KR20180026311A - Anti-aging Composition Including Dendranthema zawadskii Extract - Google Patents

Abstract

The present invention relates to a cell aging inhibiting composition including a Chrysanthemum zawadskii Herbich var. lucidum (Nakai) T. Lee extract as an effective component. More particularly, the present invention relates to health functional food and a pharmaceutical composition containing a Chrysanthemum zawadskii Herbich var. lucidum (Nakai) T. Lee extract. The Chrysanthemum zawadskii Herbich var. lucidum (Nakai) T. Lee extract of the present invention can treat or prevent cell aging-related diseases and inflammatory diseases.

Description

(Anti-aging Composition Including Dendranthema zawadskii Extract)

The present invention relates to a composition for inhibiting cell senescence comprising Ulleung chrysanthemum extract as an active ingredient, and to a composition for inhibiting cell senescence comprising Ulleung chrysanthemum extract useful for aging-related diseases.

The delay in the aging process, without degenerative disease, healthy and long-lasting without disease is an ongoing effort to find a substance that inhibits aging by the wind of all people. Oxidation by active oxygen species such as superoxide, hydrogen peroxide, hydroxyl group and singlet oxygen is one of the factors promoting aging. Free radicals generated by the metabolites of reactive oxygen species act on proteins, biomembranes, and DNA in vivo and cause oxidative damage. Many studies have been conducted on antioxidant components to suppress these effects, Researches are being actively conducted at home and abroad to search for new materials having action. However, since the aging phenomenon is not only caused by oxidation but is known to be influenced by numerous factors such as telomerase, cell replication ability, gene damage and recovery ability, it is necessary to evaluate the antioxidative effect of natural products and drugs, A method of using aging is needed.

Cell senescence is a phenomenon in which normal somatic cells can no longer divide after a certain number of divisions, contributing to the aging of individuals and tissues, and is an important mechanism for inhibiting abnormal cell proliferation and cancer formation. Cell senescence is caused by the shortening of the telomere at the end of the chromosome due to repeated somatic cell division, the increase of the activity of the cancer gene or the cancer suppressor gene, the excessive oxidative stress, the cytotoxic substance such as ultraviolet ray or radiation, And the like.

Aging cells increase SA -? - gal (senescence-associated? -Galatosidase) activity with morphological features such as increased shape, flattening, increased heterochromatin in the nucleus, (IGFBPs), interleukin-6 (interleukin-6), and transcriptional growth factor (IGFBPs), as well as increased amounts of proteins that inhibit cell growth such as p53, p16, ? , transforming growth factor (TGF-?), and interferon (Interferon).

Cell senescence not only contributes to the aging of individuals and tissues, but also plays an important role in the pathogenesis of various diseases. Aging cells are frequently observed in inflammatory lesions such as rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin injured tissue, and arteriosclerotic vascular tissue. In addition, cell senescence is observed in proliferative hyperplasia, hepatitis, and liver cancer.

As the aging cells accumulate, the aging cells do not divide well. Therefore, not only the damaged tissues are properly restored but also the enzymes that decompose the surrounding tissues or the inflammatory cytokines are secreted, accelerating the damage of the tissues. Contributes to the pathogenesis.

Since cellular aging is considered to be an essential cause of aging, efforts have been made to develop methods for delaying cellular aging. Among them, some researches have been conducted to search for substances capable of regulating cell senescence and to utilize them for the prevention and treatment of diseases.

Some of the substances known to regulate cell senescence include resveratrol, which is known to increase SIRT1 activity, and retinoic acid, which is used to delay cellular aging in keratinocytes. In addition, studies have been reported on the identification of substances capable of regulating cell senescence from plant extracts and their use in cosmetics and health functional foods.

On the other hand, Dendranthema is distributed in Europe and America including Korea, and especially in Dendranthema I have zawadskii . lucidum (Nakai) JHPark) is located in the mountain area of Sungbong in Ulleungdo, Gyeongbuk province. Leaves are deeply and relatively thinly split, with lenticular lanceolate, thick, luminous and petiole-like. Previous studies on Ulleung chrysanthemum have shown that the antioxidant effect of Ulleung chrysanthemum is superior to DPRKH radical scavenging ability and ABTS radical scavenging ability in flowers, leaves and stems of Ulleung chrysanthemum (Ureung chrysanthemum 2, Antioxidative effects of 80% ethanol extracts of two species of Dendranthema spp., Korean J. Plant Res., 23 (1): 47, 2010). However, the results of inhibiting the cell senescence of Ulleung chrysanthemum extract were not reported.

The present invention relates to a method for the production of < RTI ID = I have zawadskii . lucidum ) extract as an active ingredient.

Yet another object of the present invention is to provide a health functional food comprising a composition for inhibiting cell senescence comprising Ulleung chrysanthemum extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition comprising a composition for inhibiting cell senescence comprising Ulleung chrysanthemum extract as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above-mentioned problems of the present invention,

Dendranthema I have zawadskii . lucidum ) extract as an active ingredient.

According to another preferred embodiment of the present invention, the extract may be extracted from the ground part of Ulleung chrysanthemum.

According to another preferred embodiment of the present invention, the Ulleung chrysanthemum extract may inhibit SA-β-galactosidase activity.

According to another preferred embodiment of the present invention, the Ulleung chrysanthemum extract may inhibit the aging of fibroblasts.

According to another preferred embodiment of the present invention, the Ulleung chrysanthemum extract may inhibit inflammation-related factors.

According to another preferred embodiment of the present invention, the Ulleung chrysanthemum extract may be extracted with at least one solvent selected from the group consisting of water, alcohol, ethyl acetate, chloroform and hexane.

According to another preferred embodiment of the present invention, the composition may contain Ulleung chrysanthemum extract at a concentration of 2 to 10 μg / ml.

In addition, the present invention provides a health functional food comprising the composition for inhibiting cell senescence comprising the above-mentioned Ulleung chrysanthemum extract as an active ingredient.

The present invention also provides a pharmaceutical composition comprising the composition for inhibiting cell senescence comprising the above-described Ulleung chrysanthemum extract as an active ingredient.

The present invention relates to a composition for inhibiting cell senescence comprising Ulleung chrysanthemum extract as an active ingredient. The Ulleung chrysanthemum extract composition, a health functional food and a pharmaceutical composition containing the composition can treat or prevent cell senescence-related diseases. Particularly, inflammatory diseases such as blood inflammation, rheumatoid arthritis, osteoarthritis, hepatitis, chronic arteriosclerosis, or geriatric diseases.

Fig. 1 is a photograph of Ulleung chrysanthemum.
2 is a graph showing the results of MTT analysis of Ulleung chrysanthemum extract in fibroblast (HDF) (Doxo: doxorubicin 0.5 μM (27.18 μg / ml), 5: Ulleung chrysanthemum extract 5 μg / ml, μg / ml, 20: Ulleung chrysanthemum extract 20 μg / ml).
3 is a graph showing the results of MTT analysis of Ulleung chrysanthemum extract in fibroblasts (Doxo: doxorubicin 0.5 μM (27.18 μg / ml), 2: Ulleung chrysanthemum extract 2 μg / ml, 4: Ulleung chrysanthemum extract 4 μg / ml, 8: 8 μg / ml of Ulleung chrysanthemum extract, and 5 mM (815.97 μg / ml) of N-acetyl-L-cysteine).
FIG. 4 is a graph (A) and an optical microscope photograph (B) showing the inhibitory activity of SA-β-gal activity of Ulleung chrysanthemum extract on fibroblasts (HDF).
FIG. 5 is a graph (A) and an optical microscope photograph (B) showing the inhibitory activity of SA-β-gal activity inhibition in low concentration of Ulleung chrysanthemum extract in fibroblasts (HDF).
FIG. 6 is a graph showing the inhibitory effect of ROS caused by senescence in aged animal aortic blood vessels of Ulleung chrysanthemum extract. FIG.
Fig. 7 is an electrophoresis (A) and graph (B) showing that Ulreung chrysanthemum extract inhibits the transcription of NF-κB, an inflammation-related substance in the aortic blood vessels of aging animals.
FIG. 8 is an electrophoresis (A) and graph (B: COX-2, C: iNOS) showing that the extract of Ulreung chrysanthemum suppresses the expression of inflammation-related proteins COX-2 and iNOS in the aortic blood vessels of aged animals .
Fig. 9 is a graph showing the inhibitory effects of Ulleung chrysanthemum extract and other herb extracts on cell senescence.

Hereinafter, the present invention will be described in more detail.

As described above, studies on the antioxidative and anti-inflammatory activities of six Asteraceae plants (Korean Patent Laid-Open Publication No. 2014-0088504) are known, and a study on the radical scavenging ability of Ulleung chrysanthemum (Woo et al. Plant Res., 23 (1): 47, 2010). However, no studies have been conducted on the anti-inflammatory, anti-aging, and wrinkle and elasticity enhancement of Ulleung chrysanthemum extract.

Accordingly, the present invention provides a composition for inhibiting cell senescence comprising Ulleung chrysanthemum extract as an active ingredient.

The term " child face "in the present invention means a child-like face of an aged person, which means that the aging of the skin is suppressed, the skin is elastic, and there is no or little wrinkles.

The Ulleung chrysanthemum composition may be useful for the prevention of diseases such as aging-related diseases such as skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostatic hyperplasia or liver cancer.

In the present invention, MTT assay was performed on the composition containing the Ulleung chrysanthemum extract as an active ingredient, and it was confirmed that the Ulleung chrysanthemum extract had low cytotoxicity, and the cell viability was increased in the test group treated with Ulleung chrysanthemum extract. It was confirmed that SA-beta-gal activity was inhibited. Thus, it was confirmed that the extract of Ulreung chrysanthemum directly has an effect of inhibiting cell senescence, in particular, improving wrinkles of skin and improving skin elasticity.

The scientific name of Ulleung chrysanthemum in the present invention is Dendranthema I have zawadskii . lucidum (Nakai) JH Park or Chrysanthemum I have zawadskii . It is also called lucidum (Nakai) Y.Lee, and the tinnitus is also called island nationalization, mystic chrysanthemum, or Ulleung ryukcho (see Fig. 1).

It is preferable that the Ulleung chrysanthemum extract is extracted from the upper part of Ulleung chrysanthemum. The above ground part refers to a part of the upper part of the upper part of the plant which is not the root part in the plant, and in detail, it may be stem, leaf and flower.

The Ulleung chrysanthemum extract may be extracted with a solvent such as water, alcohol, ethyl acetate, chloroform or hexane, or a mixture thereof, more preferably a lower alcohol having from 1 to 4 carbon atoms. More preferably, it may be ethanol. The extract may be applied by a general solvent extraction method using the above-mentioned solvent, or it may be a fraction purified by using column chromatography. The method for producing the extract according to the present invention can be applied to any plant extraction method known to those of ordinary skill in the art. For example, extraction with water, alcohol or mixed solvent, extraction with hot water or room temperature But is not limited thereto.

 The composition containing the extracted Ulleung chrysanthemum extract as an active ingredient showed little cytotoxicity in fibroblasts (HDF) and significantly increased cell proliferation (see FIGS. 2 and 3).

In addition, we have found the anti-aging effect of Ulreung chrysanthemum extract. In order to prove this, the collagenase inhibitory activity evaluation, the elastase inhibitory activity evaluation, the DPPH activity evaluation, the Xanthine oxidase inhibition The activity of inhibiting cell senescence can be confirmed by various methods such as activity evaluation. Among the most common methods, SA -? - gal activity evaluation test (Dimri et al., A biomarker that identifies senescent human cells in culture and aging skin in vivo , Proc Natl Acad Sci 92 (1995): 9363-9367). As a result, it was confirmed that SA-β-gal activity was inhibited and cell senescence was inhibited (see FIGS. 4 and 5). In particular, it can be confirmed that the Ulleung chrysanthemum extract has a remarkably superior cytostatic effect in fibroblasts.

In addition, the composition containing the extract of Ulleung chrysanthemum as an active ingredient inhibited the formation of ROS in vascular tissues (see Fig. 6), confirming the transcriptional repression inhibitory effect of NF-κB, which is an inflammatory expression-related substance in vascular tissues ) And that inflammation-related proteins COX-2 and iNOS in vascular tissues were significantly inhibited (see FIG. 8). This suggests that the Ulrethrae chrysanthemum extract of the present invention can be applied not only to cell senescence but also to the treatment of inflammatory diseases. More preferably an aging inflammatory disease or vascular inflammation.

The composition for inhibiting cell senescence of the present invention may contain the Ulleung chrysanthemum extract at a concentration of 2 to 10 μg / ml. The solvent of the composition is not particularly limited as long as it does not interfere with the cell aging inhibitory effect of Ulreung chrysanthemum extract, and more preferably water, ethanol, propyl alcohol, DMSO, and the like.

As shown in FIGS. 4 and 5, the extract of Ulleung chrysanthemum showed an inhibitory effect on cell senescence, and the extract of Ulreung chrysanthemum with 5-20 .mu.g / ml showed significant reduction of senescence in fibroblasts, / ml of Ulreung chrysanthemum extract significantly reduced the aging of fibroblasts.

In the cytotoxicity test, Ulleung chrysanthemum extract was not cytotoxic even at a concentration of 10 μg / ml. Therefore, the concentration of the Ulleung chrysanthemum extract composition having no cytotoxic effect and exhibiting excellent cell senescence inhibitory effect may be more preferably 2 to 10 μg / ml.

The present invention also provides a health functional food comprising the composition for inhibiting cell senescence comprising the Ulleung chrysanthemum extract as an active ingredient. The kind of health functional food including the composition for inhibiting cell senescence comprising the extract of Ulreung chrysanthemum according to the present invention as an active ingredient is not particularly limited and examples thereof include meat, sausage, bread, chocolate, candy, snack, confection, pizza, Noodles, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes.

The health food is used together with other foods or food additives in addition to the Ulleung chrysanthemum extract, and can be suitably used according to a conventional method. For example, in order to prevent the aging of cells containing the Ulleung chrysanthemum extract as an active ingredient, Ulleung chrysanthemum extract is included as an active ingredient, and calcium, And then cooled to room temperature to prepare a beverage. In addition, the health supplement for prevention of cell senescence comprising the Ulleung chrysanthemum extract as an active ingredient is a nutritional supplement component (vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinamide), oligosaccharide , 50% ethanol and purified water were mixed and formed into granules, dried in a vacuum drier, passed through 12 to 14 mesh to uniformly prepare granules, extruded in suitable amounts to prepare tablets or powders, or hard capsules To obtain a hard capsule product.

 The effective dose of the Ulleung chrysanthemum extract contained in the health food may be used in accordance with the effective dose of the pharmaceutical composition, and the amount of the effective ingredient to be mixed may be suitably determined according to the purpose of use such as prevention or therapeutic treatment. In the case of long-term consumption intended for health and hygiene purposes or for health control purposes, it may be below the above range.

The present invention also provides a pharmaceutical composition comprising the composition for inhibiting cell senescence comprising the Ulleung chrysanthemum extract as an active ingredient.

The pharmaceutical composition can be administered to mammals such as rats, mice, livestock, humans, and the like through various routes including oral, transdermal, subcutaneous, intravenous or muscular. In addition, the pharmaceutical composition comprising the composition for inhibiting cell senescence comprising the extract of Ulreung chrysanthemum according to the present invention as an active ingredient can be formulated into various formulations. In the case of formulation, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient (e.g., starch, sucrose, lactose and gelatin) Can be mixed and prepared. In addition to simple excipients, lubricants can also be used. Examples of liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, glycerol, gelatin and the like may be used.

The dosage of the pharmaceutical composition may be varied depending on the age, sex, condition of the subject, the degree of absorption of the active ingredient in the body, the rate of inactivation and the rate of excretion, and the drugs used in combination. A daily dose that can be used without side effects can be administered in single or divided doses in an amount of 0.1 to 20 mg / kg.

When a pharmaceutical composition comprising a composition for inhibiting cell senescence comprising the extract of Ulleung chrysanthemum according to the present invention as an active ingredient is administered to a human body, it can be safely used because it has fewer side effects than other synthetic drugs because it is a natural extract .

The pharmaceutical composition and the health functional food can be used for treating or preventing a cell senescence-related disease and particularly for preventing or treating inflammatory diseases or geriatric diseases such as blood inflammation, rheumatoid arthritis, osteoarthritis, hepatitis and chronic arteriosclerosis , But is not limited thereto. In addition, the cosmetic composition may be useful for improving wrinkles and elasticity.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

1. Preparation of Ulleung chrysanthemum extract composition

On July 30, 2012, the Dendranthema I have zawadskii. lucidum (Nakai) JHPark) was used. The sample was dried and pulverized, extracted with ethanol at room temperature and then filtered. The solvent in the extract was removed using a vacuum concentrator, and water was removed from the freeze dryer and used in the experiment. The extract obtained from 100 g of the dried sample was 7.52 g, and the yield was 7.52%.

The obtained Ulleung chrysanthemum extract was dissolved in a solvent DMSO at a concentration of 100 μg / ml to prepare a Ulleung chrysanthemum extract composition.

2. Cell culture

Human dermal fibroblasts (HDFs) were plated at 1 × 10 ⁵ cells in a 100 mm diameter culture dish using a fibroblast culture medium (FGM-2, Lonza, Switzerland) containing 1% antibiotic, 37 in a 5% carbon dioxide incubator. If 80-90% of the cells were grown on the bottom of the culture dish, the cells were treated with 2.5 x trypsin-EDTA solution (Lonza, Switzerland) and subcultured.

The population doubling (PD) was examined by the following equation.

PD = log ₂ F / log ₂ I (F = number of cells last, I = number of cells first)

The fibroblasts used in the experiment were those having a number of divisions of 8 or less.

1. Cell aging induction and Ulleung chrysanthemum extract treatment

Doxorubicin (Sigma, USA) is known to kill cells at high concentrations but induce cell senescence at low concentrations. To induce senescence of the cells cultured by the method of Example 1-2, 150,000 fibroblasts were dispensed into a 100 mm diameter culture dish and cultured in a 37, 5% carbon dioxide incubator for 3 days. Then, the cell culture medium was removed , Cells were washed twice with fibroblast culture medium containing 1% antibiotic and treated with 0.5 μM doxorubicin for 4 hours.

Cells treated with 0.5 μM doxorubicin for 4 hours were dispensed into fibroblast culture medium containing 1% antibiotic in a 50 ml tube at a rate of 5,000 cells / ml and 100 μl in each well of a 96-well culture container. Finally, 500 fibroblasts per well were dispensed and cultured in 37, 5% CO2 incubator for one day. 100 μl of fibroblast culture medium containing 1% antibiotic was further added to each well, and the Ulleung chrysanthemum extract of the above-mentioned Example was treated. Ulleung chrysanthemum extracts were treated at 2, 4, 5, 8 or 20 μg / ml, respectively. The cells were then cultured for 3 days and stained with SA-β-gal.

As a negative control, DMSO was used as a negative control. To the positive control group, 5 mM (815.97 μg / ml) NAC was treated for 3 days and then toxicity was evaluated by MTT analysis.

2. Evaluation of cytotoxicity of Ulleung chrysanthemum extract- MTT  analysis

After the cells were cultured in a 37, 5% carbon dioxide incubator for 3 days after the treatment, the degree of cell growth was confirmed by MTT method. 50 μl of 0.1% MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide) solution (Sigma, USA) was added to each well of a 96- After incubation, the culture solution and MTT solution were removed, and 100 μl of DMSO was added to dissolve the crystals. The absorbance was measured at 550 nm using a microplate reader. The percentages (% The results are shown in Table 1, FIG. 2, and FIG.

Experimental group and control group Cell viability (% of Doxo) of fibroblasts (HDF) Negative control (Doxo 0.5 [mu] M + DMSO) 100 ± 2.6 Doxo 0.5 μM + Ulleung chrysanthemum 5 μg / ml 94.8 ± 2.7 Doxo 0.5 μM + Ulleung chrysanthemum 10 μg / ml 80.7 ± 2.7 Doxo 0.5 μM + Ulleung chrysanthemum 20 μg / ml 74.8 ± 3.1

Experimental group and control group Cell viability (% of Doxo) of fibroblasts (HDF) Negative control (Doxo 0.5 [mu] M + DMSO) 100 ± 2.6 Positive control (Doxo 0.5 uM + NAC 5 mM) 96.63 ± 1.00 Doxo 0.5 μM + Ulleung chrysanthemum 2 μg / ml 98.32 + - 0.69 Doxo 0.5 μM + Ulleung chrysanthemum 4 μg / ml 97.84 ± 2.91 Doxo 0.5 μM + Ulleung chrysanthemum 8 μg / ml 94.81 ± 0.28

As shown in Table 1 and FIG. 2, in the fibroblasts, the cell survival rate was 94.8 ± 2.7% at 5 μg / ml and 80.7 ± 2.7% at 10 μg / ml Cell viability and cell viability of 74.8 ± 3.1% at 20 μg / ml.

The results of the experiment with Ulleung chrysanthemum extract of lower concentration are shown in Table 2 and FIG. The cell viability was 98.32 ± 0.69% at 2 μg / ml, 97.84 ± 2.91% at 4 μg / ml, and 94.81 ± 0.28% at 8 μg / ml.

That is, as shown in FIG. 2 and FIG. 3, it can be confirmed that the cytotoxicity was little or insignificant at a concentration of 2 to 10 μg / ml.

1. Cell aging induction and Ulleung chrysanthemum extract treatment

In the same manner as in Example 2-1, 150,000 fibroblasts were dispensed into a 100 mm diameter culture dish, and the cells were cultured in a 37%, 5% carbon dioxide incubator for 3 days. Then, the cell culture was removed and the cells were treated with 1% After washing twice with the fibroblast culture medium, 0.5 μM doxorubicin was treated for 4 hours.

Cells treated with 0.5 μM doxorubicin for 4 hours were dispensed into a 50 ml tube in a fibroblast culture medium containing 1% antibiotic at 5,000 cells / ml and 100 μl in each well of a 96-well culture container. Finally, 500 fibroblasts per well were dispensed and cultured in 37, 5% CO2 incubator for one day. 100 μl of fibroblast culture medium containing 1% antibiotic was further added to each well, and the Ulleung chrysanthemum extract of the above-mentioned Example was treated. Ulleung chrysanthemum extracts were treated with 2, 4, 5, 8, or 20 μg / ml, respectively. The cells were then cultured for 3 days and stained with SA-β-gal.

Meanwhile, DMSO, a negative control, was treated with 5 mM (= 815.97 ug / ml) NAC as a positive control.

2. SA -? - Galactosidase active staining

The cells were cultured in 37, 5% CO2 incubator for 3 days after the treatment, and the effect of Ulleung chrysanthemum extract on cell senescence inhibition was investigated by SA-β-gal activity staining. β-galactosidase is exoglycosidase that hydrolyzes β-glycosidic bonds formed between galactose and organic matter. As aging progresses, a large amount of β-galactosidase accumulates in lysosomes of cells, bromo-4-chloro-3-indolyl- [beta] D-galactopyranoside. At this time, cells aged by SA-β-gal staining are stained cytoplasm in blue.

The Ulleung chrysanthemum extract was treated for 3 days, the cells were washed twice with phosphate buffer, the cells were fixed with 3.7% paraformaldehyde for 1 minute, and the fixative solution was removed. 5 mM potassium ferricyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2, X-gal 1 mg / ml) in a 24 well culture vessel Was added, and then wrapped in silver foil and reacted at 37 for 18 hours. The cells were washed twice with phosphate buffered saline (PBS), washed twice with phosphate buffered saline, and then stained blue with an optical microscope. The degree of SA-β-gal activity was determined by counting the number of cells stained blue in the cytoplasm among 50 cells, and the percentage of SA-β-gal activity in DMSO-treated group was 100% Respectively. These experimental results are shown as mean ± deviation in Table 2 or Figures 3 and 4, and statistical significance was determined by the Newman-keuls method.

Experimental group and control group SA-β-gal staining (%) in fibroblasts Negative control (Doxo 0.5 [mu] M + DMSO) 100 ± 0 Doxo 0.5 μM + Ulleung chrysanthemum 5 μg / ml 29.99 + - 8.84 Doxo 0.5 μM + Ulleung chrysanthemum 20 μg / ml 11.62 + - 0.60

Experimental group and control group SA-β-gel staining in fibroblasts (%) Negative control (Doxo 0.5 [mu] M + DMSO) 100 ± 0 The positive control (Doxo 0.5 mu M + 5 mM NAC) 82.07 ± 5.05 Doxo 0.5 μM + Ulleung chrysanthemum 2 μg / ml 61.19 + - 4.93 Doxo 0.5 μM + Ulleung chrysanthemum 4 μg / ml 54.81 + - 4.11 Doxo 0.5 μM + Ulleung chrysanthemum 8 μg / ml 39.35 + 2.46

As a result, SA-β-gal activity was 29.99 ± 8.84% at 5 μg / ml, and SA-β-gal was 11.62 ± 0.60% at 20 μg / ml in the fibroblasts Lt; / RTI >

The results of the experiment with lower concentration of Ulleung chrysanthemum extract are shown in FIG. The activity of SA-β-gal was 61.19 ± 4.93% at 4 μg / ml, 54.81 ± 4.11% and 39.35 ± 2.46% at 8 μg / ml at the concentration of 2 μg / Of SA-β-gal activity.

That is, as shown in FIG. 4 and FIG. 5, the extract of Ulreung chrysanthemum showed an excellent SA-β-gal activity reduction at a concentration of 2 to 20 μg / ml and showed excellent antioxidant activity.

1. An aging animal occurs in vascular tissue ROS  Inhibition

In order to analyze whether ROS produced in vascular tissues of aging animals was effectively inhibited, the ROS inhibitory effect of Ulleung chrysanthemum extract treatment was measured at the age of 15 months. A 5 - month - old rat was used as a negative control and a 15 - month - old rat was used as a positive control. The experimental group was divided into three groups: one group was administered 0.2 mg / kg three times daily for 1 week and the other group was administered 1 mg / kg three times daily for 1 week. A total of six experimental lets were used for each group, and the results were shown as the average of ROS inhibition values.

ROS measurements in vascular tissues were measured by the DCFDA method (LeBel et al., 1992). The final concentration of 25 μM DCFDA solution was added to an optimal volume of 250 μL of tissue homogenate and the change in fluorescence per minute was measured using a fluorescence microplate reader (Synergy HT, BIO-TEK, USA) at an excitation wavelength of 500 nm and an emission wavelength of 536 nm Respectively.

As a result, as shown in FIG. 6, a high ROS was observed in the vascular tissue of the 15-month-old rat, but it was found that the ROS generation was lower in the group treated with the Ullung chrysanthemum extract than in the 5-month-old rat. This means that the extract of Ulreung chrysanthemum effectively inhibits the generation of ROS in blood vessels and inhibits aging of cells.

2. In aging animal blood vessel tissue Inflammatory protein  Confirm

In order to analyze whether the expression of inflammatory proteins was effectively inhibited in the vascular tissues of aged animals, the amount of inflammatory protein expression was measured according to the treatment concentration of Ulleung chrysanthemum extract at the age of 15 months. A 5 - month - old rat was used as a negative control and a 15 - month - old rat was used as a positive control. Experimental groups were divided into three groups: one group was administered 0.2 mg / kg three times daily for 1 week and the other group was administered 1 mg / kg three times daily for 1 week. A total of six experimental lets were used for each group and the results were shown as mean values.

To measure the expression level of inflammatory proteins, a tissue homogenate solution containing a certain amount of protein was prepared and diluted with gel loading buffer (0.125M Tris-HCl (pH 6.8), 4% SDS, 10% 2-mercaptoethanol, 0.2% bromophenol blue) were mixed and heated at 80 for 5 minutes. Each sample was electrophoresed at 110 V for 2 hours in a 6-10% SDS-polyacrylamide mini-gel. Proteins were transferred to PVDF (polyvinylidene difluoride) membrane using tobinbuffer (25 mM Tris-HCl, 192 mM glycin, 20% methanol) and reacted with the primary antibody for 5 hours at room temperature. Washed three times with washing buffer and reacted with horseradish - peroxidase conjugated secondary antibody for 30 minutes at room temperature. After washing three times with wash buffer, phospholipid produced by adding ECL detection material was photographed with a chemiluminescence imaging system (Davinch-chemi, I-point, Korea) to confirm the signal (Habib A et al., 1993).

As a result, as shown in FIG. 7, in the aged rats, the result of 134 was shown, but in the group administered with 0.2 mg / kg of Ulleung chrysanthemum extract, about 110 was obtained, and 1 mg / kg In the group, about 78 results were obtained. In other words, it was confirmed that the transcription of NF-? B, an inflammation-related substance, was inhibited by the concentration of Ulleung chrysanthemum extract.

In addition, as shown in Fig. 8, it was confirmed that the extract of Ulreung chrysanthemum effectively inhibited the inflammatory expression-related proteins COX-2 and iNOS. In particular, the expression level of COX-2 protein is significantly lower than that of COX-2 expression in young 5-month-old rats (see FIG. 8C). This suggests that Ulleung chrysanthemum extract may be an effective remedy or remedy for inflammatory diseases.

1. Cell culture

Human umbilical vein endothelial cells (HUVECs) were cultured in a 100 mm diameter culture dish using a vascular endothelial cell culture medium (FGM-2, Lonza, Switzerland) containing 10% fetal bovine serum and 1% Were dispensed at 1 × 10 ⁵ , and then cultured in a 37% 5% carbon dioxide incubator. If 80-90% of the cells were grown on the bottom of the culture dish, the cells were treated with 2.5 x trypsin-EDTA solution (Lonza, Switzerland) and subcultured. At this time, EGM-2 culture medium was used for the culture.

The population doubling (PD) was examined by the following equation.

PD = log ₂ F / log ₂ I (F = number of cells last, I = number of cells first)

The fibroblasts used in the experiment were those having a number of divisions of 8 or less.

2. Cell aging induction and Ulleung chrysanthemum extract treatment

In the same manner as in Example 2-1, 150,000 HUVECs (Human Umbilical Vein Endothelial Cells) were dispensed into a 100 mm diameter culture dish and cultured for 3 days in a 37, 5% carbon dioxide incubator. Cells were washed twice with EMDM medium containing 1% antibiotic and treated with 0.5 μM doxorubicin for 4 hours.

Cells treated with 0.5 μM doxorubicin for 4 hours were seeded in a 50 ml tube in an EGM-2 culture medium containing 10% fetal bovine serum and 1% antibiotic at a concentration of 5,000 cells / ml, and 100 μl of each cell was dispensed into each well of a 96- Respectively. Finally, 500 HUVECs per well were dispensed and cultured in 37, 5% CO2 incubator for one day. 100 μl of EGM-2 culture medium containing 1% antibiotic was further added to each well, and the Ulleung chrysanthemum extract of the above-mentioned Example was treated. Ullung chrysanthemum extract was treated with 20 μ / ml. The cells were then cultured for 3 days and stained with SA-β-gal.

Meanwhile, DMSO, a negative control, was treated with 5 mM (= 815.97 ug / ml) NAC as a positive control.

3. Staining of SA-β-galactosidase activity

The cells were cultured in 37, 5% CO2 incubator for 3 days after the treatment, and the effect of Ulleung chrysanthemum extract on cell senescence inhibition was investigated by SA-β-gal activity staining. β-galactosidase is exoglycosidase that hydrolyzes β-glycosidic bonds formed between galactose and organic matter. As aging progresses, a large amount of β-galactosidase accumulates in lysosomes of cells, bromo-4-chloro-3-indolyl- [beta] D-galactopyranoside. At this time, cells aged by SA-β-gal staining are stained cytoplasm in blue.

The Ulleung chrysanthemum extract was treated for 3 days, the cells were washed twice with phosphate buffer, the cells were fixed with 3.7% paraformaldehyde for 1 minute, and the fixative solution was removed. 5 mM potassium ferricyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2, X-gal 1 mg / ml) in a 24 well culture vessel Was added, and then wrapped in silver foil and reacted at 37 for 18 hours. The cells were washed twice with phosphate buffered saline (PBS), washed twice with phosphate buffered saline, and then stained blue with an optical microscope. The degree of SA-β-gal activity was determined by counting the number of cells stained blue in the cytoplasm among 50 cells, and the percentage of SA-β-gal activity in DMSO-treated group was 100% Respectively. These experimental results are shown in Table 5 or FIG. 6 as mean ± deviation, and statistical significance was determined by Student's t-test method.

≪ Comparative Example 1 > Confirmation of cell aging inhibitory effect of various herb extracts

The extract was prepared and treated in the same manner as in Example 4, except that Acanthopanax senticosus, Seocheong Rhizoma, and Pocheon Rhizoma were used.

At this time, the extracts of Ulreung chrysanthemum, Mt., Mt. Seoheung, and Pocheon were DZ, G2, G3 and G4, respectively.

Experimental group and control group SA-β-gel staining in fibroblasts (%) Negative control (Doxo 0.5 [mu] M + DMSO) 76.5 ± 6.3 The positive control (Doxo 0.5 mu M + 5 mM NAC) 49.0 ± 3.9 Doxo 0.5 μM + Ulleung chrysanthemum 20 μg / ml 61.2 ± 3.1 Doxo 0.5 μM + 20 μg / ml acid clot 62.3 ± 6.2 Doxo 0.5 μM + Seoheung Chrysanthemum 20 μg / ml 61.4 ± 4.0 Doxo 0.5 μM + perianum 20 μg / ml 66.9 ± 3.4

As a result, as shown in Fig. 9, it was confirmed that the extract of Ulreung chrysanthemum showed an inhibitory effect on cell senescence increased by about 5% ~ 7% p as compared with that of Pocheon Rhizoma extract. In other words, the extract of Ulreung chrysanthemum is superior to the extracts of Acanthopanax senticosus, Seoheung Kwangseo and Pocheon Rhizoma extracts.

< Formulation example  1> Sanje  Produce

60 mg of Ulleung chrysanthemum extract, 100 mg of lactose and 10 mg of talc were mixed and filled into airtight bags to prepare powders.

< Formulation example  2> Preparation of tablets

10 mg of Ulrell chrysanthemum extract, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and tableted according to a conventional preparation method.

< Formulation example  3> Preparation of capsules

10 mg of Ulreung chrysanthemum extract, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and filled in gelatin capsules according to the conventional capsule preparation method to prepare capsules.

< Formulation example  4> Preparation of injection

10 mg of Ulreung chrysanthemum extract per 1 ampoule (2 ml), sterilized distilled water suitable for injection, and a pH adjuster suitable amount were prepared according to the usual injection preparation method.

< Formulation example  5> Liquid  Produce

20 mg of Ulleung chrysanthemum extract, 10 g of isomerized sugar, and 5 g of mannitol were dissolved in an appropriate amount of purified water to prepare an appropriate amount of purified water. The mixture was adjusted to 100 ml with purified water, The solution was packed in a bottle and sterilized to prepare a liquid preparation.

Hereinafter, examples of the formulation of the health functional food including the Ulrethrae chrysanthemum extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.

< Formulation Example 6 > Manufacture of Health Functional Foods

Ulleung Chrysanthemum extract 40 mg, a suitable amount of vitamin mixture (Vitamin A acetate 70 μg, vitamin E 1.0 mg, vitamin B ₁ 0.13 mg, Vitamin B ₂ 0.15 mg, Vitamin B ₆ 0.5 mg, Vitamin B ₁₂ 0.2 ㎍, Vitamin C 10 mg, (1 mg of biotin, 1.7 mg of nicotinic acid amide, 50 mg of folic acid, 0.5 mg of calcium pantothenate) and an appropriate amount of inorganic compound (ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium phosphate monobasic 15 mg, 55 mg of calcium, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were mixed to prepare a granule, and a health food was prepared according to a conventional method.

Claims (9)

A composition for inhibiting cell senescence comprising an extract of Ulmung chrysanthemum (Dendranthema zawadskii var. Lucidum) as an active ingredient.
The composition for suppressing cell senescence according to claim 1, wherein the Ulleung chrysanthemum extract is extracted from the ground part of Ulleung chrysanthemum comprising stem, leaf and flower.
The composition for inhibiting cell senescence according to claim 1, wherein the Ulleung chrysanthemum extract has an effect of inhibiting SA-β-galactosidase activity and improving wrinkles and skin elasticity.
The composition for inhibiting cell senescence according to claim 1, wherein the Ulleung chrysanthemum extract inhibits the aging of human dermal fibroblasts (HDFs).
The composition for inhibiting cell senescence according to claim 1, wherein the Ulleung chrysanthemum extract is extracted with at least one solvent selected from the group consisting of water, alcohol, ethyl acetate, chloroform and hexane.
The composition for inhibiting cell senescence according to claim 1, wherein the composition comprises Ulleung chrysanthemum extract at a concentration of 2 to 10 μg / ml.
A health functional food comprising a composition for inhibiting cell senescence comprising the extract of Ulreung chrysanthemum according to any one of claims 1 to 6 as an active ingredient.
[Claim 7] The health functional food for inhibiting cell senescence according to claim 7, wherein the health functional food is in the form of a capsule, tablet, granule, powder or beverage.
A pharmaceutical composition comprising a composition for inhibiting cell senescence comprising Ulleung chrysanthemum extract according to any one of claims 1 to 6 as an active ingredient.

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