KR102440743B1 - Cosmetic composition for reducing skin wrinkles, improving skin elasticity and antioxidation containing the complex extracts of Diospyros Kaki Leaf, Scutellaria Baicalensis, Cimicifuga Dahurica and Sophora Japonica Flower - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin condition comprising, as an active ingredient, complex extracts of Diospyros kaki leaves, Scutellariae radix, Cimicifuga dahurica, and Styphnolobium japonicum flowers. More specifically, the present invention relates to a cosmetic composition that exhibits excellent antioxidant activity and is highly effective in ameliorating skin wrinkles and improving skin elasticity, by containing complex extracts of Diospyros kaki leaves, Scutellariae radix, Cimicifuga dahurica, and Styphnolobium japonicum flowers, wherein the complex extracts are prepared by an ultrasound-assisted vacuum extraction method at low temperatures.

Description

Cosmetic composition for reducing skin wrinkles, improving skin elasticity and antioxidation containing the complex extracts of Diospyros Kaki Leaf , Scutellaria Baicalensis, Cimicifuga Dahurica and Sophora Japonica Flower}

The present invention relates to a cosmetic composition for skin wrinkle improvement, elasticity improvement and anti-oxidation containing a complex extract of persimmon leaf, gold, eye horseradish and horseradish flower as an active ingredient, specifically, persimmon leaf, gold produced by ultrasonic vacuum low temperature extraction It relates to a cosmetic composition that contains a complex extract of , eye horseback riding and horseradish flowers to exhibit excellent skin wrinkle, elasticity improvement and antioxidant activity.

The skin is histologically composed of three layers: the epidermis, dermis, and subcutis. And because it is the most external, it plays the most important role in the aspect of skin aging.

The components that make up the stratum corneum, the outermost layer of the epidermis, are keratinocytes and skin lipids, which are responsible for the barrier function of the skin. It can be called a function.

However, when the skin is exposed to external environments such as stress, sunlight, air pollution, and tobacco, reactive oxygen species are generated, lipid peroxide is generated, and skin constituent proteins such as collagen and elastin are transformed. In addition, as the activity of skin cells decreases with age, the ability to produce collagen and elastin decreases. formation is promoted. In addition, as Glycosaminoglycans (GAGs), which are responsible for moisturizing the skin in the dermis layer, decrease due to skin aging, the skin becomes dry and features of aging skin such as pigmentation and decreased elasticity appear.

And aging of human skin appears as the number of fibroblasts decreases with increasing age. Various signal systems are activated by UV rays, climate, smoking, etc., and skin components are damaged and skin aging proceeds. The amount of collagen in the skin damaged by UV rays is reduced because the expression of matrix metalloproteinase (MMPs) in the skin increases by UV rays, and these MMPs play an important role in the occurrence of skin photoaging (Fisher et al. , 1999). MMPs are secreted from skin cells (keratinocytes, fibroblasts) and break down most of the protein components that make up the extracellular matrix (ECM) and basement membrane (BM), thereby destroying the connective tissue that maintains skin elasticity. It is known to cause wrinkles, loss of elasticity, and sagging of the skin.

In addition to collagen, it is reported that many studies are being conducted on elastin, an elastic fiber, and elastase, an enzyme involved in elastin decomposition, in addition to collagen (Antonicelli et al., 2007; Isenburg et al.) ., 2004; Seite et al., 2006). In particular, it has been reported that the expression of tropoelastin mRNA, a precursor of elastin, is also increased in keratinocytes of the human skin epidermis by chronic UV irradiation, and elastase in wrinkle formation through selective elastase activity inhibition. role has also been reported.

As interest in beauty and health, such as skin aging, is rapidly increasing with the improvement of living standards, research to develop functional cosmetics is becoming active. A), AHA (a-hydroxy acid), adenosine, etc. are widely used as substances that increase the synthesis of collagen, but are known to be unstable to light and heat and to irritate the skin.

Accordingly, the present inventors have endeavored to manufacture a cosmetic excellent in skin wrinkle improvement, elasticity improvement and antioxidant effect from safe natural materials, and thus completed the present invention.

(0001) Republic of Korea Patent Publication No. 10-2013-0023606 (2013.03.08) (0002) Republic of Korea Patent Publication No. 10-2011-0112231 (2011.10.12) (0003) Republic of Korea Patent Publication No. 10-2019-0070081 (2019.06.20.) (0004) Republic of Korea Patent Publication No. 10-2019-0089748 (2019.07.31)

An object of the present invention is to provide a cosmetic composition that contains persimmon leaf, gold, nun-seed horse, and rhododendron flower complex extract as an active ingredient, thereby exhibiting excellent skin wrinkle improvement, elasticity improvement and antioxidant effect.

According to the present invention, in order to achieve the above object, a cosmetic composition containing 0.1 to 30% by weight based on the total weight of the composition, as an active ingredient, is a composite extract of persimmon leaf, gold, nun-seed horse and rhododendron produced by ultrasonic vacuum low temperature extraction. is provided

The ultrasonic vacuum low-temperature extraction is to put a mixture of persimmon leaves, gold, nun-seed horses, and rhododendron flowers in an airtight container with an extraction solvent, vacuum-packed, and ultrasonically extracted for 1-5 hours, and then extracted at 30-60 ℃ for 1-10 days. It is characterized in that it is done.

At this time, as the extraction solvent, at least one selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol, dipropylene glycol and butylene glycol one can be used

The mixture is made by mixing persimmon leaf, gold, eye-catching horseradish, and flower tree flower in a ratio of 1 to 10:1 to 10:1 to 10:1 to 10, respectively, based on their dry weight, preferably 1-3: It is made by mixing in a ratio of 1~3:1~2:1~3.

The cosmetic composition is characterized in that it is used for skin wrinkle improvement, elasticity improvement and antioxidant.

The complex extract of persimmon leaf, gold, nun horseback riding, and rhododendron flower according to the present invention has excellent skin wrinkle and elasticity improvement effects, and thus can be usefully used as a cosmetic composition for skin wrinkle improvement, elasticity improvement and antioxidant.

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

The present invention is technically characterized in that the complex extract of persimmon leaf, gold, nun-seed horse and rhododendron flower produced by ultrasonic vacuum low-temperature extraction method exhibits excellent skin wrinkle, elasticity improvement and antioxidant activity.

As a food recipe, a vacuum low-temperature recipe in which food contained in a sealed plastic bag is heated in lukewarm water for a long time to cook is known. It has the advantage of being able to prevent contamination that may occur during the cooking process through vacuum, preserve the inherent flavor and moisture of the ingredients, and minimize the destruction of nutrients. In the present invention, the extract was prepared by applying this method to the preparation of the active ingredient persimmon leaf, gold, nun-seed horse, and rhododendron flower complex extract.

Persimmon tree ( Diospyros kaki Thunb ) is mainly distributed in the central and southern regions of Korea. In addition to the reproductive Shija, the leaves, Shihwa, and corpses have been used as folk remedies since ancient times. Among them, persimmon leaves are mainly used as tea, and it is recorded in Donguibogam that it is effective in preventing and treating vascular diseases as well as in purpura (vascular inflammation). Persimmon leaves contain various substances such as vitamin C, amino acids, nucleic acids, phenolic compounds, and free sugar. As flavonoids of persimmon leaves, glycoside forms such as astragalin, a glycoside of kaempferol, myricitrin, and isoquercitrin, a glycoside of myricetin, exist. Persimmon leaves have a high total phenolic compound content of 0.1 to 5.8% compared to other plants, and have excellent antioxidant and antibacterial properties, and are known to be involved in superoxide dismutase (SOD) activity. In addition, pharmacological effects have been verified for maintaining skin elasticity and preventing colds and geriatric diseases, strongly suggesting the possibility that various substances can affect human physiological mechanisms. Phenolic compounds are reported to be effective not only as antioxidants but also as antibacterial, anti-allergic, caries prevention, heart disease and diabetes prevention, and include flavonoids, phenolic acid, phenyl propanoid, quinones, and the like.

Gold ( Scutellaria baicalensis ) is a dicotyledonous plant, a perennial plant belonging to the family Lamiaceae (Labiatae/Lamiaceae), and the height reaches 20-26cm. The root is mainly used medicinally. The appearance of this plant is columnar-semi-tubular or flat, 5-25cm long and 5-30mm in diameter, and the outer surface is yellowish-brown with coarse and distinct vertical wrinkles, and there are traces of side roots and brown bark. Fragments remain. In oriental medicine, it is called ginseng (子芩), a little (條芩), and pyeongeum (片芩), and its taste is bitter, and its nature is cold and non-toxic. ), detoxification), blue-heat control (淸熱燥濕, cooling heat and drying moisture), and hemostasis. In oriental medicine, gold is used to remove impurities from living products and cut into pieces of a certain size. In addition, golden vinegar (炒, processed by roasting medicinal herbs with heat) is mainly used to stabilize the fetal spirit by easing the cold nature of gold. Alcohol (酒), which is widely used as a liquid supplement, has an ascending property and thus elevates the efficacy of medicinal substances to the upper part of the human body. Conversely, when gold is used as a dongbyeon (童便) candle, it acts on the lower part of the human body (Chinese herbal medicine, Kiyoung Kim et al., Shinilsangsa; herbal medicine and clinical application, Hoyoung Choi et al., Youngrimsa). In addition, the golden extract contains flavonoid-based components such as Chrysin, Baicalin, Baicalein, Wogonin, and the like.

Snow horse riding ( Cimicifuga Dahurica ) is a perennial plant belonging to the buttercup family, also called kimeokgari, and the rhizomes of snow horse riding and its animals and plants are used medicinally. The rhizomes are coarse and uneven, 6-8 cm long and 10-25 cm in diameter. The outer surface is brown or black, and there are several large stem marks on the upper part of the rhizome, and many root residues are attached. It contains ferulic acid as a physiologically active ingredient of eye horse riding, and for the effect, it has been reported that eye horse riding extract regulates insulin secretion. It is known to inhibit vascular smooth muscle contraction by inhibiting the inflow of calcium.

The Sophora Japonica Flower is a flower of the Japanese rhododendron, which is a deciduous broad-leaved arboreous tree belonging to the legume family. It is known to be effective in diseases of the circulatory system such as arteriosclerosis, intestinal bleeding, uterine bleeding, gum inflammation, swelling, burns, high blood pressure, cerebral hemorrhage, stroke, paralysis of the limbs, hemorrhoids, and toothache. It contains Troxerutin as an active ingredient of the flower of the sycamore tree.

According to the present invention, there is provided a cosmetic composition containing 0.1 to 30% by weight based on the total weight of the composition, as an active ingredient, a complex extract of persimmon leaf, gold, nun-seungma, and rhododendron produced by ultrasonic vacuum low-temperature extraction.

In the present invention, the ultrasonic vacuum low-temperature extraction is performed by placing a mixture of persimmon leaves, gold, nun-seed horse and rhododendron flowers in an airtight container together with an extraction solvent, vacuum-packed, and ultrasonically extracted for 1 to 5 hours, then 1 to 10 at 30-60 ° C. It is done by extracting during the day.

At this time, as the extraction solvent, at least one selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol, dipropylene glycol and butylene glycol one can be used

According to one embodiment of the present invention, the complex extract is a mixture of persimmon leaf, gold, nun-seed horse and rhododendron flowers, put in an airtight container with water as an extraction solvent, and vacuum-packed, followed by ultrasonic extraction at room temperature for 1 to 5 hours, It is prepared by extraction at a low temperature of 30 to 60 ° C for 1 to 10 days.

The mixture is made by mixing persimmon leaf, gold, eye-catching horseradish, and flower tree flower in a ratio of 1 to 10:1 to 10:1 to 10:1 to 10, respectively, based on their dry weight, preferably 1-3: It is made by mixing in a ratio of 1~3:1~2:1~3. When persimmon leaf, gold, snow horse riding horseradish, and rhododendron flowers are mixed and extracted in a ratio of 1:2:2:3, respectively, based on their dry weight, it is most preferable because it shows the best skin wrinkle, elasticity improvement and antioxidant activity.

The complex extracts of persimmon leaf, golden, nun-seed horse and rhododendron flowers prepared by the ultrasonic vacuum low temperature extraction method have excellent elastase inhibition, collagen biosynthesis promoting effect and MMP-1 production inhibitory effect (Test Examples 2, 3, 4) and Since it exhibits DPPH Free Radical scavenging effect (Test Example 5), the cosmetic composition containing it as an active ingredient can be usefully used for skin wrinkle improvement, elasticity improvement and antioxidant.

The complex extract as an active ingredient is contained in an amount of 0.1 to 30% by weight based on the total weight of the cosmetic composition.

The cosmetic composition of the present invention may be prepared in any conventionally prepared formulation, for example, lotion, cream, essence, cleansing foam, cleansing water, pack, body lotion, body oil, body gel, shampoo, conditioner, hair conditioners, hair gels, foundations, lipsticks, mascaras, makeup bases, and the like.

[Example]

Hereinafter, the present invention will be described in detail through the following examples and test examples, but the present invention is not limited by these examples.

Preparation Example 1: Preparation of persimmon leaf extract

1 kg of dried persimmon leaves and 5 times the weight of 70% hydrous ethanol as an extraction solvent were added, and heated to 80° C. to 100° C. in an extractor equipped with a cooling condenser (Cosmos-660, Kyungseo Machinery), and extracted 3 times for 2 hours each.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a reduced pressure concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40 ℃ ~ 50 ℃, and then using a spray dryer (B-290, BUCHI), the inlet temperature was 180 ℃, the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) 200 g of the title extract powder was obtained.

Preparation Example 2: Preparation of golden extract

1 kg of dried golden root and 5 times the weight of 70% hydrous ethanol as an extraction solvent were added, and heated to 80°C~100°C in an extractor equipped with a cooling condenser (Cosmos-660, Kyungseo Machinery), and extracted 3 times for 2 hours each.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a reduced pressure concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40 ℃ ~ 50 ℃, and then using a spray dryer (B-290, BUCHI), the inlet temperature was 180 ℃, the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the titled extract powder.

Preparation Example 3: Preparation of an extract from the eye horse riding horse

1 kg of dried nunja horse root and 5 times the weight of 70% hydrous ethanol as an extraction solvent were added, and heated to 80° C. to 100° C. in an extractor equipped with a cooling condenser (Cosmos-660, Gyeongseo Machinery), and extracted 3 times for 2 hours each.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a reduced pressure concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40 ℃ ~ 50 ℃, and then using a spray dryer (B-290, BUCHI), the inlet temperature was 180 ℃, the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the titled extract powder.

Preparation Example 4: Preparation of extract

1 kg of dried sycamore tree flowers and 5 times the weight of 70% hydrous ethanol as an extraction solvent were added, heated to 80°C~100°C in an extractor equipped with a cooling condenser (Cosmos-660, Kyungseo Machinery), and extracted 3 times for 2 hours each.

After extraction by the above method, the mixture was left at room temperature for 3 days, and the precipitate was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Then, using a reduced pressure concentrator (Coolace CCA-1100, EYELA), it was concentrated at a temperature of 40 ℃ ~ 50 ℃, and then using a spray dryer (B-290, BUCHI), the inlet temperature was 180 ℃, the inhaler (Aspirator) Drying under the conditions of 100% efficiency (35㎤/hour), 25% pump efficiency (7.5ml/min), Nozzle Cleaner 4 and Rotameter: 30mm (357 liters/hour) to obtain the titled extract powder.

Preparation Examples 5-7: Preparation of complex extracts from persimmon leaves, gold, eye horses, and rhododendron flowers

After mixing persimmon leaf, gold, noun horseradish and flower tree flower in the weight ratio shown in Table 1 below, washed with purified water and dried persimmon leaf, gold, noun horseradish and flower tree flower 1 kg and 70% hydrous ethanol 5 as an extraction solvent The weight of the pear was added and heated to 80~100℃ in an extractor with a cooling condenser (Cosmos-660, Kyungseo Machinery), and extracted 3 times for 2 hours each.

After extracting the mixture of persimmon leaf, gold, nun-seed horse, and rhododendron flowers by the above method, the extract was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. And after concentrating at a temperature of 40 ℃ ~ 50 ℃ using a reduced pressure concentrator (product name: Coolace CCA-1100, manufacturer: EYELA), and then using a spray dryer (model name: B-290, BUCHI company product) using the inlet (inlet) ) Temperature: 180 ℃, Aspirator Efficiency: 100% (35㎤/hour), Pump Efficiency: 25% (7.5ml/min), Nozzle Cleaner 4 and Rotameter: It was dried under the conditions of 30 mm (357 liters/hour) to obtain the title extract powder.

weight ratio Preparation 5 Preparation 6 Preparation 7 persimmon leaves One One One Gold 3 2 2 snow riding One 2 2 painting tree flower One One 3

Examples 1-3: Preparation of complex extracts of persimmon leaf, gold, nun-eun horseback riding and horse chestnut flower by ultrasonic vacuum low temperature extraction method

After mixing persimmon leaf, gold, noun horseradish and flower tree flower in the weight ratio shown in Table 2 below, washed with purified water and dried persimmon leaf, gold, noun horseradish and flower tree flower mixture 1 kg and 5 times the weight of purified water as an extraction solvent was placed in an airtight container, vacuum-packed, and extracted with an ultrasonic extractor for about 2 hours. After that, cold extraction was performed at 45° C. for 72 hours.

After extracting the mixture of persimmon leaf, gold, nun-seed horse, and rhododendron flowers by the above method, the extract was filtered with 300 mesh filter paper, and the precipitate was filtered twice with Edventec No. 5 filter paper and Whatman GFC 150 mm filter paper. And after concentrating at a temperature of 40 ℃ ~ 50 ℃ using a reduced pressure concentrator (product name: Coolace CCA-1100, manufacturer: EYELA), and then using a spray dryer (model name: B-290, BUCHI company product) using the inlet (inlet) ) Temperature: 180 ℃, Aspirator Efficiency: 100% (35㎤/hour), Pump Efficiency: 25% (7.5ml/min), Nozzle Cleaner 4 and Rotameter: It was dried under the conditions of 30 mm (357 liters/hour) to obtain the title extract powder.

weight ratio Example 1 Example 2 Example 3 persimmon leaves One One One Gold 3 2 2 snow riding One 2 2 painting tree flower One One 3

Test Example 1: Confirmation of cytotoxicity

Cell viability measurement experiment (MTT assay)

The MTT assay method is performed after the MTT [3-(4,5-dimethythiasol-2-yl)-2,5-diphenyl tetrazolium bromide] reagent is absorbed into the cells and then formsazan by succinate dehydrogenase in the mitochondria. ), the accumulation of this substance in cells means mitochondrial activity, broadly, cell activity, and is a representative method of measuring cell viability.

To check the expression of each gene, cells were aliquoted with 200 μl medium in 96 wells at a density of 1×10 ⁵ cell/well and then 5% CO ₂ , After culturing in an incubator at 37°C for 24 hours, the medium was discarded, washed with PBS, and the same amount of medium and samples dissolved in PBS were treated in each well according to various concentrations from 0.1% to 5.0%, and cultured for 24 hours. 4 hours before the end of incubation, 20 μl of 5 mg/ml MTT dissolved in PBS was added to each well, shielded from light with aluminum foil, and incubated in an incubator under 5% CO ₂ and 37° C. conditions for 3 hours. After removing the culture medium, adding 200 μl of DMSO solution, incubating at 37° C. for 1 hour, using an ELISA reader, absorbance was measured and compared at 570 nm. Cell viability was calculated by the following formula, and the results are shown in Table 3 below.

Cell viability (%) = Absorbance of the sample added group / Absorbance of the control group × 100

Sample Use Concentration
(μg/ml) Cell viability (%) 0 50 100 500 1,000 Preparation Example 1 100 101 101 100 100 Preparation 2 101 100 101 100 100 Preparation 3 100 100 100 100 100 Preparation 4 100 101 100 101 100 Preparation 5 101 100 101 100 100 Preparation 6 100 100 100 100 100 Preparation 7 100 101 100 100 100 Example 1 101 101 101 100 101 Example 2 101 100 101 100 100 Example 3 100 100 100 101 100 control
Adenosine 100 100 100 101 101

As can be seen in Table 3, it was confirmed that neither of the samples of Preparation Examples and Examples showed cytotoxicity, so there was no problem in safety.

Test Example 2: Confirmation of Elastase Activity Inhibition

10 ul of a solution in which the test sample and the elastase enzyme solution (1,000 units/mL, Sigma, USA) were mixed in 2.5 wt% agar containing 0.1 wt% Elastin-Congo red (Sigma, USA) and reacted at room temperature for 10 minutes After each injection, it was incubated at 37°C for 18 hours. When a transparent zone is formed around Elastin-Congo red agar due to the activity of elastase after the end of culture, the elastase activity inhibitory effect can be measured by measuring the diameter of the halo.

While only elastase is added dropwise to an agar medium containing elastin, solutions having concentrations of 0.5, 1.0, and 2.0%, respectively, of Preparation Examples and Examples are sampled and added dropwise together with elastase, halo appearing on the medium. The elastase inhibitory activity of the sample was measured by measuring the diameter of the sample, and as a control, Retinol crystal was added dropwise together with elastase for comparison. The test results are shown in Table 4 below.

sample concentration
(%) Halo diameter(cm) 0.5 1.0 2.0 Preparation Example 1 1.8 1.7 1.7 Preparation 2 1.7 1.5 1.8 Preparation 3 1.5 1.3 1.3 Preparation 4 1.4 1.3 1.4 Preparation 5 1.5 1.3 1.1 Preparation 6 1.5 1.3 1.1 Preparation 7 1.5 1.4 1.1 Example 1 1.2 1.1 1.0 Example 2 1.2 1.1 1.1 Example 3 1.1 1.0 0.8 Elastase 1.8 Retinol crystal 0.8

The larger the halo diameter, the greater the decomposition of elastin. It was found to have elastase inhibitory activity. The composite extracts of Examples 1 to 3 prepared by the vacuum low temperature extraction method showed more excellent inhibitory activity, and the composite extract of Example 3 showed the most excellent effect.

Test Example 3: Confirmation of collagen biosynthesis promoting effect

Fibroblasts were inoculated at a cell concentration of 5×10 ⁵ in IMDM medium supplemented with 10% FBS, and cultured at 37° C., 5% CO ₂ in an incubator for 24 hours. After culturing for 24 hours, the dilution solution prepared by diluting the extracts of Preparation Examples and Examples to concentrations of 0.5, 1.0 and 2.0%, respectively, was added, followed by incubation under the same conditions for 18 hours, followed by UV irradiation for 1 hour. This puts stress on the cells. Thereafter, fibroblasts were recovered using Trizol reagent (invitrogen, USA), mRNA was extracted, and cDNA was synthesized through a series of processes. The amount of gene expression was confirmed through electrophoresis by amplifying the PROCOLLAGEN TYPE I gene region from the synthesized cDNA, and gene amplification was performed using a thermal cycler (GenePro, Hangzhou bioer tech., CHINA), 10x taq polymerase buffer, 10 mM dNTP, 10 Mix pmol primer (F: AGC CAG CAG ATC GAG AAC AT, R: TCT TGT CCT TGG GGT TCT TG) and taq polymerase, add distilled water to adjust to 50 uL, 95 degrees 5 minutes 1 cycle/95 degrees 1 minute/51 degrees It was carried out under the conditions of 2 min/72 °C 1 min 28 cycle amplification, and reaction at 72 °C for 5 min. The expression rate of the generated procollagen was calculated according to the following formula, and as a control, normal fibroblasts not treated with the sample and not irradiated with UV were used.

Procollagen expression rate (%) = B/A × 100

A: Amount of procollagen expression in the control group

B: Amount of procollagen expression in sample-treated and UV-irradiated fibroblasts

Sample concentration (%) procollagen expression rate 0.5 1.0 2.0 Preparation Example 1 32.2 46.5 59.6 Preparation 2 35.6 39.1 46.5 Preparation 3 41.2 49.5 60.3 Preparation 4 45.6 56.8 69.5 Preparation 5 48.6 59.5 65.3 Preparation 6 34.6 41.3 62.3 Preparation 7 50.2 60.3 75.3 Example 1 70.3 81.2 89.6 Example 2 75.6 82.3 91.3 Example 3 152.3 160.5 180.2

As can be seen in Table 5, the composite extract of Example exhibited a more excellent collagen biosynthesis promoting effect compared to the sample of Preparation Example.

Test Example 4: Confirmation of MMP-1 production inhibitory effect

Fibroblasts, which are normal human skin cells (Korea Cell Line Bank, Korea), were inoculated in a 48-well microplate (Nunc. Denmark) to 1st 10 ⁶ cells per well, DMEM medium (Sigma, USA) and conditions at 37°C. After culturing for 24 hours in a serum-free DMEM medium after adding a diluted solution prepared by diluting the extracts of Preparation Examples and Examples in dimethyl sulfoxide to a concentration of 100, 500, and 1,000 μg/ml for 48 hours cultured. After incubation, the supernatant of each well was collected and the amount of newly synthesized MMP-1 (μg/ml) was measured using an MMP-1 assay kit (Amersham, USA). TGF-β (10 mg/ml, Roche, USA) was used as a positive control for inhibition of MMP-1 production. The inhibition rate of MMP-1 production was calculated according to the following formula, and the results are shown in Table 6 below.

MMP-1 production inhibition rate (%) = [1-(B/A)] × 100

A: Amount of MMP-1 in the control group

B: Amount of MMP-1 in the experimental group

sample concentration
(μg/ml) MMP-1 production inhibition rate (%) 100 500 1,000 Preparation Example 1 21.3 39.6 45.6 Preparation 2 24.6 35.6 46.8 Preparation 3 25.8 34.7 49.5 Preparation 4 31.2 38.2 51.2 Preparation 5 29.5 41.2 50.6 Preparation 6 33.6 39.4 52.6 Preparation 7 32.5 46.5 53.6 Example 1 31.2 48.9 68.9 Example 2 34.5 50.6 71.5 Example 3 41.2 71.5 90.4 TGF-β 44.3 75.6 94.6

Test Example 5: Measurement of DPPH free radical scavenging ability

DPPH free radical scavenging activity was measured according to the method of Nanjo et al. (1996). After mixing 30 μl of 60 μM DPPH (1,1-diphenyl-2-picrylhydrazyl) solution with 30 μl of sample, transfer the reaction solution to 100 μl quartz capillary tube and after 2 minutes electron spin resonance (ESR) spectrometer (JES-FA, JEOL) Ltd., Tokyo, Japan). The experimental conditions are as follows. magnetic field, 336.5 ± 5 mT; power, 5 mW; modulation frequency, 9.41 GHz; amplitude, 1×1,000; sweep time, 30 s; temperature, 298 K. The DPPH radical scavenging ability was calculated by the difference in the relative radical signal peak heights of H and H ₀ . The results are shown in Table 7 below. Ascorbic acid was used as a control.

Sample concentration (%) Radical scavenging activity (%) 0.1 0.5 One Preparation Example 1 40 49 61 Preparation 2 43 52 62 Preparation 3 38 55 64 Preparation 4 37 59 68 Preparation 5 42 56 61 Preparation 6 43 58 65 Preparation 7 47 55 60 Example 1 56 62 71 Example 2 68 74 80 Example 3 70 85 92 Ascorbic acid 95 97 98

As can be seen in Table 7, the composite extracts of Examples 1 to 3 prepared by the vacuum low temperature extraction method showed more excellent inhibitory activity, and the composite extract of Example 3 showed the best antioxidant effect.

Formulation Example 1: Preparation of skin lotion

According to the composition of Table 8 below, 1 kg of skin lotion containing the complex extract of Examples of the present invention was prepared in a conventional manner.

Raw material Content (wt%) Complex extract (Example 3) 1.0 glycerin 3.0 butylene glycol 2.0 propylene glycol 2.0 Polyoxyethylene hydrogenated castor oil 1.0 ethanol 10.0 triethanolamine 0.1 antiseptic a very small amount pigment a very small amount Spices a very small amount Purified water remaining amount

Formulation Example 2: Preparation of Nutrient Lotion

According to the composition of Table 9 below, 1 kg of nutrient lotion containing the complex extract of Examples of the present invention was prepared in a conventional manner.

Raw material Content (wt%) Complex extract (Example 3) 1.0 beeswax 1.0 Polysorbate 60 1.5 Sorbitan Sesquiolate 0.5 liquid paraffin 10.0 Sorbitan Stearate 1.0 lipophilic monostearate glycerin 0.5 stearic acid 1.5 Glyceryl Stearate/PEG-400 Stearate 1.0 propylene glycol 3.0 carboxy polymer 0.1 triethanolamine 0.2 antiseptic a very small amount pigment a very small amount Spices a very small amount Purified water remaining amount

Test Example 6: Confirmation of formulation stability

For the formulation prepared in the above formulation example, store and observe for 12 weeks ( discoloration, discoloration, and separation), and stability was confirmed. The results are shown in Table 10.

temperature condition Stability check (discoloration, discoloration and separation) Formulation Example 1 Formulation Example 2 Room temperature (25°C) 0 0 Refrigeration (4℃) 0 0 constant temperature (50℃) 0 0

< Formulation stability grade >

0: No change 1: Minor change 2: Change 3: Extreme change

Claims (5)

After mixing persimmon leaves, gold, snow horseshoes, and rhododendron flowers in a ratio of 1:2:2:3 based on their dry weight, the mixture is placed in an airtight container with an extraction solvent, vacuum-packed, and ultrasonically extracted for 1 to 5 hours, A cosmetic composition containing 0.1 to 30% by weight based on the total weight of the composition, as an active ingredient, of a complex extract of persimmon leaf, golden, nun-seed horse, and rhododendron flowers prepared by extraction at 30-60° C. for 1 to 10 days. delete According to claim 1, wherein the extraction solvent is selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol, dipropylene glycol and butylene glycol A cosmetic composition, characterized in that at least one. delete The cosmetic composition according to claim 1, wherein the cosmetic composition is for skin wrinkle improvement, elasticity improvement, and antioxidant use.