A method for extracting chlorogenic acid from blueberry leaves

Abstract

The present invention belongs to the technical field of plant active ingredient extraction, and specifically discloses a method for extracting chlorogenic acid from blueberry leaves. The method for extracting chlorogenic acid from blueberry leaves of the present invention first mixes blueberry leaves with a solvent and cellulase, and ultrasonically treats to obtain an extract; the extract is centrifuged, and the supernatant is collected, and the supernatant is concentrated to obtain a crude blueberry leaf polyphenol concentrate; the crude blueberry leaf polyphenol concentrate is dissolved and separated and purified using a medium-pressure preparative liquid chromatograph to obtain chlorogenic acid. The present invention turns waste blueberry leaves into treasure, obtains high-purity chlorogenic acid through appropriate conditions, and the purification time is short and the operation is simple.

Description

Technical Field

[0001] The invention belongs to the technical field of plant active ingredient extraction, and particularly relates to a method for extracting chlorogenic acid from blueberry leaves. Background Art

[0002] Blueberry is a shrub native to North America, Europe, and Asia. Its fruit is a nutritious, antioxidant-rich superfood. Blueberry leaves, as part of the blueberry plant, are also rich in nutrients. Research has shown that chlorogenic acid, an important polyphenolic compound, possesses multiple biological activities, including antioxidant, anti-inflammatory, antibacterial, antidiabetic, lipid-lowering, and antihypertensive properties. It also has potential pharmacological effects, such as anti-tumor, cardiovascular disease, liver disease, and obesity. It is widely used in health foods, cosmetics, and food additives. However, in current production, blueberry leaves are often treated as waste and not effectively utilized, resulting in a significant waste of resources. Therefore, research on methods for extracting chlorogenic acid from blueberry leaves to obtain chlorogenic acid products is crucial for the research and development of natural medicines and functional foods. Summary of the Invention

[0003] The object of the present invention is to provide a method for extracting chlorogenic acid from blueberry leaves, which can extract high-purity chlorogenic acid from blueberry leaves, and has short purification time and simple operation.

[0004] To achieve the above objectives, the present invention adopts the following technical solutions.

[0005] The present invention provides a method for extracting chlorogenic acid from blueberry leaves, comprising the steps of:

[0006] S1, mixing blueberry leaves with a solvent and cellulase, and performing ultrasonic treatment to obtain an extract;

[0007] S2, centrifuging the extract, collecting the supernatant, and concentrating the supernatant to obtain a crude blueberry leaf polyphenol concentrate;

[0008] S3, dissolving the crude blueberry leaf polyphenol concentrate and separating and purifying it using a medium-pressure preparative liquid chromatography to obtain chlorogenic acid;

[0009] Wherein, the medium pressure preparative liquid chromatograph adopts SNAP Ultra C 18 The chromatographic column is eluted with a mobile phase consisting of a mobile phase A and a mobile phase B, wherein the mobile phase A is water and the mobile phase B is acetonitrile.

[0010] As an embodiment of the present invention, the elution is gradient elution. Preferably, the gradient elution is performed in such a manner that the volume percentage of mobile phase A decreases from 95% to 42% with the total volume of mobile phase A and mobile phase B being 100%, and the balance being mobile phase B.

[0011] As a further preferred embodiment of the present invention, the gradient elution method is: with the total volume of mobile phase A and mobile phase B as 100%, from 0 to 3 minutes, the volume percentage of the mobile phase A decreases from 95% to 60%, and the remainder is mobile phase B; from 3 to 18 minutes, the volume percentage of the mobile phase A decreases from 60% to 52%, and the remainder is mobile phase B; from 18 to 25 minutes, the volume percentage of the mobile phase A decreases from 52% to 48%, and the remainder is mobile phase B; from 25 to 30 minutes, the volume percentage of the mobile phase A decreases from 48% to 42%, and the remainder is mobile phase B.

[0012] As an embodiment of the present invention, the medium pressure preparative liquid chromatograph adopts SNAP Ultra C 18 30g chromatography column.

[0013] As an embodiment of the present invention, the injection volume of the medium-pressure preparative liquid chromatograph is 4 to 8 mL, preferably 5 to 6 mL, and more preferably 6 mL.

[0014] As an embodiment of the present invention, the flow rate of the mobile phase of the medium pressure preparative liquid chromatograph is 5 to 15 mL / min, preferably 8 to 10 mL / min, and more preferably 10 mL / min.

[0015] As an embodiment of the present invention, the detector used by the medium-pressure preparative liquid chromatograph is a UV detector with a detection wavelength of 280 nm.

[0016] As an embodiment of the present invention, during separation and purification by the medium-pressure preparative liquid chromatography, the elution time of chlorogenic acid is 13.3 min, and chlorogenic acid is collected.

[0017] As an embodiment of the present invention, in step S1, the amount of cellulase added is 0.2% to 0.8%, preferably 0.4% to 0.7%, and more preferably 0.6%, based on the weight of the blueberry leaves.

[0018] As an embodiment of the present invention, in step S1, the blueberry leaves are preferably blueberry leaf powder, which is prepared by grinding fresh shade-dried blueberry leaves and passing them through an 80-mesh sieve to obtain blueberry leaf powder.

[0019] As an embodiment of the present invention, in step S1, the solvent is an ethanol aqueous solution, wherein the volume fraction of ethanol in the ethanol aqueous solution is preferably 40% to 80%, more preferably 40% to 60%, and even more preferably 50%.

[0020] As an embodiment of the present invention, in step S1, the amount of solvent used is: 10-50 mL of solvent is added per gram of blueberry leaf powder, preferably, 30-50 mL of solvent is added per gram of blueberry leaf powder, and more preferably, 40 mL of solvent is added per gram of blueberry leaf powder.

[0021] As an embodiment of the present invention, in step S1, the temperature of ultrasonic treatment is 50-60°C, the treatment time is 30-60 minutes, the ultrasonic power is 600W, and the ultrasonic frequency is 40kHz.

[0022] As an embodiment of the present invention, in step S3, the solvent used to dissolve the crude blueberry leaf polyphenol concentrate is methanol, and the amount of methanol used is: 30 to 50 mL per gram of the crude blueberry leaf polyphenol concentrate, preferably 40 mL per gram of the crude blueberry leaf polyphenol concentrate.

[0023] As an embodiment of the present invention, in step S3, after the blueberry leaf polyphenol crude concentrate is dissolved, it is first filtered through a membrane and then separated and purified using a medium-pressure preparative liquid chromatography. Preferably, the membrane used for membrane filtration is a 0.22 μm filter membrane.

[0024] As an embodiment of the present invention, the extraction method provided by the present invention further comprises the step of analyzing and detecting chlorogenic acid, preferably by high performance liquid chromatography.

[0025] Further preferably, the detection conditions of the high performance liquid chromatography method are: using a Hypersil GOLD chromatographic column, model 250×4.6 mm, 5 μm; and / or,

[0026] Column temperature: 35°C; and / or,

[0027] The detector is a UV detector with a detection wavelength of 280 nm; and / or,

[0028] Injection volume: 10 μL; and / or,

[0029] Mobile phase flow rate: 1.0 mL / min; and / or,

[0030] Mobile phase A is acetonitrile, mobile phase B is 0.2% (volume percentage) formic acid aqueous solution; and / or,

[0031] The gradient elution method is: the total volume of mobile phase A and mobile phase B is 100%,

[0032] 0-10 min, 15%-50% mobile phase A, the balance is mobile phase B;

[0033] 10-20 min, 50-100% mobile phase A, the balance is mobile phase B;

[0034] 20-25 min, 100% A;

[0035] 25-27 min, 100%-15% mobile phase A, the balance is mobile phase B;

[0036] 27~40min, 15% A.

[0037] The present invention provides a method for extracting chlorogenic acid from blueberry leaves. The method first extracts blueberry leaf polyphenols from the blueberry leaves to obtain a crude extract. The crude extract is then separated and purified using medium-pressure preparative liquid chromatography to obtain chlorogenic acid. The present invention also analyzes the isolated chlorogenic acid using high-performance liquid chromatography (HPLC), which is simple to operate and provides highly reproducible results.

[0038] This invention transforms blueberry leaves into valuable resources, not only reducing environmental pollution but also, more importantly, yielding high-purity chlorogenic acid under suitable conditions. The purification process is also quick, requiring only approximately 13.3 minutes, significantly reducing the purification time required. The extraction method is simple to operate, and the purified chlorogenic acid is of high purity, promising broad application prospects in the health food and pharmaceutical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a graph showing the relationship between different amounts of cellulase added and the polyphenol content in the supernatant obtained in Example 1 of the present invention;

[0040] Figure 2 is a graph showing the relationship between different concentrations of ethanol aqueous solutions and the polyphenol content in the obtained supernatant in Example 1 of the present invention;

[0041] Figure 3 This is a graph showing the relationship between different mixing ratios of blueberry leaf powder and ethanol aqueous solution and the polyphenol content in the obtained supernatant in Example 1 of the present invention;

[0042] Figure 4 This is a medium-pressure preparative liquid chromatogram obtained in Example 2 of the present invention;

[0043] Figure 5 This is a high performance liquid chromatography test chart obtained in Example 2 of the present invention;

[0044] Figure 6 This is a medium-pressure preparative liquid chromatogram obtained under the conditions of comparative mobile phase 1 in Test Example 1 of the present invention;

[0045] Figure 7This is a medium-pressure preparative liquid chromatogram obtained under the conditions of comparative mobile phase 2 in Test Example 1 of the present invention;

[0046] Figure 8 This is a medium-pressure preparative liquid chromatogram obtained in Experimental Example 2 of the present invention;

[0047] Figure 9 This is a medium-pressure preparative liquid chromatogram obtained in Experimental Example 3 of the present invention;

[0048] Figure 10 This is a medium-pressure preparative liquid chromatogram obtained in Test Example 4 of the present invention. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is described in further detail below. It should be understood by those skilled in the art that the specific embodiments described are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0050] It should be noted that, unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available conventional products.

[0051] The following is a detailed explanation through examples.

[0052] Example 1

[0053] This example provides a method for preparing a crude blueberry leaf polyphenol concentrate, comprising the following steps:

[0054] (1) Grind fresh, shade-dried blueberry leaves and pass them through an 80-mesh sieve. The sieved residue is the blueberry leaf powder.

[0055] (2) mixing the blueberry leaf powder obtained in step (1) with an ethanol aqueous solution and cellulase, and ultrasonically treating the mixture for 1 h at a temperature of 55° C., an ultrasonic power of 600 W, and an ultrasonic frequency of 40 kHz to obtain an extract;

[0056] (3) The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was collected;

[0057] (4) The solvent in the supernatant was removed by rotary evaporation, and then the supernatant was dried at 70°C to obtain a crude blueberry leaf polyphenol concentrate.

[0058] This example investigates the relationship between the polyphenol content in the supernatant obtained using different concentrations of ethanol-water solutions, different mixing ratios of blueberry leaf powder and ethanol-water solutions, and different amounts of cellulase added. The details are as follows.

[0059] When investigating the relationship between the polyphenol content in the supernatant obtained using ethanol aqueous solutions of different concentrations, the volume fraction of ethanol in the ethanol aqueous solution was set to 40%, 50%, 60%, 70%, and 80%, respectively; at this time, the mixed material-liquid ratio of blueberry leaf powder and ethanol aqueous solution was fixed at 1:40 g / mL, and the amount of cellulase added was set to 0.6% of the mass of the blueberry leaf powder.

[0060] When investigating the relationship between different mixing ratios of blueberry leaf powder and ethanol aqueous solution and the polyphenol content in the obtained supernatant, the material-liquid ratio of blueberry leaf powder and ethanol aqueous solution (i.e., the volume ratio of blueberry leaf powder to ethanol aqueous solution) was set to 1:10 g / mL, 1:20 g / mL, 1:30 g / mL, 1:40 g / mL, and 1:50 g / mL, respectively; at this time, the volume fraction of ethanol in the ethanol aqueous solution was 50%, and the amount of cellulase added was set to 0.6% of the mass of the blueberry leaf powder.

[0061] To investigate the relationship between different amounts of cellulase added and the polyphenol content in the resulting supernatant, the cellulase addition levels were set at 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% of the blueberry leaf powder mass. The blueberry leaf powder and ethanol-water mixture ratio was fixed at 1:40 g / mL, and the volume fraction of ethanol in the ethanol-water solution was 50%.

[0062] The amount of blueberry leaf powder used during the investigation was 1.00 g. According to the method provided in this example, experiments were conducted under different conditions.

[0063] The relationship between different cellulase addition amounts and the polyphenol content in the obtained supernatant is shown in Figure 1 As shown, Figure 1 The results showed that when the amount of cellulase added was 0% of the mass of blueberry leaf powder (i.e., when no cellulase was added), the polyphenol content in the supernatant was only 108.64 mg / g, and the chlorogenic acid content was 5.75 mg / g. As the amount of cellulase added increased, the polyphenol content in the supernatant also increased significantly. When the amount of cellulase added was 0.6%, the polyphenol content in the supernatant reached its maximum value of 159.72 mg / g, and the chlorogenic acid content was 8.45 mg / g. However, when the amount of cellulase added exceeded 0.6%, the polyphenol content in the supernatant began to decrease. Therefore, when extracting blueberry leaf polyphenols, the amount of cellulase added is preferably 0.4% to 0.7% of the mass of blueberry leaf powder, and the optimal amount of cellulase added is 0.6% of the mass of blueberry leaf powder.

[0064] The relationship between the polyphenol content in the supernatant obtained by using different concentrations of ethanol aqueous solution is shown in Figure 2 As shown, Figure 2The results showed that the polyphenol content in the supernatant increased significantly with increasing ethanol volume fraction in the ethanol-water solution. At a 50% ethanol volume fraction, the polyphenol content in the supernatant reached a maximum of 172.59 mg / g, along with a chlorogenic acid content of 9.13 mg / g. However, when the ethanol volume fraction exceeded 50%, the polyphenol content in the supernatant began to decrease. Therefore, when extracting blueberry leaf polyphenols, the ethanol volume fraction in the ethanol-water solution is preferably between 40% and 60%, with the optimal ethanol volume fraction being 50%.

[0065] The relationship between the mixing ratio of blueberry leaf powder and ethanol water solution and the polyphenol content in the obtained supernatant is shown in Figure 3 As shown, Figure 3 The results showed that the polyphenol content in the supernatant increased significantly with increasing solvent content. When 40 mL of solvent was added per gram of blueberry leaf powder, the polyphenol content in the supernatant reached its maximum value, reaching 162.20 mg / g, and the chlorogenic acid content was 8.58 mg / g. However, when the amount of solvent added exceeded 40 mL per gram of blueberry leaf powder, the polyphenol content in the supernatant began to decrease. Therefore, when extracting blueberry leaf polyphenols, the optimal amount of solvent added per gram of blueberry leaf powder is 30-50 mL, with the optimal amount being 40 mL per gram of blueberry leaf powder.

[0066] Example 2

[0067] This embodiment provides a method for extracting chlorogenic acid from blueberry leaves, comprising the following steps:

[0068] (1) Grind fresh, shade-dried blueberry leaves and pass them through an 80-mesh sieve. The sieved residue is the blueberry leaf powder.

[0069] (2) 1.00 g of blueberry leaf powder was mixed with an ethanol aqueous solution and cellulase, wherein the volume fraction of ethanol in the ethanol aqueous solution was 50%, the material-liquid ratio of blueberry leaf powder to ethanol aqueous solution was 1:40 g / mL, and the amount of cellulase added was 0.6% of the mass of the blueberry leaf powder. After mixing, the mixture was ultrasonically treated for 1 h at a temperature of 55°C, an ultrasonic power of 600 W, and an ultrasonic frequency of 40 kHz to obtain an extract;

[0070] (3) The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was collected. The polyphenol content in the supernatant was 159.72 mg / g after testing.

[0071] (4) removing the solvent from the supernatant by rotary evaporation, and then drying at 70°C to obtain a crude blueberry leaf polyphenol concentrate;

[0072] (5) separating and purifying the crude blueberry leaf polyphenol concentrate obtained in step (4), dissolving the crude blueberry leaf polyphenol concentrate in methanol, wherein the amount of methanol is as follows: adding 40 mL of methanol per gram of crude blueberry leaf polyphenol concentrate, performing membrane filtration after dissolution, and using a 0.22 μm filter membrane to collect the filtrate;

[0073] (6) The obtained filtrate was separated and purified by medium pressure preparative liquid chromatography; the medium pressure preparative liquid chromatography was performed by SNAP Ultra C 18 30 g chromatographic column, injection volume of 6 mL, mobile phase flow rate of 10 mL / min;

[0074] Gradient elution of mobile phase: the mobile phase used for elution consists of mobile phase A and mobile phase B, mobile phase A is water, and mobile phase B is acetonitrile; the gradient elution method is: the total volume of mobile phase A and mobile phase B is 100%,

[0075] From 0 to 3 minutes, the volume percentage of mobile phase A decreases from 95% to 60%, and the remainder is mobile phase B;

[0076] From 3 to 18 minutes, the volume percentage of mobile phase A decreases from 60% to 52%, with the remainder being mobile phase B;

[0077] From 18 to 25 minutes, the volume percentage of mobile phase A decreases from 52% to 48%, with the remainder being mobile phase B;

[0078] After 25 to 30 minutes, the volume percentage of mobile phase A decreased from 48% to 42%, and the remainder was mobile phase B.

[0079] The detector used in the medium-pressure preparative liquid chromatograph was a UV detector with a detection wavelength of 280 nm. During the separation and purification, the elution time of chlorogenic acid was 13.3 min, and the chlorogenic acid was collected.

[0080] The collected chlorogenic acid was analyzed and detected by high performance liquid chromatography. The detection conditions of the high performance liquid chromatography were as follows:

[0081] A Hypersil GOLD column (250 × 4.6 mm, 5 μm) was used; the column temperature was 35°C; a UV detector was used at a detection wavelength of 280 nm; the injection volume was 10 μL; the mobile phase flow rate was 1.0 mL / min; the mobile phase A was acetonitrile, and the mobile phase B was a 0.2% (volume percentage) formic acid aqueous solution; the mobile phase gradient elution method was: the total volume of mobile phase A and mobile phase B was 100%,

[0082] 0-10 min, 15%-50% mobile phase A, the balance is mobile phase B;

[0083] 10-20 min, 50-100% mobile phase A, the balance is mobile phase B;

[0084] 20-25 min, 100% A;

[0085] 25-27 min, 100%-15% mobile phase A, the balance is mobile phase B;

[0086] 27~40min, 15% A.

[0087] The medium pressure preparative liquid chromatogram is shown in Figure 4 As shown, the HPLC detection diagram is shown in Figure 5 As shown, the test results show that the purity of the chlorogenic acid monomer in the collected chlorogenic acid is 94.98%. It was also measured that 32.08 mg of chlorogenic acid was isolated from 150 mg of crude blueberry leaf polyphenol extract, with an extraction rate of 21.39%.

[0088] Test Example 1

[0089] This test example mainly investigated the mobile phase used in separation and purification by medium-pressure preparative liquid chromatography.

[0090] The test subjects were all the filtrate obtained in step (5) of Example 2 of the present invention, and the filtrate was separated and purified by medium pressure preparative liquid chromatography, and the medium pressure preparative liquid chromatography was performed by SNAP Ultra C 18 30g chromatographic column, injection volume of 6mL, mobile phase flow rate of 10mL / min. The only difference from step (6) in Example 2 of the present invention is that the mobile phase composition used for separation and purification by medium-pressure preparative liquid chromatography is different, but the mobile phase gradient elution method is the same.

[0091] The mobile phase compositions of the medium pressure preparative liquid chromatographs used for comparison are:

[0092] Comparative mobile phase 1: consists of mobile phase A1 and mobile phase B1, mobile phase A1 is water, mobile phase B1 is methanol;

[0093] Comparative mobile phase 2: consists of mobile phase A2 and mobile phase B2, mobile phase A2 is 0.2% (volume percentage) formic acid aqueous solution, and mobile phase B2 is acetonitrile.

[0094] The filtrate obtained in step (5) of Example 2 of the present invention was separated and purified using a medium-pressure preparative liquid chromatograph under the conditions of comparative mobile phase 1 and comparative mobile phase 2, and chlorogenic acid was collected.

[0095] The obtained medium pressure preparative liquid chromatograms are shown in Figure 6 and Figure 7The test results showed that the purity of the chlorogenic acid monomer in the collected chlorogenic acid was 81.74% and 86.39%, respectively. This comparison shows that the composition of the mobile phase used during separation and purification by medium-pressure preparative liquid chromatography significantly affects the separation effect. When mobile phase A is water and mobile phase B is acetonitrile, the separation and purification effect is optimal, with the purity of the chlorogenic acid monomer exceeding 90%. Therefore, the optimal mobile phase is determined to be an acetonitrile / water solution.

[0096] Test Example 2

[0097] This experimental example mainly investigated the injection volume during separation and purification using a medium-pressure preparative liquid chromatography.

[0098] The test object is the filtrate obtained in step (5) of Example 2 of the present invention, and the medium pressure preparative liquid chromatography is used for separation and purification. The medium pressure preparative liquid chromatography uses SNAP Ultra C 18 30 g chromatographic column, the only difference from step (6) in Example 2 of the present invention is that the injection volume of the medium-pressure preparative liquid chromatography separation and purification is 8 mL, and other conditions are the same.

[0099] Under the condition of an injection volume of 8 mL, the filtrate obtained in step (5) of Example 2 of the present invention was separated and purified using a medium-pressure preparative liquid chromatograph, and chlorogenic acid was collected.

[0100] The obtained medium pressure preparative liquid chromatogram is shown in Figure 8 The test results showed that the purity of the chlorogenic acid monomer in the collected chlorogenic acid was 88.21%. Comparison shows that the injection volume used in the separation and purification of medium-pressure preparative liquid chromatography also has a significant impact on the separation effect. The separation effect deteriorates when the injection volume increases.

[0101] Test Example 3

[0102] This test example mainly investigated the mobile phase flow rate during separation and purification using a medium-pressure preparative liquid chromatograph.

[0103] The test object is the filtrate obtained in step (5) of Example 2 of the present invention, and the medium pressure preparative liquid chromatography is used for separation and purification. The medium pressure preparative liquid chromatography uses SNAP Ultra C 18 The 30 g chromatographic column is different from step (6) in Example 2 of the present invention only in that the mobile phase flow rate of the medium pressure preparative liquid chromatograph for separation and purification is 15 mL / min, and other conditions are the same.

[0104] The filtrate obtained in step (5) of Example 2 of the present invention was separated and purified using a medium-pressure preparative liquid chromatograph at a mobile phase flow rate of 15 mL / min, and chlorogenic acid was collected.

[0105] The obtained medium pressure preparative liquid chromatogram is shown in Figure 9 The test results showed that the purity of the chlorogenic acid monomer in the collected chlorogenic acid was 79.31%. Comparison shows that the flow rate of the mobile phase used in the separation and purification of medium-pressure preparative liquid chromatography also has a significant impact on the separation effect. The separation effect deteriorates when the flow rate increases.

[0106] Test Example 4

[0107] This test example mainly investigated the chromatographic columns used in separation and purification by medium-pressure preparative liquid chromatography.

[0108] The test subjects all used the filtrate obtained in step (5) of Example 2 of the present invention, and were separated and purified using a medium-pressure preparative liquid chromatograph. The mobile phase composition, mobile phase gradient elution method, injection volume, and mobile phase flow rate of the medium-pressure preparative liquid chromatograph were the same as those in step (6) of Example 2 of the present invention. The only difference was that the chromatographic column selected for separation and purification by the medium-pressure preparative liquid chromatograph was different.

[0109] The chromatographic column of the medium pressure preparative liquid chromatograph used for comparison is: ODS C 18 Chromatographic column.

[0110] Using ODS C 18 The filtrate obtained in step (5) of Example 2 was separated and purified by medium-pressure preparative liquid chromatography, and chlorogenic acid was collected. The obtained medium-pressure preparative liquid chromatogram is shown in Figure 10 The test results showed that the purity of the chlorogenic acid monomer in the collected chlorogenic acid was 82.49%. Compared with the results, it can be seen that the selection of chromatographic column for separation and purification by medium-pressure preparative liquid chromatography has a significant impact on the separation effect. The chromatographic column is SNAP Ultra C 18 The separation and purification effect was the best when the purity of chlorogenic acid monomer reached more than 90%. Therefore, the best chromatographic column was determined to be SNAP Ultra C 18 .

[0111] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. The embodiments of this application and the features in the embodiments may be combined with each other in any manner unless there is a conflict.

Claims

1. A method for extracting chlorogenic acid from blueberry leaves, characterized in that: Including steps: S1. Mixing blueberry leaves with a solvent and cellulase, and ultrasonically treating the mixture to obtain an extract; the amount of the cellulase added is 0.4% to 0.7% based on the weight of the blueberry leaves; the solvent is an ethanol-water solution, and the volume fraction of ethanol in the ethanol-water solution is 40% to 60%; S2, centrifuging the extract, collecting the supernatant, and concentrating the supernatant to obtain a crude blueberry leaf polyphenol concentrate; S3, dissolving the crude blueberry leaf polyphenol concentrate and separating and purifying it using a medium-pressure preparative liquid chromatography to obtain chlorogenic acid; Wherein, the medium pressure preparative liquid chromatograph adopts SNAP Ultra C 18 30g chromatography column; The mobile phase used for elution consists of mobile phase A and mobile phase B, wherein mobile phase A is water and mobile phase B is acetonitrile; The elution is a gradient elution, and the gradient elution method is as follows: with the total volume of mobile phase A and mobile phase B being 100%, from 0 to 3 minutes, the volume percentage of mobile phase A decreases from 95% to 60%, and the remainder is mobile phase B; from 3 to 18 minutes, the volume percentage of mobile phase A decreases from 60% to 52%, and the remainder is mobile phase B; from 18 to 25 minutes, the volume percentage of mobile phase A decreases from 52% to 48%, and the remainder is mobile phase B; from 25 to 30 minutes, the volume percentage of mobile phase A decreases from 48% to 42%, and the remainder is mobile phase B; The injection volume is 5-6 mL; and / or the flow rate of the mobile phase is 8-10 mL / min.

2. The extraction method according to claim 1, wherein The detector used in the medium-pressure preparative liquid chromatograph is a UV detector with a detection wavelength of 280 nm.

3. The extraction method according to claim 2, wherein During separation and purification by medium-pressure preparative liquid chromatography, the elution time of chlorogenic acid was 13.3 min, and chlorogenic acid was collected.

4. The extraction method according to claim 1, wherein In step S1, the amount of cellulase added is 0.6% based on the weight of the blueberry leaves.

5. The extraction method according to claim 1, wherein In step S3, the solvent used to dissolve the crude blueberry leaf polyphenol concentrate is methanol, and the amount of methanol used is: 30 to 50 mL per gram of the crude blueberry leaf polyphenol concentrate.

6. The extraction method according to claim 5, wherein In step S3, after the crude blueberry leaf polyphenol concentrate is dissolved, it is first filtered through a membrane and then separated and purified using a medium-pressure preparative liquid chromatography; the filter membrane used for membrane filtration is a 0.22 μm filter membrane.

7. The extraction method according to claim 1, wherein The extraction method further comprises the step of analyzing and detecting chlorogenic acid by high performance liquid chromatography, wherein the detection conditions of the high performance liquid chromatography are: using a Hypersil GOLD chromatographic column, model 250×4.6 mm, 5 μm; and / or, Column temperature: 35°C; and / or, The detector is a UV detector with a detection wavelength of 280 nm; and / or, Injection volume: 10 μL; and / or, Mobile phase flow rate: 1.0 mL / min; and / or, Mobile phase A: acetonitrile, mobile phase B: 0.2% formic acid in water; and / or, The gradient elution method is: the total volume of mobile phase A and mobile phase B is 100%, 0-10 min, 15%-50% mobile phase A, the balance is mobile phase B; 10-20 min, 50-100% mobile phase A, the balance is mobile phase B; 20-25 min, 100% A; 25-27 min, 100%-15% mobile phase A, the balance is mobile phase B; 27~40 min, 15% A.

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