KR102491188B1 - Manufacturing method of the strawberry stem extracts and Anti-oxidant and anti-inflammatory composition, Health functional foods containing the same and Cosmetics using the same - Google Patents

Abstract

The present invention relates to an antioxidant and anti-inflammatory composition and health functional food and cosmetics using the same, and more specifically, to a composition containing an unripe strawberry pulp extract as an active ingredient, and health functional food and/or cosmetics using the same. The antioxidant and anti-inflammatory composition of the present invention contains an unripe strawberry pulp extract as an active ingredient, wherein the unripe strawberry pulp extract is a lower alcohol extract having 1 to 4 carbon atoms.

Description

Antioxidant and anti-inflammatory composition, health functional food containing the same, and cosmetics containing the same {Manufacturing method of the strawberry stem extracts and Anti-oxidant and anti-inflammatory composition, Health functional foods containing the same and Cosmetics using the same}

The present invention relates to an antioxidant and anti-inflammatory composition using an extract of immature strawberry pulp, and to health functional foods and cosmetics using the same.

Humans experience an aging phenomenon in which physiological functions deteriorate over time, and one of the indicators that can determine the degree of biological aging is the skin. Skin, as the primary barrier of the human body, protects various organs in the body from changes in temperature and humidity, ultraviolet rays and pollutants from the external environment, and plays an important role in maintaining body homeostasis such as regulating body temperature. Since the skin is always exposed to the external environment, it can be said that many of the skin changes due to aging are due to the influence of external factors. In particular, inflammatory reactions to external factors, active oxygen, and ultraviolet rays are considered to be the main causes.

Active oxygen is involved in skin aging as well as biological aging by destroying the antioxidant defense system by leading skin cell and tissue damage, and causes various diseases and accelerates aging by causing functional damage to various organs. In addition, continuous oxidative stress causes damage to biocomponents such as activation of proteolytic enzymes, cutting of collagen and elastin, which are elastic fibers, and cutting of hyaluronic acid. bring

Inflammatory response is a mechanism to regenerate damaged areas caused by chemical substances, bacterial infections, and physical actions that cause organic changes in the body. It is caused by the release of vasoactive substances such as inflammatory components, and if such an inflammatory reaction continues, it rather promotes mucosal damage, thereby causing various diseases such as redness, fever, swelling, pain, and functional disorders. Enzymes secreted as a result of an inflammatory reaction in the skin destroy and damage surrounding cells or tissues, which causes skin aging such as destruction of collagen fibers and elastic fibers of the skin. Therefore, in order to protect the skin from various causes of skin aging, it is necessary to soothe and protect the skin by suppressing active oxygen and alleviating skin irritation and inflammation.

Cultivated strawberry (Fragaria ananassa Duch.) is a perennial plant belonging to the Rosaceae family that has a long cultivation period and is a labor-intensive crop, but can be grown at low temperatures and can be harvested from November to May through low-temperature facility cultivation. Cultivated strawberries contain flavonoids such as quercetin-7-xyloside, fragarin, procyanidin B3, and 1-O-p-coumaroylglucose. ), 2-heptanol or ellagic acid. As a result, many studies on diseases such as anti-inflammatory, anti-cancer, and kidney diseases using strawberries are being conducted. In addition, because of its excellent fragrance and color, it is currently used as a raw material for manufacturing jams, syrups, juices, etc. Research on various antioxidants and anti-inflammatory agents using strawberries and development of related products are continuing, but product development that satisfies consumer satisfaction is insufficient.

Japanese Laid-Open Patent No. 2006-089410 (published on April 6, 2006) Korean Patent Publication No. 2019-0045191 (published on 2019.05.02)

The present invention is to provide an extract containing a large amount of antioxidant and anti-inflammatory active ingredients processed from immature strawberry fruits that have not matured due to fallen fruit or weather damage.

In addition, the present invention is to provide a health functional food, cosmetics using the extract.

In order to solve the above problems, the antioxidant and anti-inflammatory composition of the present invention contains immature strawberry pulp extract as an active ingredient, and the immature strawberry pulp extract is a lower alcohol extract having 1 to 4 carbon atoms.

In addition, the present invention relates to a method for preparing an immature strawberry pulp extract, comprising the steps of preparing strawberry powder by washing and drying the immature strawberry pulp and then pulverizing it; A second step of mixing the strawberry powder and the lower alcohol aqueous solution having 1 to 4 carbon atoms and then stirring at 20 to 30 ° C. for 18 to 30 hours; Step 3 of separating the supernatant and the precipitate by filtering the extract obtained in step 2; It may be prepared by carrying out a process comprising: performing vacuum concentration on the supernatant to obtain a concentrate, followed by lyophilization to obtain an alcohol extract.

The immature strawberry pulp extract of the present invention has low cytotoxicity and contains a large amount of polyphenols, so it has excellent electron donating activity and ABTS radical scavenging activity, so it has excellent antioxidant properties, inhibits NO production and iNOS and COX-2 proteins It has excellent anti-inflammatory properties by suppressing expression, and it can help increase the profits of strawberry farmers by utilizing discarded strawberry fruits and immature strawberry fruits.

1 is a result of measuring electron donating ability (DPPH assay, 1-1-diphenyl-2-picryl-hydrazyl) of immature strawberry pulp ethanol extracts performed in Experimental Example 1.
2 is a result of measuring ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity of immature strawberry pulp ethanol extracts performed in Experimental Example 1.
3 is a result of measuring cytotoxicity (or cell viability) performed in Experimental Example 2.
4 is a result of NO production inhibition measurement conducted in Experimental Example 3.
5 is a result of measuring inhibition of iNOS and COX-2 protein expression conducted in Experimental Example 4.

Hereinafter, the present invention will be described in more detail through a method for preparing the unripe strawberry pulp extract, which is a material for the antioxidant and anti-inflammatory composition of the present invention.

The present invention uses immature strawberry pulp (or immature strawberry), and immature strawberry pulp refers to a pure Korean word for 'unripe fruit'. The proper harvesting time of strawberries is determined by the degree of coloration, and the immature period during the growth period of strawberries is a state in which the degree of coloration of the fruit has not occurred. It is classified as a young strawberry of about 30 years old and is called immature.

The pulp extract of immature strawberry of the present invention may be an alcohol extract of immature strawberry.

Immature strawberry pulp alcohol extract is prepared by preparing strawberry powder by washing and drying immature strawberry pulp (immature strawberry) and then pulverizing; After mixing the strawberry powder and the aqueous alcohol solution, a second step of performing stirring at 20 to 30 ° C, preferably 22 to 27 ° C for 18 to 30 hours; Step 3 of separating the supernatant and the precipitate by filtering the extract obtained in Step 2; It can be prepared by performing a process including; and performing vacuum concentration on the supernatant to obtain a concentrate, followed by lyophilization to obtain an alcohol extract.

In the first step of the alcohol extract manufacturing process, the aqueous alcohol solution may use an aqueous solution of a lower alcohol having 1 to 4 carbon atoms, preferably an aqueous ethanol solution, and more preferably an aqueous ethanol solution having a concentration of 65 to 80% by volume. there is. When using an aqueous ethanol solution, if the concentration of the aqueous ethanol solution is less than 65% by volume, the content of the physiologically active active ingredient in the extract may be low, and if the concentration exceeds 80% by volume, there may be a problem with a drop in extraction efficiency. It is preferable to use

In the manufacturing process of the immature strawberry pulp alcohol extract, the above three steps may be repeated 1 to 3 times. In addition, the vacuum concentration in step 4 may be performed by a general vacuum concentration method used in the art, and the method is not particularly limited. In addition, the freeze-drying may also be performed by a general freeze-drying method used in the art.

The unripe superheated strawberry extract and/or alcohol extract of the present invention prepared in this way has excellent antioxidant activity and anti-inflammatory effect.

The immature strawberry pulp extract of the present invention may have a total polyphenol content of 1.50 to 3.50 mg tanninc acid/g, preferably 1.70 to 3.50 mg tanninc acid/g, and more preferably 1.80 to 3.20 mg tanninc acid/g. can

In addition, when the DPPH radical scavenging activity of the immature strawberry pulp extract of the present invention is measured, when the concentration of the extract is 1000 ppm (μg/ml), the DPPH radical scavenging activity according to the following formula 1 is 88% or more, preferably 89.00% or more , More preferably, it may be 90.0 to 95.00%.

[Equation 1]

DPPH radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%

The absorbance of the sample in Equation 1 was measured at 517 nm using a spectrophotometer after mixing 3 ml of an ethanol solution containing 60 μM DPPH with 0.5 ml of immature strawberry pulp extract and reacting in the dark for 15 minutes. 0.5 ml of distilled water was mixed with 3 ml of an ethanol solution containing 60 µM DPPH, reacted in the dark for 15 minutes, and then measured at 517 nm using a spectrophotometer.

In addition, when measuring the ABTS radical scavenging activity of the immature strawberry pulp extract of the present invention, when the concentration of the extract is 1000 ppm (μg/ml), the ABTS radical scavenging activity based on the following formula 2 is 90% or more, preferably 92% or more , More preferably, it may be 92.0 to 98.0%.

[Equation 2]

ABTS radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%

The absorbance of the sample in Equation 2 was obtained by mixing 150 μl of immature strawberry pulp extract and 3 ml of ABTS solution, vortexing for 30 seconds, reacting at 25 ° C for 3 minutes, and measuring the absorbance at 734 nm. 150 μl and 3 ml of ABTS solution were mixed, vortexed for 30 seconds, reacted at 25° C. for 3 minutes, and absorbance was measured at 734 nm.

In addition, the immature strawberry pulp extract of the present invention, when measuring the NO inhibitory ability, when the extract concentration is 500 ppm (μg / ml), the NO inhibitory ability measured according to the following formula 3 is 20% or more, preferably , Preferably it may be 23.0 ~ 32.0%.

[Equation 3]

NO inhibition ability (%) = measured NO level of control (%) - measured NO level of extract (%)

The NO inhibition ability of Equation 3 was determined by treating RAW 264.7 cells with 10 μg/ml of LPS (Lipopolysaccharide), then treating immature strawberry pulp extract at a certain concentration 2 hours later, incubating for 18 hours, and measuring the amount of NO in the culture medium. The NO measurement level of the control group is 100%, and the NO measurement level of the extract is a value obtained by converting the measured NO amount to % based on the control group.

In addition, when the immature strawberry pulp extract of the present invention has an extract concentration of 500 ppm (μg/ml), the iNOS expression inhibition ability measured according to Equation 4 below may be 80% or more, preferably 85.0 to 95.0%, and more Preferably it may be 88.0 to 95.0%.

[Equation 4]

iNOS expression inhibition ability (%) = iNOS expression rate of negative control group (%) - iNOS expression rate of extract (%)

In Equation 4, the negative control group is the iNOS expression rate measured after treatment with the stimulant LPS alone, the iNOS expression rate of the extract is the iNOS expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the negative control group It is a value converted to % as a standard.

In addition, when the immature strawberry pulp extract of the present invention has an extract concentration of 500 ppm (μg/ml), the COX-2 expression inhibitory ability measured according to the following formula 5 may be 30.0% or more, preferably 31.0 to 40.0% , More preferably, it may be 32.0 to 38.0%.

[Equation 5]

COX-2 expression inhibition ability (%) = COX-2 expression rate of control (%) - COX-2 expression rate of extract (%)

In Equation 5, the negative control group is the COX-2 expression rate measured after treatment with the stimulant LPS alone, and the iNOS expression rate of the extract is the COX-2 expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the measured COX-2 expression rate. 2 It is a value obtained by converting the expression level to % based on the negative control group.

Using the composition containing the immature strawberry pulp extract as an active ingredient of the present invention, it is possible to provide health functional foods or inner beauty products by processing various foods such as sweets, beverages, candies, capsules, and nutritional supplements.

In addition, it can be used as a cosmetic for various cosmetics such as skin cosmetics, scalp cosmetics, and hair cosmetics.

In the above, the present invention has been described with a focus on embodiments, but this is only an example and does not limit the embodiments of the present invention, and those skilled in the art to which the embodiments of the present invention belong will appreciate the essential characteristics of the present invention. It will be appreciated that various modifications and applications not exemplified above are possible within a range that does not deviate. For example, each component specifically shown in the embodiment of the present invention can be modified and implemented. And differences related to these modifications and applications should be construed as being included in the scope of the present invention as defined in the appended claims.

[Example]

Example 1: Preparation of immature strawberry pulp ethanol extract

Immature strawberry pulp was prepared by removing the calyx after collecting all ripe fruits of strawberries by May 2021 in Cheongju, Chungcheongbuk-do.

Next, it was washed and dried with hot air, and then crushed to prepare strawberry powder.

Next, the strawberry powder was added to an aqueous ethanol solution having a concentration of 70% by volume. At this time, the mixing ratio of unripe strawberry powder and aqueous ethanol solution was 1:10 by weight.

Next, after stirring at 25 ° C. for 24 hours, the supernatant and the precipitate were separated by primary filtration with a non-woven fabric, and the filtration and separation were repeated three times.

At this time, filtration and separation were performed by filtration using a vacuum pump and filter paper (Whatman No.5, No. 4, No.2).

Next, the supernatant separated from the precipitate was concentrated under reduced pressure using a rotary vacuum evaporator (EYEKA, Japan) to obtain a concentrate, and then freeze-dried and stored at -20 ° C to obtain immature strawberry pulp. An ethanol extract was obtained.

Experimental Example 1: Total polyphenol content measurement

The total polyphenol content of the immature strawberry pulp alcohol extract of Example 1 was measured by modifying the Folin-Denis method as follows, and the results are shown in Table 1 and FIG. 1 below.

The measurement method was to dilute the extract in distilled water, mix 2ml and 50% Folin-ciocalteu reagent, and then leave it at room temperature for 3 minutes. Thereafter, 2 ml of 10% Ca ₂ CO ₃ was added, mixed, and allowed to stand at room temperature for 1 hour, and the absorbance was measured at 700 nm.

division Example 1 Total polyphenolic content (mg tannic acid/g) 1.78±0.94

In general, there is a high correlation between the content of phenolic components and free radicals, and as the phenol content increases, it shows a very good effect on physiological activities such as antioxidant. After measuring the polyphenol content of each sample, the content per gram of sample indicated the amount of

As shown in Table 1, the ethanol extract of immature strawberry pulp showed a high polyphenol content of 1.78±0.94 mg/g.

Experimental Example 2: Measurement of antioxidant effect

As the antioxidant effect of the immature strawberry pulp alcohol extract of Example 1, electron donating ability and ABTS radical scavenging activity were measured as follows.

(1) Measurement of electron donating ability

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity test as an electron donating ability was performed by modifying the Blois MS method, and 3 ml of 60 μM DPPH (in EtOH) was added to 0.5 ml of immature strawberry pulp extract (sample). After mixing and reacting in a dark room for 15 minutes, measurement was performed at 517 nm using a spectrophotometer. In addition, the DPPH scavenging ability was calculated based on Equation 1 below, and the results are shown in Table 2 and FIG. 1 below.

At this time, distilled water was used as the negative control group, and ascorbic acid (Vit.C) was used as the positive control group.

[Equation 1]

DPPH radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance}}×100%

The absorbance of the sample in Equation 1 was measured at 517 nm using a spectrophotometer after mixing 3 ml of an ethanol solution containing 60 μM DPPH with 0.5 ml of immature strawberry pulp extract and reacting in the dark for 15 minutes. 0.5 ml of distilled water was mixed with 3 ml of an ethanol solution containing 60 µM DPPH, reacted in the dark for 15 minutes, and then measured at 517 nm using a spectrophotometer.

Concentration (ppm) 5 10 50 100 500 1,000 Positive control group (Vit.C) 48.22% 88.34% 92.95% 93.46% 93.21% 93.04% Example 1 3.94% 7.41% 34.06% 57.6% 90.68% 90.7%

Looking at the measurement results of Table 2, the DPPH radical scavenging ability of Example 1 showed more than 90% from 500 ppm or more. Through this, it was confirmed that the immature strawberry pulp ethanol extract of the present invention had excellent DPPH radical scavenging activity.

(2) Measurement of ABTS radical scavenging ability

ABTS radical scavenging ability was measured according to Nicoletta's method 3). 5 ml of 7 mM ABTS and 88 μl of 140 mM K ₂ S ₂ O ₈ were mixed and reacted in the dark for 14 to 16 hours. This was mixed with ethanol (absolute ethanol) in a ratio of 1:88 to prepare an ABTS solution having an absorbance value of 0.7±0.002 at 734 nm.

150 μl of immature strawberry pulp extract (sample) and 3 ml of ABTS solution were mixed, vortexed for 30 seconds, reacted at room temperature for 2.3 minutes, and absorbance was measured at 734 nm. In addition, the ABTS radical scavenging ability was calculated based on Equation 2 below, and the results are shown in Table 3 and FIG. 2 below.

At this time, distilled water was used as the negative control group, and ascorbic acid (Vit.C) was used as the positive control group.

[Equation 2]

ABTS radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance}}×100%

The absorbance of the sample in Equation 2 was obtained by mixing 150 μl of immature strawberry pulp extract and 3 ml of ABTS solution, vortexing for 30 seconds, reacting at 25 ° C for 3 minutes, and measuring the absorbance at 734 nm. 150 μl and 3 ml of ABTS solution were mixed, vortexed for 30 seconds, reacted at 25° C. for 3 minutes, and absorbance was measured at 734 nm.

Concentration (ppm) 5 10 50 100 500 1,000 Positive control group (Vit.C) 54.16% 100% 100% 100% 100% 100% Example 1 5.6% 11.37% 43.67% 71.36% 94.95% 93.36%

Looking at the measurement results of Table 3, the ABTS radical scavenging ability of Example 1 was 90% or more from 500 ppm or more, and there was no further effect of increasing the ABTS radical scavenging ability at a concentration of 1,000 μg / ml. Through this, it was confirmed that the immature strawberry pulp ethanol extract of the present invention had excellent ABTS radical scavenging activity.

Experimental Example 3: Measurement of cytotoxicity

MTT assay was performed to confirm the toxicity of immature strawberry pulp extract to cells when exposed to cells.

First, after dispensing RAW264.7 cells to 1 × 10⁴ cells/well in a 96-well cell culture medium, samples dissolved in DMSO (dimethyl sulfoxide) were added to suit each concentration after 24 hours. After removing the medium after 24 hours, 40 μl of MTT (2-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide) reagent (in PBS 2.5 mg/ml) was added to the well, and the cell culture medium ( After reacting for 4 hours in the incubator), the supernatant was removed again.

Next, after dispensing 100 μl of DMSO and completely dissolving it, the absorbance was measured at 540 nm using a spectrophotometer, and the results are shown in FIG. 3 .

As a result of measuring the cytotoxicity of RAW264.7, Example 1 (ethanol extract) confirmed that a high cell viability of 90% or more was exhibited at a concentration of 500 μg/ml, and the cell viability was relatively low at 1000 μg/ml, which was weak. indicates cytotoxicity.

Based on these results, the protein expression measurement test of NO, iNOS, and COX-2 was conducted below using the extract having the highest concentration of 500 μg/ml in the range where cell viability is not reduced.

Experimental Example 4: Anti-inflammatory measurement

(1) Measurement of nitric oxide (NO) inhibition

In order to determine whether production of NO, a representative factor of the inflammatory response, was inhibited, RAW 264.7 cells were treated with 10 μg/ml of LPS (Lipopolysaccharide), and then the extract was treated at each concentration after 2 hours. After culturing for 18 hours, the amount of NO in the culture medium was measured, and the results are shown in FIG. 4 .

NO inhibitory activity measurement, the amount of NO in the supernatant (supernatant) of RAW 264.7 cells was measured as nitrite (nitrite) and nitrate (nitrate). A grease reagent, which is a safe form after reduction with nitrate for nitrite, was used, and when the confluence of RAW264.7 cells dispensed at 1 × 10⁴cells/well in a 96-well cell culture medium was 80%, After washing twice with PBS (phosphate buffered saline) and culturing for more than 18 hours using a serum-free medium, the samples were treated by concentration and cultured for 1 hour, treated with 1 μg/ml of LPS (lipopolysaccharide), and then cultured for 24 hours. incubated for hours. The supernatant of the culture was reacted with a grease reagent, and then the absorbance was measured at 540 nm using an ELISA reader, and the NO production rate was expressed as a percentage.

It was confirmed that there was a difference between the LPS-only treatment group and the LPS-untreated group, and when the extract was treated by LPS and concentration, Example 1 (ethanol extract) confirmed that NO production decreased in a concentration-dependent manner. At this time, the NO inhibition ability was measured based on Equation 3 below.

At a concentration of 500 μg/ml, Example 1 confirmed an inhibitory effect of 25.01%.

[Equation 3]

NO inhibition ability (%) = measured NO level of control (%) - measured NO level of extract (%)

The NO inhibition ability of Equation 3 was obtained by treating RAW 264.7 cells with 10 μg/ml of LPS, then treating immature strawberry pulp extract at a certain concentration 2 hours later, incubating for 18 hours, and then measuring the amount of NO in the culture medium, The NO measurement level of the control group is 100%, and the NO measurement level of the extract is a value obtained by converting the measured NO amount to % based on the control group.

(2) Measurement of iNOS and COX-2 expression inhibition by western blot

The anti-inflammatory effect of Example 1 (ethanol extract) on the protein expression levels of iNOS and COX-2, which are mediators of inflammation, was confirmed, and the results are shown in FIG. 5 A (iNOS expression) and B (COX-2 expression) shown in

To measure the protein expression level, RAW264.7 cells were suspended in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (fetal bovine serum), and then cultured in a 6-well cell culture medium so that the cell number was 2×10 ⁵ cells/well. Divided by ml, and cultured for 24 hours in a 37°C, 5% CO ₂ incubator. Next, the cells were cultured by treating the stimulant, LPS, for 2 hours after exchanging with a new DMEM medium, and then treating the cells with strawberry stem extract at each concentration and cultured. After 24 hours, to measure protein expression, the medium was removed, washed with cold PBS, and protein was extracted from the cell lysates by adding Protease & Phosphatase Single-Use inhibitor Cocktail 100X to RIPA buffer. Protein was quantified (BCA protein kit), electrophoresed on 10% SDS-PAGE, transferred to a membrane, and blocked in 5% skim milk in TBST for 2 hours did The primary antibody was diluted (3% skim milk in TBST), overnight at 4°C, and washed three times with TBST (Tris buffered saline with tween 20). Secondary antibodies were measured by incubation at room temperature for 2 hours.

The protein expression of iNOS and COX-2 was confirmed using an enhanced chemiluminescence (ECL) solution (Amersham, Pittsburgh, PA, USA). 1:1000 diluted beta-actin (Santa Cruz Biotechnology, Santa Cruz, California, USA) was used as an internal control (9).

The negative control group (Con) was treated with stimulant LPS alone instead of immature strawberry pulp ethanol extract, and the normal group was not treated with stimulant LPS and immature strawberry pulp extract.

In addition, the ability to inhibit iNOS expression was calculated based on Equation 4 below, and the ability to inhibit COX-2 expression was calculated based on Equation 5.

[Equation 4]

iNOS expression inhibition ability (%) = iNOS expression rate of negative control group (%) - iNOS expression rate of extract (%)

The ability to inhibit iNOS expression in Equation 4 is obtained by measuring the iNOS expression level, the iNOS expression level of the negative control group is 100%, and the iNOS expression level of the extract is the value obtained by converting the measured iNOS expression level to % based on the control group.

[Equation 5]

COX-2 expression inhibition ability (%) = COX-2 expression rate of negative control group (%) - COX-2 expression rate of extract (%)

The ability to inhibit COX-2 expression in Formula 5 is measured by the amount of COX-2 expression, the COX-2 expression rate of the control group is 100%, and the COX-2 expression rate of the extract is based on the measured COX-2 expression level of the control group. It is a value converted to %.

Referring to FIG. 5, RAW 264.7 cells were treated with extracts at concentrations of 10, 50, 100, and 500 μg/ml, and the expression level of each protein was measured. It was confirmed that the protein expression of iNOS and COX-2 was inhibited by 89.65% and 33.56%, respectively.

Through this, it was confirmed that the immature strawberry pulp extract of the present invention had excellent anti-inflammatory properties.

Through the above examples and experimental examples, it was confirmed that the immature strawberry alcohol extract of the present invention has excellent antioxidant and anti-inflammatory effects, and the extract of the present invention is suitable for application to various health functional food materials and / or cosmetics was able to confirm

Simple modifications or changes of the present invention can be easily performed by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (10)

delete delete delete delete delete delete Contains immature strawberry pulp extract as an active ingredient,
The immature strawberry pulp extract is an ethanol aqueous solution extract with a concentration of 65 to 80% by volume,
The immature strawberry pulp extract had a total polyphenol content of 1.50 to 3.50 mg tanninc acid/g,
When the concentration of immature strawberry pulp extract was 1000 ppm, the DPPH radical scavenging activity measured according to the following formula 1 was 90.0 to 95.00%, and the ABTS radical scavenging activity measured according to the following formula 2 was 92.0 to 98.0%,
When the immature strawberry pulp extract had an extract concentration of 500 ppm, the NO inhibition ability measured according to the following formula 3 was 23.0 to 32.0%, and the iNOS expression inhibitory ability measured according to the following formula 4 was 85.0 to 95.0%, and the following formula An antioxidant and anti-inflammatory composition characterized in that the COX-2 expression inhibitory ability measured according to 5 is 31.0 to 40.0%;
[Equation 1]
DPPH radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
The absorbance of the sample in Equation 1 was measured at 517 nm using a spectrophotometer after mixing 3 ml of an ethanol solution containing 60 μM DPPH with 0.5 ml of immature strawberry pulp extract and reacting in the dark for 15 minutes. 0.5 ml of distilled water was mixed with 3 ml of an ethanol solution containing 60 µM DPPH, reacted in the dark for 15 minutes, and then measured at 517 nm using a spectrophotometer.
[Equation 2]
ABTS radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
The absorbance of the sample in Equation 2 was obtained by mixing 150 μl of immature strawberry pulp extract and 3 ml of ABTS solution, vortexing for 30 seconds, reacting at 25 ° C for 3 minutes, and measuring the absorbance at 734 nm. 150 μl and 3 ml of ABTS solution were mixed, vortexed for 30 seconds, reacted at 25° C. for 3 minutes, and absorbance was measured at 734 nm.
[Equation 3]
NO inhibition ability (%) = measured NO level of control (%) - measured NO level of extract (%)
The NO inhibition ability of Formula 3 was measured by treating RAW 264.7 cells with 10 μg/ml of LPS (Lipopolysaccharide), then treating immature strawberry pulp extract at a certain concentration 2 hours later, incubating for 18 hours, and then measuring the amount of NO in the culture medium. The NO measurement level of the control group is 100%, and the NO measurement level of the extract is a value obtained by converting the measured NO amount to % based on the control group.
[Equation 4]
iNOS expression inhibition ability (%) = iNOS expression rate of negative control group (%) - iNOS expression rate of extract (%)
In Equation 4, the negative control group is the iNOS expression rate measured after treatment with the stimulant LPS alone, the iNOS expression rate of the extract is the iNOS expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the negative control group It is a value converted to % as a standard.
[Equation 5]
COX-2 expression inhibition ability (%) = COX-2 expression rate of negative control group (%) - COX-2 expression rate of extract (%)
In Equation 5, the negative control group is the COX-2 expression rate measured after treatment with the stimulant LPS alone, and the iNOS expression rate of the extract is the COX-2 expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the measured COX-2 expression rate. 2 It is a value obtained by converting the expression level to % based on the negative control group. A first step of preparing strawberry powder by washing and drying immature strawberry pulp and then pulverizing it;
A second step of mixing the strawberry powder and 65 to 80% by volume aqueous ethanol solution at a weight ratio of 1:10, followed by stirring at 22 to 27° C. for 18 to 30 hours;
Step 3 of separating the supernatant and the precipitate by filtering the extract obtained in step 2;
Performing a process comprising: performing a vacuum concentration of the supernatant to obtain a concentrate, followed by freeze-drying to obtain an immature strawberry pulp extract;
The above 3 steps are repeated 1 to 3 times,
The immature strawberry pulp extract had a total polyphenol content of 1.50 to 3.50 mg tanninc acid/g,
When the concentration of immature strawberry pulp extract was 1000 ppm, the DPPH radical scavenging activity measured according to the following formula 1 was 90.0 to 95.00%, and the ABTS radical scavenging activity measured according to the following formula 2 was 92.0 to 98.0%,
When the immature strawberry pulp extract had an extract concentration of 500 ppm, the NO inhibition ability measured according to the following formula 3 was 23.0 to 32.0%, and the iNOS expression inhibitory ability measured according to the following formula 4 was 85.0 to 95.0%, and the following formula A method for producing an immature strawberry pulp extract, characterized in that the COX-2 expression inhibitory ability measured according to 5 is 31.0 to 40.0%;
[Equation 1]
DPPH radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
The absorbance of the sample in Equation 1 was measured at 517 nm using a spectrophotometer after mixing 3 ml of an ethanol solution containing 60 μM DPPH with 0.5 ml of immature strawberry pulp extract and reacting in the dark for 15 minutes. 0.5 ml of distilled water was mixed with 3 ml of an ethanol solution containing 60 µM DPPH, reacted in the dark for 15 minutes, and then measured at 517 nm using a spectrophotometer.
[Equation 2]
ABTS radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
The absorbance of the sample in Equation 2 was obtained by mixing 150 μl of immature strawberry pulp extract and 3 ml of ABTS solution, vortexing for 30 seconds, reacting at 25 ° C for 3 minutes, and measuring the absorbance at 734 nm. 150 μl and 3 ml of ABTS solution were mixed, vortexed for 30 seconds, reacted at 25° C. for 3 minutes, and absorbance was measured at 734 nm.
[Equation 3]
NO inhibition ability (%) = measured NO level of control (%) - measured NO level of extract (%)
The NO inhibition ability of Formula 3 was measured by treating RAW 264.7 cells with 10 μg/ml of LPS (Lipopolysaccharide), then treating immature strawberry pulp extract at a certain concentration 2 hours later, incubating for 18 hours, and then measuring the amount of NO in the culture medium. The NO measurement level of the control group is 100%, and the NO measurement level of the extract is a value obtained by converting the measured NO amount to % based on the control group.
[Equation 4]
iNOS expression inhibition ability (%) = iNOS expression rate of negative control group (%) - iNOS expression rate of extract (%)
In Equation 4, the negative control group is the iNOS expression rate measured after treatment with the stimulant LPS alone, the iNOS expression rate of the extract is the iNOS expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the negative control group It is a value converted to % as a standard.
[Equation 5]
COX-2 expression inhibition ability (%) = COX-2 expression rate of negative control group (%) - COX-2 expression rate of extract (%)
In Equation 5, the negative control group is the COX-2 expression rate measured after treatment with the stimulant LPS alone, and the iNOS expression rate of the extract is the COX-2 expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the measured COX-2 expression rate. 2 It is a value obtained by converting the expression level to % based on the negative control group. A health functional food comprising the composition of claim 7. A cosmetic comprising the composition of claim 7.

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