Method for separating and purifying oleuropein by means of single column chromatography

Abstract

The present application belongs to the field of chemical engineering. Disclosed is a method for separating and purifying oleuropein by means of single column chromatography. The method comprises: performing extraction, involving: placing olive leaf powder into a non-woven fabric tea bag, and soaking and extracting same to obtain an extracting solution; preparation of an extract, involving: performing suction filtration on the extracting solution under reduced pressure, then performing rotary evaporation to obtain a residual solution, and draining same until the water content is less than or equal to 5%, so as to obtain an extract; performing sample stirring, involving: dissolving the extract, then adding silica gel thereto, and performing rotary evaporation to remove a solvent; performing column packing, involving: packing the silica gel into a chromatographic column; performing elution, involving: performing gradient elution on the chromatographic column by using an eluent at a flow speed of 5-100 mL / min; performing collection, involving: collecting the chromatographic solution with an HPLC content of more than 90% to obtain a collected solution; and performing concentration. In the present application, by controlling the drying degree (water content) of the extract (the water content not exceeding 5%), the purity of oleuropein can reach 90% or above only by means of one instance of column chromatography, and the yield (oleuropein / olive leaf powder) can reach 25-32%; and a solvent for column chromatography can be recycled.

Description

A method for separating and purifying oleuropein by single column chromatography Technical Field

[0001] The present application belongs to the field of chemical engineering and relates to a method for separating and purifying oleuropein by single column chromatography. Background Art

[0002] Oleuropein is a natural plant extract primarily derived from olive leaves. It is a non-toxic polyphenolic compound with a natural secoiridoid glycoside skeleton. Studies have shown that oleuropein exhibits multiple effects, including anti-inflammatory, antifungal, antiviral, antioxidant, anticancer, and hypoglycemic properties. Its antioxidant capacity can reduce the oxidation of low-density lipoprotein (LDL), preventing the development of coronary heart disease and atherosclerosis. It also maintains the viability of skin cells and promotes collagen metabolism and synthesis. It is currently being used in the pharmaceutical, health food, and cosmetics industries, and its application prospects are promising.

[0003] Currently, methods for extracting and purifying oleuropein include macroporous adsorption resins, column chromatography, and recrystallization. However, obtaining high-purity oleuropein requires a combination of two or more purification methods, resulting in a complex and cumbersome process. Furthermore, achieving purity of 90% is still difficult, and the yield is low, making it unsuitable for industrial production.

[0004] Currently, there is a need for a method for separating and purifying oleuropein with high efficiency and relatively simple process, which can ensure that the extracted oleuropein has high purity and high yield.

[0005] Summary of the Invention

[0006] To solve the above problems, a method for separating and purifying oleuropein by single column chromatography is provided. The method only requires one column chromatography to obtain oleuropein with a purity of more than 90% and a yield (oleuropein / olive leaf powder) of 25-32%.

[0007] The specific technical solutions of this application are as follows:

[0008] The present application provides a method for separating and purifying oleuropein by single column chromatography, comprising:

[0009] Step 1: Extraction: Put the olive leaf powder into a non-woven tea bag, soak and extract to obtain an extract;

[0010] Step 2, preparing an extract: filtering the extract under reduced pressure and then rotary evaporating the extract to obtain a residual liquid, which is then dried to a water content of less than or equal to 5% to obtain an extract;

[0011] Step 3: Mix the sample: Dissolve the extract, add silica gel, and remove the solvent by rotary evaporation;

[0012] Step 4: Column loading: Load the silica gel into the chromatography column;

[0013] Step 5: Elution: gradient elution of the chromatography column with an eluent;

[0014] Step 6: Collecting: Collecting the chromatographic solution with a HPLC content greater than 90% to obtain a collected solution;

[0015] Step 7: Concentration: The collected solutions are combined and subjected to rotary evaporation to obtain a concentrated residual solution, which is then dried to obtain oleuropein;

[0016] Step 8: Recovering the solvent: adding a certain amount of deionized water to the liquid collected after rotary evaporation, and then performing liquid separation, drying, and atmospheric distillation.

[0017] Optionally, in step one, the soaking solution is a methanol aqueous solution or an ethanol aqueous solution with a concentration of 75-85%; and the extraction method includes ultrasonic extraction.

[0018] Optionally, the volume mass ratio (mL / g) of the added methanol aqueous solution or ethanol aqueous solution to the olive leaf powder is (20-30):1;

[0019] Optionally, ultrasonic extraction conditions: ultrasonic frequency of 50-100w, temperature of 30-50°C, extraction time of 0.5-1h.

[0020] Optionally, in step 2, the temperature of the rotary evaporation is 30-50° C.; and the residual liquid is dried by any of the following methods:

[0021] (1) The residual liquid is azeotropically desolvated with water using an azeotropic solvent, and then the oil pump is used to dry the solvent;

[0022] (2) The residual liquid is evaporated to dryness using a diaphragm pump or oil pump at 30-50°C;

[0023] (3) Deionized water is added to the residual liquid, with the volume ratio of the residual liquid to deionized water being 1 / 2-1 / 5, and freeze-dried for 6-12 hours.

[0024] Optionally, the azeotropic solvent with water is one or more of anhydrous ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, ethyl acetate, methyl acetate, benzene, toluene, n-pentane, n-hexane, and cyclohexane.

[0025] Optionally, in step 3, the extract is dissolved in methanol or ethanol; and rotary evaporated at 30-50°C.

[0026] Optionally, the volume ratio of methanol or ethanol to extract is (1-2):1, the silica gel used is 100-400 mesh, and the weight of the silica gel added is 0.8-1.2 times the weight of the olive leaf powder.

[0027] Optionally, during the column packing process in step 4, the silica gel is 300-400 mesh silica gel, and the amount of silica gel added is 30-50 times the weight of the olive leaf powder.

[0028] Optionally, during the column packing process in step 4, the ratio of the chromatography column diameter to the height is 1 / 1.5-1 / 2.0, and the ratio of the sample layer to the column height is 1 / 30-1 / 60.

[0029] Optionally, the eluent in the elution process of step five is a mixture of one or more of ethyl acetate, methanol or ethanol, and deionized water; the elution method is gradient elution, and the flow rate of the eluent is 5-100 mL / min.

[0030] Optionally, the deionized water added in step eight is 15-30% of the volume of the collected eluent, and the desiccant is anhydrous sodium sulfate, anhydrous magnesium sulfate, or anhydrous calcium chloride.

[0031] The beneficial effects of this application include but are not limited to:

[0032] 1. In the process of separating and purifying oleuropein, the present application controls the dryness (water content) of the extract (water content does not exceed 5%), and achieves oleuropein purity of over 90% with a yield within the range of 25-32% using only one column chromatography. When the water content of the extract exceeds 5%, the purity and yield of the subsequent product are both reduced. This overcomes the technical difficulties in the prior art of requiring the combined use of two or more purification methods to obtain oleuropein of higher purity, where the purity is still difficult to reach 90%, and the yield is relatively low.

[0033] It is known to those skilled in the art that it is difficult to achieve an oleuropein purity of 90% or more, and even an increase of 0.1% is difficult to achieve. The present application not only ensures an oleuropein purity of 90% or more but also ensures a relatively high yield (about 30%), and only requires one column chromatography to obtain. This achieves a technical effect that is unattainable in the prior art.

[0034] However, it is beyond the imagination of those skilled in the art to improve the purity and yield of oleuropein by controlling the water content of the extract within the range of 0-5%.

[0035] 2. The present application only requires one column chromatography to obtain oleuropein with a purity of more than 90% and a yield (oleuropein / olive leaf powder) of 25-32%, which solves the problems of complex existing purification process and low yield; the present method is simple in process, low in cost, and suitable for industrial production.

[0036] 3. The solvent used in the column chromatography of this application can be recycled and reused. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0038] FIG1 is the HPLC spectrum of oleuropein of the present application. DETAILED DESCRIPTION

[0039] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0040] Unless otherwise specified, the raw materials and catalysts in the examples of this application were purchased from commercial sources.

[0041] Example 1 A method for separating and purifying oleuropein by single column chromatography

[0042] Step 1, extraction: 10 g of olive leaf powder was placed in a non-woven tea bag, and the bag was immersed in 300 mL of 85% methanol solution. Ultrasonic extraction was performed at an ultrasonic frequency of 53 W and a temperature of 50° C. for 0.5 h to obtain an extract;

[0043] Step 2: Prepare an extract: Filter the extract under reduced pressure using a sand core funnel, wash with 30 mL of 85% methanol solution, and remove the solvent by rotary evaporation at 50°C to obtain a residual liquid. Add 200 mL of anhydrous ethanol to the residual liquid to remove the solvent by azeotropic distillation, and then pump dry to obtain an extract; the water content of the extract does not exceed 5%;

[0044] Step 3: Mix the sample: add 2 times the volume of methanol to the extract, dissolve it, add 10g of 100-200 mesh silica gel, and remove the solvent by rotary evaporation at 50℃;

[0045] Step 4: Column Packing: 300 g of 300-400 mesh silica gel was loaded into the chromatography column with a column diameter to column height ratio of 1 / 1.6 and a sample layer to column height ratio of 1 / 30.

[0046] Step 5, elution: gradient elution was performed on the column using 500 mL of ethyl acetate, 475 mL of ethyl acetate / methanol / water (90 / 5 / 0.15), and 1100 mL of ethyl acetate / methanol / water (75 / 5 / 0.15) as eluents, with a flow rate of 5 mL / min.

[0047] Step 6. Collection: Collect the eluted chromatographic solution with ethyl acetate / methanol / water (75 / 5 / 0.15), 40 mL per bottle, and detect the chromatographic solution by TLC and HPLC. Collect the chromatographic solution with HPLC content greater than 90%;

[0048] HPLC test conditions: Column model: C18-4 (4.6 × 250 mm × 5 μm), developing solvent ratio: acetonitrile: water (containing 0.1% trifluoroacetic acid) = 20:80, column temperature: 25°C, flow rate: 1 mL / min, detection time: 45 min; detection wavelength: 254 nm (same below). The results are shown in Figure 1 below.

[0049] Step 7: Concentration: Combine the collected solutions and perform rotary evaporation at 50°C. The concentrated residue is pumped dry with an oil pump to obtain a white solid with a yield of 31.5% and a purity of 91.6%.

[0050] Step 8: Recover the solvent: After rotary evaporation of the eluent, add 300 mL of deionized water to the recovered liquid, separate the liquid, add anhydrous sodium sulfate to the upper liquid, dry it, filter it, and distill the filtrate to collect the fraction at 76-78°C. A total of 1280 mL of ethyl acetate was collected and can be reused.

[0051] Example 2 A method for separating and purifying oleuropein by single column chromatography

[0052] Step 1, extraction: 100 g of olive leaf powder was placed in a non-woven tea bag, and the bag was immersed in 3000 mL of 80% ethanol solution. Ultrasonic extraction was performed at an ultrasonic frequency of 60 W and a temperature of 50° C. for 0.5 h to obtain an extract;

[0053] Step 2: Prepare an extract: Filter the extract under reduced pressure using a sand core funnel, wash with 300 mL of 80% ethanol solution, and remove the solvent by rotary evaporation at 50°C to obtain a residual liquid. Add 500 mL of anhydrous ethanol and deionized water to the residual liquid and freeze-dry for 12 hours to obtain an extract; the water content of the extract does not exceed 5%;

[0054] Step 3: Mix the sample: add ethanol twice the volume of the extract, dissolve it, add 120g of 100-200 mesh silica gel, and remove the solvent by rotary evaporation at 50℃;

[0055] Step 4: Column Packing: 3000 g of 300-400 mesh silica gel was loaded into the chromatography column with a column diameter to column height ratio of 1 / 1.7 and a sample layer to column height ratio of 1 / 60.

[0056] Step 5, elution: gradient elution was performed on the column using 6 L ethyl acetate, 4.75 L ethyl acetate / ethanol / water (90 / 5 / 0.5), and 11 L ethyl acetate / ethanol / water (75 / 5 / 0.5) as eluents, respectively, at a flow rate of 18 mL / min;

[0057] Step 6: Collect: Collect the eluted chromatographic solution with ethyl acetate / ethanol / water (75 / 5 / 0.15), 500 mL per bottle, and detect the chromatographic solution by TLC and HPLC. Collect the chromatographic solution with HPLC content greater than 90%;

[0058] Step 7: Concentration: Combine the collected solutions and perform rotary evaporation at 40°C. The concentrated residue is pumped dry with an oil pump to obtain a white solid with a yield of 28.6% and a purity of 90.9%.

[0059] Step 8: Recover the solvent: After rotary evaporation of the eluent, add 3 L of deionized water to the recovered liquid, separate the liquid, add anhydrous sodium sulfate to the upper liquid, dry it, filter it, and distill the filtrate to collect the fraction at 76-78°C. A total of 13.2 L of ethyl acetate was collected and can be reused.

[0060] Example 3 A method for separating and purifying oleuropein by single column chromatography

[0061] Step 1, extraction: 100 g of olive leaf powder was placed in a non-woven tea bag, and the bag was immersed in 3000 mL of 85% methanol solution. Ultrasonic extraction was performed at an ultrasonic frequency of 55 W and a temperature of 50° C. for 0.5 h to obtain an extract;

[0062] Step 2: Prepare the extract: Filter the extract under reduced pressure using a sand core funnel, wash with 300 mL of 85% methanol solution, and remove the solvent by rotary evaporation at 50°C to obtain a residual liquid, which is then further rotary evaporated using an oil pump to dryness to obtain an extract; the water content of the extract does not exceed 5%;

[0063] Step 3: Mix the sample: add 2 times the volume of the extract to methanol, dissolve it, add 120g of 100-200 mesh silica gel, and remove the solvent by rotary evaporation at 50℃;

[0064] Step 4: Column Packing: 3000 g of 300-400 mesh silica gel was loaded into the chromatography column with a column diameter to column height ratio of 1 / 1.5 and a sample layer to column height ratio of 1 / 60.

[0065] Step 5, elution: gradient elution was performed on the column using 6 L ethyl acetate, 4.75 L ethyl acetate / methanol / water (90 / 5 / 0.5), and 12 L ethyl acetate / methanol / water (75 / 5 / 0.5) as eluents, with a flow rate of 18 mL / min;

[0066] Step 6: Collect: Collect the eluted chromatographic solution with ethyl acetate / methanol / water (75 / 5 / 0.5), 500 mL per bottle, and detect the chromatographic solution by TLC and HPLC. Collect the chromatographic solution with HPLC content greater than 90%;

[0067] Step 7: Concentration: Combine the collected solutions and perform rotary evaporation at 40°C. The concentrated residue is pumped dry with an oil pump to obtain a white solid with a yield of 27.8% and a purity of 90.8%.

[0068] Step 8: Recover the solvent: After rotary evaporation of the eluent, add 3 L of deionized water to the recovered liquid, separate the liquid, add anhydrous sodium sulfate to the upper liquid, dry it, filter it, and distill the filtrate to collect the fraction at 76-78°C. A total of 13.5 L of ethyl acetate was collected and can be reused.

[0069] Example 4 A method for separating and purifying oleuropein by single column chromatography

[0070] Furthermore, the present application also conducted a screening experiment on the water content of the extract prepared in step 2 of the above embodiment, and the remaining steps were the same as in embodiment 1.

[0071] The settings of different water contents of the extract and the effects of different water contents on the separation and purification of oleuropein can be seen in Table 1.

[0072] Table 1

[0073] As shown in Table 1, when the water content of the extract does not exceed 5% (less than or equal to 5%), the purity of the prepared oleuropein can reach 90.1% and the yield can reach 28.6%. When the water content of the extract exceeds 5%, the purity and yield of the subsequent product are reduced. In addition, Table 1 is only part of the experimental data of the experiment of this application. During the experiment, this application found that when the water content of the extract does not exceed 5%, the purity of oleuropein can reach more than 90%, and the yield is within the range of 25-32%.

[0074] It is known to those skilled in the art that it is difficult to achieve an oleuropein purity of 90% or more, and even an increase of 0.1% is difficult to achieve. The present application not only ensures an oleuropein purity of 90% or more but also ensures a relatively high yield (about 30%), and only requires one column chromatography to obtain. This achieves a technical effect that is unattainable in the prior art.

[0075] In addition, all drying methods that can achieve a water content of 0-5% for the extract of the present application are within the scope of protection of the present application.

[0076] The foregoing is merely an embodiment of the present application, and the scope of protection of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the technical ideas and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A method for separating and purifying oleuropein by single-column chromatography, characterized in that, Including: Step 1, extraction: Load olive leaf powder into a non-woven tea bag, soak and extract to obtain an extract. Step 2, preparation of extract paste: Filter the extract under reduced pressure and then perform rotary evaporation to obtain a residual liquid, dry it until the water content is less than or equal to 5% to obtain the extract paste. Step 3, sample mixing: Dissolve the extract paste and add silica gel, then rotary evaporate to remove the solvent. Step 4, column packing: Pack the silica gel into a chromatography column. Step 5, elution: Perform gradient elution on the chromatography column with an eluent. Step 6, collection: Collect the chromatographic solution with an HPLC content greater than 90% to obtain a collected solution. Step 7, concentration: Combine the collected solutions, perform rotary evaporation to obtain a concentrated residual liquid, and dry it to obtain oleuropein.

2. The method according to claim 1, wherein In Step 1, the soaking solution is an aqueous methanol solution or an aqueous ethanol solution with a concentration of 75 - 85%; the extraction method includes ultrasonic extraction.

3. The method according to claim 2, wherein The volume-mass ratio (mL / g) of the added aqueous methanol solution or aqueous ethanol solution to the mass of the olive leaf powder is (20 - 30):

1. Ultrasonic extraction conditions: The ultrasonic frequency is 50 - 100 w, the temperature is 30 - 50 °C, and the extraction time is 0.5 - 1 h.

4. The method according to claim 1, wherein In Step 2, the temperature of rotary evaporation is 30 - 50 °C; and the residual liquid is dried by any one of the following methods: (1) The residual liquid is subjected to azeotropic dehydration with an azeotropic water-carrying solvent to remove the solvent, and then dried with an oil pump. (2) The residual liquid is rotary evaporated to dryness at 30 - 50 °C with a diaphragm pump or an oil pump. (3) Deionized water is added to the residual liquid, the volume ratio of the residual liquid to the deionized water is 1 / 2 - 1 / 5, and freeze-drying is performed for 6 - 12 h.

5. The method according to claim 4, characterized in that The azeotropic water-carrying solvent used is one or several of anhydrous ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, ethyl acetate, methyl acetate, benzene, toluene, n-pentane, n-hexane, cyclohexane.

6. The method according to claim 1, characterized in that In Step 3, the extract paste is dissolved in methanol or ethanol; rotary evaporation is performed at 30 - 50 °C.

7. The method according to claim 6, characterized in that, The volume-mass ratio of the methanol or ethanol used to the extract paste is (1 - 2):1, the silica gel used is 100 - 400 mesh, and the added weight of the silica gel is 0.8 - 1.2 times the weight of the olive leaf powder.

8. The method according to claim 1, wherein During the column packing process in Step 4, the silica gel is 300 - 400 mesh silica gel, and the added amount of the silica gel is 30 - 50 times the weight of the olive leaf powder; and / or, the ratio of the diameter to the height of the chromatography column is 1 / 1.5 - 1 / 2.0, and the ratio of the sample layer to the column height is 1 / 30 - 1 / 60.

9. The method according to claim 1, wherein In the elution process of Step 5, the eluent is a mixture of one or more of ethyl acetate, methanol or ethanol, deionized water; the flow rate of the eluent is 5 - 100 mL / min.

10. The method according to claim 1, wherein This method further includes Step 8: Solvent recovery: Add deionized water to the collected eluent, separate the liquid, dry it, and perform atmospheric distillation to obtain a recovered solvent that can be reused; Preferably, the added deionized water is 15 - 30% of the volume of the collected eluent, and the drying agent is anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium chloride.

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