WO2021133006A1 - Composition, for reducing skin wrinkles or preventing skin aging, containing dried and roasted radish extract and functional health food comprising composition - Google Patents

Abstract

Provided are a composition for reducing skin wrinkles or preventing skin aging without causing irritation to a human body, and a functional health food comprising the composition, the composition comprising dried and roasted radish as an active ingredient.

Description

A composition for improving skin wrinkles or preventing skin aging containing roasted radish extract and health functional food comprising the composition

The present invention relates to a composition comprising a radish extract as an active ingredient and a health functional food comprising the composition. In particular, dried radish dried radish is roasted and processed to enhance the antioxidant effect of radish, and without irritation to the human body. It relates to a composition capable of improving skin wrinkles or preventing skin aging, and a health functional food containing the composition.

As the human skin ages, wrinkles, blemishes, freckles, etc. are generated, and its elasticity decreases. The cause of skin aging is a decrease in the number of fibroblasts and a decrease in their action, resulting in a decrease in the amount of fiber component (collagen) synthesized, intrinsic aging (natural aging) caused by loss of moisture in the skin, and harmful effects caused by continuous UV exposure externally It is divided into photoaging by action.

That is, endogenous aging is an unavoidable aging phenomenon with the passage of time, and its clinical characteristics are relatively mild, and include fine wrinkles, dry skin, and decreased elasticity.

In addition, the clinical characteristics of photoaging are more severe and observed earlier than intrinsic aging, and it is mainly caused by ultraviolet rays in sunlight. That is, when the skin is exposed to ultraviolet rays, free radicals are generated in the body, and the free radicals generated in the body cause damage to skin cells and tissues, increase melanin production, and keratinization of the epidermis. In addition, as the stratum corneum is damaged, the thickness of the stratum corneum increases, and the collagen-producing ability of the dermis layer is reduced by ultraviolet rays. As a result, the dermal layer becomes thin and thick and deep wrinkles are generated.

On the other hand, the skin is composed of the epidermis, dermis, and subcutaneous tissue from the outside. The epidermis prevents the invasion of external stimuli or pathogens, regulates body temperature and moisture, and the dermis is composed of connective tissue, and is made up of collagenous fibers and some elastic fibers. It functions to keep the skin supple and elastic. In addition, the subcutaneous tissue is a layer of connective tissue, and the fat cells present here serve as a buffer to preserve and protect heat.

Collagen, a fibrous tissue that generally gives the skin flexibility, has the greatest effect on the formation of skin wrinkles, and is a major component that accounts for about 70% of the dermal tissue. As the synthesis of collagen fibers rapidly decreases, skin aging progresses, while collagen fibers are deformed by the external environment, and wrinkles are created and deepened in the skin. Collagenase, which is a collagen degrading enzyme, is involved in the decomposition of collagen.

Recently, there is a method for improving skin wrinkle by external methods such as cosmetics as a cause and improvement method for skin wrinkle formation. Foods that can improve skin health by reducing the cause of skin damage or aging while improving intestinal health with food or specific food ingredients Research to find the material is ongoing.

In addition, although there are antioxidants in the human body, the antioxidant system is disabled by age, pollution, UV rays, and stress, and the concentration of active oxygen increases, causing fatal oxygen toxicity to the body. is widely distributed in the animal and plant kingdoms.

In other words, antioxidants such as many phenolic compounds, flavonoids, tocopherol, vitamin C, and selenium in fruits and vegetables delay or prevent oxidation of fat, and play an important role in anti-aging by preventing and delaying cancer and cardiovascular diseases. do

On the other hand, radish (Radish, Raphanus sativus L.) is a vegetable of the Cruciferae family and has a special spicy taste because it contains volatile sulfur-containing components. Radish contains more free amino acids, sugar, calcium and phosphorus than other vegetables. Nabok, the root of radish, is effective in relieving phlegm, cough, dysentery, etc., and it is recorded in the classics that it is effective in relieving poisoning of seafood or noodles. And the diastase contained in radish is effective in promoting digestion, food poisoning, and relieving hangover, and rapine is known as a component with antibacterial action against bacteria, fungi, and parasites. In addition to this, radish has physiological activities such as diuretic action, bowel action, blood sugar strengthening, anti-inflammatory and hemostasis, and has been widely used in folklore.

Various developments utilizing the antioxidant properties of radish (Radish, Raphanus sativus L.) are being made. For example, Korean Patent No. 10-1706817 (registered on Feb. 8, 2017) has anti-inflammatory and antioxidant properties containing radish extract. Disclosed is an animal feed composition for animal feed composition, and Korean Patent No. 10-1338190 (registered on Dec. 2, 2013) discloses an antioxidant and thrombosis prevention or food and pharmaceutical composition containing Bordeaux radish grinding solution, and is registered in Korea. Patent No. 10-1685251 (registered on December 5, 2016) discloses a cosmetic composition for antioxidation, skin astringency and wrinkle improvement comprising an extract of turnip, turnip radish or turnip callus as an active ingredient, and Korean Patent Registration No. 10- No. 0976619 (registered on Aug. 11, 2010) discloses an increase in the content of ingredients excellent in antioxidant effect by pressure-fried radish radish.

In contrast, the present inventors have completed the present invention by confirming that the roasted radish processed radish can be usefully used for improving skin wrinkles or preventing or improving skin aging while conducting research to expand the utility of radishes.

The present invention, in consideration of the prior art described above, not only enhances the antioxidant effect of dried radish by roasting and processing dried radish, but also improves skin wrinkles or prevents skin aging without irritation to the human body, and a composition thereof To provide health functional food containing

The composition for improving skin wrinkles or preventing skin aging according to the present invention is characterized in that it contains the roasted radish extract as an active ingredient.

In addition, the roasted radish extract of the composition for skin wrinkle improvement or skin aging prevention according to the present invention is an extract extracted by roasting dried radish radish to a moisture content of 3% by weight at 200° C. for 30 minutes.

In addition, the composition for improving skin wrinkles or preventing skin aging according to the present invention is characterized in that it is for application to the skin.

In addition, the composition for improving skin wrinkles or preventing skin aging according to the present invention is characterized in that it is for oral administration.

In addition, the health functional food according to the present invention is characterized in that it comprises the above-described composition for improving skin wrinkles or preventing skin aging and drinking water (water).

The composition containing the roasted radish extract according to the present invention as an active ingredient is effective in improving skin wrinkles or preventing skin aging.

The health functional food containing the roasted radish extract and drinking water (water) according to the present invention is effective in improving skin wrinkles or preventing skin aging.

1 is a representative image of the back skin of a mouse for observing skin redness and keratinization according to the lapse of time in the skin application test group.

2 is a graph measuring the thickness of the skin layer according to the lapse of the period of the skin application test group.

3 is a representative image of the back skin of a mouse for observing skin redness and keratinization over the course of the oral administration experimental group.

Figure 4 is a graph measuring the thickness of the skin thickness according to the period of the oral administration experimental group.

5 is a graph measuring skin hydration (skin moisture) according to the lapse of the period of the oral administration experimental group.

6 is a graph measuring the degree of transdermal water loss.

7 is a histological image of the skin of a mouse, etc. for histopathological observation according to the lapse of the period of the skin application test group.

8 is a graph showing the thickness of the epidermis and the thickness of the dermis according to the period of the skin application test group.

9 is a histological image of the skin of a mouse, etc. for histopathological observation according to the period of the oral administration experimental group.

10 is a graph measuring the thickness of the epidermis and the thickness of the dermis according to the period of the oral administration experimental group.

11 is a graph measuring changes in the concentrations of type I collagen, type III collagen, catalase, GPX and SOD according to the lapse of time in the skin application test group.

12 is a graph measuring changes in the concentration of IL-8, an inflammatory cytokine, according to the period of the oral administration experimental group EEK.

13 is a graph measuring the change in the concentration of TNF-α.

14 is a graph measuring changes in the concentration of type I collagen and type III collagen according to the period of the oral administration experimental group.

The composition for improving skin wrinkles or preventing skin aging according to the present invention is characterized in that it contains the roasted radish extract as an active ingredient.

The roasted dried radish extract is preferably an extract of dried radish dried radish at a temperature of 180 to 200 ° C. for 15 to 30 minutes, and dried radish dried to a moisture content of 3% by weight at 200 ° C. for 30 minutes. An extract obtained by hot water extraction of one radish is more preferable.

In addition, the composition for improving skin wrinkles or preventing skin aging according to the present invention may be for oral administration for skin application.

In addition, the health functional food according to the present invention is characterized in that it comprises the above-described composition for improving skin wrinkles or preventing skin aging and drinking water (water).

Hereinafter, specific embodiments of the present invention will be described in detail with reference to the drawings.

[Example]

Example 1: Preparation of samples

1.1. daikon radish production

Radish (Raphanus sativus L.) was purchased from Jeju Agricultural Products Market on December 2018 and used as a sample. After removing the soil and fine roots of the purchased radish, washed clean, cut the skin into 1.2 × 4.5 × 0.6 cm, put it in a hot air dryer, and dried the hot air at 55 ° C. for 24 hours to prepare dried radish. The moisture content of the dried radish was measured to be 3% by weight based on the total weight of the dried radish.

1.2 Preparation of stir-fried radish radish

500 g of dried radish prepared in Example 1.1 were subjected to a stir-frying device, including moisture content (3 wt%, 5.9 wt%, 26 wt%, 27 wt%), roasting temperature (180, 200°C), And the roasting time (15 to 30 minutes) was varied, and the roasted radish dried radish was cooled at room temperature and then stored refrigerated until use.

1.3 Preparation of hot water extract

After adding 2 L of distilled water to 100 g of the dried radish (1 type) prepared in Example 1.1 and 100 g of the roasted radish (9 types) prepared in Example 1.2, high-temperature pressure extraction was performed for 15 minutes at 121 ° C. The extract was extracted with a depth filter. After primary filtration using a nonwoven fabric and secondary filtration with a vibrating membrane filter (0.45㎛), freeze-dried and stored in a low-temperature refrigerator at ^{7 o C.} 1].

division	Description of the sample
sample 1	Extract extracted after roasting radish radish containing 5.9% by weight of moisture at 200°C for 15 minutes
sample 2	Extract extracted after roasting radish radish containing 5.9% by weight of moisture at 200°C for 20 minutes
sample 3	Extract extracted after roasting radish radish containing 27% by weight of moisture at 200°C for 15 minutes
sample 4	Extract extracted after roasting radish radish containing 26% by weight of moisture at 200°C for 25 minutes
sample 5	Extract extracted after roasting radish radish containing 26% by weight of moisture at 200°C for 30 minutes
sample 6	Extract extracted after roasting radish radish containing 3% by weight of moisture at 200°C for 30 minutes
sample 7	Extract extracted after roasting radish radish containing 3% by weight of moisture at 200°C for 20 minutes
sample 8	Extract extracted after roasting radish radish containing 3% by weight of moisture at 180°C for 30 minutes
sample 9	Extract extracted after roasting radish radish containing 3% by weight of moisture at 180°C for 20 minutes
sample 10	Extract extracted from dried radish containing 3% by weight of moisture in an unroasted state

Example 2: Antioxidant effect test and establishment of extraction process

As described above, the antioxidant properties of radish (Radish, Raphanus sativus L.) are known, and it is known that the content of ingredients excellent in antioxidant effect in the pressurized roasted radish is known to increase when the radish is pressure-fried.

Therefore, in the present invention, the most effective extraction process was established by measuring total polyphenols related to antioxidant effect and measuring DPPH radical scavenging ability.

2.1. Total polyphenol determination

10 μl of Folin-Ciocalteau reagent and 200 μl of 2% sodium carbonate (Na ₂ CO ₃ ) were sequentially added to 10 μl of each of the 10 extract samples according to [Table 1], and then left at room temperature for 30 minutes. After that, the absorbance was measured at 750 nm. The total polyphenol content was calculated from the calibration curve prepared by analyzing tannic acid as a standard material and analyzing it in the same way as the sample. As a result of analyzing the total polyphenol content of 10 extract samples, the following [Table 2] ] was the same.

division	Total polyphenols (mg/100g)
sample 1	160.23
sample 2	160.93
sample 3	132.53
sample 4	163.26
sample 5	162.27
sample 6	298.22
sample 7	195.48
sample 8	210.55
sample 9	122.88
sample 10	27.90

As shown in [Table 2] above, compared to the dried radish radish extract (Sample 10) having a moisture content of 3% by weight that was simply dried at a temperature of 55° C. in warm air and not subjected to stir-frying treatment, the dried radish was dried at 180 to In the dried radish extract (Samples 1 to 9) roasted at 200° C. for 15 to 30 minutes, the total polyphenol content in the extract was significantly higher. In particular, dried radish with a moisture content of 3 wt % was roasted at 200° C. for 30 minutes. In the case of the extract (Sample 6), it was found that the total polyphenol content increased by 10.7 times or more compared to the extract (Sample 10) extracted without roasting radish dried radish having a moisture content of 3% by weight, and the roasted radish samples ( Among samples 1 to 9), it was found that the content of total polyphenols was remarkably high in the extract (Sample 6) extracted by roasting radish dried radish having a water content of 3 wt% at 200° C. for 30 minutes.

2.2 Measurement of DPPH radical scavenging ability

The electron donating ability to DPPH was measured by the Bondet method, and after adding 2 μl of each of the 10 extract samples according to <Table 1> to 198 μl of 300 μM DPPH/EtOH, it was left at 37° C. for 30 minutes and then UV/ Absorbance was measured at 517 nm using a visible spectrophotometer. The electron donating ability of each sample was obtained in % by the following formula.

Scavenging activity (%)={1-(absorbance of extract-added group/absorbance of extract-free group)}×100

_{In addition, for each of the 10 extract samples according to [Table 1], the IC 50} (mg/ml), which is the concentration of the extract sample required to reach the highest DPPH radical scavenging ability (%), was obtained [Table 3] appeared like

division	IC 50 (mg/ml)
sample 1	76.02
sample 2	90.11
sample 3	53.45
sample 4	37.06
sample 5	88.28
sample 6	36.38
sample 7	52.26
sample 8	51.05
sample 9	81.83
sample 10	590.90

As shown in [Table 3] above, compared to the dried radish radish extract (Sample 10) having a moisture content of 3% by weight that was simply dried at a temperature of 55° C. in warm air and not subjected to stir-frying treatment, the dried radish was dried at 180 to 180 to _{The IC 50} value was found to be significantly lower in the radish extract (Samples 1 to 9) roasted at 200° C. for 15 to 30 minutes. In particular, in the case of the extract (Sample 6) extracted by roasting dried radish having a water content of 3% by weight at 200°C for 30 minutes, IC _{50 compared to the extract (Sample 10) extracted without roasting radish having a moisture content of 3% by weight.} The value decreased to 1/16 level, indicating that the sulfation effect was greatly increased. Among the roasted dried radish extract samples (Samples 1 to 9), dried radish having a moisture content of 3 wt% was roasted at 200°C for 30 minutes. In the treated extract (Sample 6), the IC ₅₀ value was found to be low.

2.3. Establishment of extraction process

As shown in [Table 2] and [Table 3] above, in the case of extracts obtained by roasting radish radish at a temperature of 180 to 200° C. for 15 to 30 minutes (Samples 1 to 9), dried radish radish without roasting treatment It was found that the content of total polyphenols was significantly higher than that of the extract (Sample 10), and the IC ₅₀ value was significantly lower.

That is, it can be interpreted that the antioxidant efficacy of the radish is significantly increased when the dried radish is roasted at a temperature of 180 to 200° C. for 15 to 30 minutes.

In particular, in the extract (Sample 6) extracted by roasting radish radish having a water content of 3% by weight at 200° C. for 30 minutes, the total polyphenol content was the highest and the IC ₅₀ value was the lowest. That is, it can be interpreted that the antioxidant effect of the extract extracted by roasting dried radish dried to a moisture content of 3% by weight according to Example 1.1 at 200° C. for 30 minutes according to Example 1.2 is the most remarkable.

Therefore, as the dried radish extract according to the present invention, an extract of dried radish dried at a temperature of 180 to 200° C. for 15 to 30 minutes can be preferably used, and further, dried radish dried to a moisture content of 3% by weight at 200° C. An extract obtained by hot-water extraction of radish dried for 30 minutes may be more preferably used.

In addition, in the following examples, "dried radish" or "dried radish extract" refers to an extract of dried radish dried to 3% by weight of water content and not roasted, and "roasted radish radish extract" " or "roasted radish extract" refers to a dried radish dried radish to a moisture content of 3% by weight and roasted for 30 minutes at 200° C. or an extract of radish radish.

Example 3; Changes in composition and measurement of 5-Hydroxymethyl-2-furfural (5-HMF) content of roasted radish radish

3.1. reducing sugar

After adding 2L of distilled water to each 100g of dried radish and roasted radish, extracted at 121°C for 15 minutes by high-temperature pressure extraction, first filtered using a depth filter and non-woven fabric, and second with a vibrating membrane filter (0.45㎛) Take 0.1 ml of the filtered extract, add 0.3 ml of 3,5-dinitrosalicyclic acid (DNS) reagent, boil for 10 minutes, cool, add 2 ml of distilled water to dilute, and measure the absorbance at 540 nm. As a result of obtaining the reducing sugar content, the reducing sugar content of the dried radish extract was measured to be 325 mg/g, respectively, and the reducing sugar content of the roasted radish extract was measured to be 310 mg/g. In other words, it was found that reducing sugar was reduced by about 5% in the process of roasting dried radish dried radish with roasted radish.

3.2 Free Amino Acids

The content of free amino acids in the dried radish radish extract and the roasted radish radish extract was analyzed with an amino acid automatic analyzer (L-8800 Amino acid auto analyzer, Hitachi, Japan) after each sample was filtered with a 0.45 μm membrane filter. The content of each amino acid in the common system was calculated by comparing the standard solution (Ajinomoto-Takara Co., Japan) with an amino acid calibration mixture. A total of 16 amino acids to be analyzed include Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, He, Leu, Tyr, Lys, His, Arg, and Pro.

In general, free amino acids are known to participate in non-enzymatic browning reactions by reacting with sugars during the heat treatment process during food processing, as the content of free amino acids is high as a precursor of volatile compounds. As a result of this experiment, 18 kinds of amino acids were analyzed in dried radish extract and roasted radish radish, and their contents are shown in [Table 4] below.

free amino acids	Dried radish (mg/kg)	Roasted radish (mg/kg)
L-Aspartic acid	176.51	55.06
L-Threonine	55.84	26.42
L-Serine	89.45	35.56
L-Glutamic acid	1,322.34	807.88
L-proline	206.31	83.91
L-Glycine	63.57	37.29
L-Alamine	181.49	58.14
L-Valine	63.75	19.74
L-Isoleucine	34.77	10.83
L-Leucine	20.76	13.66
L-Thyrosine	20.76	0
L-Phenylalanine	5.05	2.69
1-Methyl-L-Histidine	21.24	4.25
L-Lysine	14.74	4.38
L-Arginine	204.37	49.22
L-Cysteine	37.62	38.20
L-Methionine	3.37	2.38
L-Tryptophan	0	6.57
Total	2,521.94	1,256.28

As shown in [Table 4] above, the total free amino acids contained in the dried radish was 2,521.94 mg/kg, and the most abundant free amino acid was glutamic acid, accounting for 52% of the total amino acids, and other The content of amino acids was insignificant.

On the other hand, the total free amino acid content of the roasted radish was 1,256.28 mg/kg, and the free amino acid content decreased by about 50% during the roasting process of the dried radish.

3.3. Measurement of 5-Hydroxymethyl-2-furfural (5-HMF) content

5-Hydroxymethyl-2-furfural (5-HMF) content was quantified by HPLC. 5 g of each freeze-dried sample of dried radish extract and roasted radish extract were completely dissolved in 50 ml of distilled water, and 10 ml of them were filtered through a 0.45 μm membrane filter and used as a sample for HPLC analysis. LC-18 (4.6 mm×150 mm) was used as the analytical column, methanol-water (10:90, v:v) was used as the mobile phase, and the elution rate was 1.0 ml/min per minute. The concentration was calculated from the area by comparing the peak obtained by injecting 20 μl of the sample into the detector (HP1100, UV280 nm) with the peak obtained from the standard 5-HMF (Sigma-Aldrich Co., USA), and the result is as follows. It is shown in [Table 5].

	dried radish	Stir-fried dried radish
Free amino acid (mg/kg)	2,521.94	1,256.28
Reducing sugar (mg/g)	325	310
5-HMF (mg/100g)	0	7.44

As shown in [Table 5] above, 5-HMF was not detected in dried radish, but 5-HMF was measured to be 7.44 mg/100g in roasted radish.

3.4. sintering

The Maillard reaction is a reaction that occurs when sugars and amino acids are condensed (amino-carbonyl reaction), and free amino acids and reducing sugars participate in the reaction.

That is, as described above, as a result of comparing the reducing sugar and free amino acid contents of the dried radish and the roasted radish, it can be confirmed that the reducing sugar and free amino acids decreased during the pressure-roasting of the dried radish. It can be confirmed that the Maillard reaction occurred during the roasting process of the dried radish.

In addition, the color of the roasted radish was dark brown and showed a significant difference in chromaticity from that of the dried radish, which is thought to be due to the formation of melanoidin pigment by the Maillard reaction during the roasting process.

In addition, as described above, as a result of measuring 5-Hydroxymethyl-2-furfural (5-HMF), an intermediate product of the Maillard reaction, it was not analyzed in the dried radish, but a significant amount of 5-HMF was detected in the roasted radish. became

That is, the Maillard reaction product has strong antioxidant activity, and in particular, it has been reported that 5-HMF, an intermediate product, has physiological activities such as inhibition of nitric oxide production and inhibition of tyrosinase. It can be seen that antioxidants are produced by Maillard reaction during the roasting process of dried radish radish.

Example 4: Efficacy of skin dermal cell proliferation and SOD (Superoxide dismutase)-like activity

4.1. Cells used in the experiment and cell culture

Human dermal fibroblasts (PromoCell, USA) were _{cultured in an incubator at 37°C, 5% CO 2} , and Dulbecco's modified Eagal medium (FBS) containing 100 units/ml penicilin-streptomycin and 5% fetal bovine serum (FBS) as a culture medium DMEM) was used. Cells are cultured while changing the culture medium 2-3 times a week, and when cell differentiation reaches the maximum on day 6-7, wash the cells with phosphate buffered saline (PBS) and then trypsion-EDTA solution (0.05% trypsin and 0.02% EDTA). After separating the adherent cells with a mixed solution), centrifugation was performed to collect the cells, put them in a medium, mixed them well with a pipette to distribute the cells evenly, and dispensed them in several barrels, and then they were used for subculture while stored in liquid nitrogen. Cells used in the experiment were subcultured in the same manner as above, and each passage number was recorded during culture. When the passage number was 10 or more, new cells were taken out from the liquid nitrogen tank and cultured again to be used as experimental cells.

4.2. Efficacy of skin dermal cell proliferation by roasted radish extract

The human skin dermis cells, human dermal fibroblasts (HDFs, PromoCell, USA) a 96-well plate in (5x10 ³ cells / well) seeding with 5x10 ³ cells / well and in DMEM (10% FBS, 1% penicillin streptomycin) medium After culturing for 24 hours, the dried radish extract and the roasted radish extract were treated for 24 hours by concentration, and the cell proliferation effect was measured using the WST-1 assay (water-soluble tetrazolium salt-1) kit (Cayman, USA). did.

Cell proliferation effect (%) = [(absorbance of experimental group - absorbance of control group) / absorbance of control group] × 100

The cell viability results obtained above are shown in [Table 6] below.

Treatment concentration (μg/ml)		Cell viability (%)
dried radish radish extract	100	90±0.7
	200	100±0.87
	300	89±0.95
	500	87±1.38
Roasted radish extract	100	100±0.7
	200	94±0.84
	300	95±0.87
	500	110±1.57

As shown in [Table 6] above, it was found that there was no cytotoxicity by the addition of the dried radish radish extract and the roasted radish radish extract. In addition, the dried radish extract treated group showed 89% and 87% cell viability effects at 300 and 500 μg/ml, respectively, and in the case of the stir-fried radish extract treated group, 95% and 110% at 300 and 500 μg/ml, respectively. of the cell viability effect, the cell viability increased by about 6% and 23% in the case of the roasted dried radish extract treatment group compared to the dried radish extract treatment group.

4.3. Superoxide dismutase (SOD)-like activity

Human dermal fibroblasts (HDFs, PromoCell, USA), which are human skin dermal cells, ^{were cultured at 1×10 6} cells in a 6-well plate, and dried radish extract and roasted radish extract were treated for 24 hours at different concentrations. After cell disruption, SOD-like activity was measured using a superoxide dismutase activity kit (Cayman, USA), as shown in [Table 7] below.

Treatment concentration (μg/ml)		Relative SOD-like activity
dried radish radish extract	unprocessed	One
	100	1.18±0.7
	200	1.22±0.7
	300	1.24±0.4
	500	1.25±0.6
Roasted radish extract	unprocessed	One
	100	1.20±0.7
	200	1.23±1.38
	300	1.27±1.38
	500	1.28±0.6

As shown in [Table 7] above, both the dried radish extract and the stir-fried radish extract increased the SOD-like activity in a concentration-dependent manner. However, in the dried radish-treated group, the relative SOD-like activity of 1.24 and 1.25 at 300 and 500 μg/ml, respectively, were shown, while in the roasted dried radish-treated group, the relative SOD-like activity was 1.27 and 1.28 at 300 and 500 μg/ml, respectively. SOD-like activity was shown. That is, it was found that the relative SOD-like activity effect increased by about 2.4% and 2.4% in the case of the roasted dried radish-treated group compared to the dried radish-treated group.

Example 5: Animal test

5.1. laboratory animal

Species and strains of experimental animals were purchased from Orient Bio (address: 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do) as female SKH-1 hairless specific pathogen-free (SPF) mice.

In the case of the SKH-1 hairless mouse used in this test, a number of research results have been reported on skin and skin aging studies including photoaging, and the pathological mechanisms of various skin diseases are well known based on the previously reported research results, Since skin aging evaluation criteria have been established, it has the advantage of being suitable for photoaging research by UV irradiation.

After obtaining the experimental animals, they were quarantined and acclimatized in the animal room for 7 days, and only healthy animals were used for the test by observing general symptoms during the acclimatization period.

The mice used in this test were subjected to a temperature of 23±3℃, relative humidity of 50±10%, illumination time of 12 hours (08:00 on - 20:00 off), ventilation frequency of 10-20 times/hr, and illuminance of 150-300Lux. Each was acclimatized and bred in the laboratory animal lab of Konkuk University College of Veterinary Medicine, and all test methods in this test were approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University.

In addition, the experimental animals were housed in a polycarbonate breeding box (240W × 390L × 175H mm) for acclimatization, quarantine and administration, and observation periods, each of 6 mice or less, and identification of individuals was performed by labeling using an ear punch and by breeding box. The individual identification card display method was used.

5.2 Animal testing methods

5.2.1. animal anesthesia

After placing the experimental animals in the induction chamber, anesthesia was induced by setting the oxygen flowmeter to 0.9L/min and setting the isoflurane concentration to 3.5%. After induction of anesthesia, the oxygen flowmeter was maintained at 4~0.8L/min using a mask, and the anesthesia was maintained by setting the isoflurane concentration to 1.5%. During anesthesia, the core body temperature was maintained at 37-38°C using a warming mat.

5.2.2. Application of test substance and UV irradiation

200 μl of each test substance per mouse is applied on the back skin of animals 6 times a week, for a total of 6 weeks. UV rays were irradiated under animal anesthesia, and the head including eyes was irradiated with UV rays after blocking UV rays.

The UV irradiator installs a UV lamp that generates UV rays of 280-320 nm (peak 313 nm) in the cabinet that can be connected to the anesthesia machine, and the intensity of UV rays located at the bottom of the cabinet is measured through a UVmeter to obtain 1 MED (week 1) and 2 Apply at MED (weeks 2-6) intensity. UV irradiation was carried out 3 times a week, 2 hours after application of the test substance, and the distance between the skin of the mouse's back and the UV lamp was 30 cm or more.

The skin application test group and treatment for each experimental group are shown in [Table 8] below.

experimental group	Mouse skin application test
Normal control (NC)	no-UV-irradiation, no treatment
Vehicle control (VC)	UV-irradiation, 200 μl/mouse of propylene glycol;ethanol:water=5:3:2 topical treatment
radish extract * topical tretment (RE-T)	UV-irradiation, 200 μl/mouse of radish extract in vehicle (500㎍/㎍) topical treatment
radish extract * topical tretment with drinking water (RE-TW)	UV-irradiation, 200 μl/mouse of radish extract in vehicle (500㎍/㎍) topical treatment with 0.5% concentration of radish extract into drinking water

* : Roasted radish dried radish extract

5.2.3. Oral administration of test substance and UV irradiation

The test substance was orally administered at a dose of 10 ml/kg per mouse 6 times a week, for a total of 6 weeks. UV rays were irradiated under animal anesthesia, and the head including eyes was irradiated with UV rays after blocking UV rays.

The UV irradiator installs a UV lamp that generates UV rays of 280-320 nm (peak 313 nm) in the cabinet that can be connected to the anesthesia machine, and the intensity of UV rays located at the bottom of the cabinet is measured through a UVmeter to obtain 1 MED (week 1) and 2 Apply at MED (weeks 2-6) intensity. UV irradiation was carried out 3 times a week, 2 hours after application of the test substance, and the distance between the skin of the back of the mouse and the UV lamp was 30 cm or more.

The oral administration experimental group and treatment for each experimental group are shown in [Table 9] below.

experimental group	mouse oral administration test
Normal control (NC)	no-UV-irradiation, no treatment
Vehicle control (VC)	UV-irradiation, 10ml/kg sterilized water for injection per oral (P.O)
radish extract * topical tretment (RE-T)	UV-irradiation, 10㎖/kg radish extract in sterilized water for injection (200㎖/kg) P.O
radish extract * topical tretment with drinking water (RE-TW)	UV-irradiation, 10ml/kg radish extract in sterilized water for injection (200ml/kg) P.O with 0.5% concentration of radish extract into drinking water

* : Roasted radish dried radish extract

5.3. Observation and inspection items

5.3.1. Visual observation of the skin application and oral administration experimental group

For visual observation, skin findings were photographed through a stereomicroscope of the results observed once a week during the entire test period, and prior to irradiation with each test substance and UV light on the day. For skinfold thickness, a certain area of the skin on the back was selected using a caliper, and the skin was lifted to measure the skin thickness.

5.3.2. Histopathological observation of skin application and oral administration experimental group

Samples were prepared in the 3rd and 6th weeks of the application of the test substance and the start of UV irradiation, an overdose of tribromoethanol (Avertin, 250mg/kg) was anesthetized, and euthanized by cervical dislocation. Two of the application sites were excised and treated with formalin. After fixation, tissue slides were prepared. Each sample was observed under a microscope after hematoxlyin-esoin (HE) staining to compare and evaluate the formation of keratin, changes in the thickness of the epidermis and dermis, and changes in the dermal collagen layer.

5.3.3. Molecular biological evaluation of the skin application group

Put the skin tissue (back skin tissue) of the test animal into 100 mM phosphate buffer (pH 7.4) and homogenize it using a tissue homogenizer (Omni) to obtain tissue homogenate, and then centrifuge at the strength recommended by each commercially available measurement kit. After separation to obtain a supernatant, protein concentrations (Collagen Type I, Collagen Type III) and antioxidant enzyme activity (Catalase colorimetric activity, Superoxide Dismutase (SOD) activity, Glutathione Peroxidase (GPX) activity) were measured.

For the measurement of skin protein concentration, Collagen Type I ELISA Kit (Cat. No. MBS775915, MyBioSource) and Collagen Type Ⅲ ELISA Kit (Cat. No. MBS775917, MyBioSource) were used, and for the measurement of antioxidant enzyme activity, Catalase colorimetric activity kit ( Cat. No. EIACATC, Invitrogen), Superoxide Dismutase (SOD) colorimetric activity kit (Cat. No. EIASODC, Invitrogen) and Glutathione Peroxidase Assay Kit (Cat. No. 703102, Cayman chemical) were used, and Bradford protein assay method was used. The protein concentration was calculated and corrected.

5.3.4. Oral administration hematological and molecular biological evaluation

Venous blood of experimental animals was collected from the embryonic vena cava, coagulated in a serum-separating tube (BD Bioscience) for 2 hours, and centrifuged at 1300xg, 4°C for 10 minutes to separate the serum to separate inflammatory mediators (IL-8, TNF- α) was measured, and the measurement of inflammatory mediators in the blood was measured using an IL-8 ELISA kit (Cat. No. MBS7606860, MyBioSource), and a TNF-α Immunoassay kit (Cat. No. MTA00B, R&D Systems).

In addition, the skin tissue of the back of the test animal was placed in 100 mM phosphate buffer (pH 7.4) and homogenized using a tissue homogenizer (Omni) to obtain tissue homogenate, and then centrifuged at the strength recommended by each commercially available measurement kit to collect the supernatant. After obtaining the protein concentration (Collagen Type I, Collagen Type Ⅲ) was measured.

For the measurement of skin protein concentration, Collagen Type I ELISA Kit (Cat. No. MBS775915, MyBioSource) and Collagen Type Ⅲ ELISA Kit (Cat. No. MBS775917, MyBioSource) were used, and the protein concentration was obtained and corrected by Bradford protein assay method. .

5.3.5. statistical method

For statistical analysis of the obtained data, one-way analysis of variance (ANOVA) and Bonferroni's post hoc test were performed. The risk rates of the assay were set at 5% and 1%.

5.4. result

5.4.1. Visual observation results of the skin application group

From the visual observation of the skin condition, skin redness and keratin formation were observed in the UV irradiation test group (VC, RE-T, RE-TW) except for the normal control group (NC) in the first week of irradiation, and the test substance applied group (RE-TW) T, RE-TW), it was observed that redness and keratinization decreased over the test period (see [Fig. 1]).

In addition, as a result of skinfold thickness measurement, no significant difference was observed due to the application of the test substance. However, in the test group (RE-TW) in which the test substance was applied and drinking water was supplied, the skin thickness increased during the entire test period. It was observed to show the lowest value (see [Fig. 2]).

5.4.2. Visual observation results of the oral administration experimental group

In the macroscopic observation of the skin condition, skin redness and skin keratin formation were observed in the UV irradiation test groups (VC, RE-TPO, RE-TPOW) except for the normal control group (NC) at the first week of UV irradiation. On the other hand, it is observed that the skin redness symptoms of the UV irradiation test group (VC, RE-TPO, RE-TPOW) are alleviated over time from 3 to 6 weeks, compared to the excipient control group (VC), the test substance applied group ( RE-TPO, RE-TPOW) was observed to have a faster remission rate, and the degree of keratin formation was observed to be less in the test substance application group (RE-TPO, RE-TPOW) than in the excipient control group (VC) ([Fig. 3]).

In addition, as a result of skinfold thickness measurement, it was observed that the skin thickness of the excipient control group (VC) gradually increased over the test period, but in the case of the test substance application group (RE-TPO, RE-TPOW), it was observed at the 1st week. Compared to that, it was observed that the skin thickness decreased at the 3rd and 6th weeks. In particular, the test substance application group (RE-TPO, RE-TPOW) showed a significantly lower value than the excipient control group (VC) at the 6th week at the end of the test. was observed to show (see [Fig. 4]).

In addition, as a result of measuring skin moisture and transepidermal water loss (TEWL), a decrease in skin moisture was also observed in the normal control group (NC) over the test period, and UV irradiation test group (VC, RE-TPO, In the case of RE-TPOW), the test substance application group (RE-TPO, RE-TPOW) had higher skin moisture than the excipient control group (VC), and in particular, the test substance application group (RE-TPO, RE-TPOW) was orally administered with drinking water (RE). -TPOW) was observed to be very similar to that of the normal control group (NC) as the skin moisture level was improved at the 6th week, the time at which the test was terminated (see [Fig. 5]).

On the other hand, in the measurement of transdermal moisture loss, no significant difference was observed in the UV irradiation test group (VC, RE-TPO, RE-TPOW) according to the application of the test substance, and all test groups showed a similar degree of transdermal moisture loss. was observed to show (see [Fig. 6]).

5.4.3. Histopathological observation results of the skin application group

As a result of skin histopathological observations at 3 and 6 weeks, the excipient control group (VC) showed markedly proliferation and keratinization of the epidermal layer during the entire test period compared to the normal control group (NC), and the test substance applied group (RE- T, RE-TW) was observed to be thinner than the excipient control group (VC) (see [Fig. 7] and [Fig. 8]).

On the other hand, in the dermal layer thickness analysis, the normal control group (NC) also showed a decrease in the dermal layer thickness over time, but the test substance applied groups (RE-T, RE-TW) showed a normal dermal layer thickness during the entire test period (NC). It was observed to be thicker than the excipient control group (VC). In particular, the test substance application group (RE-T) showed a statistically significant (p<0.05) increase in the dermal layer thickness compared to the excipient control group (VC) at week 6 (see [Fig. 7] and [Fig. 8]).

5.4.4. Histopathological observation results of the oral administration experimental group

As a result of skin histopathological observations at 3 and 6 weeks, it was observed that the epidermal layer was thicker than that of the normal control group (NC) in all of the UV irradiation test groups (VC, RE-TPO, RE-TPOW) during the entire test period. Among the groups (VC, RE-TPO, RE-TPOW), in particular, in the case of the test group (RE-TPOW), in which the test substance was orally administered with drinking water, the thinnest epidermal layer thickness among the UV irradiation test groups at the 6th week at the end of the test was shown. On the other hand, in terms of the dermal layer thickness, it was observed that the dermal layer thickness was increased in the test substance application group (RE-TPO, RE-TPOW) compared to the excipient control group (VC) at the 6th week at the end of the test ([Fig. 9] and [Fig. 10]).

5.4.5. Molecular biological evaluation results of the skin application group

As a result of observing the change in skin protein concentration in the skin tissue of the resected application site by ELISA, the concentration of type I collagen and type III collagen in the excipient control group (VC) was observed to show almost similar concentrations at the 3rd and 6th weeks, but , In the case of the test substance application group (RE-T, RE-TW), it was observed that the concentration increased over time, and in the case of the test substance application group (RE-T, RE-TW) compared to the excipient control group (VC), the type High concentrations of I collagen and type III collagen were observed.

In addition, as a result of analyzing the antioxidant enzymes catalase, GPX, and SOD, the excipient control group (VC) and the test substance application group (RE-T, RE-TW) were observed to be similar in catalase concentration, but GPX (glutathione peroxidase) activity in the test substance application group (RE-T, RE-TW) compared to the excipient control group (VC) was observed to be all higher at week 6, and also in the SOD concentration, the test substance application group and drinking water supply were concurrently applied (RE). -TW) was observed to show a higher trend compared to the excipient control group (VC) (see [Fig. 11]).

5.4.6. Hematological and molecular biological evaluation results of the oral administration experimental group

The results of measuring the concentration change of inflammatory cytokines (IL-8, TNF-α) in the blood are as follows,

First, as a result of measuring the change in the concentration of IL-8 in the blood, it was observed that the concentration of IL-8 was higher in the excipient control group (VC) at the 3rd and 6th weeks compared to the normal control group (NC), and the test substance was applied. In the case of the group (RE-TPO, RE-TPOW), it was observed that the IL-8 concentration was lower than that of the excipient control group (VC) during the entire test period. In particular, the test substance oral administration group (RE-TPO) showed a lower concentration than that of the normal control group (NC) during the entire test period (see [Fig. 12]).

In addition, as a result of measuring the change in the concentration of TNF-α in the blood, it was found that the lowest concentration was exhibited in the test group (RE-TPOW), in which the test substance was orally administered with drinking water among all test groups at the 6th week, the end of the test. (See Fig. 13).

In addition, in the observation of changes in the concentration of type I collagen and type III collagen protein in the excised skin tissue, the excipient control group (VC) was observed to show lower values during the entire test period compared to the normal control group (NC), but the test substance applied group In the case of (RE-TPO, RE-TPOW), an increase in type I collagen and type III collagen was observed at the 6th week compared to the 3rd week, and the levels were similar to those of the normal control group (NC) (see [Fig. 14]).

As described above, looking at the animal test results, it was observed that when the roasted radish extract of the present invention was applied to the skin topically, it was observed that the skin damage caused by UVB ultraviolet rays was suppressed.

That is, as a result of visual observation, it was observed that skin keratin formation was alleviated in the test substance application group (RE-T, RE-TW) compared to the excipient control group (VC). In the case of the excipient control group (VC), the thickness of the epidermal layer exhibited by proliferation was similar during the entire test period, but in the case of the test substance application group (RE-T, RE-TW), the thickness of the epidermal layer increased over time from 3 weeks to 6 weeks. was observed to decrease, and the thickness of the epidermal layer was observed to be thinner than that of the excipient control group (VC).

On the other hand, no significant difference was observed between the test groups in the evaluation of the skin thickness by visual observation, but as a result of the analysis of the dermal layer thickness that can affect the skin thickness, compared to the normal control group (NC) and the excipient control group (VC), The thickness of the dermal layer was observed to be thick in the test substance application group (RE-T, RE-TW), and in particular, the dermal layer thickness at the 6th week of the test substance application group (RE-T) was observed to be the thickest among all test groups.

In addition, as a result of protein concentration analysis using ELISA for skin tissue, it was observed that the concentration of type I collagen and type III collagen in the test substance application group (RE-T, RE-TW) was higher than that in the excipient control group (VC). It is judged to be consistent with the results of the normal analysis, and also in the evaluation of antioxidant enzymes that can exhibit photoaging inhibitory efficacy, GPX in the test substance application group (RE-T, RE-TW) was increased at the 6th week in the excipient control group (VC). was observed to be higher than

Therefore, the skin application of the roasted radish radish extract helps to increase the GPX antioxidant enzyme activity in skin photoaging caused by UVB ultraviolet irradiation, and maintains the thickness of the dermal layer by inhibiting the decomposition of type I collagen and type III collagen, which are the main proteins of the dermal layer of the skin. and it was confirmed to exhibit the effect of reducing wrinkle formation.

In addition, as a result of animal experiments, it was observed that when the roasted radish extract of the present invention was orally administered, skin damage caused by UVB ultraviolet rays was suppressed.

That is, as a result of visual observation, it was observed that clinical symptoms such as skin redness and keratin formation were alleviated in the test substance application group (RE-TPO, RE-TPOW) compared to the excipient control group (VC), and compared to the excipient control group (VC). In the case of the test substance application group (RE-TPO, RE-TPOW), it was observed that the skin moisture content was high.

In addition, keratinization relief is related to the reduction in the thickness of the epidermal layer of the skin in the test substance application group (RE-TPO, RE-TPOW) in histopathological analysis. In particular, the test substance orally administered with drinking water (RE-TPOW) A decrease in the thickness of the epidermal layer was clearly observed.

In addition, in the dermal layer thickness analysis result, it was observed that the normal control group (NC) also showed a decrease in the dermal layer due to aging, and in the case of the test substance application group (RE-TPO, RE-TPOW), compared to the excipient control group (VC), at the end of the test An increase in the thickness of the dermal layer was observed.

In addition, the reduction in the thickness of the dermal layer due to photoaging is caused by an increase in inflammatory mediators due to the generation of reactive oxygen species (ROS) by UVB ultraviolet rays, and collagenase (MMPs) secreted by dermal fibroblasts by these inflammatory mediators. In the test group to which the roasted radish extract according to the present invention was applied, the concentrations of IL-8 and TNF-α, which are inflammatory mediators in the blood, were observed to be low, and the concentrations of type I collagen and type III collagen proteins were observed to be high. It was confirmed that it was consistent with the results of histopathological analysis.

That is, when the roasted radish extract according to the present invention was orally administered, or the roasted radish extract was orally administered with drinking water, the reduction of inflammatory mediators in the blood by UVB ultraviolet irradiation was shown, thereby inhibiting the degradation of type I collagen and type III collagen protein. Thus, it was confirmed that there was an effect of improving skin wrinkles and improving skin photoaging, such as maintaining the thickness of the dermis and maintaining the homeostasis of the epidermal layer, thereby improving skin moisture.

In addition, in the test group (RE-TPOW), in which the roasted radish extract was orally administered with drinking water, only the roasted radish extract was orally administered in skin moisture, epidermal layer thickness, and TNF-α concentration (RE-TPOW). TPO) was found to be superior.

Therefore, when the stir-fried radish extract is mixed with drinking water (water) and provided as a drink or other food, it is naturally expected to be effective in improving skin wrinkles or preventing skin aging.

Claims (5)

1. A composition for improving skin wrinkles or preventing skin aging, comprising roasted radish radish extract as an active ingredient.
2. According to claim 1,

   The roasted radish extract is a composition for improving skin wrinkles or preventing skin aging, characterized in that it is an extract extracted by roasting dried radish to a moisture content of 3% by weight at 200° C. for 30 minutes.
3. 3. The method of claim 1 or 2,

   The composition is a composition for improving skin wrinkles or preventing skin aging, characterized in that it is for application to the skin.
4. 3. The method of claim 1 or 2,

   The composition is a composition for improving skin wrinkles or preventing skin aging, characterized in that it is for oral administration.
5. A health functional food comprising the composition of claim 1 or 2 and drinking water (water).

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