Method for refining 8-O-4, 8-O-4-dehydrotriferulic acid from ginseng medicinal material

Abstract

The invention relates to the technical field of compound preparation, in particular to a method for refining 8-O-4, 8-O-4-dehydrotriferulic acid from a ginseng medicinal material, which comprises the following steps: S100, extraction: taking a certain amount of medicinal ginseng, crushing the medicinal ginseng into particles, extracting for a plurality of times by using 80% ethanol, and concentrating the extracting solution to obtain an extract concentrated solution; s200, enrichment: adding a certain amount of AB-8 type macroporous adsorption resin into the extract concentrated solution prepared in the step S100 for static adsorption for a certain time, placing the AB-8 type macroporous adsorption resin on a glass column, sequentially eluting with pure water and 10% ethanol until effluent is colorless, eluting with 30% ethanol until the effluent is colorless to obtain an eluent rich in a target compound, and collecting the eluent rich in the target compound. And concentrating the eluent until no alcohol exists, so as to obtain the extract. The refining method provided by the invention is good in repeatability and stability, simple and convenient to operate and easy to realize automatic control.

Description

Technical Field

[0001] The invention relates to the technical field of compound preparation, and in particular to a method for refining 8-O-4,8-O-4-dehydrotriferulic acid from ginseng medicinal materials. Background Art

[0002] Modern medical research has shown that ginseng has a variety of biological and pharmacological properties, such as immune stimulation, anti-cancer, anti-emetic, antioxidant, etc. These properties are closely related to the chemical components in ginseng, such as ginsenosides, ginseng polysaccharides, alkaloids, free amino acids, polyphenols, and volatile compounds such as limonene.

[0003] At present, there are more studies on ginsenosides, but relatively less knowledge on polyphenols. Some countries have conducted some studies in the early stage. For example, it was found that polyphenols, especially p-coumaric acid and vanillic acid, rather than ginsenosides, play a role in scavenging NO in ginseng. Phenolic compounds in raw sun-dried ginseng extracts show strong depigmentation, improved oxidative stress and reduced atherosclerosis properties, and have a strong inhibitory effect on the oxidation of rat liver microsomal lipids.

[0004] It is now found that the content of 8-O-4,8-O-4-dehydrotriferulic acid in ginseng polyphenols is more than 10 times that of p-coumaric acid, which is the main substance of ginseng polyphenols. Existing technical research extracts more total saponins and total polysaccharides from ginseng. Some known extraction methods use low-temperature plasma-complex enzyme extraction methods. The price of complex enzymes is high and they are easily inactivated. The temperature control requirements during the experiment are strict. Because the optimal temperature of the enzyme is within a very small range, changes in conditions may lead to enzyme inactivation, and the structure of polyphenols may change due to the action of the enzyme. In addition, this method extracts total polyphenols.

[0005] Therefore, it is of great significance to conduct in-depth research on 8-O-4,8-O-4-dehydrotriferulic acid and purify 8-O-4,8-O-4-dehydrotriferulic acid from ginseng. Summary of the invention

[0006] The present invention aims at the deficiencies in the prior art and provides the following technical solutions:

[0007] A method for refining 8-O-4,8-O-4-dehydrotriferulic acid from ginseng medicinal materials comprises the following steps:

[0008] S100, extraction: taking a certain amount of medicinal ginseng, crushing it into particles, extracting it with 80% ethanol several times, and then concentrating the extract to obtain an extract concentrate;

[0009] S200, enrichment: adding a certain amount of AB-8 macroporous adsorption resin to the extract concentrate prepared in S100 for static adsorption for a certain period of time, then placing the AB-8 macroporous adsorption resin on a glass column, eluting with pure water and 10% ethanol in sequence until the effluent is colorless, and then eluting with 30% ethanol until the effluent is colorless to obtain an eluate rich in target compounds, and then concentrating the eluate until it is alcohol-free to obtain an extract concentrate;

[0010] S300, purification: pumping the extract prepared in S200 into a medium-pressure column, eluting with a certain amount of pure water and methanol in turn, collecting the methanol eluate, concentrating, and freeze-drying to obtain freeze-dried powder;

[0011] S400, purification: take the lyophilized powder obtained in S300, dissolve it in 30% acetonitrile containing 0.1% formic acid to prepare a 25 mg / ml solution, filter it through a microporous filter membrane, and then inject the filtered solution, 2 ml each time, use the elution system solution to elute the chromatographic column, control the flow rate at 60 L / min, monitor online with ultraviolet light, collect the eluate containing 8-O-4,8-O-4-dehydrotriferulic acid, and concentrate the collected eluate under reduced pressure to about 500 ml, and freeze-dry to obtain the refined target product.

[0012] As an improvement of the above technical solution, in S100, the particle size of the ginseng after crushing is 0.17-0.32 mm.

[0013] As an improvement of the above technical solution, in S100, 80% ethanol is used for extraction twice, and the 80% ethanol used for each extraction is 10 times the weight of the medicinal ginseng. The extract is concentrated 10 times to obtain an extract concentrate.

[0014] As an improvement of the above technical solution, in S200, the amount of AB-8 macroporous adsorption resin used is 0.5 times the mass of the extract concentrate.

[0015] Beneficial effects of the present invention:

[0016] Without causing changes in the structure of polyphenols, the monomer component 8-O-4,8-O-4-dehydrotriferulic acid with a purity greater than 98% was obtained by using macroporous resin enrichment and preparative high performance liquid chromatography as separation means. The refining method has good repeatability and stability, and is simple, convenient and easy to realize automatic control. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A flow chart for the purification of 8-O-4,8-O-4-dehydrotriferulic acid from ginseng;

[0018] Figure 2 is the chemical formula of 8-O-4,8-O-4-dehydrotriferulic acid purified from ginseng;

[0019] Figure 3 It is the HPLC chromatogram of 8-O-4,8-O-4-dehydrotriferulic acid;

[0020] Figure 4 It is the MS spectrum of 8-O-4,8-O-4-dehydrotriferulic acid;

[0021] Figure 5 It is the 1H-NMR spectrum of 8-O-4,8-O-4-dehydrotriferulic acid;

[0022] Figure 6 8-O-4,8-O-4-dehydrotriferulic acid 13 C-NMR spectrum;

[0023] Figure 7 It is the IR spectrum of 8-O-4,8-O-4-dehydrotriferulic acid;

[0024] Figure 8 It is the UV spectrum of 8-O-4,8-O-4-dehydrotriferulic acid;

[0025] Fig. 9 is a high performance liquid chromatography analysis chart of the extract concentrate obtained by the first extraction in S100;

[0026] Fig.10 is a high performance liquid chromatography analysis chart of the extract concentrate obtained by the second extraction in S100;

[0027] Fig.11 This is a high performance liquid chromatography analysis chart of the extract concentrate obtained by the third extraction in S100. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] Existing technical studies have been conducted on extracting total saponins and total polysaccharides from ginseng. Some known extraction methods use low-temperature plasma-complex enzyme extraction methods. The complex enzyme is expensive and easily inactivated. The temperature control during the experiment requires strict control because the optimal temperature of the enzyme is within a very small range. Changes in conditions may lead to enzyme inactivation, and the structure of polyphenols may change due to the action of the enzyme. In addition, this method extracts total polyphenols.

[0030] See attached Figure 1-Figure 11In order to purify 8-O-4,8-O-4-dehydrotriferulic acid from ginseng, a method for purifying 8-O-4,8-O-4-dehydrotriferulic acid from ginseng medicinal materials is provided, comprising the following steps:

[0031] S100, extraction: taking a certain amount of medicinal ginseng, crushing it into particles with a particle size of 0.17-0.32 mm, extracting it several times with 80% ethanol, and then concentrating the extract to obtain an extract concentrate;

[0032] Preferably, the particle size is 0.24 mm.

[0033] S200, enrichment: add a certain amount of AB-8 macroporous adsorption resin to the extract concentrate prepared in S100 for static adsorption for a certain period of time, then place the AB-8 macroporous adsorption resin on a glass column, and elute with pure water and 10% ethanol in sequence until the effluent is colorless, and then elute with 30% ethanol until the effluent is colorless to obtain an eluate rich in target compounds, and then concentrate the eluate until it is alcohol-free to obtain an extract concentrate.

[0034] Specifically, 80% ethanol is used for extraction twice, and the 80% ethanol used for each extraction is 10 times the weight of the medicinal ginseng. The extract is concentrated 10 times to obtain an extract concentrate.

[0035] Preferably, the amount of AB-8 macroporous adsorption resin used is 0.5 times the mass of the extract concentrate.

[0036] S300, purification: pump the extract prepared in S200 into a medium-pressure column, elute with a certain amount of pure water and methanol in turn, collect the methanol eluate, concentrate, and freeze-dry to obtain freeze-dried powder.

[0037] Among them, the filler in the medium-pressure column is Daisogel-C18, 40-60 μm, and the column size is 100×460 mm.

[0038] S400, purification: take the lyophilized powder obtained in S300, dissolve it in 30% acetonitrile containing 0.1% formic acid to prepare a 25 mg / ml solution, filter it through a microporous filter membrane, and then inject the filtered solution, 2 ml each time, use the elution system solution to elute the chromatographic column, control the flow rate at 60 L / min, monitor online with ultraviolet light, collect the eluate containing 8-O-4,8-O-4-dehydrotriferulic acid, and concentrate the collected eluate under reduced pressure to about 500 ml, and freeze-dry to obtain the refined target product.

[0039] Among them, the refining is carried out by using preparative high performance liquid chromatography as the separation means and acetonitrile-0.5% formic acid solution (35:65) as the elution system, and finally the monomer component 8-O-4,8-O-4-dehydrotriferulic acid with a purity higher than 98% is obtained, thus achieving the refining.

[0040] In order to further illustrate the technical solution of the present application, the ethanol volume fraction in S100 was adjusted and compared.

[0041] Specifically, 10 times the volume fraction of 0-95% ethanol solution was used for heating and reflux extraction twice, each time for 3 hours, and the extract was concentrated under reduced pressure until there was no alcohol to obtain an extract concentrate. The obtained extract concentrate was analyzed by high performance liquid chromatography to obtain Table 1:

[0042] Ethanol volume fraction (%) 0 30 50 80 95 Content (μg / ml) 0.76 1.41 2.22 8.25 32.96 Concentrate volume (ml) 1890 1300 940 360 90 Total content (mg) 1.44 1.83 2.09 2.97 2.97

[0043] As can be seen from Table 1, when the volume fraction of ethanol is 80% to 95%, the quality of the extract concentrate is optimal, and when 80% ethanol is used, the material consumption is less, so it is the most preferred.

[0044] In order to further illustrate the technical solution of the present application, the masses of different multiples of 80% ethanol solution extract concentrate in S100 were compared.

[0045] Specifically, 6-12 times the amount of 80% ethanol solution was used for heating and reflux extraction twice, each time for 3 hours, and the extract was concentrated under reduced pressure until there was no alcohol taste to obtain an extract concentrate. The extract concentrate was analyzed by high performance liquid chromatography to obtain Table 2:

[0046] Solvent multiple times 6 times 8 times 10 times 12 times Content (μg / ml) 11.47 9.74 8.25 7.07 Concentrate volume (ml) 210 270 360 430 Total content (mg) 2.41 2.63 2.97 3.04

[0047] As can be seen from Table 2, when the dosage of 80% ethanol solution is 10 to 12 times, the content of the extract is optimal; but when the dosage of 80% ethanol solution is 10, the consumption is significantly reduced, so it is the most preferred.

[0048] In order to further illustrate the technical solution of the present application, a comparative explanation is given on the number of extractions in S100.

[0049] Specifically, 10 times the amount of 80% ethanol is used for heating and reflux extraction 1-3 times, each time for 3 hours, and each extract is concentrated under reduced pressure until there is no alcohol taste to obtain an extract concentrate, and the extract concentrate is analyzed by high performance liquid chromatography to obtain Figure 9-10 .

[0050] There was basically no chromatographic peak of the target substance in the third extraction, so two extractions were sufficient.

[0051] In order to further illustrate the technical solution of the present application, a comparative explanation is given on the extraction time in S200.

[0052] Specifically, 10 times the amount of 80% ethanol was used for heating and reflux extraction twice, each time for 1-4 hours, and the extract was concentrated under reduced pressure each time until there was no alcohol taste, to obtain an extract concentrate, and the extract concentrate was analyzed by high performance liquid chromatography to obtain Table 3:

[0053] Each extraction time (h) 1 2 3 4 Content (μg / ml) 5.11 7.00 8.25 8.31 Concentrate volume (ml) 360 360 360 360 Total content (mg) 1.84 2.52 2.97 2.99

[0054] It can be seen from Table 3 that the content extracted after 3 hours of extraction is basically the same as that after 4 hours of extraction, but the time difference is one hour. Therefore, in general, the extraction quality and efficiency of 3 hours of extraction are the best.

[0055] In order to further illustrate the technical solution of the present application, a comparative description is given of the enrichment process in S200.

[0056] Specifically, the extract concentrate was enriched on macroporous resin AB-8, and eluted with water, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 95% ethanol solutions in sequence until the effluent was colorless. The results were analyzed by high performance chromatography and obtained as shown in Table 4:

[0057] Ethanol volume fraction (%) 0 10 20 30 40 50 60 70 80 95 Peak area 0 0 20092 381762 0 0 0 0 0 0

[0058] It can be seen from Table 4 that 95% of 8-O-4,8-O-4-dehydrotriferulic acid is concentrated in the 30% ethanol eluent, and 5% is concentrated in the 20% ethanol eluent, so the elution effect using the 30% ethanol eluent is the best.

[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them.

Claims

1. A method for refining 8-O-4,8-O-4-dehydrotriferulic acid from ginseng medicinal materials, characterized in that: The following steps are involved: S100, extraction: taking a certain amount of medicinal ginseng, crushing it into particles, extracting it with 80% ethanol several times, and then concentrating the extract to obtain an extract concentrate; S200, enrichment: adding a certain amount of AB-8 macroporous adsorption resin to the extract concentrate prepared in S100 for static adsorption for a certain period of time, then placing the AB-8 macroporous adsorption resin on a glass column, eluting with pure water and 10% ethanol in sequence until the effluent is colorless, and then eluting with 30% ethanol until the effluent is colorless to obtain an eluate rich in target compounds, and then concentrating the eluate until it is alcohol-free to obtain an extract concentrate; S300, purification: pumping the extract prepared in S200 into a medium-pressure column, eluting with a certain amount of pure water and methanol in turn, collecting the methanol eluate, concentrating, and freeze-drying to obtain freeze-dried powder; S400, purification: take the lyophilized powder obtained in S300, dissolve it in 30% acetonitrile containing 0.1% formic acid to prepare a 25 mg / ml solution, filter it through a microporous filter membrane, and then inject the filtered solution, 2 ml each time, use the elution system solution to elute the chromatographic column, control the flow rate at 60 L / min, monitor online with ultraviolet light, collect the eluate containing 8-O-4,8-O-4-dehydrotriferulic acid, and concentrate the collected eluate under reduced pressure to about 500 ml, and freeze-dry to obtain the refined target product.

2. The method for refining 8-O-4,8-O-4-dehydrotriferulic acid from ginseng medicinal materials according to claim 1, characterized in that: In S100, the ginseng is crushed to have a particle size of 0.17-0.32 mm.

3. The method for refining 8-O-4,8-O-4-dehydrotriferulic acid from ginseng medicinal materials according to claim 1, characterized in that: In S100, 80% ethanol is used for extraction twice, and the 80% ethanol used for each extraction is 10 times the weight of the medicinal ginseng. The extract is concentrated 10 times to obtain an extract concentrate.

4. The method for refining 8-O-4,8-O-4-dehydrotriferulic acid from ginseng medicinal materials according to claim 1, characterized in that: In S200, the amount of AB-8 macroporous adsorption resin used is 0.5 times the mass of the extract concentrate.

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