KR20050089241A - Antioxidant composition containing extracts or compounds derived from rubus coreanus - Google Patents

Abstract

The present invention relates to an antioxidant composition comprising the bokbunja tree extract or a compound derived therefrom. In particular, the present invention provides an antioxidant composition comprising a methanol crude extract of bokbunja tree root or bokbunja ethyl acetate extract as an active ingredient, which is harmless to human body and can replace the existing synthetic antioxidant.

Description

Antioxidant composition comprising bokbunja tree extract or a compound derived therefrom {ANTIOXIDANT COMPOSITION CONTAINING EXTRACTS OR COMPOUNDS DERIVED FROM RUBUS COREANUS}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an antioxidant composition comprising a bokbunja tree extract or a compound derived therefrom, and more particularly to an antioxidant composition comprising methanol extract or bokbunja ethyl acetate extract of the bokbunja tree root as an active ingredient. .

[Private Technology]

All living organisms have a fundamental self-defense mechanism against the injury of free radicals as they use oxygen to generate the energy needed to sustain life. Free radicals that are not dissociated from the defense mechanisms of tissues are known to be involved in physiological changes such as aging or to a wide range of biological phenomena such as cancer, arthritis, and diabetes (Halliwell B. 1991. Drug antioxidant). effects.Drugs 42: 596-605). Radicals, known as free radicals are most stable form as the three polymerase reduced-oxygen superoxide anion radical ^{(superoxide anion radical; O 2-)} , hydrogen peroxide (H ₂ O _2), hydroxyl radical (OH ^-), lipid peroxides ( ROOH) and free radicals (ROO ·, RO ·), etc., produced here, and oxidative stress caused by free radicals in the living body when oxidized lipids of active oxygen are not normally eliminated It is the cause of many adult diseases such as cancer and cancer (Pryored, 1984). In addition, oxygen in the living body provides opportunities for the generation of free radicals, causing damage to proteins, enzymes, and DNA, and attacking polyunsaturated fatty acids, which are the constituents of the cell's biofilm, and lipid peroxidation or oxidative degradation products cause diseases in the tissue of DNA or RNA (Kim Sam-in. 1996. Search for Antioxidant Substances in Agricultural and Marine Products. Dankook University. Master Thesis 1996).

In addition, according to recent studies, diseases such as aging and adult diseases are known to be due to the presence of free radicals, and studies on antioxidants capable of regulating free radicals are being actively conducted. Antioxidants include enzyme-based Superoxide dismutase (SOD), catalase and glutathione peroxide, and natural antioxidants include tocopherol, ascorbic acid, Carotenoids, flavonoids and glutathione; synthetic antioxidants such as tert-butyl-4-hydroxyanisole (BHA), tert-butyl-4-hydroxytoluene (BHT), and troxol-C; In addition, various kinds of antioxidants are being developed (Kitahara K, Matsumoto Y, Ueda HA and Ueoka R. 1992. Chem. Pharm. Bull 40: 2208-2209; Hatano T. 1995. Natural Medicines 49: 357-363).

An object of the present invention is to provide an antioxidant extract extracted from bokbunja tree.

It is another object of the present invention to provide an antioxidant compound derived from bokbunja tree.

In order to achieve the above object, the present invention provides an antioxidant composition comprising (a) methanol crude extract of the bokbunja tree root or (b) bokbunja ethyl acetate extract as an active ingredient.

In another aspect, the present invention provides an antioxidant composition comprising 3,4-dihydric benzoic acid as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to an antioxidant composition comprising an extract of Bokbunja (Rubus coreanus) or an antioxidant compound derived therefrom as an active ingredient.

The bokbunja tree extract of the present invention can be prepared by extracting and / or fractionating certain parts of the bokbunja tree, such as fruit, leaves, branches, roots, stems, seeds and flowers, with water or an organic solvent.

The organic solvent may be a low-cost alcohol having 1 to 5 carbon atoms or alcohol dilution water, hexane, chloroform, ethyl acetate and acetone, the low alcohol may be ethanol, methanol, butanol, propanol and isopropanol, but is not limited thereto. . Preferably, the mixture is extracted with methanol or after methanol extraction, hexane, chloroform, ethyl acetate, and butanol are sequentially added, and the layers are separated by each solvent.

The extraction and layer separation method is usually carried out by this method, for example, by adding water or an organic solvent in a weight ratio of 1: 1 to 100 to an extract, a pulverized product or a powder thereof, and then extracting the extract or the separated layers. It can be carried out. Extraction and separation temperature may be 4 to 30 ℃, but is not limited thereto. The extract and the separator may then be filtered under reduced pressure and concentrated in vacuo to remove the solvent.

Bokbunja tree extract of the present invention exhibits various antioxidant activities, depending on the extraction site and maturity of the plant. For example, the antioxidant activity of a methanol crude extract to extract the extracted portions of the various bokbunja tree in methanol, in the saengyeop and seeds in the IC ₅₀ in the original root is very excellent as 8.52 ㎍ / ㎖ whereas IC ₅₀ is very approximately 95 ㎍ / ㎖ over low. In addition, the fruit of the bokbunja tree showed different antioxidant activities depending on the maturity. IC ₅₀ was 14.42 at the beginning, 15.52 ㎍ / ml at the middle stage and 39.61 ㎍ / ml at the end stage. In other words, as the fruit matures, antioxidant activity decreases.

In addition, the bokbunja tree extract of the present invention shows a significant antioxidant activity difference depending on the extraction solvent. For example, methanol extract of Bokbun and hexane extract, chloroform extract, ethyl acetate extract and water extract layered by sequentially adding hexane, chloroform and ethyl acetate to the crude extract are IC ₅₀ 39.61,>100,> 100, 7.33. , 48.27 and 19.58 μg / ml. That is, bokbunja ethyl acetate extract has the best antioxidant activity.

In addition, the present invention provides an antioxidant compound, 3,4-dihydroxybenzoic acid (3,4-Dihydroxybenzoic acid) from the bokbunja. The 3,4-dihydroxybenzoic acid has an IC ₅₀ of 1.26 µg / ml, and has excellent antioxidant properties compared to vitamin C (2.14 µg / ml) and BHA (4.35 µg / ml), which are known antioxidants.

Therefore, the bokbunja tree extract of the present invention or the antioxidant compound derived therefrom can be used as a prophylactic or therapeutic agent for diseases caused by the oxidative power of free radicals, in particular adult diseases and aging.

Thus, the present invention provides an antioxidant composition comprising the above-described bokbunja tree extract or an antioxidant compound derived therefrom as an active ingredient. The bokbunja tree extract is preferably a methanol crude extract or bokbunja ethyl acetate extract of the bokbunja tree root, the compound is 3,4-dihydroxybenzoic acid.

The antioxidant composition of the present invention may include each of the active ingredients alone, and may further include a pharmacologically acceptable carrier or excipient according to the formulation, method of use, and purpose of use. When provided as a mixture, the active ingredient may be included in the antioxidant composition of 0.1 to 99.9% by weight, it is usually included in an amount of 0.001 to 10% by weight.

The carrier or excipient includes water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, which may be used one or more, but are not limited to these, and are common carriers And excipients are both available. In addition, when formulating an antioxidant composition, it may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

The antioxidant composition of the present invention can be used as a food, a food additive, a pharmaceutical or a health food. When used as a medicament, it can be used orally or parenterally, but preferably oral administration. Since the formulation of the composition may vary depending on the method of use, it is not limited thereto. Examples of formulations include Plasters, Granules, Lotions, Powders, Syrups, Liquids and Solutions, Ointments, Fluidextract, Emulsions, Suspions, Infusions, Tablets, Injections, Capsules and Pills.

The dosage of the antioxidant composition of the present invention may be appropriately adjusted according to the use and method of the composition, for example, from 0.01 mg to 5000 mg based on one active ingredient, to be administered 1 to 5 times per day. Can be.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the protection scope of the present invention is not limited to the following examples.

Example 1 Preparation of Methanol Crude Extracts by Regions of Bokbunja Trees

Leaves, berries (early, middle, late), branches, roots, and seeds of the bokbunja trees grown in Gochang-gun, Jeonbuk were collected, and extracts were prepared from the respective samples. Fruits were classified into early, middle and late stages, and the fruits of immature fruit were classified as early and average weight of about 0.7g / piece. Fruits representing the middle, which is dark red of the terminal family of the end weight was used to classify the fruit with the average weight 2.0g / dogs.

Each part of the collected bokbunja trees was dried well, ground and ground to a powder. 50 g of each powder sample was taken into a 500 ml Erlenmeyer flask, 300 ml of methanol was poured, shaken well, and left to stand at room temperature in the dark. After 3 days, the mixture was filtered under reduced pressure and concentrated under reduced pressure at 40 ° C. using a rotary vacuum concentrator (EYELA autojack NAJ-160, Japan) to obtain a crude methanol extract.

Experimental Example 1: Antioxidant Activity Assay

Each extract or compound prepared in Examples 1 to 4 was carried out by the following method to determine the antioxidant activity.

After taking 10 mg of each extract or compound and applying 10 ml of methanol, the stock solution was diluted to 100, 50, 25, 12.5, 10, 5, 2.5 and 1.25 [mu] l per ml of methanol. 2 ml of each of the dilutions and 0.5 ml of 1.5 x 10 ^-4 M / ml (methanol) DPPH (Sigma, St. Louis, Mo, USA) solution were evenly mixed and allowed to stand at room temperature for 30 minutes, followed by 520 nm The optical density (OD) was measured. As a control, BHA, a natural antioxidant, and vitamin C, a natural antioxidant, were used. The result of antioxidant activity was shown as IC ₅₀ , which is the concentration of the sample showing a 50% decrease in absorbance compared to the control group, and the experiment result was repeated three times to calculate the average. The results are shown in Table 1 below.

Bokbunja Tree Extract of Example 1 IC ₅₀ (μg / ml) Bokbunja tree area Fruit early 14.42 Middle fruit 15.52 The end of fruit 39.61 Leaf 96.69 Deciduous 24.03 Root 8.52 Side roots 15.21 Seed 〉 100 Branch 10.19 Control Vitamin c 1.09 BHA 5.73

Bokbunja tree showed very different antioxidant activity according to the extract of each part. That is, the best antioxidant activity was observed in the roots of bokbunja, especially in the roots, while the antioxidant activity of the bokbunja trees was very low. In addition, different antioxidant capacities were observed depending on the extraction time of the bokbunja tree. The leaves of bokbunja tree had very low antioxidant activity, while the leaves showed high antioxidant activity, and also showed different antioxidant activities depending on the harvest time.

Example 2: Preparation of Bokbunja Organic Solvent Extract

A brief manufacturing process is shown in FIG.

2-1. Bokbunja Hexane Extract

20 g of bokbunja methanol crude extract, prepared using the bokbunja fruit (terminal) of Example 1, was dissolved in 800 ml of distilled water, placed in a 2 L separatory filter, and then mixed with the same amount of hexane. Layered into layers. After about 30 minutes, only the hexane layer was obtained using another separatory filter, and 800 mL of fresh hexane was poured into the water layer, and the second hexane layer was extracted in the same manner. The hexane layer was collected and concentrated by a vacuum concentrator to prepare a bokbunja hexane extract.

2-2. Bokbunja Chloroform Extract

After the 2-1 experiment, chloroform was added to the remaining water layer in the same manner to prepare a bokbunja chloroform extract.

2-3. Bokbunja Ethyl Acetate Extract

Ethyl acetate was added to the remaining water layer after the 2-2 experiment in the same manner to prepare a bokbunja ethyl acetate extract.

2-4. Bokbunja Butanol Extract

Butanol was added to the remaining water layer after the 2-3 experiment in the same manner to prepare a bokbunja butanol extract.

2-5. Bokbunja Water Extract

The water layer remaining after the 2-4 experiment was obtained and concentrated by a vacuum concentrator to prepare a bokbunja water extract.

Example 3: Preparation of organic solvent extract of bokbunja juice or gourd

45 kg of bokbunja sold at the Gochang Bokbunja test site in Jeonbuk was compressed to prepare 21 L of juice and gourd (the rest of the bokbunja except for juice).

Bokbunja juice was separated by adding hexane, chloroform and ethyl acetate in the same manner as in Example 2, and concentrated in vacuo to give a hexane extract, chloroform extract, ethyl acetate extract and water extract. A brief manufacturing process is shown in FIG.

Bokbunja gourd was prepared in the same manner as in Example 1 as a crude methanol extract. The methanol crude extract was layered by adding hexane, chloroform and ethyl acetate in the same manner as in Example 2, and concentrated in vacuo to give a hexane extract, a chloroform extract, an ethyl acetate extract and a water extract. A brief manufacturing process is shown in FIG.

Example 4: Isolation and Identification of Antioxidative Compounds from Bokbunja

Ethyl acetate extract of Bokbunja juice prepared in Example 3 was fractionated by chromatography.

After dissolving 10 g of ethyl acetate extract of bokbunja juice in acetone, adsorbing on silica gel, blowing off the solvent, finely grinding the silica gel adsorbed on the sample, and then silica gel column (φ10 × 100) directly filled with 100% ethyl acetate. cm) and hexane and ethyl acetate as elution solvent 1:99, 2:98, 3:97, 5:95, 10:90, 20:80, 30:70, 50:50, 100% ethyl Ten fractions of sequential elution were obtained as BE1 to BE10 after flowing in a concentration gradient of acetate volume ratio. The elution fractions BE1 to BE10 were each concentrated in vacuo and used as the following antioxidant activity assay samples.

In addition, the BE7 fraction was further subjected to GC-MS it was confirmed that the 3,4-dihydric benzoic acid of the formula (Fig. 3).

(Formula 1)

Experimental Example 2: Antioxidant Activity Assay

Each extract or compound prepared in Examples 2 to 4 was carried out by the following method to determine the antioxidant activity.

10 mg of each extract or compound was taken up and 10 ml of methanol, and the stock solution was diluted to 100, 50, 25, 12.5, 10, 5, 2.5 and 1.25 [mu] l per 1 ml of methanol. 2 ml of each of the dilutions and 0.5 ml of 1.5 x 10 ^-4 M / ml (methanol) DPPH (Sigma, St. Louis, Mo, USA) solution were evenly mixed and allowed to stand at room temperature for 30 minutes, followed by 520 nm The optical density (OD) was measured. As a control, BHA, a natural antioxidant, and vitamin C, a natural antioxidant, were used. The result of antioxidant activity was shown as IC ₅₀ , which is the concentration of the sample showing a 50% decrease in absorbance compared to the control group, and the experiment result was repeated three times to calculate the average. The results are shown in Tables 2 to 5 below.

Bokbunja Organic Solvent Extract of Example 2 IC ₅₀ (μg / ml) Organic solvent Hexane > 100 chloroform > 100 Ethyl acetate 7.33 Butanol 48.27 water 19.58 Control Vitamin c 2.09 BHA 5.56

Among the bokbunja organic solvent extracts of Example 2, ethyl acetate extract was the best antioxidant activity, IC ₅₀ was confirmed to be 7.33 ㎍ / ㎖. Bokbunja water extract also showed antioxidant activity.

Organic Solvent Extract of Bokbunja Juice of Example 3 IC ₅₀ (μg / ml) Organic solvent juice Hexane layer - Chloroform layer > 100 Ethyl acetate layer 49.14 Water layer 53.19 foil Hexane layer 26.40 Chloroform layer 22.53 Ethyl acetate layer 36.48 Water layer 20.35 Control Vitamin c 5.10 BHA 1.93

In the organic solvent extracts of the bokbunja juice, the antioxidant activity of the ethyl acetate extract was the best, but was lower than the antioxidant activity of the bokbunja ethyl acetate extract described in Table 2.

Chromatographic fractions of Example 4 IC ₅₀ (μg / ml) BE1 34.8 BE2 > 100 BE3 > 100 BE4 > 100 BE5 > 100 BE6 19.8 BE7 11.9 BE8 13.6 BE9 19.6 BE10 22.6 Vitamin c 4.3 BHA 2.4

In a total of 10 fractions obtained by chromatography of ethyl acetate extract of Bokbunja juice of Example 3, the BE7 fraction showed the best antioxidant activity.

compound IC ₅₀ (μg / ml) 3,4-dihydroxybenzoic acid 1.26 Vitamin c 2.14 BHA 4.35

In Example 4, 3,4-dihydroxybenzoic acid isolated and identified from ethyl acetate extract of Bokbunja juice showed very good antioxidant activity, and also had higher activity than the existing antioxidants, vitamin C and BHA. Confirmed.

As mentioned above, the antioxidant composition comprising methanol extract or bokbunja ethyl acetate extract of the bokbunja tree root of the present invention as an active ingredient is harmless to the human body and is used as a substitute for the existing synthetic antioxidants. It can be used as a prophylactic or therapeutic agent for diseases caused by oxidative power, especially adult diseases and aging.

Figure 1 shows the process of extracting the crude extract of Bokbunja tree methanol with an organic solvent.

2 illustrates a process of extracting bokbunja juice with an organic solvent.

Figure 3 is a GC-MS chromatogram of BE7 eluate isolated from ethyl acetate extract of bokbunja juice.

Claims (4)

(a) Methanol crude extract of the root of Rubus coreanus or (b) Bokbunja ethyl acetate extract comprising ethyl acetate fraction separated by sequentially adding hexane, chloroform and ethyl acetate to the crude extract of Bokbunja. Antioxidant composition comprising as an active ingredient. The composition of claim 1, wherein said root is one root or side root. Antioxidant composition comprising 3,4-dihydric benzoic acid as an active ingredient. The composition according to any one of claims 1 to 3, wherein the antioxidant composition is used as a food, a food additive, a medicament or a health supplement.