A method for extracting and purifying high-purity oleuropein from olive leaves

Abstract

The invention discloses a method for extracting and purifying high-purity oleuropein from olive leaves. The olive leaves are crushed, and then ethanol is added and heated under reflux for extraction to obtain a crude oleuropein extract; the crude oleuropein extract is enriched by macroporous adsorption chromatography to obtain an oleuropein enrichment; the oleuropein enrichment is separated and purified by gel column chromatography to obtain a crude oleuropein product; the crude oleuropein product is separated and purified by ODS medium-pressure column chromatography to obtain a refined oleuropein product; and the refined oleuropein product is freeze-dried to obtain high-purity oleuropein (purity>98%). The method for extracting and purifying oleuropein of the present invention is simple in process, short in cycle, suitable for large-scale production, and has high product purity, which can meet the demand for high-purity oleuropein in industries such as medicine.

Description

Technical Field

[0001] The invention belongs to the field of natural product extraction and separation, and relates to a method for extracting and purifying high-purity oleuropein from olive leaves. Background Art

[0002] Olive (Olea europaea L.) is a common evergreen tree in the Oleaceae family, native to the Mediterranean. Olive leaves have been used in folk medicine in Mediterranean countries for thousands of years. Olive leaf extract has numerous benefits, including anti-inflammatory, antibacterial, antioxidant, antiviral, anti-tumor, and hypoglycemic and lipid-lowering properties. The pharmacological activity of olive leaf primarily stems from its polyphenolic compounds, such as oleuropein, luteolin, quercetin, and rutin, with oleuropein being the most abundant.

[0003] Oleuropein is a polyphenol compound with a natural secoiridoid glycoside skeleton, composed of hydroxytyrosol, enolic acid and glucose molecules. Its molecular formula is C 25 H 32 O 13 The molecular weight is 540.43. It is easily soluble in ethanol, acetone, glacial acetic acid, and 5% NaOH solution. It is soluble in water, butanol, ethyl acetate, and butyl acetate. It is almost insoluble in ether, petroleum ether, chloroform, and carbon tetrachloride. It will decompose when exposed to air and sunlight. It should be stored in a sealed container at low temperature and away from light.

[0004] Oleuropein has antioxidant, anti-inflammatory, antibacterial, antiviral, anticancer, antitumor and blood sugar-lowering effects. As oleuropein moves from the cosmetics field to the pharmaceutical field, the market demand for high-purity oleuropein has increased significantly. However, most of the existing production processes have defects such as complicated steps, long cycles, high solvent consumption, high requirements for equipment and personnel operation, small preparation volume, and unsuitability for industrial production, and can no longer meet market demand.

[0005] For example, the patent "A method for extracting high-purity oleuropein from olive leaves" uses a 60-80% ethanol aqueous solution to soak olive leaves for 40-55 hours, adds 0.5% to 1% cellulase by weight of the olive leaves, and incubates at 40-50°C for 4-6 hours. After incubation, the mixture is refluxed for 1.5-2.5 hours. The olive leaf residue is then filtered out, and the ethanol aqueous solution is removed to obtain a concentrate. The concentrate is diluted with water, and 2% to 3% diatomaceous earth by weight of the olive leaves is added and mixed evenly. The mixture is allowed to stand and filtered. The filtrate is initially purified by passing it through a macroporous resin column to obtain crude oleuropein. The crude oleuropein is then purified by silica gel column chromatography to obtain high-purity oleuropein. This method is cumbersome and not conducive to practical production.

[0006] For example, the patent "A Method for Isolating High-Purity Oleuropein Using Macroporous Resin Combined with Preparative Liquid Chromatography" combines macroporous resin enrichment with preparative liquid chromatography to rapidly isolate and prepare oleuropein with a purity exceeding 90% from olive leaf extract. Another patent, "A Method for Isolating and Preparing High-Purity Oleuropein," extracts olive leaves through water extraction, followed by enrichment with macroporous adsorption resin, and then separates and purifies using high-speed countercurrent chromatography and preparative liquid chromatography to obtain oleuropein with a purity exceeding 98%. Both methods can produce high-purity oleuropein, but the yield is small and unsuitable for large-scale production. Summary of the Invention

[0007] In view of the deficiencies in the prior art, the present invention provides a method for extracting and purifying high-purity oleuropein from olive leaves;

[0008] A method for extracting and purifying high-purity oleuropein from olive leaves comprises the following steps:

[0009] (1) Extraction: The olive leaves are crushed to obtain olive leaf crude powder, and the olive leaf crude powder is added to ethanol and heated under reflux to obtain oleuropein crude extract;

[0010] (2) Enrichment: The crude oleuropein extract obtained in step (1) is enriched by macroporous adsorption chromatography to obtain an oleuropein enriched product;

[0011] (3) Purification: The oleuropein concentrate obtained in step (2) is separated and purified by gel column chromatography to obtain a crude oleuropein product;

[0012] (4) Repurification: The crude oleuropein obtained in step (3) is separated and purified by ODS medium-pressure column chromatography to obtain refined oleuropein; the refined oleuropein is freeze-dried to obtain high-purity oleuropein.

[0013] In step (1), the olive leaves are dried leaves of olea europaea, which are crushed and passed through a 40-mesh sieve to obtain olive leaf coarse powder, and the olive leaf coarse powder is reflux extracted with 80% ethanol, and the weight ratio of the olive leaf coarse powder to the ethanol is 1:5-10; the reflux extraction refers to heating to 60-70°C and reflux extraction 3 times, each time for 2 hours, to obtain oleuropein crude extract, and the content of oleuropein in the oleuropein crude extract is 10-13%.

[0014] In step (2), the crude oleuropein extract is enriched by macroporous adsorption chromatography, which means that the crude oleuropein extract is dissolved in methanol and then mixed with a macroporous resin, and after the methanol evaporates, the sample is loaded, and eluted with different eluents in sequence, and the liquid obtained after elution is collected and rotary evaporated to obtain an oleuropein enrichment. The content of oleuropein in the oleuropein enrichment is 18-20%; the macroporous adsorption resin is any one of AB-8, D-101, HPD-100, X-5 or D3520; the operating conditions for enrichment using the macroporous adsorption resin are as follows: the mass ratio of the oleuropein crude extract to the sample-mixed macroporous adsorption resin is 1:1.5, and the sample loading method is dry loading; the mass ratio of the oleuropein crude extract to the column-packed macroporous adsorption resin is 1:10, and the column packing method is wet packing; the elution with different eluents in sequence refers to elution with 30% ethanol, 60% ethanol and 90% ethanol in sequence, with an elution volume of 4-5 BV; the liquid obtained after elution with 60% ethanol is collected, and the oleuropein enrichment is rotary evaporated.

[0015] In step (3), the oleuropein concentrate is separated and purified by gel column chromatography under the following operating conditions: the oleuropein concentrate is dissolved in 50% ethanol, the supernatant is sampled after centrifugation, and eluted with 50% ethanol. The eluted liquid is collected according to the color band, and the eluate collected by the second color band is rotary evaporated to obtain a crude oleuropein product. The oleuropein content in the crude oleuropein product is 55-60%.

[0016] In step (3), when dissolving the oleuropein concentrate, the mass ratio of the oleuropein concentrate to 50% ethanol is 1:(1.5-3); after dissolution, the centrifugation conditions are a speed of 12000 r / min and a time of 5 minutes; when eluting with 50% ethanol, the flow rate is 8-10 seconds / drop. The eluate collected by the second color band is subjected to rotary evaporation to obtain a crude oleuropein product.

[0017] In step (4), the crude oleuropein product is separated and purified by ODS medium-pressure column chromatography, which means that the crude oleuropein product is mixed with ODS and then loaded, and then gradient eluted with acetonitrile solution. The eluate is collected according to the liquid phase detection result, the solvent is removed by rotary evaporation, and freeze-dried to obtain oleuropein with a purity of >98%.

[0018] In step (4), the medium-pressure chromatographic column filler is ODS filler (50 μm, YMC, Japan); the mobile phase conditions are 20%-40% acetonitrile gradient elution; the flow rate is 15 ml / min; the detection wavelengths are 254 nm and 280 nm; the running time is 4 h; and the mass ratio of crude oleuropein to sample-mixed ODS is 1:1.5.

[0019] In step (4), the rotary evaporation to remove the solvent is specifically performed at a temperature of 30-40°C, the evaporation process is equipped with a cooling water circulation system, and the temperature of the condensed water is below 0°C; the freeze-drying operation conditions are: vacuum degree 2-8mp, temperature -20°C. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to specific embodiments. The scope of protection claimed in the present invention includes but is not limited to the following description.

[0021] Example 1

[0022] (1) Weigh 200 g of olive leaves, crush them, and pass them through a 40-mesh sieve to obtain oleuropein crude powder. Add 2 L of 80% ethanol solution and reflux extract three times for 2 h each time. The combined extracts are rotary evaporated to recover ethanol to obtain a crude oleuropein extract. The oleuropein content in the crude oleuropein extract is 11%.

[0023] (2) The crude oleuropein extract was dissolved in methanol and mixed with 1.5 times macroporous resin. After the methanol evaporated, the sample was dry-loaded and then eluted with 30%, 60%, and 90% ethanol solutions in sequence. The elution volume was 4-5 BV. The liquid obtained after elution with 60% ethanol was collected and rotary evaporated to obtain an oleuropein enrichment. The oleuropein content in the oleuropein enrichment was 19%.

[0024] (3) The oleuropein concentrate was dissolved in 50% ethanol, centrifuged, and the supernatant was sampled and eluted with 50% ethanol. The eluted liquid was collected according to the color band, and the eluate collected by the second color band was rotary evaporated to obtain a crude oleuropein product. The oleuropein content in the crude oleuropein product was 57%.

[0025] (4) The crude oleuropein product was mixed with ODS and then loaded onto the sample. The sample was then gradient eluted with acetonitrile solution. The eluate was collected according to the liquid phase detection results, the solvent was removed by rotary evaporation, and the sample was freeze-dried to obtain high-purity oleuropein. The purity of the obtained oleuropein was >98%. The medium-pressure chromatographic column was filled with ODS (50 μm, YMC, Japan), and the mobile phase conditions were 20%-40% acetonitrile gradient elution; the flow rate was 15 ml / min; the detection wavelengths were 254 nm and 280 nm; the run time was 4 h; the mass ratio of the crude oleuropein product to the mixed ODS was 1:1.5.

[0026] A total of 800 mg of oleuropein was obtained, and the purity of the obtained oleuropein was >98%.

[0027] Example 2

[0028] (1) Weigh 300 g of olive leaves, crush them, and pass them through a 40-mesh sieve to obtain oleuropein crude powder. Add 5 L of 80% ethanol solution and reflux extract three times for 2 h each time. The combined extracts are rotary evaporated to recover ethanol to obtain a crude oleuropein extract. The oleuropein content in the crude oleuropein extract is 13%.

[0029] (2) The crude oleuropein extract was dissolved in methanol and mixed with 1.5 times macroporous resin. After the methanol evaporated, the sample was dry-loaded and then eluted with 30%, 60%, and 90% ethanol solutions in sequence. The elution volume was 4-5 BV. The liquid obtained after elution with 60% ethanol was collected and rotary evaporated to obtain an oleuropein enrichment. The oleuropein content in the oleuropein enrichment was 18%.

[0030] (3) The oleuropein concentrate was dissolved in pure methanol, and after centrifugation, the supernatant was sampled and eluted with pure methanol. The eluted liquid was collected according to the color band, and the eluate collected in the second color band was rotary evaporated to obtain a crude oleuropein product. The oleuropein content in the crude oleuropein product was 55%.

[0031] (4) The crude oleuropein product was mixed with ODS and then loaded onto the sample. The sample was then gradient eluted with acetonitrile solution. The eluate was collected according to the liquid phase detection results, the solvent was removed by rotary evaporation, and the sample was freeze-dried to obtain high-purity oleuropein. The medium-pressure chromatographic column used in this step was filled with ODS filler (50 μm, YMC, Japan), the mobile phase conditions were 20%-40% acetonitrile gradient elution; the flow rate was 15 ml / min; the detection wavelengths were 210 nm and 254 nm; the run time was 5 h; the mass ratio of the crude oleuropein product to the mixed ODS was 1:2. A total of 613 mg of oleuropein was obtained, and the purity of the obtained oleuropein was >98%.

Claims

1. A method for extracting and purifying high-purity oleuropein from olive leaves, characterized in that: The following steps are involved: (1) Extraction: After the olive leaves are crushed, ethanol is added and heated under reflux to obtain the crude oleuropein extract; (2) Enrichment: The crude oleuropein extract is enriched by macroporous adsorption chromatography to obtain an oleuropein enriched product; the macroporous adsorption resin used is any one of AB-8, D-101, HPD-100, X-5 or D3520, the mass ratio of the crude oleuropein extract to the sample-mixed macroporous adsorption resin is 1:1.5, the eluent is selected from 30% ethanol, 60% ethanol and 90% ethanol, and the elution volume is 4-5 BV; (3) Purification: The oleuropein concentrate was separated and purified by gel column chromatography to obtain a crude oleuropein product; the operation steps were as follows: the oleuropein concentrate was dissolved with 50% ethanol or methanol, the supernatant was sampled after centrifugation, and eluted with 50% ethanol or pure methanol, and the eluted liquid was collected according to the color band. The eluate collected by the second color band was subjected to rotary evaporation to obtain the crude oleuropein product; (4) Repurification: The crude oleuropein product was separated and purified by ODS medium-pressure column chromatography to obtain refined oleuropein product; the operation steps were as follows: the crude oleuropein product was mixed with ODS and loaded on the sample, and gradient eluted with acetonitrile solution. The eluate was collected according to the liquid phase detection results, and the solvent was removed by rotary evaporation and freeze-dried to obtain oleuropein with a purity of >98%; the ODS medium-pressure chromatography column used was filled with ODS filler, 50 μm, the mobile phase conditions were 20%-40% acetonitrile gradient elution, the flow rate was 15 ml / min, the detection wavelengths were 210 nm, 254 nm, 280 nm, the running time was 4 h or 5 h, and the mass ratio of crude oleuropein product to mixed ODS was 1:1.5 or 1:

2.

2. A method for extracting and purifying high-purity oleuropein from olive leaves according to claim 1, characterized in that: In the step (1), the olive leaves are dried olive leaves that are crushed and then passed through a 40-mesh sieve to obtain olive leaf coarse powder; the weight ratio of the olive leaf coarse powder to ethanol is 1:5 to 1:

10.

3. A method for extracting and purifying high-purity oleuropein from olive leaves according to claim 1, characterized in that: In the step (2), the enrichment operation is as follows: the crude oleuropein extract is dissolved in an organic solvent and then mixed with a macroporous resin. After the organic solvent evaporates, the crude oleuropein extract is loaded into a column with the macroporous adsorption resin, the sample is loaded, and the column is eluted with an eluent. The liquid obtained after the elution is collected and subjected to rotary evaporation to obtain an oleuropein enrichment.