A method for extracting high-purity okanine from snow chrysanthemum and extract

Abstract

The present invention discloses a method for extracting high-purity oxanthene from snow chrysanthemum. In view of the problems in the prior art of extracting oxanthene from snow chrysanthemum, such as low yield and low purity of the obtained oxanthene, the present invention adds an acid-water solution extraction and hydrolysis step before macroporous resin column chromatography, so that oxanthene derivatives are converted into oxanthene, and the oxanthene concentration in the extract is increased by more than 10 times, thereby improving the oxanthene yield and reducing the difficulty of subsequent purification and production costs; in addition, the present invention optimizes process parameters such as macroporous resin column chromatography, crystallization, and recrystallization to effectively extract high-purity oxanthene, and the purity of the obtained oxanthene can reach more than 90%. The organic solvent used in the extraction is easy to remove from the product and avoids solvent residue, and the operation is safe.

Description

Technical Field

[0001] The present invention relates to a method for extracting high-purity okanin from snow chrysanthemum and okanin extract obtained by adopting the method. Background Art

[0002] Snow chrysanthemum, also known as two-color coreopsis (Coreopsis tinctoria.Nutt.), is an annual herb. Its main active ingredients include sugars, flavonoids, saponins, alkaloids, tannins, anthraquinones, and macromolecular pigment compounds.

[0003] Okanin is a flavonoid compound found in snow chrysanthemum, which has good antioxidant, anti-inflammatory, anti-aging, and anti-photoaging effects. In vitro studies have shown that okanin can significantly inhibit TLR4 expression caused by LPS and iNOS expression induced by LPS, and can also inhibit the production and mRNA expression of IL-6 and TNF-α stimulated by LPS, thereby having a good anti-inflammatory effect. In addition, some researchers have reported that okanin has a strong protective effect against UVB-induced cell damage. Because okanin has excellent antioxidant, anti-inflammatory, and anti-aging activities, it has broad prospects for application in the fields of medicine and skin care products. With the deepening of pharmacological research on okanin, the preparation process of okanin has also received more and more attention from researchers. However, okanin has more phenolic hydroxyl groups and a more complex stereo environment. The synthetic route obtained by chemical synthesis is cumbersome, costly, and the purity is difficult to guarantee.

[0004] Currently, okanin is mainly isolated and extracted from plants in the Asteraceae family. However, the content of okanin in Asteraceae plants is relatively low, ranging from 0.1% to 0.3%. Furthermore, the compounds in snow chrysanthemum are complex, so the purification of okanin is extremely difficult, with low yields and high production costs. Currently, there are several main methods for extracting okanin: water extraction, alcohol extraction followed by macroporous resin column chromatography, alcohol extraction followed by silica gel column chromatography, etc. These methods either have simple processes but low okanin content, or high okanin content but complicated processes and the use of large amounts of flammable and toxic organic solvents. Furthermore, the yield of okanin prepared by these methods from dry medicinal materials is not high, ranging from 0.01% to 0.05% based on dry snow chrysanthemum, resulting in the snow chrysanthemum medicinal material not being fully utilized.

[0005] The invention patent with patent number CN201310064628.3 discloses a method for extracting oxanine from snow chrysanthemum. After the snow chrysanthemum is extracted with alcohol, it is extracted with n-hexane and ethyl acetate. The extract is chromatographed on a silica gel column. After crystallization and recrystallization, oxanine is extracted. This method has a complicated process, a long production cycle and high production costs.

[0006] Patent number CN202211457503.2 discloses a method for preparing a snow chrysanthemum extract. After alcohol extraction of the snow chrysanthemum, the extract is subjected to macroporous resin column chromatography. The resulting snow chrysanthemum extract has an octanin content of 10%. A disadvantage of this method is that the snow chrysanthemum contains a large variety of active ingredients, making it difficult to isolate a single substance. The resulting substance is of low purity, resulting in low octanin purity.

[0007] The invention patent with patent number CN202111135486.6 discloses a method for preparing snow chrysanthemum extract, which uses water extraction, concentration drying and other methods to extract octanin. The octanin content in the final snow chrysanthemum crude extract is only 0.5%.

[0008] Therefore, the development of an industrially scalable process for preparing okanin would be of significant significance. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the defects of low purity and yield of okanthene extracted from snow chrysanthemum in the prior art, and to provide an extraction method for improving the yield of okanthene.

[0010] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0011] A method for extracting high-purity okanin from snow chrysanthemum comprises the following steps:

[0012] S1. Alcohol extraction: After the medicinal material is crushed, it is extracted with a solvent for 1 to 4 times, each extraction time is 0.2 hours to 5 hours, the extracts are combined, and the extracts are concentrated under reduced pressure to obtain the snow chrysanthemum alcohol extract; the solvent is one or a mixed solution of water, ethanol, and methanol; preferably, when the solvent is ethanol-water solution, the volume fraction of ethanol-water solution is 60 to 90%, preferably, the volume fraction of ethanol-water solution is 90%;

[0013] The amount of solvent added is the mass of medicinal materials: the mass of solvent = 1:5-1:30, and the extraction temperature is 30-100℃;

[0014] Preferably, the ratio of medicinal material mass to solvent mass is 1:10-15;

[0015] Preferably, the extraction temperature is 80°C;

[0016] S2, acid-water extraction and hydrolysis: add an acid-water solution with a mass concentration of 0.05%-4% to the snow chrysanthemum alcohol extract obtained in S1, stir, and filter to obtain an oxanin hydrolyzate; after acid hydrolysis, the oxanin derivatives (such as mariglycoside) in the snow chrysanthemum can be converted into oxanin, thereby improving the extraction rate of oxanin. Preferably, the acid in the acid-water solution is any one or more of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, oxalic acid, citric acid, malic acid or maleic acid; among them, hydrochloric acid is highly volatile and corrosive, and has high requirements for preparation equipment and process; nitric acid is easy to make explosive, difficult to obtain, and easily causes soil eutrophication, and is difficult to discharge sewage; phosphoric acid easily causes soil eutrophication, and is difficult to discharge sewage; acetic acid is highly volatile, has high requirements for the preparation environment, has low conversion, and requires a long hydrolysis time, which is not conducive to industrial production; oxalic acid is toxic and easily remains in the product, which is not conducive to the subsequent development and application of the product; preferably, the acid is sulfuric acid or hydrochloric acid; more preferably, the acid is sulfuric acid.

[0017] When the concentration of the acid aqueous solution is too low, the conversion rate of the oxanide derivative will be low and the conversion cannot be complete, resulting in a low yield of oxanide; when the concentration of the acid aqueous solution is too high, it may damage the structure of oxanide, resulting in a low yield of oxanide. In addition, when the acid concentration is very high, the requirements for production equipment and production environment are high, which greatly increases the difficulty of production and has low production feasibility.

[0018] Preferably, the concentration of the acid is 0.1-1%. Under this concentration requirement, the conversion rate of the okanin derivative is high, the requirements for production equipment and production environment are low, and the production feasibility is greatly increased.

[0019] In one embodiment, the temperature of the heating hydrolysis is 70-100°C, and the hydrolysis time is 1 to 8 hours; when the temperature is not high enough or the time is insufficient, the conversion rate of the oxanide derivative will be low and it will not be completely converted, resulting in a low yield of oxanide; when the temperature is too high or the time is too long, the structure of oxanide will be destroyed, resulting in a decrease in the yield of oxanide.

[0020] Preferably, the temperature for heating and hydrolysis is 80-95° C., and the hydrolysis time is 2 to 5 hours.

[0021] In one embodiment, the amount of the acid solution added is the mass of the medicinal material: the volume of the acid solution = 1:1 to 1:10, preferably 1:5 to 1:8.

[0022] S3. Macroporous resin column chromatography separation: The okanin hydrolyzate is subjected to macroporous resin separation, followed by gradient elution with alcohol-water solutions of varying volume fractions. The eluate is collected and dried to obtain a crude okanin extract having a purity greater than 40%;

[0023] The adsorption of oxanin by macroporous resin is a physical adsorption based on van der Waals forces. Oxanin is a weakly acidic substance. In an acidic environment, oxanin exists in molecular form and has excess electrons that easily generate van der Waals forces with the active groups of the macroporous resin, making it easily adsorbed and having a high adsorption capacity. In this application, an acid aqueous solution with a mass concentration of 0.05%-4% can achieve the highest adsorption capacity, further improving the extraction rate of oxanin.

[0024] Macroporous resin is a type of artificially synthesized organic high molecular polymer adsorbent with a porous three-dimensional structure, which can be used for physical adsorption due to its huge surface area. Macroporous resin has the advantages of good adsorption selectivity, simple regeneration, high physicochemical stability, mild desorption conditions and low cost, and is widely used in industrial production. The principle of using macroporous resin to separate and purify oxanin is to utilize the surface adsorption of macroporous resin and its hydrogen bond formation with the phenolic hydroxyl group of oxanin to selectively adsorb oxanin, and then use solvent molecules to form stronger hydrogen bonds with oxanin to elute oxanin, thereby achieving the purpose of purification and refinement.

[0025] The macroporous resin is LX-100B, LSA100, XDA-6, D101, AB-8, HPD-300 or X-5. Preferably, it is D101 or AB-8;

[0026] Preferably, the column volume is 1 to 1.5 L / BV, the ratio of the macroporous adsorption resin column diameter to the column height is 1:10 to 12, and the sample loading rate is 1 to 1.2 BV / h; gradient elution is performed using alcohol aqueous solutions with different volume fractions, and the eluate is collected to obtain three fractions, namely Fr.1 to Fr.3; Fr.3 is concentrated and dried to obtain a crude okanin extract with a purity greater than 40%;

[0027] The alcohol aqueous solution is preferably an ethanol aqueous solution or a methanol aqueous solution;

[0028] The eluents for each gradient during gradient elution are:

[0029] Gradient I, the eluent is purified water or a 5-10% alcohol aqueous solution, the amount used is 5-10 BV; preferably purified water;

[0030] Gradient II, the eluent is an ethanol aqueous solution with a volume fraction of 10-40%, the amount used is 5-15BV; preferably 30%;

[0031] Gradient III, the eluent is an ethanol aqueous solution with a volume fraction of 40-95%, the amount used is 5-15BV; preferably 60-70%;

[0032] The purpose of gradient elution is to elute flavonoids of different polarities successively with different alcohol-water solutions. If the alcohol concentration is too low or the amount of eluent is too little, the target component cannot be eluted completely. If the alcohol concentration is too high or the amount of eluent is too large, the separation effect of different flavonoids is poor, which will affect further crystallization and purification.

[0033] S4. Crystallization: Add the crude extract of oxalin into the solvent, heat and dissolve it, and then cool it down. Stir and crystallize it at 5°C and a stirring speed of 20-50 r / min for 6h-12h, then centrifuge and filter to collect the filter cake and filtrate. After drying the filter cake, the crude oxalin with a content of more than 60% oxalin is obtained.

[0034] The solvent can also be added to the crude okanin obtained in S4, heated to dissolve, cooled, stirred and crystallized at 5°C and a stirring speed of 20-50 r / min for 6h-12h, and then centrifuged and filtered to collect the filter cake and filtrate. The okanin content of the dried filter cake is greater than 80% of the refined okanin.

[0035] Preferably, the solvent for crystallization and recrystallization is one or more of water, ethanol, ethyl acetate, methanol, n-butanol, and acetone, preferably water or ethanol; when the solvent is ethanol-water solution, the volume concentration is 50% to 80%, preferably 60%.

[0036] The beneficial effects achieved by the present invention are as follows: the present invention improves the method for extracting high-purity oxanine from snow chrysanthemum, and adds an acid-water solution extraction and hydrolysis step before macroporous resin column chromatography, so that the oxanine derivative is converted into oxanine, and the oxanine concentration in the extract is increased by more than 10 times, thereby improving the oxanine yield and reducing the subsequent purification difficulty and production cost; by improving the process parameters such as macroporous resin column chromatography, crystallization, and recrystallization, the purity of the obtained oxanine product can reach more than 90%; at the same time, the organic solvent used in this application is safe to operate, easy to remove from the product and avoid solvent residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0038] Figure 1 It is the Okanian structure diagram;

[0039] Figure 2 is the HPLC chromatogram of the snow chrysanthemum alcohol extract obtained in S1 in Example 1;

[0040] Figure 3 is the HPLC chromatogram of the okanin hydrolyzate obtained in S2 in Example 1;

[0041] Figure 4 is the HPLC chromatogram of the crude extract of Okanin obtained in S3 in Example 1;

[0042] Figure 5 is the HPLC chromatogram of the crude okanin obtained in S4 in Example 1;

[0043] Figure 6 This is the HPLC chromatogram of the fine product of Okanin obtained in S5 in Example 1;

[0044] Figure 7 This is the HPLC chromatogram of the snow chrysanthemum extract of Comparative Example 1;

[0045] Figure 8 This is the HPLC chromatogram of the snow chrysanthemum extract of Comparative Example 2. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0047] Example 1

[0048] A method for extracting high-purity okanin from snow chrysanthemum comprises the following steps:

[0049] S1, get 1 kilogram of snow chrysanthemum, and grind into powder; The snow chrysanthemum powder is placed in an extraction container, 12L volume fraction is added to the container and it is 90% ethanol aqueous solution, reflux extraction 2 hours, separate ethanol extract, add the volume fraction of 10L and it is 90% ethanol aqueous solution and reflux extraction 1.5 hours, combine extracts, obtain snow chrysanthemum extract after concentrating and drying;

[0050] S2, add 5L of 0.1% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 95°C for 3h, and cool to room temperature to prepare an okanin hydrolyzate;

[0051] S3. Then apply AB-8 macroporous adsorption resin for separation (resin loading amount is 1.5 kg, column volume is 1 L / BV, and the ratio of macroporous adsorption resin column diameter to column height is 1:10), and the loading speed is 1BV / h; after the loading is completed, use 5BV purified water, 5BV 30% ethanol aqueous solution by volume, and 5BV 70% ethanol aqueous solution by volume for gradient elution in sequence, and the flow rate of gradient elution is 1-1.5BV / h. Collect 70% ethanol aqueous solution, concentrate and dry to obtain 28g of crude extract of okanin.

[0052] The crude extract of octanin was determined by HPLC to contain 58.5% octanin, and the yield from the dry medicinal material was 2.8% (yield calculation formula = weight of crude extract of octanin / weight of the input snow chrysanthemum medicinal material).

[0053] S4. Take the crude extract of okanin, add 10 mL of 60% ethanol aqueous solution, heat to dissolve, cool to precipitate and crystallize, filter with suction, collect the crystals, wash with water, and dry in the shade to obtain 17 g of crude okanin.

[0054] The crude product of okanaline was determined by HPLC, and the content of okanaline was 77.6%, and the yield from the dry herb was 1.7%;

[0055] S5. Take the crude product of okanin, add 10 mL of 60% ethanol aqueous solution, heat to dissolve, cool to precipitate and crystallize, filter with suction, collect the crystals, wash with water, and dry in the shade to obtain 11 g of refined okanin.

[0056] The HPLC method was used to determine the content of refined oxaline, which was 92.1% and the yield from dry medicinal materials was 1.1%.

[0057] Example 2

[0058] The difference between this embodiment and embodiment 1 is that the concentration of the sulfuric acid aqueous solution used in step S2 is different:

[0059] S2, add 5L of 0.05% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 95°C for 3h, and cool to room temperature to prepare an okanin hydrolyzate;

[0060] After S3 treatment, crude extract of okanaline was obtained. The okanaline content of the crude extract was determined by HPLC and was 54.6%. The yield from dry medicinal materials was 2.2%.

[0061] Example 3

[0062] The difference between this embodiment and embodiment 1 is that the concentration of the sulfuric acid aqueous solution used in step S2 is different:

[0063] S2, add 5L of 1% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 95°C for 3h, and cool to room temperature to prepare an okanin hydrolyzate;

[0064] After S3 treatment, a crude extract of okanaline was obtained. The crude extract of okanaline was determined by HPLC, and the content of okanaline was 56.1%. The yield from dry medicinal materials was 2.4%.

[0065] Example 4

[0066] The difference between this embodiment and embodiment 1 is that the temperature of hydrolysis heating in step S2 is different:

[0067] S2, add 5L of 0.1% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 80°C for 3h, and cool to room temperature to prepare an okanin hydrolyzate;

[0068] After S3 treatment, crude extract of okanaline was obtained. The crude extract of okanaline was determined by HPLC, and the content of okanaline was 52.6%, and the yield from dry medicinal materials was 2.7%.

[0069] Example 5

[0070] The difference between this embodiment and embodiment 1 is that the temperature of hydrolysis heating in step S2 is different:

[0071] S2, add 5L of 0.1% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 70°C for 3h, and cool to room temperature to prepare an okanin hydrolyzate;

[0072] After S3 treatment, crude extract of okanaline was obtained. The crude extract of okanaline was determined by HPLC, and the content of okanaline was 43.5%, and the yield from dry medicinal materials was 1.9%.

[0073] Example 6

[0074] The difference between this embodiment and embodiment 1 is that the time of hydrolysis heating in step S2 is different:

[0075] S2. Add 5 L of 0.1% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 95° C. for 2 h, and cool to room temperature to prepare an okanin hydrolyzate;

[0076] After S3 treatment, crude extract of okanaline was obtained. The crude extract of okanaline was determined by HPLC, and the content of okanaline was 56.8%, and the yield from dry medicinal materials was 2.7%.

[0077] Example 7

[0078] The difference between this embodiment and embodiment 1 is that the time of hydrolysis heating in step S2 is different:

[0079] S2, add 5L of 0.1% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 95°C for 5h, and cool to room temperature to prepare an okanin hydrolyzate;

[0080] After S3 treatment, crude extract of okanaline was obtained. The crude extract of okanaline was determined by HPLC, and the content of okanaline was 58.9%. The yield from dry medicinal materials was 2.8%.

[0081] Example 8

[0082] The difference between this embodiment and embodiment 1 is that the amount of sulfuric acid aqueous solution added in step S2 is different:

[0083] S2. Add 2 L of 0.1% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 95° C. for 3 h, and cool to room temperature to prepare an okanin hydrolyzate;

[0084] After S3 treatment, a crude extract of okanaline was obtained. The crude extract of okanaline was determined by HPLC, and the content of okanaline was 52.2%. The yield from dry medicinal materials was 2.1%.

[0085] Example 9

[0086] The difference between this embodiment and embodiment 1 is that the amount of sulfuric acid aqueous solution added in step S2 is different:

[0087] S2, add 8L of 0.1% sulfuric acid aqueous solution to the snow chrysanthemum extract, stir, centrifuge and filter, heat and hydrolyze the resulting filtrate at 95°C for 3h, and cool to room temperature to prepare an okanin hydrolyzate;

[0088] After S3 treatment, crude extract of okanaline was obtained. The crude extract of okanaline was determined by HPLC, and the content of okanaline was 58.7%. The yield from dry medicinal materials was 2.8%.

[0089] Example 10

[0090] The difference between this embodiment and embodiment 1 is that the acid aqueous solution used in step S2 is different:

[0091] S2, adding the snow chrysanthemum extract to 6 L of a 0.1% hydrochloric acid aqueous solution, stirring, centrifuging and filtering, heating the resulting filtrate at 80° C. for 4 h, and cooling to room temperature to obtain an okaninic acid solution;

[0092] After S3 treatment, crude extract of okanaline was obtained. The crude extract of okanaline was determined by HPLC, and the content of okanaline was 53.7%. The yield from dry medicinal materials was 2.7%.

[0093] Example 11

[0094] The difference between this embodiment and embodiment 1 is that the acid aqueous solution used in step S2 is different:

[0095] S2, adding the snow chrysanthemum extract to 8 L of a 2% citric acid aqueous solution, stirring, centrifuging, heating the resulting filtrate at 95 ° C for 8 h, and cooling to room temperature to obtain an okanic acid solution;

[0096] After S3 treatment, a crude extract of okanin was obtained. The crude extract of okanin was determined by HPLC, and the content of okanin was 48.8%, and the yield from dry medicinal materials was 2.0%.

[0097] Compared with sulfuric acid in Example 1, citric acid is a weak acid and its conversion ability is weaker than that of sulfuric acid. Although the temperature is 95°C and the hydrolysis time is increased to 8h, the content and yield of the product are significantly lower than those in Example 1.

[0098] Example 12

[0099] The difference between this embodiment and embodiment 1 is that the specific parameters of separation and elution in step S3 are different:

[0100] S3, the last D101 macroporous adsorption resin separation (resin loading: 1.5 kg, column volume: 1.5 L / BV, macroporous adsorption resin column diameter:column height ratio: 1:12), sample loading rate: 1.2 BV / h;

[0101] After the loading is completed, 5BV of purified water, 7BV of 20% ethanol aqueous solution, and 8BV of 60% ethanol aqueous solution are used in sequence at a flow rate of 1-1.5BV / h. The 60% ethanol aqueous solution is collected and concentrated and dried to obtain 25g of crude extract of okanin.

[0102] After S3 treatment, crude extract of okanain was obtained. The crude extract of okanain was determined by HPLC method. The content of okanain was 61.3%, and the yield from dry medicinal materials was 2.2%.

[0103] Example 12 uses an ethanol solution with a volume fraction of 60%, and the elution concentration is reduced, which can prevent other impurities from being eluted, so the content of okanamin is increased. However, the elution ability of the 60% volume fraction ethanol aqueous solution is weak, so the yield is reduced.

[0104] Example 13

[0105] The difference between this embodiment and embodiment 1 is that the specific parameters of separation and elution in step S3 are different:

[0106] S3, one HPD-300 macroporous adsorption resin separation (resin loading: 1 kg, column volume: 1 L / BV, macroporous adsorption resin column diameter: column height ratio: 1:10), sample loading rate: 1 BV / h;

[0107] After the loading is completed, 5BV of purified water, 5BV of 30% ethanol aqueous solution, and 5BV of 80% ethanol aqueous solution are used in sequence at a flow rate of 1-1.5BV / h. The 80% ethanol aqueous solution is collected and concentrated and dried to obtain 32g of crude extract of okanin.

[0108] After S3 treatment, crude extract of okanain was obtained. The crude extract of okanain was determined by HPLC method. The content of okanain was 50.6%, and the yield from dry medicinal materials was 3.2%.

[0109] Comparing Example 13 with Example 1, it was found that when the volume fraction of 70% ethanol solution was adjusted to 80% ethanol solution, more impurities would be eluted, so the content of okanaline was reduced and the yield was increased.

[0110] Example 14

[0111] The difference between this embodiment and embodiment 1 is that the specific parameters of steps S4 to S5 are different:

[0112] S4. Take the crude extract of okanin provided in Example 1, add 10 mL of 70% ethanol aqueous solution, heat to dissolve, cool to precipitate and crystallize, filter with suction, collect the crystals, wash with water, and dry in the shade to obtain crude okanin.

[0113] The crude product of okanaline was determined by HPLC, and the content of okanaline was 73.9%, and the yield from the dry herb was 1.55%;

[0114] S5. Take the crude product of okanin, add 10 mL of 70% ethanol aqueous solution, heat to dissolve, cool to precipitate and crystallize, filter, collect the crystals, wash with water, and dry in the shade to obtain 13 g of refined okanin.

[0115] The HPLC method was used to determine the content of refined oxaline, which was 91.1% and the yield from dry medicinal materials was 1.3%.

[0116] HPLC detection method

[0117] Preparation of test solution:

[0118] 25 mL of solvent was added to the test solution for extraction. The solvent was anhydrous ethanol. The extraction method was a common ultrasonic extraction method. The extraction time was 30 min. After the extraction was completed, the solution was filtered using a 0.45 μm filter membrane to obtain the test solution.

[0119] HPLC analysis:

[0120] Octadecylsilane bonded silica gel was used as a filler, an organic solvent was used as mobile phase A, and an aqueous phase was used as mobile phase B for gradient elution. 10 μL of the test solution was injected into an ultra-high performance liquid chromatography instrument for analysis. The organic solvent was selected from acetonitrile. The gradient elution conditions were as follows: phase A was acetonitrile phase, and phase B was water (0.1% formic acid). The HPLC chromatogram was recorded.

[0121] The elution procedure is as follows:

[0122] Time (minutes) Mobile phase A (%) Mobile phase B (%) Detection wavelength 0～11 10→18 90→82 350nm 11～30 18→20 82→80 350nm 30～40 20 80 350nm

[0123] Comparative Example 1

[0124] This comparative example provides a method for extracting a crude extract of okanin from snow chrysanthemum. The specific steps are similar to those in Example 1, except that step S2 is not included. The method comprises the following steps:

[0125] A1, get 1 kilogram of snow chrysanthemum, and be ground into powder; Snow chrysanthemum powder is put into extraction container, adding 12L volume fraction is 60% ethanol aqueous solution in container, reflux extraction 2 hours, separate ethanol extract, adding the volume fraction of 10L again is 60% ethanol aqueous solution reflux extraction 1.5 hours, merge extract, obtain snow chrysanthemum extract after concentrating and drying;

[0126] A2. Then apply AB-8 macroporous adsorption resin for separation (column volume is 1L / BV, the ratio of macroporous adsorption resin column diameter to column height is 1:10) at a loading speed of 1BV / h. After the loading is completed, gradient elution is performed with 5BV of purified water, 5BV of 30% ethanol aqueous solution by volume, and 5BV of 70% ethanol aqueous solution by volume. The flow rate of the gradient elution is 1-1.5BV / h. The 70% ethanol aqueous solution is collected and concentrated and dried to obtain 20g of crude extract of okanin.

[0127] The crude extract of oxaline was determined by HPLC, and the content of oxaline was 1.2%.

[0128] Comparative Example 2

[0129] This comparative example provides a method for extracting a crude extract of okanin from snow chrysanthemum. The specific steps are similar to those in Example 1, except that an acidic solution is added to the solvent in step S1. The method comprises the following steps:

[0130] B1, get 1 kilogram of snow chrysanthemum, and be ground into powder; Snow chrysanthemum powder is put into extraction container, adding 12L volume fraction in container is 60% ethanol aqueous solution, reflux extraction 2 hours, separation ethanol extract, adding the volume fraction of 10L again is 60% ethanol aqueous solution reflux extraction 1.5 hours, combined extract, obtains the snow chrysanthemum extract after concentrating and drying; Wherein, hydrochloric acid is added in the 60% ethanol aqueous solution, makes hydrochloric acid concentration reach 0.1% of ethanol solution weight percentage;

[0131] B2. Then apply AB-8 macroporous adsorption resin for separation (column volume is 1L / BV, the ratio of macroporous adsorption resin column diameter to column height is 1:10), and the loading speed is 1BV / h; after the loading is completed, use 5BV purified water, 5BV 30% ethanol aqueous solution by volume fraction, and 5BV 70% ethanol aqueous solution by volume fraction for gradient elution in sequence, and the flow rate of gradient elution is 1-1.5BV / h. Collect 70% ethanol aqueous solution, concentrate and dry to obtain 28g of crude extract of okanin.

[0132] The crude extract of oxaline was determined by HPLC, and the content of oxaline was 0.86%.

[0133] Comparative Example 3

[0134] This comparative example provides a method for extracting a crude extract of okanin from snow chrysanthemum. The specific steps are similar to those in Example 1, except that the parameters of the gradient elution in step S3 are different. The method comprises the following steps:

[0135] S3, then apply AB-8 macroporous adsorption resin for separation (column volume is 1 L / BV, the ratio of macroporous adsorption resin column diameter to column height is 1:10) at a loading rate of 1 BV / h;

[0136] After the loading is completed, 3BV of water, 3BV of 50% ethanol, and 2BV of 80% ethanol are used for elution in sequence at a flow rate of 2.5BV / h. The 80% ethanol aqueous solution is collected and concentrated to dryness to obtain 15g of crude extract of okanin.

[0137] The crude extract of oxaline was determined by HPLC, and the oxaline content was 46.5%, and the yield from the dry herb was 1.9%;

[0138] Compared with Example 1, the difference between Comparative Example 3 is that the eluent volume, eluent concentration and elution flow rate are different during the gradient elution process; by using the eluent volume, eluent concentration and elution flow rate in Comparative Example 3, the content and yield of the obtained oxanin are greatly reduced.

[0139] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for extracting high-purity okanin from snow chrysanthemum, characterized in that: The steps include: S1. Alcohol extraction The snow chrysanthemum is crushed and then subjected to alcohol extraction to obtain the snow chrysanthemum alcohol extract; S2. Acid-water extraction and hydrolysis Adding an acid aqueous solution with a mass concentration of 0.05% to 4% to the snow chrysanthemum alcohol extract obtained in S1, stirring, heating at 70 to 100° C. for acid hydrolysis for 1 to 8 hours, and filtering to obtain an okanin hydrolyzate; S3, macroporous resin column chromatography separation The okanin hydrolyzate obtained in S2 is subjected to macroporous resin separation, and then gradient eluted with an alcohol-water solution, and the eluate is collected to obtain a crude okanin extract; S4, Crystallization The crude extract of okanin is crystallized to obtain an okanin product.

2. The method for extracting high-purity okanin from snow chrysanthemum according to claim 1, wherein: The acid in the acid solution in S2 is any one or more of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, oxalic acid, citric acid, malic acid or maleic acid; The amount of acid solution added is the mass of medicinal materials: mass of acid solution = 1:1 to 1:

10.

3. The method for extracting high-purity okanin from snow chrysanthemum according to claim 1, wherein: During the alcohol extraction of S1, an ethanol aqueous solution with a volume fraction of 60% to 90% was used as a solvent for extraction; The amount of solvent added is the mass of medicinal materials: mass of solvent = 1:5~1:30, and the extraction temperature is 30-100℃.

4. The method for extracting high-purity okanin from snow chrysanthemum according to claim 3, characterized in that: During the alcohol extraction in S1, the number of extractions is 1 to 4 times, and each extraction lasts 0.2 to 5 hours.

5. The method for extracting high-purity okanin from snow chrysanthemum according to claim 1, characterized in that: In S3, the column volume is 1 to 1.5 L / BV, the ratio of the macroporous adsorption resin column diameter to the column height is 1:10 to 12, and the loading speed is 1 to 1.2 BV / h.

6. The method for extracting high-purity okanin from snow chrysanthemum according to claim 5, characterized in that: When the alcohol-water solution in S3 is gradient eluted, Gradient I: the eluent is purified water or 5-10% alcohol aqueous solution, the dosage is 5-10 BV; Gradient II, the eluent is 10-40% ethanol aqueous solution, the dosage is 5-15 BV; Gradient III, the eluent is an ethanol aqueous solution with a volume fraction of 40-95%, and the dosage is 5-15BV.

7. The method for extracting high-purity okanin from snow chrysanthemum according to claim 6, characterized in that: The alcohol aqueous solution is an ethanol aqueous solution or a methanol aqueous solution.

8. The method for extracting high-purity okanin from snow chrysanthemum according to claim 1, wherein: During crystallization, a solvent is added, heated to dissolve, and cooled. Under the conditions of 5° C. and a stirring speed of 20 to 50 r / min, the mixture is stirred for crystallization for 6 to 12 hours, and then centrifuged and filtered to collect the filter cake and filtrate.

9. The method for extracting high-purity okanin from snow chrysanthemum according to claim 8, characterized in that: Crystallization is carried out using 50% to 80% ethanol aqueous solution as solvent.

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