure at 60°C, and crush to obtain the Citrus aurantium total flavonoid extract.

[0036] Example 3

[0037] A method for simultaneously preparing two dihydroflavonoid glycoside monomer compounds using industrial chromatography technology (process flow see Figure 2 ), wherein the dihydroflavonoid glycoside monomer compounds are naringin and neohesperidin, comprising the following steps:

[0038] Take the total flavonoid glycosides extract of Citrus aurantium obtained in Example 1 (its HPLC chromatogram is shown in Figure 1 ), wherein the content of naringin is about 15.0% (W / W), and the content of neohesperidin is about 20.0% (W / W), 5.0% (V / V) ethanol aqueous solution is added to dissolve it and prepare a concentration solution of 105 mg / ml, and filter to obtain a sample solution.

[0039] 100L of the sample solution was injected into an industrial dynamic axial compression column system, the industrial chromatographic filler was UniSil30-100 (C18), ethanol: water: trifluoroacetic acid solution (35:65:0.3, V / V / V) was the eluent, the elution flow rate was 10.0CV / h, the UV detection wavelength was 283nn, and the reference wavelength was 210nm. According to the peak time of the neohesperidin and naringin components and the height of the chromatographic peak, the start and end time of the eluent collection of the prepared naringin was determined to be 145-175min, and the start and end time of the eluent collection of the prepared neohesperidin was determined to be 225-260min, and the eluent containing the target component was concentrated to dryness under reduced pressure at 65°C and then vacuum dried at 45°C for 24h to obtain 1338.75g of a naringin monomer compound with a purity of 99.69% (its HPLC chromatogram is shown in Figure 3 ; 1836.47 g of new hesperidin monomer compound with a purity of 99.65% was obtained (its HPLC chromatogram is shown in Figure 4 ).

[0040] Example 4

[0041] A method for simultaneously preparing two dihydroflavonoid glycoside monomer compounds using industrial chromatography technology (process flow see Figure 2 ), wherein the dihydroflavonoid glycoside monomer compounds are naringin and neohesperidin, comprising the following steps:

[0042] Take the total flavonoid glycoside extract obtained in Example 2, in which the naringin content is about 40.0% (W / W) and the neohesperidin content is about 45.0% (W / W), add 8.0% (V / V) ethanol aqueous solution to dissolve it and prepare a 90 mg / ml concentration solution, filter it, and obtain a sample solution.

[0043] 120L of sample solution is injected into an industrial dynamic axial compression column system, the industrial chromatographic filler is CG161M, ethanol: water: trifluoroacetic acid solution (45:55:0.3, V / V / V) is the eluent, the elution flow rate is 7.5CV / h, the ultraviolet detection wavelength is 283nn, and the reference wavelength is 210nm. According to the peak time of the neohesperidin and naringin components and the height of the chromatographic peak, the collection start and end time of the prepared naringin eluate is determined to be 145-175min, and the collection start and end time of the prepared neohesperidin eluate is determined to be 225-260min, and the eluate containing the target component is concentrated to dryness at 65°C under reduced pressure and then vacuum dried at 45°C for 24h to obtain 3672.13g of naringin monomer compound with a purity of 99.58%; and 4106.25g of neohesperidin monomer compound with a purity of 99.65% is obtained.

[0044] 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 simultaneously preparing two dihydroflavonoid glycoside monomer compounds using industrial chromatography technology, wherein the dihydroflavonoid glycoside monomer compounds are naringin and neohesperidin, The following steps are involved: Take the total flavonoid glycoside extract, add an appropriate amount of 2.0%-10.0% V / V ethanol aqueous solution to dissolve it and prepare a solution of a certain concentration, filter it, and prepare a sample solution; inject the sample solution into an industrial dynamic axial compression column system according to a certain injection volume, the industrial dynamic axial compression column system filler is UniSil 30-100 or CG161M reverse phase filler, and then elute with a mixed solution of ethanol, water, and trifluoroacetic acid with a volume ratio of 35-45:65-55:0.3, the elution flow rate is 5.0-10.0CV / h, the injection volume is 100-120L, and the separation of each component is detected by a dual-wavelength ultraviolet online detector, the detection wavelength is 283nm, and the reference wavelength is 210nm, and the eluate of each component is collected, and the collection start and end time of the eluate of the two monomer compounds of naringin and neohesperidin are 145-175min and 225-260min respectively, and then they are concentrated and dried respectively; The preparation method of the total flavonoid glycoside extract is as follows: Take Citrus aurantium immaturus or Citrus aurantium fructus, cut into slices or crush, add 4-15 times the weight of water, decoct several times 1, 2 or 3 times, decoct for 1-3 hours each time, combine the decoctions, filter, pass the filtrate through a non-polar macroporous resin, elute with water and ethanol below 20% V / V in sequence to remove impurities, then elute with 25%-90% V / V ethanol and 0.5%-2% sodium hydroxide, collect the 25%-90% V / V ethanol eluate, concentrate under reduced pressure at 50°C-70°C to a fluid extract with a relative density of 1.20-1.40, dry under reduced pressure at 60-80°C, and crush.

2. The method according to claim 1, wherein the total flavonoid glycoside extract is a total flavonoid glycoside extract of Citrus aurantium and / or Citrus aurantium. in, The content of naringin in the total flavonoid glycoside extract is 15.0%-40.0% W / W; the content of neohesperidin is 20.0%-45.0% W / W.

3. The method according to claim 1, wherein the ethanol aqueous solution is a 5.0%-8.0% V / V ethanol aqueous solution.

4. The method according to claim 1, wherein the concentration of the solution is 90-105 mg / mL.

5. The method according to claim 1, wherein the volume ratio of the mixed solution of ethanol, water and trifluoroacetic acid is 35:65:0.3, 40:60:0.3 or 45:55:0.

3. The method according to claim 1 , wherein the elution flow rate is 7.5-10.0 CV / h.

7. The method according to claim 1, wherein the concentration is reduced pressure concentration at a concentration temperature of 60-70°C.

8. The method according to claim 1, wherein the drying is vacuum drying at a drying temperature of 40-50°C.

9. The method according to claim 1, wherein the concentration temperature is 65°C; and the drying temperature is 45°C.

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