KR100490315B1 - Uncaria sinensis extract showing anti-aging effect on cells and cosmetics comprising the same - Google Patents

Abstract

The present invention relates to an extract for inhibiting aging against cells and to an anti-aging functional cosmetic and food additive containing the same as an active ingredient, the extract for extract of the present invention has a strong antioxidant activity and a cytoprotective effect against oxidative stress. Since it is effective in inhibiting the aging of skin cells by suppressing the shortening of the telomere length, it can be usefully used in cosmetics and food additives and the like, which exhibits an anti-aging effect.

Description

Uncaria sinensis extract showing anti-aging effect on cells and cosmetics comprising the same}

The present invention relates to an extract for inhibiting aging against cells and to an anti-aging functional cosmetic and food additive containing the same as an active ingredient, the extract for extract of the present invention has a strong antioxidant activity and a cytoprotective effect against oxidative stress. Since it is effective in inhibiting the aging of skin cells by suppressing the shortening of the telomere length, it can be usefully used in cosmetics and food additives and the like, which exhibits an anti-aging effect.

Much research has been conducted on aging to date, but the exact mechanism has not yet been determined due to various phenomena and complex features. However, numerous phenomenological studies have raised some hypotheses about aging, among which free radicals from metabolic processes contribute to aging and telomeres in the chromosome ends. It is increasingly accepted that gradual disappearance ceases cell division and eventually dies (Harman, D., Free radical theory of aging: Role of free radicals in the origination and evolution of life, aging and disease processes, 3- 49. Alan R Liss, New York ( 1986); Harley, CB, Futcher, AB, Greider, CW telomeres shorten during aging of human fibroblasts Nature, 345:. 458-460, (1990)). In other words, free radicals generated in the course of normal metabolism increase oxidative stress in the cell, causing various diseases and aging, and shortening the length of the telomer that protects the chromosome prevents the cells from splitting and enters the plateau phase. Eventually, aging and cell death.

Telomeres consist of a unique structure in which the TTAGGG sequence is repeated as the terminal part of the eukaryotic chromosome. Especially, in the part of guanine (G), the chromosome is stabilized by forming a very stable G-quartet structure through hydrogen bonding with each other. Plays an important role in protection However, as cell division continues, telomeres become shorter in length and lose their protective function, resulting in instability, lumping, or loss of chromosomes, which eventually no longer divide and lead to apotosis. Harley et al. Reported that telomeres shortened with passage of normal human cells, and Vaziri et al. Reported that cells obtained from older people had shorter telomere lengths and fewer cell divisions even when cultured (Harley). , CB, Futcher, AB, Greider, CW Telomeres shorten during aging of human fibroblasts., Nature , 345 : 458-460, (1990); Vaziri, H., Schachter, F., Uchida, I., Wei, L. , Zhu, X., Effros, R., Cohen, D., Harley, CB Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes., Am. J. Hum. Genet ., 52 : 661-667, ( 1993). In addition, recent studies have reported that oxidative stress affects telomere length shortening (Furumoto, K., Inoue, E., Nagao, N., Hiyama, E., Miwa, N., Age -dependent telomere shortening is slowed down by enrichment of intracellular vitamin C via suppression of oxidative stress.Life Sci ., 63 : 935-948, (1998); Von Zglinicki, T., Role of oxidative stress in telomere length regulation and replicative senescence ..., Ann NY Acad Sci , 908:. 99-110, (2000)). Since the shortening of the telomere length is recognized to have a direct effect on the aging and lifespan of the cells, it is expected that substances or extracts capable of suppressing the shortening of the telomere length may inhibit cell aging. There are many other hypotheses, but these hypotheses do not contradict each other but complement each other to explain aging.

Meanwhile, Uncaria sinensis (Rubiaceae) has been used as an antihypertensive and antipyretic sedative in Chinese medicine (Shanghai Science and Technology Publishing House, Chinese Medicine Dictionary, Small Hakwan, p. 1485, (1985); Lee Sang-in, Herbology, Pharmacist, p. 471, (1975)), the main component of which has been reported as an alkaloid of tetracyclic or pentacyclic heteroyohimbine type, oxyindole type (Haginiwa, J., Sakai, S). ., Aimi, N., Yamanaka, E., Shinma, N. Studies of plant containing indole alkaloids.On the alkaloids of Uncaria rhynchophylla Miq.Yakugaku Zasshi , 93 : 448, (1973); Nozoye, T., Shibanuma, Y , Shigehisa, A. Studies on Uncaria alkaloids.Separation of rhinchophylline and corynoxeine.Yakugaku Zasshi , 95 : 758 (1975)). It is reported that tetracyclic oxyindole-based alkaloids such as corynoxeine, rhynchophylline, isochorynoxeine, and isorhynchophylline have hypotensive effects due to Ca ²⁺ antagonism. Endo, K., Oshima, Y., Kikuchi, H., Koshihara, Y., Hikino, H. Hypotensive principles of Uncaria hooks.J. Med. Plant Res ., 49 : 188, (1983); Joji, Y., Shuji, M., Hisashi, M., Goro, K. Screening for calcium antagonists in natural products.The active principles of Uncariae ramulus et uncus.Nippon Yakurigaku Zasshi , 90 : 133, (1987)), Has been reported to protect neuronal damage caused by glutamate (Shimada, Y., Goto, H., Itoh, T., Sakakibara, I., Kubo, M., Sasaki, H., Terasawa, K. Evaluation of the protective effects of alkaloids isolated from the hooks and stems of Uncaria sinensis on glutamate-induced neuronal death in cultur ed cerebellar granule cells from rats.J. Pharm. Pharmacol ., 51 : 715-722, (1999)). However, there have been no specific reports of the use of light bulbs in connection with the inhibition of cellular aging.

Accordingly, the present inventors have tried to find a substance that can suppress the oxidative stress telomere length shortening and skin aging inhibitory activity, as a result, the extract of the light bulbs extracted with alcohol has a strong antioxidant activity cell oxidative stress The present invention was completed by revealing a protective action, significantly shortening the length of telomeres, and providing a cosmetic and food additive for inhibiting cell aging using the same.

It is an object of the present invention to provide a composition for inhibiting aging of cells and the like and extracts for cell aging and food additives for inhibiting cell aging containing the same as an active ingredient.

In order to achieve the above object, the present invention has an anti-aging effect on the cells, and provides an extract of the morning-light and the like extracted by using alcohol as an extraction solvent.

In addition, the present invention provides a cosmetic for inhibiting cell aging containing the extract, such as light bulbs.

In another aspect, the present invention provides a food additive for inhibiting cell aging containing the extract as an active ingredient.

In another aspect, the present invention provides a cell aging inhibitor containing the extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention acts to inhibit the aging of the cells, and provides an extract of the morning-light and the like extracted by using alcohol as an extraction solvent.

The light bulb extract having an anti-aging effect of the present invention is prepared by extracting the light bulbs in a conventional manner using an organic solvent such as alcohol. In the above, alcohol, methanol, ethanol, propanol or butanol may be used, of which ethanol is preferred.

The present inventors measured the inhibitory activity against intracellular oxidation and telomere shortening, which are known to be the cause of aging, and showed significant results in each experiment when the light bulb extract of the present invention was treated. Specifically, the DPPH radical scavenging and lipid peroxidation inhibition experiments showed strong antioxidant activity with IC ₅₀ values of 26.5 and 15.1 ㎍ / ml, respectively (see Table 1 and Table 2 ), and lipid peroxide model using t-BuOOH. In the present invention, when treated with a concentration of 3-30 ㎍ / ㎖, the extract of the present invention showed a cell protective action of 70.6 to 86.0% (see Fig. 1 ). In addition, when the DNA chain cleavage was induced by irradiating UV B, 3 ㎍ / ml of the crude light bulb extract was significantly inhibited, and DNA chain cleavage was significantly inhibited. Was clearly inhibited (see FIGS . 2 and 3 ). In the telomere shortening inhibition experiment for inhibiting skin aging, the lifespan of human dermal keratinocytes was extended by 2.05 times as compared to the control group which was not administered to the control group of 3 ㎍ / ml of the light bulbs ( FIG. 5 and FIG . 5) . 6 ). The prolonged lifespan of these cells was due to the inhibition of the shortening of telomere DNA of keratinocytes, resulting in a Southern blot of DNA extracted from cells of various ages cultured continuously for 32-72 days. southern blot) results (see FIG. 4 ). The total number of cells generated by cell division per one keratinocyte was increased by 50,000 times by treating the extract of the light bulb of the present invention (see FIG. 7 ).

In addition, the present invention provides a cosmetic for inhibiting cell aging containing the extract, such as light bulbs.

The kind of cosmetic in the above, but there are nutrition cream, lotion and skin, but is not necessarily limited thereto.

In the preparation of cosmetics containing the extract of the light bulb of the present invention, the light bulb extract of the present invention is added to the cosmetic composition usually contained in an amount of 1 to 15% by weight, preferably 2 to 10% by weight.

In another aspect, the present invention provides a food additive for inhibiting cell aging containing the extract as an active ingredient.

In the case of using the light bulb extract of the present invention as a food additive, the light bulb extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the extract of the light bulb of the present invention is added in an amount of 0.1 to 15% by weight, preferably 0.2 to 10% by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. Is sure.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

In another aspect, the present invention provides a cell aging inhibitor containing the extract as an active ingredient.

The cell aging inhibitor of the present invention may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsions, It can be formulated into syrups, other liquids.

Specifically, the cell aging inhibitor of the present invention may be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, preparations or granules, emulsions, hard or soft capsules, syrups or It is formulated with elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the cell aging inhibitor of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. To formulate into parenteral formulations, the bulbous extract of the present invention is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

The dosage of the active ingredient according to the present invention is appropriately selected according to the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, but in the case of oral administration in general Adults can be administered once to several times in the amount of 2 to 200 mg of the extract of the composition of the present invention per kilogram of body weight per day, it is preferable to administer in an amount of 10 to 100 mg.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Extracts

Jogu, etc. used in the present invention was used by the appraisal purchased from Jinhyeong Trading Co., Ltd. in Seoul, and the confirmed sample is stored in the herbal medicine laboratory of Korea New Drug Magnetic Light Research Institute. First, after properly slicing the bulbs, 3 l of 95% EtOH was added to 1 kg of bulbs and extracted for 2 weeks at room temperature. The extracts were completely dried and weighed.

As a result, 250 g of light bulb extract was obtained.

Experimental Example 1 Measurement of Antioxidant Activity of Extracts

<1-1> Determination of Scavenging Activity of DPPH Radicals

Dissolve 10 μl of the bulbous bulb extract of the present invention in DMSO in a 96 well plate, add 190 μl of a 2.0 × 10 ⁻⁴ M DPPH (Sigma) solution dissolved in ethanol, and react for 30 minutes at room temperature. The absorbance of the liquid was measured at 517 nm. As a control, 10 μl of DMSO was used instead of the sample, and the concentration of the sample that eliminates DPPH radicals by 50% was set to IC ₅₀ . At this time, the IC ₅₀ value was determined by the regression line obtained by performing three experiments using five different concentrations.

A _control : Absorbance of the control without added sample

A _sample : absorbance of the reaction sphere

As a result, the DPPH radical scavenging activity of the extract of Joong-gu etc. showed an IC ₅₀ value of 26.5 ㎍ / ml, and the radical scavenging activity of α-tocopherol (IC ₅₀ 26.0 ㎍ / ml) was used as a positive control. It was confirmed to represent ( Table 1 ).

Antioxidant Activity of the Extracts of Light Bulbs against DPPH Radicals DPPH radical scavenging activity IC ₅₀ (µg / ml) Early EtOH Extract 26.5 α-tocopherol 26.0

<1-2> Measurement of lipid peroxidation inhibitory activity

10 μl of bulbous extract, 740 μl of 50 mM KP buffer (pH 7.4, KH ₂ PO ₄ -KOH), 50 μl of rat brain homogenate (10 mg protein / ml), 0.1 mM 100 μl of FeSO ₄ · 7H ₂ O and 100 μl of ascorbic acid at a concentration of 1 mM were added, followed by reaction at 37 ° C. for 30 minutes. After the reaction was stopped by adding 250 µl of 20% TCA (Sigma) aqueous solution to the reaction solution, 250 µl of 1% TBA (Sigma) dissolved in 50 mM NaOH solution was added and heated at 95 ° C for 5 minutes. After centrifugation at 9,000 rpm for 10 minutes, the supernatant was taken and the absorbance was measured at 532 nm. Lipid peroxidation inhibitory activity of each sample was calculated as the degree of reduced production of MDA (malonedialdehyde) to the control, the concentration to inhibit the lipid peroxidation reaction 50% was set to IC ₅₀ . At this time, the IC ₅₀ value was determined by the regression line obtained by performing three experiments using five different concentrations.

A _control : Absorbance of the control without added sample

A _sample : absorbance of the reaction sphere

A _blank : Absorbance of blank without sample and FeSO ₄ · 7H ₂ O / ascorbic acid solution

As a result, in the measurement of lipid peroxidation inhibitory activity using the mouse brain homogenate, the IC ₅₀ value was 15.1 μg / ml, and the α-tocopherol used as a positive control showed a strong inhibitory activity similar to that of (IC ₅₀ 16.0 μg / ml). It was confirmed ( Table 2 ).

Antioxidant Activity of the Extracts of Cellulose Root on Lipid Peroxidation Lipid peroxidation inhibitory activity IC ₅₀ (㎍ / mL) Early EtOH Extract 15.1 α-tocopherol 16.0

Experimental Example 2 Measurement of Inhibition of Apoptosis by Peroxidation

HaCaT cell lines, human skin keratinocytes, were grown to log phase and then inoculated in 24-well plates and incubated for 24 hours. A light bulb extract containing 10% FBS and MEM was added to the cells, incubated for 2 hours, and washed. T-BuOOH (tert-butyl-hydroperoxide) dissolved in 1 ml of 0.02% ethanol-HBSS (Gibco Co., Ltd.) was administered to the cells for 24 hours to induce peroxidation, and then the cells were treated with 5% M ethidium homodimer (Et). ₂ , Millipore) was reacted for 30 minutes, and apoptosis was measured using a Millipore microplate fluorometer Cytofluor 2350 (excitation: 485 nm, emission: 645 nm) (Fujiwara M, Nagao N., Monden K., Misumi M., Kageyama K., Yamamoto K., Miwa N., Free Rad.Res. , 27 : 97-104, (1996)).

As a result, the survival rate of HaCaT cells, which are human skin keratinocytes, was reduced to 28.0% for 24 hours by 700 μM t-BuOOH treatment, but 70.6 to 86.0% when the bulbous bulb extract was treated at a concentration of 3 to 30 μg / ml. It was confirmed that the cell survival rate of the present invention, such as extracts of the present invention showed a significant cytoprotective effect ( Fig. 1 ).

Experimental Example 3 Measurement of Cytoprotective Effect on UV Irradiation

Human skin keratinocytes were cultured in serum-free K-GM medium (Kurabo), and then inoculated in 24-well plates to 0.5-4 × 10 ⁴ cells / 2 cm ² and incubated for 18 hours. . After inoculation with crude extract, the cells were incubated for 2 hours and washed with PBS. After UV irradiation of the cells using the Spectronics UV transilluminator EBF-260 (maximum wavelength, 312 nm; a half-peak intensity range, 297-328 nm), the UV-irradiated cells were incubated for 1-7 hours and 5 μM Et ₂ (Millipore) was administered. After 30 minutes, the degree of fluorescence was measured using a Millipore microplate fluorometer Cytofluor 2350 (excitation: 485 nm, emission: 645 nm). HaCaT cells, which are human skin keratinocytes, were irradiated with UV B of 35 mJ / cm ² and the DNA chains of the nucleus were cut off and quantitated using the TUNEL method (Kanatate K., Nagao N., Sugimoto M., Kageyama K. , Fujimoto T., Miwa N., Cellular and Molecular Biology Research , 41 : 561-567, (1995)). In addition, irradiation of HaCaT cell line with UV A of 45 mJ / cm ² produces hydrogen peroxide and various peroxides to increase intracellular oxidative stress, which is determined by fluorescence graph quantitation using CDCFH-DA, a redox fluorescent dye precursor. Was carried out. At this time, cells debris and cell membranes that were destroyed appear bright under fluorescence microscopy when stained with Et ₂ (Samsung Co., Ltd. Chemical, Fragrance, Nar, Tokyo, 93-144, (1999)).

As a result, when pre-treatment of the light bulbs extract at a concentration of 3 ㎍ / ㎖, DNA chain cleavage by UV B was significantly reduced ( Fig. 2 ), and even when the UV A irradiation, the extracts of 3 ㎍ / ㎖ In the case of treatment, it was confirmed that the increase in intracellular oxidative stress was significantly suppressed ( FIG. 3 ).

Experimental Example 4 Measurement of Telomere Shortening Inhibition Activity

In order to measure the telomeres shortening inhibitory activity of the extracts of the present invention, NHEK- was prepared by separating fibroblasts, another main component of the skin, from the human neonatal epidermis and treating with uridine bromide. F cells were used and cultured in DMEM medium containing 10% of FBS.

<4-1> Length measurement of telomeres by Southern blot

Genomic DNA was extracted from cells of each age using an isoquic nucleic acid extraction kit (ORCA Research Inc.), and the extracted DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Store at 4 ° C. Add 2 µl of 10 × H buffer (TaKaRa), 2 µl of extracted genomic DNA solution and 15 µl of tertiary distilled water to 1.5 µl tube to make 19 µl total, and then limit the enzyme Hinf I (6 U / µl, TaKaRa). ) 1 μl was added and reacted at 37 ° C. for 3 to 4 hours, followed by electrophoresis. The gel plate was prepared so that the concentration of agarose (type I, Sigma) gel was 1% in the bridge part and 0.8% in the bed part (Marisol KS-8405, 20 × 14 cm), where 1 × Boyer buffer (50 mM Tris -HCl, 20 mM sodium acetate, 2 mM EDTA, 18 mM NaCl, pH 8.0). As a marker, 0.5 µg of a 1 kb DNA ladder (GIBCO BRL) was used. 3 µl of loading buffer was added and electrophoresis was performed at 35 V / cm for 20 hours. After electrophoresis was completed, staining with EtBr of 2 ㎍ / ㎖ concentration for 15 minutes, which was confirmed in a UV trans-illuminator. The gel was then shaken for 15 minutes in 0.25 N HCl and washed twice with distilled water. The washed gel was immersed in a denatured solution (0.2 N NaOH, 0.6 M NaCl), shaken for 25 minutes at room temperature, and washed three times with distilled water. A blotting device filled with 6 × SSC was placed in the order of nitrocellulose membrane Optitran BA-S 85 (Schleicher & Schuel), 3 MM noji, paper towels, glass plates, weights (2 kg) and blotted overnight. After blotting, the membrane filter was immersed in 3 × SSC, lightly dehydrated and the wells positioned in the UV transilluminator. Baked at 80 ° C. overnight at 80 ° C., prehybridization at 65 ° C. using denatured salmon sperm DNA (Wako), and then hybridization buffer (1 × Denhan). solution, 1 M NaCl, 50 mM Tris-HCl, 10 mM EDTA, 0.1% SDS, 50 μg / ml of denatured salmon sperm DNA) and 50 using ³² P-labeled (TTAGGG) ₄ at the 5′-end. Hybridization reaction was carried out at ℃. The membrane was immersed in the wash solution (4 x SSC / 0.1% SDS) and shaken at 55 ° C. for 15 minutes. After the film was dried, the film was covered with a wrap, placed in a sensitized cassette together with an X-ray film (Scientific Imaging Film, Kodak), and subjected to autoradiography overnight at -80 ° C. The position of the wells was marked by combining the positions of the developed film and the film, and the density peaks of TRFs were detected by laser densitometer UltroScan XL (Pharmacia) to calculate the mobility (Furumoto K., Inoue E., Nagao N.). , Hiyama E., Miwa N., Life Science, 63 : 935-948, (1998); 三 羽 信 比 古, ビ オ 抗 酸化劑 プ ロ ビ タ ミ ン C, ガ, ガ ン 老化 の 防御 と 實用 化 硏 究,フ レ グ ラ ン ス ジ ャ, ナ ル company, Tokyo, 93-144, (1999)).

As a result, in the control cells not treated with the extract of the light bulb of the present invention, the telomere DNA was gradually reduced from 12.0 kb when the PDL 2.8 to 7.9 kb when the PDL 14.8. In contrast, in the case of continuous administration of the light bulb extract of the present invention, the shortening of the telomeres was suppressed ( FIGS. 4 and 6 ). That is, in the case of the cells administered with the early morning light extract, the shortening rate of telomeres was 159 bp / PDL on average, which was observed to be slower than 342 bp / PDL of the control group ( FIG .

<4-2> Measuring the Effects on Cell Aging

In order to measure the effect of the extracts of the present invention on the aging of NHEK-F cells, keratinocytes, cells were first cultured continuously at a dilution ratio of 1: 8. Cells were grown to a population of about 3 PDL (population doubling level) without the addition of the light bulb extract, and then 3 μg / ml of the light bulb extract was administered during incubation. Medium exchange was performed every 3 days with the addition of freshly prepared DMEM-10% FBE containing the same concentration of crude bulb extract (Furumoto K., Inoue E., Nagao N., Hiyama E., Miwa N., Life). Science, 63 : 935-948, (1998); 羽 羽 信 比 古, 抗酸 化 比 プ ロ ビ タ ミ ン C, ガ, ン, 老化 の 防御 と 硏 究 化 硏 究, Fragrasja, Nar, Tokyo, 93- 144, (1999).

As a result, the lifespan of NHEK-F cells, which are human skin keratinocytes, was extended to PDL 30.5 in the case of cells to which the light bulb extract was added compared to the control group without the light bulb extract of the present invention (PDL 14.9) ( FIG. 5 ). . From the above results, it can be seen that the life span of the cells was extended by about 2.05 times by the light bulb extract of the present invention when compared with the maximum PDL of the control group.

Cutaneous keratinocytes and fibroblasts are commonly known to be damaged by ultraviolet radiation, lipid peroxidation, dryness and mechanical injury and all factors exerting cell disruption on skin cells. The division of the new cells can continue as long as the telomere DNA is maintained above the critical DNA length (estimated to be 7.9 to 8.4 kb for human skin keratinocytes). It can be seen that it is possible to prolong the period at which cell division capacity is terminated by slowing down the rate of reaching the threshold by slowing the rate of reaching the threshold ( FIG. 6 ).

Experimental Example 5 Influence of Human Skin Keratinocytes on Lifetime Cell Supply Ability

The number of cells that a cell supplies throughout life is called "lifetime cell supply capacity." The present inventors measured the effect of the extract of the light bulb of the present invention on the lifetime cell supply ability of NHEK-F cells, which are human skin keratinocytes.

As a result, NHEK-F cells, which are human skin keratinocytes, provide a total number of cells of 3.02 × 10 ⁴ per ancestral cell throughout the life of the cell from the first cell division to the final cell division. In the case of administering the extracts of the present invention, the cell supply capacity of the cells was found to be 1.51 × 10 ⁹ . From the above results, it was confirmed that the increase in life-time cell supply capacity was shown to be 50,000 times higher when added to the extract of crude light ( Fig. 7 ).

Intracellular peroxidation can provide single stranded cleavage of telomere DNA and damage genes encoding enzymes that increase the length of telomeres. As such, through the scavenging of the peroxide damaging DNA, the extract of the present invention can prevent the shortening of the length of the telomere DNA, which can be effective in improving the cell supply capacity of human skin keratinocytes and preventing skin aging. , The composition of the present invention may be usefully used in the development of skin anti-aging cosmetics and the like.

Preparation Example 1 Preparation of Cosmetics Containing Extracts, such as Headache

The inventors of the present invention prepared cosmetics containing the extract of the present invention as an active ingredient, as shown in Tables 3 to 5 .

<1-1> Preparation of nutrition cream

Raw material name Quantity (kg) Cetostearyl alcohol 2.80 Smuggling 2.60 Stearic acid 1.40 Lipophilic Monostearic Acid Glycerin 2.00 Fiji-100 Stearate 1.00 Sesquioleate Sorbate 1.40 Jojoba oil 4.00 Squalane 3.80 Polysorbate 60 1.10 Macadamia oil 2.00 Tocopherol Acetate 0.20 Methyl Polysiloxane 0.40 Eichparaben 0.10 Propylparaben 0.10 Euxyl K-400 0.10 1,3-butylene glycol 7.00 Methylparaben 0.05 glycerin 6.00 d-pandenol 0.20 Light bulb extract 3.50 Cereal Extract 2.00 Brown algae extract 1.50 Angelica Extract 0.80 Ian Extract 2.60 Bark Extract 0.80 March extract 3.00 Persimmon Leaf Extract 1.20 Centella Extract 0.80 Triethanolamine 0.20 pt 41891 0.20 pH ₂ O 47.15 Sum 100

<1-2> Preparation of Lotion

Raw material name Quantity (kg) Cetostearyl alcohol 1.60 Stearic acid 1.40 Lipophilic Monostearic Acid Glycerin 1.80 Fiji-100 Stearate 2.60 Sesquioleate Sorbate 0.60 Squalane 4.80 Macadamia oil 2.00 Jojoba oil 2.00 Tocopherol Acetate 0.40 Methyl Polysiloxane 0.20 Eichparaben 0.10 Propylparaben 0.10 1,3-butylene glycol 4.00 Methylparaben 0.10 Xanthan Gum 0.10 glycerin 4.00 d-pandenol 0.15 Allantoin 0.10 Light bulb extract 2.60 Angelica Extract 2.50 Green Tea Extract 0.20 Ian Extract 2.00 Bark Extract 1.20 Brown algae extract 3.40 March extract 3.60 Persimmon Leaf Extract 1.20 Centella Extract 0.80 Carbona (2% aq.Sol) 4.00 Triethanolamine 0.15 ethanol 3.00 pt 41891 0.10 pH ₂ O 49.20 Sum 100

<1-3> Preparation of Skin

Raw material name Quantity (kg) Propylene glycol 8.00 Methylparaben 0.10 Xanthan Gum 0.10 Allantoin 0.10 Dipotassium glycyrrhinate 0.10 Ethylenediaminetetradiacetic acid sodium 0.05 Fiji 60 Hydrogenated Castioyl 0.10 Light bulb extract 5.00 Brown algae extract 3.60 Angelica Extract 2.40 Ian Extract 2.40 Green Tea Extract 1.00 Bark Extract 2.40 March extract 4.00 Witch Hazel Extract 3.50 Centella Extract 1.00 Imidazolidinylurea 0.10 Triethanolamine 0.10 ethanol 2.50 pt 4291 0.10 pH ₂ O 63.35 Sum 100

Preparation Example 2 Preparation of Foods and Beverages Containing Extracts of Light Bulbs

The present inventors prepared the food or beverage composition containing the above-mentioned extracts of the light bulbs as an active ingredient as follows.

<2-1> Preparation of Chewing Gum

Gum base 20%

Sugar 76.36-76.76%

Headlight Lamp Extract 0.24 ~ 0.64%

1% fruit flavor

Water 2%

Chewing gum was prepared using conventional methods using the above composition and content.

<2-2> Preparation of Candy

50 to 60% sugar

Starch syrup 39.26-49.66%

Headlight Lamp Extract 0.24 ~ 0.64%

Orange flavor 0.1%

Candy was prepared using conventional methods with the above composition and content.

<2-3> Preparation of Biscuits

Force Class 1 88 kg

Gravity First Class 76.4 ㎏

16.5 kg per white

2.5 kg of salt

2.7 kg of glucose

Palm shortening 40.5 kg

5.3 kg of ammo

Medium kg 0.6 kg

0.55 kg sodium bisulfite

Rice flour 5.0 kg

Vitamin B1 0.003 kg

0.003 kg of vitamin B2

Milk Flavor 0.16 ㎏

71.1 kg of water

Whole milk powder 4 kg

Substitute powder 1 kg

0.1 kg of calcium phosphate

Spray salt 1 kg

25 kg of spray oil

Cocktail Lamp Extract 0.1 ~ 0.5 ㎏

Biscuits were prepared using conventional methods with the above composition and content.

<2-4> Preparation of Ice Cream

Milkfat 10.0%

Non-fat solids 10.8%

Sugar 12.0%

Starch syrup 3.0%

Emulsifying stabilizer (span) 0.5%

Spices (Strawberries) 0.15%

Water 63.31-62.91%

Headlight Lamp Extract 0.24 ~ 0.64%

Ice cream was prepared using conventional methods using the above composition and content.

<2-5> Preparation of Drink

522 mg of honey

Chioctosanamide 5 mg

Nicotinamide 10 mg

Riboflavin Sodium Hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Morning Glory Extract 0.48-1.28 mg

200 ml of water

With the above composition and content, drinks were prepared using conventional methods.

<2-6> preparation of sausage

Pork 63.6%

Chicken 27.5%

Starch 3.5%

Soy Protein 1.7%

Saline 1.62%

Glucose 0.5%

Other additives (glycerine) 0.94-1.34%

Headlight Lamp Extract 0.24 ~ 0.64%

Sausages were prepared using conventional methods using the above composition and content.

<2-7> Preparation of Chocolate

Sugar 34.36-34.76%

Cocoa Butter 34%

Cocoa Mass 15%

Cocoa Powder 15%

Lecithin 0.5%

0.5% vanilla

Headlight Lamp Extract 0.24 ~ 0.64%

With the above composition and content, chocolate was prepared using conventional methods.

Preparation Example 3 Preparation of Cell Aging Inhibitor Containing Extracts of Crude Oil as Active Ingredients

The present inventors prepared the food or beverage composition containing the above-mentioned extracts of the light bulbs as an active ingredient as follows.

<3-1> Manufacturing method of injection liquid

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

100 g of crude bulb lamp extract, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water. The solution was bottled and sterilized by heating at 20 ° C for 30 minutes.

The components of the injection solution are as follows.

Extract of light bulbs, etc. ··········· 1

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ··················

<3-2> Manufacturing method of syrup

Syrup containing the crude extract of the present invention as an active ingredient 2% (weight / volume) is prepared by the following method.

The bulbous extract, saccharin, and sugar were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to make 100 ml.

The components of the syrup are as follows.

Extract of light bulbs, etc ... 2 g

Saccharin: 0.8 g ················

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ·····················

<3-3> Manufacturing method of tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

It was mixed with 250 g of bulbous extract, 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

250 g of light bulbs, extracts ... ········ 250 g

Lactose ········ 175.9 g

Potato starch ········· 180 g

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch · 160 g

Talc · 50 g

Magnesium stearate 5 g

As described above, the light bulb extract of the present invention exhibits a cytoprotective effect of oxidative stress with a strong antioxidant activity, inhibits the shortening of the telomere length and is effective in inhibiting the aging of the cells, and thus exhibits a cosmetic anti-aging effect. It can be usefully used for health food for cell aging inhibition.

1 is a graph showing the cytoprotective action of the extract of the light bulb of the present invention against apoptosis induced by lipid peroxide in HaCaT cells, which are human skin keratinocytes,

Figure 2 is a photograph showing the inhibitory effect of the bulbous extract of the present invention on the cleavage of the DNA induced by UV B,

A: control group, B: UV B treatment group, C: UV B / light bulb extract treatment group

Figure 3 is a photograph showing the scavenging effect of the light bulb extract of the present invention on the intracellular antioxidant stress produced by UV A,

A: control group, B: UV A treatment group, C: UV A / light bulb extract treatment group

FIG. 4 is a Southern blot photograph showing the effect of telomeres on the PDL of successively passaged NHEK-F cells when the bulbous extract of the present invention was treated or not,

5 is a graph showing the effect of the bulbous extract of the present invention on the lifespan of NHEK-F cells, keratinocytes derived from the skin envelope of a human newborn,

○: control group, □: extract treatment group

Figure 6 is a graph showing the effect of the length of telomeres DNA on the lifespan (PDL) of NHEK-F cells treated or untreated with the light bulb extract of the present invention,

○: control group, □: extract treatment group

Figure 7 is a graph showing the effect of the morninglight extract of the present invention on the life cell supply capacity of human skin keratinocytes.

Claims (7)

Cosmetics for inhibiting skin aging containing alcohol extracts, such as, for example. The cosmetic for inhibiting skin aging according to claim 1, wherein the alcohol is ethanol. The cosmetic for inhibiting skin aging according to claim 1 or 2, wherein the extract inhibits skin cell damage caused by ultraviolet rays. The cosmetic for inhibiting skin aging according to claim 1 or 2, wherein the extract inhibits the shortening of the telomeres to extend the lifespan of skin cells and to improve the life supply of life cells. The cosmetic for inhibiting skin aging according to claim 1 or 2, wherein the cosmetic is selected from the group consisting of nourishing cream, lotion and skin. delete delete