阅读说明：本技术 大麻二酚的提取工艺及大麻二酚或大麻提取物在制备预防或者治疗bph的药物中的应用 (Extraction process of cannabidiol and application of cannabidiol or cannabis extract in preparation of drugs for preventing or treating BPH ) 是由 温尧林 申凯 贾远敏 于 2020-11-05 设计创作，主要内容包括：本发明涉及植物提取与纯化领域,具体提供了一种大麻二酚的提取工艺及大麻二酚或大麻提取物在制备预防或者治疗BPH的药物中的应用,该大麻二酚的提取工艺包括重结晶步骤,所述重结晶步骤包括：取工业大麻花叶经过浸提步骤、吸附脱色步骤、大孔树脂层析柱步骤和反向柱层析步骤处理后得到的粗品与70-99vt％的醇水溶液混合,加热溶解,趁热过滤,取滤液冷却结晶,固液分离,干燥,得到大麻二酚纯品；其中,所述粗品与70-99vt％的醇水溶液的质量比为1:2-5；通过该重结晶步骤能够在保证高纯度的基础上显著提高大麻二酚的收率,且无需使用正己烷等有机溶剂,仅采用醇和水,提高生产过程的安全性以及提取得到的大麻二酚纯品的安全性。(The invention relates to the field of plant extraction and purification, and particularly provides a cannabidiol extraction process and application of cannabidiol or cannabis extract in preparation of a medicine for preventing or treating BPH (BPH), wherein the cannabidiol extraction process comprises a recrystallization step, and the recrystallization step comprises the following steps: mixing a crude product obtained by treating industrial hemp flowers and leaves through an extraction step, an adsorption decoloration step, a macroporous resin chromatographic column step and a reverse column chromatography step with 70-99 vt% of an alcohol-water solution, heating for dissolving, filtering while hot, taking filtrate for cooling and crystallizing, carrying out solid-liquid separation, and drying to obtain a pure cannabidiol product; wherein the mass ratio of the crude product to 70-99 vt% alcohol-water solution is 1: 2-5; the recrystallization step can remarkably improve the yield of the cannabidiol on the basis of ensuring high purity, and only alcohol and water are adopted without using organic solvents such as n-hexane and the like, so that the safety of the production process and the safety of the extracted cannabidiol pure product are improved.)

1. An extraction process of cannabidiol, which is characterized by comprising a recrystallization step, wherein the recrystallization step comprises the following steps: mixing a crude product obtained by treating industrial hemp flowers and leaves through an extraction step, an adsorption decoloration step, a macroporous resin chromatographic column step and a reverse column chromatography step with 70-99 vt% of an alcohol-water solution, heating for dissolving, filtering while hot, taking filtrate for cooling and crystallizing, carrying out solid-liquid separation, and drying to obtain a pure cannabidiol product;

wherein the mass ratio of the crude product to 70-99 vt% alcohol-water solution is 1: 2-5.

2. The process for the extraction of cannabidiol as claimed in claim 1, wherein the crude product is mixed with 95-99% vt% aqueous alcohol and prior to cooling crystallization further comprises the step of diluting the filtrate with an aqueous solution; the mass ratio of the crude product to the aqueous solution is 1: 0.1-3.

3. The cannabidiol extraction process of claim 2, wherein the aqueous solution is selected from pure water, distilled water, water for injection, or an alkaline solution; preferably, the alkaline solution is an aqueous solution containing 0.1 to 0.5 wt% of sodium hydroxide.

4. The process for extracting cannabidiol as claimed in any one of claims 1 to 3, wherein the temperature of heating for dissolution in the recrystallisation step is 50-85 ℃ for 10-20 min.

5. The cannabidiol extraction process as claimed in any one of claims 1 to 4, wherein in the recrystallisation step, cooling crystallisation is carried out under stirring at a temperature of-20 to 4 ℃, at a speed of 20 to 100rpm, for a period of 20 to 28 hours.

6. The cannabidiol extraction process of any one of claims 1-5, wherein the leaching step comprises extracting dried industrial cannabis sativa leaves with 80-95 vt% aqueous alcohol at 70-95 ℃ for 1-3 times, combining the extracts, and concentrating to obtain extract; or, the leaching step comprises extracting industrial hemp flower and leaf with water at 90-95 deg.C for 1-3 times, adding 80-95 vt% alcohol water solution, extracting at 55-75 deg.C for 1-3 times, mixing extractive solutions, and concentrating to obtain extract;

the adsorption decoloring step comprises the steps of diluting the extract obtained in the leaching step with water, adding active carbon accounting for 5-10 wt% of the mass of the industrial hemp leaves, uniformly mixing, decoloring at 50-65 ℃ for 0.5-1h, filtering, and collecting decolored liquid;

the macroporous resin chromatographic column comprises the steps of taking the decolorized solution treated in the adsorption decolorization step, washing impurities with 2-4BV of water or 30-50 vt% of alcohol water solution, then resolving with 3-4BV of 70-95% of alcohol water solution, collecting resolved solution, and concentrating;

and the reverse column chromatography step comprises the steps of taking the analytic solution obtained in the step of macroporous resin chromatography column, washing impurities with 2-4BV of 50-70 vt% alcohol water solution, then analyzing with 2-4BV of 85-95 vt% alcohol water solution, collecting the analytic solution, concentrating to obtain an extract, standing for precipitation, and collecting solids to obtain a crude product.

7. Use of cannabidiol and/or cannabis extract in the manufacture of a medicament for the prevention or treatment of prostatic hyperplasia.

8. Use according to claim 7, characterized in that the cannabis extract is prepared by a process comprising the following steps: extracting the dried industrial hemp flowers and leaves with 80-95 vt% alcohol-water solution at 70-95 deg.C for 1-3 times, mixing extractive solutions, and concentrating to obtain extract;

or, the method comprises the following steps: extracting industrial hemp flower and leaf with water at 90-95 deg.C for 1-3 times, adding 80-95 vt% alcohol water solution, extracting at 55-75 deg.C for 1-3 times, mixing extractive solutions, and concentrating to obtain extract.

9. The use according to claim 7 or 8, wherein the medicament comprises cannabidiol and/or cannabis extract as active ingredients, and further comprises a pharmaceutically acceptable carrier.

10. The use according to claim 9, wherein the medicament is a gel, cream, tablet, capsule, powder, mixture, pill, granule, solution, syrup, decoction, suppository, aerosol, patch, ointment, injection, spray, liniment, tincture, wet compress, paste, lotion or sustained-release preparation;

the pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, polymeric matrix materials, and film-forming materials.

Technical Field

The invention relates to the field of plant extraction and purification, in particular to a cannabidiol extraction process and application of cannabidiol or cannabis extract in preparation of a medicine for preventing or treating BPH.

Background

Benign Prostatic Hyperplasia (BPH) is a common frequently encountered disease in older men, with increasing incidence increasing with age, usually initially after age 40, with incidence rates of greater than 50% by age 60 and up to 83% by age 80, and has become the most common urological disorder that affects extensively the health and quality of life of older men.

In the case of BPH, the prostate volume increases due to the massive proliferation of prostatic stroma and epithelial cells, and the Lower Urinary Tract Symptoms (LUTS) such as frequent urination, urgent urination, urge incontinence, and dysuria are accompanied by the Bladder neck obstruction (BOO) phenomenon in more than 50% of patients, which has been a major manifestation affecting the quality of life of BPH patients. When BOO occurs, the bladder detrusor muscle function is impaired, which is expressed as a process from compensation to decompensation; the phenomenon of Detrusor Instability (DI) is particularly prominent, namely: during the storage period when the bladder is full, the detrusor muscle undergoes spontaneous involuntary contraction which cannot be actively inhibited.

The clinical treatment means for BPH mainly comprise: surgical treatment and pharmaceutical treatment. BPH patients are generally older, have weak physiological functions and have no surgical indications; other drugs for inhibiting prostatic hyperplasia have certain toxic and side effects, and especially have certain influence on the immune system. The search for new active substances against prostatic hyperplasia from Chinese medicine extracts has been a hot spot of pharmaceutical research.

Cannabidiol (CBD) which is a non-addictive medicinal component in industrial cannabis sativa has biological activities of resisting convulsion, vomiting, spasm, anxiety, sedation, inflammation, insomnia, oxidation and stabilization, and is widely applied to a plurality of fields such as medicines, health products, foods and daily chemicals. The drug epididex developed from cannabidiol has been approved by the FDA in the united states for the treatment of two rare but very serious epileptic disorders, Lennox-Gastaut syndrome and Dravet syndrome; the american beverage, megahead coca cola, is intended to develop a cannabidiol beverage that is said to have a market value of $ 2.6 billion in the united states by 2022; a facial mask containing cannabidiol, marketed by the world-wide brand of cosmetics, ashyland, inc. At present, no report is available about the application of cannabidiol in treating prostatic hyperplasia.

At present, cannabidiol is generally obtained by directly extracting dried industrial hemp leaves by an organic solvent extraction method or a supercritical carbon dioxide extraction method. In order to improve the purity of cannabidiol products, chinese patent document CN11147093A discloses a method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves, which comprises the steps of leaching, adsorption and desorption by macroporous resin, reverse phase chromatography and crystallization, although the method can improve the purity of cannabidiol, the amount of extracted cannabidiol is small, so that the extraction yield of cannabidiol is low, the yield of cannabidiol is sacrificed in order to improve the purity of cannabidiol, and n-hexane organic solvents are required to be used in the process of recrystallizing cannabidiol, and the use of the organic solvents can damage the environment and affect the safety of products.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects of low product and operation safety and low extraction yield caused by the fact that a high-purity cannabidiol product is selected to be recrystallized by using an n-hexane organic solvent in the extraction process of cannabidiol in the prior art, so that the extraction process of cannabidiol is provided.

The invention provides a cannabidiol extraction process, which comprises a recrystallization step, wherein the recrystallization step comprises the following steps: mixing a crude product obtained by subjecting industrial hemp flowers and leaves to an extraction step, an adsorption decoloration step, a macroporous resin chromatographic column step and a reverse column chromatography step with 70-99 vt% of an alcohol-water solution, heating to dissolve, filtering while hot, cooling and crystallizing a filtrate, carrying out solid-liquid separation, and drying to obtain a pure cannabidiol product; wherein the mass ratio of the crude product to 70-99 vt% alcohol-water solution is 1: 2-5.

Preferably, the mass ratio of the crude product to the 70-99 vt% aqueous alcohol solution is 1: 2-3.

In the invention, the alcohol aqueous solution is a mixed solution of alcohol and water, the adopted alcohol can be ethanol or methanol, preferably ethanol, and is safer and more environment-friendly, and the 70-95 vt% alcohol aqueous solution means that the volume percentage of the alcohol contained in the alcohol aqueous solution is 70-95%. The dissolution process can also be accelerated by stirring.

Wherein, the hot filtration means that the filtration is carried out before the temperature of the solution is obviously reduced after the heating and dissolving step is finished, and the temperature of the solution is not lower than 30 ℃ when the filtration is carried out while the solution is hot.

Preferably, when the crude product is mixed with 95-99 vt% of alcohol aqueous solution, before cooling and crystallizing, the method further comprises the step of diluting the filtrate by using the aqueous solution, and the mass ratio of the crude product to the aqueous solution is 1: 0.1-3. The mixing can be accelerated in the process of dilution by adopting a stirring mode. The aqueous solution can also be added in a slow dropwise manner or added in 3-7 times.

Further, the aqueous solution is selected from water or an alkaline solution; the water can be pure water, distilled water, water for injection, etc.; the alkaline solution can be conventional alkaline solutions such as aqueous solution containing sodium hydroxide, aqueous solution containing potassium hydroxide, etc., and preferably, the alkaline solution is aqueous solution containing 0.1-0.5 wt% of sodium hydroxide; namely, an aqueous solution containing sodium hydroxide is adopted, and the mass percent of the sodium hydroxide is 0.1-0.5%.

Preferably, in the recrystallization step, the temperature for heating and dissolving is 50-85 ℃ for 10-20 min.

Preferably, in the recrystallization step, the mixture is cooled and crystallized under stirring, the temperature is-20 to 4 ℃, the rotation speed is 20 to 100rpm, and the time is 20 to 28 hours.

Further, the leaching step comprises the steps of extracting the dried industrial hemp flowers and leaves by using 80-95 vt% alcohol-water solution at 70-95 ℃ for 1-3 times, combining the extracting solutions, and concentrating to obtain the extract. Preferably, 8-12 times of the alcohol-water solution is added each time; extracting for 0.5-3 hr each time.

Further, the leaching step may also include extracting industrial hemp flower and leaf with water at 90-95 deg.C for 1-3 times, adding 80-95 vt% alcohol water solution, extracting at 55-75 deg.C for 1-3 times, mixing extractive solutions, and concentrating to obtain extract. Preferably, 8-15 times of water is added each time, and the extraction is carried out for 1-2h each time; adding 8-12 times of alcohol water solution each time, and extracting for 1-3 hr each time.

Further, the adsorption decoloring step comprises the steps of diluting the extract obtained in the leaching step with water, adding active carbon accounting for 5-10% of the mass of the industrial hemp leaves, uniformly mixing, decoloring at 50-65 ℃ for 0.5-1h, filtering, and collecting decolored liquid. Adding the solution diluted until the ratio of the total volume of the solution to the mass of the industrial hemp flowers and leaves is 3-4L: 1 kg.

Further, the macroporous resin chromatographic column comprises the steps of taking the decolorized solution treated in the adsorption decolorization step, washing impurities by using 2-4BV of water or 30-50% of alcohol water solution, then resolving by using 3-4BV of 70-95% of alcohol water solution, collecting resolved solution, and concentrating.

Further, the reverse phase column chromatography step comprises the steps of taking the analytic solution obtained in the step of macroporous resin chromatography column, washing with 2-4BV of 50-70 vt% alcohol water solution, then analyzing with 2-4BV of 85-95 vt% alcohol water solution, collecting the analytic solution, concentrating to obtain an extract, standing for precipitation, and collecting solid to obtain a crude product.

The reverse phase column chromatography step employs a conventional reverse phase column, such as a C18 column; the macroporous resin chromatographic column step adopts conventional macroporous resin chromatographic columns, such as AB-8 chromatographic column and D101 chromatographic column.

The invention also provides application of cannabidiol or cannabis extract in preparation of a medicament for preventing or treating prostatic hyperplasia.

The hemp extract is an industrial hemp flower and leaf extract.

The hemp extract is obtained by extracting industrial hemp flowers and leaves according to a conventional extraction method or respectively according to a conventional extraction method; or the effective part of the extract is obtained by refining and purifying process;

preferably, the conventional extraction method comprises one or more of immersion extraction, decoction extraction, reflux extraction, percolation extraction, ultrasonic extraction and steam distillation; the extraction solvent comprises water or 20-95 vt% ethanol solution; the refining and purifying process comprises one or more of water extraction and alcohol precipitation, extraction, silica gel chromatographic column separation and macroporous resin column separation.

More preferably, the preparation method of the cannabis extract comprises the following steps: extracting the dried industrial hemp flowers and leaves with 80-95 vt% alcohol-water solution at 70-95 deg.C for 1-3 times, mixing extractive solutions, and concentrating to obtain extract;

or, the method comprises the following steps: extracting industrial hemp flower and leaf with water at 90-95 deg.C for 1-3 times, adding 80-95 vt% alcohol water solution, extracting at 55-75 deg.C for 1-3 times, mixing extractive solutions, and concentrating to obtain extract.

The medicine takes cannabidiol and/or cannabis extract as active ingredients and also comprises a pharmaceutically acceptable carrier.

Further, the medicine is gel, cream, tablet, capsule, powder, mixture, pill, granule, solution, syrup, soft extract, suppository, aerosol, emplastrum, ointment, injection, spray, liniment, tincture, wet dressing, paste, lotion or sustained and controlled release preparation.

Further, the pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, polymeric matrix materials, and film-forming materials.

The technical scheme of the invention has the following advantages:

1. according to the extraction process of cannabidiol provided by the invention, industrial cannabis sativa leaves are subjected to extraction, adsorption and decoloration, macroporous resin chromatographic column and reverse column chromatography to obtain a crude product, the crude product is mixed with 70-99 vt% of alcohol-water solution, the mixture is heated and dissolved, the hot mixture is filtered, the filtrate is cooled and crystallized, and solid-liquid separation is carried out to obtain a cannabidiol pure product, the mass ratio of the crude product to the 70-99 vt% of alcohol-water solution is controlled to be 1:2-5, the recrystallization step can obviously improve the yield of cannabidiol on the basis of ensuring high purity, only normal hexane and other organic solvents are used, and the safety of the production process and the safety of the extracted cannabidiol pure product are improved;

when the concentration of the ethanol is too high, the content of the cannabidiol in the mixed solution is remarkably reduced, so that the yield of the target product is reduced; when the concentration of the ethanol is too low, the cannabidiol cannot be separated from other impurities, so that the purity of the target product is reduced. According to the method, the crude product and the alcohol-water solution in a mass ratio of 1:2-5 are selected, 70-95 vt% of the alcohol-water solution is adopted to be heated and dissolved, and the hot filtering and cooling crystallization are combined, so that the extraction of the cannabidiol in the industrial cannabis leaves is facilitated, the residual impurities in the extracting solution can be obviously reduced, and the yield of the cannabidiol is obviously improved on the basis of ensuring high purity.

2. According to the extraction process of the cannabidiol provided by the invention, researches show that when the crude product is mixed with 95-99 vt% of alcohol-water solution, more cannabidiol can be extracted by adding the water solution for dilution and limiting the mass ratio of the crude product to the water solution to be 1:0.1-3, so that higher yield is obtained.

3. According to the extraction process of cannabidiol provided by the invention, the aqueous solution can be water or an alkaline solution with the pH of 8-10; preferably, more cannabidiol can be extracted using an aqueous solution containing 0.1-0.5 wt% sodium hydroxide as the alkaline solution, resulting in higher yields.

4. According to the extraction process of the cannabidiol provided by the invention, in the recrystallization process, the temperature for heating and dissolving is controlled to be 50-85 ℃, the time is 10-20min, the solubility of the cannabidiol can be obviously improved, and more insoluble pigments and impurities are removed by combining with a hot filtering step, so that higher yield and purity are obtained.

5. In the recrystallization step, the cannabidiol is cooled and crystallized under stirring, the temperature is-20-4 ℃, the rotating speed is 20-100rpm, and the time is 20-28 h; more cannabidiol can be separated out through the cooling crystallization step, so that the yield of the cannabidiol is further improved.

6. The application of the cannabidiol or the cannabis extract in the preparation of the medicine for preventing or treating the prostatic hyperplasia provided by the invention is found through researches that the cannabidiol and the cannabis extract with medium and high doses have an inhibition effect on the increase of the prostate volume of a rat caused by testosterone propionate, the cannabidiol and the cannabis extract with each dose have a reduction effect on the index of each leaf of the prostate, particularly the index of the abdominal leaf of the prostate can be remarkably reduced, the SOD activity can be improved, and the MDA content can be reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing HE staining of prostate ventral lobes of a blank group of rats in Experimental example 1 of the present invention;

FIG. 2 is a graph showing HE staining of ventral prostate lobes of a rat model group in Experimental example 1 of the present invention;

FIG. 3 is a graph showing HE staining of ventral prostate lobes of rats of the administration group 1 in Experimental example 1 of the present invention;

FIG. 4 is a graph showing HE staining of ventral prostate lobes of 4 rats in the administration group in Experimental example 1 of the present invention;

FIG. 5 is a high performance liquid chromatogram of a pure cannabidiol prepared in example 1 of Experimental example 1 of the present invention.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

The vt% ethanol aqueous solution is the volume percentage of ethanol in the mixed solution formed by mixing ethanol and water. The% alcohol aqueous solution or vt% alcohol aqueous solution means the volume percentage of alcohol in a mixed solution formed by mixing alcohol and water.

Example 1

The embodiment provides a cannabidiol extraction process, which comprises the following steps:

(1) leaching: taking 1kg of fresh industrial hemp flower leaves (with cannabidiol content of 0.97%), adding 12 times of water by mass, heating to 95 ℃, extracting for 1.5h, filtering, adding 10 times of water by mass into residues, extracting for 1.5h at 95 ℃, filtering, adding 10 times of 80% ethanol aqueous solution by mass into residues, extracting for 2 times at 75 ℃ for 2h each time, combining extracting solutions (with cannabidiol content of 12.83%) and concentrating under reduced pressure at 55 ℃ until the volume of the solution is 2L to obtain an extract A.

(2) An adsorption decoloring step: diluting the extract A with water to 3L, adding 303 active carbon (50 g active carbon is added corresponding to 1kg raw material) accounting for 5% of the fresh industrial hemp flower leaf mass, stirring for 10min, mixing uniformly, heating to 60 deg.C for decolorizing for 1h, filtering with titanium rod filter after decolorizing, and collecting decolorized solution;

(3) macroporous resin chromatographic column: passing the decolorized solution through an AB-8 chromatographic column, eluting with 30%, 70% and 95% ethanol at a sample loading flow rate of 2BV/h, collecting 70% of eluted parts with each gradient of 3BV, and concentrating at 50-55 ℃ until no alcohol smell exists to obtain an eluent;

(4) reverse column chromatography step: passing the eluate through C18 column, eluting with 50%, 70%, 85%, 95% ethanol, each gradient is 3BV, collecting the 85% ethanol eluate, concentrating to obtain extract, cooling to room temperature, standing to precipitate, and collecting solid to obtain crude product;

(5) a recrystallization step: heating the crude product to 60 ℃ by using 2 times of 95 vt% ethanol aqueous solution by mass, dissolving for 15min (namely the mass ratio of the crude product to the 95 vt% ethanol aqueous solution is 1:2), filtering while hot, adding 0.25 times of 0.1 wt% sodium hydroxide aqueous solution (the mass ratio of the crude product to the 0.1 wt% sodium hydroxide aqueous solution is 1:0.25) into the filtrate, diluting, cooling the mixed solution at 0 ℃ and 50rpm for crystallization for 24h, performing suction filtration, and performing vacuum drying on the solid at 35 ℃ to obtain the target product, namely, the cannabidiol pure product.

Example 2

The embodiment provides a cannabidiol extraction process, which comprises the following steps:

(1) leaching: taking 1kg of fresh industrial hemp flower and leaf (the content of cannabidiol is 1%), adding 15 times of mass water, heating to 95 ℃, extracting for 1h, filtering, adding 8 times of mass water into residue, extracting for 2h at 95 ℃, filtering, adding 8 times of mass 90% ethanol aqueous solution into residue, extracting for 2 times at 70 ℃ for 1h each time, combining extracting solutions, and concentrating under reduced pressure at 55 ℃ until the volume of the solution is 2L to obtain an extract A;

(2) an adsorption decoloring step: diluting the extract obtained in the step (1) to 4L with water, adding activated carbon accounting for 8% of the fresh industrial hemp flower and leaf mass (namely adding 80g of activated carbon corresponding to 1kg of raw material), stirring for 10 minutes, uniformly mixing, heating to 60 ℃, decoloring for 30min, filtering the mixed solution through a titanium rod filter after decoloring, and collecting a decolored solution;

(3) macroporous resin chromatographic column: passing the decolorized solution through a D101 chromatographic column (macroporous resin adsorption column), eluting with 30%, 80% and 95% ethanol at a flow rate of 2BV/h, collecting 80% of the eluted parts with each gradient of 3BV, and concentrating at 50-55 deg.C until no alcohol smell exists to obtain an eluate;

(4) reverse column chromatography step: passing the eluate through C18 column, eluting with 50%, 70%, 85%, and 95% ethanol, each gradient is 3BV, collecting 85% eluate, concentrating to obtain extract, cooling to room temperature, and standing to obtain precipitated solid as crude product;

(5) and (2) a recrystallization step, heating the crude product to 70 ℃ by using 99% ethanol aqueous solution with the mass of 4 times, dissolving for 20min, filtering while hot, adding the filtrate into 0.5 wt% sodium hydroxide aqueous solution with the mass of 3 times while stirring, averagely adding the solution for 7 times, then cooling and crystallizing for 20h under the condition that the temperature is 4 ℃ and the rotating speed is 20rpm, carrying out suction filtration on crystals, and carrying out vacuum drying at 35 ℃ to obtain the target product, namely, the pure cannabidiol.

Example 3

The embodiment provides a cannabidiol extraction process, which comprises the following steps:

(1) leaching: cutting 1kg of fresh industrial hemp flower and leaf (with cannabidiol content of 1%), drying at 120 deg.C for 3h, adding 12 times of 95% ethanol, extracting at 65 deg.C for 3h for 1 time, collecting ethanol extractive solution (with cannabidiol content of 15.24%), and concentrating at 55 deg.C under reduced pressure to obtain 2L solution to obtain extract;

(2) an adsorption decoloring step: diluting the extract with water to 3.5L, adding activated carbon (100 g activated carbon corresponding to 1kg raw material) 10% of fresh industrial hemp flower and leaf, mixing well (stirring for 10min), heating to 60 deg.C for decolorizing for 30min, filtering the mixed solution with titanium rod filter, and collecting decolorized solution;

(3) macroporous resin chromatographic column: passing the decolorized solution through an AB-8 chromatographic column, eluting with pure water, 50% and 95% ethanol at a flow rate of 2BV/h, collecting 95% of eluted parts, and concentrating at 50-55 deg.C until no alcohol smell exists to obtain an eluent;

(4) reverse column chromatography step: passing the eluate through a C18 column, eluting with 50%, 70%, 85% and 95% ethanol at each gradient of 3BV, collecting 85% of the eluate, concentrating to obtain extract, cooling to room temperature, and standing to obtain precipitated solid, i.e. crude product;

(5) a recrystallization step: heating the crude product to 85 ℃ by using 70% ethanol in an amount which is 5 times that of the crude product for dissolving for 10min, filtering while the crude product is hot, collecting filtrate, heating again to 55 ℃ for dissolving, filtering while the crude product is hot, collecting filtrate while stirring, adding 0.12 times of aqueous solution containing 0.5 wt% of sodium hydroxide in mass into the filtrate while stirring, averagely adding the aqueous solution into the filtrate in 3 times, cooling and crystallizing for 28h under the conditions that the temperature is minus 20 ℃ and the rotating speed is 100rpm, carrying out suction filtration on crystals, and carrying out vacuum drying at 35 ℃ to obtain the target product, namely, the pure cannabidiol.

Example 4

This example provides a cannabidiol extraction process, which differs from example 1 only in the recrystallization step, which is:

heating the crude product to 60 ℃ by using 2 times of 95 vt% ethanol aqueous solution by mass, dissolving for 15min, filtering while hot, taking the filtrate, standing at 0 ℃, cooling, crystallizing for 24h, performing suction filtration, taking the solid, and performing vacuum drying at 35 ℃ to obtain the target product, namely, the cannabidiol pure product.

Example 5

This example provides a cannabidiol extraction process, which differs from example 4 only in the recrystallization step, which is:

heating the crude product to 60 ℃ by using 2 times of 95 vt% ethanol aqueous solution by mass, dissolving for 15min, filtering while the solution is hot, adding 0.25 times of pure water by mass into the filtrate for dilution, standing the mixed solution at the temperature of 0 ℃, cooling and crystallizing for 24h, carrying out suction filtration, taking the solid, and carrying out vacuum drying at the temperature of 35 ℃ to obtain the target product, namely, the cannabidiol pure product.

Example 6

This example provides a cannabidiol extraction process, which differs from example 4 only in the recrystallization step, which is:

heating the crude product to 60 ℃ by using 2 times of 95 vt% ethanol aqueous solution by mass, dissolving for 15min, filtering while the solution is hot, adding 0.25 times of 0.1 wt% sodium hydroxide aqueous solution into the filtrate for dilution, standing the mixed solution at the temperature of 0 ℃, cooling and crystallizing for 24h, carrying out suction filtration, taking the solid, and carrying out vacuum drying at the temperature of 35 ℃ to obtain the target product, namely the cannabidiol pure product.

Example 7

This example provides a cannabidiol extraction process, which differs from example 4 only in the recrystallization step, which is:

heating the crude product to 60 ℃ by using 2 times of 95 vt% ethanol aqueous solution by mass, dissolving for 15min, filtering while hot, taking the filtrate, cooling and crystallizing for 24h at the temperature of 0 ℃ and the rotation speed of 50rpm, carrying out suction filtration, taking the solid, and carrying out vacuum drying at 35 ℃ to obtain the target product, namely the pure cannabidiol.

Comparative example 1

This comparative example provides a cannabidiol extraction process, differing from example 4 only in the recrystallisation step, which is:

the crude product was taken and dissolved in 10ml of n-hexane at 25 ℃. After the dissolution is finished, cooling to-40 ℃, preserving heat for 48h, centrifuging, and vacuum drying at 35 ℃ to obtain the target product cannabidiol pure product.

Comparative example 2

This comparative example provides a cannabidiol extraction process, differing from example 4 only in the recrystallisation step, which is:

heating the crude product to 60 ℃ by using 2 times of 50 vt% ethanol aqueous solution by mass, dissolving for 15min, filtering while hot, taking the filtrate, standing at 0 ℃, cooling, crystallizing for 24h, performing suction filtration, taking the solid, and performing vacuum drying at 35 ℃ to obtain the target product, namely, the cannabidiol pure product.

Comparative example 3

This comparative example provides a cannabidiol extraction process, differing from example 4 only in the recrystallisation step, which is:

heating the crude product to 60 ℃ by using 1 time of 95 vt% ethanol aqueous solution by mass, dissolving for 15min, filtering while hot, taking the filtrate, standing at 0 ℃, cooling, crystallizing for 24h, performing suction filtration, taking the solid, and performing vacuum drying at 35 ℃ to obtain the target product, namely, the cannabidiol pure product.

Comparative example 4

This comparative example provides a cannabidiol extraction process, differing from example 4 only in the recrystallisation step, which is:

heating the crude product to 60 ℃ by using 2 times of 95 vt% ethanol aqueous solution by mass, dissolving for 15min, standing at room temperature, filtering, taking the filtrate, standing at 0 ℃, cooling, crystallizing for 24h, performing suction filtration, taking the solid, and performing vacuum drying at 35 ℃ to obtain the target product, namely the pure cannabidiol.

Experimental example 1

1. Experimental methods

The high performance liquid chromatography is adopted to respectively determine the purity and the yield of the pure cannabidiol product and the content of cannabidiol in the industrial cannabis mosaic and cannabis extract, and the specific method is as follows:

(1) preparation of standard solution

Precisely weighing 3.75mg cannabidiol reference substance, adding 25ml methanol for dissolving, and making into standard solution containing 150 μ g/ml cannabidiol reference substance.

Weighing 2.5mg of tetrahydrocannabinol control accurately, adding 50ml of methanol for dissolving, and making into standard solution containing 50 μ g/ml of tetrahydrocannabinol control.

(2) Preparation of test solution

Preparing a solution of a sample to be detected of a pure cannabidiol product: the pure cannabidiol prepared in each example or comparative example is precisely weighed to be 7.57mg, placed in a 50mL volumetric flask, dissolved in methanol to a constant volume to be calibrated, shaken up, filtered by an organic filter membrane of 0.45 μm, and a subsequent filtrate is taken to obtain a sample solution of the pure cannabidiol to be tested.

Preparing a solution of a sample to be detected of industrial hemp flower leaves: weighing 0.75g of crushed industrial hemp flowers and leaves, adding 50mL of methanol, performing ultrasonic extraction for 1h, centrifuging the extracting solution at 12000rpm for 10min, taking the supernatant, adding methanol to a constant volume of 50mL, precisely transferring 1mL, putting the solution into a 10mL volumetric flask (with a dilution multiple of 10 times), adding methanol to a constant volume of scales, shaking up, filtering with a 0.45 mu m organic filter membrane, and taking the subsequent filtrate to obtain the sample solution to be detected of the industrial hemp flowers and leaves.

Preparing a sample solution to be detected of an industrial hemp flower and leaf extracting solution: weighing the industrial hemp flower and leaf extract, recording the total weight, precisely transferring 3mL, placing in a 100mL volumetric flask (with the dilution ratio of 100/3 times), adding methanol to a constant volume to scale, shaking up, filtering with a 0.45 mu m organic filter membrane, and taking the subsequent filtrate to obtain a sample solution of the industrial hemp flower and leaf extract.

(3) Chromatographic conditions and System suitability test

The apparatus Agilent1260DAD, chromatographic column XDB-C18(250mm, 4.6mm,5 μm), mobile phase-acetonitrile-0.1 vt% aqueous ammonia solution (aqueous solution containing 0.1 vt% aqueous ammonia) the peak area of the sample was tested using the following gradient elution procedure, the detection wavelength was 220nm, the column temperature was 35 ℃, the flow rate was 1ml/min, and the sample injection amount was 10 μ l.

TABLE 1 gradient elution Table

(4) Assay method

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and calculating.

(5) The method adopts an external standard one-point method to calculate according to the following formula

The concentration of cannabidiol in each group of samples to be tested is the standard preparation concentration x (peak area of sample to be tested/peak area of standard);

the purity (%) of cannabidiol in the sample to be detected is (the concentration of cannabidiol in the sample to be detected is multiplied by the volume of the sample multiplied by the dilution ratio)/the weighing multiplied by 100%;

the total yield (%) of cannabidiol in the cannabidiol pure product/the mass of cannabidiol contained in industrial cannabis sativa leaves × 100%.

2. Results of the experiment

TABLE 2 purity and yield results table

As can be seen from the table above, compared with comparative examples 1 to 5, the yield of the cannabidiol products prepared in examples 1 to 7 of the present application is significantly improved, and the purity is over 98.5%, so that the yield is greatly improved on the basis of ensuring high purity.

Experimental example 2

1. Laboratory animal

Male healthy SD (Sprague-Dawley) rats, SPF grade, 6-8 weeks old, body weight 200g + -20 g.

2. Reagent and medicine

And (3) testing a sample: extract a prepared according to the process of example 1, cannabidiol pure product prepared according to the process of example 2;

testosterone propionate: purchased from alatin corporation, specifications: 5 g/bottle;

positive drug: finasteride (specification: 5 mg/tablet, lot No. 294051, Hangzhou Moshadong pharmaceutical Co., Ltd.).

3. Content of the experiment

SD rats are adaptively raised for 5 days, 6 rats are randomly treated as a blank group by a sham operation, the rest rats are subjected to respiratory anesthesia by isoflurane under the aseptic condition, bilateral testicles are removed through scrotum, an operation opening is opened, and iodophor is sterilized. Free diet, single cage feeding, recovery for one week. On the 8 th day of castration, the castrated rats are randomly divided into 6 groups according to the body weight, each group comprises 6 animals, namely a model group, a positive control group and administration groups 1-4, the grouping and administration conditions are shown in the following table 1, the castration is performed by gavage for 1 time from the 9 th day of castration in the morning, and the administration period is 21 days; during the administration period, the model group and the administration medicines 1-4 are simultaneously injected with testosterone propionate for model making by subcutaneous injection, the injection dose is 2.0 mg/kg/day, the solvent is commercial olive oil, the injection volume is 1mL/kg, and the model making period is 21 days.

TABLE 1 grouping and administration

After 24h of the last administration, the rats were sacrificed by neck-breaking, the leaf tissues of the prostate (ventral and dorsal leaves) were rapidly removed, the surface fluid was removed by suction with filter paper, the adipose tissues were peeled off, the wet weight thereof was weighed with an electronic balance, and the prostate index of the rats, which is the mass (g) of each leaf of the prostate/the body weight (g), was calculated according to the following formula. Then, the sample is put into a metering tube containing physiological saline to measure the volume. Tissue homogenate is made by a tissue homogenizer on the same mass and part of the tissue, and the contents of MDA and SOD in the tissue are respectively determined by adopting an MDA kit and an SOD kit of Nanjing Biotechnology establishment company according to the kit specification.

The prostate ventral lobes of the rats of the blank group, the model group, the administration group 1 and the administration group 4 are fixed in 10% formalin solution to be sliced into normal paraffin, and then the rat prostate histopathology sections are observed under the optical microscope after HE staining. The paraffin section comprises the following specific steps: (1) taking prostate abdominal leaves, placing the prostate abdominal leaves into a fixing solution (10% formalin), fixing for more than 24h, taking out tissue blocks from the fixing solution, placing the tissue blocks on clean filter paper, and completely sucking the surface liquid; (2) ethanol dehydration of tissue blocks: sequentially soaking in 75% ethanol (5h), 85% ethanol (2h), 95% ethanol (2h), 100% ethanol I (40min), and 100% ethanol II (35 min); (3) transparent fixation of tissue blocks: sequentially soaking in xylene I (2-10min, tissue observation at any time, transparent edge to prevent penetration) and xylene II (10 min); (4) sequentially soaking the tissue blocks in paraffin I (60min) and paraffin II (60 min); (5) embedding (including embedding wax should be the same as the wax that was last waxed).

4. Data statistics and analysis

And (4) recording and statistically analyzing the data by using Excel. The measurement data in the experimental results are expressed by mean ± standard deviation (mean ± SD). The software performs one-way anova on the comparison of SPASS13.0 among groups, and the comparison difference between the groups has statistical significance when P is less than 0.05.

5. Results of the experiment

(1) Effect of drugs on prostate volume and prostate index of testosterone propionate-induced prostate hyperplasia rats

TABLE 2 Effect of drugs on prostate hyperplasia prostate index and prostate volume in rats (n ═ 6, mean. + -. SD)

Note: denotes P <0.05 compared to model control group (t-test); denotes P <0.01 compared to model control group (t-test); p <0.001 compared to model control (t-test); # denotes that P <0.001 for the model group compared to the blank group (t-test)

From the above table, it can be seen that: after 21 days of modeling, the prostate volume and prostate index of the castration model group were significantly increased (p <0.001) compared to the blank group, indicating successful modeling.

In the measurement of prostate volume, compared with the model group, the administration groups 1, 3 and 4 have inhibition effect on the increase of the prostate volume of rats caused by testosterone propionate, and have statistical difference (P < 0.05); the administration group 2 has a certain inhibition effect on the increase of the volume of the rat prostate caused by testosterone propionate, but has no statistical significance, which shows that the medicament has a certain dosage dependence on the inhibition effect on the increase of the volume of the rat prostate caused by testosterone propionate.

In the prostate index measurement, compared with a model group, the positive medicine group and each administration group have the effect of reducing the index of each prostate lobe, and particularly can obviously reduce the index of prostate ventral lobe; the prostate ventral lobe index of the administration groups 1, 3 and 4 is remarkably reduced (P is less than 0.01), the prostate ventral lobe index of the administration group 2 (low-dose cannabidiol group) is remarkably reduced (P is less than 0.05), and the medicament has an inhibition effect on rat prostate hyperplasia and is dose-dependent.

(2) Effect of drugs on testing of MDA and SOD content in prostate tissue

TABLE 3 Effect of drugs on MDA and SOD content assay in prostate tissue (n ═ 6, mean. + -. SD)

Note: denotes P <0.05 compared to model control group (t-test); denotes P <0.01 compared to model control group (t-test); p <0.001 compared to model control (t-test); # denotes that P <0.001 for the model group compared to the blank group (t-test)

As can be seen from Table 3: the SOD activity in prostate gland tissue of mice (model group) with prostate hyperplasia treated by testosterone propionate is obviously reduced (p is less than 0.001), and the MDA content is obviously increased (p is less than 0.001), which indicates that the model building is successful. The SOD activity and MDA content in the mouse prostate tissue are improved after administration, which shows that the medicament can effectively improve the SOD activity and MDA content reduction caused by prostatic hyperplasia induced by testosterone propionate.

(3) Rat prostate abdominal lobe histopathology section for treating prostatic hyperplasia caused by testosterone propionate

As shown in figure 1, the blank group shows that the prostate has regular shape of the large gland, the glandular epithelial cells are in a monolayer shape and are arranged normally, a small part of the glandular epithelial cells have small papillary endothelia, a small amount of secretion is contained in the glandular cavity, the glandular cavity is not expanded, and the stroma has no fibrous tissue hyperplasia.

As shown in figure 2, the model group shows that the number of prostate glands of the rat is increased and dense, new glands are proliferated, parts of the glands are expanded, epithelia are in a high column shape, some of the glands form a pseudo-multilayer structure, more papillary bulges are protruded into cavities, interstitial fluid is gathered, and a large amount of fibrous tissues are proliferated.

As shown in FIG. 3, the administration group 1 (hemp extract group) had enlarged partial glandular cavities, fewer glands than the model group, and a reduced number of hyperplasia of glands, and a small part of papillary protruded into the cavities, and a smaller number of prostate glandular cavities than the model group, and half of prostate glandular cavities were regular in morphology, and epithelial cells were mostly cubic columnar epithelium, with effusion in the stroma and partial glandular cavities, and a small amount of fibroplasia in the stroma.

As shown in fig. 4, in the group 4 (high-dose cannabidiol group), the number of hyperplasia of glands was reduced, papillary processes were significantly reduced, the prostate cavities were half morphologically regular, epithelial cells were mostly cubic columnar epithelium, and exudate was present in the interstitium and part of the glandular cavities, but the glands were not of the same size, and some were larger but were much better than the model group.

The results show that the cannabidiol or the cannabis extract has a good effect of inhibiting the hyperplasia of prostate.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.