KR100791282B1 - Pimpinella komarovii extracts having whitening activity - Google Patents

Abstract

An extract of Pimpinella komarovii is provided to secure excellent whitening effect and excellent antioxidative and anti-inflammatory activities. And a composition comprising the same as an effective ingredient is provided. An extract of Pimpinella komarovii with whitening activity is characterized in that it is prepared by hot-wind drying washed Pimpinella komarovii at a temperature of 40 deg.C, crushing the dried Pimpinella komarovii, dipping the crushed material of the Pimpinella komarovii in 70% ethanol with agitating for 2-3 days at room temperature, filtering the leachate using a filter to prepare a crude extract, and fractioning the crude extract using n-hexane, ethylacetate, and butanol in sequence. A composition for whitening, improving and preventing inflammatory diseases or antioxidizing comprises the extract of Pimpinella komarovii as an effective ingredient.

Description

Extract of roe deer sprouts showing whitening activity {PIMPINELLA KOMAROVII EXTRACTS HAVING WHITENING ACTIVITY}

1 is a manufacturing process of the roe deer extract of the present invention and the caffeic acid methyl ester separated therefrom

Figure 2 is a graph of HPLC analysis results for roe deer extract of the present invention

Figure 3 is a graph of HPLC analysis results for the roe deer ethyl acetate fraction of the present invention

Figure 4 is a graph of the HPLC analysis results for the roe deer butanol fraction of the present invention

Figure 5 is a graph of HPLC analysis results for a single substance (caffeic acid methyl ester) derived from roe deer sprouts of the present invention.

6 is an LC-MS spectrum of a single substance (caffeic acid methyl ester) derived from roe deer sprout of the present invention.

7 is a 1H-NMR spectrum of a single substance (caffeic acid methyl ester) derived from roe deer sprout of the present invention.

8 is a 13C-NMR spectrum of a single substance (caffeic acid methyl ester) derived from roe deer sprouts of the present invention.

Figure 9 is a H-H COSY spectrum for a single substance (caffeic acid methyl ester) derived from roe deer sprouts of the present invention

10 is a caffeic acid methyl ester structure of roe deer sprouts of the present invention

11 is a graph showing the results of cytotoxicity test of Melan-a cells against roe deer extract of the present invention

12 is a graph showing the results of the melanin synthesis inhibitory to the roe deer extract of the present invention

A: Roe deer yam extract, B: Roe deer yam extract

13 is a graph showing the results of cell tyrosinase inhibitory effect on Melan-a cells to the roe deer extract of the present invention

A: Roe deer yam extract, B: Roe deer yam extract

14 is an anti-inflammatory effect in RAW293.7 cells to the roe deer extract of the present invention

A: cell NO assay result, B: cell MTT assay result

The present invention relates to a roe deer extract extract showing whitening activity.

Human skin color is affected by the presence or absence of pigments such as melanin, hemoglobin or carotenoids produced by melanocytes, which are pigment cells present in the epidermis, and the thickness and reflectivity of the skin.

Among these, melanin pigment, which is the most important factor in determining the skin color, is typified by tyrosinase, an amino acid that is normally present in the human body, and is dopa- ted by tyrosinase, an enzyme in melanocytes. The complex oxidation process leads to melanin, a dark brown polymer.

Melanin synthesis is promoted by factors such as UV exposure, melanoma and hyperpigmentation disease.

In addition, the biosynthesis of melanin pigment is controlled by various enzymes, including tyrosinase enzymes, among which tyrosinase is the initial biosynthesis process of tyrosine (Trosin) as a substrate to L-dopaquinone It acts on the oxidation of dihydroxyindoles.

Therefore, the search for tyrosinase activity inhibitors is an important part of the development of the whitening agent, and as the tyrosinase inhibitors which are still known, hydroquinone, 4-hydroxyanisole, ascorbic acid ( ascorbic acid derivatives, kojic acid, azelaic acid, corticosteroids, retinoids, arbutin, catechin, etc. There is a difficulty in using it as a problem.

Accordingly, in recent years, researches using natural products, rather than artificial substances, in whitening raw materials, functional foods, and functional cosmetics using natural plants have been actively conducted.

In Korean Registered Patent Publication No. 10-0542751 (composition with gompi extract with tyrosinase inhibitory activity or phlotantanins isolated therefrom), gompi extract or a compound of phlorotannins isolated therefrom plays a key role in melanin biosynthesis process It acts on tyrosinase, which is an enzyme, and effectively inhibits its enzymatic activity, and thus, there is disclosed a composition for preventing browning, such as a whitening cosmetic composition or food for preventing melanin overproduction.

In addition, Korean Patent Publication No. 10-0602684 (a cosmetic composition for whitening containing the forget-me-not extract) as an active ingredient, skin whitening that the forget-me-not extract inhibits melanin production in melanocytes, inhibits tyrosinase activity, and also has antioxidant activity. A cosmetic composition for cosmetics is disclosed.

On the other hand, Pimpinella komarovii is a perennial herb belonging to the mountain family family, which grows in Jeju Island, North Korea, Hamgyongnamdo, and East Siberia.

Korean Registered Patent Publication No. 10-0291089 (Method for manufacturing meat and fish odor remover using yam and fish odor remover) extracts yam sprouts extracted with diethyl ether solvent, and dehydrated and filtered with anhydrous sodium sulfate. The use of meat and fish odors as a remover is disclosed.

In addition, Korean Patent Publication No. 10-0624664 (hair cosmetic composition for the control of dandruff growth and scalp itch improvement), extracting fruit juice, thick pepper, and sesame seed with an organic solvent to produce a cosmetic composition with improved scalp itch and dandruff. Is open to the public.

As the above inventions, conventionally, extracts using sesame sprouts ( Pimpinella ) are used as cosmetic compositions for improving scalp itch and dandruff or as deodorants of meat and fish, and bronchial bronchus in some species of Pimpinella The research on physiological activity of Pimpinella komarovii is insufficient, as it is known to be used for medicines, spices, and foods for treating asthma and gastrointestinal disorders, antipyretics, antispasmodics, expectorants, and anti-tussives.

An object of the present invention is to provide a roe deer extract having excellent whitening activity and excellent antioxidant and anti-inflammatory activity.

In addition, an object of the present invention is to provide a composition comprising the roe deer extract of the present invention as an active ingredient.

It is also an object of the present invention to provide a caffeic acid methyl ester separated from the roe deer extract of the present invention, and to provide a composition comprising the separated substance as an active ingredient.

The present invention relates to a roe deer extract extract showing whitening activity.

The roe deer extract of the present invention is dried after rinsing the roe roe sprout at 40 ° C. for 3 days, and then pulverized, and immersing the roe deer pulverized product in 70% ethanol and stirring at room temperature for 2-3 days, The crude extract was prepared by filtration with a filter, and the crude extract was fractionated sequentially using solvents having different polarities, n-hexane, ethyl acetate and butanol, to prepare an extract fraction.

In addition, the present invention consists in preparing a composition comprising the roe deer extract as an active ingredient.

In addition, the present invention consists in preparing a composition comprising the caffeic acid methyl ester isolated from the roe deer extract as an active ingredient.

On the other hand, the roe deer ( Pimpinella komarovii ) of the present invention is a perennial herb belonging to the mountain family, native to Jeju Island, North Korea, Hamgyongnam-do, East Siberia, etc., and the flowers bloom white in August, and the leaves are revived twice. It is a three-lobed lobe, the rosettes die after growth, and the fruit is a narrow, ovoid, flat, hairless plant.

There are currently no studies on Pimpinella komarovii , but some of the species of Pimpinella are known to be used for the treatment of bronchial asthma and gastrointestinal disorders, as a repellent, antifungal, antispasmodic, expectorant, etc. have.

While the inventors of the present invention have studied natural plants having excellent physiological activity such as whitening activity and substances isolated from natural plants, whitening activity such as melanin synthesis inhibitory activity and tyrosinase inhibitory activity in extracts of roe deer sprout and single substance separated therefrom The present invention was completed by knowing that the physiological activity is excellent and excellent in antioxidant and anti-inflammatory activity.

Roe deer yam of the present invention ( Pimpinella) To determine the effect of komarovii ) extract on the cell viability of melan-a cell line, 12.5, 25 ㎍ / mL concentration of crude extract of roe deer sprout when the cell viability of control group was 100%. At 96% and 97% respectively, slightly decreased compared to the control, but 102% at 50 ㎍ / mL concentration and 107% at 100 ㎍ / mL concentration, similar to the control (Fig. 11).

As such, there was a slight difference compared to the control group, but there was no significant change.

In addition, the melanin production amount was measured to measure the whitening activity of the roe deer extract of the present invention, and the inhibition rate against tyrosinase activity was measured.

The crude extract of the present invention was treated with various concentrations up to 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml for 3 days, and then the melanin production was measured. As a control, melazove 30 uM (Pacific Co., Ltd.) showed 77% melanogenesis inhibitory activity, and 11%, 23%, 42 when treated to concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml, respectively. %, 62% showed inhibitory activity (FIG. 12A).

Therefore, the crude extract of roe deer bran showed a tendency to inhibit melanin production as the dose was increased in melan-a cells, and it was statistically significant. The ethyl acetate fraction was 48% in the roe deer extract extract. The highest effect was shown in% (FIG. 12B).

In addition, the inhibition of the final melanin amount after the treatment of the roe deer extract was measured tyrosinase activity in melan-a cells because it indicates that it is related to the activity of enzymes involved in melanogenesis and synthesis.

As a result, it showed 75% tyrosinase inhibitory activity when treated with melazove 30 uM (Pacific Co., Ltd.), and 12.5 ㎍ / mL, 25 ㎍ / mL, 50 ㎍ / mL and 100 ㎍ / mL concentrations of roe deer extract 45%, 59%, 66%, 74% of inhibitory activity, respectively (FIG. 13A).

Therefore, the crude extracts of roe deer sprouts showed a tendency of inhibiting tyrosinase activity with increasing treatment concentration in melan-a cells and showed statistical significance. The extract fraction of roe deer sprouts was 25 ㎍ in ethyl acetate fraction. (74%), 50 ㎍ (78%) showed a high inhibitory effect (Fig. 13B).

In addition, the antioxidant activity of the roe deer extract of the present invention was tested.

The most specific mechanism of antioxidants reacts with free radicals. The free radical scavenging action is used as a measure of antioxidant activity in plants or as a measure of inhibiting aging in the human body by donating electrons to free radicals. DPPH free radical scavenging activity was measured by measurement.

As a result, IC ₅₀ of commercialized ascorbic acid showed 8.23 ㎍ with high active radical inhibition effect.

And showed an IC ₅₀ is low effect 227.32 ㎍ of roe chamnamul crude extract, deer chamnamul ethyl acetate fraction IC ₅₀ is 79.76 ㎍, butanol fraction is the IC ₅₀ 161.33 ㎍ / ㎖) layer is twice or more active oxygen than the crude extract It showed an inhibitory effect.

Inhibition of nitric oxide production of roe deer extract showed that the crude extract, water fraction, ethyl acetate fraction and butanol fraction showed very low inhibition rate, but IC ₅₀ was 123.53 in hexane fraction.

In addition, Xanthine oxidase inhibition and peroxide scavenging activity were measured to show that the xanthine oxidase inhibitory effect was very low in the fraction containing ethanol crude ethanol crude extract, but the peroxide scavenging activity was ₅₀ 30.79 µg / ml, the ethyl acetate fraction is IC ₅₀ at 8.97 µg / ml, butanol fraction IC ₅₀ at 14.03 µg / ml, hexane fraction IC ₅₀ at 151.33 µg / ml, and water fraction at IC ₅₀ > 400 µg / ml. It showed a high inhibitory effect on the ethyl acetate fraction as ml (Table 4).

Therefore, it was found that the roe deer extract of the present invention is excellent in antioxidant activity.

In addition, the anti-inflammatory activity was measured by measuring the cell NO inhibitory effect and cytotoxicity inhibitory effect in RAW264.7cell with respect to the roe deer extract of the present invention, IC ₅₀ value was 75 ㎍ showed a relatively high inhibitory effect (Fig. 14A). ).

In addition, MTT assay showed no cytotoxicity until 100 ㎍ / ㎖ treatment (Fig. 14B).

On the other hand, the single material separated from the roe deer extract of the present invention was confirmed that the molecular weight of 194 through LC-MS (Fig. 6).

Further, in ¹ H-NMR, δ 7.54 (d, J = 15.8 Hz, H-8), 7.02 (d, J = 1.9 Hz, H-2), 6.93 (dd, J = 8.3, 1.9 Hz, H-5 ), 6.76 (d, 8.3 Hz, H-6), 6.25 (d, 15.8, H-7), 3.75 (s, OMe) (FIG. 7), and ¹³ C-NMR is δ 169.93 (C-9), 149.99 (C-4), 147.12 (C-7), 147.06 (C-4), 127.66 (C-1), 123.03 (C-6), 116.59 (C-5), 115.12 (C-2), 114.79 (C-8) and 51.97 (OMe) (FIG. 8), H and CO are Huped 8 and 7, and H and 6H are coupled in HH COSY (FIG. 9).

As a result of the analysis, the material isolated from the roe deer extract of the present invention was identified as Caffeic acid methyl ester (FIG. 10).

Therefore, since a single substance isolated from the roe deer sprout extract of the present invention was found to be caffeic acid methyl ester, it can be separated using a variety of conventional methods when separating the roe deer sprout-derived caffeic acid methyl ester in large quantities.

On the other hand, the roe deer extract of the present invention can be utilized in various ways, such as food additives, feed additives, beverage compositions, cosmetic compositions.

The whitening composition comprising the roe deer extract of the present invention and the caffeic acid methyl ester separated therefrom as an active ingredient can be prepared.

In addition, the present invention can prepare a composition for preventing or improving inflammation or an antioxidant comprising a roe deer sprout extract showing the physiological activity as described above and a caffeic acid methyl ester separated therefrom as an active ingredient.

At this time, the composition of the present invention may be prepared in various forms such as pills, capsules, granules, suspensions.

On the other hand, when preparing a composition containing the roe deer extract of the present invention and the caffeic acid methyl ester separated therefrom as an active ingredient, the addition amount can be variously adjusted according to the use and form of the composition.

Hereinafter, the roe deer extract of the present invention and the material separated therefrom will be described in detail through Examples and Experimental Examples, but these are not intended to limit the scope of the present invention.

<Example 1> roe deer extract extract of the present invention

Pimpinella komarovii , native to Jeju Island, was collected and prepared.

The prepared roe deer starch outpost (全 草) was washed with running water and hot-air dried at 40 ° C. for 3 days.

100 g of dried roe deer outpost were crushed with a grinder, which was immersed in 70% ethanol and stirred at room temperature for 2-3 days.

The leachate was filtered with a filter, and the leaching and filtration of the present invention was prepared by repeating the above leaching and filtration two to three times.

10 g of the crude roe deer extract was dissolved in distilled water, n-hexane was added thereto, fractionated into an n-hexane layer and an aqueous layer, and this was repeated twice to obtain a hexane layer.

Then, ethyl acetate was added to the aqueous layer, the mixture was partitioned into ethyl acetate and the aqueous layer, and then repeated three times to obtain an ethyl acetate layer. Butanol was added to the aqueous layer to separate the butanol layer and the aqueous layer and then repeated three times. To obtain a butanol layer.

The fractions were concentrated under reduced pressure, respectively, to prepare 1.1 g of roe deer hexane fraction, 3.0 g of ethyl acetate fraction, 3.4 g of butanol fraction, and 2.5 g of water fraction.

<Experimental Example 1> Separation experiment of a single substance from the roe deer extract of the present invention

Crude extract and extract fractions of the roe deer sprout prepared in Example 1 of the present invention were prepared.

High performance liquid chromatography (HPLC) analysis was performed on the crude extracts and extracts.

The analysis conditions were as follows.

<Table 1> HPLC apparatus conditions for the analysis of roe deer sprouts

HPLC Waters Alliance 2695 system Column Sunfire ^TM C18 5μm 4.6 × 250mm Detecter Photodiode Array Detector Flow rate 1.0 ml / min Injection Volume 10 μl Column temperature 25 ℃ Sample Temperature 10 ℃

TABLE 2 Mobile phase conditions for HPLC gradient elution

Program order Time Flow H2O Acetonitrile Curve One 1.00 95.0 5.0 2 5.00 1.00 95.0 5.0 6 3 10.00 1.00 90.0 10.0 6 4 30.00 1.00 80.0 20.0 6 5 40.00 1.00 80.0 20.0 6 6 50.00 1.00 70.0 30.0 6 7 60.00 1.00 70.0 30.0 6 8 70.00 1.00 50.0 50.0 6 9 80.00 1.00 50.0 50.0 6 10 81.00 1.00 0.0 100.0 6 11 90.00 1.00 0.0 100.0 6 12 91.00 1.00 95.0 5.0 6 13 100.00 1.00 95.0 5.0 6

As a result, as shown in FIG. 2, the peak 1 shown in 46 components was isolate | separated and described as a reference substance.

It was confirmed that the material was aggregated in the ethyl acetate fraction layer through HPLC chromatography for the roe deer extract and each extract fraction of the present invention (FIG. 3).

However, in the butanol fraction layer, peak 1 was very weak at 46 min RT (FIG. 4).

Thus, the ethyl acetate layer was eluted with Prep-LC for 50 minutes at a solvent H ₂ O / Acetonitril (90/10) with a gradient to obtain pure peak 1.

In addition, single material was analyzed by HPLC to confirm purity (FIG. 5).

Experimental Example 2 Analysis of a Single Substance Isolated from the Roe Deer Sprout Extract of the Present Invention

As a result of negative confirmation using LC-MS (ESI-MS, m / z 193 [MH]) to confirm the molecular weight of the single substance separated in Experimental Example 1, it was confirmed that the molecular weight of the separated substance was 194 (Fig. 6).

In addition, ¹ H-NMR (CD ₃ OD, 400 MHz) was confirmed by NMR analysis. As a result, trans structures were obtained at 7.54 ppm (d, J = 15.8 Hz, H-8) and 6.25 ppm (d, 15.8, H-7). It can be confirmed that it has, 7.02ppm (d, J = 1.9Hz, H-2), 6.93ppm (dd, J = 8.3, 1.9Hz, H-5), 6.76ppm (d, 8.3Hz, H-6 In the benzene ring, 3.75ppm (s, OMe), singlet can be expected to have a form of O atom with a large electronegativity attached to a methyl group (FIG. 7).

¹³ C-NMR (CD ₃ OD, 100MHz) confirmed that the ester group at 169.93ppm (C-9), 149.99ppm (C-4), 147.06ppm (C-3), 127.66ppm (C -1), 123.03ppm (C-6), 116.59ppm (C-5), 115.12ppm (C-2) were predicted as aromatic form (Fig. 8).

In addition, it was confirmed that the C group of the methyl group attached to the O atom at 51.97 ppm (OMe) (Fig. 8).

Thus, in summary, at ¹ H-NMR, δ 7.54 (d, J = 15.8 Hz, H-8), 7.02 (d, J = 1.9 Hz, H-2), 6.93 (dd, J = 8.3, 1.9 Hz, H-5), 6.76 (d, 8.3 Hz, H-6), 6.25 (d, 15.8, H-7), 3.75 (s, OMe), and ¹³ C-NMR is δ 169.93 (C-9), 149.99 (C-4), 147.12 (C-7), 147.06 (C-4), 127.66 (C-1), 123.03 (C-6), 116.59 (C-5), 115.12 (C-2), 114.79 ( C-8), 51.97 (OMe).

In H-H COSY it was confirmed that 8 H and 7 H are coupling, and 5 H and 6 H are coupling (FIG. 9).

Therefore, summarizing the above results, as shown in Table 3 below, the material separated from the roe deer extract of the present invention was identified as caffeic acid methyl ester (Caffeic acid methyl ester) (FIG. 10).

On the other hand, the above substance was confirmed in the same as the results reported by the ivy (Parthenocissue tricuspidata) (Saleem et al, 2004) Caffeic acid methyl ester (Caffeic acid methyl ester).

<Table 3> NMR Analysis Result Data

C H One 127.66 2 115.12 7.02 (1H, d , 1.9 Hz) 3 147.06 4 149.99 5 116.59 6.93 (1H, dd , 8.3,1.9 Hz) 6 123.03 6.76 (1H, d , 8.3 Hz) 7 147.12 6.25 (1H, d , 15.8 Hz) 8 114.79 7.54 (1H, d , 15.8 Hz) 9 169.93 O-Me 51.97 3.75 (3H, s )

Example 2 Mass Production of Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

The roe deer crude crude extract prepared by the method of Example 1 of the present invention was prepared.

In order to separate a large amount of caffeic acid methyl ester derived from roe deer sprouts, sephadex LH-20 was first charged into a glass column and 1 kg of the crude extract was added.

Methanol / dichloromethane (30/70) was flowed here and it isolate | separated into the molecular weight (194) size of the caffeic acid methyl ester.

Thereafter, 5 g of the caffeic acid methyl ester material was separated by flowing the developing solvent methanol and dichloromethane at a ratio of 1: 3 for 100 minutes by using medium pressure liquid chromatography (MPLC).

<Example 3> Manufacture of pills using roe deer extract

After the lyophilized roe deer extract extract prepared by the method of Example 1 of the present invention was prepared as a powder.

200 g of water and 100 g of honey were put in 1 kg of the prepared powder, kneaded, put into a pill manufacturing apparatus to make a ring shape, and then put into a drier and dried at 30 ° C. for 12 hours to prepare a ring.

Example 4 Preparation of Capsules Using Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

The caffeic acid methyl ester isolated by the method of Example 2 of the present invention was prepared.

Capsules were prepared in a conventional manner using the prepared material.

Example 5 Preparation of a Beverage Composition Containing Extract of Roe Deer Sprouts

A roe deer extract was prepared by the method of Example 1 of the present invention.

20 g of roe deer extract, 1 g of vitamin B2, 6 g of nicotinic acid amide, 10 g of citric acid amide, 30 g of citric acid, 2 kg of liquid fructose, and 70 ml of herbal flavor were added to the extract of the roe deer extract of the present invention. Prepared beverage composition.

Example 6 Preparation of a Beverage Composition Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

The roe deer sprout-derived caffeic acid methyl ester isolated by the method of Example 2 of the present invention was prepared.

10 g of prepared substance per 1 liter of purified water, 4 g of vitamin B2, 6 g of vitamin B6, 10 g of nicotinic acid amide, 30 g of citric acid, 2 kg of liquid fructose, and 70 ml of herb flavor were added to the caffeic acid methyl ester derived from roe deer sprout of the present invention. A beverage composition containing was prepared.

<Example 7> Preparation of the whitening cosmetic composition containing the roe deer extract

1. Preparation of skin composition for whitening

A roe deer extract was prepared by the method of Example 1 of the present invention.

In preparing a conventional skin composition, 2% by weight of the extract was added to prepare a skin composition for whitening.

2. Manufacture lotion composition for whitening

A roe deer extract was prepared by the method of Example 1 of the present invention.

In preparing a conventional lotion composition, 2 wt% of the extract was added to prepare a lotion composition for whitening.

Example 8 Preparation of a Whitening Cosmetic Composition Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

1. Cream for whitening

A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

In preparing a conventional cream composition, 2 wt% of the above material was added to prepare a cream composition for whitening.

2. Preparation of whitening essence composition

A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

In preparing a typical essence composition, 1 wt% of the above material was added to prepare a whitening essence composition.

Example 9 Preparation of Cosmetic Composition for Amelioration and Treatment of Inflammatory Disease Containing Roe Deer Fructus Extract

1. Preparation of essence composition for improvement and treatment of inflammatory disease

A roe deer extract was prepared by the method of Example 1 of the present invention.

In preparing a typical essence composition, 2% by weight of the extract was added to prepare an essence composition for improving and treating inflammatory diseases.

2. Preparation of lotion composition for improving and treating inflammatory diseases

A roe deer extract was prepared by the method of Example 1 of the present invention.

In preparing a conventional lotion composition, the extract was added 3% by weight to prepare a lotion composition for improving and treating inflammatory diseases.

Example 10 Preparation of Cosmetic Composition for the Improvement and Treatment of Inflammatory Disease Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

1. Preparation of cream for improving and treating inflammatory diseases

A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

In preparing a typical cream composition, 3 wt% of the above substance was added to prepare a cream composition for improving and treating inflammatory diseases.

2. Preparation of essence composition for improvement and treatment of inflammatory disease

A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

In preparing a typical essence composition, by adding 3% by weight of the above substances to prepare an essence composition for the improvement and treatment of inflammatory diseases.

Example 11 Preparation of a Food Additive Containing Roe Deer Sprout Extract

The roe deer sprout extract prepared by the method of Example 1 of the present invention was prepared as a powder after lyophilization.

2 wt% of the above powder was used to prepare a conventional food.

Example 12 Preparation of a Food Additive Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

2 wt% of the above-mentioned substance was used to prepare a conventional food.

Example 13 Preparation of Feed Additives Containing Roe Deer Sprout Extract

The roe deer sprout extract prepared by the method of Example 1 of the present invention was prepared as a powder after lyophilization.

2 wt% of the above powder was added to a conventional livestock feed.

Example 14 Preparation of a Feed Additive Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

Caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

2 wt% of the above material was added to a conventional livestock feed.

<Experiment 3> Cytotoxicity test on the roe deer extract of the present invention

1. Material Preparation

The melan-a cells used in the present invention are cells derived from the embryo of C57BL as a non-tumorigenic mouse melanocyte cell line of non-tumorigenic mice.

melan-a cells were treated with PRMI containing 10% fetal bovine serum (FBS, Gibco), 1% Antibiotic-Antimycotic (Gibco), 100nM TPA, L-glutamine and sodium bicarbonate 37 ° C., 10% CO ₂ using 1640 medium (Gibco) Cultured in a cell culture phase.

2. Cytotoxicity Experiment

Roe deer yam of the present invention ( Pimpinella) To evaluate the effect of komarovii ) extract on cell viability of melan-a cell line, cytotoxicity experiments were performed.

Inoculate melan-a cells in 24 well plates at 1 × 10 ⁵ cells per well and for 24 hours at 37 ° C., 10% CO ₂ After cultivation in the cell incubator, the roe deer extract was incubated for 3 days at the concentration of 12.5 ㎍ / ㎖, 25 ㎍ / ㎖, 50 ㎍ / ㎖ and 100 ㎍ / ㎖ in each well.

200 μl of the MTT solution prepared at a concentration of 2 mg / ml PBS was added thereto, followed by incubation for 4 hours under the same culture conditions.

The culture solution was removed, and 200 μl of DMSO was added to each well, and the absorbance was measured at 570 nm with an ELISA reader (μQuant, USA).

As a result, as shown in FIG. 9, when the cell viability of the control group was 100%, 12.5 and 25 µg / ml concentrations of crude extracts of the roe deer were 96% and 97%, respectively, slightly decreased compared to the control group. 102% at μg / ml concentration and 107% at 100 μg / ml concentration were similar to the control group.

As such, there was a slight difference compared to the control group, but there was no significant change.

Experimental Example 4 Melanin Biosynthesis Inhibitory Activity of the Roe Deer Sprout Extract of the Present Invention

Melan-a cell line synthesizes melanin as melanocytes derived from mouse.

The crude roe deer extract of the present invention was treated with various concentrations up to 12.5 µg / ml, 25 µg / ml, 50 µg / ml and 100 µg / ml for 3 days, and then the melanin production was measured.

Inoculate melan-a cells in 24 well plates at 1 × 10 ⁵ cells per well and for 24 hours at 37 ° C., 10% CO ₂ After culturing in a cell culture medium, the roe deer extracts were sampled at concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml in each well for 3 days, and then the medium was removed. Washed twice.

500 μl of 1N NaOH was added to each well and dissolved at 56 ° C. for 30 minutes, and then absorbance was measured at 405 nm with an ELISA reader (μQuant, USA).

As a result of the control, melazove 30 uM (Pacific Pacific) treated group showed 77% melanin inhibitory activity, and treated to 12.5 ㎍ / mL, 25 ㎍ / mL, 50 ㎍ / mL and 100 ㎍ / mL, respectively. 11%, 23%, 42%, and 62% of the cells showed inhibitory activity (Fig. 10A).

Therefore, the extract of roe deer yam extract showed a tendency to inhibit melanin production as the dose was increased in melan-a cells, and it was statistically significant.

Melanin inhibitory effect of the fraction showed the highest effect of the ethyl acetate fraction 48% (Fig. 10B).

Experimental Example 5 Experimental Study of Intracellular Tyrosinase Inhibitory Activity of the Roe Deer Fructus Extract of the Present Invention

Inhibition of the final melanin amount after the crude extract of roe deer sprouts was related to the activity of enzymes involved in melanin synthesis. Therefore, tyrosinase activity was measured in melan-a cells.

Inoculate melan-a cells in a 24 well plate at 1 × 10 ⁵ cells per well, 37 ° C., 10% CO ₂ for 24 h. After culturing in a cell incubator, the roe deer sprout extract was treated to each well at concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml, and then the cells of the well were centrifuged to obtain cell precipitates. Lysis buffer (0.1 M sodium phosphate buffer, 0.2 mM PMSF, 1% Triton X-100) was added.

After standing on ice to destroy cells and centrifugation, the supernatant was taken and used for enzyme activity measurement.

Each sample was added to the reaction solution (12.5 mM L-Dopa, 1.5 mM L-Tyrosine, 67 mM sodium phosphate buffer (pH6.8)) and reacted at 37 ° C for 1 hour, followed by absorbance at 405 nm using an ELISA reader. Measured.

As a result, it showed 75% tyrosinase inhibitory activity when treated with melazove 30 uM (Pacific Co., Ltd.), and 12.5 ㎍ / mL, 25 ㎍ / mL, 50 ㎍ / mL and 100 ㎍ / mL concentrations of the roe deer extract were treated. 45%, 59%, 66%, 74% of inhibitory activity, respectively (FIG. 11A).

Therefore, the crude extracts of roe deer were found to have a tendency of inhibiting tyrosinase activity with increasing treatment concentration in melan-a cells and showed statistical significance.

In addition, the cell tyrosinase inhibitory effect on the extract fraction of the roe deer sprout of the present invention showed a high inhibitory effect of 25 ㎍ (74%), 50 ㎍ (78%) in the ethyl acetate fraction (Fig. 11B).

Experimental Example 6 Anti-inflammatory Activity Test of Roe Deer Sprout Extract of the Present Invention

The cell NO inhibitory effect and cytotoxicity inhibitory effect in RAW264.7 cells were measured for the roe deer extract of the present invention.

Raw264.7 cell culture was maintained by adding 10% FBS, 1% streptomycin / penicillin (stereptomycine / penicillin) to DMEM medium.

Cell concentration 1.5 × 10 ⁵ cell / ml was seeded in a 24 well plate and incubated for 18 hours.

The sample was incubated for 24 hours after 100 μg / ml and 1 μg / ml of LPS (lipopolysaccaride).

NO production was measured by mixing 100 μl of Griess reagent (1% sulfanilamide, 0.1% naphylethylene diamine in 2.5% phosphoric acid) and 100 μl of the culture solution for 10 minutes.

And absorbance was measured using an ELISA reader at 540 nm.

As a result, the IC ₅₀ value was 75 µg, which showed a relatively high inhibitory effect.

In addition, MTT assay showed no cytotoxicity until 100 ㎍ / ㎖ treatment.

Experimental Example 7 Antioxidant Activity of the Roe Deer Sprout Extract of the Present Invention

1. DPPH radical scavenging activity

The most specific mechanism of antioxidants is the reaction with free radicals. The free radical scavenging action is used as a measure of the antioxidant effect in plants by inhibiting aging in the human body by donating electrons to free radicals.

Electron donating ability was measured by DPPH free radical scavenging method by Blosis method.

First, 100 μl of various concentrations of samples dissolved in methanol were dispensed into 96 well plates, and 0.4 mM DPPH solution was added in the same amount to react for 10 minutes at room temperature, and then measured at 517 nm.

At this time, ascorbic acid (ascrbic acid), trolox (trolox) was used as a control.

DPPH radical scavenging activity was calculated from the following equation, and the absorbance of DPPH was expressed as the concentration of the 50% sample (IC ₅₀ ).

DPPH radical scavenging activity (%) = (A _control -A _sample ) / A _control × 100

A _sample = absorbance of the reaction solution to which the sample was added

A _control = absorbance of reaction solution with methanol instead of sample

As a result, IC ₅₀ of commercialized ascorbic acid showed 8.23 ㎍ with high active radical inhibition effect.

And showed an IC ₅₀ is low effect 227.32 ㎍ of roe chamnamul crude extract, deer true herbs ethyl acetate fraction IC ₅₀ is 79.76 ㎍, butanol fraction is the IC ₅₀ layer 161.33 ㎍ / ㎖) is twice as active than the crude extract It showed oxygen suppression effect.

2. Measurement of Nitirc oxide scavenging activity

Nitrogen compounds are highly cytotoxic and when a large amount of NO is produced, they cause indirect effects such as nitrozation and nitration and oxidative reactions, resulting in harmful effects.

In this experiment, the inhibitory activity of nitric oxide production of the roe deer extract of the present invention was measured using qucertin as a control.

The NO scavenging activity was measured by measuring the amount of nitrite using sodium nitroprusside (SNP) that produces nitric oxide.

Samples were added to 200 μl of 10 mM SNP solution by concentration, and reacted at 25 ° C. for 3 hours. Then, the same amount of Griess reagent (2.5% phophoric acid, 1% sulfanilamide, 0.1% naphylethylendiamine) was added for 10 minutes Absorbance was measured at 540 nm.

Nitric oxide scavenging activity was expressed as the concentration of the sample (IC ₅₀ ) that appears when the absorbance was reduced by 50%.

As a result, the inhibition rate was very low in the crude extract, water fraction, ethyl acetate fraction, butanol fraction, but the IC ₅₀ was 123.53 in the hexane fraction.

3. Xanthine Oxidase Inhibition and Peroxide Scavenging Activity

The production of uric acid by xantihine / xanthine oxidase was measured by increased absorbance at 290 nm.

The amount of superoxide was measured by the nitroblue tetrazolium (NBT) reduction method.

The reaction solution (0.5mM xanthine, 200 mM phosphate buffer (pH7.5) 1mM EDTA) was added to 50mU / ml xanthine oxidase to induce the production of uric acid (uric acid).

Superoxide scavenging activity was reacted by adding 0.5mM NBT to the reaction solution.

Xanthine oxidase inhibition and superoxide scavenging activity were expressed as the concentration of the sample (IC ₅₀ ) which appeared when the absorbance of the produced uric acid and peroxide decreased by more than 50%.

As a result, the inhibitory effect of xanthine oxidase was very low in the fraction containing the ethanol extract of roe deer ethanol.

However, peroxide scavenging activity deer chamnamul crude extract of IC ₅₀ is 30.79 ㎍ / ㎖, ethyl acetate fraction IC ₅₀ is 8.97 ㎍ / ㎖, IC ₅₀ of the butanol fraction was 14.03 ㎍ / ㎖, IC ₅₀ of the hexane fraction was 151.33 ㎍ / Ml, water fraction showed a high inhibitory effect on the ethyl acetate fraction as IC ₅₀ > 400 μg / ml.

<Table 3> Antioxidant Activity Test Results of Roe Deer Sprout Extract and Extract Fraction

division DPPH (IC ₅₀ ) NO (IC ₅₀ ) Xanthine oxidase inhibitory activity (IC ₅₀ ) Peroxide Scavenging Activity (IC ₅₀ ) Crude extract 227 > 400 - 30.79 Hexane fraction > 400 123.52 219 151.33 Ethyl acetate fraction 79.7 > 400 125.78 8.97 Butanol fraction 161.33 > 400 187.93 14.03 Water fraction > 400 13.53 > 400 > 400 Control Asco 8.23 Qucertin 13.53 - Allo 0.028

According to the present invention, a roe deer extract having excellent whitening activity and excellent antioxidant and anti-inflammatory activity is provided.

In addition, there is provided a composition comprising the roe deer extract of the present invention as an active ingredient.

In addition, there is provided a caffeic acid methyl ester separated from the roe deer extract of the present invention, a composition comprising the separated substance as an active ingredient.

Claims (7)

Roe deer roe extract, which has whitening activity, characterized in that it is extracted from Pimpinella komarovii . The method of claim 1, The roe deer extract is hot-dried at 40 ° C. for 3 days after washing the roe deer sprout, and then pulverized. The roe deer crushed powder is immersed in 70% ethanol and stirred for 2 to 3 days at room temperature, followed by filtration. The crude extract is prepared by filtration, and the crude extract is sequentially fractionated using n-hexane, ethyl acetate, butanol, which are solvents having different polarities, to prepare an extract fraction. A roe deer extract showing whitening activity. The method of claim 1, Roe deer bran extract is characterized by showing melanin biosynthesis inhibitory activity or tyrosinase inhibitory activity, A roe deer extract showing whitening activity. Roe deer Pimpinella komarovii ) Whitening composition comprising the extract as an active ingredient. Roe deer Pimpinella komarovii ) A composition for improving and preventing inflammatory diseases comprising an extract as an active ingredient. Roe deer Pimpinella komarovii ) Antioxidant composition comprising the extract as an active ingredient. Roe deer Pimpinella komarovii ) Whitening composition comprising caffeic acid methyl ester isolated from the extract as an active ingredient.

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