Solvents and methods for efficient separation of high purity oleuropein

Abstract

The present application relates to a kind of solvent and method for separating high purity oleuropein with high efficiency.The mixed solvent is selected from: (1) the mixed solvent consisting of n-butanol, dichloromethane, methanol and water, wherein the volume ratio of n-butanol, dichloromethane, methanol and water is 1: (5-15): (1-5): (5-15), preferably 1: (5-10): (1-2): (5-10);(2) the first solvent, the first solvent is the upper layer after the mixed solvent of (1) is left to separate;(3) the second solvent, the second solvent is the lower layer after the mixed solvent of (1) is left to separate;And (4) the combination of first solvent and second solvent.The present application also includes the method for separating oleuropein using the mixed solvent.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and purification, and specifically relates to a solvent and method for efficiently separating high-purity oleuropein. Background Technology

[0002] Oleuropein is a schizocarpine ether terpenoid compound found in olive oil (Olive oleifera). European olive tree L . The content of this herb is relatively high in the leaves, generally ranging from 2% to 5% by weight in dried leaves, with high-quality varieties reaching over 6%. It possesses excellent pharmacological effects, including anti-tumor, anti-inflammatory, neuroprotective, antioxidant, and hypoglycemic properties. Therefore, it has attracted numerous domestic scholars to study its extraction and purification methods.

[0003] Existing purification methods include: 1. After solvent extraction of olive leaves, oleuropein is enriched using macroporous resin, and then purified by silica gel column chromatography or gel column chromatography, medium-pressure chromatography, and high-performance liquid chromatography (e.g., methods CN115925759 and CN117801040); 2. After solvent extraction of olive leaves, the solvent is recovered to obtain olive leaf extract, which is then purified using a solvent system of n-butanol, ethyl acetate, methanol, and water as the two-phase solvent for HSCCC (high-speed countercurrent chromatography) separation (method CN114085258). Method 1 uses multiple organic solvents, and gel chromatography, medium-pressure chromatography, and high-performance liquid chromatography are expensive, making them suitable only for scientific research. Method 2 directly purifies olive leaf extract using HSCCC, but it cannot efficiently separate oleuropein from its structural analogues, resulting in a very low yield of high-purity oleuropein.

[0004] In view of the shortcomings of existing purification technologies for oleuropein, the present invention aims to provide a method that is simple to operate, low in cost, and can obtain oleuropein with a purity of ≥98% with high efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a method for efficiently separating high-purity oleuropein. Specifically, it relates to a method for separating high-purity oleuropein from olive leaf extract, and particularly to a method for achieving high-purity oleuropein separation through dynamic pH adjustment during high-speed countercurrent chromatography.

[0006] The first aspect of this invention provides a mixed solvent selected from: (1) A mixed solvent composed of n-butanol, dichloromethane, methanol and water, wherein the volume ratio of n-butanol, dichloromethane, methanol and water is 1:(5-15):(1-5):(5-15), preferably 1:(5-10):(1-2):(5-10); (2) The first solvent is the upper layer of the mixed solvent described in (1) after it has been allowed to stand and separate into layers; (3) A second solvent, wherein the second solvent is the lower layer after the mixed solvent of (1) has been allowed to stand and separate into layers; and (4) The combination of the first solvent and the second solvent.

[0007] In one or more embodiments, the second solvent further contains polyacrylic acid; preferably, the concentration of polyacrylic acid in the second solvent is 0.05-0.2 wt%.

[0008] In one or more embodiments, the second solvent comprises a second solvent with a pH of 3.5-4.0 and / or a second solvent with a pH of 7.5-8.0.

[0009] A second aspect of the present invention provides a method for isolating oleuropein, the method comprising the steps of: (1) Provide olive leaf extract containing oleuropein; (2) The oleuropein in the olive leaf extract was separated by high-speed countercurrent chromatography; In this process, a mixed solvent consisting of n-butanol, dichloromethane, methanol, and water is used as the two-phase solvent for high-speed countercurrent chromatography separation. The upper phase of the mixed solvent is the stationary phase, and the lower phase of the mixed solvent is the mobile phase. Step (2) includes: (a) In the initial stage of high-speed countercurrent chromatography separation, from 0 to 40 min, the first mobile phase is used for elution; wherein, the first mobile phase is the lower phase adjusted to pH 3.5-4.0 with acid, and the first mobile phase also contains polyacrylic acid; (b) During the intermediate stage of high-speed countercurrent chromatography separation at min 40-70, a second mobile phase is used for elution; wherein the second mobile phase is the lower phase adjusted to pH 7.5-8.0 with alkali, and the second mobile phase also contains polyacrylic acid; (c) During the high-speed countercurrent chromatography separation phase from 70 to 110 min, the lower phase is used for elution, and the eluent containing oleuropein is collected.

[0010] The present invention has the following beneficial effects: 1. In the method of this invention, during the initial stage of HSCCC purification, the mobile phase is acidic, which can separate the sesquiterpenoid components from other components. During the middle stage of purification, the mobile phase is alkaline. Increasing the pH increases the partition coefficient of oleuropein in the mobile phase. Since its analogues have fewer polar groups, the increase in their partition coefficient in the mobile phase is not significant. Therefore, increasing the pH can improve the separation degree of oleuropein and its analogues. Furthermore, during the middle stage of purification, the PAA in the mobile phase can further widen the difference in partition coefficients between oleuropein and its analogues, further improving the separation effect. Under the dual effects of pH control and PAA, the purification effect is significantly improved. Compared with the conventional HSCCC method, the yield of high-purity oleuropein (purity greater than 98%) is increased by more than 200%.

[0011] 2. The method of this invention makes full use of the volatile properties of formic acid and ammonia. Adjusting the pH of the first mobile phase with formic acid, adjusting the pH of the second mobile phase with ammonia, and using the initial mobile phase (the mobile phase before sample injection) in the later stage of purification can ensure that the collected oleuropein fraction is free of other impurities after solvent recovery. Attached Figure Description

[0012] Figure 1 This is the purity test result of oleuropein in Example 1. Detailed Implementation

[0013] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0014] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0015] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0016] In this document, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0017] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0018] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0019] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0020] To achieve efficient separation of high-purity oleuropein, this invention improves the HSCCC method. The core of this improvement lies in the innovative integration of the intelligent adsorption aid polyacrylic acid (PAA) and dynamic pH adjustment into the HSCCC separation process. The basic principle is that when polyacrylic acid (PAA) is added to the mobile phase, under acidic conditions, the carboxyl groups in PAA are not ionized. The carboxyl groups within the molecule form hydrogen bonds, causing the PAA molecular chain to coil into clusters, with the carboxyl groups encapsulated internally. At this point, the interaction between PAA and polar molecules such as oleuropein is extremely weak, while van der Waals forces exist with smaller polar molecules. The components are mainly separated in HSCCC using traditional solvent partitioning. Sesquicyclic ether terpenes can be effectively separated from smaller polar molecules, flavonoids, polyphenols, etc., but the separation of oleuropein (CAS number 32619-42-4), which is also a sesquicyclic ether terpenes, and its analogues (such as ligstroside, CAS number 35897-92-8) is relatively poor. Under alkaline conditions, the carboxyl group of PAA forms a salt. Due to the lack of intramolecular hydrogen bonding, the PAA molecular chain unfolds and forms hydrogen bonds with oleuropein and its analogues. The strength of hydrogen bonds between the PAA molecular chain and different molecules varies (e.g., ligstroside, which has one less phenolic hydroxyl group than oleuropein, has a weaker interaction with PAA), resulting in greater differences in the partition coefficients of each molecule in the stationary and mobile phases, thus allowing for better separation of oleuropein and its analogues.

[0021] In this article, High-Speed ​​Countercurrent Chromatography (HSCCC) refers to a liquid-liquid partition chromatography technique that does not require a solid support. Its core lies in the two-way hydrodynamic equilibrium formed by two immiscible solvent systems in a spiral tube. Separation is achieved by the different partition coefficients of the components in the analytes in the two phases (upper and lower phases). Before separation using HSCCC, a two-phase solvent system needs to be prepared. This system consists of two or more solvents mixed in a certain proportion, allowed to stand at a constant temperature, and naturally separate into two immiscible liquid layers. The upper phase is the denser layer located at the top, and the lower phase is the denser layer located at the bottom. In the actual separation process, the two-phase solvent system can be selected according to the polarity of the analytes.

[0022] In this paper, during high-speed countercurrent chromatography (HSCCC), the centrifugal force generated by the high-speed planetary rotation of the spiral tube retains one phase (either the upper or lower phase) within the column as the stationary phase. The stationary phase provides a static partitioning environment for the components in the mixture. The other phase, propelled by a pump, passes through the stationary phase retained within the column, serving as the mobile phase. The samples to be separated are typically dissolved in a small amount of the mobile phase or a mixture of the two phases and injected into the HSCCC for separation.

[0023] In this paper, conventional methods, such as the external standard method, can be used to quantify the isolated oleuropein. In some embodiments, ultraviolet absorption spectrometry is used to determine the content of oleuropein in the solution; preferably, detection is performed at a wavelength of 220-240 nm, more preferably at a wavelength of 230 nm.

[0024] The present invention provides a mixed solvent, wherein the mixed solvent is composed of n-butanol, dichloromethane, methanol and water, wherein the volume ratio of n-butanol, dichloromethane, methanol and water is 1:(5-15):(1-5):(5-15), preferably 1:(5-10):(1-2):(5-10); preferably, the volume ratio of n-butanol, dichloromethane, methanol and water is 1:(5-8):(1-2):(5-8), more preferably 1:6:1.5:6.

[0025] This invention provides a first solvent, which is the upper layer after the mixed solvent described herein has been allowed to stand and separate. The first solvent can be used as the stationary phase in the high-speed countercurrent chromatography method for separating oleuropein according to this invention.

[0026] The present invention also provides a second solvent, which is the lower layer after the mixed solvent described herein has been allowed to stand and separate. This second solvent can be used as the mobile phase in the high-speed countercurrent chromatography method for separating oleuropein according to the present invention.

[0027] The present invention provides a solvent combination comprising the first solvent and the second solvent described herein.

[0028] This invention provides a first mobile phase, wherein the first mobile phase is a second solvent adjusted to a pH of 3.5-4.0 with an acid, and the first mobile phase further contains polyacrylic acid. The acid can be any acid conventionally used in the art for pH adjustment. Preferably, the acid is selected from one or more of formic acid, acetic acid, hydrochloric acid, and sulfuric acid, with formic acid being preferred. In some embodiments, the first mobile phase contains 0.05-0.2 wt% polyacrylic acid (PAA), for example 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.12 wt%, 0.15 wt%, 0.18 wt%, 0.2 wt%, or within any range of two values, preferably 0.1-0.15 wt%, 0.05-0.1 wt%, or 0.08-0.12 wt%.

[0029] This invention provides a second mobile phase, wherein the second mobile phase is a second solvent adjusted to a pH of 7.5-8.0 with an alkali, and the second mobile phase further contains polyacrylic acid. The alkali can be any alkali conventionally used in the art for pH adjustment. Preferably, the alkali is selected from one or more of sodium carbonate, potassium carbonate, triethylamine, pyridine, and ammonia, with ammonia being the most preferred. In some embodiments, the second mobile phase contains 0.05-0.2 wt% polyacrylic acid (PAA), for example 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.12 wt%, 0.15 wt%, 0.18 wt%, 0.2 wt%, or within any range of two values, preferably 0.1-0.15 wt%, 0.05-0.1 wt%, or 0.08-0.12 wt%.

[0030] The present invention provides a third mobile phase, which is a second solvent that is not pH adjusted and does not contain polyacrylic acid, i.e., the original lower layer.

[0031] The present invention provides a mobile phase combination, which includes any two or all three of the first mobile phase, the second mobile phase and the third mobile phase described herein.

[0032] This invention provides a method for isolating oleuropein, the method comprising the steps of: (1) Provide olive leaf extract containing oleuropein; (2) The oleuropein in the olive leaf extract was separated by high-speed countercurrent chromatography; In this process, a mixed solvent consisting of n-butanol, dichloromethane, methanol, and water is used as the two-phase solvent for high-speed countercurrent chromatography separation. The upper phase of the mixed solvent is the stationary phase, and the lower phase of the mixed solvent is the mobile phase. Step (2) includes: (a) In the initial stage of high-speed countercurrent chromatography separation, from 0 to 40 min, the first mobile phase is used for elution; wherein, the first mobile phase is the lower phase adjusted to pH 3.5-4.0 with acid, and the first mobile phase also contains polyacrylic acid; (b) During the intermediate stage of high-speed countercurrent chromatography separation at min 40-70, a second mobile phase is used for elution; wherein the second mobile phase is the lower phase adjusted to pH 7.5-8.0 with alkali, and the second mobile phase also contains polyacrylic acid; (c) During the high-speed countercurrent chromatography separation phase from 70 to 110 min, the lower phase is used for elution, and the eluent containing oleuropein is collected.

[0033] In some embodiments, in step (2), before step (a), the stationary phase is first pumped into the separation column of the high-speed countercurrent chromatography (HSCCC). After the stationary phase fills the separation column and runs stably, the mobile phase is then injected. After the two phases have run stably, the mixture containing the olive leaf extract is injected into the HSCCC. In some embodiments, in step (2), the sample injected into the HSCCC is a mixture containing the olive leaf extract. Preferably, the content of the olive leaf extract in the mixture is 5-20 g / L, for example, 5 g / L, 10 g / L, 15 g / L, 20 g / L, or within a range of any two values, such as 10-15 g / L. In some embodiments, the solvent of the mixture is the lower phase (i.e., the mobile phase) as described in any embodiment herein; that is, after the two phases have run stably, the olive leaf extract is dissolved in the mobile phase and injected into the HSCCC. In this article, the content of oleuropein in the mixture containing the olive leaf extract can be ≤20 g / L, such as ≤15 g / L, ≤12 g / L, ≤10 g / L, ≤8 g / L or ≤6 g / L, such as 3-10 g / L or 5-8 g / L.

[0034] In this invention, the inventors discovered that using a mixed solvent composed of n-butanol, dichloromethane, methanol, and water as a two-phase solvent system can achieve highly efficient and high-purity separation of oleuropein from olive leaf extract. The volume ratio of n-butanol, dichloromethane, methanol, and water can be 1:(5-15):(1-5):(5-15), preferably 1:(5-10):(1-2):(5-10); more preferably, the volume ratio of n-butanol, dichloromethane, methanol, and water is 1:(5-8):(1-2):(5-8), and more preferably 1:6:1.5:6.

[0035] In some implementations, controlling the pH of the first mobile phase between 3.5 and 4.0 (e.g., 3.5-3.7 or 3.7-4.0) can effectively suppress the ionization of PAA, allowing the separation of schizocarpine ethers from other components, thereby achieving high-efficiency and high-purity separation of oleuropein from olive leaf extract.

[0036] In some implementations, the pH of the second mobile phase is controlled at 7.5-8.0 (e.g., 7.5-7.6 or 7.6-8.0), which allows the carboxyl groups of PAA to form salts and prevents oleuropein from becoming unstable under excessively alkaline conditions. This allows oleuropein to be separated from other secoiridoids, thereby achieving high-efficiency and high-purity separation of oleuropein from olive leaf extract.

[0037] In step (a), the pH of the first mobile phase can be adjusted using a conventional organic or inorganic acid, such as one or more of formic acid, acetic acid, hydrochloric acid, and sulfuric acid. Preferably, the acid is formic acid. When formic acid is used to adjust the pH of the first mobile phase, since formic acid is more volatile than other acids, a separate acid removal process is not required. The collected eluent containing oleuropein can simply be used to recover the solvent, without introducing any other impurity removal processes.

[0038] In step (b), the pH of the second mobile phase can be adjusted using a conventional organic or inorganic base, such as one or more of sodium carbonate, potassium carbonate, triethylamine, pyridine, and ammonia. Preferably, the base is ammonia. When ammonia is used to adjust the pH of the second mobile phase, since ammonia is more volatile than other bases, a separate alkali removal process is not required. The collected eluent containing oleuropein can simply be used to recover the solvent, without introducing any other impurity removal processes.

[0039] In some embodiments, step (c) uses an unadjusted mobile phase for elution; that is, the mobile phase in step (c) is the lower phase of a mixed solvent consisting of n-butanol, dichloromethane, methanol, and water. Preferably, the mobile phase used in step (c) does not contain polyacrylic acid.

[0040] In this document, the molecular weight of the polyacrylic acid used in steps (a) and (b) can be conventionally selected based on the solubility of polyacrylic acid in the first and second mobile phases. All molecular weights of polyacrylic acid mentioned herein refer to the number-average molecular weight. In some embodiments, the (number-average) molecular weight of the polyacrylic acid is 1000-2000.

[0041] In some embodiments, the first mobile phase contains 0.05-0.2 wt% polyacrylic acid (PAA), for example 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.12 wt%, 0.15 wt%, 0.18 wt%, 0.2 wt%, or within any two of these values, preferably 0.1-0.15 wt%, 0.05-0.1 wt%, or 0.08-0.12 wt%. The inventors have found that controlling the PAA content in the first mobile phase within the range defined herein is beneficial for achieving efficient and high-purity separation of oleuropein. Too low a PAA content prevents effective separation of oleuropein, while too high a PAA content hinders its solubility in the first mobile phase.

[0042] In some embodiments, the second mobile phase contains 0.05-0.2 wt% polyacrylic acid (PAA), for example 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.12 wt%, 0.15 wt%, 0.18 wt%, 0.2 wt%, or within any two of these values, preferably 0.1-0.15 wt%, 0.05-0.1 wt%, or 0.08-0.12 wt%. The inventors have found that controlling the PAA content in the second mobile phase within the range defined herein is beneficial for achieving efficient and high-purity separation of oleuropein. Too low a PAA content prevents effective separation of oleuropein, while too high a PAA content hinders its dissolution in the second mobile phase.

[0043] In some implementations, during the initial stage of high-speed countercurrent chromatography separation (0-40 min), the pH of the mobile phase is adjusted to 3.5-4.0 with formic acid to obtain a first mobile phase. This first mobile phase also contains 0.05-0.2 wt% PAA (number average molecular weight 1000-2000). Under these conditions, flavonoids, polyphenols, and other low-polarity impurities migrate rapidly with the mobile phase and are quickly separated from the secoirid ether terpenoid components.

[0044] In some implementations, during the intermediate stage (40-70 min) of high-speed countercurrent chromatography separation, the pH of the mobile phase is adjusted to 7.5-8.0 with ammonia to obtain a second mobile phase containing 0.05-0.2 wt% PAA (number average molecular weight 1000-2000). Under these conditions, oleuropein and its analogues are effectively separated due to the significant difference in their partition coefficients in the stationary and mobile phases.

[0045] In some implementation schemes, during the later stages of high-speed countercurrent chromatography (70-110 min), a mobile phase without pH adjustment is used. Under these conditions, the separation degree of oleuropein and its analogues decreases somewhat, but they have already been effectively separated in the middle of purification and will not mix again. Switching to the initial mobile phase (i.e., the mobile phase before the olive leaf extract is injected) during the later stages of purification can displace the PAA-containing mobile phase. The collected oleuropein fraction does not contain PAA, eliminating the need for further separation of PAA and oleuropein, thus reducing purification steps.

[0046] Those skilled in the art can routinely select the rotation speed of the stationary phase and the flow rate of the mobile phase in high-speed countercurrent chromatography, as long as it does not adversely affect the separation of oleuropein. In actual separation, the rotation speed of the stationary phase and / or the flow rate of the mobile phase can be selected according to the size of the instrument. In this paper, a TBE-300C type instrument is used to implement the high-speed countercurrent chromatography separation method. In some embodiments, in step (2), the rotation speed of the stationary phase in high-speed countercurrent chromatography is 400-2000 rpm, preferably 800-1000 rpm, more preferably 800-900 rpm, 900-1000 rpm or 850-950 rpm. In some embodiments, in step (2), the flow rate of the mobile phase in high-speed countercurrent chromatography can be 1-5000 rpm, preferably 1-10 mL / min, more preferably 1-5 mL / min, 5-10 mL / min or 3-8 mL / min.

[0047] In this paper, conventional methods for extracting active ingredients from natural products (such as solvent extraction) can be used to extract oleuropein from olive leaves. In some embodiments, step (1) includes: (A) extracting oleuropein from olive leaves using a solvent to obtain an olive leaf extract. Preferably, the solvent is a mixture of ethanol and water, wherein the volume fraction of ethanol in the mixture is preferably 50-75%. Preferably, the extraction temperature is the reflux temperature of the solvent. In some embodiments, the solvent is a mixture of ethanol and water, and the extraction temperature is 70-90°C or 80-85°C. Preferably, in step (A), the amount of solvent used is 10-15 times the weight of the olive leaves, preferably 10-12 times or 12-15 times. Preferably, in step (A), the extraction time is 1-2 h, such as 1-1.5 h or 1.5-2 h. Preferably, the olive leaf extract is filtered to obtain a clear olive leaf extract. The olive leaf extract can be filtered using methods conventional in the art, such as using a filter membrane with a pore size of 3 micrometers, 4 micrometers, or 5 micrometers. In some embodiments, the solvent in the clarified olive leaf extract is removed to obtain an olive leaf extract containing oleuropein.

[0048] Further, step (1) also includes: (B) concentrating the clarified olive leaf extract, using resin adsorption, desorption, and solvent removal to obtain an olive leaf extract containing oleuropein.

[0049] In this paper, the clarified olive leaf extract is concentrated using conventional methods. Specific temperatures and pressures can be selected based on the solvent in the olive leaf extract to remove some of the solvent. In some embodiments, the extract solution is ethanol and water, and the extract is concentrated under reduced pressure at 50-60 °C until the volume fraction of ethanol in the solution is less than 10%, yielding a concentrate. Preferably, the concentrate is filtered and then adsorbed using a resin. For example, a filter membrane with a pore size of 0.5-1.0 micrometers (e.g., 0.5 micrometers, 0.6 micrometers, 0.8 micrometers, 1.0 micrometers, or within any range of two values, such as 0.5-0.8 micrometers) can be used for filtration.

[0050] In some embodiments, the resin used to adsorb iridoid glycosides in the concentrate can be a nonpolar or weakly polar macroporous resin, such as HPD-100, NKA-9, D101, or AB-8 macroporous adsorption resins. Preferably, the ratio of the amount of resin to the weight of olive leaves in step (A) is (0.8-1.2):1, more preferably (0.8-1):1 or (1-1.2):1. Preferably, the resin adsorption temperature is 15-30°C, such as 20-25°C. Preferably, the resin adsorption time is 0.5-4 hours, such as 1-2 hours. After resin adsorption, the resin is collected by filtration for desorption.

[0051] In some embodiments, the desorption includes eluting the adsorbed resin with an elution solvent to obtain an eluent containing iridoid glycosides. Preferably, the elution solvent is an aqueous ethanol solution; for example, a 50-80% (volume fraction) aqueous ethanol solution, a 60-80% (volume fraction) aqueous ethanol solution, or a 50-60% (volume fraction) aqueous ethanol solution. Preferably, the mass of the elution solvent is 5-8 times, such as 5-7 times or 7-8 times, the mass of the resin added before adsorption. Preferably, the elution temperature is 15-30°C, such as 20-25°C. Preferably, the elution time is 0.5-4 h, such as 0.5-1.5 h or 1-2 h. Preferably, the eluent is filtered, the filtrate is collected, and the solvent is recovered to obtain olive leaf extract; preferably, filtration is performed using a 0.3-0.5 micrometer filter membrane (such as 0.3 micrometer, 0.35 micrometer, 0.4 micrometer, 0.45 micrometer, 0.5 micrometer, or within any range of two values).

[0052] In this paper, oleuropein can be initially separated from other components through resin adsorption and desorption, thereby increasing the content of iridoid glycosides such as oleuropein and improving the efficiency of HSCCC separation in step (2). Alternatively, the olive leaf extract obtained by removing the solvent from the concentrate can be directly subjected to HSCCC separation without resin adsorption and desorption, but this will increase impurities and lower the purification efficiency of HSCCC.

[0053] In some implementations, in step (c), the eluent containing oleuropein is collected in segments.

[0054] In some embodiments, the method further includes elution buffers containing oleuropein with a purity ≥98%.

[0055] In some embodiments, the method further includes the step of removing the solvent from the combined eluent containing oleuropein. Those skilled in the art can select a suitable removal method based on the physical properties of the solvent to be removed. In some embodiments, the solvent is removed under conditions of 40-50°C and -0.1-0.01 MPa.

[0056] In the method of this invention, after removing the solvent from the eluent containing oleuropein, high-purity oleuropein can be obtained. In some embodiments, the yield of oleuropein obtained by the method of this invention reaches 80% or more, for example, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, or 93% or more, based on the content of oleuropein contained in the olive leaf extract. In some embodiments, the purity of oleuropein obtained by the method of this invention reaches 98.5% or more, for example, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99.0% or more, 99.1% or more, 99.2% or more, or 99.3% or more.

[0057] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0058] In this article, the ethanol content expressed as a percentage in ethanol solutions refers to the volume fraction of ethanol.

[0059] In this article, the D101 macroporous adsorption resin was produced by Xi'an Hanyu Resin Technology Co., Ltd.

[0060] In this article, the grade of polyacrylic acid is P822497.

[0061] Experimental Example 1: Preparation of Olive Leaf Extract

[0062] Take 200 g of olive leaves and place them in a 5 L round-bottom flask. Add 2400 g of 50%-75% ethanol aqueous solution and reflux for 1.5 h. After extraction, filter through a 5 μm pore size polypropylene membrane to obtain a clear olive leaf extract.

[0063] The olive leaf extract was concentrated under reduced pressure at 50-60 °C until the ethanol concentration was less than 10%, yielding a concentrated solution. The concentrated solution was clarified by filtration through a 0.8 μm polypropylene membrane, and then 200 g of D101 macroporous adsorption resin was added. Adsorption was carried out by stirring at room temperature for 2 h, followed by filtration, and the macroporous adsorption resin was collected. The macroporous adsorption resin was eluted with 1400 g of 60% ethanol at room temperature for 1 h. The eluent was filtered through a 0.45 μm polypropylene membrane, the filtrate was collected, and the solvent was recovered, yielding 21.5 g of olive leaf extract.

[0064] Experimental Example 2: Purity / Content Analysis

[0065] Chromatographic conditions: Column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); Column temperature: 30 ℃; Mobile phase: Acetonitrile: Water = 25:75; Flow rate: 1.0 mL / min; Detection wavelength: 230 nm.

[0066] Accurately weigh 20.0 mg of the olive leaf extract obtained in Example 1 into a 25 mL volumetric flask, dissolve it in 50% methanol and dilute to the mark, shake well to obtain the test solution. Take 10 μL of the test solution for liquid chromatography analysis, and calculate the oleuropein content using the external standard method. The results show that the oleuropein content in the olive leaf extract of Example 1 is 43.7 wt%.

[0067] In this article, the purity of oleuropein in the solution (such as the fraction containing oleuropein collected by the HSCCC method) is equal to the peak area of ​​oleuropein in liquid chromatography divided by the total peak area of ​​all peaks.

[0068] Example 1: pH-controlled HSCCC purification of oleuropein (1)

[0069] Take 200 mg of the sample from Example 1 (containing 87.4 mg of oleuropein) and prepare a solution with 15 mL of mobile phase. Purify oleuropein using a TBE-300C type HSCCCC (Shanghai Tongtian).

[0070] A solvent system consisting of n-butanol, dichloromethane, methanol, and water was used as the two-phase solvent for HSCCC separation, with the upper phase serving as the stationary phase and the lower phase as the mobile phase. The volume ratio of n-butanol, dichloromethane, methanol, and water was 1:5:1:5. The stationary phase was first pumped into the HSCCC separation column. After the column was filled with the stationary phase, the instrument was run at a speed of 800 rpm. Once the flow stabilized, the mobile phase was injected. After both phases had stabilized, the sample was injected. During subsequent purification, the mobile phase flow rate was 5 mL / min, and the detection wavelength was 230 nm.

[0071] In the initial purification phase (0-40 min), the mobile phase pH was adjusted to 3.7 with formic acid and contained 0.1% PAA (molecular weight 1000-2000). In the middle purification phase (40-70 min), the mobile phase pH was adjusted to 7.6 with ammonia and contained 0.1% PAA (molecular weight 1000-2000). In the later purification phase (70-110 min), an unadjusted mobile phase without PAA was used. Once the detector detected the presence of oleuropein, fractions containing oleuropein were collected (8-10 mL per fraction).

[0072] The purity of oleuropein in each fraction was determined using the method in Experiment 2. Fractions with a purity ≥ 98% were combined and the solvent was removed by evaporation at 40-50℃ and a pressure of -0.1-0.01 MPa to obtain 79.5 mg of high-purity oleuropein, with a yield of 91.0% (79.5 mg / 87.4 mg) and a purity of 99.0%.

[0073] Example 2: pH-controlled HSCCC purification of oleuropein (2)

[0074] Take 200 mg of the sample from Example 1 (containing 87.4 mg of oleuropein) and prepare a solution with 15 mL of mobile phase. Purify oleuropein using a TBE-300C type HSCCCC (Shanghai Tongtian).

[0075] A solvent system consisting of n-butanol, dichloromethane, methanol, and water was used as the two-phase solvent for HSCCC separation, with the upper phase serving as the stationary phase and the lower phase as the mobile phase. The volume ratio of n-butanol, dichloromethane, methanol, and water was 1:6:1.5:6. The stationary phase was first pumped into the HSCCC column. After the column was filled with the stationary phase, the instrument was run at a speed of 900 rpm. Once the flow stabilized, the mobile phase was injected. After both phases had stabilized, the sample was injected. During subsequent purification, the mobile phase flow rate was 5 mL / min, and the detection wavelength was 230 nm.

[0076] In the initial purification phase (0-40 min), the mobile phase pH was adjusted to 3.7 with formic acid and contained 0.1% PAA (molecular weight 1000-2000). In the middle purification phase (40-70 min), the mobile phase pH was adjusted to 7.6 with ammonia and contained 0.1% PAA (1000-2000). In the later purification phase (70-110 min), an unadjusted mobile phase without PAA was used. Once the detector detected the presence of oleuropein, fractions containing oleuropein were collected (8-10 mL per fraction).

[0077] The purity of oleuropein in each fraction was determined using the method in Experiment 2. Fractions with a purity ≥ 98% were combined and the solvent was removed by evaporation at 40-50℃ and a pressure of -0.1-0.01 MPa to obtain 81.4 mg of high-purity oleuropein, with a yield of 93.1% (81.4 mg / 87.4 mg) and a purity of 99.3%.

[0078] Example 3: pH-controlled HSCCC purification of oleuropein (3)

[0079] Take 200 mg of the sample from Example 1 (containing 87.4 mg of oleuropein) and prepare a solution with 15 mL of mobile phase. Purify oleuropein using a TBE-300C type HSCCCC (Shanghai Tongtian).

[0080] A solvent system consisting of n-butanol, dichloromethane, methanol, and water was used as the two-phase solvent for HSCCC separation, with the upper phase serving as the stationary phase and the lower phase as the mobile phase. The volume ratio of n-butanol, dichloromethane, methanol, and water was 1:10:2:10. The stationary phase was first pumped into the HSCCC separation column. After the column was filled with the stationary phase, the instrument was run at a speed of 1000 rpm. Once the flow stabilized, the mobile phase was injected. After both phases had stabilized, the sample was injected. During subsequent purification, the flow rate of the mobile phase was 5 mL / min, and the detection wavelength was 230 nm.

[0081] In the initial purification phase (0-40 min), the mobile phase pH was adjusted to 3.7 with formic acid and contained 0.1% PAA (molecular weight 1000-2000). In the middle purification phase (40-70 min), the mobile phase pH was adjusted to 7.6 with ammonia and contained 0.1% PAA (molecular weight 1000-2000). In the later purification phase (70-110 min), an unadjusted mobile phase without PAA was used. Once the detector detected the presence of oleuropein, fractions containing oleuropein were collected (8-10 mL per fraction).

[0082] The purity of oleuropein in each fraction was determined using the method described in Experiment 2. Fractions with a purity ≥ 98% were combined and the solvent was removed by evaporation at 40-50℃ and a pressure of -0.1-0.01 MPa to obtain 78.2 mg of high-purity oleuropein, with a yield of 89.5% (78.2 mg / 87.4 mg) and a purity of 98.9%.

[0083] Comparative Example 1: Conventional HSCCC purification of oleuropein

[0084] Take 200 mg of the sample from Example 1 (containing 87.4 mg of oleuropein) and prepare a solution with 15 mL of mobile phase. Purify oleuropein using a TBE-300C type HSCCCC (Shanghai Tongtian).

[0085] A solvent system consisting of n-butanol, dichloromethane, methanol, and water was used as the two-phase solvent for HSCCC separation, with the upper phase serving as the stationary phase and the lower phase as the mobile phase. The volume ratio of n-butanol, dichloromethane, methanol, and water was 1:6:1.5:6. The stationary phase was first pumped into the HSCCC column. After the column was filled with the stationary phase, the instrument was run at a speed of 900 rpm. Once the flow stabilized, the mobile phase was injected. After both phases had stabilized, the sample was injected. During subsequent purification, the mobile phase flow rate was 5 mL / min, and the detection wavelength was 230 nm.

[0086] Once the detector detects the presence of oleuropein, fractions containing oleuropein are collected (8-10 mL per fraction).

[0087] The purity of oleuropein in each fraction was determined using the method described in Experiment 2. Fractions with a purity ≥ 98% were combined and the solvent was removed by evaporation at 40-50℃ and a pressure of -0.1-0.01 MPa to obtain 24.8 mg of high-purity oleuropein, with a yield of 28.4% (24.8 mg / 87.4 mg) and a purity of 98.2%.

[0088] Comparative Example 2, pH Adjustment - Conventional HSCCC Purification of Oleuropein (without adsorption aids)

[0089] Take 200 mg of the sample from Example 1 (containing 87.4 mg of oleuropein) and prepare a solution with 15 mL of mobile phase. Purify oleuropein using a TBE-300C type HSCCCC (Shanghai Tongtian).

[0090] A solvent system consisting of n-butanol, dichloromethane, methanol, and water was used as the two-phase solvent for HSCCC separation, with the upper phase serving as the stationary phase and the lower phase as the mobile phase. The volume ratio of n-butanol, dichloromethane, methanol, and water was 1:6:1.5:6. The stationary phase was first pumped into the HSCCC column. After the column was filled with the stationary phase, the instrument was run at a speed of 900 rpm. Once the flow stabilized, the mobile phase was injected. After both phases had stabilized, the sample was injected. During subsequent purification, the mobile phase flow rate was 5 mL / min, and the detection wavelength was 230 nm.

[0091] In the initial purification phase (0-40 min), the pH of the mobile phase was adjusted to 3.7 with formic acid, without adding PAA. In the middle purification phase (40-70 min), the pH of the mobile phase was adjusted to 7.6 with ammonia, without adding PAA. In the later purification phase (70-110 min), the mobile phase without pH adjustment was used. After the detector showed a signal of oleuropein, the fractions containing oleuropein were collected in fractions (8-10 mL per fraction).

[0092] The purity of oleuropein in each fraction was determined using the method in Experiment 2. Fractions with a purity ≥ 98% were combined and the solvent was removed by evaporation at 40-50℃ and a pressure of -0.1-0.01 MPa to obtain 47.3 mg of high-purity oleuropein, with a yield of 54.1% (47.3 mg / 87.4 mg) and a purity of 98.4%.

[0093] Comparative Example 3: Purification of oleuropein with only adsorption aid added, without pH adjustment.

[0094] Take 200 mg of the sample from Example 1 (containing 87.4 mg of oleuropein) and prepare a solution with 15 mL of mobile phase. Purify oleuropein using a TBE-300C type HSCCCC (Shanghai Tongtian).

[0095] A solvent system consisting of n-butanol, dichloromethane, methanol, and water was used as the two-phase solvent for HSCCC separation, with the upper phase serving as the stationary phase and the lower phase as the mobile phase. The volume ratio of n-butanol, dichloromethane, methanol, and water was 1:6:1.5:6. The stationary phase was first pumped into the HSCCC column. After the column was filled with the stationary phase, the instrument was run at a speed of 900 rpm. Once the flow stabilized, the mobile phase was injected. After both phases had stabilized, the sample was injected. During subsequent purification, the mobile phase flow rate was 5 mL / min, and the detection wavelength was 230 nm.

[0096] During the initial and middle stages of purification (0-70 min), the mobile phase contained 0.1% PAA (molecular weight 1000-2000). During the later stages of purification (70-110 min), the initial mobile phase was switched back, and after the detector showed a signal of oleuropein, the fractions containing oleuropein were collected in fractions (8-10 mL per fraction).

[0097] The purity of oleuropein in each fraction was determined using the method described in Experiment 2. Fractions with a purity ≥ 98% were combined and the solvent was removed by evaporation at 40-50℃ and a pressure of -0.1-0.01 MPa to obtain 59.6 mg of high-purity oleuropein, with a yield of 68.2% (59.6 mg / 87.4 mg) and a purity of 98.8%.

[0098] Comparative Example 4

[0099] The method of Example 2 was repeated, except that the ratio (volume ratio) of n-butanol, dichloromethane, methanol and water was 1:4:1.5:4.

[0100] After purification and removal of solvent, 34.7 mg of oleuropein was obtained, with a yield of 39.7% (34.7 mg / 87.4 mg) and a purity of 98.2%.

[0101] Comparative Example 5

[0102] The method of Example 2 was repeated, except that the concentration of PAA in the mobile phase was 0.02 wt%.

[0103] After purification and removal of solvent, 66.7 mg of oleuropein was obtained, with a yield of 76.3% (66.7 mg / 87.4 mg) and a purity of 98.5%.

Claims

1. A mixed solvent, selected from: (1) A mixed solvent composed of n-butanol, dichloromethane, methanol, and water, wherein, The volume ratio of n-butanol, dichloromethane, methanol and water is 1:(5-15):(1-5):(5-15), preferably 1:(5-10):(1-2):(5-10); (2) The first solvent is the upper layer of the mixed solvent described in (1) after it has been allowed to stand and separate into layers; (3) A second solvent, wherein the second solvent is the lower layer after the mixed solvent of (1) has been allowed to stand and separate into layers; and (4) A combination of the first solvent and the second solvent; Preferably, the second solvent further contains polyacrylic acid; preferably, the concentration of polyacrylic acid in the second solvent is 0.05-0.2 wt%. Preferably, the second solvent includes a second solvent with a pH of 3.5-4.0 and / or a second solvent with a pH of 7.5-8.

0.

2. A method for isolating oleuropein, the method comprising the steps of: (1) Provide olive leaf extract containing oleuropein; (2) The oleuropein in the olive leaf extract was separated by high-speed countercurrent chromatography; in, A mixed solvent consisting of n-butanol, dichloromethane, methanol, and water is used as the two-phase solvent for high-speed countercurrent chromatography separation. The upper phase of the mixed solvent is the stationary phase, and the lower phase of the mixed solvent is the mobile phase. Step (2) includes: (a) In the initial stage of high-speed countercurrent chromatography separation, from 0 to 40 min, the first mobile phase is used for elution; wherein, the first mobile phase is the lower phase adjusted to pH 3.5-4.0 with acid, and the first mobile phase also contains polyacrylic acid; (b) During the intermediate stage of high-speed countercurrent chromatography separation at min 40-70, a second mobile phase is used for elution; wherein the second mobile phase is the lower phase adjusted to pH 7.5-8.0 with alkali, and the second mobile phase also contains polyacrylic acid; (c) During the high-speed countercurrent chromatography separation phase from 70 to 110 min, the lower phase is used for elution, and the eluent containing oleuropein is collected.

3. The method as described in claim 2, characterized in that, In step (2), the sample injected into the high-speed countercurrent chromatography is a mixture containing the olive leaf extract, wherein the content of the olive leaf extract in the mixture is 5-20 g / L; and / or, the content of oleuropein in the mixture containing the olive leaf extract is ≤20 g / L. Preferably, the solvent of the mixture is the lower phase.

4. The method as described in claim 2, characterized in that, In step (2), the volume ratio of n-butanol, dichloromethane, methanol and water in the two-phase solvent used for high-speed countercurrent chromatography separation is 1:(5-15):(1-5):(5-15), preferably 1:(5-10):(1-2):(5-10).

5. The method as described in claim 2, characterized in that, In step (a), the acid is selected from one or more of formic acid, acetic acid, hydrochloric acid and sulfuric acid, preferably formic acid; and / or, in step (b), the base is selected from one or more of sodium carbonate, potassium carbonate, triethylamine, pyridine and ammonia, preferably ammonia.

6. The method as described in claim 2, characterized in that: The polyacrylic acid has a number-average molecular weight of 1000-2000; and / or, The first mobile phase contains 0.05-0.2 wt% polyacrylic acid; and / or, The second mobile phase contains 0.05-0.2 wt% polyacrylic acid; and / or, The mobile phase used in step (c) does not contain polyacrylic acid.

7. The method as described in claim 2, characterized in that, Step (1) includes: (A) extracting oleuropein from olive leaves using a solvent to obtain olive leaf extract; preferably, the solvent is a mixture of ethanol and water; preferably, the extraction temperature is the reflux temperature of the solvent; preferably, in step (A), the amount of solvent used is 10-15 times the weight of the olive leaves; preferably, in step (A), the extraction time is 1-2 h; preferably, filtering the olive leaf extract to obtain a clear olive leaf extract; preferably, removing the solvent from the clear olive leaf extract to obtain the olive leaf extract containing oleuropein.

8. The method as described in claim 7, characterized in that, Step (1) further includes: (B) concentrating the clarified olive leaf extract, using resin adsorption, desorption, and solvent removal to obtain an olive leaf extract containing oleuropein.

9. The method as described in claim 8, characterized in that: The resin is a non-polar or weakly polar macroporous resin; and / or, The ratio of the amount of resin used to the weight of olive leaves in step (A) is (0.8-1.2):1; and / or, The resin adsorption temperature is 15-30℃; and / or, The resin adsorption time is 0.5-4 hours; and / or, After resin adsorption, the resin is collected by filtration and used for desorption; and / or, The desorption process includes eluting the adsorbed resin with an elution solvent; preferably, the elution solvent is an aqueous ethanol solution; preferably, the mass of the elution solvent is 5-8 times the mass of the resin added before adsorption; preferably, the elution temperature is 15-30°C; preferably, the elution time is 0.5-4 hours; preferably, the eluent is filtered, the filtrate is collected, the solvent is recovered, and olive leaf extract is obtained.

10. The method as described in claim 2, characterized in that, In step (c), the eluent containing oleuropein is collected in segments; Preferably, the method further includes an eluent containing oleuropein with a purity ≥98%; Preferably, the method further includes removing the solvent from the combined eluent containing oleuropein to obtain oleuropein.

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