KR102119795B1 - Anti-inflammatory Composition Comprising Broccoli Extract - Google Patents

Abstract

The present invention is an anti-inflammatory composition that can be safely used as a food material and can be safely used, and further increases the anti-inflammatory activity of broccoli, which is excellent in suppressing inflammation, and can be useful for prevention, suppression, treatment, and symptom improvement of inflammatory diseases. More particularly, it relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases, including an extract of broccoli heat-treated at 100 to 150°C as an active ingredient.

Description

Anti-inflammatory composition comprising broccoli extract

The present invention is an anti-inflammatory composition that can be safely used as a food material and can be safely used, and further increases the anti-inflammatory activity of broccoli, which is excellent in suppressing inflammation, and can be useful for prevention, suppression, treatment, and symptom improvement of inflammatory diseases. It is about.

Inflammatory diseases are reactions in the human body, including infection, trauma, and immunological reactions. There are acute inflammatory symptoms such as fever, redness, edema, and pain, and in the second half of the inflammatory process, cells such as migration of leukocytes to the site of inflammation and cytokines, degrading enzymes, bioactive lipid intermediates, transient reactive oxygen species, and lymphocyte production My change happens. Among chemical mediators of inflammatory reactions, prostaglandins (PG) and nitric oxide (NO) are known to be important mediators of carcinogenesis and inflammation.

Macrophages express iNOS or COX-2, a type of cyclooxygenase (COX), by stimulation that causes inflammation, such as mitogen, cytokine or bacterial LPS. COX is an enzyme that produces PG in the body, and has been found to exist as two isomers, COX-1 and COX-2. Of these, COX-1 is an enzyme that is involved in the production of PGs that are involved in cell protection and regulation in the gastrointestinal mucosa, platelets, and kidneys under normal conditions, whereas COX-2 has a very low concentration in normal cells, but is an inflammation-related cell. Is induced by various stimuli (cytokine, endotoxin, mitogen) and is involved in the production of PGs that are involved in pain or inflammation. Nitric oxide synthase (NOS) is involved in the regulation of vascular tone, neurotransmission, bacterial and cancer cell death and homeostasis. Neuronal NOS (Neuronal NOS) and endothelial NOS (endothelial NOS) exist under normal conditions, while iNOS (NOS type2) is induced by LPS, cytokines, and the like. High concentrations of nitrogen oxides (NO) occur in physiological reactions such as circulatory shock, inflammation, and carcinogenesis. According to the accumulated data, it is known that COX-2 and iNOS are involved in many inflammatory processes and cause various cancers, and thus play an important role in inflammation and cancer induction. Therefore, it is believed that if these inflammatory factors can be adjusted, the LPS-induced inflammatory response may be improved.

Nonsteroidal antiinflammatory drugs (NSAIDs), which are the most commonly used drugs that control inflammation or pain, exhibit pharmacological action by inhibiting cyclooxygenase (COX), which produces PG in the body. However, in addition to COX-2, which is involved in the production of PGs that are involved in the inflammatory response, COX-1 is also inhibited, so side effects such as gastrointestinal disorders and bleeding are also present. Therefore, there is a need to develop a new inflammatory prophylaxis/therapeutic agent that has no side effects and can selectively inhibit the activity of inflammatory genes and/or related substances.

Broccoli has been used for food since the Roman era and contains glucopanin and sulforaphane as the main ingredients. It has been reported to show antibacterial activity against Helicobacter pylori, the cause of gastric ulcer, and to reduce the risk of cancer. In addition, it contains vitamin C, which is twice that of lemon, and it contains beta-carotene, vitamin B1, vitamin B2, vitamin E, potassium, and calcium. Published Patent Nos. 10-2017-0114773 and 10-2013-0087174 confirm that the extract of broccoli exhibits anti-inflammatory activity, and a cosmetic composition for reducing skin inflammation and a composition for preventing or treating inflammatory diseases using the same Post it.

Patent Publication No. 10-2017-0114773 Patent Publication No. 10-2013-0087174

The present invention is to provide an anti-inflammatory composition that can be safely used for a long period of time without side effects and a side effect of increasing the anti-inflammatory activity of broccoli, including a useful effect on the human body, and can be utilized in the pharmaceutical, cosmetic, and food fields. The purpose.

The present invention for achieving the above object relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising an extract of broccoli heat-treated at 100 to 150°C as an active ingredient.

The heat treatment is to heat the broccoli. When the temperature is too low, the effect of increasing the anti-inflammatory effect due to the heat treatment is negligible. When the temperature is too high, carbonization occurs. By heat treatment, the yield of broccoli extract increased slightly, and in the case of broccoli extract, the expression of iNOS, an infectious agent increased by LPS stimulation, decreased to 44%, whereas the heat treated broccoli extract decreased by 24%. . The expression of IL-1β, a type of pro-inflammatory cytokine, was also reduced to 52% by the broccoli extract, but decreased to 24% by the treatment of the heat-treated broccoli extract, confirming that the anti-inflammatory activity was significantly increased by heat treatment. Could. The expression inhibitory effect of the heat-treated broccoli extract was superior to that of the broccoli extract without heat treatment for the pro-inflammatory mediators COX-2 or other pro-inflammatory cytokines IL-6 and TNF-α. The extract of the heat-treated broccoli of the present invention does not show cytotoxicity and can be safely used, and it can be further confirmed that AST and ALT are effectively reduced in an experiment with an acute hepatitis animal model induced by drugs (APAP).

In the following examples, only the results of heat treatment at 140 to 150° C. were described, but also when the heat treatment was performed at 100 to 140° C., an increase in anti-inflammatory activity could be confirmed, and the lower the temperature, the longer the heat treatment was required. The heat treatment time was preferably treated for 2 to 12 hours in consideration of the sample size or the heat treatment temperature.

For the heat treatment of the broccoli, raw broccoli as well as dried broccoli or frozen broccoli may be used for easy storage. In addition, an extract may be prepared by extracting the heat-treated broccoli as it is or after drying or freezing. For efficient extraction, it is more preferable to chop them, or extract them after grinding or grinding. Extraction may be any of the methods used for extraction of natural products in the art, for example, cold extraction, heat extraction, ultrasonic extraction, supercritical extraction, etc., using a juice or solvent that does not use a separate solvent. Can be used, but is not limited thereto. In the case of extraction using a solvent, it is preferable to use water or an alcohol of C1 to C4 or a mixture thereof. In addition, in order to further concentrate the active ingredient having anti-inflammatory activity, the extract may be further fractionated. The extract obtained by extraction using juice or a solvent can be used by itself, concentrated, or dried. It is natural that the drying method may also use a conventional method such as spray drying, heat drying, and freeze drying.

The inflammatory disease may be, for example, one or more selected from the group consisting of rhinitis, sinusitis, otitis media, pneumonia, gastritis, small intestine colitis, colitis, arthritis, hepatitis, dermatitis and sepsis.

The composition of the present invention may be prepared by a method known in the pharmaceutical field for use as a medicament for the prevention and treatment of inflammatory diseases, or a pharmaceutically acceptable carrier or excipient. , Diluents and the like. The composition of the present invention can be formulated and used as a formulation for oral or parenteral administration. The dosage of the active ingredient according to the present invention can be appropriately selected according to the absorbance of the active ingredient in the body, the form of the preparation, the age, sex and condition of the patient, the degree of symptoms, etc., and the dosage may be administered once a day. , It can be administered several times. The typical dosage is 0.001 mg/kg·day to 10 g/kg·day. Since the composition of the present invention can be safely used without toxicity and side effects, it can be safely used even for a long period of time for prevention purposes.

In addition, the present invention relates to a health functional food composition for preventing and improving symptoms of an inflammatory disease comprising an extract of broccoli heat-treated at 100 to 150°C as an active ingredient. Detailed description of the heat-treated broccoli extract of the present invention can be applied in the same manner as in the description of the pharmaceutical composition.

The heat-treated broccoli extract may be added to the health functional food of the present invention, preferably 0.01 to 100% by weight. The effective dose of the health food composition of the present invention may be used in accordance with the effective dose of the pharmaceutical composition, but may be below the above range for long-term intake for health and hygiene purposes or for health control purposes, The active ingredient has been taken as a food material for a long time, and since there is no problem in terms of safety, it may be used in an amount exceeding the above range. The health functional food of the present invention includes tablets, capsules, pills or liquids, etc. As foods to which the composition of the present invention can be added, for example, various foods, beverages, gums, teas, sweets, There are alcoholic beverages, chocolate, vitamin complexes, and health functional foods.

The present invention also relates to a cosmetic composition for alleviating skin inflammation comprising an extract of broccoli heat-treated at 100 to 150°C as an active ingredient, and can be usefully used for alleviating skin inflammation caused by atopic dermatitis, acne, or sunburn. . In order to use the composition of the present invention as a cosmetic, ingredients commonly used in cosmetic compositions may be further included. For example, an antioxidant, a stabilizer, a solubilizing agent, vitamins, pigments, and fragrances may be used. Carriers and the like. The cosmetic composition of the present invention may contain an extract of heat-treated broccoli in a proportion of 0.1 to 100% by weight. In addition, other useful ingredients may be further included in addition to the heat-treated broccoli extract.

The cosmetic composition of the present invention can be prepared in any formulation that is conventionally prepared in the art, for example, nutrition cream, massage cream, essence, eye cream, face wash, cleansing foam, mask pack, foundation, powder , May be manufactured in the form of lotion, but is not limited thereto. The cosmetic composition of the present invention may be used alone or in combination, or may be used in combination with other cosmetic compositions other than the present invention. In addition, the cosmetic composition having an excellent effect for alleviating skin inflammation according to the present invention can be used according to a conventional method of use, and the number of uses can be varied according to the user's skin condition or taste.

As described above, according to the composition of the present invention, safety for the human body has been established using broccoli, which has been used for a long time as a food material, and the anti-inflammatory effect is greatly improved even with a simple heat treatment process to prevent, treat, and alleviate inflammatory diseases. It can be useful as a pharmaceutical composition, health functional food composition and cosmetic composition for.

1 is an evaluation graph for the cytotoxicity of the heat-treated broccoli extract.
Figure 2 is an evaluation graph for the anti-inflammatory efficacy of the heat-treated broccoli extract.
3 is an evaluation graph for ALT of the heat-treated broccoli extract in an acute hepatitis animal model.
4 is an evaluation graph for AST of the heat-treated broccoli extract in an acute hepatitis animal model.
5 is an H&E stain image of liver tissue for evaluating the efficacy of heat-treated broccoli extract in an acute hepatitis animal model.

Hereinafter, the present invention will be described in more detail with reference to the attached examples. However, these examples are merely examples for easily explaining the contents and scope of the technical spirit of the present invention, and the technical scope of the present invention is not limited or changed thereby. It will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical spirit of the present invention based on these examples.

[Example]

Preparation Example: Preparation of heat-treated broccoli extract (HBE)

Broccoli was purchased from the agricultural and marine products market, washed, and then cut to a size of about 0.5 cm × 0.5 cm × 0.5 cm and lyophilized. The lyophilized sample was designed to withstand pressures of 10 kg/cm2 or more, and placed in an inner compartment container of a heat treatment device (Jisco, Seoul, Korea), and water was added to the container in the outer compartment at 140 to 150°C for 6 hours Heat treatment. The apparatus can prevent the carbonization of the sample by direct heat transfer due to the water contained in the container of the outer compartment, without the sample directly contacting the water.

After the heat-treated sample was cooled, it was ground using a grinder, and 10 times (v/v) distilled water was added thereto, followed by extraction at 60°C for 2 hours. The extract was filtered and lyophilized. The yield of the lyophilized extract was 4.5% (w/w) compared to raw broccoli.

Comparative Example: Preparation of broccoli extract (BE)

The extract was prepared by the same method as in Production Example, except that the heat treatment step was omitted. The yield of the lyophilized extract was 3.0% (w/w) compared to raw broccoli.

Example 1: Cytotoxicity evaluation

To measure cytotoxicity, RAW 264.7 cells in a 96 well plate were added at a concentration of 5 × 10 ³ cells/100 μL. I was busy. RAW 264.7 cells were pre-sale from the Korean cell line bank, 37° C. using Dulbecco's Modified Eagle's Medium (DMEM) with 5% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycn (P/S) added. , Cultured in a 5% CO ₂ environment. Broccoli extract prepared in Preparation Example or Comparative Example was added to the dispensed cells at a concentration of 125, 250, 500 or 1,000 µg/ml, treated for 24 hours, and then the cell viability was measured using the MTS assay method.

1 is a graph showing the results, it was confirmed that both broccoli extract and heat-treated broccoli extract do not exhibit cytotoxicity and can be safely used.

Example 2: Evaluation of anti-inflammatory activity of broccoli extract

The anti-inflammatory activity of broccoli extract was evaluated as an effect on the expression of pro-inflammatory mediators iNOS and COX-2 and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). To this end, RAW 264.7 cells were dispensed into a 6-well plate at a concentration of 5×10 ⁵ cells/100 μL, and then 0.1 μg/mL LPS was treated. After 30 minutes of LPS treatment, broccoli extract (BE) or heat-treated broccoli extract (HBE) was treated at a concentration of 1 mg/ml and cultured for 18 hours. Thereafter, RNA was extracted according to the manufacturer's manual using a TRIzol reagent (Invitrogen), and expression of pro-inflammatory mediators and cytokines was measured by qRT-PCR using the primers in Table 1 below. GAPDH was used as a loading control.

Figure 2 is a graph showing the results, broccoli extract (BE) showed an excellent inhibitory effect on the proinflammatory mediator iNOS and COX-2 and IL-1β, a proinflammatory cytokine, increased by stimulation of LPS . The pro-inflammatory cytokines TNF-α and IL-6 were also weaker than iNOS, COX-2, and IL-1β, but also showed inhibitory effects. The heat-treated broccoli extract (HBE) had better inhibitory effects on the expression of all the pro-inflammatory mediators and pro-inflammatory cytokines than the broccoli extract. In particular, it was confirmed that the suppression effect of iNOS and TNF-α was significantly increased.

Example 3: Evaluation of anti-inflammatory efficacy in APAP-induced acute inflammation-induced mouse model

Acetaminophen (APAP) is a relatively safe drug used for fever and pain relief. However, it has been recently known that a large amount of white blood cells flows in when a large amount of APAP is administered to a living body, causing acute hepatitis. Thus, in a mouse model in which acute hepatitis was induced by APAP, anti-inflammatory efficacy was evaluated by the effect of broccoli extract on the treatment of hepatitis.

Male BALB/c mice with a weight of 19 to 22 g, aged 6 to 8 weeks of age, were purchased from Dongyang Biotechnology (Gyeonggi-do) under certain conditions (temperature: 21±2℃, relative humidity: 60±10%, in a non-specific pathogen facility) Contrast: 12 hours light/dark cycle) was used after purifying for a week. During the experiment, water and feed were free. The experimental group was divided into 6 normal groups (untreated group), negative control group (APAP only treatment), positive control group (APAP+NAC treatment) and sample administration group (APAP+HBE treatment).

The positive control group was orally administered with NAC 75 mg/kg, which is known to have the effect of treating acute hepatitis caused by APAP, for 7 days daily, and the sample administration group was orally administered with 500 mg/kg of the heat-treated broccoli extract prepared in the Examples. Did. After 2 hours of oral administration of the sample to the positive control group and the sample administration group, APAP for acute hepatitis induction was intravenously administered to all groups except the normal group at a concentration of 400 mg/kg.

After 24 hours of sample administration, blood was collected through the tail vein, and the ALT and AST levels in the blood were measured using an assay kit (Asan Pharmaceutical) using an enzyme reaction with a substrate from the collected blood. 3 and 4, respectively, as a result of measuring blood ALT and AST levels, it was confirmed that the heat-treated broccoli extract was effective for acute hepatitis caused by APAP.

After blood collection, mice were sacrificed, liver tissue was extracted, embedded in paraffin, and stained with H&E. 5 is a corresponding staining image, A is a normal group, B is a negative control group, C is a positive control group, D is a sample administration group. As can be seen in FIG. 5, severe hepatocyte damage was observed by APAP, but it was confirmed that such damage was significantly reduced by NAC or heat-treated broccoli extract.

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Claims (6)

In a closed container divided into two or more spaces,
Add water to the at least one space,
Put broccoli in another space without water,
A pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising an extract of broccoli pre-heated at 100-150° C. as an active ingredient in a state capable of preventing carbonization by direct heat transfer without direct contact with water.
According to claim 1,
The pre-heat treatment time is 2 hours to 12 hours, characterized in that the pharmaceutical composition for the prevention and treatment of inflammatory diseases.
According to claim 1,
The extract is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that extracted using water or alcohols of C1 ~ C4 or a mixture thereof.
The method according to any one of claims 1 to 3,
The inflammatory disease is rhinitis, sinusitis, otitis media, pneumonia, gastritis, small intestine colitis, colitis, arthritis, hepatitis, dermatitis and sepsis, prevention or treatment of inflammatory diseases characterized in that at least one selected from the group consisting of.
In a closed container divided into two or more spaces,
Add water to the at least one space,
Put broccoli in another space without water,
A health functional food composition for preventing and improving symptoms of inflammatory diseases, including an extract of broccoli pre-heated at 100 to 150°C as an active ingredient in a state capable of preventing carbonization by direct heat transfer without directly contacting water.
In a closed container divided into two or more spaces,
Add water to the at least one space,
Put broccoli in another space without water,
A cosmetic composition for alleviating skin inflammation comprising an extract of broccoli pre-heated at 100 to 150°C as an active ingredient in a state that can prevent carbonization by direct heat transfer without directly contacting water.

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