Composition for protecting nerve cells comprising supercritical extract of cannabis sativa

Abstract

The present invention relates to a composition for protecting nerve cells, comprising a supercritical extract of Cannabis sativa and, more specifically, to a composition for protecting nerve cells, comprising a supercritical extract of Cannabis sativa, thereby exhibiting an antioxidant effect and a nerve cell death inhibitory effect.

Description

Neuroprotective composition containing cannabis supercritical extract

[0001] The present invention relates to a neuroprotective composition comprising a cannabis supercritical extract.

[0002]

[0003] Oxidative stress is known as one of the major causes of neurological disorders and can lead to various neurological problems by causing damage to cells and tissues. Oxidative stress occurs when unstable molecules called reactive oxygen species (ROS) are excessively produced within the body. While ROS are generated during normal cellular metabolic processes, their numbers can increase due to environmental factors, aging, inflammation, and toxic substances. Excessive ROS damage cellular components such as cell membranes, proteins, and DNA, and nerve cells are particularly vulnerable to oxidative stress due to their high metabolic activity and energy consumption. Although the brain accounts for approximately 20% of total oxygen consumption, its antioxidant defense system is relatively weak; consequently, there is a high likelihood of cell damage occurring due to an excess of ROS, leading to the development of various neurological disorders.

[0004] Representative neurodegenerative diseases involving such oxidative stress include Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). As average human life expectancy increases and the elderly population grows rapidly, degenerative brain diseases such as dementia and Alzheimer's disease, caused by the degeneration of nerve cells due to aging, are emerging as a social issue. In particular, the incidence of dementia, such as Alzheimer's disease, is rising sharply among the population aged 65 and older. Neurological diseases such as Alzheimer's, Parkinson's, stroke, and ALS significantly reduce an individual's quality of life, and prevention and management are becoming increasingly important due to the rapid and complex progression of these diseases. According to the World Health Organization (WHO), the number of dementia patients worldwide is expected to more than triple by 2050, rising from the current approximately 50 million to 150 million. Consequently, although research into the development of new technologies and treatments is actively underway, there is currently a lack of suitable treatments.

[0005]

[0006] Against this background, while researching substances exhibiting neuroprotective effects, the inventors completed the present invention by developing a neuroprotective composition capable of inhibiting apoptosis caused by oxidative stress by including a cannabis supercritical extract.

[0007] Accordingly, the object of the present invention is to provide a neuroprotective composition comprising a cannabis supercritical extract.

[0008] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative brain diseases comprising a cannabis supercritical extract.

[0009] Another objective of the present invention is to provide a functional food composition for improving cognitive function or memory comprising a cannabis supercritical extract.

[0010]

[0011] To solve the above-mentioned problem, the present invention provides a neuroprotective composition comprising a cannabis supercritical extract.

[0012] In the above composition, the hemp may be any one selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.

[0013] In the above composition, the cannabis supercritical extract may be extracted from one or more selected from the group consisting of cannabis flower buds, whole plant, roots, stems, leaves, bark, fruits, and seeds.

[0014] In the above composition, the cannabis supercritical extract may be included at a concentration of 0.5 to 8 μg / ml.

[0015] In the above composition, the cannabis supercritical extract may be extracted using supercritical carbon dioxide as a solvent and supercritical methanol or supercritical ethanol as a co-solvent.

[0016] In the above composition, supercritical carbon dioxide and the auxiliary solvent may be used in a ratio of 6:1 (v / v) to 10:1 (v / v).

[0017] In the above composition, the cannabis supercritical extract may be prepared by a method comprising the steps of: (a) obtaining a cannabis sample by drying cannabis flower buds at 30 to 70 ℃ for 24 to 120 hours; (b) decarbonizing the cannabis sample at 100 to 200 ℃ for 15 to 60 minutes; and (c) supercritical extracting the decarbonized cannabis sample at a pressure of 200 to 400 bar and a temperature of 30 to 80 ℃.

[0018] In addition, the present invention provides a composition for the prevention or treatment of degenerative brain diseases comprising any one of the above compositions.

[0019] In addition, the present invention provides a functional food composition for improving cognitive function or memory, comprising any one of the above compositions.

[0020]

[0021] The composition of the present invention exhibits an antioxidant effect and, by inhibiting the death of nerve cells, can exhibit a neuroprotective effect.

[0022] Based on these antioxidant effects, neuronal cell death inhibitory effects, and neuroprotective effects, the pharmaceutical composition of the present invention can prevent or treat degenerative brain diseases.

[0023] Based on these antioxidant effects, neuronal cell death inhibition effects, and neuroprotective effects, the food composition of the present invention can improve cognitive function and memory.

[0024]

[0025] Figure 1 shows the DPPH assay results of cannabis supercritical extract, cannabis Soxhlet extract, and cannabis room temperature extract.

[0026] Figure 2 confirms the neuroprotective effect of cannabis supercritical extract (CSCE) at various concentrations and shows the results of confirming whether neuronal cell death induced by glutamate is inhibited.

[0027] Figure 3 confirms the neuroprotective effect of cannabis room temperature extract (CRE) at various concentrations and shows the results of confirming whether neuronal cell death induced by glutamate is inhibited.

[0028] Figure 4 is a graph of the results of the Morris water maze experiment, showing the results of confirming whether memory was improved in the control group not treated with scopolamine, the scopolamine-treated group, the cannabis supercritical extract (CSCE) 10 mg / kg-treated group, the cannabis supercritical extract (CSCE) 20 mg / kg-treated group, and the red ginseng extract 100 mg / kg-treated group as a positive control.

[0029] Figure 5 is a process flow diagram of cannabis supercritical extraction.

[0030]

[0031] The present invention relates to a neuroprotective composition comprising a cannabis supercritical extract, a pharmaceutical composition for the prevention or treatment of degenerative brain diseases comprising the same, and a functional food composition for improving cognitive function or memory comprising the same.

[0032] The present invention provides a neuroprotective composition comprising a cannabis supercritical extract.

[0033] Cannabis is a type of plant belonging to the genus Cannabis, and there are various varieties, the major varieties of which include Cannabis sativa, Cannabis indica, and Cannabis ruderalis. Cannabis contains pharmacological components such as cannabidiol (CBD) and tetrahydrocannabinol (THC).

[0034] Supercritical extraction refers to a method of extracting active ingredients using supercritical fluids. A supercritical fluid is defined as a substance existing under conditions above the critical point pressure and temperature, where the distinction between liquid and gas is difficult. Supercritical fluids possess unique characteristics distinct from ordinary liquids or gases; while their density is similar to that of a liquid, their viscosity is similar to that of a gas. Due to their liquid-like density, they have high solubility, and due to their gas-like low viscosity, their diffusion and penetration rates are rapid. Supercritical extraction is an extraction method that utilizes these properties of supercritical fluids.

[0035] In a neuroprotective composition according to one embodiment of the present invention, the cannabis may be any one selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis. For example, it may be Cannabis sativa.

[0036] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted from one or more selected from the group consisting of cannabis flower buds, whole plant, roots, stems, leaves, bark, fruits, and seeds. For example, it may be extracted from flower buds.

[0037] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be included at a concentration of 0.5 to 8 μg / ml, 0.5 to 7 μg / ml, 0.5 to 6 μg / ml, 0.5 to 5 μg / ml, or 0.625 to 5 μg / ml.

[0038] A neuroprotective composition according to one embodiment of the present invention may have a neuroprotective effect by inhibiting the death of neurons. Additionally, the death of neurons may be induced by oxidative stress.

[0039] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted using one or more supercritical fluids selected from the group consisting of supercritical carbon dioxide, supercritical methanol, or supercritical ethanol as a solvent. For example, it may be extracted using supercritical carbon dioxide as a solvent.

[0040] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted using supercritical carbon dioxide as a solvent and supercritical methanol or supercritical ethanol as an auxiliary solvent.

[0041] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted using supercritical carbon dioxide and an auxiliary solvent in a ratio of 6:1 (v / v) to 10:1 (v / v). For example, it may be extracted using supercritical carbon dioxide and an auxiliary solvent in a ratio of 8:1 (v / v).

[0042] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted at a pressure of 200 to 400 bar, 250 to 350 bar, or 280 to 320 bar. For example, it may be extracted at a pressure of 300 bar.

[0043] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted at a temperature of 30 to 80 ℃, 40 to 70 ℃, or 40 to 60 ℃. For example, it may be extracted at a temperature of 50 ℃.

[0044] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be extracted using supercritical carbon dioxide as a solvent at a pressure of 300 bar and a temperature of 50 ℃.

[0045] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be prepared by a method comprising the following steps:

[0046] (a) A step of obtaining a hemp sample by drying hemp flower buds at 30 to 70 ℃ for 24 to 120 hours,

[0047] (b) a step of decarbonizing the hemp sample at 100 to 200 ℃ for 15 to 60 minutes, and

[0048] (c) A step of supercritical extraction of the above decarboxylated hemp sample at a pressure of 200 to 400 bar and a temperature of 30 to 80 ℃.

[0049] In a neuroprotective composition according to one embodiment of the present invention, the cannabis supercritical extract may be prepared by a method comprising the following steps:

[0050] (a) A step of obtaining a hemp sample by drying hemp flower buds at 55°C for 72 hours,

[0051] (b) a step of decarbonizing the hemp sample at 140°C for 15 to 60 minutes, and

[0052] (c) A step of supercritical extraction of the above decarboxylated hemp sample at a pressure of 300 bar and a temperature of 50 ℃ for 10 to 1 hour.

[0053] In step (c) above, supercritical extraction may be performed using supercritical carbon dioxide as a solvent and supercritical methanol or supercritical ethanol as a co-solvent. Preferably, extraction may be performed using supercritical carbon dioxide and a co-solvent in a ratio of 6:1 (v / v) to 10:1 (v / v). For example, extraction may be performed using supercritical carbon dioxide and a co-solvent in a ratio of 8:1 (v / v).

[0054] FIG. 5 is a process flow diagram of a cannabis supercritical extraction for producing a cannabis supercritical extract of the present invention, and is composed of a CO₂ cylinder (1), an auxiliary solvent cylinder (1'), a meter (2), a pre-cooler (3), a CO₂ pump (4), an auxiliary solvent pump (4'), a preheater (5), an extraction vessel (6), an internal basket loading section (7), a pressure gauge (8), a back pressure regulator (9), a product collection valve (10), a cooling medium circulator (11), a heating medium circulator (12), and a data logger (13).

[0055] The present invention provides a pharmaceutical composition for the prevention or treatment of degenerative brain diseases comprising a cannabis supercritical extract.

[0056] The present invention provides a pharmaceutical composition for the prevention or treatment of degenerative brain diseases comprising the neuroprotective composition of the present invention.

[0057] In a pharmaceutical composition according to one embodiment of the present invention, the same applies to the cannabis supercritical extract of the present invention, unless it contradicts the previously mentioned matters.

[0058] In a pharmaceutical composition according to one embodiment of the present invention, the cannabis supercritical extract may be included at a concentration of 0.5 to 8 μg / ml, 0.5 to 7 μg / ml, 0.5 to 6 μg / ml, 0.5 to 5 μg / ml, or 0.625 to 5 μg / ml.

[0059] In a pharmaceutical composition according to one embodiment of the present invention, the degenerative brain disease may be any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, stroke, cerebral hemorrhage, multiple sclerosis, amyotrophic lateral sclerosis, Pick's disease, and Creutzfeldt-Jakob disease.

[0060] A pharmaceutical composition according to one embodiment of the present invention may further include a suitable carrier, excipient, and diluent, etc., that are commonly used in the manufacture of pharmaceutical compositions. The carrier may be used without limitation as long as it is known in the art, such as a buffer, preservative, analgesic, solubilizing agent, isotonic agent, stabilizer, base, lubricant, etc.

[0061] A pharmaceutical composition according to one embodiment of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods. Furthermore, it may be used in the form of external skin preparations such as ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or gels. When formulating, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, humectants, disintegrants, and surfactants.

[0062] The dosage of a pharmaceutical composition according to one embodiment of the present invention can be appropriately adjusted according to the form and purpose of use, the patient's condition, the type and severity of symptoms, etc.

[0063] The present invention provides a method for preventing or treating degenerative brain disease, comprising the step of administering a cannabis supercritical extract according to one embodiment of the present invention to a subject requiring treatment.

[0064] A preventive or therapeutic method according to one embodiment of the present invention may be to administer a cannabis supercritical extract in a therapeutically effective amount.

[0065] The present invention provides a method for preventing or treating degenerative brain disease, comprising the step of administering a pharmaceutical composition according to one embodiment of the present invention to a subject in need.

[0066] A method for prevention or treatment according to one embodiment of the present invention may be to administer a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0067] In this specification, "subject requiring treatment" may mean mammals, including humans, and "administration" means providing a specific substance to the subject by any appropriate method. "Therapeutic effective dose" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects, and this may be determined by a person skilled in the art based on factors including the patient's sex, age, weight, health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration, release rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. It is desirable to apply a specific therapeutic effective dose for a specific patient differently based on various factors and similar factors well known in the medical field, including the specific composition, including the type and degree of response to be achieved and whether other preparations are used in some cases, the patient's age, weight, general health status, sex and diet, time of administration, route of administration and release rate of the composition, duration of treatment, and drugs used with or concurrently with the specific composition.

[0068] The present invention provides a use of a cannabis supercritical extract according to one embodiment of the present invention for protecting nerve cells.

[0069] The present invention provides a use of a cannabis supercritical extract according to one embodiment of the present invention for the prevention or treatment of degenerative brain diseases.

[0070] The present invention provides a use of a cannabis supercritical extract according to one embodiment of the present invention in the manufacture of a drug for the prevention or treatment of degenerative brain diseases.

[0071] The present invention provides a use of a pharmaceutical composition according to one embodiment of the present invention for the prevention or treatment of degenerative brain diseases.

[0072] The present invention provides a use of a pharmaceutical composition according to one embodiment of the present invention in the manufacture of a drug for the prevention or treatment of degenerative brain diseases.

[0073] The present invention provides a functional food composition for improving cognitive function or memory, comprising a supercritical cannabis extract.

[0074] The present invention provides a functional food composition for improving cognitive function or memory, comprising the neuroprotective composition of the present invention.

[0075] In a food composition according to one embodiment of the present invention, the same applies to the cannabis supercritical extract of the present invention, unless it contradicts the previously mentioned matters.

[0076] In a food composition according to one embodiment of the present invention, the cannabis supercritical extract may be included at a concentration of 0.5 to 8 μg / ml, 0.5 to 7 μg / ml, 0.5 to 6 μg / ml, 0.5 to 5 μg / ml, or 0.625 to 5 μg / ml.

[0077] A food composition according to one embodiment of the present invention may include conventional food additives, and unless otherwise specified, suitability as a food additive is determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety. In addition, a food composition according to one embodiment of the present invention may include a suitable carrier commonly used in the manufacture of food compositions.

[0078] A food composition formulation according to one embodiment of the present invention is prepared according to a conventional method and can be encapsulated after drying with a carrier, or formulated into the form of powder, granules, pills, tablets, capsules, candy, syrup, and beverage.

[0079] A food composition according to one embodiment of the present invention can be prepared as any form of food. For example, the food may be any one selected from the group consisting of drinks, meat, sausage, bread, candies, snacks, noodles, ice cream, dairy products, soup, isotonic drinks, beverages, alcoholic drinks, chewing gum, tea, and vitamin complexes.

[0080] The present invention provides a use of a food composition according to one embodiment of the present invention for protecting nerve cells.

[0081] In the present invention, the matters mentioned regarding the cannabis supercritical extract, pharmaceutical composition, food composition, preventive or therapeutic method, and use are applied equally unless they contradict each other.

[0082] Hereinafter, to specifically explain the present invention, manufacturing examples and experimental examples will be described in detail.

[0083]

[0084] Preparation Example

[0085] Preparation Example 1. Pretreatment of hemp raw material and preparation of dried hemp sample

[0086] The cannabis (Cannabis Sativa L., Purple 52 variety) used in this study was cultivated at N.Thing, a legally licensed farm in Korea. The cannabis was cultivated and harvested under specific conditions, and the harvested flower buds were dried to remove moisture. The flower buds were dried using a dryer at 55°C for 72 hours. This process ensured the production of a uniform sample, making it suitable for storage and use. The dried sample was finely ground using a small grinder (RT-08, Hanil Electric, Seoul, South Korea) and stored in a sealed glass container under refrigerated conditions at 4°C.

[0087]

[0088] Preparation Example 2. Decarbonation Process

[0089] Decarboxylation of cannabis samples was performed as a pretreatment process to remove carboxyl groups in order to activate cannabinoids. This process was carried out using an oven maintained at 140°C. Depending on the desired level of activation, the samples were treated for a duration between 15 and 60 minutes. The samples were heat-treated at 140°C for the set duration, and after decarboxylation was completed, they were collected for a subsequent extraction process.

[0090]

[0091] Preparation Example 3. Particle Size Analysis

[0092] Particle size distribution was evaluated using a sieve separator (CISA BA 200 N, CISA Cedaceria Industrial, Barcelona, ​​Spain) and seven types of standard sieves (45 μm, 100 μm, 150 μm, 180 μm, 250 μm, 300 μm, 500 μm; crystal sieve, Gunpo, South Korea). 8 g of decarboxylated sample was placed in the sieve separator and operated at a vibration amplitude of 2 mm. This process was repeated at 10-minute intervals until the particle size distribution became uniform. The weight of each fraction was measured using a precision balance (OHAUS Adventurer - Model AR2140), and the particle size distribution was evaluated based on these measurements.

[0093]

[0094] Preparation Example 4. Preparation of hemp supercritical extract through supercritical carbon dioxide extraction

[0095] Supercritical CO₂ extraction was used to extract cannabinoids from cannabis. In addition, to establish optimal extraction conditions, extraction efficiency was evaluated by adjusting various conditions, such as extraction time and the use of auxiliary solvents. The extraction conditions were set to a pressure of 300 bar, a temperature of 50 ℃, and an extraction time of 10 minutes to 1 hour. The solvent used was either pure supercritical CO₂ or a mixture of supercritical CO₂ and ethanol or methanol as auxiliary solvents. For extraction, 8 g of cannabis sample was loaded into a cylindrical inner basket measuring 187 mm in length and 13 mm in diameter. One end of the basket was constructed with a connecting joint, while the other end was filled with 0.2 g of glass fiber to prevent sample loss due to gravity and to facilitate the flow of supercritical CO₂. The cylindrical basket was mounted concentrically inside a cylindrical extraction vessel measuring 465 mm in length and 50 mm in diameter. One end of the extraction vessel was connected to a metering pump, and the other end was connected to an outlet and a back pressure regulator (BPR). The temperature of the extraction vessel was maintained at 50°C using a circulating water bath. The CO₂ flow rate was set to 8 mL / min using a high-pressure metering pump. CO₂ with a purity of 99.5% was used, and the pressure was raised to 300 bar using a high-pressure pump and then regulated via the BPR. The extract obtained during extraction passed through the BPR and flowed into a separator, where the pressure inside the separator was maintained at 30 bar via another BPR. After the extraction was completed, the extract remaining in the piping was collected by washing with an ethanol or methanol auxiliary solvent using a pump. When an auxiliary solvent was used, it was mixed with supercritical CO₂ at an 8:1 (v / v) ratio and injected in liquid form via a high-pressure pump.

[0096]

[0097] Preparation Example 5. Preparation of cannabis Soxhlet extract as a control

[0098] Cannabis Soxhlet Extract (CSE) was prepared by the following process. First, a condenser was attached to the top of the Soxhlet extraction apparatus, and a round-bottom flask containing 300 ml of 99.5 wt% ethanol was connected to the bottom. 8 g of finely ground cannabis sample was placed in a cellulose extraction thimble (CET Cellulose Extraction Thimbles, CHMLAB GROUP, Barcelona, ​​Spain), and this extraction thimble was inserted into the Soxhlet extraction apparatus. The round-bottom flask at the bottom of the Soxhlet extraction apparatus was set to be positioned on an Extraction Multi Heating Mantle (Misung Scientific Co., Ltd., Seoul, South Korea), and a recirculating chiller (Jeio Tech, Daejeon, South Korea) was operated to allow cooling water to circulate through the condenser. The heating mantle was set to the maximum temperature, and extraction was carried out for 3 hours at a solvent temperature of 78.4 °C. After the extraction was completed, the power to the heating mantle was turned off, and the round-bottom flask was separated to obtain the extract. Subsequently, the obtained extract was subjected to decarboxylation at 140 °C for 1 hour to finally obtain the hemp syllet extract.

[0099]

[0100] Preparation Example 6. Preparation of room-temperature cannabis extract as a control

[0101] Cannabis room temperature extract (CRE) was prepared by the following process. 8 g of finely ground cannabis sample was placed in a batch extractor and extracted three times at room temperature. The first extraction was performed in 150 mL of 99.5 wt% EtOH for 24 hours, the second extraction in 150 mL of 99.5 wt% EtOH for 24 hours, and the third extraction in 100 mL of 99.5 wt% EtOH for 24 hours.

[0102]

[0103] Experimental Example

[0104] Experimental Example 1. Comparison of Antioxidant Activity of Cannabis Extract (CE) According to Extraction Method

[0105] A DPPH assay was performed to evaluate the antioxidant efficacy of Cannabis Supercritical CO₂ Extract (CSCE). To confirm the superiority of the supercritical extract, Cannabis Soxhlet Extract (CSE) and Cannabis Room Temperature Extract (CRE) were prepared as controls.

[0106] Specifically, a DPPH solution stock was prepared by dissolving DPPH [DPPH (2,2-diphenyl-1-picrylhydrazyl)2 powder in 80% methanol. Then, cannabis extracts obtained by each method were prepared in 96 wells at concentrations of 46.87, 93.75, 187.5, 375, 750, 1500, and 3000 μg / ml, treated with the DPPH solution, and reacted at room temperature for 30 minutes. Afterward, absorbance was measured at 570 nm, and DPPH radical scavenging activity was compared based on the corresponding values.

[0107] As a result, it was confirmed that the cannabis supercritical extract (CSCE) exhibited higher antioxidant activity than the cannabis Soxhlet extract (CSE) and the cannabis room temperature extract (CRE) (Fig. 1). In particular, when compared to the cannabis room temperature extract (CRE), the cannabis supercritical extract (CSCE) showed a significant difference at all concentrations.

[0108]

[0109] Experimental Example 2. Neuroprotective Efficacy of Cannabis Supercritical CO₂ Extract (CSCE)

[0110] To confirm the neuroprotective effects of the supercritical fluid extract (CSCE) of cannabis, an analysis of neuronal viability, a biomarker specified in the Ministry of Food and Drug Safety’s guidelines for the functional evaluation of health functional foods, was performed. The neuroprotective effects of the room-temperature extract (CRE) of cannabis were also evaluated as a control group.

[0111] Specifically, the effect of cannabis supercritical extract on the inhibition of neuronal cell death from oxidative stress was investigated. Immortalized mouse hippocampal cell line HT-22 cells were seeded into a 96-well plate and cultured in DMEM for 24 hours. Subsequently, the cells were standardized by starvation in serum-free medium for 24 hours, and then treated with 35 μM glutamate, cannabis supercritical extract, and cannabis room temperature extract at concentrations of 0.625, 1.25, 2.5, 5, and 10 μg / ml, respectively, and cultured for 22 hours. Afterward, 10 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) solution was added and reacted for 2 hours, and cell viability was confirmed by measuring absorbance at 540 nm.

[0112] As a result, as shown in Figure 2, it was confirmed that the cannabis supercritical extract exhibited neuroprotective efficacy by inhibiting glutamate-induced neuronal death in proportion to its concentration. On the other hand, the cannabis room temperature extract did not show a significant neuroprotective effect (Figure 3). From this, it was confirmed that the cannabis supercritical extract (CSCE) protects neurons more effectively.

[0113]

[0114] Experimental Example 3. Memory-improving efficacy of Cannabis Supercritical CO₂ Extract (CSCE)

[0115] To confirm the efficacy of supercritical cannabis extract (CSCE) in improving cognitive function, the Morris water maze test was performed as presented in the Ministry of Food and Drug Safety’s Guidelines for Functional Evaluation of Health Functional Foods ('May help improve cognitive function / memory' section).

[0116] To determine the effect of cannabis supercritical extract on alleviating scopolamine-induced memory impairment, female C57BL / 6 mice were administered a vehicle (control, scopolamine) and 10 mg / kg or 20 mg / kg of cannabis supercritical extract, and 100 mg / kg of red ginseng extract as a positive control. Mice other than the control group were administered scopolamine intraperitoneally at a concentration of 1 mg / kg / day, followed by behavioral experiments. Circular tanks were filled with opaque water mixed with paint, and the tanks were divided into four quadrants with a refuge placed in one of them. The escape latency of the experimental animals to reach the refuge was measured (maximum 60 seconds).

[0117] As a result, it was confirmed that in the memory impairment group administered scopolamine, the time taken to escape to the refuge remained at a maximum, and on the 5th day, the memory impairment improved slightly. It was confirmed that the group treated with 20 mg / kg of cannabis supercritical extract showed an effect similar to the positive control group, and the group treated with 10 mg / kg of cannabis supercritical extract showed results similar to the control group not treated with scopolamine (Fig. 4). From these results, it was confirmed that the cannabis supercritical extract prepared in the above preparation example alleviates memory impairment caused by scopolamine.

[0118]

[0119] [National R&D projects that supported this invention]

[0120] [Project ID] 1545027852

[0121] [Assignment No.] 122036032HD030

[0122] [Ministry Name] Ministry of Agriculture, Food and Rural Affairs

[0123] [Project Management (Specialized) Agency Name] Korea Institute of Planning and Evaluation for Food, Agriculture and Forestry Technology

[0124] [Research Project Name] Technology Commercialization Support

[0125] [Research Project Title] Development of Production Technology for Cannabis for Food and Pharmaceuticals through Vertical Integration of Cultivation, Extraction, and Separation / Purification

[0126] [Name of Project Performing Organization] Bobsnu Co., Ltd.

[0127] [Research Period] April 1, 2022 ~ December 31, 2024

[0128]

[0129] [Explanation of the symbol]

[0130] 1: CO₂ cylinder 1': Auxiliary solvent cylinder

[0131] 2: Meter 3. Pre-cooler

[0132] 4. CO₂ Pump 4'. Auxiliary Solvent Pump

[0133] 5. Preheater 6. Extraction vessel

[0134] 7. Internal basket loading section 8. Pressure gauge

[0135] 9. Back pressure regulator 10. Product collection valve

[0136] 11. Cooling medium circulator 12. Heating medium circulator

[0137] 13. Data Logger

Claims

1. A neuroprotective composition comprising a cannabis supercritical extract.

2. A neuroprotective composition according to claim 1, wherein the cannabis is any one selected from the group consisting of Cannabis sativa, Cannabis indica, and Cannabis ruderalis.

3. A neuroprotective composition according to claim 1, wherein the cannabis supercritical extract is extracted from one or more selected from the group consisting of cannabis flower buds, whole plant, roots, stems, leaves, bark, fruits, and seeds.

4. A neuroprotective composition according to claim 1, wherein the cannabis supercritical extract is included at a concentration of 0.5 to 8 μg / ml.

5. A neuroprotective composition according to claim 1, wherein the cannabis supercritical extract is extracted using supercritical carbon dioxide as a solvent and supercritical methanol or supercritical ethanol as a co-solvent.

6. A neuroprotective composition according to claim 5, wherein the supercritical carbon dioxide and the auxiliary solvent are used in a ratio of 6:1 (v / v) to 10:1 (v / v).

7. In Claim 1, the cannabis supercritical extract is (a) A step of obtaining a hemp sample by drying hemp flower buds at 30 to 70 ℃ for 24 to 120 hours, (b) a step of decarbonizing the hemp sample at 100 to 200 ℃ for 15 to 60 minutes, and (c) A neuroprotective composition prepared by a method comprising the step of supercritical extraction of the above decarboxylated hemp sample at a pressure of 200 to 400 bar and a temperature of 30 to 80 ℃.

8. A composition for the prevention or treatment of degenerative brain diseases comprising the composition of any one of claims 1 to 7.

9. A functional food composition for improving cognitive function or memory, comprising the composition of any one of claims 1 to 7.

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