CN113372354A

CN113372354A - Preparation method of violaxanthine

Info

Publication number: CN113372354A
Application number: CN202110574064.2A
Authority: CN
Inventors: 文怀秀; 苑祥; 陶燕铎; 邵贇; 梅丽娟
Original assignee: Northwest Institute of Plateau Biology of CAS
Current assignee: Northwest Institute of Plateau Biology of CAS
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2021-09-10

Abstract

The invention discloses a preparation method of violaxanthine, which comprises the following steps: 1) crushing the fennel fruits, adding ethanol for extraction, filtering, and concentrating into thick paste with the relative density of 1.1-1.3; 2) loading the thick paste obtained in the step 1) into a macroporous resin column, eluting with water and ethanol in sequence, collecting ethanol elution parts, concentrating and drying to obtain an illicium tenuipes extract; 3) and (3) taking the fine-fruited caraway extract obtained in the step 2), adding methanol for dissolving, filtering, separating the filtrate by using preparative high performance liquid chromatography, collecting chromatographic peak fractions with the highest response values in the preparative chromatogram, concentrating and drying to obtain the caraway. The purity of the violaxylamine prepared by the preparation method is more than 98 percent.

Description

Preparation method of violaxanthine

Technical Field

The invention relates to the technical field of natural medicinal chemistry, and particularly relates to a preparation method of violaxanthine.

Background

Hypecoum leptocarpum hook.f. et Thoms.) is a plant of Carum of Papaveraceae, is one of the authentic species of the traditional Tibetan medicine, and has the effects of treating cold and fever, pneumonia and cough, fever of febrile infectious diseases, hepatitis, cholecystitis, arthralgia, sore throat, conjunctival congestion, food poisoning and the like. Due to the lack of chemical reference substances, the quality control of the Tibetan medicine standard issued by the current ministry of health only has a microscopic identification item.

The research on chemical components of the hypecoum leptocarpum which can be found in the literature at present reports twenty-several alkaloid compounds including hypecorine (hypecorine), hypecolone (hypecorine), Protopine (Protopine), coptisine (coptisine), allocryptopine (allocryptopine), violamide (corydam-ine), levorotatory N-methyltetrahydroberberine (N-methylanadine), orthokeratodine (hyperethine) and the like, but the research on a large-scale preparation method and a chemical control product of a monomer compound is rarely reported, and particularly, the research on the scopolamine which can be used as a control product and is separated and extracted from the hypecoum leptocarpum in a large-scale manner is not reported.

Zhang Ying et al, "alkaloid composition analysis of Viola yedoensis Makino (J.). plant resource and Environment bulletin, 2008,17(4):67-69 provide a preparation method of Viola yedoensis Makino, which is obtained by extracting and separating Viola yedoensis Makino from Viola yedoensis Makino of Viola of Papaveraceae, wherein the used separation and purification reagents of petroleum ether, chloroform, ammonia water and the like are toxic, have pungent smell, are serious in environmental pollution and are not beneficial to industrial popularization and use, and meanwhile, the obtained Violamine has insufficient purity and cannot be used for calibrating the content of raw materials or preparations containing Violamine yedoensis Makino, so that a method for preparing monomer alkaloid in Illicium henryi in a large scale with simple process is urgently needed to be established.

Disclosure of Invention

In order to solve the problems, the invention provides a preparation method of violaxanthine, which comprises the following steps:

1) pulverizing fructus Anisi Stellati, extracting with ethanol, filtering, and concentrating to obtain soft extract;

2) loading the thick paste obtained in the step 1) into a macroporous resin column, eluting with water and ethanol in sequence, collecting ethanol elution parts, concentrating and drying to obtain an illicium tenuipes extract;

3) dissolving the fine extract of the caraway in methanol, filtering, separating the filtrate by preparative high performance liquid chromatography, collecting the fraction of the chromatographic peak with the highest response value in the preparative chromatogram, concentrating and drying to obtain the final product;

the conditions for preparing the high performance liquid chromatography are as follows:

stationary phase: c18 bonded silica gel column; mobile phase: acetonitrile water solution with volume fraction of 15-50%; the flow rate is 40-800 ml/min.

Furthermore, the adding amount of the ethanol in the step 1) is 7-13 times of the amount of the hypecoum leptocarpum in w/w and g/g, and preferably 10 times of the amount of the ethanol in w/w and g/g.

Further, the ethanol is 60-80% ethanol, preferably 70% ethanol.

Further, the extraction in the step 1) is reflux extraction, and the extraction is carried out for 3 times in total, and each time lasts for 2 hours; the relative density of the thick paste is 1.1-1.3.

Further, the macroporous resin column in the step 2) is a D101 type macroporous resin column, and the column diameter ratio is as follows: 1/20-1/5.

Further, the step 2) sequentially uses water and 10-90% ethanol for elution, and preferably sequentially uses water and 70% ethanol for elution.

Furthermore, the using amount of the water is 3-10 times of the column volume, and the using amount of the 70% ethanol is 3-20 times of the column volume; preferably, the amount of water is 5 column volumes and the amount of 70% ethanol is 5 column volumes.

Further, the concentration of the solution obtained in the step 3) after the methanol solution is added is 1-10 mg/mL; the filtration is carried out by using a 0.45 mu m microporous membrane; the conditions for preparing the high performance liquid chromatography are as follows: stationary phase: GL-200mm DAC dynamic column using C18 HCE with 10 μm aperture as filler; mobile phase: 30% volume fraction acetonitrile water solution; flow rate: 800 ml/min; the detection wavelength is 254 nm.

Further, the concentration in the step 1), the step 2) and the step 3) is reduced pressure concentration, and the specific parameters are that the vacuum degree is 50-250mbar and the temperature is 40-60 ℃.

The invention also provides the violaxanthine prepared by the method, and the purity of the violaxanthine is more than 98%.

The invention finally provides an application of the violaxanthine in preparation of a reference substance of isoquinoline alkaloid.

Compared with the prior art, the preparation method of the violaxanthine has the following advantages:

(1) the invention has simple and efficient process and high product purity

The extraction and elution solvents used in the invention can be recycled; the chromatographic materials (macroporous resin and C18 column material) can be reused. The recycled and reused separation material ensures that the average cost is low in the separation process, the separation efficiency is high, and the purity of the target product is more than 98 percent.

(2) The technical means adopted by the invention can realize large-scale production

The invention has low requirement on raw materials, wide distribution of the medicinal materials of the hypecoum leptocarpum, easy planting and easy batch preparation. The ethanol extraction is easy to operate; macroporous resin is adopted for enrichment, and the eluent for removing impurities is pure water, so that the cost is low; the part of the solvent washed by alcohol can be recycled, thus being beneficial to large-scale production; the refining part adopts a dynamic axial compression column, is a large-scale preparation chromatographic system, can realize large-scale production, and ensures the high purity of the product.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a DAC separation chromatogram of the present invention;

FIG. 2 shows the peak mass spectra of ESI-and ESI + molecular ions of viologen compounds isolated according to the present invention;

FIG. 3 is a 1H NMR nuclear magnetic spectrum of a viologen-substituted chemical control isolated according to the present invention;

FIG. 4 is 13C NMR nuclear magnetic spectrum of a violonyemide chemical reference substance separated according to the invention

FIG. 5 is a structural diagram of the compound scoulerine of the present invention;

FIG. 6 is an HPLC chromatogram of violaxanthine obtained by the present invention

FIG. 7 is the ultraviolet spectrum of violamine inscription obtained by the present invention

Detailed Description

Example 1 preparation of Oriolamine according to the invention

1. Taking 1kg of dried hypecoum leptocarpum medicinal material, crushing, adding 70% ethanol with the weight of 10 times w/w and g/g, reflux extracting for 3 times, 2h each time, filtering, combining filtrates to obtain filtrate A, and concentrating the filtrate under reduced pressure to thick paste with relative density of 1.1 under the conditions of vacuum degree of 50-250mbar and temperature of 40-60 ℃ to obtain a hypecoum leptocarpum crude extract sample;

2. taking the ethanol crude extract of the hypecoum leptocarpum in the step 1, adsorbing the sample by a D101 type macroporous resin column with the column diameter ratio of 1/20-1/5, eluting by 5 times of column water and 5 times of column volume of 70% ethanol in sequence, discarding a water washing part, collecting an elution part of the 70% ethanol, and concentrating and drying under reduced pressure at 40-60 ℃ under the vacuum degree of 50-250mbar to obtain an alkaloid refined extract sample of the hypecoum leptocarpum;

3. dissolving the fine extract of the caraway fruticosa alkaloid obtained in the step 2) with methanol to prepare a solution with the concentration of 1-10mg/mL, filtering with a 0.45-micrometer microporous filter membrane to obtain a filtrate B, carrying out large-scale preparative chromatography on the filtrate, separating by using a reverse phase separation column, detecting by using an ultraviolet detector with the detection wavelength of 254nm, collecting a chromatographic peak fraction with the highest response value in a prepared chromatogram, and drying the chromatographic peak fraction at 40-60 ℃ under reduced pressure under the condition of the vacuum degree of 50-250mbar to obtain the violaxanthin, wherein the purity of the violaxanthin is higher than 98% after analysis;

wherein, the working parameters of the large-scale preparative chromatography are as follows: a GL-200mm DAC dynamic column with C18 HCE with the pore diameter of 10 mu m as a filler is used as a stationary phase, and acetonitrile aqueous solution with the volume fraction of 30% is used as a mobile phase; flow rate: 800 ml/min; the detection wavelength is 254 nm.

The DAC separation chromatogram of the prepared violaxanthine is shown in figure 1, and the molecular ion peak mass spectrogram, 1H NMR nuclear magnetic spectrum, 13C NMR nuclear magnetic spectrum and the structural formula chart of the violaxanthine are shown in figures 2-5.

The advantageous effects of the present invention are described below by way of test examples.

Test example 1 Octophylline detection test

The violaxylamine prepared in example 1 was dissolved in methanol to give a 1mg/mL solution, which was filtered through a 0.45 μm microporous membrane and then analyzed for purity by HPLC. The stationary phase adopts a C18 Xcharge (250mm multiplied by 4.6mm,5 μm) analytical column; mobile phase: 0-45min, 5-95% acetonitrile-water; flow rate: 1 mL/min; column temperature: 30 ℃; operating time: 45 min; detection wavelength: 254 nm. The purity of the violamine is higher than 98.31% measured by an area normalization method, meets the requirement that the purity of a reference substance is higher than 98%, and can be used as the reference substance, and specific HPLC chromatograms and ultraviolet spectrograms are shown in figures 6 and 7.

In conclusion, the preparation method of the violamine carvifolia has low cost and high separation efficiency, can realize large-scale production, and the purity of the violamine carvifolia prepared by the method is more than 98%, and can be used as a reference substance.

Claims

1. The preparation method of the violaxanthine is characterized by comprising the following steps:

stationary phase: c18 bonded silica gel column; mobile phase: acetonitrile water solution with volume fraction of 15-50%; flow rate: 40-800 ml/min.

2. The method of claim 1, wherein: the adding amount of the ethanol in the step 1) is 7-13 times of the amount of the hypecoum leptocarpum in terms of w/w and g/g, and preferably 10 times of the amount of the ethanol in terms of w/w and g/g.

3. The method of claim 2, wherein: the ethanol is 60-80% ethanol, and preferably 70% ethanol.

4. The method of claim 1, wherein: the extraction in the step 1) is reflux extraction, and is carried out for 3 times, and each time lasts for 2 hours; the relative density of the thick paste is 1.1-1.3.

5. The method of claim 1, wherein: the macroporous resin column in the step 2) is a D101 type macroporous resin column, and the column diameter ratio is as follows: 1/20-1/5.

6. The method of claim 1, wherein: and 2) sequentially eluting with water and 10-90% ethanol, preferably sequentially eluting with water and 70% ethanol.

7. The method of claim 6, wherein: the using amount of the water is 3-10 times of the column volume, and the using amount of the 70% ethanol is 3-20 times of the column volume; preferably, the amount of water is 5 column volumes and the amount of 70% ethanol is 5 column volumes.

8. The method of claim 1, wherein: the concentration of the solution after being dissolved by adding methanol in the step 3) is 1-10 mg/mL; the filtration is carried out by using a 0.45 mu m microporous membrane; the conditions for preparing the high performance liquid chromatography are as follows: stationary phase: GL-200mm DAC dynamic column using C18 HCE with 10 μm aperture as filler; mobile phase: 30% volume fraction acetonitrile water solution; flow rate: 800 ml/min; the detection wavelength is 254 nm.

9. The method of claim 1, wherein: the concentration in the steps 1), 2) and 3) is reduced pressure concentration, and the specific parameters are that the vacuum degree is 50-250mbar and the temperature is 40-60 ℃.

10. An orchitamine prepared by the method of any one of claims 1 to 9, wherein: its purity is greater than 98%.

11. Use of violaxylamine according to claim 10 in the preparation of a control of isoquinoline alkaloids.