Composition for preventing and treating inflammatory diseases comprising cannabis root extract and extract improved through complex processing as active ingredients

Abstract

The present invention provides a composition for preventing and treating inflammatory diseases, comprising, as active ingredients, a cannabis root extract having significantly increased antioxidant and anti-inflammatory activities compared to conventional hot water extraction extracts due to a special processing process, and an extract improved through complex processing.

Description

Composition for the prevention and treatment of inflammatory diseases comprising hemp root extract and an extract improved through complex processing as active ingredients

[0001] The present invention relates to a pharmaceutical composition for treating and preventing inflammatory diseases, and more particularly, to a composition for treating and preventing inflammatory diseases comprising a cannabis root extract and an extract improved through complex processing as active ingredients.

[0002] Hemp refers to industrial hemp (Cannabis sativa L.) with a hallucinogenic substance tetrahydrocannabinol (THC) content of 0.3% or less. In Korea, cannabis, which is classified as a narcotic, and hemp, which is industrial hemp, are not distinguished, and hemp is regulated based on the 'Narcotics Control Act', with the peeled seeds, roots, and stems of mature cannabis and their products being exceptions. According to data on the status of domestic hemp cultivation, the hemp cultivation area increased 2.7-fold from 31.1 ha to a total of 88.5 ha in the past five years, and the amount of hemp harvested also increased from 924,030 to 1,433,270 plants, and research on industrial utilization of hemp byproducts is also increasing. Among these hemp by-products, hemp seed meal, which is the remaining product after extracting hemp oil, has a protein content of about 33%, making it a viable substitute for DDGS (distillers' grains) with a protein content of 25-30%, or soybean meal with a protein content of about 45%, which are widely used as feed ingredients. It is also known to be more than 20% cheaper. In addition, a study conducted by the Swedish University of Agricultural Sciences several years ago reported that feeding hemp meal to 40 Swedish red and white cattle for 5 months resulted in a significant increase in nutritional components in milk. However, by-products such as hemp roots and stems, which have low commercial utility, are being discarded in their entirety. In this regard, Korean Patent No. 2393416 discloses an anti-diabetic composition containing a hemp root extract.

[0003] However, the above prior art does not have excellent antioxidant and anti-inflammatory effects, and is therefore unsuitable for use as a material for compositions, health functional foods, pharmaceuticals, over-the-counter drugs, and cosmetics having anti-inflammatory effects.

[0004] The present invention is intended to solve various problems including the above-mentioned problems, and provides a composition for preventing and treating inflammatory diseases, which comprises as active ingredients a cannabis root extract with significantly increased antioxidant and anti-inflammatory activities compared to a conventional hot water extract through a special processing process and an extract improved through complex processing.

[0005] According to one aspect of the present invention, a pharmaceutical composition for preventing and treating inflammatory diseases is provided, containing a cannabis (Cannabis sativa L.) root extract as an active ingredient.

[0006] According to another aspect of the present invention, a cosmetic composition for improving inflammatory skin disease is provided, which comprises a cannabis (Cannabis sativa L.) root extract as an active ingredient.

[0007] According to another aspect of the present invention, an antioxidant health functional food is provided, which comprises a cannabis (Cannabis sativa L.) root extract as an active ingredient.

[0008] According to another aspect of the present invention, there is provided a step of manufacturing powder by washing and drying hemp roots with distilled water and then grinding them with a grinder:

[0009] High-heat and pressurized steam treatment step of treating the above powder with high heat and pressurized steam and then leaving it to dry:

[0010] Step of sterilizing the powder that has completed the above high-temperature and pressurized steam treatment process by heating it at a high temperature:

[0011] Fermentation step of adding distilled water to the above sterilized powder and then treating it with a Lactobacillus strain to ferment it:

[0012] A step of sterilizing the powder that has completed the above fermentation process by heating it at a high temperature and then treating it with ethanol to produce an ethanol extract; and

[0013] A method for producing a pharmaceutical composition for preventing and treating inflammatory diseases is provided, comprising the steps of filtering the ethanol extract, concentrating it using an evaporator, and then freeze-drying it to obtain an extract in powder form.

[0014] As described above, the composition for preventing and treating inflammatory diseases comprising the hemp root extract of the present invention and the improved extract through composite processing as active ingredients exhibits significantly higher antioxidant and anti-inflammatory efficacy compared to the extract manufactured by a simple hot water extraction method in the past through a pressurized and high-temperature process and a fermentation process using hemp roots, a by-product that is entirely discarded due to low commercial utility, and therefore can be utilized as a material for health functional foods, pharmaceuticals, quasi-drugs, and cosmetics for preventing and treating inflammatory diseases. In addition, since it utilizes a discarded by-product, it can be said to have very high industrial utility value. Of course, the scope of the present invention is not limited by these effects.

[0015] Figure 1 is a schematic diagram schematically showing the conventional hot water extraction method and the manufacturing process of the hemp root extract of the present invention.

[0016] Figure 2 is a photograph showing the properties of a dried powder of a hemp root extract manufactured according to the manufacturing process of Figure 1. It shows a simple hot water extract (①), an extract manufactured without processing (②), and extracts manufactured after pressurized and high-temperature processing of the present invention (③, ④, ⑤).

[0017] Figure 3a is a graph showing the results of DPPH scavenging activity analysis, confirming the antioxidant efficacy of the cannabis root extract of the present invention. CSR-W; simple hot water extract, CSR-Nt; alcohol extract, CSR-4SD; alcohol extract after drying and pressurizing steaming four times, CSR-F-Lr; alcohol extract after fermentation, CSR-4SD-F-Lr; alcohol extract after drying and pressurizing steaming four times and fermentation.

[0018] Figure 3b shows the antioxidant effect of the hemp root extract of the present invention, and the IC of DPPH 50 This graph shows the results of the analysis. CSR-W; simple hot water extract, CSR-Nt; alcohol extract, CSR-4SD; alcohol extract after drying and pressurizing steam 4 times, CSR-F-Lr; alcohol extract after fermentation, CSR-4SD-F-Lr; alcohol extract after drying and pressurizing steam 4 times and fermentation.

[0019] Figure 4 is a graph showing the results of analyzing the TPC and TTC contents of the hemp root extract of the present invention. The TPC and TTC contents increased by approximately 40% or more in the CSR-4SD-F-Lr group.

[0020] Figure 5 is a graph showing the results of analyzing the cell viability of the cannabis root extract of the present invention using the mouse macrophage cell line RAW264.7.

[0021] Figure 6 is a graph showing the results of analyzing the inhibitory efficacy of the cannabis root extract of the present invention on nitric oxide (NO) production using the mouse macrophage cell line RAW264.7. Nitric oxide was reduced by approximately 50% or more in the four non-toxic cannabis root extract concentrations of 250-500 μg / ml and the CSR-4SD-F-Lr group.

[0022] Figure 7 is a graph showing the results of analyzing the iNOS expression inhibitory efficacy of the cannabis root extract of the present invention using the mouse macrophage cell line RAW264.7. At non-toxic concentrations of 250-500 μg / ml of four cannabis root extracts, iNOS expression in the CSR-4SD-F-Lr group decreased to the level of the LPS-untreated group.

[0023] Figure 8 is a graph showing the results of analyzing the TNF-α expression inhibitory efficacy of the cannabis root extract of the present invention using the mouse macrophage cell line RAW264.7.

[0024] Figure 9 is a graph showing the results of analyzing the IL-6 expression inhibitory efficacy of the cannabis root extract of the present invention using the mouse macrophage cell line RAW264.7.

[0025] Definition of terms:

[0026] The term "industrial hemp (Cannabis sativa L.)" used in this document refers to a type of cannabis that can be used industrially. It is native to Central Asia and is mainly cultivated as a fiber plant in tropical and temperate regions with humid and mild climates. Commercial hemp refers to hemp that contains little or no THC, the narcotic ingredient, and is used for various purposes in various fields, such as textiles, construction, paper, cosmetics, and food. For example, hemp fiber is more durable and has a higher production rate than commonly used fibers, and hemp paper is stronger and more durable than regular wood paper, and is an environmentally friendly material that can protect forests.

[0027] The term "cannabis byproducts" used in this document refers to byproducts produced during the cannabis production process, including parts such as cannabis leaves, stems, and roots. Cannabis byproducts can be used for a variety of industrial purposes. Depending on the cannabis species and harvest time, cannabis stems can be harvested in their entirety, including the upper portion, lower portion, or entire stem. While their uses vary slightly depending on the species, they can generally be used in therapeutics, food additives, plastic products, and paper products. Furthermore, cannabis roots, as the primary component of the cannabis plant, possess a variety of beneficial properties and can be used as health foods and pharmaceuticals.

[0028] Detailed description of the invention:

[0029] According to one aspect of the present invention, a pharmaceutical composition for preventing and treating inflammatory diseases is provided, containing a cannabis (Cannabis sativa L.) root extract as an active ingredient.

[0030] In the above pharmaceutical composition, the extract may be a composition prepared by a preparation method selected from the group consisting of:

[0031] i) Ethanol extraction method for extracting hemp root powder with ethanol;

[0032] ii) A high-temperature and high-pressure steam ethanol extraction method in which hemp root powder is treated with high heat and high-pressure steam, sterilized with high heat, and then extracted with ethanol;

[0033] iii) A high-temperature fermentation ethanol extraction method in which hemp root powder is sterilized at high heat, fermented by treating it with a Lactobacillus strain, and then extracted with ethanol; and

[0034] iv) A high-temperature and high-pressure steam fermentation ethanol extraction method in which hemp root powder is treated with high heat and high-pressure steam, sterilized with high heat, treated with a Lactobacillus strain, fermented, and then extracted with ethanol.

[0035] In the above pharmaceutical composition, the Lactobacillus strain is Lactobacillus plantarum, Lactobacillus rhamnosus

[0036] rhamnosus), Lactobacillus acidophilus and Lactobacillus casei.

[0037] In the pharmaceutical composition, the extract may be included in an amount of 0.1 to 10 parts by weight based on the total weight of the composition, and the inflammatory disease may be rhinitis, allergic conjunctivitis, epidemic conjunctivitis, hepatitis, bronchitis, laryngitis, tonsillitis, thyroiditis, laryngitis, encephalitis, myelitis, pneumonia, gastritis, colitis, cystitis, pancreatitis, cystitis, synovitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, psoriasis, pruritus, prurigo, seborrheic dermatitis, acne, irritant dermatitis, or atopic dermatitis, but is not limited thereto.

[0038] According to another aspect of the present invention, a cosmetic composition for improving inflammatory skin disease is provided, which comprises a cannabis (Cannabis sativa L.) root extract as an active ingredient.

[0039] According to another aspect of the present invention, an antioxidant health functional food is provided, which comprises a cannabis (Cannabis sativa L.) root extract as an active ingredient.

[0040] According to another aspect of the present invention, there is provided a step of manufacturing powder by washing and drying hemp roots with distilled water and then grinding them with a grinder; a step of treating the powder with high heat and pressurized steam and then drying it by leaving it to dry;

[0041] Step of sterilizing the powder that has completed the above high-temperature and pressurized steam treatment process by heating it at a high temperature:

[0042] Fermentation step of adding distilled water to the above sterilized powder and then treating it with a Lactobacillus strain to ferment it:

[0043] A step of sterilizing the powder that has completed the above fermentation process by heating it at a high temperature and then treating it with ethanol to produce an ethanol extract; and

[0044] A method for producing a pharmaceutical composition for preventing and treating inflammatory diseases is provided, comprising the steps of filtering the ethanol extract, concentrating it using an evaporator, and then freeze-drying it to obtain an extract in powder form.

[0045] In the above manufacturing method, the high-temperature and pressurized steam treatment step can be repeated 2 to 6 times under the conditions of a temperature of 95 to 100°C, a pressure of 10 to 20 psi, and a time of 20 to 40 minutes. In addition, the sterilization step can be performed by a heating process at 110 to 130°C for 90 to 120 minutes, and the fermentation step can be performed in a stirred incubator at the conditions of 35 to 40°C and 50 to 70 rpm for 5 to 10 days. In addition, the sterilization can be performed by heating at 110 to 130°C for 10 to 30 minutes after the fermentation process, and the step of preparing an ethanol extract can be prepared by treating 30 to 90% ethanol for 2 to 5 days.

[0046] In the above manufacturing method, extraction was performed using ethanol as a solvent, but extraction may also be performed by treating with water, C1 to C4 lower alcohols, or mixtures thereof, and the lower alcohols may be butylene glycol, methanol, ethanol, or propanol. In addition, the drying process used in the above manufacturing method may be natural drying, hot air drying, or freeze drying.

[0047] In the above manufacturing method, the Lactobacillus strain may be selected from the group consisting of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Lactobacillus casei, and preferably Lactobacillus rhamnosus.

[0048] In the above manufacturing method, the high-temperature and pressurized steam treatment process and the fermentation process can enhance the antioxidant and anti-inflammatory effects and increase the total phenol and tannin contents, thereby exhibiting improved efficacy. In addition, although the above manufacturing method is a manufacturing method that applies both the high-temperature and pressurized steam treatment process and the fermentation process, extracts that do not apply them or that selectively apply one of the two processes, that is, an extract produced by an ethanol extraction method in which cannabis root powder is extracted with ethanol, an extract produced by an ethanol extraction method after high-temperature and pressurized steam treatment in which cannabis root powder is treated with high heat and pressurized steam, sterilized with high heat, and then extracted with ethanol, or an extract produced by a high-temperature fermentation and ethanol extraction method in which cannabis root powder is sterilized with high heat, treated with a Lactobacillus spp. strain, fermented, and then extracted with ethanol, also exhibited improved antioxidant and anti-inflammatory effects.

[0049] The cosmetic composition of the present invention may have a dosage form selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, an oil, a patch, a mask, and a spray. In addition, it may include one or more additives used in the formulation of the cosmetic composition. For example, the additives may include 1,3-butylene glycol, soybean phospholipid choline, sphingosine, cholesterol, Tween 80, phytosphingosine, salicylic acid, a skin moisturizer (humectant), an emollient, a natural oil, a natural extract, keratin, a lipoid, an absorbable substance, a stratum corneum ceramide, an epidermal lipid acid membrane fatty acid, cholesteryl ester, ethanol, distilled water, and the like.

[0050] The antioxidant health functional food of the present invention may additionally contain food additives, and its suitability as a "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Examples of the items listed in the "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kohlrabi pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0051] Health functional foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements. In addition, the health functional foods may contain, in addition to the active ingredient, the extract of the common angelica tree, various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. In addition, the health functional food of the present invention may have a formulation of a pill, tablet, hard capsule, soft capsule, granule, paste, gel, paste, liquid, powder, jelly, liquid, chewing tablet, bar, or film.

[0052] The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered through various routes such as intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, and percutaneous absorption.

[0053] In addition, the composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered in a dosage of 0.1 mg / kg to 1 g / kg, and more preferably in a dosage of 1 mg / kg to 500 mg / kg. Meanwhile, the dosage can be appropriately adjusted depending on the age, sex, and condition of the patient.

[0054] The pharmaceutical composition of the present invention may be formulated into a suitable form with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline solution, aqueous glucose, and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition, the cell therapy composition according to the present invention may appropriately include suspending agents, solubilizing agents, stabilizers, isotonic agents, preservatives, antiadsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, antioxidants, and the like, if necessary, depending on the administration method or formulation. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].

[0055] Reactive oxygen species (ROS) are generated during the body's energy metabolism. These reactive oxygen species react with cellular components, causing DNA denaturation, lipid oxidation, and protein degradation. Synthetic substances such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been developed as antioxidants. However, they can cause side effects such as allergic reactions, hyperactivity, enzyme synthesis disorders, endocrine disruption, and reproductive toxicity. Therefore, active research is being conducted to explore antioxidants derived from natural products. In addition, the inflammatory response is the process of repairing and regenerating damaged areas due to invasion that causes substrate changes in the body and tissues, such as physical actions, chemical contact, and bacterial infections. Local vasoactive substances such as histamine, serotonin, bradykinin, and prostaglandins are released, increasing vascular permeability and inducing inflammation. LPS, a major component of the outer wall of Gram-negative bacteria, stimulates macrophages, which induces the production of nitric oxide (NO) in the process of converting L-arginine into L-citrulline by the enzyme iNOS (inducible nitric oxide synthase). There are three types of NO synthase (NOS): nNOS (neuronal NOS), eNOS (endothelial NOS), and iNOS (inducible NOS). Excessive production of NO by iNOS can cause harmful effects on the body, such as pathological vasodilation, cytotoxicity, and tissue damage, and a decrease in iNOS expression can lead to the alleviation of inflammation.

[0056] Meanwhile, industrial hemp is used for various purposes in the industrial field. In the medical industry, industrial hemp, which contains little or no THC, a narcotic component, is utilized to extract components such as CBD, or cannabidiol, due to their high therapeutic activity and can be applied in various medical fields. Among the byproducts of industrial hemp, roots and stems are largely discarded due to their low utility. Therefore, the inventors of the present invention conducted research to develop antioxidant and anti-inflammatory pharmaceutical materials for industrial use of hemp root extracts. Since conventional hot water extracts of hemp roots are relatively ineffective, the inventors of the present invention developed a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing hemp root extracts, which have high industrial utility, as an active ingredient through a specific processing process of hemp roots. Specifically, the hemp root byproducts were repeatedly subjected to high heat and pressurized steam treatment to increase their surface area, and a microbial fermentation process was applied to produce a hemp root extract with significantly higher antioxidant and anti-inflammatory activities than conventional simple hot water and alcohol extracts (Figs. 1 and 2). Four types of cannabis root extracts (CSR-Nt; alcohol extract, CSR-4SD; alcohol extract after drying and autoclaving four times, CSR-F-Lr; alcohol extract after fermentation, CSR-4SD-F-Lr; alcohol extract after drying and autoclaving four times, and fermentation) with different processing methods, excluding simple hot water extract and ethanol extract, showed no toxicity at a concentration of 500 μg / ml and increased DPPH radical scavenging activity. In addition, at concentrations of 250-500 μg / ml of the four cannabis root extracts, nitric oxide (NO) in the CSR-4SD-F-Lr group decreased by about 50% or more, and the contents of TPC and TTC also increased by about 40% or more. In addition, at concentrations of 250-500 μg / ml of cannabis root extract, iNOS expression in the CSR-4SD-F-Lr group decreased to the level of the LPS-untreated group.As described above, the cannabis root extract of the present invention exhibits superior antioxidant and anti-inflammatory effects compared to simple hot water extracts and alcohol extracts, and therefore can be used as a health functional food, pharmaceutical, over-the-counter drug, and cosmetic material for treating inflammatory diseases.

[0057] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention.

[0058] Example 1: Preparation of hemp root extract

[0059] 1-1: Preparation of alcohol extract

[0060] The present inventors prepared an alcohol extract (CSR-Nt) by grinding cannabis roots. Specifically, debris such as soil contained in cannabis roots were washed with triple-distilled water using a surfactant and left at room temperature for several days to completely dry. The dried cannabis roots were then ground to a diameter of approximately 0.2 cm or less using an electric grinder to produce powder. Subsequently, a heating process was performed at 121°C for 100 minutes. The extract was extracted for 3 days using 70% ethanol as a solvent. The ethanol extract was then filtered using a 0.1-0.5 μm filter paper, concentrated using an evaporator, and freeze-dried to obtain a quantifiable powder extract.

[0061] 1-2: Production of alcohol extracts through pressurized and high-temperature processes

[0062] The present inventors prepared an alcohol extract (CSR-4SD) through a pressurized and high-heat process. Specifically, debris such as soil contained in the cannabis root was washed with triple-distilled water using a surfactant and left at room temperature for several days to completely dry. Thereafter, the dried cannabis root was ground using an electric grinder to a diameter of approximately 0.2 cm or less to produce a powder. Subsequently, the high-heat and high-pressure steam treatment at 98°C and 15 psi for 30 minutes was repeated four times, followed by leaving at 60°C for 3 to 6 hours, and an additional heating process at 121°C for 100 minutes. Subsequently, 70% ethanol was used as a solvent and extracted for 3 days. The ethanol extract was then filtered using a 0.1-0.5 μm filter paper, concentrated using an evaporator, and freeze-dried to obtain a quantifiable powder extract.

[0063] 1-3: Production of alcohol extract through fermentation process

[0064] The present inventors produced an alcohol extract (CSR-F-Lr) that went through a fermentation process. Specifically, debris such as soil contained in the hemp root was washed with triple-distilled water using a surfactant and left at room temperature for several days to completely dry. Thereafter, the dried hemp root was ground using an electric grinder to a diameter of approximately 0.2 cm or less to produce powder. The processed hemp root was fermented using Lactobacillus rhamnosus. Specifically, triple-distilled water was first added to the processed hemp root, and a 1 M citric acid solution was prepared and adjusted to pH 4.0. Thereafter, the L. rhamnosus strain was inoculated into the solution at a concentration of 0.3%, and the fermentation process was performed for 7 days in a shaker incubator at 37°C and 60 rpm in a state sealed from the outside air. After the above fermentation process, sterilization was performed by heating at 121°C for 20 minutes, and extraction was performed for 3 days using 70% ethanol as a solvent. Thereafter, the ethanol extract was filtered using a 0.1-0.5 μm filter paper, concentrated using an evaporator, and lyophilized to obtain a quantifiable powder extract.

[0065] 1-4: Preparation of alcohol extract through two-step processing

[0066] The present inventors produced an extract (CSR-4SD-F-Lr) by grinding hemp roots and then performing a special two-step processing process. Specifically, debris such as soil contained in the hemp roots were washed with triple-distilled water using a surfactant and left at room temperature for several days to dry completely. Thereafter, the dried hemp roots were ground using an electric grinder to a diameter of approximately 0.2 cm or less to produce powder. Subsequently, high-heat and pressurized steam treatment at 98°C and 15 psi for 30 minutes was repeated four times, followed by leaving at 60°C for 3 to 6 hours, and an additional heating process at 121°C for 100 minutes. Thereafter, the processed hemp roots were fermented using Lactobacillus rhamnosus. Specifically, triple-distilled water was first added to the processed hemp roots, and a 1 M citric acid solution was prepared to adjust the pH to 4.0. Thereafter, L. rhamnosus strain was inoculated at a concentration of 0.3% and the fermentation process was performed for 7 days at 37℃, 60 rpm in a shaker incubator in a sealed state with no outside air. After the fermentation process, it was sterilized by heating at 121℃ for 20 minutes, and extracted for 3 days using 70% ethanol as a solvent. After that, the ethanol extract was filtered using a 0.1-0.5 μm filter paper, concentrated using an evaporator, and lyophilized to obtain a quantifiable powder extract. In addition, a simple hot water extract was used as a control for antioxidant and anti-inflammatory experiments using the four extracts prepared above.

[0067] Example 2: DPPH radical scavenging activity

[0068] The present inventors analyzed the antioxidant efficacy of the cannabis root extract of the present invention. Specifically, five samples were prepared in 70% ethyl alcohol at concentrations of 0, 1.25, 2.5, 5, and 10 mg / mL, respectively, and 2,2-Diphenyl-1-picylhydazyl (DPPH) from Sigma-Aldrich was prepared in pure ethyl alcohol at a concentration of 300 μM. Then, 100 μL of the prepared sample and 900 μL of DPPH solution were mixed in a 1.5 mL E-tube, incubated in a dark room without light for 30 minutes, and the absorbance was measured at 517 nm using an ELISA reader. The experiment was repeated three times under the same experimental conditions. As a result, the cannabis root extract showed a significantly increased DPPH radical scavenging activity and IC 50 The values ​​were found to have increased by about two times or more (Figures 3a and 3b).

[0069] Example 3: Analysis of total phenol and total tannin content

[0070] The inventors of the present invention analyzed the total phenol and tannin contents of the cannabis root extract of the present invention. Specifically, to confirm the total phenol content, the Folin-Ciocalteu method was referred to, and five samples were prepared at a concentration of 10 mg / mL each. 40 μL of the prepared sample was dispensed into a 5 mL test tube, and 200 μL of Sigma-Aldrich's Folin-Ciocalteu solution and 1160 μL of distilled water were added and mixed. The mixture was then incubated at room temperature for 3 minutes, 600 μL of 2% sodium carbonate solution was added, and then incubated in the dark for an additional 2 hours. After that, 100 ㎕ of the cultured mixed solution was dispensed into a 96-well plate, and the absorbance was measured at 765 nm with an ELISA reader, and various concentrations (0, 0.5, 1, 2.5, 5, and 10 mg / mL) of gallic acid were used for the standard calibration curve experiment. Next, to confirm the total tannin content, the Folin-Denis method was referred to, and five samples were prepared at a concentration of 10 mg / mL each, and 15 ㎕ of the prepared sample was dispensed into each 15 mL test tube, and the volume was adjusted to 7 mL with distilled water. After that, 0.5 mL of Folin-Denis solution and 1 mL of sodium carbonate solution were added and mixed, and distilled water was added until the final volume became 10 mL. Then, 100 ㎕ of the cultured mixed solutions were dispensed into 96-well plates, and the absorbance was measured at 700 nm using an ELISA reader, and various concentrations of tannic acid (0, 0.5, 1, 2.5, 5, and 10 mg / mL) were used for the standard calibration curve experiment. As a result, compared to the control group (simple hot water extraction), the total phenol content (49.33±1.27) and tannin content (64.26±0.47) were found to increase by about 40% or more in the CSR-4SD-F-Lr group that was treated with high heat and pressurized steam once and then fermented twice (Fig. 4).The results of the analysis of total phenol and total tannin content are summarized in Table 1 below.

[0071] Total phenol and tannin content TPCTTCCSR-Nt32.89±0.5646.15±0.31CSR-4SD34.66±0.7154.60±0.27CSR-F-Lr45.25±0.9758.26±0.72CSR-4SD-F-Lr49.33±1.2764.26±0.47

[0072] Example 4: Cell viability analysis

[0073] The inventors of the present invention analyzed cell viability according to treatment with the cannabis root extract of the present invention. Specifically, to evaluate the stability of the extract, the cannabis root extract was added to mouse macrophage RAW264.7 cells at a concentration of 100 to 500 μg / ml, and after culturing for 24 hours, the cell growth rate was analyzed using the WST assay. As a result, compared to the control group, four cannabis root extracts (CSR-Nt, CSR-4SD, CSR-F-Lr, and CSR-4SD-F-Lr) with different manufacturing processes showed almost no cell death at a concentration of 500 μg / ml (Fig. 5).

[0074] Example 5: Evaluation of anti-inflammatory efficacy

[0075] The present inventors evaluated the anti-inflammatory efficacy of the cannabis root extract of the present invention through Griess analysis and Western blot protein analysis. Specifically, the Griess analysis was performed by seeding mouse macrophage RAW264.7 cells at a density of 50,000 cells / ml in a 96-well plate, replacing the medium with serum-free medium after 24 hours, and pretreating the cells with the cannabis root extract of the present invention at various concentrations (0, 250, and 500 μg / ml) together with 1 μg / ml of lipopolysaccarides (LPS), an inflammatory substance, for 24 hours. Thereafter, nitric oxide (NO) released from the cells was measured for absorbance at 540 nm using an Elisa reader using the Griess reaction method, and the same experimental conditions were repeated three times. Analysis of iNOS, an inflammatory mediator protein, was performed using Western blot protein analysis. Specifically, mouse macrophage RAW264.7 cells were seeded in 6-well plates at a density of 50,000 cells / ml, and replaced with serum-free medium after 24 hours. After 24 hours, the cells were treated with cannabis root extract at various concentrations (0, 250, and 500 μg / ml), and pretreated with inflammatory substance, lipopolysaccarides (LPS), at a concentration of 1 μg / ml for 24 hours. Afterwards, the inhibitory effect of cannabis root extract treatment on the expression of the inflammatory mediator iNOS was analyzed through Western blot. As a result, compared to the control group, the CSR-4SD-F-Lr group showed a decrease in nitric oxide (NO) production by approximately 50% or more at non-toxic concentrations of 250 and 500 μg / ml (Fig. 6), and iNOS expression was reduced to the level of the LPS-untreated group, indicating a strong inhibition of iNOS expression (Fig. 7).

[0076] Example 6: Analysis of inhibition of proinflammatory cytokine production

[0077] The inventors of the present invention performed the inhibitory effect of the hemp root extract of the present invention on the production of proinflammatory cytokines TNF-α and IL-6 by referring to the previous literature (Yang JHet et al., Immunopharmacol Immunotoxicol, 33(2):373-379, 2011). Specifically, RAW 264.7 cells (1.5×10 5 (cells / well) were pre-cultured in a 24-well plate, replaced with DMEM medium, and the hemp root extract of the present invention (250 or 500 μg / ml) was pre-treated to the cells for 1 hour. Afterwards, 1 μg / ml of LPS was treated to each well and cultured for 24 hours at 37°C and 5% CO2 conditions. The supernatant was then collected and the amount of pro-inflammatory cytokines TNF-α and IL-6 was measured using an ELISA kit (R&D System, USA).

[0078] As a result, the production of TNF-α was significantly increased in the cell supernatant treated with LPS, but when treated with the cannabis root extract of the present invention, the production of TNF-α was decreased in a concentration-dependent manner, and in particular, the CSR-4SD-F-Lr group showed the highest inhibitory efficacy at 24.06 pg / mL (Fig. 8). In addition, as a result of analyzing the production of IL-6, the production of IL-6 in the group treated with the cannabis root extract was decreased in a concentration-dependent manner compared to the control group, and similarly, the CSR-4SD-F-Lr group showed the highest inhibitory efficacy at 29.90 pg / mL (Fig. 9). The above results suggest that the cannabis root extract of the present invention exhibits excellent anti-inflammatory activity by reducing proinflammatory cytokines induced by LPS.

[0079] In conclusion, the hemp root extract of the present invention showed an approximately 2-fold increase in DPPH radical scavenging activity and an approximately 40% or greater increase in TPC and TTC contents after undergoing several high-heat and pressurized steam treatments and fermentation processes. In addition, it inhibited the production of nitric oxide (NO) induced by LPS in mouse macrophage cell line RAW264.7 cells, significantly reduced the protein expression level of iNOS, and effectively suppressed the production of proinflammatory cytokines TNF-α and IL-6, exhibiting excellent antioxidant and anti-inflammatory effects. Therefore, the hemp root extract of the present invention can be utilized as a pharmaceutical material with an anti-inflammatory effect or a functional cosmetic material.

[0080] While the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

1. A pharmaceutical composition for the prevention and treatment of inflammatory diseases, containing cannabis (Cannabis sativa L.) root extract as an active ingredient.

2. In paragraph 1, The above extract is a composition prepared by a preparation method selected from the group consisting of: i) Ethanol extraction method for extracting hemp root powder with ethanol; ii) A high-temperature and high-pressure steam ethanol extraction method in which hemp root powder is treated with high heat and high-pressure steam, sterilized with high heat, and then extracted with ethanol; iii) A high-temperature fermentation ethanol extraction method in which hemp root powder is sterilized at high heat, fermented by treating it with a Lactobacillus strain, and then extracted with ethanol; and iv) A high-temperature and high-pressure steam fermentation ethanol extraction method in which hemp root powder is treated with high heat and high-pressure steam, sterilized with high heat, treated with a Lactobacillus strain, fermented, and then extracted with ethanol.

3. In paragraph 2, A composition wherein the above Lactobacillus strain is selected from the group consisting of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Lactobacillus casei.

4. In paragraph 1, A composition comprising the extract in an amount of 0.1 to 10 parts by weight based on the total weight of the composition.

5. In paragraph 1, The composition of the above inflammatory disease is rhinitis, allergic conjunctivitis, epidemic conjunctivitis, hepatitis, bronchitis, laryngitis, tonsillitis, thyroiditis, laryngitis, encephalitis, myelitis, pneumonia, gastritis, colitis, cystitis, pancreatitis, cystitis, synovitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, psoriasis, pruritus, prurigo, seborrheic dermatitis, acne, irritant dermatitis or atopic dermatitis.

6. A cosmetic composition for improving inflammatory skin disease, comprising cannabis (Cannabis sativa L.) root extract as an active ingredient.

7. A cosmetic composition for improving inflammatory skin disease, comprising cannabis (Cannabis sativa L.) root extract as an active ingredient.

8. Step of manufacturing powder by washing and drying the hemp roots with distilled water and then grinding them in a grinder: High-heat and pressurized steam treatment step of treating the above powder with high heat and pressurized steam and then leaving it to dry: Step of sterilizing the powder that has completed the above high-temperature and pressurized steam treatment process by heating it at a high temperature: Fermentation step of adding distilled water to the above sterilized powder and then treating it with a Lactobacillus strain to ferment it: A step of sterilizing the powder that has completed the above fermentation process by heating it at a high temperature and then treating it with ethanol to produce an ethanol extract; and A method for producing a pharmaceutical composition for preventing and treating inflammatory diseases, comprising the step of filtering the ethanol extract, concentrating it using an evaporator, and then freeze-drying it to obtain an extract in powder form.

9. In paragraph 8, A manufacturing method in which the above high-temperature and pressurized steam treatment step is repeated 2 to 6 times under the conditions of a temperature of 95 to 100°C, a pressure of 10 to 20 psi, and a time of 20 to 40 minutes.

10. In paragraph 8, A manufacturing method, wherein the above Lactobacillus strain is selected from the group consisting of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Lactobacillus casei.

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