KR102461951B1 - Optimization for supercritical extraction of hemp with response surface methodology - Google Patents

Abstract

The present invention relates to the optimization of Cannabis sativa supercritical extraction using response surface analysis. The present invention can provide optimal extraction conditions that can increase the content of cannabidiol (CBD), which is effective for treating rare and incurable diseases, and the present invention can contribute to medical development by providing extracts with high CBD.

Description

{Optimization for supercritical extraction of hemp with response surface methodology}

The present invention relates to the optimization of cannabis supercritical extraction using a response surface analysis method.

Cannabis is also known as marijuana. Hemp is an annual herb native to Central Asia. In Korea, it has been used as a raw material for hemp clothes since ancient times. Cannabis is a substance obtained from the leaves and flowers of this hemp and contains more than 400 chemical substances. Among them, about 60 cannabinoids such as cannabidiol (CBD) and cannabinol (CBN) are included.

Smoking cannabis can improve mood, relieve tension, increase appetite, and more. On the other hand, the mouth is dry and the eyes are red. Long-term exposure to marijuana causes side effects such as short-term memory and loss of kinesthetic sensation.

If a woman smokes marijuana, the ovaries either do not produce eggs or produce immature eggs. As a result, the menstrual cycle becomes irregular. If marijuana is used during pregnancy, chemicals such as cannabinoids enter the bloodstream and into the placenta, which can affect the fetus and cause premature birth.

The most psychoactive substance among cannabinoids in cannabis is delta-9 tetrahydrocannabinol, abbreviated as THC. Therefore, it can be said that the more cannabis containing more THC, the greater the harm done to the human body. Even a few hundred micrograms (μg) of THC can cause hallucinations.

In addition, THC is known to impair male reproductive function. THC acts on the pituitary gland to suppress the production of enough male hormones, resulting in a decrease in sperm count. THC can break and damage chromosomes. This damage to the reproductive cells can result in infertility or abnormal births.

On the other hand, cannabidiol (CBD) is an ingredient extracted from hemp, a type of hemp, and although the exact cause is unknown, it affects the brain by preventing the breakdown of chemicals in the brain that affect pain, mood, and mental function. expected to affect In addition, cannabidiol may block some of the psychoactive (hallucinating) effects of delta-9-tetrahydrocannabinol (THC).

Currently, there is no domestically approved product as a drug containing cannabidiol, but for rare or intractable treatment for which there is no alternative treatment in Korea, cannabis-based medicines that have been approved overseas can be imported and used for self-treatment purposes. have. In Korea, cannabidiol-containing drugs are orphan drugs, and cannot be obtained like general drugs. Individuals submit an application for handling approval, diagnosis, medical record, and medical opinion to the Korea Orphan Drug Center and receive approval for handling. It is available through the center.

As such, the use of cannabis with a double-edged sword is a reality that cannot be suppressed simply for the treatment of rare and intractable diseases, despite its addictiveness and health harm such as hallucinations. It is urgent to prepare a method for increasing the content of diol (CBD).

This patent was carried out on behalf of the order placed by Chuncheon City, and the research project name is 'R&D and Industrialization of Natural Materials (Hemp), 2020~2021', and the research period is 2020.03.01.~2021.12.31.

Korean Patent Application Laid-Open No. 10-2019-0084036 (2019.07.15) discloses a method for selective extraction of cannabinoids from plant sources. Korean Patent Application Laid-Open No. 10-2021-0086104 (2021.07.08) discloses a method for continuously producing cannabinol from a cannabis plant and its use.

An object of the present invention is to provide an extract having a high CBD content through optimization of cannabis supercritical extraction using a response surface analysis method in order to solve the above problems.

The present invention extracts the dried pulverized hemp by using a carbon dioxide supercritical extraction method, wherein the carbon dioxide supercritical extraction method uses carbon dioxide as an extraction solvent, a pressure of 25 to 50 Mpa, and a temperature of 35 to 70 ° C. for a minute It provides a method for producing a cannabis supercritical extract, characterized in that it is carried out.

In the method for producing a cannabis supercritical extract of the present invention, the dried pulverized hemp is preferably dried hemp at 25-50 ° C. for 24 to 72 hours to obtain a dried hemp product; and step B of pulverizing the dried hemp product obtained in step A to obtain a dried hemp product obtained in step B;

In the method for producing a hemp supercritical extract of the present invention, the hemp is, preferably, cherry Blossom Hemp .

On the other hand, the present invention provides a hemp supercritical extract obtained by supercritical extraction of carbon dioxide for 90 to 120 minutes at a pressure of 25 to 50 Mpa and a temperature of 35 to 70 ° C.

According to the present invention, it is possible to present optimal extraction conditions that can increase the content of cannabidiol (CBD) effective for treatment of rare and intractable diseases, and the present invention can contribute to pharmaceutical development by providing an extract with high CBD. have.

1 is a graph showing the change of the dependent variable with respect to the independent variable when extracting the extract of the present invention using the response surface analysis method.

The present invention extracts the dried pulverized hemp by using a carbon dioxide supercritical extraction method, wherein the carbon dioxide supercritical extraction method uses carbon dioxide as an extraction solvent, a pressure of 25 to 50 Mpa, and a temperature of 35 to 70 ° C. for a minute It provides a method for producing a cannabis supercritical extract, characterized in that it is carried out. At this time, the supercritical extraction is more preferably for 90 to 120 minutes at a pressure of 35 to 50 Mpa, a temperature of 52.5 to 70 ℃ It is good to be done

According to an embodiment of the present invention, when supercritical extraction of cannabis is performed under the above conditions during supercritical extraction, it is possible to obtain an extract having an effectively increased content of CBD.

Therefore, the cannabis supercritical extract prepared by the present invention can increase the content of cannabidiol (CBD) effective for rare and intractable treatment, thereby providing a substance effective for rare and intractable treatment.

On the other hand, the response surface analysis method is a regression analysis method that finds the optimal conditions for factors that give optimal response values from the change pattern of response variables according to the change of factor level by assuming a quadratic model. useful way. In the present invention, by utilizing the response surface analysis method, it was possible to find the optimal conditions for extracting the extract having high CBD.

On the other hand, supercritical extraction is an extraction method using a supercritical fluid, also referred to as supercritical fluid extraction. This is a technology that combines the principles of traditional distillation and extraction technology. The range of applications is wide, including separation of isomers and heat-denatured mixtures, which were difficult to separate with existing technologies, purification of high molecular substances, separation of active ingredients such as medicines and fragrances from natural plants, and development of energy-saving, pollution-free processes. It is one of the cutting-edge separation technologies that are actively commercialized.

In the method for preparing the supercritical extract of the present invention, the supercritical fluid may be, for example, carbon dioxide, water, ethane, ethylene, propane, or the like. Among them, carbon dioxide has a low critical point (31.1° C., 73.8 atm) and is non-toxic, so it is easy to extract and separate useful substances, so its use is preferable. In the present invention, supercritical carbon dioxide is used.

In the method for producing the cannabis supercritical extract of the present invention, the cannabis supercritical extract preferably contains 25 g or more of cannabidiol (CBD), and more preferably, the content of CBD is 25~ It may be 70 g, and more preferably, the content of CBD may be 60 to 70 g.

In the method for producing a cannabis supercritical extract of the present invention, the dried pulverized hemp is preferably dried hemp at 25-50 ° C. for 24 to 72 hours to obtain a dried hemp product; and step B of pulverizing the dried hemp product obtained in step A to obtain a dried hemp product obtained in step B;

At this time, if the hemp is dried under a temperature condition of 25 ° C. or less and a time of less than 24 hours, it is difficult to obtain a pulverized product in the following step because the drying of the hemp is not smooth, and the temperature condition exceeding 50 ° C. and 72 hours If it is dried for an excessive amount of time, excessive heat is given to the hemp, which can burn hemp, which is undesirable because it can cause denaturation of useful components contained in hemp.

In the present invention, the pulverization may be pulverized using other known methods, and the pulverization method is not limited. However, it would be preferable to grind to a known size known to be suitable for performing supercritical extraction.

In other words, the particle size plays a decisive role in the process affected by internal material movement resistance because the pulverization into small-sized particles in supercritical extraction shortens the diffusion of the solvent. Due to the severe resistance of the carbon dioxide passing through it, it will not show an even distribution, and a channeling phenomenon will occur that passes through a certain section. In this case, since the supercritical carbon dioxide does not come into contact with the material to be extracted, the efficiency and yield of the process may decrease, but in this process, grinding using an experimental grinder is sufficient.

In the method for producing a hemp supercritical extract of the present invention, the hemp is, preferably, cherry Blossom Hemp . Cherry Blossom Hemp is a highly cherry and berry-flavoured CBD strain that induces powerful relaxation, moodiness and relief. Cherry Blossom Hemp is very light green in color and covered with crystallized tree grooves, giving the appearance of white-green buds overlapping. The dense, sticky buds have quite a few orange hairs, but they are mostly hidden under the large calyx.

The exact pedigree of the Cherry Blossom hemp is not known, but it is known that it may be a variant of Cherry Wine or Cherry Pie , first crossed with Berry Blossom in the rugged hills of Colorado. Looking at the phytocannabinoid profile of Cherry Blossom hemp, it showed 18.50% total plant cannabinoids and 1.85% total secondary plant cannabinoids (CBC, THC-A), in the mid to high range of +15% CBD content. It can be used as a treatment for pain and inflammation.

On the other hand, the present invention provides a hemp supercritical extract obtained by supercritical extraction of carbon dioxide for 90 to 120 minutes at a pressure of 25 to 50 Mpa and a temperature of 35 to 70 ° C. At this time, the conditions of supercritical extraction are more preferably using carbon dioxide at a pressure of 35 to 50 Mpa and a temperature of 52.5 to 70° C. for 90 to 120 minutes. It is good to be done

According to this embodiment, when supercritical extraction of cannabis is performed under the above conditions during supercritical extraction, it is possible to obtain an extract with an effective increase in the content of CBD.

In the present invention, the cannabis supercritical extract preferably contains 25 g or more of cannabidiol (CBD), more preferably, the content of CBD may be 25 to 70 g, even more preferably The content of CBD may be 60-70 g.

Hemp is an annual plant of the genus Cannabis and is native to Central Asia. It has been cultivated for centuries as a textile raw material or as a treatment drug for asthma and pain in a wide area. The hemp stem is used as a raw material for fiber, the fruit is used as a spice or herbal medicine, and the seeds are used to obtain seasoning or oil. Cannabis sativa.L is a narcotic substance obtained from the leaves and flowers of cannabis, also called marijuana. Cannabis is a substance that is strongly regulated by law in Korea because resistance appears when continuously administered and withdrawal symptoms are caused when it is stopped, and it has recently been partially approved for medical use only.

Cannabis has long been abused as a substance that induces euphoria and hallucinations. A common use is to dry it and smoke it. In modern times, it is mainly used for hedonistic or medical purposes. Smoking is currently illegal in most countries around the world and its cultivation and distribution are strictly controlled, although some countries have relaxed such restrictions.

Cannabis smoking is partially permitted in some countries and states in the United States, and medical use is permitted in some countries, such as Canada. For medical purposes, the use of cannabinoids has been approved for the suppression of vomiting and pain in cancer patients, relief of stiffness in multiple sclerosis, and treatment of intractable convulsions. Korea is a country that strictly regulates the use of marijuana, and even if a Korean smokes in a country where marijuana is legal, those caught smoking will be punished according to Korean law. Recently, the prescription of CBD for the treatment of rare diseases has been partially approved in Korea.

Cannabidiol (CBD) is a cannabis-based drug used to treat rare and intractable diseases, such as epilepsy and multiple sclerosis, for which there is no alternative treatment in Korea. After obtaining approval for handling at the Korea Orphan Essential Drug Center, limited supply can be obtained through the center.

There are a total of two types of cannabidiol-containing products approved for handling in Korea, and there is a single agent and one combination agent each. Cannabidiol single drug treats seizures related to Lennox-Gastaut syndrome or Dravet syndrome in patients 2 years of age or older. Orphan drugs include Epidiolex ^® , an oral liquid. In addition, cannabidiol combination drugs include cannabidiol and tetrahydrocannabinol (THC, delta-9-tetrahydrocannabinol) combination drugs, which do not respond adequately to other anti-spasticity drugs and cause spasticity-related symptoms in the initial treatment stage of this drug. It is used to improve symptoms of moderate to severe spasticity in adults with clinically significant improvement in multiple sclerosis (MS) patients. Orphan prescription drugs include Satibex ^® in oral spray formulation.

Hereinafter, the configuration of the present invention will be described in detail through the following examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of technical ideas equivalent thereto.

[Example 1: Preparation of cannabis supercritical extract using response surface analysis]

The cannabis supercritical extract was prepared by optimizing the cannabis supercritical extraction conditions using the response surface analysis method. For this purpose, the hemp variety used the outpost (above) of cherry blossom hemp, dried in hot air at 35° C. for 48 hours, and then pulverized using a knife mile.

Then, 1 kg of the cherry blossom hemp pulverized product (dry sample) was extracted using the supercritical extraction method. In order to set the maximum condition for CBD extraction as the optimal condition, the independent variables for using the response surface analysis method were set to pressure 20-50 MPa, temperature 35-70 ° C, and time 60-120 minutes, and the dependent variable was [CBD +CBD-A] extraction amount.

Specific extraction conditions are shown in Table 1 below, and the cannabis supercritical extracts extracted by the extraction conditions listed in numerical order are listed in the same numerical order as the extraction conditions. That is, the cannabis supercritical extract extracted by 'Extraction Condition No. 1' becomes 'Extract 1'.

Detailed conditions for cannabis supercritical extraction for response surface analysis Exp.
No. SFE-CO ₂ Parameters Pressure(Mpa) Temperature(℃) Time(min) One 50 70 90 2 20 35 90 3 35 70 4 35 52.5 90 5 35 35 120 6 50 35 90 7 35 52.5 90 8 20 70 90 9 35 52.5 90 10 35 35 11 20 52.5 12 50 52.5 120 13 35 70 120 14 50 52.5 15 20 52.5 120

[Experimental Example 1: Evaluation of CBD and THC extraction yield of the extract extracted in Example 1]

In order to evaluate the extraction yield of CBD and THC of the extract extracted in Example 1, HPLC analysis was performed. To this end, each extract (extracts 1 to 15) was diluted to a concentration of 1 mg/ml using an ethanol solvent, and then used for component analysis. The component analysis results are shown in Tables 2 to 3 and FIG. 1 below.

Extraction yield for response surface analysis, CBD extraction optimization result Exp.
No. SFE-CO ₂ Parameters SFE-CO ₂ extract yield [CBD+CBD-A] yield [THC+THC-A] yield Pressure
(Mpa) Temperature
(℃) Time
(min) g/100g ¹⁾ mg/g extract ²⁾ g extract ³⁾ mg/g extract ⁴⁾ g extract ⁵⁾ One 50 70 90 16.8 298.54 50.15 15.44 2.59 2 20 35 90 2.2 631.95 13.90 26.16 0.58 3 35 70 10.3 413.07 42.55 20.70 2.13 4 35 52.5 90 402.80 59.61 19.59 2.90 5 35 35 120 3.6 239.78 8.63 12.42 0.45 6 50 35 90 10.8 511.28 55.22 23.39 2.53 7 35 52.5 90 14.5 414.64 60.12 21.22 3.08 8 20 70 90 8.2 689.47 56.54 34.26 2.81 9 35 52.5 90 14.9 402.63 59.99 19.91 2.97 10 35 35 3.1 418.81 12.98 18.69 0.58 11 20 52.5 7 521.23 36.49 23.16 1.62 12 50 52.5 120 11 557.91 61.37 25.30 2.78 13 35 70 120 16.8 413.49 69.47 21.45 3.60 14 50 52.5 9.2 385.45 35.46 19.04 1.75 15 20 52.5 120 9.3 304.56 28.32 16.29 1.51 Optimal conditions 47.27 60.45 105.45 - - 68.25 - -

1) Yield of solid content extraction per 100 g of raw hemp

2) Extraction yield of CBD and CBD-A (extraction yield per 1 g of solid content)

3) Extraction amount of CBD and CBD-A (Amount extracted per 1 kg of hemp)

4) Extraction yield of THC+THC-A (extraction yield per 1 g of solid content)

5) Extracted amount of THC+THC-A (extracted amount per 1kg of hemp)

As a result of the experiment, the optimal supercritical extraction conditions were 47.27 MPa, 60.45 ° C, 105.45 minutes, [CBD+CBD-A] 68.25 g under the conditions, showing the highest CBD extraction yield and the most optimized result.

In addition, according to FIG. 1 , all of the regression equations of the independent variable for the dependent variable showed a value close to 1 with an R ² value of 0.93 or higher, thereby securing the reliability of the response model. 1 is a graph showing the change of the dependent variable with respect to the independent variable when extracting the extract of the present invention using the response surface analysis method.

Predicted values and verification results for optimal extraction conditions Response variables
(g extract) Predicted values experimental values [CBD+CBD-A] 68.25 68.48±0.60

As a result of the verification, the experimental values showed similarity compared with the predicted values for the optimization condition, confirming that the optimum condition of the present invention is substantially accurate.

Claims (4)

The dried pulverized product of cherry blossom hemp is extracted using the carbon dioxide supercritical extraction method,
The cherry blossom hemp ( cherry Blossom Hemp ) dry pulverized product, cherry blossom hemp ( cherry Blossom Hemp ) by drying at 35 ° C. for 48 hours to obtain a cherry blossom hemp ( cherry Blossom Hemp ) step A to obtain a dried product; and pulverizing the dried cherry blossom hemp obtained in step A to obtain a dried cherry blossom hemp ( cherry blossom hemp ) step B;
The carbon dioxide supercritical extraction method,
Using carbon dioxide as an extraction solvent, a pressure of 47.27 Mpa, a temperature of 60.45 ℃ for 105.45 minutes By performing, cherry blossom hemp ( cherry Blossom Hemp ) Cannabidiol (cannabidiol) from cherry blossom hemp ( cherry Blossom Hemp ), characterized in that the content of cannabidiol (cannabidiol) is extracted per 1 kg of dry pulverized product is 60 to 70 g How to extract in high yield. delete delete delete

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