KR20150024606A - Composition for inhibiting cellular senescence comprising extracts of Anisi Stellati Fructus or threo-1(4'-methoxyphenyl)-1,2-propandiol isolated from the same - Google Patents

Abstract

The present invention relates to a composition for inhibiting cellular senescence comprising extracts of Anisi stellati fructus or threo-1(4′-methoxyphenyl)-1,2-propandiol isolated from the same as an active ingredient. More specifically, provided is a composition for inhibiting cellular senescence derived from Adriamycin. The composition can be used for treating diseases such as skin aging, rheumatic arthritis, ostarthritis, hepatitis, chronic skin injury, artery hardening, prostatic hyperplasia, liver cancer, etc by inhibiting cellular senescence of fibroblast or umbilical vein endothelial cell. Also, the composition can be utilized for developing antiaging functional foods, anti-vein aging drugs, and cosmetics which can inhibit cellular senescence.

Description

(4'-methoxyphenyl) -1,2-propanediol as an active ingredient isolated from an octagonal fennel extract or isolated therefrom (Composition for inhibiting cellular senescence comprising extracts of Anisi Stellati Fructus or threo-1 (4'-methoxyphenyl) -1,2-propanediol isolated from the same}

The present invention relates to the use of an octopus fennel extract or threo-1 (4'-methoxyphenyl) -1,2-propanediol isolated therefrom as an active ingredient To a composition for inhibiting cell senescence.

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.

The octagonal fennel is an evergreen tree belonging to Illiciaceae. Leaves are horny, and the leaves are oval or elliptical obovate, with a flat and transparent margin. Flower is one liquid each, 7 ~ 12 petal pieces arranged in various circumferences of the stomach and the inside circumference is pink or crimson. The pharmacological action of octagonal fennel is inhibition of tubercle bacillus and Bacillus thuringiensis on octagonal fennel. Alcohol extract is known to inhibit Staphylococcus aureus, Pneumococcus and Diphtheriae bacteria. In addition, It is used for the treatment of vomiting, anorexia, anemia of abdominal pain, and low back pain.

Korean Patent Laid-Open No. 10-2012-0010984 discloses a composition for preventing or treating an allergic disease which contains an octagonal fennel extract as an active ingredient, wherein the extract of Illicium verum is a compound 48/80, (TNF-a, IL-4, and IL-8) by increasing the secreted histamine secreted from mast cells and decreasing the amount of allergen-induced cytokines (PMA and A23187) There is no mention of the inhibition of umbilical vein endothelial cells or fibroblast aging as in the invention.

It is an object of the present invention to provide a method of producing an octagonal fennel extract or threo-1 (4'-methoxyphenyl) -1,2-propanediol, And a composition for inhibiting cell senescence.

To achieve the above object, the inventors of the present invention investigated whether the four monoclonal antibodies isolated from octopus fenugreek extract and octopus fenugrea inhibited cell senescence in human vascular endothelial cells and fibroblasts. As a result, in human vascular endothelial cells, octanol-fructose butanol extract and ethyl acetate extract were used. In human fibroblasts, octachloro-ethyl acetate extract, hexane extract and ASF-2 (threo-1 (4'-methyoxyphenyl) Confirming the cell aging inhibitory effect and completed the present invention.

(4'-methoxyphenyl) -1,2-propanediol represented by the following formula (1) separated from an octagonal fennel extract or threo-1 propanediol] as an active ingredient.

≪ Formula 1 >

Specifically, the octopus fennel extract was prepared by adding ethyl acetate (EtOAc) to a distilled water layer fractionated by adding distilled water and n-hexane to methanol extract of octopus fennel and fractionating the extracted ethyl acetate (EtOAc) fraction Is an extract.

Specifically, the octopus fennel extract was prepared by adding ethyl acetate (EtOAc) to a distilled water layer fractionated by adding distilled water and n-hexane to methanol extract of octopus fennel, adding butanol to the fractionated distilled water layer And the fraction is an extracted butanol fraction extract. More specifically, the cell is an umbilical vein endothelial cell.

Specifically, the octopus fennel extract is an extract of hexane (n-hexane) fraction extracted by adding distilled water and n-hexane to an octagonal fennel methanol extract, and more specifically, the cell extract is a fibroblast .

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 comprises the octopus fennel extract or threo-1 (4'-methoxyphenyl) -1, 2-propanediol, and the like. Such pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia But are not limited to, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, , Mineral oil, and the like, but are 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 present inventors have found that an octagonal fennel extract or threo-1 (4'-methoxyphenyl) -1,2-propanediol separated therefrom can be added to adriamycin Inhibits cell senescence by cell division and inhibits the replication senescence due to cell division. 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 octopus fennel extract.
Figure 2 shows the chemical composition of a single component separated from octagonal fennel.
FIG. 3 shows cytotoxicity of octopus fennel extract and inhibition of cell senescence by adriamycin in human umbilical vein endothelial cells. A, Cytotoxic effect of octopus 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 octopus fennel extract was treated with 10, 100 ug / ml, and SA-β-gal activity 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 cytotoxicity and anti-aging effect of adriamycin on human umbilical vein endothelial cells from octopal-folic acid butanol extract. A, shows the cytotoxic effect of butanol extract of octopus ficus. 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 octopal-fructose butanol extract was treated with 1-10 ug / ml, and SA-β-gal staining was performed 3 days later. 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 cytotoxicity of the octachromatic ethyl acetate extract in human umbilical vein endothelial cells and the cytotoxic effect of adriamycin on cell senescence. A, indicating the cytotoxic effect of the octachromethane 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, the extracts of octagonal ethylacetate were treated with 1-10 .mu.g / ml, and SA-.beta.-gal 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. 6 shows the inhibitory effect of octanol-fructose butanol extract on the reproduction cell senescence in human umbilical vein endothelial cells. A, indicating the cytotoxic effect of the octanol-fructose butanol extract on the replicating senescent 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. The replanted senescent cells were treated with 1-10 ug / ml of octopal-fructose butanol extract and stained with SA-β-gal for 3 days. 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. 7 shows the effect of the octachromatic ethyl acetate extract on inhibition of cell senescence in human umbilical vein endothelial cells. A, the cytotoxic effect of the octachromatic ethyl acetate extract 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 .mu.g / ml of octa-fructooligosaccharide ethyl acetate extract and subjected to SA-.beta.-gal active staining three days later. 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. 8 shows cytotoxicity of single component of octopus folium and inhibition of cell senescence by adriamycin in human umbilical vein endothelial cells. A, Cytotoxic effect of single component of octagonal fennel. 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, ASF-1 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. * p < 0.05 vs. DMSO.
Fig. 9 shows cytotoxicity of octopus fennel extract and inhibition of cell senescence by adriamycin in human fibroblasts. A, Cytotoxic effect of octopus 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 octopus fennel extract was treated with 10 ug / ml and after 3 days, SA-β-gal activity staining was performed. 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 octachromate ethyl acetate extract in human fibroblasts and the cytotoxic effect of adriamycin on cell senescence. A, indicating the cytotoxic effect of the octachromethane ethyl acetate extract. The extracts were treated with 1-10 ug / ml for 3 days, and cytotoxic effect was investigated by MTT assay. B and C, SA-β-gal active staining and SA-β-gal active staining percentage. After treatment with adriamycin, the extract was treated with 1-10 .mu.g / ml, and SA-.beta.-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 cytotoxicity of octane-fennel hexane extract in human fibroblasts and the cytotoxic effect of adriamycin on cell senescence. A, indicating the cytotoxic effect of octane extract of hexane. The extracts were treated with 1-10 ug / ml for 3 days, and cytotoxic effect was investigated by MTT assay. B and C, SA-β-gal active staining and SA-β-gal active staining percentage. After treatment with adriamycin, the extract was treated with 1-10 ug / ml of octane-fennel hexane extract and subjected to SA-β-gal staining three days later. 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. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.
Fig. 12 shows the effect of the octachromethane ethyl acetate extract on the inhibition of cell senescence in human fibroblasts. A, the cytotoxic effect of the octachromatic ethyl acetate extract 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. Repeated aging cells were treated with 1-10 ug / ml of octopus extract and subjected to SA-β-gal staining three days later. 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. 13 shows the effect of inhibiting the senescence of the cloned cells of the octane-fennel hexane extract in human fibroblasts. A, indicating the cytotoxic effect of the Octane Fennel hexane extract on the replicating senescent 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. The replanted senescent cells were treated with 1-10 ug / ml of octane-fennel hexane extract and subjected to SA-β-gal staining three days later. 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.
14 shows cytotoxicity of a single component of octopus felis and inhibition of cell senescence by adriamycin in human fibroblasts. A, Cytotoxic effect of single component of octagonal fennel. 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, each component 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. * p < 0.05 vs. DMSO.
Fig. 15 shows the results obtained from human fibroblasts using an octagonal fennel single component ASF-2 (4'-methoxyphenyl) -1,2-propanediol ] Exhibit cytotoxic effects by adriamycin. A and B, SA-β-gal active staining and SA-β-gal active staining percentage. After adriamycin treatment, ASF-2 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, ASF-2 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 &lt; 0.05 or ** p < 0.01 vs. DMSO.
16 shows the effect of ASF-2 (4'-methoxyphenyl) -1,2-propanediol) on the production of active oxygen in human fibroblasts. propanediol]. A, active oxygen flow cytometry. B, the average amount of active oxygen. The fibroblasts were treated with adriamycin, treated with ASF-2 at 10 ug / ml, 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 &lt; 0.05 or ** p &lt; 0.01 vs. DMSO.
FIG. 17 is a graph showing the effect of ASF-2 (tre-1 (4'-methoxyphenyl) -1,2-propanediol) [threo-1 (4'-methoxyphenyl) Exhibit an inhibitory effect on aging. A, SA-β-gal active dye images. B, SA-β-gal active staining percentage. C and ASF-2. Transfected senescent cells were treated with 1-10 μg / ml of ASF-2 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 &lt; 0.05 or ** p &lt; 0.01 vs. DMSO.

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> Octagonal fennel  Extraction Separation and Chemical Composition of a Single Component

1. Octagonal Fennel Extract and Single Component Separation

9kg of dried octagonal fennel was extracted three times with MeOH: H ₂ O = 9: 1 (12.0 L) at about 70 ° C for 12 hours while refluxing. The extract was filtered under reduced pressure and concentrated to obtain 1.13 kg of MeOH extract. The MeOH extract in distilled water (1.5 L) and n - performing an operation twice that fraction with hexane (n -hexane) layer from the aqueous layer and n - - hexane (n -hexane) n in a fraction funnel was added (1.5 L) hexane in butanol (n -butanol), H ₂ O net - a layer (n -hexane) was concentrated under reduced pressure and n-hexane (n -hexane) and a fraction was obtained, ethyl acetate (EtOAc) and the aqueous layer in the same way as the back, n extracted with n - hexane (n -hexane) 255.77g fractions, 64.66g EtOAc fraction, n - butanol (n -butanol) fraction 246.78g, H ₂ O to give a fraction 254.84g. N of which - with silica gel column (silica gel column) butanol (n -butanol) 246.787g fractions were isolated as follows. column (length 80cm, 8cm diameter) on silica gel (No.9385, 230-400mesh, Merck ) to about 25 cm filled with n - butanol (n -butanol) = eluted (elution) to a 3L fixed bed of 100% (stationary phase was homogenized, and 246.78 g of the sample was adsorbed on 450 g of silica gel (No. 7734, 70-230 mesh, Merck) and loaded on a column. Since n - starting butanol (n -butanol) = mobile phase elution (mobile phase elution) to 100% n -butanol: AcOH: H 2 O = 100: 1: 1, 50: 1: 1, 10: 1: 1 (BASF1-7) was prepared by flash column chromatography, which was performed by increasing the polarity in the order of 4: 1: 1 and eluting the column with a constant pressure. . Fraction After loading 20 g of BASF-2 (10.9 g) onto 20 g of silica gel (No. 7734, 70-230 mesh, Merck) and loading it with a column of 4 cm in diameter and 60 cm in length filled with silica gel (No. 9385, 230-400mesh, Merck) loading. The solvent used was saturated with a column of CH ₂ Cl ₂ : CH ₃ OH: H ₂ O = 15: 2: 0.1 and then 12: 2: 0.1, 10: 2: 0.1, 8: 2: : 3: 0.1, 4: 6: 0.1. Finally, the column was washed with CH ₃ OH: H ₂ O = 100: 0.1. Fraction BASF-2-4 (625.3 mg) was further subjected to column chromatography. The used column was eluted with 1 L of MeOH: H ₂ O = 1: 1 mixed solvent in a reverse phase (particle size; 40 to 63 μm) column (length 75 cm, diameter 2 cm) And made uniform. The sample was dissolved in a mixed solvent of MeOH and H ₂ O and was loaded on a column. After that, the ratio of MeOH was gradually increased to elute the ASF-1 mixture containing two enantiomers (46.2 mg) and ASF-2 (38.4 mg), a diastereomer of ASF-1, were obtained. ASF-3 (20.7 mg) and ASF-4 (18.4 mg) were separated by column chromatography using the same reverse-phase column as that of Fraction BASF-2-11 (657.5 mg). The chemical structure of the compounds was determined and compared with the literature by using spectroscopic methods such as ( ¹ H, ¹³ C, DEPT, ¹ H- ¹ H COZY, HMQC, and HMBC) 1).

2. Physico-chemical properties of separated materials

Octagonal fennel n - compounds of the four kinds of separation in butanol (n -butanol) fraction as a base material ^{(1 H, 13 C, DEPT} , 1 H- 1 H COSY, HMQC, HMBC) various spectroscopic analysis methods, such as (4'-methoxyphenyl) -1,2-propanediol], ASF-1 (erythro-1 (4'-methoxyphenyl) (4'-methoxyphenyl) -1,2-propanediol], ASF-3 (1- (4'-methoxyphenyl) Phenyl) - (1S, 2R) -propan-1-ol-2-O- beta -D-glucopyranoside) 2-O-? -D-glucopyranoside], ASF-4 (1- (4'-methoxyphenyl) - (1S, 2S) ) Was identified as [1- (4'-methoxyphenyl) - (1S, 2S) -propan-1-ol-2- O- beta -D-glucopyranoside] (FIG.

ASF- 1 [white powder, IR? _Max (KBr) cm ^-1 : 3410 (OH), 1459 (C = C stretch); ^{1 H-NMR (300 MHz,} acetone- d 6) δ: 4.50 (dd, J = 3.9 Hz, H-1), 3.82 (m, J = 6.2 Hz, H-2), 1.02 (d, J = 6.2 Hz, H-3), 4.10 (d, J = 3.7 Hz, 1-OH), 3.51 (d, J = 5.0 Hz, 2-OH), 7.29 (d, J = 8.4 Hz, H-2 '), 6.85 (d, J = 8.8 Hz , H-3 '), 6.85 (d, J = 8.4 Hz, H-5'), 7.29 (d, J = 8.4 Hz, H-6 '), 3.76 (s, H -OCH _3); ^{13 C-NMR (75 MHz,} , acetone- d 6) d: 77.7 (C-1), 71.9 (C-2), 18.0 (C-3), 135.5 (C-1 '), 128.6 (C-2 '), 113.8 (C-3 '), 159.5 (C-4 '), 113.8 (C-5'), 128.6 (C-6 '), 55.3 (OCH 3). Positive FAB-MS m / z 205 [M + Na] &lt; ⁺ &gt;.

ASF- 2 [white powder, IR? _Max (KBr) cm ^-1 : 3260 (OH), 1454 (C = C stretch); ^{1 H-NMR (600 MHz,} acetone- d 6) δ: 4.26 (dd, J = 7.3 Hz, H-1), 3.58 (m, J = 3.8 Hz, H-2), 0.93 (d, J = 6.3 Hz, H-3), 4.27 (d, J = 3.3 Hz, 1-OH), 3.82 (d, J = 3.8 Hz, 2-OH), 7.28 (d, J = 9.5, H-2 '), 6.87 (d, J = 8.7Hz, H -3 '), 6.87 (d, J = 8.7Hz, H-5'), 7.28 (d, J = 9.5, H-6 '), 3.77 (s, H-OCH ₃ ); ^{13 C-NMR (150 MHz,} , acetone- d 6) δ: 79.3 (C-1), 72.6 (C-2), 19.1 (C-3), 135.1 (C-1 '), 128.9 (C-2 '), 114.0 (3-C'), 159.9 (C-4 '), 114.0 (-C 5'), 128.9 (C-6 '), 55.3 ₍₃ OCH). Positive FAB-MS m / z 205 [M + Na] &lt; ⁺ &gt;.

ASF- 3 [yellow crystals, IR? _Max (KBr) cm ^-1 : 3397 (OH), 1458 (C = C stretch); ^{1 H-NMR (300 MHz,} acetone- d 6) d: 4.90 (d, H-1), 4.04 (d, J = 6.1 Hz, H-2), 0.92 (d, J = 7.1 Hz, H-3 ), 7.32 (d, J = 8.1 Hz, H-2 '), 6.86 (d, J = 8.1Hz, H-3'), 6.86 (d, J = 8.1 Hz, H-5 '), 7.32 (d , J = 8.1 Hz, H- 6 '), 3.75 (s, H-OCH 3), 4.63 (d, J = 7.6 Hz, H-1 "), 3.43 (dd, J = 3.9 Hz, H-2" ), 3.54 (t, J = 6.0 Hz, H-3 "), 3.47 (t, J = 3.5 Hz, H-4"), 3.41 (m, H-5 "), 3.75 (dd, J = 10.0 Hz , H-6 "); ^{13 C-NMR (75 MHz,} , acetone- d 6) δ: 74.5 (C-1), 80.5 (C-2), 15.3 (C-3), 134.0 (C-1 '), 128.5 (C-2 '), 114.0 (C-3 '), 159.5 (C-4 '), 114.0 (C-5'), 128.5 (C-6 '), 55.4 (OCH 3), 103.1 (C-1 "), 74.8 (C-2 "), 77.5 (C-3"), 71.1 (C-4 "), 77.1 (C-5"), 62.5 (C-6 ").

ASF -4 [yellow crystals, IR? _Max (KBr) cm ^-1 : 3405 (OH), 1613 (C = C stretch); ^{1 H-NMR (300 MHz,} acetone- d 6) δ: 4.43 (dd, J = 7.7 Hz, H-1), 3.85 (m, J = 12.1 Hz, H-2), 1.00 (d, J = 5.7 Hz, H-3), 7.26 (d, J = 8.3, H-2 '), 6.86 (d, J = 8.3Hz, H-3'), 6.87 (d, J = 8.3 Hz, H-5 ') , 7.26 (d, J = 8.3 , H-6 '), 3.73 (s, H-OCH 3), 4.54 (d, J = 7.5 Hz, H-1 "), 4.79 (s, H-2"), 3.45 (t, J = 6.0 Hz, H-3 ''), 3.29 (m, H-4 ''), 3.30 (m, H-5 ''), 3.70 (m, H-6 ''); ¹³ C-NMR (75 MHz, acetone- d ₆ ) ?: 79.3 (C-1), 72.6 (C-2), 19.1 '), 114.0 (C-2 '), 159.9 (C-2 '), 114.0 (C-2'), 128.9 (C-2 '), 55.3 (OCH 3), 105.4 (C-1 "), 75.3 (C-2 "), 78.4 (C-3"), 71.1 (C-4 "), 77.6 (C-5"), 62.4 (C-6 ").

< Example  2> Octagonal fennel  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 octopus extracts and compounds on cell growth rate 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 effect of single-component extracts and octopus extracts on cell senescence by adriamycin

We investigated whether the octagonal fennel extract and single compounds isolated from this extract were effective on aged cells 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, treated with an octagonal extract of fenugreek and a single component for 1 hour before treatment, and adriamycin 500 nM 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. The nitrocellulose membrane was reacted overnight in a 5% whole milk powder-TTBS solution containing the primary antibody against p53, pS6 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 each, and then the amount of p53, pS6 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 the single compound was treated with 10 ug / ml and 1 ug / ml. 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 inhibition of cell senescence of octopus fenugreek extract in human umbilical cord blood

Cytotoxicity of five octopus extracts from human umbilical cord blood was investigated. The butanol extract did not show cytotoxicity at 100 ug / ml, but the remaining extract showed cytotoxicity at 100 ug / ml. However, when these extracts were treated with 10 ug / ml, the hexane extract was cytotoxic, while the other extracts did not show cytotoxicity (FIG. 3A). Next, we investigated whether the octopus fennel extract inhibited adriamycin - induced cell senescence. Butanol extracts were treated with 100 and 10 ug / ml, and other extracts with 10 ug / ml. When 10 ug / ml of butanol extract was treated, the SA-β-gal activity increased by adriamycin treatment was significantly decreased, and the ethyl acetate extract also decreased SA-β-gal activity (FIG. 3B). However, other extracts did not inhibit SA-β-gal activity. Butanol extract and ethyl acetate extract were investigated to increase SA-β-gal activity. As a result, it inhibited SA-β-gal activity which was increased in a concentration-dependent manner by adriamycin (FIGS. 4 and 5). Therefore, it was confirmed that the butanol extract and the ethyl acetate extract have an effect of inhibiting adiamycin-induced cell senescence.

We investigated the effect of two extracts on the expression of proteins such as p53, p21, and phosphorylated S6K, which are increased during adiamycin - induced cell senescence. Butanol extract inhibited the expression of p53 and p21, which are increased by adriamycin (Fig. 4D).

Butanol extract and ethyl acetate extract inhibited cell senescence by adriamycin as well as cell senescence induced by replication. As a result, both extracts decreased SA-β-gal activity increased by repletion aging in a concentration-dependent manner (FIGS. 6 and 7).

From the above results, it was confirmed that the octanol-fructose butanol extract and the ethyl acetate extract were effective in inhibiting adipocyte-induced cell senescence and cell senescence.

(2) Cytotoxic effect and cell aging inhibition effect of single component of octopus fennel in human umbilical cord blood

Cytotoxicity of four components of octagonal fennel was investigated in human umbilical cord blood. No cytotoxicity was observed when each compound was treated at 10 ug / ml (Fig. 8A). Among these, ASF-1 was examined to inhibit cell senescence and it was confirmed that it did not affect cell senescence inhibition (Fig. 8B).

As a result, it was confirmed that the single components isolated from the octagonal fennel did not inhibit cell senescence in the cord blood cells.

(3) Cytotoxicity and inhibition of cell senescence of octopus fennel extract in human fibroblasts

The cytotoxicity of five fenugreek extracts from human fibroblasts was examined first. Water extract, butanol and ethyl acetate extract did not show cytotoxicity at 100 ug / ml, but hexane extract and methanol extract showed cytotoxicity at 100 ug / ml. However, when these extracts were treated with 10 ug / ml, only the hexane extract showed cytotoxicity, and the remaining extract did not show cytotoxicity (FIG. 9A). Next, we investigated whether the octopus fennel extract inhibited adriamycin - induced cell senescence. Water extract, butanol and ethyl acetate extract were treated with 100 and 10 ug / ml, respectively, and hexane and methanol extract with 10 ug / ml, respectively. When treated with 10 μg / ml of ethyl acetate and hexane extract, the 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 the extract of ethyl acetate and the extract of hexane inhibited cell senescence by adriamycin at 10 ug / ml. The cell aging inhibitory effects of ethyl acetate and hexane extracts were further investigated. As a result, both substances inhibited SA-β-gal activity which was increased by adriamycin in a concentration-dependent manner (FIGS. 10 and 11).

We investigated the effect of two extracts on the expression of proteins such as p53, p21, and phosphorylated S6K, which are increased during adiamycin - induced cell senescence. Ethyl acetate extract reduced p53, pS6 and p21 protein expression at a concentration of 10 ug / ml (Fig. 10D), while hexane extract reduced p21 protein expression only (Fig. 11D).

Ethyl acetate extract and hexane extract inhibited cell senescence by adriamycin as well as cell senescence induced by replication. As a result, both extracts decreased the SA-β-gal activity, which was increased by repletion aging in a concentration-dependent manner (Fig. 12, 13).

From the above results, it was confirmed that the extract of octagonal ethylacetate and the extract of hexane in human fibroblasts inhibit the aging of the cells due to adiamycin-induced cell senescence and cell division.

(4) Cytotoxic effect and cellular aging inhibition effect of single component of octopus folium in human fibroblasts

We examined the cytotoxicity of human fibroblasts on four single components (ASF-1, ASF-2, ASF-3, and ASF-4) No cytotoxicity was observed when each single component was treated at 10 ug / ml (Fig. 14A). Four of the single components inhibited adiamycin-induced cell senescence, all of which inhibited increased SA-β-gal activity by adriamycin treatment (FIGS. 14B, 14C). In order to further confirm the cell aging inhibitory effect of ASF-2 whose cell shape is most similar to that of young cells, the cell aging inhibitory effect according to the concentration was investigated. SA-β-gal activity was decreased in an ASF-2 concentration-dependent manner (FIGS. 15A and 15B). Also, when 10 μg / ml of ASF-2 was treated, expression of p53 protein was decreased (FIG. 15C). As a result of measuring the amount of ROS in the cells by adriamycin, the amount of ROS decreased when treated with 10 μg / ml of ASF-2, but was not statistically significant (FIG. 16). As a result of investigating whether ASF- inhibits cell senescence in cells induced by replication senescence, it was also observed that the SA-β-gal activity increased by replicative senescence (FIG. 17).

These results suggest that ASF-2 (threo-1 (4'-methoxyphenyl) -1,2-propanediol) isolated from the octagonal fennel can induce replication senescence due to adiamycin-induced cell senescence and cell division at a concentration of 10 ug / ml Which is known to have an inhibitory effect.

Claims (9)

(4'-methoxyphenyl) -1,2-propanediol] represented by the following formula (1) separated from the octagonal fennel extract or threo-1 A pharmaceutical composition for inhibiting cell senescence comprising as an active ingredient.
&Lt; Formula 1 &gt;

The method according to claim 1, wherein the octopus fennel extract is obtained by adding ethyl acetate (EtOAc) to a distilled water layer fractionated by adding distilled water and n-hexane to methanol extract of octopus fennel, and extracting ethyl acetate (EtOAc ) &Lt; / RTI &gt; fractions. The method according to claim 1, wherein the octopus fennel extract is obtained by adding ethyl acetate (EtOAc) to a distilled water layer obtained by adding distilled water and n-hexane to an octachromatic methanol extract, Wherein the extract is a butanol fraction extracted by fractionation. The pharmaceutical composition for inhibiting cell senescence according to claim 3, wherein the cell is an umbilical vein endothelial cell. The method according to claim 1, wherein the octopus fennel extract is an extract of hexane (n-hexane) extracted by adding fractionated water and hexane (n-hexane) to an octagonal fennel methanol extract. Composition. The pharmaceutical composition for inhibiting cell senescence according to claim 5, wherein the cell is a fibroblast. The pharmaceutical composition for inhibiting cell senescence according to claim 1, wherein the cell aging is induced by adriamycin. The pharmaceutical composition for inhibiting cell senescence according to claim 1, wherein the inhibition of cell senescence measures the inhibition of senescence-associated beta-galactosidase (SA-beta-gal) activity. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is characterized by preventing or treating any one selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostatic hyperplasia and liver cancer Or a pharmaceutically acceptable salt thereof.

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