KR101341819B1 - Anti-inflammatory pharmaceutical composition comprising an extract from colored potato peel and its active fraction - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating inflammatory diseases comprising a color potato skin extract or a fraction thereof as an active ingredient, and inhibiting the activation of inflammatory factors expressed by various stimuli, and anti-inflammatory mechanisms. Inhibiting the mediating enzymes can be expected to have an excellent effect on the prevention and treatment of inflammatory diseases.

Description

Anti-inflammatory pharmaceutical composition comprising an extract from colored potato peel and its active fraction

The present invention relates to a pharmaceutical composition for preventing and treating inflammatory diseases comprising a color potato skin extract or a fraction thereof as an active ingredient.

The biggest disease that threatens the health of modern people is cancer. It is also expressed from the inflammatory response. Inflammatory response is a series of complex physiological reactions, including edema, fever, and erythema, including enzyme-activated humoral infiltration and cellular disruption of tissue, involving various inflammatory mediators and immune cells in local blood vessels and body fluids when cells are damaged or infected by an external source of infection. Of external symptoms. In normal cases, the inflammatory response removes the infectious agent and regenerates damaged cells, thereby restoring biological function. However, if the inflammatory response persists because the antigen is not removed, the inflammatory response may be expressed as cancer.

In general, anti-inflammatory drugs are effective by blocking the action of enzymes such as cyclooxygenase (COX), the enzyme type 1 cyclooxygenase (COX-1) from the stomach and blood and skin, joints or wounds By suppressing the activity of the enzyme type 2 cyclooxygenase (COX-2) occurring at the site, etc., it plays a role in alleviating pain. COX-1 is usually expressed in the stomach and platelets, which blocks the acid-induced damage and platelets involve clotting, whereas COX-2 causes inflammation. It looks after the generation properties, prostaglandin _{E 2 (prostaglandin E 2; PGE} 2) for enhancing the sensation of pain is also involved in the generation of. COX-2 is not only produced at the site of inflammation, but also expressed in nerve cells including the spinal cord and the brain, and PGE ₂ is produced in the central nervous system. This PGE ₂ exerts the excitability of neurons. This induces a change in pain signaling, resulting in analgesic efficacy. In addition, when looking at the complex metabolic pathways leading to pain from inflammation, local inflammation occurs, creating a molecular substance called interleukin-1β (IL-1β), which stimulates nerve cells to activate COX-2. When the gene is active, the COX-2 enzyme is expressed and PGE ₂ is generated from the nerve cells to stimulate the pain. In this series of metabolic pathways, drugs that block the production of IL-1β by inhibiting IL-1β converting enzymes and drugs that inhibit the expression of COX-2 block their metabolic pathways and thus have analgesic effects. It can be confirmed that.

Accordingly, the present inventors have continuously studied natural products exhibiting anti-inflammatory activity, the outer skin extract of color potato or solvent fractions that have been discarded during food manufacturing processing exhibits excellent anti-inflammatory effect to prevent diseases related to inflammation And the discovery that it can be used for treatment has led to the completion of the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing and treating inflammatory diseases comprising a color potato (Coat potato) shell extract or a fraction thereof as an active ingredient.

It is also an object of the present invention to provide a method for producing the color potato skin fraction.

Lastly, an object of the present invention is to provide a functional food for preventing or improving inflammatory disease, which contains a color potato shell extract or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising a color potato (Extract color) shell extract or a fraction thereof as an active ingredient.

In addition, the present invention comprises the steps of (a) slicing the dry color potato shell in a dry state and extracted with water, lower alcohol of C ₁ ~ C ₄ or a mixed solvent thereof and then concentrated under reduced pressure to obtain a color potato shell extract; And (b) suspending the extract in water and then sequentially dividing the extract into hexane, chloroform, ethyl acetate and butanol.

Finally, the present invention provides a functional food for preventing or improving an inflammatory disease containing a color potato shell extract or a fraction thereof as an active ingredient.

Color potato skin extract or fractions thereof of the present invention inhibits NF-κB activity and is directly involved in inhibiting the production of NO (Nitric oxide) and PGE ₂ (Prostaglandin E ₂ ) induced by lipopolysaccaride (LPS). By inhibiting the expression of protein enzymes such as (Inducible nitric oxide synthase) and COX-2 (Cyclooxygenase-2) shows excellent anti-inflammatory effect.

1 is a diagram showing the peeling part of the color potato shell and the specification of the specimen.
Figure 2a is a diagram showing the effect on the nitrite production of color potato extract and polar and non-polar solvent fraction in RAW 264.7 cells, a mouse macrophage line induced with lipopolysaccaride (LPS).
Figure 2b is a diagram showing the effect on the production of prostaglandin E ₂ (prostagladin E ₂ ; PGE ₂ ) of color potato extract and fractions in LPS-induced RAW264.7 macrophages.
Figure 2c is a diagram showing the expression regulation of inducible nitric oxide synthase (iNOS) enzyme and COX-2 (cyclooxygenase-2) enzyme treated with color potato fraction in LPS-induced RAW 264.7 cells to be.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including the color potato skin extract or a fraction thereof as an active ingredient.

The color potato in the present invention may be a variety of varieties such as Hongyoung (Jaong), Jayoung, (Superior), but is not limited thereto, and can be used without limitation color known in the art.

Color potato (Color potato) shell extract of the present invention comprises the steps of: (a) extracting by adding an extraction solvent to the dry color potato shell; (b) filtering the extract of step (a); And (c) can be obtained through the step of preparing the extract by concentrating the filtered extract of step (b) under reduced pressure.

The color potato shell may use the entire color potato shell, and preferably, the outer shell from 10 mm to 10 mm, more preferably 5 mm, may exhibit the best anti-inflammatory activity. However, the scope of the present invention is not limited thereto.

The extraction solvent may be water, a lower alcohol of C ₁ ~ C ₄ or a mixed solvent thereof, more preferably extracted with methanol or ethanol, but is not limited thereto. The amount of the extraction solvent is preferably 1 to 30 times the dry weight of the color potato shell, and more preferably 10 to 20 times, but is not limited thereto. When the extraction temperature is preferably 10 to 150 ℃, more preferably 20 to 80 ℃, but is not limited thereto. The extraction time is preferably 1 hour to 10 days, but is not limited thereto.

The method for preparing the extract may be a conventional extraction method such as supercritical extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, ultrasonic extraction, or the like using XAD and HP-20 adsorbent resins. Method may be used, and it is preferable to heat, reflux or extract at room temperature, but it is not limited thereto. The extraction number is preferably 1 to 5 times, more preferably 3 times repeated extraction is not limited thereto.

In the above method, the decompression concentration in step (c) is preferably a vacuum rotary evaporator, but is not limited thereto. The drying is preferably, but not exclusively, reduced-pressure drying, vacuum drying, boiling drying, spray drying, room temperature drying or freeze-drying.

Meanwhile, a fraction of the color potato shell extract may be additionally extracted by using an organic solvent, wherein the organic solvent is preferably hexane, chloroform, ethyl acetate, and normal butanol, but is not limited thereto. The fraction may be any one of hexane fraction, chloroform fraction, ethyl acetate fraction, normal butanol or water fraction obtained by suspending the color potato shell extract in water and then systematically fractionating the mixture with hexane, chloroform, ethyl acetate and normal butanol. desirable. The fraction may be obtained by repeating the fractionation process from 1 to 5 times, preferably 3 times from the color potato shell extract, but preferably concentrated under reduced pressure after the fraction, but is not limited thereto.

The color potato shell extracts and fractions inhibit iF-κB activity and directly induce the inhibition of the production of NO (Nitric oxide) and PGE ₂ (Prostaglandin E ₂ ) induced by lipopolysaccaride (LPS). By inhibiting the expression of protein enzymes such as oxide synthase) and COX-2 (Cyclooxygenase-2), it has excellent anti-inflammatory effects, resulting in inflammations such as acute inflammatory diseases, rheumatoid arthritis, gastritis, colitis, nephritis, hepatitis, cancer or degenerative diseases It has excellent prophylactic and therapeutic effects on the disease.

The composition comprising the color potato skin extract or fractions thereof of the present invention preferably comprises 0.1 to 50% by weight of the color potato skin extract or fractions thereof based on the total weight of the composition, but is not limited thereto.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The composition according to the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods. . Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the patient's condition, weight, age, sex, The range varies depending on the diet, the rate of excretion, the severity of the disease, the form of the drug, the time of administration, the method of administration, the route of administration and the duration of administration. The daily dose is from 0.0001 mg / kg to 500 mg / kg, preferably from 0.001 mg / kg to 100 mg / kg in an amount when the extract, fraction or compound according to the invention is lyophilized, and daily if necessary Administration may be from one to several times.

In addition, the present invention provides a functional food for preventing or improving inflammatory diseases containing a color potato (Coat potato) shell extract or a fraction thereof as an active ingredient.

There is no particular limitation on the kind of the food. Examples of foods to which the substance may be added include drinks, meat, sausages, breads, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, Beverages, alcoholic beverages and vitamin complexes and the like, and include all of the health foods in the conventional sense.

The color potato skin extract or fractions thereof of the present invention can be added as is to foods or used with other foods or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the amount of the extract in the dietary supplement may be added to 0.1 to 90 parts by weight of the total food weight. However, in the case of prolonged intake for health and hygiene purposes or health control purposes, the amount may be below the above range.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned color potato shell extract of the present invention or fractions thereof, various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (cheese, chocolate, etc.), pectic acid and Salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the color potato skin extract or fractions thereof of the present invention may contain a pulp for producing natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0.1 to about 20 parts by weight per 100 parts by weight of the color potato skin extract or fractions thereof.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1: peeling and drying the color potato skin

Materials: Two new varieties of color potatoes (Hongyoung, Jayoung) and one of the existing varieties (Sumi) were used.

Skin: The skin was peeled from the flesh of the potato at 1 cm in height and 5 mm in thickness (depth) (see Fig. 1).

Drying: Lyophilization was carried out at -70 ° C using an cryogenic lyophilizer.

Example  2: Preparation of Ethanol Extract of Color Potato Skin

Each 100 g of the cryogenic lyophilized color potato skin (Hongyoung, Jayoung, Sumi) samples were immersed in 1 liter of ethanol and extracted under reflux for 5 hours at room temperature, and the supernatant was recovered after vacuum filtration. This process was repeated three times, followed by concentration under reduced pressure to obtain a color potato shell ethanol extract (Hongyoung: 14 g, Jayoung: 9 g, Sumi: 6 g).

Example  3: Color Potato Skin Fraction  Produce

Color potato skin (Hongyoung: 14g, Jayoung: 9g, Sumi: 6g) obtained in Example 2 was suspended in 0.5 liter of ethanol extract, followed by nucleic acid (1 liter x 3) and chloroform (1 liter x 3). Eluted with ethyl acetate (1 liter x 3) and normal butanol (1 liter x 3), and the respective fractions were concentrated in vacuo to give hexane, chloroform, ethyl acetate, and normal butanol organic solvent fractions. See Table 1).

Yield of color potato shell solvent fraction Sample Weight (g) Hongyoung
(Hongyoung) Self-help
(Jayoung) Sumy
(Superior) Hexane fraction
(Hexane fraction) 1.15 0.94 0.23 Chloroform Fraction
(Chloroform fraction) 0.75 0.58 0.08 Ethyl acetate fraction
(Ethylacetate fraction) 10.57 4.27 4.38 Normal Butanol Fraction
( n -buthanol fraction) 2.27 7.84 10.2

Experimental Example  1: Culture of Cells

RAW 264.7 cells were treated at 37 ° C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) medium containing 10% FBS and penicillin (100 μg / ml) and streptomycin (100 U / ml). Maintained and incubated.

Experimental Example  2: cytotoxicity test ( MTT Toxicity test)

To determine the degree of toxicity was measured by the MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide thiazolyl blue, Sigma, USA) test method.

Cells incubated in 96 well plates (3072, falcone, USA) for 24 hours were removed, washed with PBS buffer (phosphate buffer saline), and 100 μl of peracid and 100 μl of various concentration samples were added to each well. After incubation for 45 minutes with hydrogen water, the supernatant was removed, washed twice with PBS buffer solution, and 200 μl of the medium was incubated for 24 hours. 50 μl of 5 mg / ml MTT reagent was added to the culture solution, and after 4 hours of incubation, the supernatant was removed, and 100 μl of DMSO (Dimethylsulfoxide, Sigma, USA) was added, followed by ELISA reader (Power Wave 340, Bio-). Absorbance was measured by Tek Instruments, USA.

The experimental results, it was confirmed by hongyoung inwardly of chloroform (Chloroform) fraction> n-butanol (n -butanol) fraction> n-butanol of Sumi sheath (n -butanol) in the order of fractions independent outer shell that is cytotoxic (see table 2) .

Cytotoxicity Measurement of Solvent Fraction from Color Potato Peel Sample
(Sample) IC ₅₀ (μg / ml) kind
(Cultivar) interior
(Tuber) coat
(Peel) Ethanol extract
Ethanol extract Hongyoung > 200 > 200 Jayoung > 200 > 100 Superior > 200 > 200 Hexane fraction
(Hexane fraction) Hongyoung ≒ 200 > 100 Jayoung > 200 > 200 Superior > 200 > 100 Chloroform Fraction
(Chloroform fraction) Hongyoung ≒ 12.5 ≒ 100 Jayoung > 200 ≒ 100 Superior ≒ 100 ≒ 100 Ethyl acetate fraction
(Ethylacetate fraction) Hongyoung > 200 > 200 Jayoung > 200 > 200 Superior > 200 > 200 Normal Butanol Fraction
( n -buthanol fraction) Hongyoung > 200 ≒ 100 Jayoung > 200 ≒ 50 Superior > 200 ≒ 25

Experimental Example  3: Nitrite ( Nitrite ) And PGE ₂  Experiment

To identify anti-inflammatory effects, nitrite and PGE ₂ The experiment was performed. Nitric oxide (NO) is synthesized by various types of cells, including endothelial cells, neurons, vascular smooth muscle and hepatocytes, and is synthesized from the amino acid L-arginine by the intracellular nitric oxide synthase (NOS) and oxidized. stable and inert oxide of nitrite ions (NO ^{2 -)} and nitrate (NO ^3-), and to, the TNF-α, IL-1 ( Interleukin-1) and IL-6 and NO TNF at inflammatory ( Many cytokines such as Interleukin-6) are produced rapidly.

Nitrite accumulation, a marker of NO synthesis, was measured in culture media by the Gries reaction, and NO studies have shown that NG-monomethyl-L-arginine (NMA), similar to L-arginine This was used. 100 μl of cell culture medium was mixed with 100 μl of Gries reagent consisting of the same amount of 1% sulfanylamide dissolved in 5% phosphoric acid and 0.1% naphthylethylenediamine-hydrochloric acid and incubated for 10 minutes at room temperature.

The absorbance was then measured at 550 nm using a microplate reader (Power Wave 340, Bio-Tekinstruments, USA). Freshly made culture medium was used as a blank in all experiments and the amount of nitrite in the sample was determined from the freshly prepared sodium nitrite standard curve in the medium. PGE ₂ levels in macrophages were quantitated using PGE ₂ EIA kit (Amersham pharmacia Biotech, USA).

Experimental results, lipid polysaccharide (lipopolysaccharide; LPS, Sigma, USA) in a mouse macrophage RAW 264.7 cells, independent shell chloroform (Chloroform) fraction as n-butanol (n -butanol) in the fraction (Korea Cell Line Bank) induced by the NO ^{2 -} Significantly inhibited production in a concentration-dependent manner, and also PGE ₂ of LPS-induced RAW 264.7 cells Looking at the results produced by using the excellent NO production-inhibiting effect in Screening independent shell chloroform (Chloroform) fraction, n-butanol (n -butanol) fraction PGE ₂ The production inhibitory effect was confirmed.

According to Figure 2 PGE ₂ significantly increased by LPS It was confirmed that the production decreased in a concentration-dependent manner. However, the self-enveloping chloroform fraction of the two fractions inhibited LPS-induced PGE ₂ by 76.68% at the concentration of 100 μg / ml, the highest concentration without cytotoxicity. It was confirmed that the inhibitory effect is higher than the butanol ( n -butanol) fraction inhibited 51.43% at μg / ml (see Table 3, Figure 2a and Figure 2b).

Determination of Nitric Oxide Inhibition in RAW 264.7 Macrophages Sample
(Sample) IC ₅₀ (μg / ml) kind
(Cultivar) interior
(Tuber) coat
(Peel) Ethanol extract
Ethanol extract Hongyoung > 200 112.48 Jayoung 177.77 > 100 Superior > 200 121.54 Hexane fraction
(Hexane fraction) Hongyoung > 200 > 100 Jayoung 178.25 113.64 Superior > 200 51.55 Chloroform Fraction
(Chloroform fraction) Hongyoung > 10 52.96 Jayoung 141.04 36.15 Superior > 100 > 100 Ethyl acetate fraction
(Ethylacetate fraction) Hongyoung > 200 > 200 Jayoung > 200 > 200 Superior > 200 74.58 Normal Butanol Fraction
( n -buthanol fraction) Hongyoung 189.18 63.57 Jayoung > 200 23.15 Superior > 200 > 200

Experimental Example  4: Western Blat  Experiment( iNOS  And COX -2 experiments)

Inhibitory Effect of Syringaresinol on the Inflammatory Mediator NO and PGE ₂ and Regulation of Inducible Nitric Oxide Synthase (iNOS) and COX-2 (cyclooxygenase-2) Enzymes That Induce Inflammation To elucidate the relationship, Western blot analysis confirmed the expression level.

iNOS is synthesized by immunological or inflammatory stimuli in cells such as hepatocytes, vascular smooth muscle cells, fibroblasts or macrophages of mice, and these cells produce large amounts of NO. Cellular proteins were extracted from RAW 264.7 cells treated with control and fractions, and washed cell pellets were 5 μg / ml each of leupetin (leupeptin, Sigma, USA) and aprotin (aprotin, Sigma, USA). Extraction solution contained (composition: 50 mM HEPES pH 7.0, 250 mM sodium chloride, 5 mM EDTA, 0.1% Nonidet p-40, 1 mM phenylmethylsulfonyl fluoride (Sigma, USA), 0.5 It was suspended in mM dithiothreitol (Sigma, USA), 5 mM NaF, 0.5 mM sodium orthovanadate (Naorthovanadate) and incubated at 4 ° C. for 15 minutes. Cell debris was removed by micro centrifuge and immediately frozen at the top, and protein concentration was determined by protein assay reagent (Bio-Rad).

Cell proteins obtained from 40-50 μg of treated and untreated cell extracts were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes, which were monoclonal anti-iNOS ( Inducible nitric oxide synthase (Santa Cruz Biotechnology, USA) and COX-2 (cyclooxygenase-2, Santa Cruz Biotechnology, USA) were incubated for 4 hours with a 1: 500 dilution of 5% skim milk blotting solution. Incubated overnight at 4 ° C. The blot was washed twice with PBS buffer and incubated for 1 hour at room temperature with 1: 1000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz Biotechnology, USA). Blots were washed three times with Tween 20 / Tris buffered saline (TTBS) and measured by chemiluminescence (Amersham Life Science, USA).

As a result, it was confirmed that iNOS and COX-2 protein expression levels significantly induced by LPS stimulation were significantly inhibited when treated with 50 μg / ml, iNOS inhibited from 25 μg / ml. (See FIG. 2C).

Claims (7)

A pharmaceutical composition for preventing and treating inflammatory diseases, comprising a chloroform or normal butanol fraction of a color potato shell extract as an active ingredient.
The pharmaceutical composition for preventing and treating inflammatory diseases according to claim 1, wherein the color potato is at least one selected from Hongyoung, Jayoung and Sumi.
The method of claim 1, wherein the extract is a color potato (Color potato) shell water, C ₁ A pharmaceutical composition for preventing and treating inflammatory diseases, which is extracted with a lower alcohol of C ₄ or a mixed solvent thereof.
The pharmaceutical composition for preventing and treating inflammatory diseases according to claim 3, wherein the lower alcohol is methanol or ethanol.
delete delete Functional food for preventing or ameliorating inflammatory disease containing chloroform or normal butanol fraction of color potato skin extract as an active ingredient.

Images