A method for extracting natural products from eucommia leaf extract

Abstract

The present application relates to a kind of methods for extracting, separating anhalonid, chlorogenic acid and geniposidic acid from eucommia leaf extract, belonging to the technical field of natural product extraction.The present application adopts the combination of ceramic nanofiltration membrane and organic nanofiltration membrane, obtains the mixed solution of anhalonid, chlorogenic acid and geniposidic acid after decolorization, and separates using chromatography, concentrates using reverse osmosis membrane, and obtains pure anhalonid, chlorogenic acid and geniposidic acid by freeze-drying, with purity of about 99%.The present application adopts the combination of membrane separation and chromatography, and develops a simple, low-input, high-output and environmentally friendly technical process for purifying and refining anhalonid, chlorogenic acid and geniposidic acid from eucommia leaf extract.

Description

Technical Field

[0001] This invention relates to a method for extracting natural products from Eucommia ulmoides leaf extract, specifically a method for extracting and separating aucubin, chlorogenic acid and geniposide from Eucommia ulmoides leaf extract, belonging to the field of natural product extraction technology. Background Technology

[0002] Eucommia ulmoides, also known as eucommia bark, is a plant belonging to the genus Eucommia in the family Eucommiaceae. It is endemic to my country and possesses high economic value. Traditionally, the bark of Eucommia ulmoides is used medicinally, while the leaves have similar active ingredients and pharmacological effects, including tonifying the liver and kidneys, strengthening muscles and bones, and lowering blood pressure. Aucupin, chlorogenic acid, and geniposide are important bioactive substances and also key active components in Eucommia ulmoides leaves, possessing significant effects such as clearing damp-heat, promoting urination, relieving pain, lowering blood pressure, protecting the liver, anti-tumor, antibacterial, antiviral, antioxidant, and anti-aging properties.

[0003] Currently, the extraction, separation, and purification of active ingredients such as aucubin, chlorogenic acid, and genipin from Eucommia ulmoides leaves mostly employ macroporous resins, enzymatic hydrolysis, extraction, and chromatography. These methods have drawbacks, including relatively complex processes, long processing times, and high consumption of organic reagents. Summary of the Invention

[0004] This invention addresses the problems of complex extraction methods and high reagent consumption in existing technologies for obtaining active ingredients such as aucubin, chlorogenic acid, and geniposide. This invention utilizes nanofiltration for coarse separation of the active ingredients, followed by purification using chromatography.

[0005] The technical solution is:

[0006] A method for extracting natural products from Eucommia ulmoides leaf extract, wherein the natural products are aucubin, chlorogenic acid, and genipin; comprising the following steps:

[0007] Step 1: Crush the Eucommia ulmoides leaves, extract with water, and obtain a crude extract;

[0008] Step 2: The crude extract is filtered sequentially through a ceramic nanofiltration membrane and an organic nanofiltration membrane to obtain the permeate from the organic nanofiltration membrane.

[0009] Step 3: The permeate is concentrated using a reverse osmosis membrane and then separated by chromatographic chromatography to obtain a chromatographic solution containing aucubin, chlorogenic acid and genipin.

[0010] In step 1, the raw materials are crushed to pass through a 20-mesh sieve.

[0011] In step 1, the water extraction is performed 1-3 times, the extraction temperature is 60-90℃, the liquid-to-solid ratio is 5:1-20:1, and the extraction time is 1-4 hours.

[0012] When using ceramic nanofiltration membranes for filtration, the molecular weight cutoff of the ceramic nanofiltration membranes is 2KD-15KD.

[0013] Operating temperature: 40-60℃; operating pressure: 0.1-0.3Mpa.

[0014] When filtering using organic nanofiltration membranes, the molecular weight cutoff of the organic nanofiltration membranes used is 150-300 Da.

[0015] Operating temperature: 20-50℃; operating pressure: 0.5-1.5Mpa.

[0016] In step 3, a silica gel packed column is used in the chromatographic chromatography method.

[0017] In chromatographic chromatography, the elution process uses a 20-50 vol.% polar solvent aqueous solution.

[0018] The flow rate is 2-4 BV / h; the polar solvent is selected from one or a mixture of two of alcohol solvents and nitrile solvents.

[0019] The eluents were collected at 10-30 min, 25-50 min, and 60-90 min, respectively, and then freeze-dried to obtain pure aucubin, chlorogenic acid, and genipin.

[0020] Beneficial effects

[0021] Nanofiltration is used for the coarse separation of active ingredients, followed by purification by chromatography. This method is simple, fast, efficient, and low-cost, and has the potential for industrial application. Attached Figure Description

[0022] Figure 1 HPLC analysis of the nanofiltration membrane clear liquid of Eucommia ulmoides leaves (concentrated 5 times);

[0023] Figure 2 This involves HPLC analysis of mixed standards (1. aucubin, 2. genipin, 3. chlorogenic acid).

[0024] Figure 3 This is the HPLC chromatogram of aucubin;

[0025] Figure 4 This is the HPLC chromatogram of genipin.

[0026] Figure 5 This is the HPLC chromatogram of chlorogenic acid;

[0027] Figure 6 This is the HPLC chromatogram of the filtrate from the ceramic nanofiltration membrane. Detailed Implementation

[0028] The extraction process of the Eucommia ulmoides product of the present invention is described in detail below:

[0029] A method for extracting and separating aucubin, chlorogenic acid and genipin from Eucommia ulmoides leaf extract includes the following steps:

[0030] (1) After the raw material of Eucommia ulmoides leaves is crushed, it is extracted twice with water as solvent to obtain crude extracts of aucubin, chlorogenic acid and genipin.

[0031] (2) Membrane separation is performed using ceramic nanofiltration membranes and organic nanofiltration membranes to remove impurities, including macromolecular proteins, alkaloids, tannins, lipids, organelles, etc., and a clear solution containing aucubin, chlorogenic acid and geniposide is obtained in the permeate of the nanofiltration membrane.

[0032] (3) The supernatant is concentrated by reverse osmosis membrane to remove water, and the concentrate is loaded onto a column for product separation using chromatographic techniques.

[0033] In step 1), the Eucommia ulmoides leaf raw material is pulverized to 20 mesh.

[0034] In step 1), Eucommia ulmoides leaves are extracted with water at a temperature of 60-90℃, 1-3 times, with a liquid-to-solid ratio of 5:1-20:1.

[0035] Extraction time: 1-4 hours.

[0036] In step 2), the crude extract is separated using a ceramic nanofiltration membrane with a molecular weight cutoff of 2KD-15KD at an operating temperature of 40-60℃ and an operating pressure of 0.1-0.3Mpa, and the clear liquid A is collected.

[0037] In step 2), the supernatant A is separated by an organic nanofiltration membrane with a molecular weight cutoff of 150-300 Da at an operating temperature of 20-50℃ and an operating pressure of 0.5-1.5 MPa, and the supernatant B is collected.

[0038] The supernatant B was concentrated by reverse osmosis membrane to remove water. The concentrate was loaded onto a column for chromatographic chromatography to separate the products. In step 3), the concentrate from the reverse osmosis membrane was injected into a silica gel-packed chromatography column. The products were eluted with a 20-50% ethanol aqueous solution. Eluent A was collected starting at 10-30 min, eluent B was collected at 25-50 min, and eluent C was collected at 60-90 min. The eluents were then lyophilized to obtain pure aucubin, chlorogenic acid, and geniposide.

[0039] Example 1

[0040] Eucommia ulmoides leaves were pulverized to 20 mesh and then extracted twice with water as the solvent. The extraction temperature was 75-0℃, the liquid-to-solid ratio was 8:1, and the extraction time was 3 hours each time to obtain a crude extract. The crude extract was then filtered using a ceramic nanofiltration membrane with a molecular weight cutoff of 5KD. The liquid-to-solid temperature was 40-60℃, and the operating pressure was 0.2MPa. The filtrate from the ceramic nanofiltration membrane was then separated using an organic nanofiltration membrane with a molecular weight cutoff of 300Da. The operating temperature was 20-30℃, and the operating pressure was 0.8MPa. The permeate from the organic nanofiltration membrane was collected. A clear solution containing aucubin, chlorogenic acid, and geniposide was obtained from the permeate of the nanofiltration membrane. The supernatant was concentrated using a reverse osmosis membrane to remove water. The concentrate was then loaded onto a silica gel-packed chromatography column at a flow rate of 3 BV / h. After adsorption saturation, the products were separated using chromatographic techniques. The products were eluted with a 40% ethanol aqueous solution. Eluent A was collected from 10 to 30 minutes, eluent B from 30 to 50 minutes, and eluent C from 60 to 90 minutes. The eluents were then lyophilized to obtain pure aucubin, chlorogenic acid, and geniposide.

[0041] The permeate from the organic nanofiltration membrane was analyzed by HPLC. The chromatographic conditions were as follows: column: Shimadzu InertSustain C18 (150×4.6mm, 5μm); mobile phase: acetonitrile (A)-0.1% phosphoric acid solution (B); gradient elution (0–5 min, 5% A; 5–30 min, 5% A→10% A; 30–35 min, 10% A→20% A; 35–45 min, 20% A; 45–50 min, 20% A→50% A; 50–60 min, 50% A→5% A; 60–70 min, 5% A); flow rate: 1.0 mL / min; column temperature: 35℃; detection wavelength: 210 nm; injection volume: 10 μL.

[0042] HPLC chromatogram of nanofiltration membrane permeate as follows Figure 1 As shown. The HPLC chromatogram of the mixed standard of aucubin, chlorogenic acid, and genipin is shown below. Figure 2 As shown in the figure. The HPLC chromatograms of aucubin, chlorogenic acid, and genipin monophosphate obtained by column chromatography are shown in the figure. Figures 3-5 As shown.

[0043] The final purities of aucubin, chlorogenic acid, and genipin were 98.8%, 98.7%, and 99.2%, respectively.

[0044] Example 2

[0045] Eucommia ulmoides leaves were pulverized to 20 mesh and then extracted twice with water as solvent at an extraction temperature of 65-70℃, a liquid-to-solid ratio of 10:1, and an extraction time of 1-4 hours to obtain a crude extract. The crude extract was then filtered using a ceramic nanofiltration membrane with a molecular weight cutoff of 5KD, a feed-to-liquid temperature of 40-45℃, and an operating pressure of 0.25MPa. The filtrate from the ceramic nanofiltration membrane was then separated using an organic nanofiltration membrane with a molecular weight cutoff of 150Da, an operating temperature of 25-30℃, and an operating pressure of 1.0MPa to collect the permeate from the organic nanofiltration membrane. A clear solution containing aucubin, chlorogenic acid, and geniposide was obtained from the permeate of the nanofiltration membrane. The supernatant was concentrated using a reverse osmosis membrane to remove water. The concentrate was then loaded onto a silica gel-packed chromatography column at a flow rate of 3 BV / h. After adsorption saturation, the products were separated using chromatographic techniques. The products were eluted with a 45% ethanol-water solution. Eluent A was collected from 10 to 30 minutes, eluent B from 30 to 50 minutes, and eluent C from 60 to 90 minutes. The eluents were then lyophilized to obtain pure aucubin, chlorogenic acid, and geniposide, with purities of 99.1%, 98.9%, and 99.4%, respectively.

[0046] Example 3

[0047] Optimization of nanofiltration membrane operating parameters:

[0048] Based on the conditions of Example 1, the operating parameters of the nanofiltration membrane were optimized. The results of the determination of the contents of three effective components in the concentrated and clear solutions of Eucommia ulmoides leaf organic membrane are shown in the table below:

[0049] Determination of the content of three active ingredients in the concentrated and clear solutions of eucommia leaf organic membrane

[0050]

[0051] Note: "\" indicates that the measured value is below the detection limit.

Claims

1. A method for extracting natural products from Eucommia ulmoides leaf extract, characterized in that, Includes the following steps: Eucommia ulmoides leaves were pulverized to 20 mesh and then extracted twice with water as the solvent. The extraction temperature was 65-70℃, the liquid-to-solid ratio was 10:1, and the extraction time was 1-4 hours to obtain a crude extract. The crude extract was then filtered using a ceramic nanofiltration membrane with a molecular weight cutoff of 5KD. The liquid-to-solid temperature was 40-45℃, and the operating pressure was 0.25 MPa. The filtrate from the ceramic nanofiltration membrane was then subjected to membrane separation using an organic nanofiltration membrane with a molecular weight cutoff of 150Da. The operating temperature was 25-30℃, and the operating pressure was 1.0 MPa. The permeate from the organic nanofiltration membrane was collected. The solution was prepared by obtaining a clear solution containing aucubin, chlorogenic acid, and geniposide in the permeate of a nanofiltration membrane. The clear solution was then concentrated using a reverse osmosis membrane to remove water. The concentrate was then loaded onto a silica gel-packed chromatography column at a flow rate of 3 BV / h. After adsorption saturation, the products were separated using chromatographic chromatography. The products were eluted with a 45% ethanol aqueous solution. Eluent A was collected from 10 to 30 minutes, eluent B from 30 to 50 minutes, and eluent C from 60 to 90 minutes. The eluents were then lyophilized to obtain pure aucubin, chlorogenic acid, and geniposide.

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