Rapid preparation method of high-purity pedunculoside

Abstract

The invention belongs to the technical field of extraction and purification of natural products, and provides a rapid preparation method of high-purity pedunculoside. Optimized ethanol reflux extraction is combined with a multi-step integrated purification process, and the method specifically comprises the steps of raw material pretreatment, ethanol water solution heating reflux extraction, filter residue repeated extraction, filtrate combination and vacuum concentration, distilled water dispersion and precipitation, activated carbon decoloration, petroleum ether deoiling, methanol water solution recrystallization and the like, and finally a high-purity pedunculoside product is obtained. The process is simple and efficient, avoids complex operation of traditional column chromatography purification, has the outstanding advantages of high product purity, short operation period, low solvent consumption, easiness in large-scale amplification and the like, and is suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and purification technology, specifically relating to a rapid preparation method for high-purity holly glycosides, which is particularly suitable for the efficient extraction and purification of holly glycosides from plants such as *Ilex chinensis*. Background Technology

[0002] Ilex stevia glycoside is an important natural active ingredient with various pharmacological effects, including anti-inflammatory, antioxidant, antitumor, and cardiovascular protective properties, and is widely used in the pharmaceutical, health product, and cosmetic fields. Currently, the extraction and purification methods for Ilex stevia glycoside mostly employ solvent extraction combined with column chromatography and multiple recrystallization processes, which suffer from problems such as cumbersome operation steps, long cycle time, high organic solvent consumption, low yield, and difficulty in achieving large-scale production. Traditional processes often use silica gel column chromatography for purification, which is not only costly but also prone to loss of the target component, failing to meet the requirements of high efficiency, economy, and environmental protection for industrial production. Therefore, developing a simple, rapid, efficient, and industrially suitable method for preparing high-purity Ilex stevia glycoside is of great significance for promoting the development and utilization of related natural products. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a rapid preparation method for high-purity lecithin, aiming to solve the problems mentioned in the background art.

[0004] This invention provides a rapid preparation method for high-purity lecithin, comprising the following steps: Step 1: Select and remove impurities from the sapote, crush it to obtain sapote powder; add ethanol aqueous solution in a certain proportion, heat and reflux to extract, filter to obtain filtrate, and repeat the extraction operation on the filter residue; combine the filtrates and concentrate under reduced pressure to a thick paste, add distilled water to disperse, filter to separate and obtain the first precipitate; Step 2: Dissolve the first precipitate in an organic solvent, add activated carbon for decolorization, filter and retain the filtrate, then concentrate under reduced pressure to a paste. Add petroleum ether to the paste for defatting, and filter to obtain the second precipitate. Dissolve the second precipitate in a methanol-water solution, crystallize at low temperature overnight, centrifuge, and repeat the crystallization operation to obtain the final precipitate. Dry the final precipitate to obtain high-purity lecithin.

[0005] Furthermore, the volume fraction of ethanol in the ethanol-water solution is 85-95%, the amount of ethanol-water solution used is 4-8 times the mass of Jiubiying powder, and the heating and reflux extraction time is 2-6 hours.

[0006] Furthermore, in step one, the temperature for vacuum concentration is 45~55℃, the rotation speed is 40~50rpm, and the vacuum degree is 40~100bar.

[0007] Furthermore, in step two, the organic solvent is one of methanol, ethanol, or acetone.

[0008] Furthermore, in step two, the amount of activated carbon used is 5-10% of the mass of the first precipitate.

[0009] Furthermore, in step two, the amount of petroleum ether added is 10 to 15 times the volume of the paste.

[0010] Furthermore, in step two, the volume fraction of methanol in the methanol-water solution is 40-60%.

[0011] Furthermore, in step two, the drying temperature is 65~75℃.

[0012] Furthermore, the high-purity ilex glycoside obtained by a rapid preparation method of high-purity ilex glycoside has a ilex glycoside content of greater than 97%.

[0013] Furthermore, high-purity lecithin is used in the preparation of drugs or health products with anti-inflammatory, antioxidant, or cardiovascular protective effects.

[0014] The present invention has the following technical effects: (1) The medicinal material of Jiubiying was selected to remove impurities, crushed and weighed, and extracted by heating and reflux with ethanol aqueous solution. After filtration, the residue was extracted twice, the filtrates were combined and concentrated under reduced pressure to a thick paste, dispersed with distilled water and filtered. The precipitate was decolorized with activated carbon, deoiled with petroleum ether, and recrystallized with methanol aqueous solution to obtain high purity long-stem holly glycoside.

[0015] (2) The preparation method is simple to operate, no column chromatography is required, which greatly simplifies the purification process and reduces the production cost. By optimizing the extraction and purification process, the efficient extraction and high-purity preparation of long-stemmed holly glycosides are achieved, with a product purity of ≥97% and a yield of 4%. The process conditions are mild, the equipment is versatile, and it is suitable for large-scale industrial production. The solvents used are safe and easy to recycle, which meets the requirements of green production. Attached Figure Description

[0016] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 This is the HPLC standard curve of the lecithin standard in Example 1 of the present invention; Figure 2 This is the HPLC data chromatogram of the long-stemmed holly glycoside standard in Example 1 of the present invention; Figure 3 This is the HPLC data chromatogram of the long-stemmed holly glycoside sample extracted from *Ilex chinensis* in Example 1 of the present invention; Figure 4This is the HPLC data chromatogram of the long-stemmed holly glycoside sample extracted from *Ilex chinensis* in Comparative Example 1, which uses column chromatography in this invention. Detailed Implementation

[0017] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0019] This invention provides a rapid preparation method for high-purity lecithin, comprising the following steps: Step 1: Select and remove impurities from the sapote, crush it to obtain sapote powder; add ethanol aqueous solution in a certain proportion, heat and reflux to extract, filter to obtain filtrate, and repeat the extraction operation on the filter residue; combine the filtrates and concentrate under reduced pressure to a thick paste, add distilled water to disperse, filter to separate and obtain the first precipitate; Step 2: Dissolve the first precipitate in an organic solvent, add activated carbon for decolorization, filter and retain the filtrate, then concentrate under reduced pressure to a paste. Add petroleum ether to the paste for defatting, and filter to obtain the second precipitate. Dissolve the second precipitate in a methanol-water solution, crystallize at low temperature overnight, centrifuge, and repeat the crystallization operation to obtain the final precipitate. Dry the final precipitate to obtain high-purity lecithin.

[0020] In some embodiments, the volume fraction of ethanol in the aqueous ethanol solution is 85-95%, the amount of aqueous ethanol solution is 4-8 times the mass of the Jiubiying powder, and the heating and reflux extraction time is 2-6 hours.

[0021] In some embodiments, in step one, the temperature of vacuum concentration is 45~55℃, the rotation speed is 40~50rpm, and the vacuum degree is 40~100bar.

[0022] In some embodiments, in step two, the organic solvent is one of methanol, ethanol, or acetone.

[0023] In some embodiments, in step two, the amount of activated carbon used is 5 to 10% of the mass of the first precipitate.

[0024] In some embodiments, in step two, the amount of petroleum ether added is 10 to 15 times the volume of the paste.

[0025] In some embodiments, in step two, the volume fraction of methanol in the methanol aqueous solution is 40-60%; the amount of methanol aqueous solution used is 5-10 times the mass of the second precipitate.

[0026] In some embodiments, in step two, the centrifugation conditions are 4000 rpm for 30-60 minutes.

[0027] In some embodiments, the drying temperature in step two is 65~75°C.

[0028] In some embodiments, the high-purity ilex glycoside obtained by a rapid preparation method of high-purity ilex glycoside has a ilex glycoside content of greater than 97%.

[0029] In some embodiments, high-purity holmite glycosides are used in the preparation of drugs or health products with anti-inflammatory, antioxidant or cardiovascular protective effects.

[0030] The following detailed description of the long-stemmed holly glycoside and its preparation method provided by the present invention, with reference to the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1: The medicinal material of *Ilex chinensis* was selected, impurities and pulverized, and 500g of powder was weighed. Six times the volume of 85% ethanol was added, and the mixture was heated under reflux for 4 hours. The filtrate was obtained by filtration, and the residue was extracted twice more. The filtrates were combined. The filtrate was concentrated under reduced pressure at 50 bar, 40 rpm, and 45°C to a thick paste. The paste was dispersed in distilled water and filtered to obtain a precipitate. The precipitate was dissolved in methanol, and 10% activated carbon was added and heated at 45°C for 2 hours for decolorization. The activated carbon was separated by filtration. The filtrate was concentrated under reduced pressure at 100 bar, 40 rpm, and 40°C to a paste. Ten times the volume of petroleum ether was added, and the mixture was sonicated for 1 hour to remove oil. The precipitate was obtained by filtration. The precipitate was dissolved in five times the volume of 50% methanol by sonication for 1 hour, crystallized at low temperature overnight, and separated by centrifugation at 4000 rpm for 10 minutes. This process was repeated three times. The precipitate was dried at 70°C overnight, weighed, and stored in a dry place to obtain the *Ilex chinensis* glycoside sample, with a yield of 4%.

[0032] Referring to the detection method for the active ingredient in Jiubiying (Chinese Pharmacopoeia 2020), the content of ileoside in the obtained samples was determined. Specifically, ileoside standards of 0.075, 0.15, 0.3, 0.6, and 1.2 mg / mL were prepared using 50% methanol, and ileoside samples of 1 mg / mL were prepared for testing. The HPLC standard curve of the ileoside standards is shown below. Figure 1 As shown; the HPLC data chromatogram of the long-stemmed holly glycoside standard is as follows. Figure 2 As shown.

[0033] A C18 column was used with acetonitrile and water as the mobile phase for gradient elution. The detection temperature was 35℃ and the wavelength was 210nm. The HPLC data of the olivine glycoside sample prepared in this example were analyzed. Figure 3 As shown in the figure; the results showed that the content of ide in the obtained ide sample was 98%.

[0034] Example 2:

[0035] The medicinal material of *Ilex chinensis* was selected, impurities and pulverized, and 500g of powder was weighed. Eight times the volume of 85% ethanol was added, and the mixture was heated under reflux for 4 hours. The filtrate was obtained by filtration, and the residue was extracted twice more. The filtrates were combined. The filtrate was concentrated under reduced pressure at 60 bar, 40 rpm, and 50°C to a thick paste. The paste was dispersed in distilled water and filtered to obtain a precipitate. The precipitate was dissolved in methanol, and 8% activated carbon was added and heated at 50°C for 2 hours for decolorization. The activated carbon was separated by filtration. The filtrate was concentrated under reduced pressure at 80 bar, 40 rpm, and 40°C to a paste. Twelve times the volume of petroleum ether was added, and the mixture was sonicated for 1 hour to remove oil. The precipitate was obtained by filtration. The precipitate was dissolved in eight times the volume of 50% methanol by sonication for 1 hour, crystallized at low temperature overnight, and separated by centrifugation at 4000 rpm for 10 minutes. This process was repeated three times. The precipitate was dried at 65°C overnight, weighed, and stored in a dry place to obtain the *Ilex chinensis* glycoside sample, with a yield of 4.1%.

[0036] Referring to the detection method of active ingredients in Jiubiying in the 2020 edition of the Chinese Pharmacopoeia, the content of long-stemmed holly glycoside in the obtained sample was determined. The results showed that the content of long-stemmed holly glycoside in the obtained sample was 97.3%.

[0037] Example 3: The medicinal material of *Ilex chinensis* was selected, impurities and pulverized, and 500g of powder was weighed. Six times the volume of 90% ethanol was added, and the mixture was heated under reflux for 4 hours. The filtrate was obtained by suction filtration, and the residue was extracted twice more. The filtrates were combined. The filtrate was concentrated under reduced pressure at 50 bar, 40 rpm, and 45°C to a thick paste, dispersed with distilled water, and filtered to obtain a precipitate. The precipitate was dissolved in methanol, and 8% activated carbon was added and heated at 50°C for 2 hours for decolorization. The activated carbon was separated by suction filtration. The filtrate was concentrated under reduced pressure at 80 bar, 40 rpm, and 40°C to a paste. 15 times the volume of petroleum ether was added, and the mixture was sonicated for 1 hour to remove oil. The precipitate was obtained by suction filtration. The precipitate was dissolved in 10 times the volume of 50% methanol by sonication for 1 hour, crystallized at low temperature overnight, and separated by centrifugation at 4000 rpm for 10 minutes. This process was repeated three times. The precipitate was dried at 70°C overnight, weighed, and stored in a dry place to obtain the *Ilex chinensis* glycoside sample, with a yield of 3.8%.

[0038] Referring to the detection method of active ingredients in Jiubiying in the 2020 edition of the Chinese Pharmacopoeia, the content of long-stemmed holly glycoside in the obtained sample was determined. The results showed that the content of long-stemmed holly glycoside in the obtained sample was 97.1%.

[0039] Comparative Example 1: The medicinal material of *Ilex chinensis* was selected, impurities and pulverized, and 500g of powder was weighed. Six times the volume of 85% ethanol was added, and the mixture was heated under reflux for 4 hours. The filtrate was obtained by suction filtration, and the residue was extracted twice more. The filtrates were combined. The filtrate was concentrated under reduced pressure at 50 bar, 40 rpm, and 45°C to a thick paste, dispersed with distilled water, and filtered to obtain a precipitate. The precipitate was dissolved in methanol, and 10% activated carbon was added and heated at 45°C for 2 hours for decolorization. The activated carbon was separated by suction filtration. The filtrate was concentrated under reduced pressure at 80 bar, 40 rpm, and 40°C to a paste. Column chromatography was performed using macroporous adsorption resin D101, with gradient elution of 0%, 10%, 30%, and 100% ethanol-water solutions. The 10% eluent was collected, concentrated, dried, weighed, and stored to obtain the *Ilex chinensis* glycoside sample, with a yield of 3.2%.

[0040] Referring to the detection method for the active ingredient in Jiubiying (Chinese Pharmacopoeia 2020), the content of long-stemmed holly glycoside in the obtained sample was determined. The HPLC data chromatogram of the long-stemmed holly glycoside sample is shown below. Figure 4 As shown in the figure; the results showed that the content of ide in the obtained ide sample was 93.5%.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid preparation method for high-purity lecithin, characterized in that, Includes the following steps: Step 1: Select and remove impurities from the sapote, crush it to obtain sapote powder; add ethanol aqueous solution in a certain proportion, heat and reflux to extract, filter to obtain filtrate, repeat the extraction operation on the filter residue; combine the filtrates and concentrate under reduced pressure to a thick paste, add distilled water to disperse, filter to separate and obtain the first precipitate; Step 2: Dissolve the first precipitate in an organic solvent, add activated carbon for decolorization, filter and retain the filtrate, concentrate under reduced pressure to a paste, add petroleum ether to the paste for defatting, and filter to obtain the second precipitate. The second precipitate was dissolved in an aqueous methanol solution, crystallized overnight at low temperature, separated by centrifugation, and the crystallization operation was repeated to obtain the final precipitate; The final precipitate was dried to obtain high-purity leucine glycoside.

2. The method as described in claim 1, characterized in that: In step one, the volume fraction of ethanol in the ethanol-water solution is 85-95%, the amount of ethanol-water solution used is 4-8 times the mass of Jiubiying powder, and the heating and reflux extraction time is 2-6 hours.

3. The method as described in claim 1, characterized in that: In step one, the temperature for vacuum concentration is 45~55℃, the rotation speed is 40~50rpm, and the vacuum degree is 40~100bar.

4. The method as described in claim 1, characterized in that: In step two, the organic solvent is one of methanol, ethanol, or acetone.

5. The method as described in claim 1, characterized in that: In step two, the amount of activated carbon used is 5-10% of the mass of the first precipitate.

6. The method as described in claim 1, characterized in that: In step two, the amount of petroleum ether added is 10 to 15 times the volume of the paste.

7. The method as described in claim 1, characterized in that: In step two, the volume fraction of methanol in the methanol-water solution is 40-60%.

8. The method as described in claim 1, characterized in that: In step two, the drying temperature is 65~75℃.

9. The high-purity lecithin obtained by the method according to any one of claims 1-8, characterized in that: The content of long-stemmed holly glycoside in high-purity long-stemmed holly glycoside is greater than 97%.

10. The use of the high-purity long-stemmed holly glycoside as described in claim 9 in the preparation of drugs or health products with anti-inflammatory, antioxidant or cardiovascular protective effects.

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