Method for extracting berberine from nandina domestica and berberine extract

Abstract

The invention discloses a method for extracting berberine from nandina domestica and a berberine extract prepared by the method. The method comprises the following steps: carrying out compound enzyme enzymolysis treatment on a nandina raw material, carrying out ventilated extraction on the nandina raw material and NADES at 40-55 DEG C for 40-60 minutes, and filtering an extracting solution to remove solid impurities; and separating and purifying the filtered leaching solution, and further purifying the berberine through methods such as crystallization or recrystallization to obtain a high-purity berberine extract product. According to the method, the content and yield of berberine in the product can be remarkably improved, the content of berberine in the product can reach 98.6%, and the yield of berberine in the product can reach 85.22%.

Description

Technical Field

[0001] This invention relates to the field of natural product active ingredient extraction technology, specifically to a method for extracting berberine from Nandina domestica and the berberine extract prepared therefrom. Background Technology

[0002] Nandina domestica, a traditional medicinal plant of the Berberidaceae family, is rich in berberine, a highly active alkaloid, in its stem bark. The content can reach 1.5%–2.8% (dry weight), significantly higher than traditional sources such as Coptis chinensis. Berberine, a quaternary ammonium isoquinoline alkaloid, possesses broad-spectrum antibacterial, hypoglycemic, and anti-inflammatory pharmacological activities. It is particularly effective against drug-resistant Candida albicans (MIC50 as low as 8 μg / mL), providing a new alternative to synthetic antifungal drugs.

[0003] However, traditional organic solvent extraction methods (such as methanol and ethanol) have problems such as toxic residues, environmental pollution, and high energy consumption. Compared with the current raw materials for berberine extraction, such as Phellodendron amurense, Coptis chinensis, and Phellodendron chinense, Nandina domestica has a short growth cycle, can regenerate by pruning branches, and can be used sustainably. After Phellodendron chinense is peeled and felled, new plants need 8 to 10 years to grow. The technology for dense planting of Nandina domestica is mature, and the price of using Nandina domestica as raw material is much lower than that of Phellodendron chinense.

[0004] Existing technologies, such as supercritical CO2 extraction, are costly and have low extraction rates for polar alkaloids (e.g., berberine). Qiu Lin et al. optimized the extraction process of berberine hydrochloride from Phellodendron amurense by decocting in a solvent of 60% ethanol and 20 times the amount of water for 1 hour, and then repeating the extraction three times. Patent CN120478464A uses ultrasonic extraction with 60%~80% ethanol for 2~3 times; however, this method is time-consuming.

[0005] Zhang Delong et al. determined the optimal extraction process for Phellodendron amurense using supercritical CO2 extraction: extraction pressure of 31.00 MPa, extraction temperature of 52.00℃, extraction time of 90.00 min, and ethanol volume fraction of 94.00%. This requires the purchase of supercritical extraction equipment, resulting in high investment costs. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the technical defects of the prior art and provide a method for extracting berberine from Nandina domestica and the berberine extract prepared therefrom. This invention employs a method for extracting berberine from Nandina domestica and the berberine extract prepared therefrom, which can significantly improve the content and yield of berberine in the product, wherein the content of berberine in the product can reach 98.0%, and the yield is greater than 80.95%.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0008] A method for extracting berberine from Nandina domestica includes the following steps:

[0009] (1) Raw material processing: Nandina domestica raw material is dried and then ground into powder to obtain Nandina domestica coarse powder; the Nandina domestica coarse powder is treated by adding a compound enzyme; and then put into a reaction vessel with a porous distributor.

[0010] (2) Add a natural eutectic solvent composed of choline chloride, levulinic acid, and water, wherein choline chloride: levulinic acid = 1:1.5~2.5 (molar ratio) + 25%~40% water (w / w);

[0011] (3) In the reaction vessel, gas is continuously introduced from the bottom at a rate of 0.1~0.5V / V·m, and the control system pressure is 0.1~0.3MPa. Extraction is carried out at 40℃~55℃ for 40min~60min to obtain the extract.

[0012] (4) After centrifuging the extract, the solid and liquid components are separated;

[0013] (5) Purification: The filtrate is concentrated by evaporation under vacuum; the concentrate is added to a chromatography column containing silica gel, and the vacuum pump is turned on to allow the concentrate to pass through the chromatography column; the filtrate is eluted with a dichloromethane-acetone mixture; the eluent is collected and the solvent is reduced by evaporation to crystallize berberine.

[0014] Preferably, in step (1), the raw material of Nandina domestica is selected from one or more of the branches, leaves, roots and stems of Nandina domestica.

[0015] Preferably, in step (1), the composite enzyme is 0.5% cellulase and 0.2% pectinase. Preferably, the enzymatic hydrolysis conditions of the composite enzyme of the present invention are 45℃ for 2 hours.

[0016] Preferably, in step (1), the drying conditions for the Nandina domestica raw material are: placed in an oven at 60°C for 24 hours.

[0017] Preferably, in step (2), choline chloride: acetylpropionic acid = 1:2 (molar ratio) + 35% water (w / w).

[0018] Preferably, in step (2), the water content in the natural eutectic solvent is 20% to 40%.

[0019] Preferably, in step (3), the introduced gas includes a nitrogen-oxygen mixture, oxygen, and nitrogen. Preferably, in this application, "introducing gas" refers to introducing nitrogen.

[0020] Preferably, in step (3), the extraction temperature is 45~50℃ and the extraction time under ventilation is 40min.

[0021] Preferably, in step (4), the centrifugation speed is 4000 rpm;

[0022] Preferably, in step (5), the volume ratio of the dichloromethane-acetone mixture is 1:1.

[0023] A berberine extract was prepared using a method described above for extracting berberine from Nandina domestica.

[0024] A berberine extract was prepared using a method described above for extracting berberine from Nandina domestica.

[0025] The "%" mentioned in this invention generally refers to the percentage content by mass.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] (1) This invention uses NADES (natural eutectic solvent) to extract berberine, which improves the extraction efficiency. This solvent forms a low-temperature eutectic system (melting point <100℃) through molecular self-assembly of hydrogen bond acceptors (HBA, choline chloride) and hydrogen bond donors (HBD, levulinic acid), possessing three core advantages: A. High selective dissolution: The anionic groups of NADES generate electrostatic attraction with the berberine cations (—N+—), achieving targeted extraction; B. Thermal stability protection: The hydrogen bond network competitively disrupts the lignocellulose structure of Nandina domestica, accelerating the release of active ingredients; C. Biodegradability of NADES: The solvent is biodegradable (LD). 50 With a concentration of >5000 mg / kg and a recycling rate of ≥90%, carbon emissions are reduced by 40%.

[0028] (2) The technology of this invention deeply couples the pretreatment of compound enzyme hydrolysis with dynamic extraction in natural eutectic solvents: the hydrolysis products (small molecule sugars, amino acids) form a eutectic system with the choline chloride-levulinic acid solvent system, inhibiting the recrystallization of components; the aeration extraction avoids the secondary adsorption loss of components after enzymatic hydrolysis by dynamically updating the solvent interface concentration. This synergistic mechanism has achieved a technological breakthrough in extraction efficiency.

[0029] (3) In this invention, Nandina domestica and NADES are extracted by aeration at 45°C for 40 min. The extract is then filtered to remove solid impurities. The filtered extract is further purified by crystallization or recrystallization to obtain a high-purity product.

[0030] (4) NADES is composed of natural components choline chloride-levulinic acid, which is biodegradable; it has strong solubility: the hydrogen bond network structure can efficiently dissolve polar alkaloids; it has high stability: its antioxidant properties protect heat-sensitive components. By ventilating the DES-Nandina domestica mixed system, the bubbles collapse to generate local high pressure, which destroys the plant cell wall, controls the gas flow rate and system pressure, and improves the extraction efficiency of berberine. The extraction time is as short as 40 min, and the content of berberine can reach 98.6%, and the yield can reach 85.22%.

[0031] (5) The present invention can obtain a high concentration of berberine extract by purging the reactor for 40 minutes and then filtering. Detailed Implementation

[0032] To better understand the content of this invention, further description is provided below with reference to specific embodiments. It should be understood that these embodiments are only for further illustration of the invention and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art may make some non-essential modifications or adjustments to the invention, which still fall within the protection scope of this invention.

[0033] After drying the raw material of Nandina domestica in an oven at 60℃ for 24 hours, it was ground into powder to obtain coarse powder of Nandina domestica. The coarse powder of Nandina domestica rhizome or Nandina domestica rhizome and leaf was enzymatically hydrolyzed with 0.5% cellulase and 0.2% pectinase. The enzymatic hydrolysis conditions were 45℃ for 2 hours.

[0034] The coarse powder of Nandina domestica root and stem or coarse powder of Nandina domestica root, stem and leaves after enzymatic hydrolysis were used as raw materials in subsequent examples.

[0035] Example 1

[0036] Weigh 25g of Nandina domestica rhizome powder treated with 0.5% cellulase and 0.2% pectinase, add 500mL of NADES, choline chloride: levulinic acid = 1:2 + 35% water, and extract at 50℃ with a pressure of 0.2MPa for 1 hour. Centrifuge the extract at 4000rpm for 15 min to separate the solid and liquid phases, and filter the remaining liquid through a filter cloth. Dilute 500μL of the filtrate to 5mL, filter through a 0.45μM membrane, and the yield is 6.74mg / g. The berberine content of the raw material is 7.832mg / g, resulting in a yield of 86.06%. Evaporate the filtrate under vacuum. Add the concentrate to a chromatography column containing 350g of silica gel, turn on the vacuum pump, and pass the concentrate through the column. Elute with a dichloromethane-acetone mixture (volume ratio 1:1). The eluent was collected, and the solvent was reduced by evaporation to crystallize berberine, yielding a berberine extract with a berberine content of 98.6% and a yield of 85.22%.

[0037] Example 2

[0038] Weigh 50g of Nandina domestica rhizome powder treated with 0.5% cellulase and 0.2% pectinase, add 800mL of NADES, choline chloride: levulinic acid = 1:2 + 25% water, control the pressure in the reactor at 0.25MPa, and extract at 45℃ with aeration for 40min. Centrifuge the extract at 4000rpm for 15min to separate the solid and liquid, and filter the extract through a filter cloth. Take 500μL of the filtrate, dilute it to 5mL, filter it through a 0.45μM filter membrane, and the yield is 6.34mg / g. The berberine content of the raw material is 7.832mg / g, and the yield is 80.95%. Evaporate and concentrate the filtrate under vacuum. Add the concentrate to a chromatography column containing 350g silica gel, turn on the vacuum pump, and pass the concentrate through the chromatography column. Elute with a dichloromethane-acetone mixture (1:2 ratio). The eluent was collected, and the solvent was reduced by evaporation to allow berberine to crystallize. The collected product was sent for testing, and the berberine content was 98.3%, with a yield of 80.01%.

[0039] Example 3

[0040] Weigh 50g of Nandina domestica root, stem, and leaf powder treated with 0.5% cellulase and 0.2% pectinase, add 1000mL of NADES, choline chloride: levulinic acid = 1:2.5 + 25% water, control the pressure in the reactor at 0.3MPa, and extract at 50℃ with aeration for 60min. Centrifuge the extract at 4000rpm for 15min to separate the solid and liquid, and filter the extract through a filter cloth. Evaporate and concentrate the filtrate under vacuum. Take 500μL of the filtrate, dilute it to 5mL, filter it through a 0.45μM filter membrane, and the yield is 6.58mg / g. The berberine content of the raw material is 7.832mg / g, and the yield is 84.01%. Add the concentrated solution to a chromatography column containing 350g silica gel, turn on the vacuum pump, and pass the concentrated solution through the chromatography column. Elute with a dichloromethane-acetone mixture (1:0.5 ratio). The eluent was collected, and the solvent was reduced by evaporation to allow berberine to crystallize. The collected product was sent for testing, and the berberine content was 98.0%, with a yield of 83.51%.

[0041] Example 4

[0042] Weigh 50g of Nandina domestica root, stem, and leaf powder treated with 0.5% cellulase and 0.2% pectinase, add 1000mL of NADES, choline chloride: levulinic acid = 1:2.5 + 40% water, control the pressure in the reactor at 0.1MPa, and extract at 45℃ with aeration for 60min. Centrifuge the extract at 4000rpm for 15min to separate the solid and liquid, and filter the extract through a filter cloth. Evaporate and concentrate the filtrate under vacuum. Take 500μL of the filtrate, dilute it to 5mL, filter it through a 0.45μM filter membrane, and the yield is 6.37mg / g. The berberine content of the raw material is 7.832mg / g, and the yield is 81.33%. Add the concentrated solution to a chromatography column containing 350g silica gel, turn on the vacuum pump, and pass the concentrated solution through the chromatography column. Elute with a dichloromethane-acetone mixture (1:0.5 ratio). The eluent was collected, and the solvent was reduced by evaporation to allow berberine to crystallize. The collected product was sent for testing, and the berberine content was 98.2%, with a yield of 80.33%.

[0043] Comparative Example 1

[0044] Accurately weigh 50g of dried Nandina domestica rhizome powder treated with 0.5% cellulase and 0.2% pectinase, add 1000mL of choline chloride:fructose (5:2 molar ratio) + 15% water to form a homogeneous solution. Stir in a 45℃ constant temperature magnetic stirring water bath for 60 min (200 rpm) without aeration. After 1 h, cool to room temperature, transfer the mixture containing the raw materials to a 50mL centrifuge tube, centrifuge at 4000 r / min for 15 min at room temperature, take 500μL of the extract, dilute to 5mL, and filter through a 0.45 μm filter membrane. The results showed that the berberine yield was 1.8 mg / g, the berberine content of the raw material was 7.832 mg / g, i.e., the yield was 22.98%.

[0045] Comparative Example 2

[0046] Accurately weigh 50g of dried Nandina domestica rhizome powder treated with 0.5% cellulase and 0.2% pectinase, add 1000mL of choline chloride:fructose (5:2 molar ratio) + 15% water to form a homogeneous solution. Stir in a 50℃ constant temperature magnetic stirring water bath for 60 min (200 rpm), and aerate the solution at a pressure of 0.15MPa. After 1 h, cool to room temperature, transfer the mixture containing the raw materials to a 50mL centrifuge tube, centrifuge at 4000 r / min for 15 min at room temperature, take 500μL of the extract, dilute to 5mL, and filter through a 0.45 μm filter membrane. The results showed that the berberine yield was 2.28mg / g, the berberine content of the raw material was 7.832mg / g, i.e., the yield was 29.11%.

[0047] Comparative Example 3

[0048] Accurately weigh 50g of dried Nandina domestica rhizome powder treated with 0.5% cellulase and 0.2% pectinase, and add it to 800mL of choline chloride:lactic acid (1:2 molar ratio) + 35% water to form a homogeneous solution. Stir in a 50℃ constant temperature magnetic stirring water bath for 60 min (200 rpm), and aerate the solution at a pressure of 0.15MPa. After 1 h, cool to room temperature, transfer the mixture containing the raw materials to a 50mL centrifuge tube, centrifuge at 4000 r / min for 15 min at room temperature, take 500μL of the extract, dilute to 5mL, and filter through a 0.45 μm filter membrane. The results showed that the berberine yield was 3.48mg / g, the berberine content of the raw material was 7.832mg / g, i.e., the yield was 44.43%.

[0049] Comparative Example 4

[0050] Accurately weigh 50g of dried Nandina domestica rhizome powder treated with 0.5% cellulase and 0.2% pectinase, and add it to 800mL of 2‰ H2SO4 solution. Stir in a 60℃ constant temperature magnetic stirring water bath for 45 min (200 rpm), and aerate the solution at a pressure of 0.2MPa. After 1 h, cool to room temperature, transfer the mixture containing the raw materials to a 50mL centrifuge tube, centrifuge at 4000 r / min for 15 min at room temperature, take 500μL of the extract, dilute to 5mL, and filter through a 0.45 μm filter membrane. The results showed that the berberine yield was 2.16mg / g, the berberine content of the raw material was 7.832mg / g, i.e., the yield was 27.58%.

[0051] Comparative Example 5

[0052] Accurately weigh 25g of dried Nandina domestica rhizome powder treated with 0.5% cellulase and 0.2% pectinase, add 500mL of lactic acid:glucose = 5:1, +15% water. Stir in a 50℃ constant temperature magnetic stirring water bath for 60 min (200 rpm), and aerate the solution at a pressure of 0.3MPa. After 1 h, cool to room temperature, transfer the mixture containing the raw materials to a 50mL centrifuge tube, centrifuge at 4000 r / min for 15 min at room temperature, take 500μL of the extract, dilute to 5mL, and filter through a 0.45 μm filter membrane. The results showed that the berberine yield was 1.92mg / g, the berberine content of the raw material was 7.832mg / g, i.e., the yield was 24.51%.

[0053] Comparative Example 6

[0054] Accurately weigh 50g of untreated dried Nandina domestica rhizome powder and add it to 1000mL of a solution containing choline chloride: levulinic acid = 1:2.5 + 40% water. Stir in a 40℃ constant temperature magnetic stirring water bath for 60 min (200 rpm), and aerate the solution at a pressure of 0.15MPa. After 1 h, cool to room temperature, transfer the mixture containing the raw material to a 50mL centrifuge tube, centrifuge at 4000 r / min for 15 min at room temperature, take 500μL of the extract, dilute to 5mL, and filter through a 0.45 μm filter membrane. The results showed that the berberine yield was 4.47mg / g, the berberine content of the raw material was 7.832mg / g, and the yield was 57.07%.

[0055] Comparing Example 1, Comparative Example 2, and Comparative Example 3, among the three hydrogen bond donors (levulinic acid, fructose, and lactic acid), levulinic acid in Example 1 of this invention has the highest extraction efficiency when used as a hydrogen bond donor.

[0056] Conditions for determining berberine hydrochloride content

[0057] 1.1 Column conditions

[0058] Chromatographic column: Diamonsil C18 (200 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-0.05 mol / mL KH2PO4 (35:65), adjusted to pH 3.2 with H3PO4; flow rate: 1.0 mL / min; column temperature: 30 ℃; detection wavelength: 345 nm; injection volume: 10 μL.

[0059] 1.2 Preparation of reference solution and sample solution

[0060] Accurately weigh an appropriate amount of berberine hydrochloride reference standard dried to constant weight at 105℃, add methanol to prepare an 8 μg / mL solution, filter through a 0.45 μm microporous membrane, and collect the filtrate. Accurately measure 0.2 g of extracted berberine into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0061] 1.3 Determination of the maximum absorption wavelength of berberine hydrochloride

[0062] Accurately measure an appropriate amount of berberine hydrochloride standard into a 10 mL volumetric flask, dissolve it in methanol solution and dilute to the mark. Using methanol as a blank, perform ultraviolet scanning in the wavelength range of 200~400 nm, and determine 345 nm as the maximum absorption wavelength.

[0063] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for extracting berberine from Nandina domestica, characterized in that, Includes the following steps: (1) Raw material processing: Nandina domestica raw material is dried and then ground into powder to obtain Nandina domestica coarse powder; the Nandina domestica coarse powder is treated by adding a compound enzyme; and then put into a reaction vessel with a porous distributor. (2) Add a natural eutectic solvent composed of choline chloride, levulinic acid, and water, wherein choline chloride: levulinic acid = 1:1.5-2.5 (molar ratio) + 25%~40% water (w / w); (3) In the reaction vessel, gas is continuously introduced from the bottom at a rate of 0.1-0.5V / V·m, and the control system pressure is 0.1-0.3MPa. Extraction is carried out at 40℃-55℃ for 40-60 minutes to obtain the extract. (4) After centrifuging the extract, the solid and liquid are separated; the resulting filtrate is the berberine extract; (5) Purification: The filtrate is concentrated by evaporation under vacuum; the concentrate is added to a chromatography column containing silica gel, and the vacuum pump is turned on to allow the concentrate to pass through the chromatography column; the filtrate is eluted with a dichloromethane-acetone mixture; the eluent is collected, and the solvent is reduced by evaporation to crystallize berberine to obtain berberine extract.

2. The method for extracting berberine from Nandina domestica as described in claim 1, characterized in that, In step (1), the raw material of Nandina domestica is selected from one or more of the branches, leaves, roots and stems of Nandina domestica.

3. The method for extracting berberine from Nandina domestica as described in claim 1, characterized in that, In step (1), the compound enzyme is 0.5% cellulase and 0.2% pectinase.

4. The method for extracting berberine from Nandina domestica as described in claim 1, characterized in that, In step (1), the drying conditions for the Nandina domestica raw material are: placing it in a 60℃ oven for 24 hours.

5. The method for extracting berberine from Nandina domestica as described in claim 1, characterized in that, In step (2), choline chloride: acetylpropionic acid = 1:2 (molar ratio) + 35% water (w / w).

6. The method for extracting berberine from Nandina domestica as described in claim 1, characterized in that, In step (3), the introduced gas includes a nitrogen-oxygen mixture, oxygen, and nitrogen.

7. The method for extracting berberine from Nandina domestica as described in claim 1, characterized in that, In step (3), the extraction temperature is 45~50℃; the extraction time under ventilation is 40min.

8. The method for extracting berberine from Nandina domestica as described in claim 1, characterized in that, In step (5), the volume ratio of the dichloromethane-acetone mixture is 1:

1.

9. A berberine extract, characterized in that, Berberine was prepared using a method for extracting berberine from Nandina domestica as described in any one of claims 1 to 8.

10. A berberine extract, characterized in that, Berberine was prepared using a method for extracting berberine from Nandina domestica as described in any one of claims 1 to 8.