A method for preparing oleuropein

Abstract

The application belongs to the technical field of plant extraction, and particularly relates to a preparation method of oleuropein. The method comprises the following steps: chopping and ultrafine grinding olive leaves to obtain raw material particles, performing flash extraction, filtering to obtain a filtrate, performing membrane filtration to collect a cut-off liquid and a membrane filtration liquid, contacting the membrane filtration liquid with a resin, performing elution, concentrating and drying to obtain a dry sample, recrystallizing to obtain chlorogenic acid; concentrating the cut-off liquid, then performing centrifugal treatment to obtain a precipitate and a supernatant, uniformly mixing the precipitate with a calcium oxide solution, performing suction filtration to obtain a filtrate, and performing crystallization to obtain rutin; performing chromatography on the supernatant, performing elution, collecting an eluate, and removing a solvent to obtain hydroxyoleuropein and oleuropein. The product obtained by the preparation method has an oleuropein content of more than 70%, is water-soluble, is white in color, can be widely applied in various applicable fields, and can comprehensively utilize olive leaves and obtain multiple effective functional components from raw materials.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to a method for preparing oleuropein. Background Technology

[0002] Oleuropein is derived from the leaves of the olive tree (Olea europaea L.). The main component of the olive tree is oleuropein, an active ingredient with strong antibacterial and antiviral properties, as well as significant antioxidant capacity.

[0003] Currently, patent 202010133694.1 discloses a method for extracting oleuropein. This method simultaneously yields rutin, oleuropein, and oleanolic acid. The process includes raw material extraction, centrifugation, and macroporous resin chromatography of the supernatant to obtain oleuropein. The centrifuged residue is then recrystallized to obtain rutin and oleanolic acid.

[0004] Existing technologies produce products with poor water solubility and a pale yellow color, limiting their applications. There is an urgent need to develop a method to obtain a product containing >70% oleuropein, with good water solubility and a near-white color, suitable for wide application in various fields, while also considering economic benefits and comprehensively utilizing olive leaves to extract as many effective components as possible from the raw material. Summary of the Invention

[0005] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing oleuropein.

[0006] The objective of this invention is achieved by at least one of the following technical solutions.

[0007] The method for preparing oleuropein provided by the present invention includes:

[0008] (1) Chop fresh olive leaves, ultrafine grind them (greater than 300 mesh) to obtain powder particles smaller than 300 mesh, sieve to obtain raw material particles, then put them into a flash extractor for flash extraction, filter to collect the filtrate, pass through a membrane, and collect the retentate and the membrane-passed liquid separately.

[0009] (2) The membrane solution described in step (1) is contacted with the resin, eluted, the eluent is collected, concentrated and dried to obtain a dried sample, which is then recrystallized to obtain chlorogenic acid; the chlorogenic acid extracted in step (2) has a content greater than 80%.

[0010] (3) The retentate obtained in step (1) is concentrated to a concentration free of organic solvent, and then centrifuged to obtain a precipitate and a supernatant. The precipitate is mixed evenly with a calcium oxide solution, filtered, and crystallized to obtain rutin. The supernatant is passed through a chromatography column, eluted, and the eluent is collected. The solvent is removed to obtain hydroxyl complexol product and oleuropein product. The purity of the hydroxyl complexol product extracted in step (3) is greater than 70%; the purity of the oleuropein product extracted in step (3) is greater than 70%. In addition, the oleuropein product also contains 0.01-1% hydroxyl complexol and 0.1-2% verbascoside.

[0011] Further, the sieve used in step (1) has a mesh size of 300; the flash extraction includes: adding the raw material particles into a mixed solution of ethyl acetate and ethanol and stirring.

[0012] Furthermore, in the mixed solution of ethyl acetate and alcohol, the volume percentage concentration of ethyl acetate is 1-5%, and the volume percentage concentration of alcohol is 50-70%; the volume-to-mass ratio of the mixed solution of ethyl acetate and alcohol to the raw material particles is 4.5-6 mL / g, the alcohol is methanol or ethanol, the stirring speed is 10000-15000 rpm, and the stirring time is 3-5 minutes.

[0013] Preferably, the flash extraction in step (1) is performed three times. When the flash extraction is performed three times, each flash extraction lasts for 1 minute, and there is a 2-minute interval between two adjacent flash extractions. The filtrates after the three flash extractions are separated and combined.

[0014] Further, the membrane in step (1) is an OSN membrane (Organic Solvent Nanofiltration), the molecular weight cutoff of the OSN membrane is 200-300, and the pressure of the membrane is 290-870 PSi.

[0015] Further, the contact between the membrane solution and the resin in step (2) includes: contacting the membrane solution with the cation exchange resin to obtain effluent 1, performing a first elution with water to obtain washing solution 1, combining effluent 1 and washing solution 1 to obtain a mixture, then contacting the mixture with the anion exchange resin, performing a second elution with water until the effluent is clear and transparent, and then eluting with acetic acid solution to obtain eluent 2.

[0016] Furthermore, the cation exchange resin is of type D152 or D113, and the manufacturer of the cation exchange resin is Aimeikejian Biomedical Co., Ltd.; the weight ratio of olive leaves to cation exchange resin is 8-10:1; the volume ratio of water to cation exchange resin in the first elution with water is 1:1; the weight ratio of olive leaves to anion exchange resin is 8-10:1; the volume percentage concentration of the acetic acid solution is 0.5-2%, and the volume ratio of the acetic acid solution to the anion exchange resin is 3-5:1; the anion exchange resin is of type D941, LK98cl, or T5, and the manufacturer of the anion exchange resin is Aimeikejian Biomedical Co., Ltd.

[0017] Further, the recrystallization includes: adding the dried sample to a mixed solution of ethyl acetate and ethanol, wherein the volume ratio of ethyl acetate to ethanol is 8:2, and the mass-volume ratio of the dried sample to the mixed solution of ethyl acetate and ethanol is 1:3-5 g / mL, dissolving evenly, and then crystallizing at 8°C for 8 hours, separating the crystals and filtrate by filtration, and drying the crystals to obtain the chlorogenic acid;

[0018] Further, in step (3), the centrifugation speed is 4000-6000 rpm and the centrifugation time is 0.5-1 h; the mass percentage concentration of the calcium oxide solution is 4-5.5% and the mass-volume ratio of the precipitate to the calcium oxide solution is 1:4 g / mL; the crystallization includes: adjusting the pH of the filtrate to 6.5-7.0 and then letting it stand for 11-14 h.

[0019] Preferably, the temperature of the calcium oxide solution is 40 degrees Celsius.

[0020] Further, step (3) of the chromatography column includes: passing the supernatant through a first macroporous resin, then eluting with water until the eluent is clear and transparent, eluting with an alkaline solution of ethanol, collecting the eluent to obtain eluent 1, eluting with water until neutral, then eluting with alcohol, collecting the eluent to obtain eluent 2; the solvent removal includes: heating eluent 1 under reflux, then adjusting the pH to 6.5-7.0, then passing it through a second macroporous resin, eluting with methanol solution, collecting the eluent, concentrating and drying it to obtain hydroxyl complexol; concentrating eluent 2 to a solid content of 40-50%, adding an equal volume of organic solvent, crystallizing at 4-10℃ for 12-24h, filtering the crystals, washing, and drying to obtain oleuropein.

[0021] Further, the first macroporous resin is of type ADS-7 or lk-8 (manufactured by Xi'an Lanxiao Technology New Material Co., Ltd.); the weight ratio of olive leaves to the first macroporous resin is 0.8-1.2:1; in the alkaline solution of ethanol, the volume percentage concentration of ethanol is 30%, and the mass percentage concentration of sodium hydroxide is 0.5-1%; the volume-to-mass ratio of the alkaline solution of ethanol to the first macroporous resin is 3-4:1 ml / g; the alcohol is an ethanol solution or a methanol solution, and the volume percentage concentration of the ethanol solution or methanol solution is 6%. The content of the first macroporous resin is 0-75%; the temperature of the heating and reflux treatment is greater than 100 degrees Celsius, and the heating and reflux treatment time is 3-4 hours; the second macroporous resin is of type T28 or LK315, and the manufacturer of the second macroporous resin is Xi'an Lanxiao Technology New Material Co., Ltd.; the weight ratio of the olive leaf to the second macroporous resin is 3-5:1; the volume percentage concentration of the methanol solution is 28%; the volume-to-mass ratio of the methanol solution to the macroporous resin is 3-4:1 ml / g; the organic solvent is a mixture of ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether is 9:1.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] (1) The preparation method provided by the present invention is an innovative process technology that can simultaneously obtain the following different high-content products from one olive leaf: oleuropein, hydroxyprostol, rutin, and chlorogenic acid.

[0024] (2) The preparation method provided by the present invention adopts flash extraction technology, which fully and reasonably extracts the required effective components from olive leaves at the same time, while protecting some easily oxidized effective components from damage, and is energy-saving and efficient.

[0025] (3) The preparation method provided by this invention uses OSN (organic nanofiltration membrane) separation for further purification of content. It also considers decolorization. The purification data have been optimized to achieve maximum efficiency, reducing production costs and increasing production efficiency.

[0026] (4) The preparation method provided by this invention, using a chromatography column (continuous ion exchange resin equipment technology): optimizes various data for macroporous resin purification to achieve maximum efficiency. This reduces production costs and increases production efficiency. It significantly reduces resin usage, chemical usage, and pollution. The fully automated equipment ensures stable processes and greatly improves production efficiency.

[0027] (5) The oleuropein product obtained by the preparation method provided by the present invention has good water solubility, is off-white, has a simple process, and is suitable for large-scale production. Attached Figure Description

[0028] Figure 1 This is a chromatogram of HPLC detection results of chlorogenic acid products provided in Example 1 of the present invention;

[0029] Figure 2 This is a chromatogram of HPLC detection results of rutin products provided in Example 1 of the present invention;

[0030] Figure 3 This is a chromatogram of HPLC detection results for the hydroxyl complex alcohol product provided in Example 1 of the present invention;

[0031] Figure 4 This is a chromatogram of HPLC detection results for oleuropein products provided in Example 1 of the present invention;

[0032] Figure 5 This is a chromatogram of HPLC detection results for chlorogenic acid products provided in Example 2 of the present invention;

[0033] Figure 6 This is a chromatogram of HPLC detection results of rutin products provided in Example 2 of the present invention;

[0034] Figure 7 This is a chromatogram of HPLC detection results for the hydroxyl complex alcohol product provided in Example 2 of the present invention;

[0035] Figure 8 This is a chromatogram of HPLC detection results for olive bitter glycoside products provided in Example 2 of the present invention;

[0036] Figure 9 This is a chromatogram of HPLC detection results for chlorogenic acid products provided in Example 3 of the present invention;

[0037] Figure 10 This is a chromatogram of HPLC detection results of rutin products provided in Example 3 of the present invention;

[0038] Figure 11 This is a chromatogram of HPLC detection results for the hydroxyl complex alcohol product provided in Example 3 of the present invention;

[0039] Figure 12 The image shows the HPLC detection results of the olive bitter glycoside product provided in Example 3 of this invention. Detailed Implementation

[0040] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0041] Example 1

[0042] (1) Take 2000g of fresh olive leaves, chop them first, then pulverize them into ultrafine powder, and then sieve them (300 mesh) to obtain raw material particles.

[0043] (2) The raw material particles were placed in a flash extractor and subjected to three flash extractions using a solvent as the extraction solvent. During each flash extraction, a portion of the solvent was added to the flash extractor (i.e., the volume ratio of the solvent added to the raw material particles was 2 mL / g). The motor speed was 12000 R / min, and the extraction was performed at room temperature for one minute each time. After each extraction, there was a 2-minute rest period before the next flash extraction was performed. A total of three flash extractions were performed. The filtrate was separated, and the filtrates from the three extractions were combined to obtain the total filtrate. The solvent was a mixed solution of ethyl acetate and ethanol; in the mixed solution of ethyl acetate and ethanol, the volume percentage concentration of ethyl acetate was 1.5%, and the volume percentage concentration of ethanol was 55%.

[0044] (3) Pass the total filtrate from step (2) through an OSN membrane, wherein the OSN membrane has a molecular weight cutoff of 300 and an inlet pressure of 350 PSi. Collect the retentate and the permeate separately.

[0045] (4) The membrane-passing solution is first passed through 200g of cation exchange resin (model D152, manufactured by Aimeikejian Biomedical Co., Ltd.), with the weight ratio of olive leaves to cation exchange resin being 10:1. After passing through the resin, effluent 1 is obtained. Then, it is eluted with one volume of pure water to obtain washing solution 1. The effluent 1 and washing solution 1 are combined and collected to obtain a mixture. The mixture is then passed through 200g of anion exchange resin (model D941, manufactured by Aimeikejian Biomedical Co., Ltd.), with the weight ratio of olive leaves to anion exchange resin being 10:1. First, wash the anion exchange resin with pure water until the effluent is clear and transparent. Then, elute with acetic acid solution (4 times the volume of the anion exchange resin, 1% by volume). Collect the eluent to obtain eluent 2. Concentrate and dry the sample. Dissolve the dried sample in a mixed solvent (ethyl acetate and ethanol in a ratio of 8:2), with a mass-to-volume ratio of 1:4 g / mL. Crystallize at 8°C for 8 hours. Filter the crystals and bake them dry to obtain chlorogenic acid. HPLC analysis showed a content of 86.97%, as shown in the results below. Figure 1 As shown;

[0046] (5) Concentrate the retentate from the OSN membrane until no organic solvent remains. Centrifuge (4500 R / min) for 0.5 h. Separate the precipitate and supernatant. Dissolve the precipitate in 4 times its volume of calcium oxide solution (5% by mass, 40°C) with stirring, and filter the solution. Adjust the pH of the filtrate to 6.5-7 with 10% hydrochloric acid solution, allow it to crystallize for 12 h, and filter the crystals. Dry the sample and analyze it by HPLC to obtain rutin (HPLC content: 72.55%) (refer to...). Figure 2 (As shown).

[0047] (6) Pass the supernatant through 2000g of the first macroporous resin (model ADS-7, manufactured by Xi'an Lanxiao Technology New Material Co., Ltd.). After passing through the solution, elute with pure water until the effluent is clear and transparent. First, elute with an alkaline solution of ethanol (the volume percentage concentration of ethanol in the alkaline solution is 30%, and the mass percentage concentration of sodium hydroxide is 0.8%), the volume-to-mass ratio of the alkaline solution of ethanol to the first macroporous resin is 4:1 ml / g. Collect the eluent to obtain eluent 1, wash with pure water until neutral, and then elute the resin with a high-concentration solvent (volume percentage concentration of 60% ethanol solution) to obtain eluent 2.

[0048] (7) First, the eluent was heated to 120℃ and refluxed for 3 hours to remove ethanol. Then, the pH was adjusted to neutral (6.5) with 20% hydrochloric acid. Next, 500g of the second macroporous resin (model T28, manufactured by Xi'an Lanxiao Technology New Materials Co., Ltd.) was introduced. After resin introduction, the sample was washed with pure water until transparent. Elution was then performed with a 28% methanol solution (v / v). The eluent was collected, concentrated, and dried to obtain hydroxyl complexol. The sample content was measured to be 73.12% (high hydroxyl complexol content). (Refer to...) Figure 3 As shown.

[0049] (8) Eluent 2 was concentrated to a small amount (40% solids) and an equal volume of organic solvent (ethyl acetate and petroleum ether in a 9:1 ratio) was added directly. The mixture was crystallized at 5°C for 12 hours. The crystals were filtered. The sample was washed with anhydrous ethanol. The sample was dried to obtain 120 g, with a yield of 6%. HPLC analysis showed that the sample contained 75.49% oleuropein, 0.01% hydroxylamine, and 0.6% verbascoside (refer to...). Figure 4 (As shown).

[0050] Example 2

[0051] (1) Take 2000g of fresh olive leaves, chop them first, then pulverize them into ultrafine powder, and then sieve them (300 mesh) to obtain raw material particles.

[0052] (2) The raw material particles were placed in a flash extractor and subjected to three flash extractions using a solvent as the extraction solvent. During each flash extraction, a portion of the solvent was added to the flash extractor (i.e., the volume ratio of the solvent added to the raw material particles was 2 mL / g). The motor speed was 12000 R / min, and the extraction was performed at room temperature for one minute each time. After each extraction, there was a 2-minute rest period before the next flash extraction was performed. A total of three flash extractions were performed. The filtrate was separated, and the filtrates from the three extractions were combined to obtain the total filtrate. The solvent was a mixed solution of ethyl acetate and ethanol; in the mixed solution of ethyl acetate and ethanol, the volume percentage concentration of ethyl acetate was 1%, and the volume percentage concentration of ethanol was 65%.

[0053] (3) Pass the total filtrate from step (2) through an OSN membrane, wherein the OSN membrane has a molecular weight cutoff of 300 and an inlet pressure of 450 PSi. Collect the retentate and the permeate separately.

[0054] (4) The membrane-passing solution is first passed through 200g of cation exchange resin (model D113, manufactured by Aimeikejian Biomedical Co., Ltd.), with the weight ratio of olive leaves to cation exchange resin being 10:1. After passing through the resin, effluent 1 is obtained. Then, it is eluted with one volume of pure water to obtain washing solution 1. The effluent 1 and washing solution 1 are combined and collected to obtain a mixture. The mixture is then passed through 200g of anion exchange resin (model D941, manufactured by Aimeikejian Biomedical Co., Ltd.), with the weight ratio of olive leaves to anion exchange resin being 10:1. First, the anion exchange resin was washed with pure water until the effluent was clear and transparent. Then, it was eluted with acetic acid solution (4 times the volume of the anion exchange resin, with a volume percentage concentration of 1.5%). The eluent was collected to obtain eluent 2. The sample was concentrated and dried. The dried sample was dissolved in a mixed solvent (ethyl acetate and ethanol in a ratio of 8:2), with a mass-to-volume ratio of 1:3 g / mL. The sample was crystallized at 8 degrees Celsius for 8 hours. The crystals were filtered and dried to obtain chlorogenic acid. HPLC analysis showed a content of 91.87%, as shown in the results. Figure 5 As shown;

[0055] (5) Concentrate the retentate from the OSN membrane until no organic solvent remains. Centrifuge (4500 R / min) for 1 hour. Separate the precipitate and supernatant. Dissolve the precipitate in 4 times its volume of calcium oxide solution (5.5% by mass, 40°C) with stirring, and filter the solution. Adjust the pH of the filtrate to 6.7 with 10% hydrochloric acid solution, allow it to crystallize for 12 hours, and filter the crystals. Dry the sample and analyze it by HPLC to obtain rutin (HPLC content: 72.60%) (refer to...). Figure 6 (As shown).

[0056] (6) Pass the supernatant through 2000g of the first macroporous resin (model lk-8, manufactured by Xi'an Lanxiao Technology New Material Co., Ltd.). After passing through the solution, elute with pure water until the effluent is clear and transparent. First, elute with an alkaline solution of ethanol (the volume percentage concentration of ethanol in the alkaline solution is 30%, and the mass percentage concentration of sodium hydroxide is 0.9%), the volume-to-mass ratio of the alkaline solution of ethanol to the first macroporous resin is 3:1 ml / g. Collect the eluent to obtain eluent 1, wash with pure water until neutral, and then elute the resin with a high-concentration solvent (volume percentage concentration of 65% ethanol solution) to obtain eluent 2.

[0057] (7) First, the eluent was heated to 110℃ and refluxed for 4 hours to remove ethanol. Then, the pH was adjusted to neutral (6.7) with 20% hydrochloric acid. Next, 500g of the second macroporous resin (model T28, manufactured by Xi'an Lanxiao Technology New Materials Co., Ltd.) was introduced. After resin introduction, the sample was washed with pure water until transparent. It was then eluted with a 28% methanol solution (v / v). The eluent was collected, concentrated, and dried to obtain hydroxyl complexol. The sample content was measured to be 77.21% (high hydroxyl complexol content). (Refer to...) Figure 7 As shown.

[0058] (8) Eluent 2 was concentrated to a small amount (45% solids) and directly added to an equal volume of organic solvent (ethyl acetate and petroleum ether in a 9:1 ratio). Crystallization was carried out at 5°C for 12 hours. The crystals were filtered. The sample was washed with anhydrous ethanol. The sample was dried to obtain 118 g, with a yield of 5.9%. HPLC analysis showed that the sample contained 78.54% oleuropein, 0.02% hydroxylamine, and 0.2% verbascoside (refer to...). Figure 8 (As shown).

[0059] Example 3

[0060] (1) Take 2000g of fresh olive leaves, chop them first, then pulverize them into ultrafine powder, and then sieve them (300 mesh) to obtain raw material particles.

[0061] (2) The raw material particles were placed in a flash extractor and subjected to three flash extractions using a solvent as the extraction solvent. During each flash extraction, a portion of the solvent was added to the flash extractor (i.e., the volume ratio of the solvent added to the raw material particles was 1.5 mL / g). The motor speed was 12000 R / min, and the extraction was performed at room temperature for 1.5 minutes each time. After each extraction, there was a 2.5-minute rest period before the next flash extraction was performed. A total of three flash extractions were performed. The filtrate was separated, and the filtrates from the three extractions were combined to obtain the total filtrate. The solvent was a mixed solution of ethyl acetate and ethanol; in the mixed solution of ethyl acetate and ethanol, the volume percentage concentration of ethyl acetate was 4.5%, and the volume percentage concentration of ethanol was 50%.

[0062] (3) Pass the total filtrate from step (2) through an OSN membrane. The OSN membrane has a molecular weight cutoff of 290 and an inlet pressure of 850 PSi. Collect the retentate and the permeate separately.

[0063] (4) The membrane-passing solution is first passed through 200g of cation exchange resin (model D113, manufactured by Aimeikejian Biomedical Co., Ltd.), with the weight ratio of olive leaves to cation exchange resin being 10:1. After passing through the resin, effluent 1 is obtained. Then, it is eluted with one volume of pure water to obtain washing solution 1. The effluent 1 and washing solution 1 are combined and collected to obtain a mixture. The mixture is then passed through 200g of anion exchange resin (model LK98cl, manufactured by Aimeikejian Biomedical Co., Ltd.), with the weight ratio of olive leaves to anion exchange resin being 10:1. First, wash the anion exchange resin with pure water until the effluent is clear and transparent. Then, elute with acetic acid solution (5 times the volume of the anion exchange resin, with a volume percentage concentration of 0.8%), collect the eluent to obtain eluent 2. Concentrate and dry the sample. Dissolve the dried sample in a mixed solvent (ethyl acetate and ethanol in a ratio of 8:2), with a mass-to-volume ratio of 1:3 g / mL. Crystallize at 8 degrees Celsius for 8 hours. Filter the crystals and bake them dry to obtain chlorogenic acid. HPLC analysis showed a content of 93.19%, as shown in the results. Figure 9 As shown;

[0064] (5) Concentrate the retentate from the OSN membrane until no organic solvent remains. Centrifuge (4500 R / min) for 1 hour. Separate the precipitate and supernatant. Dissolve the precipitate in 4 times its volume of calcium oxide solution (5.5% by mass, 40°C) with stirring, and filter the solution. Adjust the pH of the filtrate to 7.0 with 10% hydrochloric acid solution, allow it to crystallize for 12 hours, and filter the crystals. Dry the sample and analyze it by HPLC to obtain rutin (HPLC content: 74.89%) (refer to...). Figure 10 (As shown).

[0065] (6) Pass the supernatant through 2000g of the first macroporous resin (model lk-8, manufactured by Xi'an Lanxiao Technology New Material Co., Ltd.). After passing through the solution, elute with pure water until the effluent is clear and transparent. First, elute with an alkaline solution of ethanol (the volume percentage concentration of ethanol in the alkaline solution is 30%, and the mass percentage concentration of sodium hydroxide is 0.9%), the volume-to-mass ratio of the alkaline solution of ethanol to the first macroporous resin is 3:1 ml / g. Collect the eluent to obtain eluent 1, wash with pure water until neutral, and then elute the resin with a high-concentration solvent (volume percentage concentration of 70% ethanol solution) to obtain eluent 2.

[0066] (7) First, the eluent was heated to 105℃ and refluxed for 3 hours to remove ethanol. Then, the pH was adjusted to 6.5 with 20% hydrochloric acid. Next, 500g of the second macroporous resin (model LK315, manufactured by Xi'an Lanxiao Technology New Materials Co., Ltd.) was introduced. After the resin was added, the sample was washed with pure water until transparent. Elution was then performed with a 28% methanol solution (v / v). The eluent was collected, concentrated, and dried to obtain hydroxyl complexol. The sample content was measured to be 79.45% (high hydroxyl complexol content). (Refer to...) Figure 11 As shown.

[0067] (8) Eluent 2 was concentrated to a small amount (50% solids) and directly added to an equal volume of organic solvent (ethyl acetate and petroleum ether in a 9:1 ratio). Crystallization was carried out at 5°C for 12 hours. The crystals were filtered. The sample was washed with anhydrous ethanol. The sample was dried to obtain 116 g, with a yield of 5.8%. HPLC analysis showed that the sample contained 80.00% oleuropein, 0.02% hydroxylamine, and 0.4% verbascoside (refer to...). Figure 12 (As shown).

[0068] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A method for preparing oleuropein, characterized in that, include: (1) Chop the olive leaves, pulverize them into ultrafine powder, sieve them to obtain raw material particles, then flash extract them, filter them to collect the filtrate, pass them through a membrane, and collect the retentate and the membrane-passed liquid separately. (2) The membrane solution described in step (1) is brought into contact with the resin, eluted, concentrated and dried to obtain a dry sample, and recrystallized to obtain chlorogenic acid; (3) Concentrate the retentate obtained in step (1), then centrifuge to obtain precipitate and supernatant. Mix the precipitate with calcium oxide solution, filter the filtrate, crystallize to obtain rutin; pass the supernatant through a chromatography column, elute, collect the eluent, remove the solvent to obtain hydroxyl complexol and oleuropein. The sieve used in step (1) has a mesh size of 300; the flash extraction includes: adding the raw material particles to a mixed solution of ethyl acetate and ethanol and stirring. The membrane used in step (1) is an OSN membrane, the molecular weight cutoff of the OSN membrane is 200-300, and the membrane pressure is 290-870 Psi. The contact between the membrane solution and the resin in step (2) includes: contacting the membrane solution with the cation exchange resin to obtain effluent 1, eluting with water for the first time to obtain washing solution 1, combining effluent 1 and washing solution 1 to obtain a mixture, then contacting the mixture with the anion exchange resin, eluting with water for the second time until the effluent is clear and transparent, and then eluting with acetic acid solution to obtain eluent 2. The recrystallization in step (2) includes: adding the dried sample to a mixed solution of ethyl acetate and ethanol, wherein the volume ratio of ethyl acetate to ethanol is 8:2 and the mass-volume ratio of the dried sample to the mixed solution of ethyl acetate and ethanol is 1:3-5 g / mL, dissolving evenly, and then crystallizing at 8°C for 8 hours, separating the crystals and filtrate by filtration, and drying the crystals to obtain the chlorogenic acid; Step (3) of the chromatography column includes: passing the supernatant through a first macroporous resin, then eluting with water until the eluent is clear and transparent, eluting with an alkaline solution of ethanol, collecting the eluent to obtain eluent 1, eluting with water until neutral, then eluting with alcohol, collecting the eluent to obtain eluent 2; the solvent removal includes: heating eluent 1 under reflux, then adjusting the pH to 6.5-7.0, passing it through a second macroporous resin, eluting with methanol solution, collecting the eluent, concentrating and drying it to obtain hydroxyl complexol; concentrating eluent 2 to a solid content of 40-50%, adding an equal volume of organic solvent, crystallizing at 4-10℃ for 12-24h, filtering the crystals, washing, and drying to obtain oleuropein.

2. The method for preparing oleuropein according to claim 1, characterized in that, In step (1), the volume percentage concentration of ethyl acetate in the mixed solution of ethyl acetate and ethanol is 1-5%, and the volume percentage concentration of ethanol is 50-70%; the volume mass ratio of the mixed solution of ethyl acetate and ethanol to the raw material particles is 4.5-6 mL / g; the stirring speed is 10000-15000 rpm; and the stirring time is 3-5 minutes.

3. The method for preparing oleuropein according to claim 1, characterized in that, In step (2), the cation exchange resin is of type D152 or D113, and the manufacturer of the cation exchange resin is Aimeikejian Biomedical Co., Ltd. The weight ratio of olive leaves to cation exchange resin is 8-10:1; the volume ratio of water to cation exchange resin in the first elution with water is 1:1; the weight ratio of olive leaves to anion exchange resin is 8-10:1; the volume percentage concentration of the acetic acid solution is 0.5-2%, and the volume ratio of the acetic acid solution to the anion exchange resin is 3-5:1; the anion exchange resin is of type D941, LK98cl, or T5, and the manufacturer of the anion exchange resin is Aimeikejian Biomedical Co., Ltd.

4. The method for preparing oleuropein according to claim 1, characterized in that, The centrifugation process in step (3) is carried out at a speed of 4000-6000 rpm for 0.5-1 h; the mass percentage concentration of the calcium oxide solution is 4-5.5%; the mass-volume ratio of the precipitate to the calcium oxide solution is 1:4 g / mL; the crystallization process includes adjusting the pH of the filtrate to 6.5-7.0 and then letting it stand for 11-14 h.

5. The method for preparing oleuropein according to claim 1, characterized in that, In step (3), the first macroporous resin is of type ADS-7 or lk-8, and the manufacturer of the first macroporous resin is Xi'an Lanxiao Technology New Material Co., Ltd.; the weight ratio of olive leaves to the first macroporous resin is 0.8-1.2:1; in the alkaline solution of ethanol, the volume percentage concentration of ethanol is 30%, and the mass percentage concentration of sodium hydroxide is 0.5-1%; the volume-to-mass ratio of the alkaline solution of ethanol to the first macroporous resin is 3-4:1 ml / g; the alcohol is an ethanol solution or a methanol solution, and the volume percentage concentration of the ethanol solution or methanol solution is 60-75%. The heating and reflux treatment temperature is greater than 100℃, and the heating and reflux treatment time is 3-4 hours; the second macroporous resin is of type T28 or LK315, and the manufacturer of the second macroporous resin is Xi'an Lanxiao Technology New Material Co., Ltd.; the weight ratio of olive leaves to the second macroporous resin is 3-5:1; the volume percentage concentration of methanol solution in the methanol elution is 28%; the volume-to-mass ratio of methanol solution to macroporous resin is 3-4:1 ml / g; the organic solvent is a mixture of ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether is 9:1.

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