KR100556187B1 - Food composition comprising extract of Rosa multiflora with antioxdative activity - Google Patents

Abstract

The present invention relates to a food composition comprising an extract of Rosa multiflora having antioxidant activity. Specifically, the present invention relates to a food composition containing procyanidin B3 and comprising brier extract having antioxidant activity. Brier extract according to the present invention and a composition comprising the same can be very useful for the treatment or prevention of diseases caused by free radicals, maintaining the quality of food and preventing damage due to oxidation of the skin.

Brier, underground, antioxidant activity, extract

Description

Food composition comprising extract of Rosa multiflora with antioxdative activity

Figure 1a shows a hydrogen nuclear magnetic resonance spectrum of the active ingredient showing antioxidant activity in the extract of the root of the brier.

Figure 1b shows the carbon nuclear magnetic resonance spectrum of the active ingredient showing antioxidant activity in the extract of the Brier root.

Figure 2a shows the result of the thin film chromatography of the brier extract according to the present invention and color development with 10% sulfuric acid (1: acetone extract, 2: water-soluble ethanol extract, 3: ethyl acetate fraction, 4: butanol fraction, 5 : Water fraction, 6: procyanidin B3, arrow indicates where procyanidin B3 is identified).

Figure 2b shows the result of spraying the 1,1-diphenyl-2-picrylhydrazyl (1,1-diphenyl-2-picrylhydrazyl, DPPH) solution after thin film chromatography of the brier extract according to the present invention (1: acetone extract, 2: water soluble ethanol extract, 3: ethyl acetate fraction, 4: butanol fraction, 5: water fraction, 6: procyanidin B3, arrow is the part where procyanidin B3 is identified).

Figure 2c shows the thin layer chromatography results for the fraction obtained by chromatography of the brier root acetone extract according to the present invention (arrow is the part where procyanidin B3 is confirmed).

The present invention relates to an extract of Rosa multiflora having antioxidant activity, a method for preparing the same, and a composition comprising the same. Specifically, the present invention provides a method for preparing a brier extract, which contains procyanidine B3 and has an antioxidant activity, extracts the basement of brier with an organic solvent, and separates and purifies by chromatography. It relates to a composition comprising an antioxidant activity.

Antioxidant activity refers to an activity that prevents the production of free radicals in vivo and prevents oxidative phenomena that cause irreparable damage to cells. The stable oxygen (triplet oxygen) is a highly reactive reactive oxygen such as superoxide radicals, hydroxy radicals, hydrogen peroxide due to enzymatic system, reducing metabolism, chemicals, pollutants, and photochemical reactions Oxygen Species (ROS), which irreversibly destroy cellular components. The action of these free radicals is antioxidant enzymes such as superoxide dismutase (SOD), catalase, peroxidase, and vitamin C, vitamin E, glutathione ( It can be minimized by the action of antioxidants such as glutathione). However, if the biological defenses are abnormal or exposed to excessive free radicals, the balance is broken and the free radicals irreversibly destroy lipids, proteins, DNA, and the like. As a result, various diseases such as aging, cancer, multiple atherosclerosis, arthritis and Parkinson's disease are caused.

Synthetic antioxidants developed so far include butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT) and nordihydro-guaiare tic acid (NDGA), and natural antioxidants include superoxide dismutase, peroxidase, catalase, Antioxidant enzymes such as glutathione peroxidase and non-enzymatic antioxidants such as tocopherol (vitamin E), vitamin C (Vitamin C, ascorbic acid), carotenoids and glutathione.

However, synthetic antioxidants are toxic in vivo and can cause allergies and tumors. On the other hand, natural antioxidants have the advantage of being safe to the living body compared to synthetic antioxidants, but the disadvantage is that the effect is weak. Therefore, there is an urgent need for the development of new natural antioxidants that are excellent in antioxidant activity and safer.

Meanwhile, procyanidin, which is known to be safe and excellent in antioxidant activity among natural antioxidants developed to date, is one of proanthocyanidins present in a wide range of plants. Proanthocyanidins are a type of condensed tannins that are condensed using flavan-3-ol or flavan-3,4-diol as structural units. Refers to a group of compounds bound by polymerization. Among these, procyanidins are catechin, epicatechin, catechin gallate, epicatechin gallate, epilocating gallate, gallocatechin gallate, or epigallocatechin gallate. It is a generic name for oligomers and polymers of dimers or more having a backbone as a basic skeleton. Procyanidins are classified into several types according to their basic backbone and binding forms. For example, procyanidin B3 has the structure of catechin- (4α → 8) -catechin, and procyanidin B1 has the structure of epicatechin- (4β → 8) -catechin. Procyanidin B2, epicatechin - (4β → 8) - has the structure of epicatechin, procyanidin B5 is epicatechin - (4β → 6) -. .. Has the structure of epicatechin (Walter Feucht et al, Z Naturforsch, 54c, 942 -945, 1999).

Procyanidins can be obtained by solvent extraction from various kinds of plants or by separation and purification by chromatography, and are known to be separated from wine or beer. Literature ( Vigna angularis Ohwi et Ohashi) (Toshiaki Ariga et al., Agricultural Biological Chemistry , Vol. 45, 2709-2712, 1981), Loblolly pine (RW Hemingway et al., Phytochemistry , Vol. 22) , 275-281, 1983) and at the root of Polygonum multiflorum (Nonaka et al., Phytochemistry , Vol. 21, 429-432, 1982), procyanidins were isolated by solvent extraction and chromatography.

Procyanidins have the ability to bind proteins and have been reported to have anti-inflammatory effects. In particular, catechin, known as green tea tannin, has been reported to have excellent anticancer effects. U.S. Patent No. 4797421 discloses the antioxidant effect of Procyanine and U.S. Pat.No.5646178 discloses the antibacterial effect of Procyanidin obtained from cranberry extract belonging to the genus Vaccinium sp. Korean Patent Laid-Open No. 2001-10154 discloses a medicament using procyanidine as an active ingredient for the prevention and treatment of diseases such as wounds, cancer metastasis, periodontal disease, and arthritis related to matrix metalloproteinases. Korean Patent Laid-Open Publication No. 1998-702533 discloses an anti-obesity agent using procyanidin isolated from tamarind seed extract as an active ingredient.

However, the antioxidant effect of the brier extract has not been reported so far, and it has not been reported that the brier extract contains procyanidins.

Therefore, the present inventors discovered a new fact that the extract extracted from the underground part of the brier ( Rosa mulgiflora) with an organic solvent while studying a new natural antioxidant has an antioxidant activity and the extract contains a procyanidin B3 compound. The present invention has been completed by preparing a brier extract having antioxidant activity and a composition comprising the extract.

Accordingly, it is an object of the present invention to provide a brier extract having antioxidant activity obtained by extracting a brier ( Rosa multiflora ) basement with an organic solvent.

Another object of the present invention (a) extracting by immersing the basement of the brier in an organic solvent; And (b) separating and purifying the extract of step (a) by chromatography to provide a method for producing a brier extract having antioxidant activity, characterized in that it comprises a step of purification.

Still another object of the present invention is to provide a composition comprising the brier extract and having an antioxidant activity.

In order to achieve the above object, the present invention provides a brier extract having antioxidant activity obtained by extracting the brier ( Rosa multiflora ) basement with an organic solvent.

In order to achieve another object of the present invention, the present invention comprises the steps of (a) extracting by immersing the base of the brier in an organic solvent; And (b) separating and purifying the extract of step (a) by chromatography.

In order to achieve another object of the present invention, the present invention provides a composition comprising the brier extract and having an antioxidant activity.

Hereinafter, the present invention will be described in detail.

Rosa multiflora is a deciduous shrub of the family Rosaceae and is widely distributed in foothills and river basins throughout the country. In oriental medicine, fruit is used as a drug called Youngsil, and it is known to be effective in insomnia, forgetfulness, decreased sexual function, and edema. It is also used as a diuretic.

The brier extract with antioxidant activity according to the present invention can be obtained by extracting the brier basement with an organic solvent. As the organic solvent, ethyl acetate, acetone, ethanol, methanol, water, and a mixed solvent thereof can be used. Preferably acetone is used. In the case of using acetone as an extraction solvent, there is an advantage that an extract containing a large amount of antioxidant active material can be obtained without further solvent fractionation as compared with the case of using water-soluble ethanol. On the other hand, in the case of using the water-soluble ethanol as the extraction solvent as a whole has the advantage of high extraction yield, a large amount of the material is too high polarity is extracted. Therefore, in order to facilitate the use of chromatography in the subsequent purification process, an ethanol extract was obtained, and then ethyl acetate and water were added to obtain an ethyl acetate fraction and a water fraction. Butanol was added to the water fraction to add water fraction and butanol. The step of obtaining fractions is further carried out. Thus, the extract extracted using ethanol or water-soluble ethanol as the extraction solvent has the advantage that it is safe to use as a food raw material. In one embodiment of the present invention, the extract of the brier root with water-soluble ethanol, and then distilled water and ethyl acetate added to the ethanol extract fractionated twice into water and ethyl acetate layer and added twice butanol to the water layer and butanol layer By fractionation, an ethyl acetate fraction, butanol fraction, and water fraction were obtained, respectively. In each of the obtained fractions, all of the active ingredients were present, but the ethyl acetate fraction was identified as the fraction having the highest antioxidant activity and easy separation of the substances. This was confirmed by the development on the TLC plate.

The ratio between the basement of the brier and the extraction solvent during extraction is not particularly limited, but the extraction solvent is applied to 1 g of the live weight of the basement of the brier. It can be used 1 to 20 times (by weight). In order to increase the extraction efficiency, it can be repeated twice using an extraction solvent 3 times to 10 times (by weight) with respect to 1 g of live weight of the brier underground.

The extraction temperature is preferably carried out at room temperature under atmospheric pressure, the extraction time depends on the extraction temperature, but 1 to 48 hours, preferably Extract from 12 hours to 24 hours. In addition, the extraction efficiency can be further increased when stirring with a shaker (shaker) during extraction.

The brier base used for extraction may use brier roots or its dry matter, and to increase the extraction efficiency, a brittle root or its dry matter may be crushed by a grinder.

The antioxidant activity of the brier extract according to the present invention prepared by the above method was investigated by antioxidant activity measurement method (Korean Society of Food and Nutrition , Food and Nutrition Experiment Handbook , 651p, 2000; Blois, MS, Nature , 4617: 1198, 1958). As a result, it was confirmed that the BHA, which is a conventional synthetic antioxidant, or tocopherol, which is a natural antioxidant, was relatively high.

Furthermore, according to the present invention, an active fraction exhibiting antioxidant activity can be obtained by separating and purifying the brier extract obtained above by chromatography. Chromatography is preferably performed once or twice using silica gel column chromatography. The active fraction obtained by silica gel column chromatography can be further subjected to ODS column chromatography to obtain a more purely purified active fraction. When the chromatography is carried out as a developing solvent may be used a mixed solvent of chloroform and methanol or a mixture of methanol and distilled water. The flow rate of the developing solvent is 5 ml / hour to 1000 ml / hour, preferably 20 ml / hour to 500 ml / hour. The fraction eluted in the chromatography may be collected in a test tube of 5 ml to 100 ml, and the active fraction showing the antioxidant activity may be obtained by measuring the antioxidant activity of the collected fraction. Antioxidant activity can be measured according to a method known in the art (Korea Food and Nutrition Society, Food and Nutrition Experiment Handbook, 651p, 2000; Blois, M. S., Nature, 4617: 1198, 1958). According to one embodiment of the present invention, the antioxidant activity of the obtained active fraction was measured, and 2.7 times and 3.8 times stronger than the antioxidant activities of the synthetic antioxidant BHA and the natural antioxidant α-tocopherol, respectively.

The present inventors investigated the physicochemical properties of the active fraction separated and purified by chromatography using hydrogen and carbon nuclear magnetic resonance spectra. As a result, it was confirmed that it is a procyanidine B3 compound having a catechin- (4α → 8) -catechin structure represented by the following formula. Therefore, the brier extract according to the present invention is characterized in that it comprises procyanidin B3 as its active ingredient.

In addition, the present invention (a) extracting by immersing the basement of the brier in an organic solvent; And (b) separating and purifying the extract of step (a) by chromatography.

In the step (a), the organic solvent may be acetone or ethanol as described above. When ethanol is used, obtaining an ethanol extract and then adding ethyl acetate and water to the ethanol extract to obtain an ethyl acetate fraction and a water fraction, and adding butanol to the water fraction to obtain a water fraction and a butanol fraction. Additionally. At this time, the extraction conditions are as described above.

In step (b), the chromatography may be sequentially subjected to silica gel column chromatography and ODS column chromatography, and the chromatography conditions are as described above.

In order to achieve another object of the present invention, the present invention provides a composition comprising the brier extract and having an antioxidant activity. More specifically, the present invention provides a pharmaceutical composition of the brier extract of the present invention as an active ingredient and having antioxidant activity.

The brier extract included in the pharmaceutical composition according to the present invention is preferably purified through column chromatography, but may also include an extract obtained by a simple solvent extraction method.

The pharmaceutical composition of the present invention can be administered orally or parenterally. Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, Intestinal, sublingual or topical administration is possible.

For such clinical administration, the pharmaceutical compositions of the present invention can be formulated into suitable formulations using known techniques. For example, in oral administration, it may be mixed with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets. For oral administration, the active compounds can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. In addition, various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques.

The effective amount of the brier extract contained in the pharmaceutical composition of the present invention is 0.1 mg / kg to 500 mg / kg. Preferably it is 1mg / kg to 100mg / kg, it may be administered once to 5 times a day. However, the final dose and frequency of administration may be adjusted to the required range according to the clinical judgment in consideration of the condition, age, etc. of the patient.

The pharmaceutical composition according to the present invention can be usefully used for the treatment or prevention of diseases caused by oxidation of cellular components by free radicals. Such diseases include, but are not limited to, aging, cancer, multiple atherosclerosis, arthritis, and Parkinson's disease, for example.

The present invention also provides a food composition comprising the brier extract of the present invention and having an antioxidant activity. Brier extract according to the present invention is excellent in antioxidant activity and can effectively prevent the smell or flavor of food caused by oxidation, rancidity of oil and fat, and discoloration of food. Accordingly, the brier extract can be used to preserve these foods or to maintain the freshness and quality of foods for a long time by blending them with various conventional foods.

The term foodstuffs includes not only typical foods, but also beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. hams). , Sausage cornbeans, etc., breads and noodles (e.g. udon, soba, ramen, spagate, macaroni, etc.), fruit juices, various drinks, cookies, candy, dairy products (e.g. butter, cheese), edible vegetable oils , Margarine, vegetable protein, retort foods, frozen foods and various seasonings (eg, miso, soy sauce, sources, etc.).

In addition, the present invention provides a cosmetic composition having an antioxidant activity comprising the brier extract of the present invention. The cosmetic composition of the present invention may be used as it is, or diluted as needed. The brier extract may be prepared in liquid or solid form using bases, adjuvants and additives commonly used in the cosmetic field. Cosmetics in liquid or solid form may include, but are not limited to, for example, cosmetics, creams, lotions, baths and the like.

Bases, adjuvants and additives conventionally used in the cosmetic field are not particularly limited and may include, for example, water, alcohols, propylene glycol, stearic acid, glycerol, cetyl alcohol and liquid paraffin.

The cosmetic composition according to the present invention is very useful for preventing damage caused by oxidation of the skin, for example, spots (brown spots), freckles, skin cracks, ultraviolet damage (sunburn), etc., and prevents oxidation of the cosmetics themselves. This can be very useful for maintaining the quality of cosmetics.

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

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

<Example 1>

Preparation of Acetone Extract from Brier Root

Brier roots were purchased at the Kyungdong Market Herbal Medicine in Seoul. Add 3000 liters of acetone to 3000 g of livestock roots (live weight) at room temperature Extraction for 24 hours. This was repeated twice and concentrated to dryness to give 92 g of acetone extract.

<Example 2>

Ethanol Extract of Brier Root

15 g of 70% ethanol was added to 3000 g of brier roots (live weight) and extracted at room temperature for 24 hours. This was repeated twice and concentrated to dryness to obtain 146.4 g of an aqueous ethanol extract.

<Example 3>

Preparation of Ethyl Acetate, Butanol and Water Fractions from Brier Roots

1000 ml of distilled water was added to the aqueous ethanol extract obtained in Example 2, and 1000 ml of ethyl acetate was added, and the mixture was partitioned twice into water and ethyl acetate layers. 600 mL of butanol was added to the water layer obtained above, and the resultant was partitioned twice into a water layer and a butanol layer. Each fraction was concentrated under reduced pressure to give 76.9 g of ethyl acetate fraction, 12.5 g of butanol fraction and 53.2 g of water fraction.

<Example 4>

Chromatography of Acetone Extract of Brier Root

The brier root acetone extract obtained in Example 1 was subjected to silica gel chromatography and ODS chromatography in order to isolate and purify the substance showing antioxidant activity. Silica gel (70-230mesh, Merck) was suspended in a chloroform and methanol solvent, filled in a glass column (10 × 15 cm), and then eluted by loading the Brier root acetone extract obtained in Example 1 above. At this time, as the developing solvent, a chloroform: methanol solvent system was used while increasing the methanol ratio step by step. The ratio of chloroform: methanol was 3: 1 and the solvent amount was 6000ml. Then, the ratio of methanol was increased to adjust the ratio of chloroform: methanol to 2: 1 and the amount of solvent to 2100ml. Only 2000ml of methanol was used. The flow rate of the elution solvent was 200 ml / hour, and the fractionation size was Fractionation was carried out at 50 ml / flask. As a result of chromatography, 170 eluted fractions were obtained, and thin layer chromatography (hereinafter referred to as TLC) was performed. After spraying with 10% sulfuric acid to confirm the distribution of the material, the fractions containing similar substances were collected and divided into 12 large fractions. TLC used a TLC plate (silica gel F ₂₄₅ , MERCK Co., Ltd.) as a stationary phase and a mixed solvent of chloroform and water (2: 1) as a mobile phase. The antioxidant activity of these 12 fractions was measured by the antioxidant activity measurement method using the free radicals (Korea Food and Nutrition Society, Food and Nutrition Experiment Handbook, 651, 2000). As a result, it was found that the fraction which exhibited the greatest antioxidant activity was the sixth fraction and the amount obtained was 6.16 g. The active fraction was once again subjected to silica gel column chromatography in the same manner as above to obtain 3.11 g of an active fraction showing antioxidant activity. This was again eluted with a mixed solvent of methanol: distilled water (methanol: distilled water = 1: 2, 1800 ml) using a glass column (4.5 × 12 cm) filled with an ODS gel (40-63 μm, Merck). 217 mg was isolated and purified.

<Example 5>

Determination of Chemical Structure of Active Ingredients in the Extracts of Brier Root

The chemical structure of the active ingredient exhibiting antioxidant activity in the extract of Brier root was investigated by hydrogen and carbon nuclear magnetic resonance spectra, and its physicochemical properties were investigated.

As a result, it was found that the active ingredient exhibiting antioxidant activity was procyanidin B3 having a structure of catechin- (4α → 8) -catechin represented by the following formula. Its physicochemical properties are as follows, hydrogen and carbon nuclear magnetic resonance spectra are as shown in Figures 1a and 1b.

1) Name of the compound: catechin- (4α → 8) -catechin (catechin- (4α → 8) -catechin)

2) Appearance of substance: Pale yellow powder

3) Molecular Weight: 578

4) Chemical structure

<Example 6>

Thin layer chromatography of brier root extract and chromatographic fraction extracted with organic solvent

Each of the brier extracts, solvent fractions and chromatography fractions obtained in Examples 1, 2, 3 to 4 were subjected to thin layer chromatography (hereinafter referred to as TLC) to extract the most procyanidin B3. Fractions were investigated. In stationary phase Using TLC plate (silica gel F ₂₄₅ , MERCK Co., Ltd.) and using mobile solvent as a mixed solvent of chloroform and water (2: 1), each extract and fraction were developed, sprayed with a 10% sulfuric acid solution with a coloring reagent, and heated. Colors may be removed by spraying a 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution, or an anti-oxidant active material may be present. A method of identifying by site was used.

As a result, when procured with 10% sulfuric acid, procyanidin B3 was identified in the acetone extract, the ethanol extract, and the ethyl acetate fraction thereof. In addition, the abundance of procyanidin B3 was estimated from the intensity of color development in the developed band. As a result, it was confirmed that procyanidin B3 is relatively present in the acetone extract and the ethyl acetate fraction (FIG. 2A). When the DPPH solution was sprayed to confirm purple discoloration, proceanidine B3 was identified in the acetone extract, the ethanol extract, and the ethyl acetate fraction thereof in the same manner as the color development with sulfuric acid (FIG. 2B).

In addition, as a result of TLC of the 12 fractions separated by chromatography in Example 4, it was confirmed that a lot of procyanidin B3 was present in the 6th fraction (FIG. 2C). This is the same as the result of measuring the antioxidant activity of the 12 fractions by the sulfated activity measurement method using free radicals in Example 4.

<Example 7>

Antioxidant Activity of Black Root Extract

The antioxidant activity of the brier root extract obtained in Example 1 and the final active material obtained in Example 4, procyanidin B3, was measured by comparing the antioxidant activities of BHA, a synthetic antioxidant, and α-tocopherol, a natural antioxidant.

Antioxidant activity was measured using the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Korea Food and Nutrition Science, Food and Nutrition Experiment Handbook , 651p, 2000; Blois, MS, Nature , 4617: 1198, 1958). 0.2 ml of an ethanol solution containing 2.0, 1.0, 0.5, 0.25, 0.125, 0.063 mg / ml of brier root extract obtained in Example 1, the final active material obtained in Example 4, BHA and tocopherol, respectively, was placed in a test tube. 0.8 ml of DPPH (0.4 mM) solution and 3 ml of ethanol were added thereto to react at room temperature for 30 minutes, and then the absorbance was measured at 517 nm. Antioxidant activity is represented by RC ₅₀ value. RC ₅₀ represents the concentration (μg / mL) required to reduce the absorbance value of 50% of the control without adding each sample.

As a result, as shown in Table 1, the antioxidant activity of the brier root extract was relatively higher than that of BHA and α-tocopherol. In addition, the antioxidative activity of procyanidin B3 isolated from the root extract of the larvae was 2.7 times and 3.8 times stronger than that of BHA and α-tocopherol, respectively.

Antioxidant Activity of Brier Root Extract and Procyanidin B3 Isolated from It sample Antioxidant Activity RC ₅₀ (μg / mL) Procyanidin B3 3.4 Brier Root Extract 7.6 BHA 9.2 α-tocopherol 13.0

Brier extract according to the present invention and a composition comprising the same can be very useful for the treatment or prevention of diseases caused by free radicals, maintaining the quality of food and preventing damage due to oxidation of the skin.

Claims (4)

A food composition having an antioxidant activity, which is obtained by fractionating an ethanol extract of a brier underground with water and ethyl acetate, and containing an ethyl acetate fraction containing procyanidin B3 of formula (I).

(I) Food composition having an antioxidant activity comprising the acetone extract of the brier basement containing procyanidin B3 of formula (I). The food composition according to claim 1 or 2, wherein the brier underground is its root or the root of root. delete

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