WO2016047823A1 - Composition for inhibiting cellular senescence containing melandrium firmum rohrbach extract or bornesitol separated from same as active ingredient - Google Patents

Abstract

The present invention relates to a composition for inhibiting cellular senescence containing a Melandrium firmum Rohrbach extract or (-)-bornesitol separated from same as an active ingredient, the provided pharmaceutical composition for inhibiting cellular senescence being derived from Adriamycin. The present invention inhibits the process of cellular senescence of fibroblasts or umbilical vein endothelial cells, and thus may be usefully employed for treating diseases related to senescence, for example, skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damage tissue, arteriosclerosis, prostatic hyperplasia, and liver cancer, etc. Further, the present invention is expected to be used for developing anti-aging functional food, anti-vascular aging drugs, and cosmetics which may inhibit cellular senescence.

Description

A composition for inhibiting cellular aging containing Jangchaechae extract or Bornecitol isolated therefrom as an active ingredient

The present invention relates to a composition for inhibiting cellular aging containing Jangchaechae extract or (-)-bornenesitol ((-)-bornesitol) separated therefrom as an active ingredient.

Normal somatic cells divide into a certain number of times and can no longer divide, resulting in cell aging. This occurs because the telomeres at the ends of the chromosomes become shorter in the process of cell division, resulting in DNA damage, which is called replication aging. Cell aging is induced not only by shortening of telomeres, but also by dysfunction of oncogenes and cancer suppressor genes, inflammatory reactions, oxidative stress, anticancer agents, ultraviolet rays and radiation. Aging cells are larger in size and flatter in shape, stop cell growth, have many signs of DNA damage in the nucleus, and secrete a variety of inflammatory proteins. Biochemically, it is known that senescence-associated β-galactosidase (SA-β-gal) activity is increased. Aging is induced by various factors, but it has been shown that aging is regulated through p53 and Rb / p16 cancer suppressor gene signaling pathways.

Cell aging may also inhibit or promote cancer, and has been suggested as an important mechanism of tissue regeneration and repair, tissue / individual aging and aging-related diseases. In addition, cell aging contributes to the pathogenesis of various aging-related diseases such as cancer, arteriosclerosis, skin aging, neurodegenerative diseases, myotropenia, osteoporosis and prostatic hyperplasia. Recent studies have shown that selective control of cell aging can regulate the development of tissues, organs, aging, health life, and age-related diseases. Telomerase deficient mice are known to be rapidly aging, and it has been shown that increasing telomerase expression in older telomere deficient mice reverses degenerative changes in tissues or organs with aging. As a result of the selective removal of p16-expressing cells, which are known to increase expression in senescent cells in a fast-aging mouse model, tissue lesions due to aging are suppressed and the occurrence of aging-related diseases is reduced. In the process of liver fibrosis in mice, aging of hepatic stellate cells appears, and aging of hepatic stellate cells is known to function to inhibit excessive liver fibrosis. If p53 activity is too high in an unregulated state, aging occurs quickly, but proper p53 activity is known to inhibit aging.

In addition, the results of research on substances that are effective in inhibiting cell aging have been reported. Drugs or single components such as vitamin C, N-acetylcysteine, NS398 and epipriederanol, inhibit this cell aging. In the rat model of rapamycin, 4,4'-diaminodiphenylsulfone has been reported to inhibit the development of aging-related diseases and to increase the lifespan.

Jangguchae is a biennial plant that has been widely distributed in Korea and used to treat various diseases. It is reported in the literature that it has been widely used in the treatment of urinary, breast cancer, gonorrhea and lactation diseases. Butanol fraction of methanol extract of Jangchaechae has been reported to cause hepatic activity including hexobarbital prolongation of sleep, elevated serum transaminase activity, and severe pathological histological changes of hepatic cells. The effect of inhibiting cell or fibroblast aging has not been reported yet.

On the other hand, Korean Patent Registration No. 10-0686260 relates to a pharmaceutical composition and health functional food for liver function improvement and liver disease treatment comprising at least one of jangguchae and Ulleungjangguchae extract as an active ingredient, jangguchae or Ulleung jangguchae extract Not only inhibits AST and ALT activity, but also significantly inhibits the amount of collagen, alpha-smooth-muscle actin and TGF-β accumulated due to fibrosis in liver tissue, which is useful for the prevention and treatment of liver disease. Although it is disclosed that it can be used as a pharmaceutical and health functional food, there is no mention of inhibiting aging of umbilical vein endothelial cells or fibroblasts as in the present invention.

It is an object of the present invention to provide a composition for inhibiting cellular aging containing Jangchaechae extract or (-)-bornenesitol ((-)-bornesitol) separated therefrom as an active ingredient.

The present invention provides a pharmaceutical composition for inhibiting cellular aging containing Jangchaechae extract or (-)-bornenesitol ((-)-bornesitol) represented by the following Chemical Formula 1 isolated therefrom as an active ingredient.

<Formula 1>

The present inventors confirmed that Jangchaechae extract or (-)-bornenesitol ((-)-bornesitol) isolated therefrom inhibits cellular senescence by adriamycin and also inhibits aging due to cell division. By inhibiting the cellular aging process, it can be usefully used for treating diseases related to aging, for example, skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damage tissue, arteriosclerosis, prostate hyperplasia and liver cancer. In addition, anti-aging functional foods, anti-vascular aging drugs, and cosmetics that can inhibit cell aging of vascular endothelial cells and fibroblasts are expected to be utilized.

Figure 1 shows an isolated schematic diagram of Jangchaechae extract.

Figure 2 shows the chemical structure of a single component separated from Jangguchae.

Figure 3 shows the cytotoxicity and inhibitory effect of cell aging by adriamycin of Jangchaechae extract in human umbilical vein endothelial cells. A, Cytotoxic effect of Jangchaechae extract. Each extract was treated with 10, 100 ug / ml, and then cultured for 3 days to examine the cytotoxic effect by MTT method. B, SA-β-gal active staining picture and SA-β-gal active staining percentage. After adriamycin treatment, each extract was treated with 10 ug / ml, and 3 days later, SA-β-gal activity staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.

Figure 4 shows the cytotoxicity of the Jangchaechae ethyl acetate extract in the human umbilical vein endothelial cells and the inhibitory effect of cell aging by adriamycin. A, Cytotoxic effect of Jangchaechae ethyl acetate extract. Each extract was treated with 1-10ug / ml, and then cultured for 3 days to examine the cytotoxic effect by MTT method. B and C, SA-β-gal active staining picture and SA-β-gal active staining percentage. After adriamycin treatment, Jangchaechae ethyl acetate extract was treated with 1-10 ug / ml, and 3 days later, SA-β-gal activity staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. D, p53, phosphorylated S6K, p21 expression. After the adriamycin treatment, the extract was treated with 1-10 ug / ml, and the expression level of each protein was examined by Western blot method. NT, untreated; Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.

Figure 5 shows the effect of inhibiting the replication cell aging of Jangchaechae ethyl acetate extract in human umbilical vein endothelial cells. A, Cytotoxic effect of Jangchaechae ethylacetate extract on cloned aging cells. Each extract was treated with 1-10ug / ml, and then cultured for 3 days to examine the cytotoxic effect by MTT method. B and C, SA-β-gal active staining picture and SA-β-gal active staining percentage. 1-10ug / ml of Jangchaechae ethyl acetate extract was applied to cloned aging cells, and 3 days later, SA-β-gal activity staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. Old, Old cells; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.

Figure 6 shows the cytotoxicity of a single component of Jangguchae in human umbilical vein endothelial cells and the inhibitory effect of cell aging by adriamycin. A, Cytotoxic effect of jang guchae single component. Each component was incubated for 10 days after 10ug / ml treatment, and the cytotoxic effect was examined by MTT method. B, SA-β-gal active staining percentage. After adriamycin treatment, each single component was treated with 10 ug / ml, and 3 days later, SA-β-gal active staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine. * p <0.05 vs DMSO.

Figure 7 shows the cytotoxicity of the jangguchae single component in human umbilical vein vascular endothelial cells and cell aging inhibitory effect by adriamycin. A, Cytotoxic effect of jang guchae single component. Each component was treated with 1ug / ml, and then cultured for 3 days, and the cytotoxic effect was examined by MTT method. B, SA-β-gal active staining percentage. After adriamycin treatment, ZGC-3 was treated with 1 ug / ml, and 3 days later, SA-β-gal activity staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine. ** p <0.01 vs DMSO.

Figure 8 shows the cytotoxicity and inhibitory effect of cell aging by adriamycin of Jangchaechae extract in human fibroblasts. A, Cytotoxic effect of Jangchaechae extract. Each extract was treated with 10, 100 ug / ml, and then cultured for 3 days to examine the cytotoxic effect by MTT method. B, SA-β-gal active staining picture and SA-β-gal active staining percentage. After adriamycin treatment, each extract was treated with 10 ug / ml, and 3 days later, SA-β-gal activity staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.

Figure 9 shows the cytotoxicity and inhibitory effect of cell aging by adriamycin of Jangchaechae hexane extract in human fibroblasts. A, showing the cytotoxic effect of jangguchae hexane extract. Each extract was treated with 1-10ug / ml, and then cultured for 3 days to examine the cytotoxic effect by MTT method. B and C, SA-β-gal active staining picture and SA-β-gal active staining percentage. After adriamycin treatment, Jangchaechae hexane extract was treated with 1-10 ug / ml, and 3 days later, SA-β-gal activity staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. D, p53, phosphorylated S6K, p21 expression. After the adriamycin treatment, the extract was treated with 1-10 ug / ml, and the expression level of each protein was examined by Western blot method. NT, untreated; Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.

Figure 10 shows the effect of inhibiting replication cell aging of Jangchaechae hexane extract in human fibroblasts. A, the cytotoxic effect of Jangchaechae hexane extract on cloned aging cells. Each extract was treated with 1-10ug / ml, and then cultured for 3 days to examine the cytotoxic effect by MTT method. B and C, SA-β-gal active staining picture and SA-β-gal active staining percentage. The cloned aging cells were treated with Jangchaechae hexane extract 1-10 ug / ml, and 3 days later, SA-β-gal activity staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. Old, Old cells; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.

Figure 11 shows the cytotoxicity of the Jangchaechae single component in human fibroblasts and the effect of inhibiting cell aging by adriamycin. A, Cytotoxic effect of jang guchae single component. Each component was incubated for 10 days after 10ug / ml treatment, and the cytotoxic effect was examined by MTT method. B, SA-β-gal active staining percentage. After adriamycin treatment, ZGC-5, ZGC-6, and ZGC-9 were treated at 10 ug / ml, and 3 days later, SA-β-gal active staining was performed. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine. * p <0.05 vs DMSO.

Figure 12 shows the effect of inhibiting cell aging by adriamycin of Jangchaechae single component ZGC-9 ((-)-bornenesitol) in human fibroblasts A. B, SA-β-gal activity Staining Photos and Percentage of SA-β-gal Active Staining After adriamycin treatment, ZGC-9 was treated at 1-10 ug / ml and SA-β-gal active staining was performed 3 days later. After repeated three or more times, the mean and standard deviation were expressed, and the expression of C, p53, phosphorylated S6K, and p21 was expressed After treatment with adriamycin, ZGC-9 was treated at 1-10 ug / ml, followed by Western blot method. The expression level of each protein was examined using: NT, untreated; Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * P < 0.05 or ** p <0.01 vs DMSO.

13 shows the effect of ZGC-9 ((-)-bornesitol) on free radical production in human fibroblasts. A, free radical flow cytometry. B, average free radicals comparison. Fibroblasts were treated with adriamycin and ZGC-9 was treated with 10 ug / ml, and the level of intracellular free radicals was examined by flow cytometry. Each experiment was repeated three times or more independently, and the mean and standard deviation were expressed. ADR, adriamycin; Y, young cells; Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.

14 shows the effect of ZGC-9 ((-)-bornesitol) on replication senescence in human fibroblasts. A, SA-β-gal active staining pictures are shown. B, SA-β-gal active staining percentage. C, ZGC-9 shows the cytotoxic effect. The cloned cells were treated with 1-10 ug / ml of ZGC-9 and subjected to SA-β-gal activity staining 3 days later. Cytotoxicity was examined by MTT method. The results were expressed as mean and standard deviation after each experiment was repeated three times or more independently. Old, Old cells; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.

Figure 15 shows the cytotoxicity of Jangchaechae single component ZGC-2 (ursolic acid) in human fibroblasts and the inhibition of cell aging by adriamycin. A, shows the cytotoxic effects of ZGC-2. , SA-β-gal activity staining pictures and SA-β-gal activity staining percentages After adriamycin treatment, ZGC-2 was treated with 1 ug / ml, and 3 days later, SA-β-gal activity staining was performed. Results were shown as mean and standard deviation after each experiment repeated three or more times: Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin (rapamycin). * p <0.05 or ** p <0.01 vs DMSO.

Figure 16 shows the effect of inhibiting cell aging by adriamycin of Jangchaechae single component ZGC-2 (ursolic acid) in human fibroblasts. A and B, SA-β-gal activity staining photos and SA-β-gal Percentage of Active Staining After adriamycin treatment, ZGC-2 was treated with 0.01-1 ug / ml and SA-β-gal active staining was performed three days later. The mean and standard deviation are shown, followed by the expression of C, p53, phosphorylated S6K and p21 After treatment with adriamycin, ZGC-2 was treated with 0.01-1 ug / ml, and the expression level of each protein was determined by Western blot. NT, untreated; Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * P <0.05 or ** p <0.01 vs DMSO.

Figure 17 shows the effect of ZGC-2 (ursolic acid) on the production of free radicals in human fibroblasts. A, free radical flow cytometry. B, average free radicals comparison. Fibroblasts were treated with adriamycin, ZGC-2 was treated with 1 ug / ml, and the level of free radicals in the cells was examined by flow cytometry, and each experiment was repeated three or more times independently, indicating the mean and standard deviation ADR, adriamycin; Y, young cells; Cont, control; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * P <0.05 or ** p <0.01 vs DMSO.

Figure 18 shows the effect of inhibiting replication cell aging of ZGC-2 (ursolic acid) in human fibroblasts. A, shows the SA-β-gal activity staining pictures. B, SA-β-gal activity staining percentage ZGC-2 was treated with 0.01-1 ug / ml of cloned aging cells and subjected to SA-β-gal activation staining 3 days later.The results were repeated three or more times independently of each experiment. Variation: Old, Old cells; DMSO, Dimethylsulfoxide; NAC, N-acetylcysteine; Rap, Rapamycin. ** p <0.01 vs DMSO.

 The inventors investigated whether Jangchaechae extract and twelve single components isolated from Jangchaechae are effective against cell aging in human vascular endothelial cells and fibroblasts. In conclusion, Jangchaechae ethyl acetate extract in human vascular endothelial cells and Jangchaechae hexane extract, ZGC-2 (ursolic acid) and ZGC-9 ((-)-bornesitol) in human fibroblasts were found to be effective in inhibiting cell aging. The invention was completed.

The present invention provides a pharmaceutical composition for inhibiting cellular aging containing Jangchaechae extract or (-)-bornenesitol ((-)-bornesitol) represented by the following Chemical Formula 1 isolated therefrom as an active ingredient.

<Formula 1>

Specifically, the jangguchae extract is hexane (n-hexane) fraction extract extracted by adding distilled water and hexane (n-hexane) to the jangguchae methanol extract and fractionated, more specifically, the cells are fibroblasts It is done.

In detail, the jangguchae extract is ethyl acetate (EtOAc) fraction extract extracted by adding ethyl acetate (EtOAc) to the distilled water layer fractionated by adding distilled water and hexane (n-hexane) to the methanol extract of Jangguchae More specifically, the cells are characterized in that the umbilical vein vascular endothelial cells.

Specifically, the cellular senescence is characterized by induced by adriamycin, the cell aging inhibition is to measure the inhibition of senescence-associated β-galactosidase (SA-β-gal) activity It features.

In the case of the pharmaceutical composition of the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the jang-guchae extract or (-)-bornenesitol ((-)-bornesitol), such a pharmaceutically acceptable Carriers to be used are commonly used in pharmaceutical preparations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrroly Don, cellulose, 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 humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like as an additive.

The pharmaceutical composition is determined by the method of administration according to the degree of symptoms of cellular aging, usually topical administration is preferred. In addition, the dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the degree of the disease, the age, sex, and weight of the patient, and may be administered once to several times daily.

The pharmaceutical composition may be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The pharmaceutical compositions may be prepared in unit dose form or formulated using pharmaceutically acceptable carriers and / or excipients or may be prepared within a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion, or may be in the form of an exercicide, extract, powder, granule, tablet, warning, lotion, ointment, or the like.

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

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 Isolation of Jangguchae Extract and Chemical Structure of a Single Component

1.Isolation of Jangguchae Extract and Single Ingredient

20.0 L of 80% methanol was added to 10.0 kg of jangguchae and extracted three times for 12 hours by reflux cooling at 60 ° C. Filter paper was laid and filtered under reduced pressure, and the filtrate was concentrated under reduced pressure to obtain 550.0 g of an extract from which methanol was removed. 550.0 g extract in after the suspension was shaken added to H ₂ O 1.0 L, the same amount of n - hexane (n -hexane), ethyl acetate (EtOAC), n - through solvent fractionation was added to butanol (n -butanol) n 75.9 g of -hexane fraction, 47.4 g of EtOAC fraction, 97.0 g of n -butanol fraction, and 329.7 g of H ₂ O fraction were obtained.

After making a 60 × 12 cm size Silica-gel (70-230mesh) column, 50.0 g of the n- hexane fraction was loaded and gradient eluted with a mobile phase solvent [EtOAC: MeOH = 100: 1 → 1: 1], followed by TLC. 32 sub fractions (HZG1 ~ 32) were obtained. Fraction HZG14 (2.5 g) was loaded onto a 50 × 4 cm size Silica-gel (70-230mesh) column, followed by gradient elution with mobile phase solvent [n-hexane: EtOAC = 50: 1 → 1: 1] to ZGC-1 (300 mg) was obtained. Fraction HZG24 (1.0 g) (2.5 g) was loaded onto a 75 x 2 cm size Silica-gel (70-230mesh) column and gradient elution with mobile phase solvent [n-hexane: EtOAC = 50: 1 → 1: 1] So ZGC-2 (30.4 mg) was obtained, fraction HZG25 (0.5 g) was loaded onto a 75 × 5 cm size Silica-gel (70-230mesh) column and gradient elution with mobile phase solvent [n-hexane: EtOAC = 50 : 1 → 1: 1] to obtain ZGC-3 (25.7 mg). Fraction HZG31 (34.2 g) was recrystallized from methanol to obtain ZGC-4 (20.0 mg). ZCC-5 (25.7 mg) was obtained by loading in a Silica-gel (70-230mesh) column having a size of 75 × 5 cm and gradient eluting with a mobile phase solvent [n-hexane: EtOAC = 50: 1 → 1: 1].

Silica-gel (70-230mesh) column of 60 × 12 cm size was made, and 90.0 g of n- butanol fraction was loaded and gradient eluted with a mobile phase solvent [CHCl ₃ : MeOH = 100: 1 → 10:30] to TLC. 20 sub fractions (BZG1 ~ 20) were obtained. Fraction BZG11 (4.2 g) was loaded onto a Silica-gel (70-230mesh) column of 50 × 4 cm size and gradient eluted with a mobile phase solvent [CHCl ₃ : MeOH = 100: 1 → 1: 1] to give ZGC-5 ( 142.5 mg) was loaded onto a 75 × 2 cm Sephadex ™ LH-20 column, and ZGC-6 (40 mg) was obtained with a solvent composition of CHCl 3: MeOH = 1: 3. Fraction BZG12 (0.3 g) was loaded onto a 75 × 2 cm Sephadex ™ LH-20 column to obtain ZGC-7 (13.5 mg) and ZGC-8 (50.5 mg) with a solvent composition of 100% MeOH. Fraction HZG13 (2.0 g) and Fraction HZG17 (30.0 g) were recrystallized from methanol to obtain ZGC-9 (50.0 mg) and ZGC-10 (69.5 mg). Fraction BZG14 (6.5 g) was loaded onto a 75 × 2 cm Sephadex ™ LH-20 column, yielding ZGC-11 (14.5 mg) and ZGC-12 (30.0 mg) with a solvent composition of 100% MeOH (FIG. 1). ). Twelve isolated single substances were determined by spectroscopic analysis methods such as MS, NMR ( ¹ H, ¹³ C, DEPT, ¹ H- ¹ H COZY, HMQC, HMBC) to determine the chemical structure of the compounds and compare them with the literature. I identified it.

2. Physical and Chemical Properties of Separated Materials

12 compounds isolated from the n -haxane fraction with n -butanol fraction of jangguchae are various spectroscopic analysis or the like as base material ^{MS, NMR (1 H, 13} C, DEPT, 1 H- 1 H COSY, HMQC, HMBC) Method, and compared with literature, ZGC-1 [α-spinasterol], ZGC-2 [ursolic acid], ZGC-3 [ergosterol peroxide], ZGC-4 [α-spinasterol-glucoside], ZGC-5 [2-methoxy-9-β-D-ribofuranosyl purine (2-methoxy-9-β-D- ribofuranosyl purine], ZGC-6 [aristeromycin], ZGC-7 [ecdysteron], ZGC-8 [polypodoaurein], ZGC-9 [(-)- Identified as Bornecitol ((-)-bornesitol], ZGC-10 [D-mannitol], ZGC-11 [vitexin], ZGC-12 [cytisoside] (FIG. 2).

ZGC-1 [colorless crystals, IR ν _max (KBr) cm ⁻¹ : 3251 (OH), 1676 (C = C stretch); ¹ H-NMR (250 MHz, pyridine- d ₅ ) δ: 5.18 (1H, dd, J = 15.0, 8.7 Hz, H-22), 5.14 (1H, t, J = 4.0 Hz, H-7), 5.08 (1H, dd, J = 15.0, 8.7 Hz, H-23), 3.84 (1H, m, H-3), 1.06 (3H, d, J = 6.5 Hz, CH ₃ -21), 0.87 (3H, d , J = 6.8 Hz, CH ₃ -26), 0.85 (3H, d, J = 6.5 Hz, CH ₃ -27), 0.82 (3H, s, CH ₃ -19), 0.59 (3H, s, CH _3- 18); ¹³ C-NMR (62.5 MHz,, pyridine- d ₅ ) d: 37.5 (C-1), 32.1 (C-2), 71.0 (C-3), 38.9 (C-4), 40.5 (C-5) , 29.5 (C-6), 117.9 (C-7), 139.6 (C-8), 49.7 (C-9), 34.4 (C-10), 21.8 (C-11), 39.6 (C-12), 43.4 (C-13), 55.3 (C-14), 23.3 (C-15), 28.2 (C-16), 55.9 (C-17), 13.2 (C-18), 12.1 (C-19), 41.1 (C-20), 21.2 (C-21), 138.6 (C-22), 129.6 (C-23), 51.3 (C-24), 37.6 (C-25), 21.5 (C-26), 19.1 ( C-27), 25.6 (C-28), 12.4 (C-29). Positive FAB-MS m / z 412 [M] ⁺ .]

ZGC-2 (colorless needles, ¹ H-NMR (250 MHz, MeOD) δ: 5.03 (1H, t, H-12), 3.05 (1H, m, H-3), 2.87 (1H, dd, J = 13.2 , 4.2 Hz, H-2), 1.84 (1H, d, J = 11.3 Hz, H-18), 1.00 (3H, s, CH ₃ -23), 0.93 (3H, d, J = 6.7 Hz, CH ₃ -30), 0.86 (3H, s, CH ₃ -26), 0.84 (3H, s, CH ₃ -27), 0.78 (3H, d, J = 6.5 Hz, CH ₃ -29), 0.73 (3H, s _{, CH 3 -24), 0.66 (} 3H, s, CH 3 -25); ¹³ C-NMR (62.5 MHz, MeOD) d: 39.8 (C-1), 27.9 (C-2), 79.7 (C-3), 38.1 (C-4), 56.7 (C-5), 19.5 (C -6), 34.3 (C-7), 40.8 (C-8), 48.9 (C-9), 40.0 (C-10), 24.1 (C-11), 126.9 (C-12), 139.6 (C- 13), 43.2 (C-14), 28.8 (C-15), 25.3 (C-16), 48.9 (C-17), 54.3 (C-18), 40.4 (C-19), 40.4 (C-20 ), 30.8 (C-21), 38.1 (C-22), 29.2 (C-23), 16.0 (C-24), 16.4 (C-25), 17.8 (C-26), 24.4 (C-27) , 181.7 (C-28), 17.7 (C-29), 21.6 (C-30).]

ZGC-3 [Needles, UV λ max (CHCl ₃ ) nm 243; v _max (KBr) cm-1 3406 (OH), 2954 (CH), 1716 (C = C), 1457 (CH ₂ ), 1045 (CO); ¹ H-NMR (CDCl ₃ , 250 MHz,) δ 0.81 (3H, s), 0.82 (3H, d, J = 6.8 Hz), 0.83 (3H, d, J = 6.8 Hz), 0.86 (3H, s) , 0.95 (3H, d, J = 6.6 Hz), 1.19 (3H, d, J = 6.6 Hz), 3.91 (1H, m, H-3), 5.13 (2H, m, H-22, H-23) , 6.20, 6.45 (each 1 H, d, J = 8.4 Hz, H-6, H-7); ¹³ C-NMR (CDCl ₃ , 62.9 MHz) δ 135.4 (C-7), 135.1 (C-23), 132.2 (C-22), 130.6 (C-6), 82.1 (C-8), 79.3 (C -5), 66.2 (C-3), 56.1 (C-17), 51.5 (C-14), 50.9 (C-4), 4.5 (C-13), 42.7 (C-24), 39.7 (C- 20), 39.2 (C-12), 36.8 (C-1, C-10), 34.6 (C-9), 33.0 (C-25), 29.9 (C-2), 28.6 (C-15), 23.3 (C-16), 20.8 (C-11), 20.5 (C-27), 19.9 (C-26), 19.6 (C-21), 18.1 (C-19), 17.5 (C-28), 12.8 ( C-18). Positive FAB-MS m / z 429 [M + H] ⁺ .]

ZGC-4 [colorless crystals, IR ν _max (KBr) cm ⁻¹ : 3395 (OH), 2938, 2864 (CH), 1457 (CH ₂ ), 1367 (CH ₃ ), 1060, 1030 (glycosidec CO); ¹ H-NMR (250 MHz, pyridine- d ₅ ) d: 5.17 (1H, dd, J = 8.1, 3.5 Hz, H-7), 5.05 (2H, m, H-22, H-23), 4.62 ( 1H, d, J = 9.6 Hz, H-1 '), 4.44 (1H, dd, J = 4.5, 9.3 Hz, H-5'), 4.30 (1H, m, H-6 '), 4.10-3.97 ( 4H, m, H-2 ', 3', 4 'and H-3), 1.07 (3H, d, J = 6.5 Hz, CH ₃ -21), 0.83-0.90 (9H, m, CH ₃ -26, 27, 29), 0.70 (3H, s, CH ₃ -19), 0.57 (3H, s, CH ₃ -18); ¹³ C-NMR (62.5 MHz, pyridine- d ₅ ) d: 37.3 (C-1), 32.2 (C-2), 71.7 (C-3), 37.3 (C-4), 40.1 (C-5), 30.0 (C-6), 117.9 (C-7), 139.5 (C-8), 49.5 (C-9), 34.5 (C-10), 21.7 (C-11), 39.6 (C-12), 43.4 (C-13), 55.3 (C-14), 23.3 (C-15), 28.9 (C-16), 60.0 (C-17), 13.1 (C-18), 12.2 (C-19), 41.2 ( C-20), 21.6 (C-21), 138.7 (C-22), 129.6 (C-23), 51.4 (C-24), 37.3 (C-25), 21.7 (C-26), 19.2 (C -27), 25.7 (C-28), 12.5 (C-29), 102.2 (C-1 '), 75.4 (C-2'), 78.7 (C-3 '), 77.0 (C-4'), 78.6 (C-5 '), 62.9 (C-6'). Positive FAB-MS m / z 574 [M] ⁺ .]

ZGC-5 (amorphous powder, IR ν _max (KBr) cm ⁻¹ : 3422 (OH), 1697 (C = C), 1470, 1264, 1109 (CN); ¹ H-NMR (250 MHz, pyridine- d ₅ ) d: 7.71 (1H, s, H-2), 7.67 (1H, s, H-8), 5.76 (1H, d, dd, J = 6.0 Hz, H-1 '), 4.48 (1H, t, J = 5.5 Hz, H-2'), 4.04 (1H, dd, J = 2.4, 5.5 Hz, H-3 '), 3.72 (1H, d, J = 2.4 Hz, H-4 '), 3.27 (1H, doublet of doublets, J = 2.2, 12.4 Hz, H-5'); ¹³ C-NMR (75 MHz, pyridine- d ₅ ) d: 164.4 (C-6), 152.2 (C-2), 150.0 (C-4), 141.0 (C-8), 102.3 (C-5), 90.2 (C-1 '), 86.1 (C-4 '), 76.0 (C-2'), 71.1 (C-3 '), 61.6 (C-5'), 49.6 (OCH ₃ ).]

ZGC-6 [amorphous powder, UV λ max (H ₂ O) nm 265; IR n _max (KBr) cm ⁻¹ : 3335, 3148 (NH ₂ ), 2929 (CH), 1666, 1604 (NH), 1209 (CN), 1037 (glycosidec CO); ¹ H-NMR (250 MHz,, pyridine- d ₅ ) d: 7.71 (1H, s, H-2), 7.67 (1H, s, H-8), 5.76 (1H, d, dd, J = 6.0 Hz , H-1 '), 4.48 (1H, t, J = 5.5 Hz, H-2'), 4.04 (1H, dd, J = 2.4, 5.5 Hz, H-3 '), 3.72 (1H, d, J = 2.4 Hz, H-4 '), 3.27 (1H, doublet of doublets, J = 2.2, 12.4 Hz, H-5'); ¹³ C-NMR (75 MHz, pyridine- d ₅ ) d: 157.7 (C-6), 157.6 (C-4), 153.2 (C-2), 140.6 (C-8), 121.4 (C-5), 90.7 (C-1 '), 87.8 (C-4 '), 75.4 (C-2'), 72.5 (C-3 '), 63.0 (C-5'). FAB-MS m / z 266 [M + H] ⁺ .]

ZGC-7 (amorphous powder, IR ν _max (KBr) cm ^-1 : 3359 (OH), 2712 (CH), 1650 (C = C), 1452 (CH ₂ ), 1321 (CH ₃ ), 1068 (COC) ; ¹ H-NMR (250 MHz, pyridine- d ₅ ) d: 5.80 (1H, d, J = 2.0 Hz, H-7), 3.93-3.60 (2H, m, H-2, H-3), 3.28 ( 1H, dd, J = 9.6, 2 Hz, H-22), 1.20 (9H, s, CH ₃ -21, CH ₃ -26, CH ₃ -27), 0.95 (3H, s, CH ₃ -19), _{0.88 (3H, s, CH 3} -18); ¹³ C-NMR (62.5 MHz, pyridine- d ₅ ) d: 37.3 (C-1), 68.5 (C-2), 68.6 (C-3), 32.5 (C-4), 50.5 (C-5), 206.4 (C-6), 122.1 (C-7), 168.0 (C-8), 35.0 (C-9), 39.2 (C-10), 21.0 (C-11), 32.8 (C-12), 49.9 (C-13), 85.2 (C-14), 31.8 (C-15), 21.0 (C-16), 51.7 (C-17), 18.0 (C-18), 24.4 (C-19), 77.9 ( C-20), 21.5 (C-21), 78.4 (C-22), 27.3 (C-23), 42.4 (C-24), 71.3 (C-25), 28.9 (C-26), 29.7 (C -27). FAB-MS m / z 479 [M ⁻ H] ⁻ .]

ZGC-8 (amorphous powder, IR ν _max (KBr) cm ^-1 : 3289 (OH), 2915 (CH), 1651 (C = C), 1471 (CH ₂ ), 1371 (CH ₃ ), 1072 (COC) ; ¹ H-NMR (250 MHz, pyridine- d ₅ ) d: 6.23 (1H, s, H-7), 4.17 (1H, m, H-3), 3.84 (1H, m, H-2), 3.66 ( 1H, m, H-22), 3.57 (3H, s, OCH ₃ ), 1.56 (3H, s, CH ₃ -21), 1.34 (6H, s, CH ₃ -26, 27), 1.19 (3H, s _{, CH 3 -19), 1.04 (} 3H, s, CH 3 -18); ¹³ C-NMR (62.5 MHz, pyridine- d ₅ ) d: 36.3 (C-1), 68.1 (C-2), 68.0 (C-3), 31.9 (C-4), 51.8 (C-5), 203.4 (C-6), 121.6 (C-7), 166.7 (C-8), 34.3 (C-9), 38.6 (C-10), 21.4 (C-11), 32.4 (C-12), 48.0 (C-13), 84.1 (C-14), 31.7 (C-15), 21.0 (C-16), 51.3 (C-17), 17.8 (C-18), 24.4 (C-19), 76.8 ( C-20), 21.6 (C-21), 77.4 (C-22), 27.4 (C-23), 42.6 (C-24), 69.5 (C-25), 29.9 (C-26), 30.0 (C -27). Positive FAB-MS m / z 495 [M + H] ⁺ .]

ZGC-9 [whitish amorphous powder, IR ν _max (KBr) cm ⁻¹ : 3434 (OH), 2954 (CH), 1652, 1431, 1055 (CO); ¹ H-NMR (250 MHz, DMSO- d ₆ ) d: 4.72 (1H, t, J = 2.1 Hz, H-2), 4.63 (1H, m, H-6), 4.54 (1H, m, H- 1), 4.49 (1H, m, H-4), 4.34 (1H, m, H-5), 3.61 (3H, s, OCH ₃ ), 2.98 (1H, t, J = 9.0, H-4); ¹³ C-NMR (62.5 MHz, DMSO- d ₆ ) δ: 84.1 (C-1), 70.3 (C-2), 71.1 (C-3), 72.6 (C-4), 72.8 (C-5), 72.2 (C-6), 59.9 (OCH ₃ ).]

ZGC-10 [white powder, mp 168 ~ 169 ℃. IR v _max (KBr) cm ^-1 : 3406 (-OH), 2908 (CH), 1072 (CO); ¹ H-NMR (250 MHz, pyridine- d ₅ ) d: 3.36 (2H, d, CH ₂ -1 or 6), 4.59 (1H, qui, CH-2 or 5), 4.85 (1H, td, CH- 3 or 4), 5.96 (1 H, OH). ¹³ C-NMR (62.5 MHz, pyridine- d ₅ ) d: 65.6 (C-1), 72.1 (C-2), 73.3 (C-3), 73.3 (C-4), 72.1 (C-5), 65.6 (C-6).]

ZGC-11 [yellowish amorphous powder, UV λ max (MeOH) nm 335, 272, 215; IR v _max (KBr) cm ^-1 : 3370 (OH), 1638 (C = C), 1354, 1096; ¹ H-NMR (250 MHz, CD ₃ OD) δ: 7.82 (1H, d, J = 8.7 Hz, H-2 ′, 6 ′), 6.92 (1H, d, J = 8.6 Hz, H-3 ′, 5 '), 6.56 (1H, s, H-3), 6.47 (1H, s, H-6), 4.90 (H, d, J = 7.2 Hz, H-1 "), 4.18 3.44 (6H, m, sugar protons); ¹³ C-NMR (62.5 MHz, CD ₃ OD) d: 183.9 (C-4), 166.2 (C-2), 164.8 (C-9), 162.8 (C-7), 162.0 (C-4 '), 158.7 (C-5), 129.4 (C-2 ', 6'), 123.0 (C-1 '), 117.0 (C-3', 5 '), 109.1 (C-10), 105.2 (C-8), 103.8 (C-3), 95.2 (C- 6), 82.6 (C-5 "), 80.1 (C-3"), 75.3 (C-1 "), 72.5 (C-2"), 71.7 (C-4 "), 62.8 (C-6") .]

ZGC-12 [yellowish amorphous powder, UV λ max (MeOH) nm 335, 270, 213; IR v _max (KBr) cm ^-1 : 3372 (OH), 1640 (C = C), 1346, 1090; ¹ H-NMR (250 MHz, CD ₃ OD) δ: 7.83 (1H, d, J = 8.7 Hz, H-2 ′, 6 ′), 6.93 (1H, d, J = 8.6 Hz, H-3 ′, 5 '), 6.57 (1H, s, H-3), 6.48 (1H, s, H-6), 7.39 (1H, s, H-2'), 5.0 (H, d, J = 7.0 Hz, H -1 "), 4.18-3.44 (6H, m, sugar protons), 3.80 (3H, s, OCH ₃ ); ¹³ C-NMR (62.5 MHz, CD ₃ OD) d: 182.1 (C-4), 166.1 (C-2), 162.8 (C-9), 162.8 (C-7), 162.5 (C-4 '), 158.5 (C-5), 129.5 (C-2 ', 6'), 123.1 (C-1 '), 117.0 (C-3', 5 '), 109.8 (C-10), 103.2 (C-8), 103.9 (C-3), 95.9 (C- 6), 82.5 (C-5 "), 77.8 (C-3"), 73.6 (C-1 "), 72.1 (C-2"), 71.5 (C-4 "), 62.3 (C-6") , 56.6 (OCH ₃ ).]

Example 2 Investigation of Cytotoxicity and Cellular Aging Efficacy of Jangchaechae Extract and Single Component

1. Experimental Materials

Human fibroblasts and umbilical vein endothelial cells were purchased from Lonza (Walkersville, MD, USA). Dubroccos-Modified Eagle's medium (DMEM), Fetal Bovine Serum, Antibiotic Solution Penicillin-Streptomycin, WelGene (Daegu, Korea), Endothelial Cell Growth Medium-2 -2, EGM-2) was purchased from Lonza (Walkersvill, MD, USA). Antibodies to p53 are described in SantaCruz Biotech, Inc. (SantaCruz, CA, USA), and antibodies against p21 and pS6 were from Cell Signaling Technology Inc. (Beverly, MA, USA). GAPDH antibody was distributed by Dr. Ki-sun Kwon, Korea Research Institute of Bioscience and Biotechnology. Adriamycin used products of Ildong Pharmaceutical Co., Ltd.

2. Cell Culture

Human fibroblasts were cultured using a DMEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin 10,000 units / ml, streptomycin 10,000 ug / ml). After dispensing 10 ⁵ , it was incubated in 37 ℃, 5% carbon dioxide incubator. When the cells grew to 80-90% at the bottom of the culture dish, trypsin-EDTA solution (2.5X) was added to separate the cells, and then passaged. Umbilical cord vascular endothelial cells were cultured in the same manner as human fibroblasts using EGM-2 as a culture medium. Each time the cells were passaged, the cell number was measured to determine how many times the cells divide. Population doubling (PD) was calculated using the formula PD = log ₂ F / log ₂ I (F = last cell number, I = first cell number). The cells used in the experiment were divided into PD <35 or PD> 75 for human fibroblasts and PD <30 or PD> 50 for cord vascular endothelial cells.

3. Induction of Cell Aging by Adriamycin Treatment

^1.5 x 10 ⁵ aliquots of human fibroblasts and umbilical vein endothelial cells were dispensed in a 100 mm diameter dish. After incubation for 3 days at 37 ℃, 5% carbon dioxide incubator, the cell culture was removed. The cells were washed twice with DMEM culture containing antibiotics. The cells were treated with 500 nM adriamycin for 4 hours, and then washed three times with DMEM culture medium containing antibiotics. Human fibroblasts were cultured in DMEM medium containing 10% fetal calf serum and 1% antibiotic, and human umbilical vein endothelial cells were cultured in EGM-2 culture. After 4 days, it was confirmed that senescence-associated β-galactosidase (SA-β-gal) activity staining induced cell aging.

4. Method of measuring 3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide; MTT)

The effect of jangguchae extract and compound on the growth rate of cells was investigated by MTT method. 0.1% MTT solution was added to 50 ul of each well of a 96 well culture vessel, and the reaction was performed at 37 ° C. and a 5% carbon dioxide incubator for 3 hours. After removing the culture solution and the MTT solution, 100 ul of dimethyl sulfoxide was added to dissolve the crystals formed. The growth rate of the cells was measured by measuring the absorbance at 550 nm using a microplate reader.

5. Investigation of the effects of Jangchaechae extract and single components on cell aging by adriamycin

We investigated whether Jangchaechae extract and 12 single compounds isolated from the extract were effective 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 96well, 12well, and 24well cell culture vessels. The fibroblasts and 500 umbilical endothelial cells were dispensed in each well in a 96 well cell culture vessel. In 24 wells, fibroblasts were divided into 3000 / well, umbilical cord endothelial cells and 5000 / well, and 12well were divided into 5000 fibroblasts / well and 7000 endothelial cells / well. Incubated at 37 ° C., 5% carbon dioxide incubator for one day. In 96 well, add 100ul of DMEM culture medium and EGM-2 medium containing 10% fetal bovine serum and 1% antibiotic, and replace 12well and 24well culture medium, extract 100ug / ml, single compound 10 treated with ug / ml. Dimethyl sulfoxide was added as a negative control and 5 mM of N-acetylcysteine and 500 nM of rapamycin were added as a positive control. After incubation in a 5% carbon dioxide incubator at 37 ° C. for 3 days, the growth rate of the cells was determined by the MTT method, and the senescence-associated β-galactosidase (SA-β-gal) activity was observed by the degree of cell aging. It was examined by staining.

6. Senescence-associated β-galactosidase (SA-β-gal) activity staining

The effect on cell aging was examined by SA-β-gal activity staining. After treatment with a single compound in a 24 well or 12 well culture vessel for 3 days, the cells were washed with phosphate buffer. After fixing the cells with 3.7% paraformaldehyde (paraformaldehyde), the fixed solution was removed and washed again with phosphate buffer. 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] were added to each well in a 24 well culture vessel. 250 ul per, 12 well culture vessel was put 500ul per well. Wrapped in tinfoil and reacted at 37 ° C. for 17 hours. After washing twice with phosphate buffer (PBS), it was stained for 1 minute with 1% Eogene solution. After washing twice with phosphate buffer solution, the cells stained blue with an optical microscope were observed. SA-β-gal activity level was expressed as a percentage (%) by measuring the number of cells stained blue in the cytoplasm out of a total of 50-100 cells.

7. Cell Protein Extraction

Each cell was dispensed into 1 × 10 ⁵ in a 60 mm culture dish and then cultured in a 37 ° C., 5% carbon dioxide incubator. After washing the cells twice with DMEM broth containing antibiotics, Jangchaechae extract and single components were treated for 1 hour before concentration, and adriamycin 500 nM for 4 hours. After removing the culture solution, it was washed twice with phosphate buffer. Cell lysis solution per culture dish [25 mM Tris-HCl (pH 7.6), 150 mM Nacl, 1% Tryton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium vanadate vanadate), 5 mM NaF, protease inhibitor or 1 mM PMSF] was added 50 ul. The plate was scraped using a cell scraper to collect the solution and cells and transferred to the microacupuncture tube. The solution was shaken every 10 minutes while reacting for 30 minutes on ice. The supernatant was transferred to a new tube by centrifugation for 15 minutes at 12,000 rpm. 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 isolated by electrophoresis on 10% SDS-polyacrylamide gel. After transferring the protein to the nitrocellulose membrane, it was reacted 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 primary antibodies against p53, pS6 or p21. After washing three times with TTBS solution three times, and reacted with a horseradish peroxidase-bound secondary antibody for 1 hour 30 minutes. After washing the membrane five times for 7 minutes with TTBS, the amount of protein 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 was used in each experiment and compared with glyceraldehyde-3-phosphate dehydrogease (GAPDH) antibody.

9. Determination of free radicals in cells

The cells were dispensed into ^1.5 × 10 ⁵ in a 100 mm culture dish and incubated for 3 days in a 37 ° C., 5% carbon dioxide incubator. The cells were washed twice with DMEM broth containing antibiotics and then treated with adriamycin 500 nM for 4 hours. The cells were washed once with phosphate buffer, treated with trypsin-EDTA solution (2.5%), and cells were separated, and then aliquoted into 1 × 10 ⁵ in a 60 mm culture dish. Incubated at 37 ° C., 5% carbon dioxide incubator for one day. The culture solution was changed and a single compound was treated with 10 ug / ml and 1 ug / ml. Dimethyl sulfoxide was added as a negative control and 5 mM of N-acetylcysteine and 500 nM of rapamycin were added as a positive control. After incubation for 3 days at 37 ℃, 5% carbon dioxide incubator, washed twice with DMEM culture medium containing antibiotics and was treated with H ₂ DCFDA 250uM for 20 minutes. Once washed with phosphate buffer solution, trypsin-EDTA solution was added to the cells were separated and transferred to the microneedle tube. The supernatant was discarded by centrifuging at 5,000 × g for 1 minute, 1ml of phosphate buffer solution containing 2% fetal calf serum was washed, and the cells were further centrifuged at 5,000 × g for 1 minute. After repeated washing twice, 1 ml of 1% paraformaldehyde was added. Intracellular ROS levels were measured using a BD FACS CantoII flow cytometer (BD Biosciences, San Jose, Calif.).

10. Results

(1) Investigation of Cytotoxicity and Cellular Aging Inhibitory Effect of Jangchaechae Extract in Human Umbilical Vessel Endothelial Cells

The cytotoxicity of five Jangchaechae extracts from human umbilical vascular endothelial cells was investigated. When each extract was treated with 10, 100 ug / ml, only butanol extract showed toxicity at 10 ug / ml and all other substances did not show cytotoxicity (FIG. 3A). We investigated whether Jangchaechae extract inhibited cell aging induced by adriamycin. Treatment with ethyl acetate extract at 10 ug / ml significantly reduced SA-β-gal activity increased by adriamycin treatment (FIG. 3B). Treatment with increasing concentration of ethyl acetate extract resulted in inhibition of SA-β-gal activity increased by adriamycin in a concentration-dependent manner (FIGS. 4A, 4B and 4C). The effect of this extract on the expression of proteins such as p53, p21, and phosphorylated S6K during cell aging induced by adriamycin was investigated. As a result, it was confirmed that the ethyl acetate extract inhibited the expression of p21 increased by adriamycin (FIG. 4D).

We investigated whether ethyl acetate extracts inhibit cell senescence not only in cell aging by adriamycin but also in cells in which senescence is induced. As a result, ethyl acetate extract reduced SA-β-gal activity increased by replication aging in a concentration-dependent manner (FIG. 5).

From the above results, it was confirmed that Jangchaechae ethyl acetate extract has an effect of inhibiting replication aging due to cell aging and cell division by adriamycin.

(2) Investigation of Cytotoxic Effect and Inhibitory Effect of Cellular Aging in Human Umbilical Vein Endothelial Cells

The cytotoxicity of 12 single components of Jangchaechae in human umbilical vascular endothelial cells was investigated. When the compounds were treated with 10 ug / ml, all of the other substances except ZGC-2 and 3 were not cytotoxic (FIG. 6A). Among them, ZGC-6, 7, 10, 11, 12 was investigated whether inhibiting cell aging by adriamycin, but the effect of inhibiting cell aging could not be observed (Fig. 6B). When the concentration of ZGC-2, 3, which was toxic at 10 ug / ml, was lowered by 1 ug / ml, no cytotoxicity was observed (FIG. 7A). Treatment with 1 ug / ml ZGC-3 did not inhibit cell senescence by adramycin (FIG. 7B).

As a result, it was confirmed that the single components isolated from Jangguchae had no effect of inhibiting cell aging in umbilical vascular endothelial cells.

(3) Investigation of Cytotoxicity and Cellular Aging Inhibitory Effect of Jangchaechae Extract in Human Fibroblasts

The cytotoxicity of five Jangchaechae extracts from human fibroblasts was first investigated. When these extracts were treated with 10 and 100 ug / ml, no cytotoxicity was observed at 10 ug / ml (FIG. 8A). We investigated whether Jangchaechae extract inhibited cell aging induced by adriamycin. When 10 ug / ml of ethyl acetate extract and hexane extract were treated, SA-β-gal activity increased by adriamycin was decreased, and hexane extract showed greater effect than ethyl acetate extract (FIG. 8B). As a result of further investigation of the effect of the concentration of hexane extract, it was observed that the concentration-dependent inhibition of SA-β-gal activity increased by adriamycin (Figs. 9A, 9B and 9C). The effect of hexane extracts on the expression of proteins such as p53, p21, and phosphorylated S6K during cell aging by adriamycin was investigated. p53, pS6 protein expression was all reduced when treated with 10 ug / ml, p21 protein was reduced from 3 ug / ml (Fig. 9D).

When the hexane extracts inhibited cell aging by adriamycin as well as cell senescence in cells induced by replication aging, concentration-dependently decreased SA-β-gal activity increased by replication aging (FIG. 10). .

From the above results, it was confirmed that Jangchaechae hexane extract has an effect of inhibiting replication aging due to cell aging and cell division by adriamycin.

(4) Investigation of Cytotoxic Effect and Inhibitory Effect of Cellular Aging in Human Fibroblast Cells

The cytotoxicity of 12 single Jangchaechae single component in human fibroblasts was investigated. Cytotoxicity was not observed in the other materials except ZGC-2 when the compounds were treated with 10 ug / ml (FIG. 11A). Among them, ZGC-5, 6, and 9 were investigated to inhibit cell aging, and it was confirmed that ZGC-9 inhibited cell aging (FIGS. 11B and 11C). When ZGC-9 was treated by concentration, SA-β-gal activity was reduced in a concentration-dependent manner (Figs. 12A, 12B). It also reduced p53 and pS6 protein expression in a concentration dependent manner (FIG. 12C). As a result of measuring the amount of intracellular ROS increased by adriamycin by treatment with ZGC-9 10 ug / ml, the amount of ROS decreased but there was no statistical significance (FIG. 13). As a result of examining whether cell senescence was inhibited even in cells in which senescence was induced, it was observed that the SA-β-gal activity increased by senescence was decreased (FIG. 14).

ZGC-2, which was toxic when treated at 10 ug / ml, did not exhibit cytotoxicity when treated at 1 ug / ml (FIG. 15A). ZGC-2 was treated at 1 ug / ml to determine how it affects cellular senescence by adriamycin. It was confirmed that cell senescence was inhibited by ZGC-2 (FIGS. 15B and 15C). ZGC-2 inhibited SA-β-gal activity and p53, p21, pS6 protein expression in a concentration-dependent manner (Figs. 16A, 16B, 16C). ZGC-2 also reduced the amount of intracellular ROS increased by adriamycin at 1 ug / ml (FIG. 17). As a result of examining whether ZGC-2 inhibited cell aging even in cells of replication aging, it was observed that the increased SA-β-gal activity was reduced by the replication aging (FIGS. 18A and 18B).

From the above results, ZGC-2 (ursolic acid) and ZGC-9 ((-)-bornesitol) isolated from Jang-chae were caused by cell aging and cell division by adriamycin at concentrations of 1 ug / ml and 10 ug / ml, respectively. It was confirmed that there is an effect that inhibits replication aging.

Claims (8)

1. A pharmaceutical composition for inhibiting cellular aging containing Jangchaechae extract or (-)-bornenesitol ((-)-bornesitol) represented by the following Chemical Formula 1 as an active ingredient.

   <Formula 1>

   
2. [Claim 2] The pharmaceutical composition for inhibiting cell aging according to claim 1, wherein the jang-guchae extract is hexane (n-hexane) fraction extract extracted by adding distilled water and hexane (n-hexane) to the jang-guchae methanol extract.
3. The pharmaceutical composition for inhibiting cellular aging according to claim 2, wherein the cells are fibroblasts.
4. According to claim 1, wherein the jangguchae extract is ethyl acetate (EtOAc) fraction extracted by adding ethyl acetate (EtOAc) to the distilled water layer fractionated by adding distilled water and hexane (n-hexane) to the jangguchae methanol extract Pharmaceutical composition for inhibiting cell aging, characterized in that the extract.
5. The pharmaceutical composition of claim 4, wherein the cells are umbilical vein vascular endothelial cells.
6. The pharmaceutical composition of claim 1, wherein the cellular senescence is induced by adriamycin.
7. The pharmaceutical composition for inhibiting cellular senescence of claim 1, wherein the inhibition of cellular senescence measures inhibition of senescence-associated β-galactosidase (SA-β-gal) activity.
8. The method of claim 1, wherein the pharmaceutical composition prevents or treats any one disease selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damage tissue, arteriosclerosis, prostate hyperplasia and liver cancer. Pharmaceutical composition for inhibiting cell aging.

Images