KR101869639B1 - Composition comprising Astilboides tabularis extracts or fraction thereof as an active ingredient and use thereof - Google Patents

Abstract

The present invention relates to a composition comprising an Astilboides tabularis extract or a fraction thereof as an active ingredient, and to a use thereof. The Astilboides tabularis extract or the fraction thereof can be used as a composition for anti-cancer, anti-inflammation, anti-obesity or skin improvement by inducing apoptosis of cancer cells, and inhibiting the production of nitric oxide, activity of tyrosinase or elastase, and differentiation of lipid precursor cells.

Description

Composition containing Astragalus membranaceus extract or fraction thereof as an active ingredient and its use (Composition comprising Astilboides tabularis extract or fraction thereof as an active ingredient and use thereof)

The invention more screens (Astilboides tabularis) it relates to a composition and use thereof containing extracts or fractions thereof as an active ingredient.

Dog screens are perennial herbaceous plants belonging to the family Angiospermae, Rosales, Saxifragaceae (Lee, 2003a) and are a northern plant designated as the endangered wild plant Class II by the Ministry of the Environment Environmental Research, 2014). It is limited in the limestone areas of the northern part of the Korean peninsula and Gangwon province, and in the Jilin and Liaoning areas of China (Fu and Hong, 1998; Korea National Arboretum, 2012).

In addition, the rhizome of the dog's screen is called a mountain leaf, and it is effective for abdominal pain, enteritis treatment or branch office, and young leaf is used for food (Ahn, 2003; Lee, 2006a). Studies on dog screening have all revealed genetic diversity (Lee, 2007), ecological characteristics of 13 populations (Ku, 2006) and their genetic characteristics. On the other hand, internationally, six compounds such as quercitrin, camperol-3-OL-rhamnoside, quercetin-3-O- beta -D-galactoside, gallic acid, verrenin and quercetin .

The oxygen contained in the atmosphere is 21%, which is an essential element for most living organisms. However, conversion of oxygen to reactive oxygen species (ROS) causes fatal toxicity (Yoo, 2011). Therefore, lipid, protein, sugar, and DNA, which are constituents of cells, irreversibly cause various diseases such as cancer, inflammation, stroke, autoimmune disease and aging (Enrique et al., 2001).

On the other hand, antioxidants are substances that prevent our bodies from aging and damaging by active oxygen. Examples include synthetic antioxidants such as natural antioxidants such as superoxide dismutase (SOD), glutathione, and peroxidase, and flavonoids and polyphenols. At present, butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA), which are synthetic antioxidants, are widely used. However, consumers are avoiding this by reporting adverse effects on the toxicity of synthetic antioxidants (Branen, 1975; Pokorny, 2007).

Inflammation is a defense mechanism of the body by infection or stimulation of metabolites in vivo (Rocca et al., 2012). Macrophage NF-kB plays an important role in the expression of inflammatory factors such as iNOS and COX-2 (O'Connell et al., 1998). Thus, substances that inhibit inflammatory factors associated with macrophage activity can be useful in a variety of inflammatory studies and therapies. Currently, various steroids and non-steroidal anti-inflammatory drugs are used as therapeutic agents for inflammation.

Cancer is one of the most common causes of death in Korea. It occurs in the following order: gastric cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, and prostate cancer. The treatment of cancer is surgical, radiation, and drug therapy. Anticancer drugs for drug therapy often interfere with the function of proteins essential for cell survival to inhibit the growth of tumors by inhibiting the replication or transcription of DNA in cells. However, this causes cytotoxicity not only in cancer cells but also in normal cells, resulting in side effects such as vomiting, alopecia, diarrhea and impaired immunity (Hofman, 2004; Kang and Liang 1997; Park et al., 1995).

Skin increases melanin to inhibit skin cell damage caused by ultraviolet light. Melanin is converted to dopaquinone via DOPA (3,4-dihydroxyphenylalanine) by tyrosinase, TRP-1 and TRP-2, and is converted to dopaquinone by autoxidation and enzyme reaction in dopaquinone. (Alaluf et al., 2001). Therefore, in the development of whitening-related products, the inhibition of enzyme activity such as tyrosinase, TRP-1 and TRP-2 has been used as the primary evaluation method (Curto et al., 1999).

The currently used whitening agents include kojic acid, arbutin, hydroquinone, and vitamin C. These substances may be decomposed, colored, adhered, odorless, (Choung et al., 2010).

Obesity increases the incidence of adult diseases such as diabetes, hypertension, hyperlipidemia and cardiovascular disease (Spiegelman and Flier, 2001). In order to reduce these adverse effects, anti-obesity drugs, which have been proven to be stable, have been reported to have various side effects such as headache, depression, fat and cognitive impairment (Ballinger and Peikin, 2002; Lee, (Jeon et al., 2014; Choi et al., 2013; Shon et al., 2013) have been under development for many years.

Accordingly, the inventors of the present invention completed the present invention by confirming that the dog screen wind extract has an anti-cancer and anti-obesity activity as well as a skin improving effect while trying to develop a therapeutic agent using natural materials having excellent stability.

It is an object of the present invention to provide a composition containing an edible screen wind extract or a fraction thereof as an active ingredient and its use.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an extract of the canola or a fraction thereof as an active ingredient.

Further, the present invention provides a health functional food for improving cancer comprising an extract of planktonia or a fraction thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises an extract of a canola or a fraction thereof as an active ingredient.

In addition, the present invention provides a health functional food for improving inflammatory diseases containing an extract of the canola or a fraction thereof as an active ingredient.

In addition, the present invention provides an anti-inflammatory external preparation for skin comprising an open-celled leaf extract or a fraction thereof as an active ingredient.

In addition, the present invention provides an anti-inflammatory cosmetic composition comprising an edible bell-shaped extract or a fraction thereof as an active ingredient.

In addition, the present invention provides an anti-obesity agent containing an extract of a dog's screen or a fraction thereof as an active ingredient.

In addition, the present invention provides an external skin preparation for anti-obesity containing an extract of bentopneumoniae or a fraction thereof as an active ingredient.

In addition, the present invention provides a health functional food for an anti-obesity containing a dog screen wind extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for skin improvement comprising an extract of bentopneumoniae or a fraction thereof as an active ingredient.

The dog screen wind extract and its fractions of the present invention induce apoptosis of cancer cells and inhibit the production of nitric oxide, tyrosinase or elastase activity, and the differentiation of lipid precursor cells, and thus the anti-cancer, anti-inflammatory, anti-obesity Or as a composition for improving skin.

FIG. 1 is a graph showing the reducing power of the extract of the canola or its fraction:
M: methanol extract;
H: n-hexane fraction;
E: ethyl acetate fraction;
W: water fraction;
B: n-butanol fraction; And
A: ascorbic acid
FIG. 2 is a graph showing the inhibitory activity against nitric oxide formation of the extract of the canola or its fraction:
M: methanol extract;
H: n-hexane fraction;
E: ethyl acetate fraction;
B: n-butanol fraction; And
W: Water fractions.
FIG. 3 is a graph showing the tyrosinase inhibitory activity of the extract of the canola or its fraction:
M: methanol extract;
H: n-hexane fraction;
E: ethyl acetate fraction;
W: water fraction;
B: n-butanol fraction; And
K: Kojic acid.
4 is a graph showing the activity of inhibiting the enzyme activity of the extract of the canola or its fraction:
M: methanol extract;
H: n-hexane fraction;
E: ethyl acetate fraction;
B: n-butanol fraction;
W: water fraction; And
UA: Ursolic acid.
FIG. 5 is a graph showing the cytotoxicity of cancer cells extracts or fractions thereof on cancer cells:
M: methanol extract;
H: n-hexane fraction;
E: ethyl acetate fraction;
B: n-butanol fraction; And
W: Water fractions.
FIG. 6 is a graph showing cytotoxicity of normal leaf cell extract or fraction thereof to normal cells:
M: methanol extract;
H: n-hexane fraction;
E: ethyl acetate fraction;
B: n-butanol fraction; And
W: Water fractions.
FIG. 7A is a FACS result showing the activity of inducing apoptosis in HCT 116 cells of the ethyl acetate or butanol fraction of the canola extract.
FIG. 7B is a graph and a graph showing changes in the expression of genes that induce apoptosis of cancer cells by the butanol fraction of the canola extract.
FIG. 7C is a graph showing changes in the expression of proteins inducing apoptosis of cancer cells by the butanol fraction of the canola extract.
Fig. 8 is a graph and graph showing the neutral lipid inhibitory activity of the dog screen wind extract or its fractions. Fig.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising an extract of the canola or a fraction thereof as an active ingredient.

The dog screen can be cultivated or commercially available, and leaves, stems, branches or roots of dog screens can be used.

The dog screen wind extract can be produced by a manufacturing method comprising the following steps.

1) preparing an extract by adding an extraction solvent to a screen;

2) filtering the extract of step 1); And

3) Concentrating the filtered extract of step 2) under reduced pressure and drying.

The extraction solvent in step 1) may be water, alcohol or a mixture thereof. The alcohol may be a C ₁ to C ₄ lower alcohol, and specifically, the lower alcohol may be ethanol or methanol. At this time, the extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight.

In addition, the step 1) may be carried out using shaking, Soxhlet extraction, reflux extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction or ultrasonic extraction. Further, the extraction may be repeated one to five times, specifically three times.

In the above production method, the vacuum concentration in the step 3) may be carried out using a vacuum decompression concentrator or a vacuum rotary evaporator. The drying may be vacuum drying, vacuum drying, boiling drying, spray drying or freeze-drying.

The fraction of the extract of the canola blossom may be prepared by a method comprising sequentially extracting the extract with an organic solvent to prepare a fraction.

Specifically, the fraction of the dog screen wind extract can be a hexane fraction, an ethyl acetate fraction, an n-butanol fraction or a water fraction obtained by successively fractionating the extract of the dog screen with hexane, ethyl acetate, n-butanol and water. In one embodiment of the invention, the fraction may be n-butanol, specifically the water saturated n-butanol fraction.

The cancer may be a colon cancer, a colon cancer, a lung cancer, a liver cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, a breast cancer, a cervical cancer or a prostate cancer.

In a specific example of the present invention, the present inventors have found that the hexane fraction, the ethyl acetate fraction, the butanol fraction and the water fraction of the canopy screen from the canopy screening methanol extract are prepared, and the extract and the fraction of the canopy screening inhibit the growth of cancer cells (See FIG. 6). In addition, the extracts and fractions induced apoptosis and increased the expression of related factors (see FIGS. 7a to 7c).

Therefore, the dog screen wind extract and fractions of the present invention can be usefully used for the treatment of cancer.

The pharmaceutical composition according to the present invention may contain 10% to 95% by weight of the extract as an active ingredient or the fraction thereof, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further comprise at least one active ingredient which exhibits the same or similar functions in addition to the above-mentioned effective ingredients.

The pharmaceutical compositions of the present invention may comprise carriers, diluents, excipients or mixtures thereof conventionally used in biological preparations. Pharmaceutically acceptable carriers can be used as long as they are suitable for delivering the composition in vivo. Specifically, the carrier is selected from Merck Index, 13th ed., Merck & Sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. If necessary, conventional additives such as an antioxidant, a buffer, a bacteriostatic agent and the like may be added.

When the composition is formulated, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient may be added.

The compositions of the present invention may be formulated into oral or parenteral formulations. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, a pill, a powder, a granule, a capsule, or a troche. The solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or a mixture thereof. In addition, the solid preparation may comprise a lubricant, examples of which include magnesium stearate, talc, and the like. On the other hand, the liquid preparation may be a tongue, a solution, an emulsion or a syrup. At this time, the liquid preparation may contain additives such as a wetting agent, a sweetener, a fragrance, a preservative, and the like.

The parenteral preparation may contain injections, suppositories, respiratory inhalation powders, aerosol formulations for spraying, powders and creams. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, and the like. As the non-aqueous solvent or suspending agent, vegetable oils such as propylene glycol and olive oil, and injectable esters such as ethyl oleate may be used.

The composition of the present invention can be administered orally or parenterally according to the desired method. Parenteral administration may include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathecal injection.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, activity of the drug, sensitivity of the patient to the drug, administration time, route of administration, duration of treatment, However, for the desired effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg / kg, specifically 0.001 to 10 mg / kg. The administration may be one time a day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered concomitantly, the administration can be sequential or simultaneous.

Further, the present invention provides a health functional food for improving cancer comprising an extract of planktonia or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

The cancer may be a colon cancer, a colon cancer, a lung cancer, a liver cancer, a stomach cancer, a stomach cancer, a bile duct cancer, a breast cancer, a cervical cancer or a prostate cancer.

In a specific example of the present invention, the inventors of the present invention found that the dog screen wind extract, the hexane fraction, the ethyl acetate fraction, the n-butanol fraction and the water fraction of the canopy screen inhibit the growth of cancer cells (see FIG. 6) , And increased the expression of factors associated therewith (see Figs. 7a to 7c). Therefore, the dog screen wind extract and fractions of the present invention can be usefully used as a health functional food for cancer.

The composition of the present invention can be added directly to food or used together with other food or food ingredients. At this time, the content of the active ingredient to be added may be determined depending on the purpose, and may generally be 0.01 to 90 parts by weight of the total food weight. The form and kind of the health functional food are not particularly limited. Specifically, the health functional food may be in the form of a tablet, a capsule, a powder, a granule, a liquid and a ring. The health functional food may contain various flavors, sweeteners or natural carbohydrates as an additional ingredient. The sweeteners may be natural or synthetic sweeteners, examples of natural sweeteners include tau martin and stevia extract. On the other hand, examples of synthetic sweeteners include saccharin and aspartame. The natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides, sugar alcohols, and the like.

The health functional food of the present invention may contain, in addition to the above-described additional ingredients, a nutrient, a vitamin, an electrolyte, a flavoring agent, a colorant, a pectin and a salt thereof, an alginic acid and its salt, an organic acid, a protective colloid thickening agent, , Glycerin, alcohol, and the like. These components may be used independently or in combination. The proportion of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises an extract of a canola or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

The inflammatory disease may be bronchitis, gastritis, arthritis, inflammatory bowel disease, multiple sclerosis, hepatitis, cholecystitis, fungal infections, ulcer, dermatitis, enteritis, pancreatitis, tendinitis or nephritis.

The pharmaceutical composition may have the characteristics as described above.

In a specific example of the present invention, the present inventors confirmed that the dog screen wind extract, the hexane fraction, the ethyl acetate fraction, the n-butanol fraction and the water fraction of the canopy screen inhibit the production of nitrogen oxides in a concentration-dependent manner, The extract or fraction thereof can be effectively used as a composition for the treatment of inflammatory diseases.

In addition, the present invention provides a health functional food for improving inflammatory diseases containing an extract of the canola or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

The inflammatory disease may be bronchitis, gastritis, arthritis, inflammatory bowel disease, multiple sclerosis, hepatitis, cholecystitis, ulcer, dermatitis, enteritis, pancreatitis, retinitis, tendinitis or nephritis.

The health functional food may have the above-described characteristics.

In a specific example of the present invention, the present inventors confirmed that the dog screen wind extract, the hexane fraction, the ethyl acetate fraction, the n-butanol fraction and the water fraction of the canopy screen inhibit the production of nitrogen oxides in a concentration-dependent manner, The extract or its fractions can be effectively used as a health food for the improvement of inflammatory diseases.

In addition, the present invention provides an anti-inflammatory external preparation for skin comprising an open-celled leaf extract or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

The anti-inflammatory skin external agent may have anti-inflammatory activity against arthritis, dermatitis or retinitis.

In a specific example of the present invention, the present inventors confirmed that the dog screen wind extract, the hexane fraction, the ethyl acetate fraction, the n-butanol fraction and the water fraction of the canopy screen inhibit the production of nitrogen oxides in a concentration-dependent manner, The extract or its fractions can be effectively used as an anti-inflammatory external preparation for skin.

The external preparation for skin of the present invention may contain a cosmetically and dermatologically acceptable medium or base. It may be any formulation suitable for external application, for example, as a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic form (liposome) / Or in the form of non-ionic fodder dispersants, in the form of creams, powders, ointments, sprays, and the like. The external preparation for skin may be prepared according to a conventional method in the art. The external preparation for skin may also be used in the form of an aerosol containing a form of foam or a compressed additive.

In addition, the external preparation of the present invention can be used in the form of a solution or suspension of a lipid, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, Such as, but not limited to, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics And may contain adjuvants conventionally used in the scientific field. These adjuvants are introduced in amounts commonly used in the cosmetic or dermatological fields.

In addition, the present invention provides an anti-inflammatory cosmetic composition comprising an edible bell-shaped extract or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

The cosmetic composition may have anti-inflammatory activity against arthritis, dermatitis or retinitis.

In a specific example of the present invention, the present inventors confirmed that the dog screen wind extract, the hexane fraction, the ethyl acetate fraction, the n-butanol fraction and the water fraction of the canopy screen inhibit the production of nitrogen oxides in a concentration-dependent manner, Extracts or fractions thereof can be usefully used as an anti-inflammatory cosmetic composition.

Specific formulations of the cosmetic composition of the present invention may be formulated as skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisturizing lotions, nutrition lotions, massage creams, nutritional creams, A soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a latex, a pressed powder, a loose powder and an eye shadow.

The cosmetic composition may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening agents and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, As used in cosmetics or in cosmetics, in the form of tablets, capsules, tablets, pills, tablets, pills, emulsions, suspensions, emulsions or emulsions, fillers, sequestering and chelating agents, preservatives, vitamins, May also contain adjuvants customarily used in the cosmetics field, such as other ingredients of cosmetics.

In addition, the present invention provides an anti-obesity agent containing an extract of a dog's screen or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

In a specific example of the present invention, the present inventors have found that the n-butanol fraction of the dog screen can inhibit the formation of fat globules in a concentration-dependent manner (see FIG. 8) Respectively.

In addition, the present invention provides an external skin preparation for anti-obesity containing an extract of bentopneumoniae or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

The external preparation for skin may have the above-described characteristics.

In a specific example of the present invention, the present inventors have found that the n-butanol fraction of the dog screen can inhibit the formation of fat globules in a concentration-dependent manner (see FIG. 8) Respectively.

In addition, the present invention provides a health functional food for an anti-obesity containing a dog screen wind extract or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

The health functional food may have the above-described characteristics.

In a specific example of the present invention, the inventors of the present invention found that the n-butanol fraction of dog breasts suppresses the formation of lipid sphere in a concentration-dependent manner (see FIG. 8) Respectively.

In addition, the present invention provides a cosmetic composition for skin improvement comprising an extract of bentopneumoniae or a fraction thereof as an active ingredient.

The dog screen wind extract or its fractions may have the above-described characteristics. Specifically, the open screen may be cultivated or commercially available, and water, an alcohol or a mixture thereof may be used as an extraction solvent for preparing an extract. The extraction solvent may be added in an amount of 1 to 10 times the dry dog screen weight. The fraction may be a fraction that is sequentially fractionated with hexane, ethyl acetate, n-butanol, or water. Specifically, it may be a fraction that is fractionated with water saturated n-butanol.

The cosmetic composition may have the above-described characteristics.

In a specific example of the present invention, the inventors of the present invention found that the fraction of the dog's breeze inhibits the activity of tyrosinase (see FIG. 3) and inhibits the activity of the elastase in a concentration-dependent manner (see FIG. 4) It was confirmed that the screen wind extract or the fraction thereof can be used as a cosmetic composition for skin improvement.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> out of screen Fraction  Produce

The present inventors obtained a crude extract of methanol from Gangwon-do Institute of Natural Environment to prepare a fraction of the crude extract of the canola.

Specifically, 113 g of methanol crude extract was suspended in distilled water and then subjected to sequential solvent fractionation with n-hexane, ethyl acetate, n-butanol (water saturated) and water. 27.4 g of n-hexane fraction (24.2%), 37.6 g of ethyl acetate fraction (33.3%), 26.6 g of n-hexane fraction (24.2%), Butanol fraction (23.5%) and 21.4 g of water fraction (19%).

< Test Example  1> Confirmation of antioxidant activity

The antioxidant activity of the dog screen wind extract or its fractions of Example 1 was confirmed by the DPPH free radical scavenging method.

Specifically, 100 μl of the peritoneal membrane extract or its fraction was dispensed into a 96-well plate at a concentration of 1, 5, or 10 μg / ml, and then 100 μl of a 0.15 mM DPPH solution was added thereto. The mixture was reacted at room temperature for 30 minutes Absorbance was measured at 515 nm using a UV / VIS spectrophotometer (V530, Jasco Co, Japan). At this time, RC ₅₀ is the concentration of the compound which reduces the value of the control group without addition of the sample by 50%. As a control group, ascorbic acid, alpha -tocopherol and tert -butylhydroxyanisole (BHA), which are antioxidants, were used.

sample DPPH radical
Scavenging activity
IC ₅₀ ([mu] g / ml) Total phenol content
(mg GAE / g) Total flavonoid content
(mg QE / g) Methanol extract 7.03 0.09 ^c 466.9 ± 0.83 ^b 102.04 + 3.88 ^b Hexane fraction 9.40 + - 0.13 ^d 302.38 ± 5.39 ^d 62.07 + - 3.35 ^c Ethyl acetate
Fraction 2.90 ± 0.08 ^a 549.70 ± 2.72 ^a 154.58 ± 1.04 ^a Butanol fraction 4.80 ± 0.07 ^b 419.15 + - 1.91 ^c 24.75 ± 0.34 ^d Water fraction 21.63 ± 0.26 ^e 78.04 ± 2016 ^e 13.69 ± 0.06 ^e BHA 6.12 ± 0.27 - - alpha -tocopherol 10.60 0.40 - - Ascorbic acid 2.19 ± 0.06 - -

a: p-value mean value P < 0.05

As a result, as shown in Table 1, the ethyl acetate fraction of the dog breeze showed an RC ₅₀ value of 2.9 μg / ml, similar to ascorbic acid (2.19 μg / ml), a powerful antioxidant, and alpha-tocopherol and BHA (Table 1)

< Test Example  2> Determination of phenol and flavonoid contents in the screen

The content of phenol and flavonoid in the extract of the canola or its fraction of Example 1 was measured by the method of Folin Ciocalteu (1996).

First, 50 쨉 l of Folin Ciocalteu (Sigma Aldrich, USA) was added to 100 쨉 l screen or fraction thereof, and stabilized at room temperature for 5 minutes. Then, 300 쨉 l of 20% sodium carbonate was added. After stabilization at room temperature for 15 minutes, 1 ml of distilled water was added, and the absorbance was measured at 725 nm using a UV / VIS spectrophotometer. The content of phenol was measured by using gallic acid as a reference material and quantified as gallic acid equivalents (GAE).

On the other hand, to measure the content of flavonoid, 100 [mu] l of canola extract or its fractions were mixed with 20 [mu] l of 10% aluminum nitrate, 20 [mu] l of 1M potassium acetate and 860 [mu] l of 80% ethanol in this order and stabilized at room temperature for 40 minutes The absorbance was measured at 415 nm. The measured values were expressed as quercetin equivalents (QE) after preparing a calibration curve using quercetin as a standard substance.

As a result, as shown in Table 2, the total phenol content of the dog leaves was the highest in the ethyl acetate fraction, and was higher in the order of methanol, n-butanol, n-hexane and water fractions. On the other hand, the flavonoid content was also highest in the ethyl acetate fraction (Table 2).

< Test Example  3> Increasing Reduction Power  effect

The reducing power of the dog screen wind extract or its fraction of Example 1 was measured by modifying the method of Oyaize (1996).

Concretely, 100 占 퐇 of the canola extract or its fractions were mixed with 100 占 퐇 of 0.2 M sodium phosphate buffer (pH 6.6) and 100 占 퐇 of 1% potassium ferricyanide at a concentration of 10, 50 or 100 占 퐂 / ml, Lt; 0 &gt; C for 20 minutes. Thereto, 100 mu l of 10% trichloroacetic acid, 400 mu l of distilled water and 10 mu l of 0.1% ferric chloride were sequentially added and mixed. The absorbance of the mixture was measured at 700 nm using a spectrophotometer.

As a result, as shown in Fig. 1, the reducing power was increased depending on the concentration of the dog leaves or the fraction thereof. In particular, the ethyl acetate fraction and the methanol fraction showed a high reducing power. The ethyl acetate fraction at a concentration of 100 μl / ml had activity similar to the positive control ascorbic acid (FIG. 1).

< Test Example  4> Confirmation of the anti-inflammatory activity of the screen

The anti-inflammatory activity of the dog screen wind extract or its fractions of Example 1 was confirmed by measuring the inhibitory activity of nitric oxide production.

Specifically, RAW 264.7 cells were seeded in 96-well plates at 5 × 10 ⁵ cells / well and cultured for 24 hours at 5% CO _{2 and} 37 ° C. After the incubation, the medium was removed and the dog leaves or their fractions were treated at a concentration of 100, 200, or 500 / / ml, and treated with 100 ng / ml of LPS after 30 minutes. This was incubated again for 24 hours, and 50 μl of the Griess reagent and 50 μl of the culture supernatant were mixed and the absorbance was measured at 540 nm with an ELISA microplate reader.

As a result, as shown in Fig. 2, the generation of nitric oxide was inhibited in all treatment groups in a concentration-dependent manner. In particular, the ethyl acetate fraction showed the most excellent effect (FIG. 2).

< Test Example  5> Confirm skin improvement effect

<5-1> Confirm skin whitening effect

The whitening effect of the dog screen wind extract or its fractions of Example 1 was confirmed by measurement of tyrosinase inhibitory activity.

Specifically, 120 μl of 8.3 mM L-DOPA, 50 μl of tyrosinase (125 U) and the extract of canine or its fraction were added to each 10 μl of each sample by concentration (10 μl) using 67 mM sodium phosphate solution (pH 6.8) 50 or 100 占 퐇 / ml). 40 [mu] l of the prepared sample was dispensed into a 96-well plate, reacted at 37 [deg.] C for 30 minutes, and the absorbance at 490 nm was measured using an ELISA reader to quantify the DOPA chromium produced. At this time, a buffer was added to the control group instead of the sample, and kojic acid was used as the positive control group. The tyrosinase inhibition rate was calculated using the following equation (1).

&Quot; (1) &quot;

Inhibition rate (%) = (1-absorbance of sample-added group / absorbance of no-addition group) X 100

As a result, as shown in FIG. 3, about 62% of the group treated with kojic acid at a concentration of 10 μg / ml and the group treated with a concentration of 50 μg / Rocinase inhibition rate was shown. In particular, it was confirmed that the group treated with the ethyl acetate fraction at a concentration of 100 占 퐂 / ml showed a high inhibition rate of 77.9% and had an excellent whitening effect (Fig. 3).

<5-2> Confirmation of wrinkle and elasticity improvement effect

The wrinkle and elasticity-improving effect of the dog screen wind extract or its fractions of Example 1 was measured by the following method.

Specifically, a sample containing the extract of the canola or its fraction of 200, 500 or 1,000 / / ml was prepared in the same manner as in <Example 5-1>. To this was added 100 mM tris-HCl buffer (pH 8.6) in which the pig's yeast elastase was dissolved at 0.25 μg / ml and N-succinyl- (L-alanine) -3-P-nitroanil 100 mM tris-HCl buffer (pH 8.6) in which N-succinyl- (L-Ala) -3-P-nitroanilide was dissolved was added. The reaction was carried out at 37 ° C for 20 minutes, and the amount of p -nitroaniline produced from the substrate was determined by measuring the absorbance at 415 nm. The inhibition rate of the elastase was calculated using the above equation (1).

As a result, as shown in Fig. 4, the ethyl acetate fraction of the dog breeze was almost ineffective, but the group treated with the other fractions showed an elastase inhibitory activity in a concentration-dependent manner. This was similar to the positive control group ursolic acid (Fig. 4).

&Lt; Test Example 6 >

<6-1> Confirmation of growth inhibitory effect of cancer cells

The water-soluble tetrazolium salt (WST) -1 assay confirmed whether or not the dog screen wind extract of Example 1 or its fraction inhibited the growth of cancer cells.

Specifically, as the cancer cells, AGS as a cancer cell in the body, CoLo 205 as a colon cancer cell, HeLa as a cervical cancer cell, MCF 7 as a breast cancer cell, PC 4 as a prostate cancer cell and HCT 116 as a colon cancer cell were distributed from a Korean cell line bank . The cells were prepared by subculturing under DMEM or RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 ° C and 5% CO ₂ . The cultured cancer cell lines were recovered using a 0.25% trypsin-EDTA solution, counted, and dispensed into a 96-well plate at a concentration of 3 × 10 ⁴ cells / ml, 100 μl per well. The cells were further cultured for 24 hours in the same conditions as above, and then the extracts or their fractions were added at a concentration of 10, 50, 100 or 200 μg / ml. After 24 hours of culture, the degree of cell proliferation was measured. At this time, HEK 293 cell line was used as a control group.

As a result, as shown in FIG. 5, when treated with the dog leaf n-butanol fraction at a concentration of 200 μg / ml, the cell death rate was high in all cancer cells. In particular, HCT 116, a colon cancer cell line, exhibited a survival rate of 14.1%, indicating that the n-butanol fraction of the canine leaf extract had an excellent growth inhibitory effect on colon cancer cell lines (FIG. 5). On the other hand, normal cells did not show a growth inhibitory effect except for the case where the ethyl acetate fraction was added at a high concentration (500 or 1,000 占 퐂 / ml) (Fig. 6).

&Lt; 6-2 > Confirmation of induction of apoptosis of cancer cells

Whether or not the dog screen wind extract of Example 1 or its fractions induced cell death of cancer cells was confirmed by FACS.

First, the HCT 116 cell line was divided into 6 well plates at a concentration of 1 × 10 ⁵ cells / well and cultured at 37 ° C. and 5% CO ₂ for 24 hours. Ethyl acetate or n-butanol fraction was added at the concentration of 10, 50, 100 or 200 ㎍ / ㎖, and cultured for another 24 hours. After incubation, FACS was performed according to the manufacturer's protocol using the Annexin V-FITC apoptosis detection kit (Biovision, UK). Specifically, 5 쨉 l of anexin and 5 쨉 l of propidium iodide (PI) were added to stain the cells, and flow cytometry was performed using FACS Calibur (488 nm laser, 633 nm laser).

As a result, as shown in FIG. 7 (a), cancer cell death was increased in a concentration-dependent manner as the concentrations of ethyl acetate and n-butanol fractions in the dog breasts increased. In particular, 74% HCT 116 cells were killed when the n-butanol fraction of the canopy was treated at a concentration of 200 μg / ml. Thus, it was confirmed that the n-butanol fraction of the dog's screen was excellent in the cell death of colon cancer cells (FIG. 7A).

<6-3> Identification of expression of cell death-related factors

Whether or not the gene expression level of the apoptosis-related factor was changed by the fraction of the dog's screen of Example 1 was confirmed by the PCR test.

Specifically, HCT 116 cells were seeded at a concentration of 3 × 10 ⁵ cells / well in a 6-well plate, and the n-butanol fraction of the dog's mouth was added at a concentration of 10, 50, 100 or 200 ppm. After 24 hours of culture, RNA was isolated according to the manufacturer's protocol using a total RNA extraction kit (Cat.17211, iNtRON, Korea). The absorbance of the separated RNA was measured at 260 nm and 280 nm to confirm the purity (1.7-2.0) and the concentration of the RNA at OD ₂₆₀ . CDNA was synthesized by using a conventional method using quantified RNA, and caspase 3, caspase 9, and p53 gene were amplified using a primer shown in Table 2 as a template to confirm the expression change.

Name of the primer The sequence (5 '- &gt; 3') SEQ ID NO: β-actin F 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3 ' SEQ ID NO: 1 β-actin R 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3 ' SEQ ID NO: 2 p53 F 5'-GCGCACAGAGGAAGAGAATC-3 ' SEQ ID NO: 3 p53 R 5'-CTCTCGGAACATCTCGAAGC-3 ' SEQ ID NO: 4 Caspase-3 F 5'-GAACTGGACTGTGGCATTGA-3 ' SEQ ID NO: 5 Caspase-3R 5'-TGTCGGCATACTGTTTCAGC-3 ' SEQ ID NO: 6 Caspase-9 F 5'-TGGACGACATCTTTGAGCAG-3 ' SEQ ID NO: 7 Caspase-9R 5'-GCAAGATAAGGCAGGGTGAG-3 ' SEQ ID NO: 8

As a result, the expression of p53 and caspase-9 gene was increased depending on the concentration of the n-butanol fraction, as shown in Fig. 7B (Fig. 7B).

<6-4> Confirmation of expression of apoptosis inducing protein

The expression of the apoptosis-inducing protein by the fraction of the open mouth of Example 1 was confirmed by immunoblotting.

Specifically, 50 쨉 l of a lysis buffer (Triton X-100 to which 0.1 M PMSF was added) was added to the HCT 116 cell line, followed by reaction at 4 째 C for 30 minutes, followed by centrifugation at 4 째 C and 13,500 rpm for 5 minutes To obtain a supernatant. The amount of protein was quantified by a spectrophotometer using a bradford assay. The quantified protein was heated at 100 ° C for 5 minutes, separated by electrophoresis using 12% sodium dodecylsulfate (SDS) -polyacrylamide gel, and then transferred to a PVDF membrane (Bio Rad, USA). The PVDF membrane was pre-treated with 5% skim milk and diluted 1: 1000 as the primary antibody, Cell Signaling Technology (USA), Cell Signaling Technology (USA) or PolyADP- (PARP, Cell Signaling Technology, USA), and reacted overnight at 4 ° C. The cells were washed three times with PBS containing 0.5% Tween-20 for 10 minutes, and then reacted with a secondary antibody (Santa Cruz Biotechnology, USA) diluted 1: 1000 for 1 hour at room temperature. After the reaction, it was washed with PBS three times for 10 minutes, and protein bands were detected using ECL (enhanced chemiluminoeseence, Amersham Life Science Corp., USA) solution.

As a result, as shown in Fig. 7C, cleaved caspase-9, cleaved caspase-3 and PARP (3), which were truncated depending on the concentration of the n- Poly ADP ribose polymerase protein expression was increased. This indicates that the caspase-3 enzyme is activated by the n-butanol fraction of the dog's screen to induce cell death (Fig. 7C).

< Test Example  7> screen Anti-obesity  Verify Active

The anti-obesity activity of the n-butanol fraction of the open mouth of Example 1 was confirmed by oil red O staining.

Specifically, 3T3-L1 cells as lipid precursor cells were cultured in DMEM medium containing 10% bovine calf serum and 1% antibiotic (penicillin / streptomycin). The cells were divided into 6-well plates at a concentration of 2 X 104 cells / well, and then cultured in a confluent medium (100% confluent). On the other hand, the n-butanol fraction of the dog breasts was treated with 10% fetal bovine serum and 1% antibiotic (penicillin / streptomycin) and 10 μg / ml insulin, 1 mM DEX (Dexamethasone, Sigma Aldrich, 50, 100 or 200 μg / ml in a DMEM medium containing 0.5 mM IBM (Isobutylmethylxanthine, Sigma Aldrich, USA). The n-butanol fraction of the prepared dog screen was added to the cells after 48 hours of culture. Cells were then cultured in DMEM medium supplemented with 10% fetal bovine serum and 10 μg / ml insulin alone to induce differentiation into adipocytes.

10% formalin (Intron, Korea) was added to differentiation-induced 3T3-L1 cells and fixed at room temperature for 1 hour. The fixed cells were completely dried, and the oily red O reagent prepared by dissolving the oil red O solution in isopropanol for 24 hours was added to the accumulated fat spheres. The stained cells were suspended in isopropanol, the oily red O stained in the cell was eluted, and the absorbance at 510 nm was measured.

As a result, as shown in Fig. 8, n-butanol as an extract of the screen folds inhibited the formation of lipids in a concentration-dependent manner (Fig. 8).

<110> KANGWON PROVINCE          KANGWON NATIONAL UNIVERSITY INDUSTRY COOPERATION FOUNDATION <120> Composition comprising Astilboides tabularis extracts or fraction          as an active ingredient and use thereof <130> 2016P-08-032 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> beta actin forward primer <400> 1 tgacggggtc acccacactg tgcccatcta 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> beta actin reverse primer <400> 2 ctagaagcat ttgcggtgga cgatggaggg 30 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p53 forward primer <400> 3 gcgcacagag gaagagaatc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p53 reverse primer <400> 4 ctctcggaac atctcgaagc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> caspase-3 forward primer <400> 5 gaactggact gtggcattga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> caspase-3 reverse primer <400> 6 tgtcggcata ctgtttcagc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> caspase-9 forward primer <400> 7 tggacgacat ctttgagcag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> caspase-9 reverse primer <400> 8 gcaagataag gcagggtgag 20

Claims (15)

A pharmaceutical composition for preventing or treating cancer comprising a fraction of methanol extract of dog breed as an active ingredient,
Wherein the fraction is an ethyl acetate fraction or an n-butanol fraction obtained by sequentially fractionating an extract of methanol with n-hexane, ethyl acetate, n-butanol and water.
delete delete The pharmaceutical composition according to claim 1, wherein the n-butanol is water-saturated butanol.
The pharmaceutical composition according to claim 1, wherein the cancer is a colon cancer, a colon cancer, a lung cancer, a liver cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, a breast cancer, a cervical cancer or a prostate cancer.
The present invention relates to a health functional food for improving cancer comprising an extract of methanol extract as an active ingredient,
Wherein the fraction is an ethyl acetate fraction or an n-butanol fraction obtained by sequentially fractionating an extract of methanol with n-hexane, ethyl acetate, n-butanol and water. delete delete delete delete delete delete delete delete delete

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