KR101698234B1 - Composition for inhibiting cellular senescence comprising extracts of Evodia officinalis or rutaecarpine or limocitrin 3-O-rutinoside isolated from the same - Google Patents

Abstract

The present invention relates to a composition for inhibiting cell senescence comprising as an active ingredient ounce oil extract or rutaecarpine or limocitrin 3-O-rutinoside isolated therefrom, wherein the adriamycin The present invention also provides a pharmaceutical composition for inhibiting cell senescence. The present invention relates to a method of treating diseases such as aging-related diseases such as skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostate hyperplasia and liver cancer by inhibiting the cell senescence process of fibroblast or umbilical vein endothelial cells . ≪ / RTI > It is also expected to be useful for development of anti-aging functional food, anti-aging drug, and cosmetics which can inhibit cell senescence.

Description

[0001] The present invention relates to compositions for inhibiting cellular senescence comprising, as an active ingredient, extracts of oyster oil or rutaecarpine or 3,3-rutinoside isolated therefrom, isolated from the same}

The present invention relates to a composition for inhibiting cell senescence comprising as an active ingredient a crude oil extract or rutaecarpine or limocitrin 3-O-rutinoside isolated therefrom.

When normal somatic cells are divided a certain number of times, they can no longer divide and become aged. This is because the telomere at the end of the chromosome is becoming shorter and shorter in the process of cell division, resulting in DNA damage. Cellular senescence is induced not only by shortening of telomere but also by dysfunction of cancer gene and cancer suppressor gene, inflammation reaction, oxidative stress, anticancer agent, ultraviolet ray and radiation. Aging cells are large in size, flatter in shape, stop cell growth, have many signs of DNA damage in the nucleus, and secrete a variety of inflammatory proteins. And biochemically it is known that senescence-associated β-galactosidase (SA-β-gal) activity increases. Although senescence is induced by a variety of factors, it has been shown that senescence is regulated through the signaling pathway of p53 and Rb / p16 tumor suppressor genes.

Cell senescence also inhibits or promotes cancer, and is suggested as an important mechanism of tissue regeneration and restoration, tissue / individual aging and aging related diseases. In addition, cellular senescence contributes to the pathogenesis of various aging-related diseases such as cancer, arteriosclerosis, skin aging, degenerative neurological diseases, myopenia, osteodystrophy, and hyperplasia of the prostate. Recent studies have shown that selective regulation of cell senescence can regulate tissue, organ aging, health life span, and the development of aging-related diseases. Telomerase-deficient mice are known to be aging rapidly, confirming that increasing telomerase expression in old telomeres-deficient mice reverses the degenerative changes of tissues or organs due to aging. In a mouse model with rapid senescence, selective removal of cells expressing p16, which is known to increase expression in senescent cells, inhibits senescence-induced tissue lesions and reduces the incidence of aging-related diseases. In the course of hepatic fibrosis in mice, aging of hepatic stellate cells occurs. It is known that aging of hepatic stellate cells inhibits excessive liver fibrosis. If the p53 activity is not properly regulated, the aging will occur quickly, but the proper p53 activity is known to inhibit aging.

Studies have also been reported on substances that have the potential to inhibit cellular senescence. Drugs or single components such as vitamin C, N-acetylcysteine, NS398 and epifriedelanol inhibit this cell senescence. In rapamycin-induced mouse models, 4,4'-diaminodiphenylsulfone has been reported to inhibit the development of aging-related diseases in small nematodes and to increase health life span.

It is native to China and is planted in Gyeongju province. Its height is 5m and hairy on young branches. In one room, fruits are used as medicines. The ingredient contains a volatile essential oil. The main ingredients are evoden, ocimene, and evodin. The alkaloids contain evodiamine and rutaecarpine. It is known to be used as an anticancer, antiparasitic agent, detoxification and diuretic agent. Anticancer activity, antiinflammatory effect, cardiovascular disease-related efficacy and cirrhosis-inhibiting effect have been reported.

Korean Patent No. 10-11812164 discloses a pharmaceutical composition for the prevention and treatment of inflammatory diseases or infectious diseases comprising quinolone alkaloid compounds derived from Evodia fructus as an active ingredient, Or cold, but there is no mention of inhibiting umbilical vein endothelial cells or fibroblast aging as in the present invention.

It is an object of the present invention to provide a composition for inhibiting cell senescence comprising as an active ingredient ounce oil extract or rutaecarpine or limocitrin 3-O-rutinoside isolated therefrom .

In order to achieve the above object, the inventors of the present invention investigated whether the eight monoclonal antibodies isolated from the crude oil extract and the crude oil were effective in inhibiting cell senescence in human vascular endothelial cells and fibroblasts. As a result, it was confirmed that the extracts of ethyl acetate in human vascular endothelial cells and the extracts of methanolic extract of oolong oil, EO-1 (rutaecarpine) and EO-8 (limocitrin 3-O-rutinoside) Thereby completing the invention.

The present invention relates to a pharmaceutical composition comprising a crude oil extract or rutaecarpine represented by the following general formula (1) separated therefrom or limocitrin 3-O-rutinoside represented by the general formula (2) A pharmaceutical composition for inhibiting cell senescence is provided. Specifically, the cell is a fibroblast.

≪ Formula 1 >

(2)

R ₁ a = OH, R ₂ = OCH ₃ Im.

Specifically, the sour milk extract is an ethyl acetate (EtOAc) fraction extract obtained by adding distilled water, n-hexane, ethyl acetate (EtOAc) and butanol to a methanol extract of sour milk, The cell is characterized by being an umbilical vein endothelial cell.

Specifically, the cell senescence is characterized by being induced by adriamycin, and inhibition of cell senescence is measured by measuring senescence-associated beta-galactosidase (SA-beta-gal) .

In the case of the pharmaceutical composition of the present invention, the pharmaceutical composition is pharmaceutically acceptable in addition to the oolong oil extract or rutaecarpine or limocitrin 3-O-rutinoside isolated therefrom. Such pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin , Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like , But is not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. as an additive.

In the above pharmaceutical composition, the administration method is determined according to the degree of symptom of cell senescence. Usually, local administration method is preferable. The dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and may be administered once to several times per day.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The pharmaceutical composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient, or may be prepared by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions, or may be in the form of elixirs, excipients, powders, granules, tablets, alerts, lotions, ointments and the like.

Meanwhile, the pharmaceutical composition may treat any one selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin injured tissue, arteriosclerosis, prostatic hyperplasia and liver cancer, but is not limited thereto.

The inventors of the present invention found that oats extract or rutaecarpine or limocitrin 3-O-rutinoside isolated therefrom inhibits cell senescence by adriamycin and also inhibits replication due to cell division And inhibited aging. By inhibiting the cell senescence process, it can be useful for treating diseases such as aging-related diseases such as skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostatic hyperplasia and liver cancer. It is also expected to be useful for the development of anti-aging functional food, anti-vascular aging drug, and cosmetics which can inhibit cell aging of vascular endothelial cells and fibroblasts.

Fig. 1 is a schematic view showing the separation of the crude oil extract.
Fig. 2 shows the chemical composition of a single component separated from flax oil.
Fig. 3 shows the cytotoxicity of oolong oil extract and the inhibitory effect of adriamycin on cell senescence in human umbilical vein endothelial cells. A, Cytotoxic effect of oolong oil extract. Each extract was treated with 10, 100 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active staining and SA-β-gal active staining percentage. After treatment with adriamycin, the crude oil extract was treated with 10, 100 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 4 shows the cytotoxicity of the ethyl acetate extract of pomegranate juice and the cytotoxic effect of adriamycin on human umbilical vein endothelial cells. A, and the cytotoxic effect of ethyl acetate extract. Each extract was treated with 1-10 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B and C, SA-β-gal active staining and SA-β-gal active staining percentage. After treatment with adriamycin, ethyl acetate extract of citrus oil was treated with 1-10 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. D, p53, phosphorylated S6K, p21. After treatment with adriamycin, the extract was treated with 1-10 ug / ml, and the degree of expression of each protein was determined by Western blotting. NT, untreated; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
FIG. 5 shows the inhibitory effect of ethyl acetate extract of oyster oil on the reproduction cell senescence in human umbilical vein endothelial cells. A, the cytotoxic effect of the ethyl acetate extract of citrus oil on the reproductive aging cells. Each extract was treated with 1-10 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B and C, SA-β-gal active staining and SA-β-gal active staining percentage. Reproductive aging cells were treated with 10 ug / ml of ethyl acetate extract of citrus milk and 3 days later, SA-β-gal active staining was performed. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Old, Old cells; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 vs. DMSO.
FIG. 6 shows the cytotoxicity of single component of folic acid and the inhibition of cell senescence by adriamycin in the umbilical vein endothelial cells of human umbilical vein. A, cytotoxic effect of single component of folate. Each component was treated with 10 ug / ml, cultured for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active stain percentage. After adriamycin treatment, EO-4 was treated with 10 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine.
Fig. 7 shows the cytotoxicity of single component of folate oil and inhibition of cell senescence by adriamycin in human umbilical vein endothelial cells. A, cytotoxic effect of single component of folate. Each component was treated with 10 ug / ml, cultured for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active stain percentage. After adriamycin treatment, EO-3 and EO-7 were treated with 1 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 8 shows the cytotoxicity of folic acid single component EO-1 in the umbilical vein endothelial cells of the umbilical vein and the cell aging inhibitory effect by adriamycin. A, EO-1. EO-1 was treated with 0.1 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active stain percentage. After adriamycin treatment, EO-1 was treated with 0.1 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine.
Fig. 9 shows cytotoxicity of oolong oil extract in human fibroblasts and inhibition of cell senescence by adriamycin. A, Cytotoxic effect of oolong oil extract. Each extract was treated with 10, 100 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active staining and SA-β-gal active staining percentage. After treatment with adriamycin, the crude oil extract was treated with 10, 100 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 10 shows the cytotoxicity of the methanol extract of oyster oil from human fibroblasts and the inhibitory effect on cell aging by adriamycin. A, indicating the cytotoxic effect of methanol extract of sour milk. Each extract was treated with 1-10 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B and C, SA-β-gal active staining and SA-β-gal active staining percentage. After treatment with adriamycin, the methanol extract of sour milk was treated with 1-10 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. D, p53, phosphorylated S6K, p21. After treatment with adriamycin, the extract was treated with 1-10 ug / ml, and the degree of expression of each protein was determined by Western blotting. NT, untreated; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 11 shows the effect of the methanol extract of oyster oil on inhibition of cell senescence in human fibroblasts. A, cytotoxic effect of methanol extract of sour milk on reproductive aging cells. Each extract was treated with 1-10 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B and C, SA-β-gal active staining and SA-β-gal active staining percentage. Reproductive aging cells were treated with 1-10 ug / ml of methanol extract of citrus milk and 3 days later, SA-β-gal active staining was performed. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Old, Old cells; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 12 shows cytotoxicity of single component of folic acid in human fibroblasts and cytotoxic effect of adriamycin on cell senescence. A, cytotoxic effect of single component of folate. Each component was treated with 10 ug / ml, cultured for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active stain percentage. After adriamycin treatment, EO-4 and EO-8 were treated with 10 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 13 shows the cytotoxic effect of adriamycin on the osmolysis of single component EO-8 (limocitrin 3-O-rutinoside) in human fibroblasts. A and B, SA-β-gal active staining and SA-β-gal active staining percentage. After treatment with adriamycin, EO-8 was treated with 1-10 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. C, p53, phosphorylated S6K, p21. After treatment with adriamycin, EO-8 was treated with 1-10 ug / ml, and the degree of expression of each protein was examined by Western blotting. NT, untreated; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Figure 14 shows the effect of EO-8 (limocitrin 3-O-rutinoside) on the production of active oxygen in human fibroblasts. A, active oxygen flow cytometry. B, the average amount of active oxygen. The fibroblasts were treated with adriamycin, treated with 10 μg / ml of EO-8, and the degree of active oxygen in the cells was examined by flow cytometry. Each experiment was repeated three times or more independently and then expressed as mean and standard deviation. ADR, adriamycin; Y, young cells; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 15 shows the effect of EO-8 [limocitrin 3-O-rutinoside] on human cell fibroblasts in inhibiting the senescence of the cloned cells. A, SA-β-gal active dye images. B, SA-β-gal active staining percentage. C and EO-8. Reproductive aging cells were treated with 1-10 μg / ml of EO-8 and stained for SA-β-gal activity 3 days later. Cytotoxicity was assessed by MTT assay. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Old, Old cells; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 16 shows the cytotoxicity of folic acid single component EO-1 [rutaecarpine] in human fibroblasts and the cytotoxic effect of adriamycin on cell senescence. Cytotoxic effects of A and B, EO-1. C and D, SA-β-gal active staining and SA-β-gal active staining percentage. After adriamycin treatment, EO-1 was treated with 0.1 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. E, p53, phosphorylated S6K, p21. After treatment with adriamycin, EO-1 was treated with 0.1 μg / ml and the degree of expression of each protein was examined by Western blotting. NT, untreated; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Figure 17 shows the effect of EO-1 [rutaecarpine] on the production of active oxygen in human fibroblasts. A, active oxygen flow cytometry. B, the average amount of active oxygen. The fibroblasts were treated with adriamycin, treated with 0.1 μg / ml of EO-1, and the degree of active oxygen in the cells was examined by flow cytometry. Each experiment was repeated three times or more independently and then expressed as mean and standard deviation. ADR, adriamycin; Y, young cells; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 18 shows the effect of EO-1 [rutaecarpine] on human cell fibroblasts in the inhibition of cell senescence. A, SA-β-gal active dye images. B, SA-β-gal active staining percentage. C and EO-1. Reproductive aging cells were treated with 0.1 μg / ml of EO-1 and subjected to SA-β-gal active staining three days later. Cytotoxicity was assessed by MTT assay. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Sour milk  Extraction Separation and Chemical Composition of a Single Component

1. Sour oil extract and single component separation

9.0 kg of crude oil was refluxed with MeOH (12 L) at about 60 ° C for 12 hours and then concentrated under reduced pressure to obtain 1.23 kg of MeOH extract. After this suspension MeOH extract the H ₂ O 1.5 L was added to the shaking, the same amount of n - hexane was added to (n -Hexane), EtOAC, n -BuOH and the solvent fraction n - hexane (n -Hexane) 73.0 g of fraction, 308.68 g of EtOAC fraction, 198.89 g of n- BuOH fraction and 630.62 g of water layer were obtained.

A 70 × 6 cm size Silica-gel (230-400 mesh) column was loaded, loaded with 26.4 g of EtOAC fraction and eluted with mobile phase solvent [ n- Hexane: EtOAC] fraction (EOE1 ~ 22). EO-1 (890 mg), EOE-15 (48.0 mg), and fraction EOE-9 were obtained by recrystallization of fraction EOE-9 (1.2 g), fraction EOE-10 (110.0 mg), fraction EOE- 2 (26.0 mg), EO-3 (10.0 mg) and EO-4 (31.0 mg). EOE-18 and fraction EOE-16 were loaded on a RP-18 column (size 60 × 2.0 cm) and eluted with mobile phase solvent [MeOH: H ₂ O] 12.0 mg) and EO-7 (20.0 mg).

A diaion column with an 80 × 10 cm size was loaded, and 130.0 g of H ₂ O fraction was loaded. The gradient elution [H ₂ O: MeOH] was confirmed by TLC using mobile phase solvent. Five subfractions (EOW1 ~ 5). Fraction EOE-4 (2 g) was loaded on a 75 x 4.0 cm RP-18 column and gradient eluted with mobile phase solvent [MeOH: H ₂ O] to give 15 subfractions (EOW4-1-15 ). EO-8 (10.0 mg) was obtained by loading fraction EOE-4-8 (200.0 mg) onto a RP-18 column of 60 × 2.0 cm size and using a mobile phase solvent [MeOH: H ₂ O] ). The chemical structure of the compounds was determined by using spectroscopic methods such as MS, NMR ( ¹ H, ¹³ C, DEPT, ¹ H- ¹ H COZY, HMQC and HMBC) Respectively.

2. Physico-chemical properties of separated materials

Eight kinds of compounds separated from EtOAC fraction and water layer of oolong oil were analyzed by various spectroscopic methods such as MS and NMR ( ¹ H, ¹³ C, DEPT, ¹ H- ¹ H COZY, HMQC, and HMBC) EO-1 [rutaecarpine], EO-2 [evodiamine], EO-3 [7-hydroxyrutaecarpine], EO-4 [ (1-Methyl-2 - [(Z) -9-pentadecenyl] -4 (1H) -quinolone), EO- EO-7 [dihydroevocarpine], EO-8 [remorse citrin-3-yl] -9-pentadecenyl] -4 (1H) -quinolone)], EO-6 [evocarpine] Limocitrin 3-O-rutinoside] (Fig. 2).

EO- 1 ( Rutaecarpine ): Pale yellow amorphous solid; _{^{1 H-NMR (CDCI 3,}} 250 MH z) 9.80 (1H, s, NH), 8.30 (1H, dd, J = 8.0,1.2 Hz, H-19), 7.77 (1H, ddd, J = 8.2, 7.0 , 1.2 Hz, H-17), 7.70 (1H, brd, J = 8.0 Hz, H-9), 7.64 (1H, brd, J = 8.2 Hz, H- 8.2, 7.0 Hz, H-11), 7.15 (1H, brdd, J), 7.27 (1H, brdd, J = = 8.0, 7.0 Hz, H-10), 4.56 (2H, t, J = 7.0 Hz, H-5), 3.22 (2H, t, J = 7.0 Hz, H-6); ¹³ C-NMR (CDCl ₃ , 62.9 MHz) 161.5 (C-5), 147.2 (C-la), 145.3 4), 127.2 (C-3a), 126.9 (C-1), 126.2 (C-3), 125.6 19.6 (C-8); Positive FAB-MS m / z 288 [M + H] &lt; ⁺ &gt;.

EO- 2 ( Evodiamine ): Pale yellow plates; _{^{1 H-NMR (CDCI 3,}} 250 MHz) 2.48 (NCH 3, s), 2.94 (2H, m), 3.25 (1H, m), 4.87 (1H, m), 7.19 (4H, m), 7.43 (2H , 7.58 (1H, d, J = 7.8 Hz), 8.10 (1H, dd, J = 1.4, 7.8 Hz), 8.32 (1H, s); ¹³ C-NMR (CDCl ₃ , 62.9 MHz) 164.8 (C-21), 150.7 (C-15), 136.7 (C-12), 126.0 (C-8), 124.0 (C-10), 123.7 ), 113.6 (C-7) , 111.3 (C-12), 68.8 (C-3), 39.5 (C-5), 37.2 (N-CH 3), 20.1 (C-6); Positive FAB-MS m / z 304 [M + H] &lt; ⁺ &gt;.

EO- 3 (7- Hydroxyrutaecarpine ): Pale yellow powder (10.0 mg); ¹ H-NMR (pyridine- d ₅ , 250 MHz) 13.3 (1H, s, 1-NH), 9.3 D, J = 8 Hz, 1H), 7.7 (1H, ddd, J = (1H, d, J = 8.5 Hz, H-16), 7.5 (1H, dd, J = 5.5, 1.5 Hz, H- Hz, H-18), 7.3 (1H, ddd, J = 8.5,7,1 Hz, H-11), 7.2 (1H, ddd, J = , dd, J = 16.8, 1.5Hz, H-6eq), 3.4 (1H, dd, J = 16.8,5.5Hz, H-6ax); _{^{13 C-NMR (pyridine- d 5}} , 62.9 MHz) 161.6 (C-21), 148.6 (C-15), 145.1 (C-3), 139.9 (C-13), 134.6 (C-17), 127.7 ( (C-18), 127.0 (C-16), 127.2 (C-18), 127.1 -10), 120.3 (C-9), 115.5 (C-7), 112.8 (C-12), 74.6 (C-5), 28.5 (C-6); Positive FAB-MS m / z 304 [M + H] &lt; + &gt;.

EO- 4 ( Wuchuyuamide 1): Amorphous powder; _{^{1 H-NMR (pyndlne- d 5}} , 250 MHz) 11.66 (1H, s, 1-NH), 8.26 (1H, dd, J = 6.4, 1.6 Hz, H-19), 7.77 (1H, d, J = (1H, m, H-11), 7.15 (1H, t, J = 7.6 Hz, H-18), 7.07 d, J = 8.4 Hz, H-16), 7.02 (1H, t, J = 7.4 Hz, H-10), 6.99 (1H, d, J = 8.0 Hz, H-12) , 7-OH), 4.84, 4.76 (each 1H, m, H-5), 3.36 (3H, s, H-22), 2.90, 2.82 (each 1H, m, H-6); _{^{13 C-NMR (pyridine- d 5}} , 62.9 MHz) 180.8 (C-2), 161.6 (C-21), 150.8 (C-3), 142.9 (C-13), 140.8 (C-15), 134.7 ( C-17), 133.3 (C-8), 129.4 (C-11), 128.5 (C-19), 124.8 -20), 114.1 (C-16), 110.2 (C-12), 75.7 (C-7), 37.5 (C-5), 36.3 (C-6), 30.4 (C-22). Positive FAB-MS m / z 352 [M + H] &lt; + &gt;.

EO -5 (1- Methyl- 2 - [(Z) -9- pentadecenyl ] -4 (1H) -quinolone ): Colorless oil; _{^{1 H-NMR (CDCI 3,}} 250 MHz) 8.5 (1 H, dd, J = 1.5 Hz, 8.0 Hz, H-5), 7.7 (1H, m, H-7), 7.6 (1H, d, J = 8.0 (1H, s, H-3), 5.3 (2H, m, olefinic proton), 3.8 (3H, s, (2H, m, H-2 '), 1.25-1.47 (16H, m, H- , m), 0.9 (3H, J = 7.0 Hz, H-15 '); ¹³ C-NMR (CDCl ₃ , 62.9 MHz) 177.5 (C-4), 154.7 (C-2), 141.7 (C-8a). (C-10), 129.9 (C-9), 126.3 (C-5), 126.2 (C-3), 34.6 ( C-1 '), 34.0 (N-CH 3), 31.8 (C-13'), 29.1 (C-7 '), 28.9 (C-6'), 28.5 (C- 2 ', 3', 6 ', 8'), 22.2 (C-12 '), 22.5 '), 14.0 (C-15'). Positive FABMS m / z 367 [M + H] &lt; ⁺ &gt;.

EO- 6 ( Evocarpine ): Colorless oil; _{^{1 H-NMR (CDCI 3,}} 250 MHz) 8.28 (1H, dd, J = 1.5, 8.0 Hz, H-5), 7.51 (1H, m, H-7), 7.33 (1H, d, J = 8.0 Hz (2H, m, olefinic proton), 3.56 (3H, s, N-H), 7.21 (1H, t, J = 8.0Hz, H-6), 6.03 (2H, m, H-2 '), 1.31-1.21 (12H, m), 2.52 (2H, t, J = 7.6 Hz, ), 0.82 (3H, J = 7.0 Hz, H-15 '); _{^{13 C-NMR (CDCI 3,}} 62.9 MHz) 177.5 (C-4), 154.5 (C-2), 141.8 (C-8a), 131.7 (C-7), 129.7 (C-8 '), 129.3 (C (C-5), 126.1 (C-4a), 122.9 (C-6), 115.2 (C-8), 110.6 _{N-CH 3), 31.7 (} C-11 '), 29.4 (C-2'), 29.1 (C-3 '), 28.9 (C-4'), 28.6 (C-5 '), 28.1 (C- 6 '), 26.9 (C-7'), 26.6 (C-10 '), 22.1 (C-12'), 13.8 (C-13 '); Positive FAB-MS m / z 340 [M + H] &lt; ⁺ &gt;.

EO- 7 ( Dihydroevocarpine ): Colorless powder; _{^{1 H-NMR (CDCI 3,}} 250 MHz) 8.38 (1H, dd, J = 1.5, 8.0 Hz, H-5), 7.60 (1H, m, H-7), 7.43 (1H, d, J = 8.0 Hz H-8), 7.30 (1H, t, J = 8.0 Hz, H-6), 6.15 (1H, s, H- J = 7.6 Hz, H-1 '), 1.60 (2H, m, H-12'), 1.21-1.36 (20H, m), 0.83 (3H, J = 7.0 Hz, H-13 '); _{^{13 C-NMR (CDCI 3,}} 62.9 MHz) 177.6 (C-4), 154.7 (C-2), 141.8 (C-8a), 131.9 (C-7), 126.4 (C-5), 126.4 (C- 4 '), 123.1 (C- 6), 115.3 (C-8), 110.9 (C-3), 34.6 (C-1'), 34.0 (N-CH 3), 31.8 (C-11 '), 29.5 -28.8 (C-3 'to 10'), 28.4 (C-2 '), 22.6 (C-12'), 14.0 (C-13 '); Positive FAB-MS m / z 342 [M + H] &lt; ⁺ &gt;.

EO- 8 ( Limocitrin 3-O- rutinoside ): Yellow solid; _{^{1 H-NMR (CD 3 OD}} , 250 MHz) 8.0 (1H, d, J = 2.5 Hz, H-2 '), 7.8 (1H, dd, J = 2.5, 8.5 Hz, H-6'), 7.7 ( 1H, d, J = 8.5 Hz, H-5 '), 6.3 (1H, s, H-6), 5.3 (1H, dd, J = 7 Hz, H- , H-rham-l '" ), 4.0 (3H, s, 3'-OCH 3), 3.9 (3H, s, 8-OCH 3), 1.1 (3H, d, J = 6.5 Hz, rham-CH 3 ); _{^{13 C-NMR (CD 3 OD}} , 62.9 MHz) 179.5 (C-4), 158.6 (C-7, 2), 157.9 (C-5), 151.0 (C-4 '), 150.4 (C-9), C-3 '), 129.1 (C-8), 124.0 (C-6'), 123.0 (C-1 '), 116.2 2 '), 105.6 (C-10), 104.4 (C-glu-1''), 102.6 (C-2 ''), 72.0 (C-4 ''), 70.8 (C-3 ''), 70.6 C-5 '"), 67.1 (C-6"), 62.1 (8-OCH 3), 56.7 (3'-OCH 3), 17.9 (C-6'"); Positive FAB-MS m / z 654.5 [M + H] &lt; ⁺ &gt;.

< Example  2> Sour milk  Investigation of cytotoxicity and inhibition of cellular senescence of extracts and single components

1. Experimental material

Human fibroblasts and umbilical vein endothelial cells were purchased from Lonza (Walkersville, MD, USA). (Penicillin-Streptomycin, WelGene (Daegu, Korea), Endothelial Cell Growth Medium-2 (DMEM), Dulbecco's Modified Eagle's Medium -2, EGM-2) were purchased from Lonza (Walkersville, MD, USA). Antibodies to p53 were obtained from Santa Cruz Biotech, Inc. (SantaCruz, CA, USA), and antibodies against p21 and pS6 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The GAPDH antibody was obtained from Dr. Kwon Sun - sun of Korea Biotechnology Research Institute. Adriamycin was manufactured by Ildong Pharmaceutical Co., Ltd.

2. Cell culture

Human fibroblasts were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotic (penicillin 10,000 цm / ml, streomycin 10,000 ug / ml) 10 ⁵ , and then cultured in a 5% carbon dioxide incubator at 37 ° C. If the cells were grown at the bottom of the culture dish by 80-90%, trypsin-EDTA solution (2.5X) was added to separate the cells, followed by subculture. Cells in umbilical cord blood were cultured in the same manner as human fibroblasts using EGM-2 as a culture medium. The number of cells was measured every time the cells were transferred, and the number of times of division of the cells was examined. The population doubling (PD) was calculated using the equation: PD = log ₂ F / log ₂ I (F = final cell number, I = initial cell number). The cells used for the experiment were PD <35 or PD> 75 in the case of human fibroblasts, and PD <30 or PD> 50 in the cord blood.

3. Induction of cell aging by treatment with adriamycin

Human fibroblasts and umbilical vein endothelial cells were plated at a density of 1.5x10 ⁵ cells in a 100 mm diameter culture dish. After culturing for 3 days at 37 ° C in a 5% carbon dioxide incubator, the cell culture medium was removed. Cells were washed twice with DMEM containing antibiotics. Cells were treated with 500 nM adriamycin for 4 hours and then washed three times with DMEM medium containing antibiotics. Human fibroblasts were cultured in DMEM containing 10% fetal bovine serum and 1% antibiotic, and human umbilical vein endothelial cells were cultured in EGM-2. After 4 days, it was confirmed that senescence-associated β-galactosidase (SA-β-gal) staining resulted in cell senescence.

4. Measurement of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT)

The effects of the extracts and compounds on the growth rate of the cells were investigated by MTT method. 0.1% MTT solution was added to each well of a 96-well culture vessel in an amount of 50 μl per well and reacted in a 5% carbon dioxide incubator at 37 ° C for 3 hours. After removing the culture solution and MTT solution, 100 μl of dimethyl sulfoxide was added to dissolve the crystals formed. The cell growth rate was measured by measuring the absorbance at 550 nm using a microplate reader.

5. Investigation of the effects of oolong oil extract and single component in cell senescence by adriamycin

We investigated the effects of osmotic oil extracts and single compounds isolated from this extract on the cells aged by adriamycin. Cells treated with adriamycin for 4 hours were separated from the culture dish with trypsin-EDTA and dispensed into 96-well, 12-well, 24-well cell culture vessels. In a 96-well cell culture container, 500 fibroblasts and 1,000 cells were placed in each well. 24 wells were plated at 3000 cells / well, cord blood cells were plated at 5000 cells / well and 12 wells were plated at 5000 cells / well and 7000 cells / well of vascular endothelial cells. And cultured in a 5% carbon dioxide incubator at 37 ° C for one day. In 96 wells, 100 μl of DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic and 100 μl of EGM-2 culture medium were added to each well. After 12 and 24-well cultures were exchanged, &lt; / RTI &gt; ug / ml. Dimethylsulfoxide was used as a negative control, and 5 mM N-acetylcysteine and 500 nM rapamycin were added as a positive control. After 3 days of incubation at 37 ° C in a 5% carbon dioxide incubator, the degree of cell growth was measured by MTT method and the degree of cell senescence was measured by senescence-associated β-galactosidase (SA-β-gal) Staining method.

6. senescence-associated β-galactosidase (SA-β-gal) active staining

The effect on cell senescence was examined by SA-β-gal active staining. After 3 days of treatment with a single compound in a 24 well or 12 well culture vessel, the cells were washed with phosphate buffer. The cells were fixed with 3.7% paraformaldehyde, and the fixative solution was removed and washed again with phosphate buffer solution. SA-β-gal staining solution [40 mM citric acid / phosphate; pH 5.8, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl ₂ , X-gal 1 mg / ml] was added to each well 250 ul per well and 500 ul per well in a 12 well culture vessel. Wrapped in silver foil and reacted at 37 캜 for 17 hours. After washing twice with phosphate buffered saline (PBS), the cells were stained with 1% ezine solution for 1 minute. After washing twice with phosphate buffer, blue-stained cells were observed under an optical microscope. The degree of SA-β-gal activity was expressed in percentage (%) by measuring the number of cells stained blue in the cytoplasm among 50-100 cells in total.

7. Cell protein extraction

Each cell was placed in a 60 mm culture dish at 1 × 10 ⁵ cells and cultured at 37 ° C. in a 5% CO 2 incubator. Cells were washed twice with DMEM medium containing antibiotics, and then treated with 500 μM adriamycin for 4 hours. After the culture medium was removed, the cells were washed twice with phosphate buffer. The cell lysate solution (25 mM Tris-HCl, pH 7.6), 150 mM Nacl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium vanadate vanadate), 5 mM NaF, protease inhibitor or 1 mM PMSF]. The cells were scraped using a cell scraper to scrape the culture dish, and then transferred to a microneedle tube. The solution was shaken every 10 minutes while reacting on ice for 30 minutes. The supernatant was transferred to a new tube by spinning at 12,000 rpm for 15 minutes. The amount of protein in the solution was quantified by bicinchoninic acid (BCA) method (Pierce Biotechnology Inc., Rockford IL, USA) using bovine serum albumin as a standard protein.

8. Western blot analysis

Protein (30 μg) was separated by electrophoresis on a 10% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and allowed to react for 30 minutes in Tween-20-Tris buffered saline (TTBS) containing 5% whole milk powder. Nitrocellulose membranes were reacted overnight in 5% whole milk powder-TTBS solution containing primary antibody against p53 or p21. After washing three times for 10 minutes with TTBS solution, the cells were reacted with a secondary antibody conjugated with horseradish peroxidase for 1 hour and 30 minutes. The membranes were washed five times with TTBS for 7 min and then the amount of p53 or p21 was measured using an enhanced chemiluminescence solution. The amount of specific protein reacted with each antibody was measured using a LAS-3000 imaging device (Fujifilm Corp., Stanford, CT, USA). The same amount of protein used in each experiment was compared using glyceraldehyde-3-phosphate dehydrogease (GAPDH) antibody.

9. Measurement of intracellular reactive oxygen (ROS) concentration

Cells were plated at ^1.5 × 10 ⁵ in a 100 mm culture dish and cultured in a 5% carbon dioxide incubator at 37 ° C. for 3 days. Cells were washed twice with DMEM medium containing antibiotics and treated with 500 nM adriamycin for 4 hours. The cells were washed once with phosphate buffer and treated with trypsin-EDTA solution (2.5%), and then the cells were divided into 1 × 10 ⁵ cells in a 60 mm culture dish. And cultured in a 5% carbon dioxide incubator at 37 ° C for one day. The culture medium was changed and a single compound was treated at each concentration. As a negative control, dimethyl sulfoxide was added as a positive control, 5 mM N-acetylcysteine and 500 nM rapamycin. After incubation for 3 days in a 5% CO 2 incubator at 37 ° C, the cells were washed twice with DMEM containing antibiotics and treated with 250 μM H ₂ DCFDA for 20 minutes. The cells were washed once with phosphate buffered saline, and the trypsin-EDTA solution was added to the cells. The supernatant was discarded at 5,000 × g for 1 minute, and the cells were washed with 1 ml of phosphate buffer solution containing 2% fetal bovine serum, and further washed at 5,000 × g for 1 minute. The washing procedure was repeated twice, followed by the addition of 1 ml of 1% paraformaldehyde. The amount of intracellular ROS was measured using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, Calif.).

10. Results

(1) Cytotoxicity and cell aging inhibitory effect of oolong oil extract in human umbilical cord blood

Cytotoxicity of five extracts of oyster oil in human umbilical cord blood was examined first. Water extract, butanol and methanol extract showed no cytotoxicity at 100 ug / ml, whereas ethyl acetate and hexane extract showed cytotoxicity at 100 ug / ml (FIG. 3A). However, when these extracts were treated with 10 ug / ml, no cytotoxicity was observed (Fig. 3A).

We investigated whether osseous oil extract inhibited adriamycin-induced cell senescence. Water extract, butanol and methanol extracts were treated with 100 and 10 ug / ml, ethyl acetate extract, and hexane extract with 10 ug / ml, respectively. When the ethyl acetate extract was treated, SA-β-gal activity increased by adriamycin treatment was decreased (FIG. 3B). However, other extracts did not inhibit SA-β-gal activity. As a result of investigating the cell aging inhibitory effect while increasing the concentration of ethyl acetate extract, it was observed that SA-β-gal activity, which is increased by adriamycin in a concentration-dependent manner, is inhibited (FIGS. 4A, 4B and 4C).

We investigated the effect of ethyl acetate extract on the expression of proteins such as p53, p21, and phosphorylated S6K, which are increased during adiamycin - induced cell senescence. As a result, it was confirmed that the expression of p53, which is increased by adriamycin, is suppressed (Fig. 4D).

Ethyl acetate extract inhibited cell senescence not only in adriamycin-induced cell senescence but also in senescence-induced cells, resulting in a dose-dependent decrease in SA-β-gal activity induced by replicative senescence (FIG. 5) .

From the above results, it was confirmed that the ethyl acetate extract of Pseudomonas aeruginosa inhibits adipocyte aging and cell senescence by cell division.

(2) Cytotoxic effect and cellular aging inhibition effect of single component of folic acid in human umbilical cord blood

Cytotoxicity of 8 single components of folic acid was investigated in human umbilical cord blood. When the compounds were each treated at 10 ug / ml, EO-4 and EO-8 alone were not cytotoxic (Fig. 6A). Among them, EO-4 was examined to inhibit cell senescence and it was confirmed that adiamycin does not inhibit cell senescence (FIG. 6B).

When the concentration of the single component was lowered to 1 ug / ml, the other compounds except EO-1 showed no cytotoxicity (Fig. 7A). EO-3 and EO-7 inhibited cell senescence and confirmed that it did not inhibit cell senescence by adriamycin (Fig. 7B).

When the concentration of EO-1 was further lowered to 0.1 ug / ml, no cytotoxicity was shown, but no cytostatic effect was observed (Figs. 8A and 8B)

(3) Cytotoxicity and cell aging inhibitory effect of oolong oil extract in human fibroblasts

The cytotoxicity of five extracts from human fibroblasts was examined first. Water extract, butanol and methanol extract did not show cytotoxicity at 100 ug / ml, but ethylacetate and hexane extract showed cytotoxicity at 100 ug / ml. However, when these extracts were treated with 10 ug / ml, no cytotoxicity was observed (Fig. 9A).

We investigated whether osseous oil extract inhibited adriamycin-induced cell senescence. Water extract, butanol and methanol extracts were treated with 100 and 10 ug / ml, ethyl acetate extract, and hexane extract with 10 ug / ml, respectively. When treated with methanol extract at 10 ug / ml, SA-β-gal activity increased by adriamycin was reduced (FIG. 9B). However, other extracts did not inhibit SA-β-gal activity. Therefore, it was confirmed that low concentration of methanol extract inhibits adiamycin-induced cell senescence. The cell aging inhibitory effect according to methanol extract concentration was further investigated. As a result, it was observed that inhibition of SA-β-gal activity, which is increased by adriamycin in a concentration-dependent manner (FIGS. 10A, 10B and 10C). The effect of methanol extract on the expression of proteins such as p53, p21, and phosphorylated S6K, which are increased during adiamycin-induced cell senescence, was examined, but it was confirmed that there was no significant change (Fig. 10D).

Methanol extract inhibited not only cell senescence by arrythmiaein but also cell senescence induced by replication-induced senescence, and decreased SA-beta-gal activity increased by replica senescence in a concentration-dependent manner (Fig. 11 ).

From the above results, it was confirmed that the methanol extract of sour milk methanol has the effect of inhibiting adipocyte aging and cell senescence due to cell division.

(4) Cytotoxic effect and cellular aging inhibition effect of single component of folic acid in human fibroblasts

To investigate the cytotoxicity of human fibroblasts on 8 single components of foliar oil. When each compound was treated at 10 ug / ml, only EO-1, EO-5, and EO-6 were cytotoxic (FIG. 12A). EO-4 and EO-8 inhibited adriamycin-induced cell senescence and EO-8 inhibited SA-β-gal activity increased by adriamycin (FIGS. 12B and 12C). The effect of EO-8 concentration on cell senescence inhibition was investigated. As a result, SA-β-gal activity was decreased in an EO-8 concentration-dependent manner (FIGS. 13A and 13B). However, there was no change in p53, p21, pS6 protein expression (Fig. 13C). After intracellular ROS changes induced by adriamycin after EO-8 treatment, the amount of ROS was decreased by EO-8 but not statistically significant (FIGS. 14A and 14B). We investigated whether EO-8 inhibits cell senescence by adriamycin as well as cell senescence induced by replication. It was observed that the SA-β-gal activity increased by replication aging in a EO-8 concentration-dependent manner (FIGS. 15A, 15B and 15C).

When EO-1 was treated at a concentration of 1, 0.1 ug / ml, cytotoxicity was observed at 1 ug / ml (Fig. 16A), but no cytotoxicity at 0.1 ug / ml (Fig. 16B). EO-1 was treated with 0.1 mg / ml to examine the effect of adriamycin on cell senescence. As a result, the expression of SA-β-gal activity and the expression of p53 and phosphorylated S6K were decreased by adriamycin. 1 &lt; / RTI &gt; ug / ml, no cytotoxicity was observed and the cell aging by adriamycin was inhibited (Fig. 16C, 16D and 16E). In addition, EO-1 also reduced the amount of ROS that was increased by the treatment with arkadimine (FIGS. 17A and 17B). It was observed that EO-1 not only inhibited cell senescence by adriamycin but also inhibited cell senescence in cells induced by transcriptional aging. As a result, it was observed that SA-β-gal activity increased by transcriptional aging (FIG. 18).

These results suggest that EO-8 (Limocitrin 3-O-rutinoside) at a concentration of 10 ug / ml and EO-1 (rutaecarpine) at a concentration of 0.1 ug / ml may inhibit cell senescence and cell division by adriamycin Which is an effective inhibitor of replication aging.

Claims (7)

A pharmaceutical composition for preventing or treating skin aging comprising, as an active ingredient, limocitrin 3-O-rutinoside represented by the formula (2) isolated from methanol extract of oyster oil, Characterized in that it inhibits fibroblastogenesis induced by myosin and inhibition of fibroblastogenesis is measured through inhibition of senescence-associated beta-galactosidase (SA-beta-gal) activity. A pharmaceutical composition for preventing or treating skin aging.

(2)

R ₁ a = OH, R ₂ = OCH ₃ Im. delete delete delete delete delete delete

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