Method for efficiently extracting cannabidiol by compounding microorganisms with industrial cannabis sativa

Abstract

The invention relates to the technical field of microorganism extraction, in particular to a method for efficiently extracting cannabidiol by compounding microorganisms with industrial cannabis sativa. According to the method for extracting cannabidiol, the cannabis plants are crushed and sieved, and low-temperature plasma treatment is adopted; adding the product and a complex microbial inoculant into water, fermenting at 30-40 DEG C for 4-8 hours, and inactivating; adding absolute ethyl alcohol, performing reflux extraction, filtering, combining filtrate, and concentrating to obtain extract; adding the magnetic molecularly imprinted microspheres into water, ultrasonically dispersing uniformly, and adding the extract into the water for ultrasonic treatment; magnetic separation: washing the microspheres with an ethanol water solution; collecting the washing liquid, loading the washing liquid to a resin column, and in the elution process, sequentially eluting and removing impurities by adopting ethanol aqueous solutions with different mass fractions to obtain a target product eluent; and carrying out molecular distillation specific adsorption on the target product eluent to remove residual tetrahydrocannabinol, collecting fractions, concentrating, crystallizing, washing and carrying out vacuum drying.

Description

Technical Field

[0001] The present invention relates to the field of microbial extraction technology, and in particular to a method for efficiently extracting cannabidiol from industrial hemp by compounding industrial hemp with microorganisms. Background Art

[0002] Industrial hemp, an important industrial raw material and medicinal plant, is rich in various bioactive components, with cannabidiol (CBD) being particularly prominent. CBD exhibits anti-inflammatory, anxiolytic, anticonvulsant, and neuroprotective properties, while being non-neurotoxic. It holds broad application prospects in biopharmaceuticals, functional foods, and other fields.

[0003] However, the extraction of CBD from industrial hemp faces multiple technical bottlenecks: on the one hand, psychoactive phenolic substances such as tetrahydrocannabinol (THC) also exist in hemp plants, which are difficult to separate; on the other hand, traditional extraction processes rely on toxic organic solvents such as petroleum ether and dichloromethane, which can easily cause environmental pollution and product residue risks, seriously restricting the sustainable development of the industry.

[0004] Currently, CBD extraction from industrial hemp primarily utilizes techniques such as solvent extraction, supercritical fluid extraction, or column chromatography. While solvent extraction is simple to operate, it poses the problem of residual solvents, and CBD is susceptible to oxidation and degradation in high temperatures or polar solvents. Supercritical CO2 extraction, while able to avoid solvent contamination, suffers from high equipment costs and limited selective separation of THC. Column chromatography, on the other hand, relies on macroporous resins or silica gel for separation, but suffers from low separation efficiency and difficulty in resin regeneration. More critically, existing processes do not thoroughly destroy the cell walls of the hemp plant, resulting in low dissolution rates of target components like CBD, necessitating repeated extractions, further increasing solvent consumption and production costs.

[0005] In recent years, bio-assisted extraction technology has gained increasing attention. Using microbial fermentation to degrade lignin and cellulose in cannabis plant cell walls can improve the release of target ingredients. However, the ability of these strains to degrade complex plant tissues is limited, and the fermentation process is susceptible to contamination by other bacteria, affecting product stability. Currently, efficient CBD extraction and separation processes remain underdeveloped. Summary of the Invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for efficiently extracting cannabidiol by compounding industrial hemp with microorganisms.

[0007] A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, comprising the following steps: S1. The cannabis plant is crushed and sieved, and treated with low-temperature plasma for 4-6 minutes at a plasma power of 50-80W and a frequency of 13.56 MHz. The product and the composite bacterial agent are added to water, the pH of the system is adjusted to 5-6, and the mixture is fermented at 30-40°C for 4-8 hours to instantly inactivate the microorganisms at high temperature. Anhydrous ethanol is then added, the mixture is refluxed and extracted for 1-3 hours, and filtered to obtain a filter residue a and a filtrate a. The filter residue a is then added to anhydrous ethanol, the mixture is refluxed and extracted for 1-2 hours, and filtered to obtain a filtrate b. The filtrate a and the filtrate b are then combined and concentrated to obtain an extract. S2. adding the magnetic molecularly imprinted microspheres to water and ultrasonically dispersing them uniformly, adding the extract thereto and ultrasonically treating for 1-2 minutes; performing magnetic separation and washing the magnetic molecularly imprinted microspheres; collecting the washing solution and loading it onto an AB-8 resin column; during the elution process, eluting with a 30-40% by mass ethanol aqueous solution to remove impurities, and then eluting with a 50-60% by mass ethanol aqueous solution to obtain an eluate of the target product; S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol, the fractions are collected and concentrated, supersaturated crystallized with ethanol at 5-12°C, washed with purified water at 0°C, and dried in vacuo.

[0008] Preferably, in S1, the mass ratio of the cannabis plant to the composite bacterial agent is 10-30:0.1-1.

[0009] Preferably, in S1, the composite bacterial agent comprises Trichoderma reesei and Aspergillus niger; wherein the viable count of Trichoderma reesei is 3-6×10 8 cfu / g, the number of viable Aspergillus niger is 1-4×10 8 cfu / g.

[0010] Preferably, in S1, the relative density of the extract at 50°C is 1.06-1.08.

[0011] Preferably, in S2, after the extract is added, the frequency of ultrasonic treatment is 40-45 kHz, and the ultrasonic temperature is 30-50°C.

[0012] Preferably, in S2, after magnetic separation, the magnetic molecularly imprinted microspheres are washed with an ethanol aqueous solution having a mass fraction of 70-80%.

[0013] Preferably, in S2, the particle size of the magnetic molecularly imprinted microspheres is 50-100 μm.

[0014] Preferably, in S2, the mass ratio of the magnetic molecularly imprinted microspheres to the extract is 5-10:20-30.

[0015] Preferably, in S2, the magnetic molecularly imprinted microspheres are prepared by the following steps: adding nano-ferroferric oxide and polyethylene glycol to water and ultrasonically dispersing them uniformly, adding 1-butyl-3-methylimidazolium chloride ionic liquid thereto, stirring under nitrogen protection for 5-15 minutes, adding methacrylic acid, ethylene glycol dimethacrylate, and azobisisobutyronitrile in sequence, stirring at 50-70° C. for 5-10 hours, washing, magnetic separation, vacuum drying, and crushing.

[0016] More preferably, the mass ratio of nano-ferrosoferric oxide, polyethylene glycol, 1-butyl-3-methylimidazolium chloride ionic liquid, methacrylic acid, ethylene glycol dimethacrylate, and azobisisobutyronitrile is 20-40:1:1-5:1-5:1-3:0.5-1.

[0017] Preferably, in S3, the fractions are collected and concentrated to a relative density of 1.1-1.15 at 50°C.

[0018] Beneficial effects: The present invention crushes the cannabis plant and combines it with low-temperature plasma treatment to precisely regulate cell membrane permeability. It then uses a composite bacterial agent to further fully decompose and promote the dissolution of cannabidiol (CBD). Compared with traditional high-pressure homogenization, this method not only significantly improves the release rate of cell contents, but also has a high cannabidiol retention rate.

[0019] The plasma treatment of the present invention, combined with the action of a composite bacterial agent, significantly increases the effective contact area of the magnetic molecularly imprinted microspheres. Pre-adsorption of the magnetic molecularly imprinted microspheres effectively alleviates the load pressure of subsequent AB-8 chromatography, effectively improving column efficiency and significantly enhancing the separation efficiency of cannabidiol. The product is extremely pure and of stable quality, making it suitable for large-scale industrial production. The magnetic molecularly imprinted microspheres used in the present invention use methacrylic acid as a functional monomer and specifically adsorb cannabidiol through an ionic liquid-enhanced π-π interaction. Further chromatography with AB-8 resin increases the purity of the final product from 68% to over 98%.

[0020] The cannabidiol extraction process of the present invention uses ethanol as the main extraction solvent throughout the process, and is combined with an improved extraction process. Experiments have shown that the yield and purity of cannabidiol are significantly improved, and no tetrahydrocannabinol component is detected, thereby ensuring the safety of the product and facilitating industrial production.

[0021] The present invention can significantly increase the yield of cannabidiol while ensuring high purity, and improves the safety of the production process and the safety of the extracted cannabidiol. The present invention uses cannabidiol products as a drug for benign prostatic hyperplasia. Tests have found that they can inhibit the increase in prostate volume in rats caused by testosterone propionate, increase SOD activity, and reduce MDA content. The present invention has a good effect in inhibiting benign prostatic hyperplasia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a comparison chart of the cannabidiol extraction rate, cannabidiol content and tetrahydrocannabinol content in the product using the extraction methods of Example 5 and Comparative Examples 1-2.

[0023] Figure 2 1 is a comparison chart of the prostate volume and prostate index of rats in the blank group, model group, positive control group, Example 5 group, and Comparative Example 2 group.

[0024] Figure 3 This is a comparison chart of the malondialdehyde content and superoxide dismutase content of rats in the blank group, model group, positive control group, Example 5 group, and Comparative Example 2 group. DETAILED DESCRIPTION

[0025] The present invention will be further explained below with reference to specific embodiments.

[0026] Example 1 A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, comprising the following steps: S1. 100 g of cannabis plants were crushed through a 40-mesh sieve and treated with low-temperature plasma at 30°C for 4 min, with a plasma power of 50 W and a frequency of 13.56 MHz. The product was mixed with 1 g of a composite bacterial agent (wherein the number of viable Trichoderma reesei was 3 × 10 8 cfu / g, the number of viable Aspergillus niger is 1×10 8 cfu / g) was added to 500 g of water, the pH of the system was adjusted to 5-6, and the mixture was fermented at 30°C for 4 h to instantaneously inactivate the microorganisms at high temperature; 800 g of anhydrous ethanol was then added, the mixture was refluxed for 1 h, and filtered to obtain a residue a and a filtrate a; the residue a was added to 300 g of anhydrous ethanol, the mixture was refluxed for 1 h, and filtered to obtain a filtrate b; the filtrate a and the filtrate b were combined and concentrated to a relative density (50°C) of 1.06 to obtain an extract; S2. Add 50 g of magnetic molecularly imprinted microspheres to 500 g of deionized water and ultrasonically disperse them uniformly. Add 200 g of the extract thereto and ultrasonically treat for 1 min at an ultrasonic frequency of 40 kHz and an ultrasonic temperature of 30°C. Perform magnetic separation and wash the magnetic molecularly imprinted microspheres with a 70% by mass ethanol aqueous solution. Collect the washing solution and load it onto an AB-8 resin column. During the elution process, use a 30% by mass ethanol aqueous solution to elute and remove impurities. Then, use a 50% by mass ethanol aqueous solution to elute to obtain an eluate of the target product. Finally, use a 95% by mass ethanol aqueous solution to elute and regenerate the chromatography column. The magnetic molecularly imprinted microspheres were prepared by the following steps: 200 g of nano-ferroferric oxide and 10 g of polyethylene glycol were added to 400 g of deionized water and ultrasonically dispersed uniformly, 10 g of [BMIM]Cl ionic liquid was added thereto, and stirred under nitrogen protection for 5 min at a stirring speed of 5000 r / min. Then, 10 g of methacrylic acid, 10 g of ethylene glycol dimethacrylate, and 5 g of azobisisobutyronitrile were added in sequence, and stirred at a temperature of 50°C for 5 h. The mixture was washed with ethanol twice, magnetically separated, vacuum dried, and crushed. S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol. The fractions are collected and concentrated to a relative density (50°C) of 1.10. The fractions are supersaturated and crystallized using ethanol at 5°C, washed with purified water at 0°C, and dried under vacuum.

[0027] Example 2 A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, comprising the following steps: S1. 300 g of cannabis plants were crushed and passed through a 40-mesh sieve. The plants were treated with low-temperature plasma at 40°C for 6 min, with a plasma power of 80 W and a frequency of 13.56 MHz. The product was mixed with 10 g of a composite bacterial agent (wherein the number of viable Trichoderma reesei was 6 × 10 8 cfu / g, the number of viable Aspergillus niger was 4×10 8 cfu / g) was added to 600 g of water, the pH of the system was adjusted to 5-6, and the mixture was fermented at 40°C for 8 h to instantaneously inactivate the microorganisms. 1000 g of anhydrous ethanol was then added, the mixture was refluxed for 3 h, and filtered to obtain a residue a and a filtrate a. The residue a was added to 500 g of anhydrous ethanol, the mixture was refluxed for 2 h, and filtered to obtain a filtrate b. The filtrates a and b were combined and concentrated to a relative density (50°C) of 1.08 to obtain an extract. S2. 100 g of magnetic molecularly imprinted microspheres were added to 1000 g of deionized water and ultrasonically dispersed uniformly. 300 g of the extract was added thereto and ultrasonically treated for 2 min at an ultrasonic frequency of 45 kHz and an ultrasonic temperature of 50° C.; magnetic separation was performed, and the magnetic molecularly imprinted microspheres were washed with an 80% by mass ethanol aqueous solution; the washing liquid was collected and loaded onto an AB-8 resin column. During the elution process, impurities were removed by elution with a 40% by mass ethanol aqueous solution, and then the target product eluate was obtained by elution with a 60% by mass ethanol aqueous solution. Finally, the column was regenerated by elution with a 98% by mass ethanol aqueous solution; The magnetic molecularly imprinted microspheres were prepared by the following steps: 400 g of nano-ferroferric oxide and 10 g of polyethylene glycol were added to 600 g of deionized water and ultrasonically dispersed uniformly, 50 g of [BMIM]Cl ionic liquid was added thereto, and the mixture was stirred under nitrogen for 15 min at a stirring speed of 10,000 r / min. Subsequently, 50 g of methacrylic acid, 30 g of ethylene glycol dimethacrylate, and 10 g of azobisisobutyronitrile were added in sequence, and the mixture was stirred at 70°C for 10 h. The mixture was washed with ethanol four times, magnetically separated, vacuum dried, and crushed. S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol, and the fractions are collected and concentrated to a relative density (50°C) of 1.15. The fractions are supersaturated and crystallized using ethanol at a temperature of 12°C, washed with purified water at a temperature of 0°C, and dried in vacuo.

[0028] Example 3 A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, comprising the following steps: S1. 150 g of cannabis plants were crushed through a 40-mesh sieve and treated with low-temperature plasma at 37°C for 5 min, with a plasma power of 60 W and a frequency of 13.56 MHz. The product was mixed with 7 g of a composite bacterial agent (wherein the number of viable Trichoderma reesei was 4 × 10 8 cfu / g, the number of viable Aspergillus niger was 3×10 8 cfu / g) was added to 520 g of water, the pH of the system was adjusted to 5-6, and the mixture was fermented at 37°C for 5 h to instantaneously inactivate the microorganisms. 950 g of anhydrous ethanol was then added, the mixture was refluxed for 1.5 h, and filtered to obtain a residue a and a filtrate a. The residue a was added to 450 g of anhydrous ethanol, the mixture was refluxed for 80 min, and filtered to obtain a filtrate b. The filtrates a and b were combined and concentrated to a relative density (50°C) of 1.07 to obtain an extract. S2. Add 90 g of magnetic molecularly imprinted microspheres to 700 g of deionized water and ultrasonically disperse them uniformly. Add 280 g of the extract thereto and ultrasonically treat for 2 min at an ultrasonic frequency of 41 kHz and an ultrasonic temperature of 45°C. Perform magnetic separation and wash the magnetic molecularly imprinted microspheres with a 73% by mass ethanol aqueous solution. Collect the washing liquid and load it onto an AB-8 resin column. During the elution process, use a 37% by mass ethanol aqueous solution to elute and remove impurities. Then, use a 52% by mass ethanol aqueous solution to elute to obtain an eluate of the target product. Finally, use a 97% by mass ethanol aqueous solution to elute and regenerate the chromatography column. The magnetic molecularly imprinted microspheres were prepared by the following steps: 250 g of nano-ferroferric oxide and 10 g of polyethylene glycol were added to 550 g of deionized water and ultrasonically dispersed uniformly, 20 g of [BMIM]Cl ionic liquid was added thereto, and the mixture was stirred under nitrogen for 12 min at a stirring speed of 7000 r / min. Subsequently, 40 g of methacrylic acid, 15 g of ethylene glycol dimethacrylate, and 9 g of azobisisobutyronitrile were added in sequence, and the mixture was stirred at 55°C for 9 h. The mixture was washed with ethanol three times, subjected to magnetic separation, vacuum dried, and crushed. S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol, and the fractions are collected and concentrated to a relative density (50°C) of 1.12. The fractions are supersaturated and crystallized using ethanol at a temperature of 10°C, washed with purified water at a temperature of 0°C, and dried in vacuo.

[0029] Example 4 A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, comprising the following steps: S1. 250 g of cannabis plants were crushed through a 40-mesh sieve and treated with low-temperature plasma at 33°C for 5 min, with a plasma power of 70 W and a frequency of 13.56 MHz. The product was mixed with 3 g of a composite bacterial agent (wherein the number of viable Trichoderma reesei was 5 × 10 8 cfu / g, the number of viable Aspergillus niger was 2×10 8 cfu / g) was added to 580 g of water, the pH of the system was adjusted to 5-6, and the mixture was fermented at 33°C for 7 h to instantaneously inactivate the microorganisms at high temperature; 850 g of anhydrous ethanol was then added, the mixture was refluxed for 2.5 h, and filtered to obtain a residue a and a filtrate a; the residue a was added to 350 g of anhydrous ethanol, the mixture was refluxed for 100 min, and filtered to obtain a filtrate b; the filtrates a and b were combined and concentrated to a relative density (50°C) of 1.07 to obtain an extract; S2. Add 70 g of magnetic molecularly imprinted microspheres to 900 g of deionized water and ultrasonically disperse them uniformly. Add 220 g of the extract thereto and ultrasonically treat for 2 min at an ultrasonic frequency of 43 kHz and an ultrasonic temperature of 35°C. Perform magnetic separation and wash the magnetic molecularly imprinted microspheres with a 77% by mass ethanol aqueous solution. Collect the washing solution and load it onto an AB-8 resin column. During the elution process, use a 33% by mass ethanol aqueous solution to elute and remove impurities. Then, use a 58% by mass ethanol aqueous solution to elute to obtain an eluate of the target product. Finally, use a 96% by mass ethanol aqueous solution to elute and regenerate the chromatography column. The magnetic molecularly imprinted microspheres were prepared by the following steps: 350 g of nano-ferroferric oxide and 10 g of polyethylene glycol were added to 450 g of deionized water and ultrasonically dispersed uniformly, 40 g of [BMIM]Cl ionic liquid was added thereto, and the mixture was stirred under nitrogen for 8 min at a stirring speed of 9000 r / min. Subsequently, 20 g of methacrylic acid, 25 g of ethylene glycol dimethacrylate, and 7 g of azobisisobutyronitrile were added in sequence, and the mixture was stirred at 65°C for 7 h. The mixture was washed with ethanol three times, magnetically separated, vacuum dried, and crushed. S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol. The fractions are collected and concentrated to a relative density (50°C) of 1.14. The fractions are supersaturated and crystallized using ethanol at 6°C, washed with purified water at 0°C, and dried under vacuum.

[0030] Example 5 A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, comprising the following steps: S1. 200 g of cannabis plants were crushed and passed through a 40-mesh sieve. The plants were treated with low-temperature plasma at 35°C for 5 min, with a plasma power of 65 W and a frequency of 13.56 MHz. The product was mixed with 5 g of a composite bacterial agent (wherein the number of viable Trichoderma reesei was 5 × 10 8 cfu / g, the number of viable Aspergillus niger was 3×10 8 cfu / g) was added to 550 g of water, the pH of the system was adjusted to 5-6, and the fermentation was carried out at 35°C for 6 hours to instantly inactivate the microorganisms at high temperature; then 900 g of anhydrous ethanol was added, the mixture was refluxed for 2 hours, and filtered to obtain a residue a and a filtrate a; the residue a was added to 400 g of anhydrous ethanol, the mixture was refluxed for 90 minutes, and filtered to obtain a filtrate b; the filtrate a and the filtrate b were combined and concentrated to a relative density (50°C) of 1.07 to obtain an extract; S2. 80 g of magnetic molecularly imprinted microspheres were added to 800 g of deionized water and ultrasonically dispersed uniformly. 250 g of the extract was added thereto and ultrasonically treated for 2 min at an ultrasonic frequency of 42 kHz and an ultrasonic temperature of 40°C. The magnetic molecularly imprinted microspheres were washed with a 75% by mass ethanol aqueous solution for magnetic separation. The washing solution was collected and loaded onto an AB-8 resin column. During the elution process, a 35% by mass ethanol aqueous solution was used to elute and remove impurities. Then, a 55% by mass ethanol aqueous solution was used to elute to obtain an eluate of the target product. Finally, a 97% by mass ethanol aqueous solution was used to elute and regenerate the chromatography column. The magnetic molecularly imprinted microspheres were prepared by the following steps: 300 g of nano-ferroferric oxide and 10 g of polyethylene glycol were added to 500 g of deionized water and ultrasonically dispersed uniformly, 30 g of [BMIM]Cl ionic liquid was added thereto, and the mixture was stirred under nitrogen for 10 min at a stirring speed of 8000 r / min. Subsequently, 30 g of methacrylic acid, 20 g of ethylene glycol dimethacrylate, and 8 g of azobisisobutyronitrile were added in sequence, and the mixture was stirred at 60°C for 8 h. The mixture was washed with ethanol three times, magnetically separated, vacuum dried, and crushed. S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol. The fractions are collected and concentrated to a relative density (50°C) of 1.13. The fractions are supersaturated and crystallized using ethanol at 8°C, washed with purified water at 0°C, and dried under vacuum.

[0031] The magnetic molecularly imprinted microspheres used above were dispersed in purified water, and the particle size of the magnetic molecularly imprinted microspheres used above was measured using a particle size analyzer. The average particle size was 75±15 μm.

[0032] The adsorption test of cannabidiol (CBD) using the magnetic molecularly imprinted microspheres used above was conducted as follows: 0.01, 0.02, 0.05, 0.10, 0.15, and 0.20 g of magnetic molecularly imprinted microspheres were accurately taken and added to a 5 mg / L cannabidiol ethanol solution, respectively. The pH value of the system was adjusted to 7.0, and the mixture was shaken in a constant temperature shaker at 25°C and 150 r / min for 15 minutes. After the reaction was completed, the system was magnetically separated, and the supernatant was taken and the concentration of residual cannabidiol was measured by liquid chromatography. It was found that when the amount of magnetic molecularly imprinted microspheres added reached 0.15 g, the adsorption amount reached the highest, reaching 26.1 mg / g; further increasing the amount of magnetic molecularly imprinted microspheres added did not further increase the adsorption amount.

[0033] The adsorbed and magnetically separated magnetic molecularly imprinted microspheres were then eluted with a 75% ethanol-water solution for 30 minutes. The cannabidiol content in the eluate was measured by liquid chromatography, and the elution rate of the magnetic molecularly imprinted microspheres was calculated to be 98.35%. This confirmed that the magnetic molecularly imprinted microspheres were able to effectively adsorb and elute cannabidiol.

[0034] Comparative Example 1 A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, comprising the following steps: S1. 200 g of cannabis plants were crushed through a 40-mesh sieve and processed using a conventional high-pressure homogenization process; the product was mixed with 5 g of a composite bacterial agent (wherein the number of viable Trichoderma reesei was 5 × 10 8 cfu / g, the number of viable Aspergillus niger was 3×10 8cfu / g) was added to 550 g of water, the pH of the system was adjusted to 5-6, and the fermentation was carried out at 35°C for 6 hours to instantly inactivate the microorganisms at high temperature; then 900 g of anhydrous ethanol was added, the mixture was refluxed for 2 hours, and filtered to obtain a residue a and a filtrate a; the residue a was added to 400 g of anhydrous ethanol, the mixture was refluxed for 90 minutes, and filtered to obtain a filtrate b; the filtrate a and the filtrate b were combined and concentrated to a relative density (50°C) of 1.07 to obtain an extract; S2. 80 g of magnetic molecularly imprinted microspheres were added to 800 g of deionized water and ultrasonically dispersed uniformly. 250 g of the extract was added thereto and ultrasonically treated for 2 min at an ultrasonic frequency of 42 kHz and an ultrasonic temperature of 40°C. The magnetic molecularly imprinted microspheres were washed with a 75% by mass ethanol aqueous solution for magnetic separation. The washing solution was collected and loaded onto an AB-8 resin column. During the elution process, a 35% by mass ethanol aqueous solution was used to elute and remove impurities. Then, a 55% by mass ethanol aqueous solution was used to elute to obtain an eluate of the target product. Finally, a 97% by mass ethanol aqueous solution was used to elute and regenerate the chromatography column. The magnetic molecularly imprinted microspheres were prepared by the following steps: 300 g of nano-ferroferric oxide and 10 g of polyethylene glycol were added to 500 g of deionized water and ultrasonically dispersed uniformly, 30 g of [BMIM]Cl ionic liquid was added thereto, and the mixture was stirred under nitrogen for 10 min at a stirring speed of 8000 r / min. Subsequently, 30 g of methacrylic acid, 20 g of ethylene glycol dimethacrylate, and 8 g of azobisisobutyronitrile were added in sequence, and the mixture was stirred at 60°C for 8 h. The mixture was washed with ethanol three times, magnetically separated, vacuum dried, and crushed. S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol. The fractions are collected and concentrated to a relative density (50°C) of 1.13. The fractions are supersaturated and crystallized using ethanol at 8°C, washed with purified water at 0°C, and dried under vacuum.

[0035] Comparative Example 2 A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, comprising the following steps: S1. 200 g of cannabis plants were crushed and passed through a 40-mesh sieve. The plants were treated with low-temperature plasma at 35°C for 5 min, with a plasma power of 65 W and a frequency of 13.56 MHz. The product was mixed with 5 g of a composite bacterial agent (wherein the number of viable Trichoderma reesei was 5 × 10 8 cfu / g, the number of viable Aspergillus niger was 3×10 8cfu / g) was added to 550 g of water, the pH of the system was adjusted to 5-6, and the fermentation was carried out at 35°C for 6 hours to instantly inactivate the microorganisms at high temperature; then 900 g of anhydrous ethanol was added, the mixture was refluxed for 2 hours, and filtered to obtain a residue a and a filtrate a; the residue a was added to 400 g of anhydrous ethanol, the mixture was refluxed for 90 minutes, and filtered to obtain a filtrate b; the filtrate a and the filtrate b were combined and concentrated to a relative density (50°C) of 1.07 to obtain an extract; S2. Add 250 g of the extract to 800 g of deionized water and ultrasonically treat for 2 min at an ultrasonic frequency of 42 kHz and an ultrasonic temperature of 40°C; load the sample onto an AB-8 resin column, and during the elution process, use a 35% ethanol aqueous solution to elute and remove impurities, then use a 55% ethanol aqueous solution to elute to obtain the target product eluate, and finally use a 97% ethanol aqueous solution to elute to regenerate the chromatography column; S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol. The fractions are collected and concentrated to a relative density (50°C) of 1.13. The fractions are supersaturated and crystallized using ethanol at 8°C, washed with purified water at 0°C, and dried under vacuum.

[0036] The cannabidiol content of cannabis plants (original flowers and leaves) is tested as follows: (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A and water was used as mobile phase B, and isocratic elution was performed at A (%):B (%) = 80:20; the detection wavelength was 210 nm; the theoretical plate number calculated based on the CBD peak should be no less than 2500; (2) Preparation of reference solution: Accurately weigh the cannabidiol reference substance and add methanol (1:1) to prepare a reference solution containing 0.1 mg of each substance per 1 mL; (3) Accurately weigh the tetrahydrocannabinol reference substance and add methanol (1:1) to prepare a solution containing 0.01 mg of the reference substance per 1 mL; (4) Take approximately 1 g of cannabis plant, accurately weigh it, add 25 mL of methanol, sonicate for 15 min, filter, add another 25 mL of methanol, sonicate for 15 min, combine the filtrates, make up to 50 mL, shake well, filter through a microporous filter membrane (0.45 μm), and take the filtrate; (5) Determination method: Accurately aspirate 10 μL of the reference solution and the test solution, inject them into the liquid chromatograph, and determine; (6) Using 1 g of cannabis plant, products were obtained according to the methods of Example 5 and Comparative Examples 1-2, and the cannabidiol and tetrahydrocannabinol in each group of products were determined by liquid chromatography; (7) Count the cannabidiol and tetrahydrocannabinol contents in each group and calculate the cannabidiol extraction rate in each group.

[0037] like Figure 1 As shown, the product obtained by the method of Example 5 has the highest cannabidiol extraction rate and cannabidiol content, and the lowest tetrahydrocannabinol content, which is better than that of Comparative Examples 1-2 (P < 0.05).

[0038] In view of the similar cannabidiol content of the products obtained in Example 5 and Comparative Example 1, the products obtained in Example 5 and Comparative Example 2 were used to conduct a rat test (male healthy SD rats, SPF grade, 6-8 weeks old, weighing 200g±20g), specifically as follows: the rats were adaptively raised for 5 days, 6 rats were randomly selected for sham surgery as a blank group, and the remaining rats were anesthetized with isoflurane breathing under sterile conditions, and the bilateral testicles were removed through the scrotum. The surgical incision was opened and disinfected with iodine tincture; the rats were free to eat and housed in single cages for one week of recovery; on the 8th day after castration, the castrated rats were divided into two groups according to their weight. The rats were divided into 4 groups, 6 in each group, namely the model group, the positive control group, the Example 5 group, and the comparative example 2 group. The grouping and administration are shown in Table 1 (an aqueous solution containing 0.1% Tween-80 and 0.5% sodium carboxymethyl cellulose was used as the solvent); the drug was administered by gavage once in the morning of the 9th day after castration, and the administration cycle was 21 days; during the administration period, the model group, the Example 5 group, and the comparative example 2 group were simultaneously subcutaneously injected with the modeling agent testosterone propionate, the injection dose was 2.0 mg / kg / day, the solvent was olive oil, the injection volume was 1 mL / kg, and the modeling cycle was 21 days.

[0039] Table 1 Grouping and drug administration

[0040] 24 hours after the last administration, the rats were killed by cervical dislocation, and the prostate tissues (ventral and dorsal lobes) were quickly removed. The surface liquid was absorbed with filter paper, and the fat tissue was peeled off. The wet weight was measured with an electronic balance, and the rat prostate index was calculated.

[0041] Prostate index = prostate mass ÷ body weight.

[0042] Then, the tissue was placed in a measuring tube containing physiological saline to measure the volume. The same mass of tissue from the same part was taken and homogenized using a tissue homogenizer. The malondialdehyde (MDA) and superoxide dismutase (SOD) levels in the tissue were determined using a kit.

[0043] like Figure 2 and Figure 3 As shown, the prostate volume and prostate index of the model group rats were the largest, while the malondialdehyde content was the highest and the superoxide dismutase content was the lowest, indicating that the model was successfully established; the prostate volume, prostate index, and malondialdehyde content of the rats in Example 5 group were lower than those in the control group 2 (P < 0.05), while the superoxide dismutase content was higher than that in the control group 2 (P < 0.05), but there was no significant difference with the positive control group (P > 0.05).

[0044] The applicant believes that this is due to the present invention's method of pulverizing the hemp plant, then combining it with low-temperature plasma treatment to precisely regulate cell membrane permeability. Furthermore, the method further decomposes the hemp plant using a composite microbial agent, promoting cannabidiol (CBD) dissolution. Compared to traditional high-pressure homogenization, this method not only significantly improves the release rate of cellular contents but also increases the retention rate of CBD. Furthermore, the plasma treatment combined with the composite microbial agent significantly increases the effective contact area of the magnetic molecularly imprinted microspheres. Pre-adsorption of the magnetic molecularly imprinted microspheres effectively alleviates the load pressure of subsequent AB-8 chromatography, effectively improving column efficiency and significantly enhancing the separation efficiency of CBD. The product is extremely pure and of stable quality, making it suitable for large-scale industrial production. The magnetic molecularly imprinted microspheres used in the present invention use methacrylic acid as a functional monomer and specifically adsorb CBD through an ionic liquid-enhanced π-π interaction. Further chromatography with AB-8 resin increases the purity of the final product from 68% to over 98%.

[0045] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for efficiently extracting cannabidiol from industrial hemp using microorganisms, characterized in that: The steps include: S1. The cannabis plant is crushed and sieved, and treated with low-temperature plasma for 4-6 minutes at a plasma power of 50-80W and a frequency of 13.56 MHz. The product and the composite bacterial agent are added to water, the pH of the system is adjusted to 5-6, and the mixture is fermented at 30-40°C for 4-8 hours to instantly inactivate the microorganisms at high temperature. Anhydrous ethanol is then added, the mixture is refluxed and extracted for 1-3 hours, and filtered to obtain a filter residue a and a filtrate a. The filter residue a is then added to anhydrous ethanol, the mixture is refluxed and extracted for 1-2 hours, and filtered to obtain a filtrate b. The filtrate a and the filtrate b are then combined and concentrated to obtain an extract. S2. adding the magnetic molecularly imprinted microspheres to water and ultrasonically dispersing them uniformly, adding the extract thereto and ultrasonically treating for 1-2 minutes; performing magnetic separation and washing the magnetic molecularly imprinted microspheres; collecting the washing solution and loading it onto an AB-8 resin column; during the elution process, eluting with a 30-40% by mass ethanol aqueous solution to remove impurities, and then eluting with a 50-60% by mass ethanol aqueous solution to obtain an eluate of the target product; S3. The target product eluate is subjected to molecular distillation to specifically adsorb and remove the remaining tetrahydrocannabinol, the fractions are collected and concentrated, supersaturated crystallized with ethanol at 5-12°C, washed with purified water at 0°C, and dried in vacuo.

2. The method according to claim 1, characterized in that In S1, the mass ratio of the cannabis plant and the composite bacterial agent is 10-30:0.1-1.

3. The method according to claim 1, characterized in that In S1, the composite bacterial agent includes Trichoderma reesei and Aspergillus niger; the viable count of Trichoderma reesei is 3-6×10 8 cfu / g, the number of viable Aspergillus niger is 1-4×10 8 cfu / g.

4. The method according to claim 1, characterized in that In S1, the relative density of the extract at 50°C is 1.06-1.

08.

5. The method according to claim 1, characterized in that: In S2, after the extract is added, the frequency of ultrasonic treatment is 40-45 kHz and the ultrasonic temperature is 30-50°C.

6. The method according to claim 1, characterized in that In S2, after magnetic separation, the magnetic molecularly imprinted microspheres are washed with an ethanol aqueous solution having a mass fraction of 70-80%.

7. The method according to claim 1, characterized in that: In S2, the mass ratio of the magnetic molecularly imprinted microspheres to the extract is 5-10:20-30, and the particle size of the magnetic molecularly imprinted microspheres is 50-100 μm.

8. The method according to claim 1, characterized in that: In S2, magnetic molecularly imprinted microspheres are prepared by the following steps: adding nano-ferroferric oxide and polyethylene glycol to water and ultrasonically dispersing them uniformly, adding 1-butyl-3-methylimidazolium chloride ionic liquid thereto, stirring for 5-15 minutes under nitrogen protection, adding methacrylic acid, ethylene glycol dimethacrylate, and azobisisobutyronitrile in sequence, stirring at 50-70°C for 5-10 hours, washing, magnetic separation, vacuum drying, and crushing.

9. The method according to claim 8, characterized in that The mass ratio of nano-ferrosoferric oxide, polyethylene glycol, 1-butyl-3-methylimidazolium chloride ionic liquid, methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile is 20-40:1:1-5:1-5:1-3:0.5-1.

10. The method according to claim 1, characterized in that: In S3, the fractions were collected and concentrated to a relative density of 1.1-1.15 at 50°C.

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