阅读说明：本技术 一种次***二酚的提取分离方法 (Extraction and separation method of hypocannabidiol ) 是由 王钲霖 刘胜贵 蒋永昌 付彬彬 李智高 孔令羽 于 2019-11-26 设计创作，主要内容包括：本发明公开了一种次大麻二酚的提取分离方法,该方法采用乙醇水溶液提取后,用有机溶剂萃取富集次大麻二酚,活性炭脱色后经聚酰胺树脂柱、中性氧化铝和键合硅胶柱纯化富集,结晶获得高纯度的次大麻二酚,本发明方法易操作,实用性强,适于工业化推广应用。(The invention discloses a method for extracting and separating hypocannabidiol, which comprises the steps of extracting and enriching the hypocannabidiol by using an organic solvent after extracting by using an ethanol aqueous solution, purifying and enriching the hypocannabidiol by using a polyamide resin column, neutral alumina and a bonded silica gel column after decoloring by using activated carbon, and crystallizing to obtain the high-purity hypocannabidiol.)

1. The extraction and separation method of the hypocannabidiol is characterized by comprising the following steps:

(1) pulverizing hemp flower and leaf as raw materials into 30-50 mesh particles, extracting with 60-80% ethanol solution under reflux for 2-3 times (1-3 hr for each time), mixing extractive solutions, concentrating under reduced pressure, and recovering ethanol to obtain ethanol extract concentrate;

(2) after the ethanol extraction concentrated solution in the step (1) is cooled to normal temperature, adding a certain amount of normal hexane for extraction, extracting the cannabidiol into an organic solvent phase layer, collecting and combining the organic solvent phase layer, and recovering the organic solvent to obtain the cannabidiol extraction concentrated solution;

(3) adding a certain amount of activated carbon into the secondary cannabidiol extraction concentrated solution in the step (2), heating and stirring, standing for 2 minutes, filtering by using a glass sand funnel, concentrating and draining the filtrate to obtain a secondary cannabidiol crude extract;

(4) adding ethanol with the mass concentration of 20-30% into the crude cannabidiol paste obtained in the step (3), heating and stirring to form a suspension, standing and cooling, taking a supernatant, passing through a polyamide resin column, eluting impurities with purified water after sample loading until an effluent liquid is colorless, then washing the polyamide resin column with an ethanol solution with the mass concentration of 30-42% until the effluent liquid is colorless, eluting with an ethanol solution with the mass concentration of 55-60% to collect cannabidiol, collecting cannabidiol in bottles, merging eluents containing cannabidiol target substances after thin-layer identification, and concentrating under reduced pressure to recover ethanol to obtain a crude cannabidiol product for polyamide chromatography;

(5) mixing the polyamide chromatography hypocannabidiol concentrate obtained in the step (4) with a neutral alumina dry method, loading the mixture on a neutral alumina column, eluting and removing impurities by using chloroform-n-hexane solution after sample loading by a dry method, then eluting hypocannabidiol by using chloroform-ethyl acetate-ethanolamine solution, collecting target object eluent containing the hypocannabidiol, and concentrating to obtain a hypocannabidiol semi-finished product with the content of more than 60%;

(6) dissolving the crude product of the cannabidiol obtained in the step (5) by using methanol, passing through an amino bonded silica gel column, a cyano bonded silica gel column or a phenyl bonded silica gel column, performing pressure column chromatography, balancing the bonded silica gel column by using a methanol solution with the mass concentration of 20%, washing 2-3 column volumes by using a methanol solution with the mass concentration of 30% -42%, then performing cannabidiol elution by using a methanol solution with the mass concentration of 52% -65%, performing thin-layer chromatography identification until no cannabidiol spots exist, performing thin-layer chromatography identification on a methanol eluent with the concentration of 52% -65%, merging the eluents containing the cannabidiol, and concentrating to obtain a pure product of the cannabidiol;

(7) and (4) dissolving the concentrate obtained in the step (6) by using cyclohexane, adding glacial acetic acid, uniformly mixing, standing, crystallizing, filtering, and drying the crystals at low temperature to obtain the finished product of the cannabidiol.

2. The method for extracting and separating cannabidiol as claimed in claim 1, wherein: in the step (1), ethanol solution which is 7-9 times of the weight of the added raw materials in the first extraction is extracted under reflux, ethanol solution which is 4-5 times of the weight of the added raw materials in the other extractions is added, the concentration temperature is 75-80 ℃, the vacuum degree is 0.06-0.078MPa, and the concentration is carried out until the volume is 1/10.

3. The method for extracting and separating cannabidiol as claimed in claim 1, wherein: and (3) adding n-hexane for extraction, wherein the adding amount of the n-hexane is 2-3 times of that of the ethanol extraction concentrated solution, the extraction times are 2-3 times, and the combined extract is concentrated to 1/5 of the volume of the ethanol extraction concentrated solution.

4. The method for extracting and separating cannabidiol as claimed in claim 1, wherein: adding active carbon in an amount of 2-8% of the cannabidiol extraction concentrate in the step (3), heating to 40-50 ℃, and stirring for 15-20 min.

5. The method for extracting and separating cannabidiol as claimed in claim 1, wherein: eluting and removing impurities by using a solution of normal hexane-chloroform, namely normal hexane and chloroform according to the volume ratio of 9: 1; then eluting the cannabidiol with chloroform-ethyl acetate-ethanolamine solution, wherein the chloroform, the ethyl acetate and the ethanolamine are mixed according to the volume ratio of 9:1: 2.

6. The method for extracting and separating cannabidiol as claimed in claim 1, wherein: the amount of glacial acetic acid added in step (7) was 0.8%.

Technical Field

The invention relates to an extraction and separation method of hypocannabinol, belonging to the field of extraction of phytochemical components.

Background

The hemp, also known as China hemp and industrial hemp, is one of the traditional Chinese medicinal materials and health food raw materials. The national ministry of health 2002 documents list fructus cannabis in a list of articles which are both medicines and foods, so that the development and utilization of fructus cannabis foods are regulated and guaranteed. This also provides conditions and support for the development of the hemp plant leaves. In recent years, as people have been increasingly aware of cannabis and have been deeply studied about the strong physiological functions of processed products thereof, it is common in various countries to release the restriction on the development of cannabis and products thereof. With the continuous enlargement of the planting area of the cannabis sativa and the annual increase of the yield of the cannabis sativa. As a byproduct of the cannabis plants, the cannabis leaves can be used as a fertilizer by burning straws, and also contain rich cannabinol substances with strong physiological activity, especially Cannabidiol (CBD). How to utilize the high added value of the hemp leaves is a technical and environmental problem which needs to be solved urgently at present. The utilization and extraction reports of cannabidiol are quite many at present, cannabidiol has fully embodied unique value in the pharmaceutical industry and the human big health industry, and the cannabidiol serving as sub-cannabidiol which only differs from cannabidiol by one methylene group has the same mother ring as two parts of cannabis structurally and has the same extractable value in cannabis flower and leaf.

The invention adopts enzymolysis, ethanol water solution extraction and column chromatography separation and purification, searches and optimizes the extraction process of the sub-cannabinol in the hemp leaves to find the optimal extraction condition and industrial feasibility of the sub-cannabinol in the hemp leaves, aims to research and develop a process suitable for industrial large-scale production, and aims to provide guidance for fully utilizing the bioactive components in the hemp leaves and realizing the comprehensive utilization of hemp resources and maximizing the benefit.

Disclosure of Invention

In order to fully utilize the bioactive components in the hemp leaves and search the availability of the hypocannabidiol in the hemp leaves, the invention provides the extraction and separation method of the hypocannabidiol with high yield and high product purity.

The method comprises the following specific steps:

(1) pulverizing folium Cannabis as raw material, making into 30-50 mesh granules, extracting with 60-80% ethanol solution under reflux for 2-3 times, each time for 1-3 hr, mixing extractive solutions, concentrating under reduced pressure, and recovering ethanol to obtain ethanol extract concentrate.

(2) And (2) after the ethanol extraction concentrated solution in the step (1) is cooled to normal temperature, adding a certain amount of n-hexane for extraction, extracting the cannabidiol into the organic solvent phase layer, collecting and combining the organic solvent phase layer, and recovering the organic solvent to obtain the cannabidiol extraction concentrated solution.

(3) And (3) adding a certain amount of activated carbon into the secondary cannabidiol extraction concentrated solution in the step (2), heating and stirring, standing for 2 minutes, filtering by using a glass sand funnel, concentrating and draining the filtrate to obtain the secondary cannabidiol crude extract.

(4) Adding 20-30% ethanol into the crude cannabidiol ointment obtained in the step (3), heating and stirring to form a suspension, standing and cooling, taking a supernatant, passing through a polyamide resin column, eluting impurities with purified water after sample loading until an effluent liquid is colorless, then washing the polyamide resin column with 30-42% ethanol solution until the effluent liquid is colorless, eluting with 55-60% ethanol solution, collecting cannabidiol, separating and collecting cannabidiol in bottles, combining eluates containing cannabidiol target substances after thin-layer identification, and concentrating under reduced pressure to recover ethanol to obtain a crude cannabidiol product for polyamide chromatography.

(5) And (3) mixing the polyamide chromatography hypocannabidiol concentrate obtained in the step (4) with a neutral alumina dry method, loading the mixture on a neutral alumina column, eluting the mixture by using chloroform-n-hexane solution to remove impurities after the sample is loaded by a dry method, eluting hypocannabidiol by using chloroform-ethyl acetate-ethanolamine solution, collecting the target object eluent containing the hypocannabidiol, and concentrating to obtain a hypocannabidiol semi-finished product with the content of more than 60%.

(6) Dissolving the crude product of the cannabidiol obtained in the step (5) with methanol, passing through an amino bonded silica gel column, a cyano bonded silica gel column or a phenyl bonded silica gel column, performing pressure column chromatography, balancing the bonded silica gel column with a methanol solution with the mass concentration of 20%, washing 2-3 column volumes with a methanol solution with the mass concentration of 30% -42%, performing cannabidiol elution with a methanol solution with the mass concentration of 52% -65%, performing thin-layer chromatography identification until no cannabidiol spots exist, performing thin-layer chromatography identification on a methanol eluent with the concentration of 52% -65%, merging the eluents containing the cannabidiol, and concentrating to obtain a pure cannabidiol product.

(7) And (4) dissolving the concentrate obtained in the step (6) by using cyclohexane, adding glacial acetic acid, uniformly mixing, standing, crystallizing, filtering, and drying the crystals at low temperature to obtain the finished product of the cannabidiol.

And (2) refluxing and extracting ethanol solution which is 7-9 times of the weight of the added raw materials in the first extraction in the step (1), extracting ethanol solution which is 4-5 times of the weight of the added raw materials in the rest extractions, concentrating at 75-80 ℃ under the vacuum degree of 0.06-0.078MPa to 1/10 volumes.

And (3) adding n-hexane for extraction in the step (2), wherein the adding amount of the n-hexane is 2-3 times of that of the ethanol extraction concentrated solution, the extraction times are 2-3 times, and the combined extract is concentrated to 1/5 of the volume of the ethanol extraction concentrated solution.

And (3) adding active carbon in an amount of 2-8% of the cannabidiol extraction concentrate in the step (3), heating to 40-50 ℃, and stirring for 15-20 min.

Eluting and removing impurities by using a solution of n-hexane-chloroform, namely n-hexane and chloroform according to a volume ratio of 9:1 in the step (5); then eluting the cannabidiol with chloroform-ethyl acetate-ethanolamine solution, wherein the chloroform, the ethyl acetate and the ethanolamine are mixed according to the volume ratio of 9:1: 2.

The amount of glacial acetic acid added in the step (7) is 0.8%.

The method has the advantages and the technical effects that:

according to the method, after alcohol extraction and concentration, n-hexane is used for extraction, the characteristic of low polarity of a target object is utilized, the target object is extracted into the n-hexane as much as possible according to the characteristic of similar intermiscibility, and meanwhile, most impurities are left in water, so that the purity of a crude extract is greatly improved; activated carbon is added into the obtained extraction concentrated solution, so that the pigment and other impurities in the extraction concentrated solution are obviously reduced, and the subsequent separation and purification are more convenient. The polyamide resin column chromatography purification basically has no dead adsorption, so that the sample is hardly lost, while the existing silica gel process has too much dead adsorption, too much sample loss and too high elution cost by using an organic solvent; the method comprises the following steps of utilizing neutral alumina to perform dead adsorption on phenolic substances, performing dead adsorption on a target object on a column, firstly enabling a solvent to pass through the column to remove impurities, and then eluting cannabidiol dead-adsorbed by the alumina by using an alkaline solvent, so that cannabidiol is effectively enriched, and a better impurity removal effect is achieved; the target product is separated by adopting fillers such as amino, cyano and the like in a pressurized and rapid flash manner, the column chromatography separation speed is higher, the sample carrying amount is larger, the number of separation degree column plates and the purity of the obtained sample are both higher than those of a C-18 column, and when the purification comparison with a gel column shows that the gel column chromatography can also obtain a high-purity compound, but the gel column chromatography speed is lower; the method has the advantages of simple operation, small loss of target substances in the whole process, complete impurity removal, high purity of the obtained product of more than 98 percent, high yield and suitability for industrial popularization and application.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.