KR19990033613A - Use of beta-glucogallin compounds as antioxidants and methods for their isolation and purification - Google Patents

Abstract

The present invention relates to a use of a beta glucogallin compound represented by the general formula (1) as an antioxidant, and a method of separating and purifying the same. More particularly, the present invention relates to a method for extracting a corresponding extract having an antioxidative activity And a method for separating and purifying a beta-glucogalline compound.

The beta-glucogallin compound and its extract of the present invention have excellent antioxidant activity and are expected to be used as a natural antioxidant for controlling active oxygen species in vivo.

Description

Use of beta-glucogallin compounds as antioxidants and methods for their isolation and purification

The present invention relates to a novel use of a beta-glucogallin compound represented by the following formula (1) as an antioxidant and a method for producing the same.

Formula 1

More particularly, the present invention relates to a method for isolating and purifying a beta glucogallgyline compound by extracting an appropriate extract having an antioxidative activity by extracting a corresponding compound with an organic solvent, The extract is expected to be used as a natural antioxidant to control active oxygen species in vivo because of its excellent antioxidant activity.

All aerobic organisms, including humans, use oxygen (O ₂ ) to metabolize energy and survive. However, in vivo oxygen, various physical, chemical, upon receiving the biological stress, superoxide anion radical ^{_{(superoxide anion radical, O 2 -}} .), Hydrogen peroxide (H ₂ O _2), hydroxyl radical ^(. Hydroxy radical, OH), etc. Of active oxygen species in the body, causing a deadly physiological disorder in the body, and in severe cases, causing disease and losing its life.

It is thought that the living body evolved by developing an antioxidative mechanism as a self-defense mechanism for removing reactive oxygen species, but the generation of reactive oxygen species beyond the protective ability of the tissue is caused by immune such as protein, DNA, enzyme and T cell It damages the factors of the system and causes various diseases. In addition, the reactive oxygen species attack the unsaturated fatty acid, which is a constituent of the cell membrane, and cause a peroxidation reaction. Thus, it is known that lipid peroxidation accumulated in vivo causes aging and various diseases (Halliwell, B., Drugs, 42: 569 J., Gebicki, JM and Stocker, R., Biochem., J. 293: 601, 1990. Neuzil, J., Gebicki, JM and Stocker, K. and Takaishi, Y., J. Act. Dxyg. Free Rad. 1993; Sies, H., ed., Oxidative stress, 1985; Steinberg, D., et al., N. Engl. J. Med. 320: 915, 1989; , 1990).

Recently, research has been actively conducted on the development of antioxidants known as substances capable of regulating reactive oxygen species, as the theory that aging and diseases of adult diseases are caused by active oxygen species has been actively studied, and superoxide dismutase, peroxidase (antioxidant enzymes such as peroxidase, catalase and glutathione peroxidase) and low-molecular-weight antioxidants derived from natural products such as tocopherol, ascorbate, carotenoid and glutathione 42, 1102, 1977; Hammerschmidt, PA and Pratt, DE, J. Food Sci. 43: 556, 1977; Pratt, DE and 2,7'-di-tert-butyl-4-hydroxytoluene (BHT) , 2,6'-di-tert-butyl-4-hyd 40, 2208, 1992; Hatano, T., Natural Medicines 49 (1995), pp. : 357, 1995; Masaki, H., et al., Biol. Pharm. Bull.

However, the use of synthetic antioxidants has become increasingly limited due to the tendency of consumers to avoid synthetic antioxidants and the use of synthetic antioxidants in large-scale animal studies (Branen, A. L., JAOCS 52: 59, 1975). Therefore, it is urgently required to develop a new natural antioxidant which is excellent in efficacy and safer.

It is a deciduous shrub belonging to Rosaceae and is used as a garden and medicinal plant in the Orient. In China, dried flowers are used as tea as well as treatment of bruises, air intoxication, cold pain in the abdomen, and menstrual hyperreflexia and diarrhea of the lady (Jiangsu Shin school, 4909, 1985). In Japan, flowers are used as anti-diarrhea, hemostatic agents (Shibata, K. ed., A cyclopedia of useful plant and plant products: Enlarged and revised edition, 612, 1957) . In Korea, folk remedies use the underground as a treatment for diabetes.

Hashimoto et al. Reported a chemical composition and a physiologically active substance in the corresponding tissues (Park et al., Journal of Pharmacopoeia, 24: 319, 1993), Hadadoko reported that tannins, catechin derivatives, flavonoids, , And terpenoid (Hashidoko, Phytochemistry 43: 535, 1996).

However, there has been no report on the active substances having antioxidative activity against the subject.

Accordingly, the inventors of the present invention have investigated antioxidative activity of dozens of medicinal plants in order to develop a new natural antioxidant which is excellent in efficacy and safety. As a result, it has been found that the extract obtained from the extract of Rosa rugosa with an organic solvent has a high antioxidative activity (Choi, JS, et al., Kor. J. Pharmacognosy 24: 299, 1993), and their physicochemical properties were investigated by separating the substances having antioxidative activity by the method of DPPH free radical scavenging The result was confirmed as beta-glucogallin, thereby completing the present invention.

The present invention aims to provide a use of a beta glucogalline compound as an antioxidant, a method for separating and purifying a beta glucogallgrin compound from a corresponding extract having an antioxidative activity and a corresponding extract.

FIG. 1 shows a mass spectrometry spectrum of a beta glucogallgyline compound isolated and purified from a corresponding extract,

2 shows the hydrogen nuclear magnetic resonance spectrum of the beta-glucogallgene compound isolated and purified from the corresponding extract,

FIG. 3 is a carbon nuclear magnetic resonance spectrum of a beta-glucogallgene compound isolated and purified from a corresponding extract.

In order to achieve the above object, the present invention provides a use of a beta glucogallgine compound represented by the following general formula (1) as an antioxidant.

Formula 1

In addition, the present invention provides an extract of Aloe vera with an antioxidative activity obtained by extracting a corresponding algae with a lower alcohol.

In addition, the present invention provides an extract of the present invention having an antioxidative activity which is extracted with methanol and then extracted again with an organic solvent such as an organic solvent such as hexane, chloroform, ethyl acetate and butanol.

The present invention also relates to a method for producing a methanol extract, which comprises immersing the above-ground top part in methanol, repeatedly extracting and concentrating the methanol extract, suspending the extract in distilled water, adding an organic solvent such as hexane, chloroform, ethyl acetate and butanol sequentially, And a method for producing the extract having the antioxidant activity.

In addition, the present invention provides a method for separating and purifying beta glucogallin by performing silica gel chromatography, ODS column chromatography, and GPC column chromatography from the above extract.

Hereinafter, the present invention will be described in detail.

The present invention provides a corresponding extract having antioxidative activity.

First, the present invention is to immerse an animal in a lower alcohol and repeatedly extract it to obtain an alcohol fraction of an animal having an antioxidative activity. In this case, it is preferable to use methanol as the alcohol, and to use the stem, the leaves, the dryness and the residue of the leaves, and the remaining residue as the applicable ground, and it is more preferable to use the leaves.

In addition, the present invention provides an extract having the antioxidative activity obtained by extracting the above-ground part with methanol and then using an organic solvent. The organic solvent includes a lower alcohol such as hexane, ethyl acetate, chloroform or butanol.

In addition, the present invention provides a method for preparing a corresponding extract having an antioxidative activity of extracting a corresponding top part with methanol and then extracting it again with an organic solvent.

In accordance with the present invention, a methanol extract of methanol and a concentrated methanol extract are used to fractionate an organic solvent to produce a butanol fraction, etc., having excellent antioxidative activity.

Specifically, the present invention extracts dried leaves of Chrysanthemum japonica collected from Chunglipo beach, Chungcheongnam-do province, to the extent that it is immersed in methanol, repeats the extraction one to five times, and then concentrates and drying it to produce a methanol extract. In addition, in the present invention, the methanol extract obtained above is suspended in distilled water, and an equal amount of a solvent is added to collect the fractions to be transferred to various solvents and concentrate under reduced pressure to prepare various solvent-extracted fractions.

Actually, the present invention uses hexane, chloroform, ethyl acetate, butanol, and the like in sequence to prepare hexane-extracted fraction, ethyl acetate-extracted fraction, butanol-extracted fraction and water-extracted fraction.

In addition, the present invention provides a pharmaceutical composition having an antioxidative activity using the above extract as an active ingredient.

Specifically, the pharmaceutical composition can be used for controlling active oxygen species in vivo to prevent and treat aging and geriatric diseases.

As a result of the investigation of the antioxidative activity using the extract of the present invention, the extract of the Korean native had a strong antioxidative activity even in the case of the crude extract. Especially, the hexane and chloroform extract fractions showed an excellent antioxidative effect.

In addition, the extract having the antioxidative activity of the present invention can be used as a functional food or a food additive, a functional beverage or a beverage additive.

The present invention also provides a use of a beta-glucogalline compound of formula (1) as an antioxidant as a garlic acid-based compound.

The present invention also provides a method for separating and purifying the above-mentioned compounds from the corresponding plants. The present invention relates to a method for separating and purifying an active substance exhibiting antioxidative activity by performing silica gel column chromatography, ODS column chromatography, and GPC column chromatography from the above extract obtained by extracting Rosa rugosa obtained from West Sea of Chungcheongnam-do with various organic solvents do.

Specifically, in the present invention, the butanol fraction obtained from the above-mentioned compaction is subjected to silica gel column chromatography. As the developing solvent, a mixed solvent of chloroform and methanol is used. In addition, the active fraction is subjected to ODS column chromatography. Distilled water and methanol are used as the developing solvent. GPC column chromatography is performed on the active fractions identified in the purification process. As the developing solvent, a mixed solvent of chloroform and methanol is used.

In the present invention, the chemical structure of the active substance exhibiting antioxidative activity separated and purified by the above procedure was examined by mass spectrometry, hydrogen and carbon nuclear magnetic resonance spectroscopy, and it was confirmed that this compound was beta glucogalline.

In order to investigate the antioxidative activity of the beta glucogallgyline compound, 1,1-diphenyl-2-picrylhydrazyl (DPPH), which is a free radical, (Choi, et al., Kor. J. Phamacogn, 24: 299-303, 1993). As a result, it was confirmed that the beta-glucogalline compound of the present invention has an antioxidative activity about 1.4 times stronger than that of tocopherol, which is well known as a natural antioxidant, and has an excellent antioxidant activity (see Table 2).

Hereinafter, the present invention will be described in detail with reference to examples.

The following examples illustrate the present invention in detail and are not intended to limit the scope of the present invention.

≪ Example 1 > Production of solvent fraction in the above-mentioned ground part

The antioxidative activities of the extracts from the seaweeds collected from Chungri beach in Chungcheongnam - do were investigated by solvent fractionation. First, 270 g of the extract was extracted twice with 10 L of 100% methanol, and then concentrated to dryness to obtain 64 g of a methanol extract.

500 ml of distilled water was added to the methanol extract, and 500 ml of nucleic acid was added thereto. 500 ml of chloroform was added to the water layer, and the mixture was subjected to solvent fractionation three times with chloroform layer and water layer. 500 ml of ethyl acetate was further added to the water layer, and solvent fractionation was performed three times with an ethyl acetate layer and a water layer. Finally, 500 ml of butanol was added to the water layer And fractionated three times into a butanol layer and a water layer.

Each of the fractions obtained above was concentrated under reduced pressure to obtain a nucleic acid fraction, chloroform fraction, ethyl acetate fraction, butanol fraction and water fraction of 4.3 g, 7 g, 8.3 g, 7 g and 37.4 g, respectively.

≪ Example 2 >

The antioxidative activity of each fraction obtained in Example 1 was measured by using an antioxidative activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH ^. ), Which is a free radical, (Choi, et al., Kor. J. Phamacogn, 24: 299-303, 1993).

4 ml of methanol containing the sample compound in concentration was placed in a glass test tube, and 1 ml of the DPPH (0.15 mM) solution was added. After reacting at room temperature for 30 minutes, absorbance was measured at 517 nm. At this time, RC ₅₀ (μg) represents the amount (μg) of sample required to reduce the absorbance value of the control without addition of the sample by 50%.

Antioxidant activity of each fraction of solvent Fraction Extract (g) Antioxidant activity (RC ₅₀ : μg) Methanol extract 64 13 Nucleic acid fraction 4.3 > 100 Chloroform fraction 7 > 100 Ethyl acetate fraction 8.3 12 Butanol fraction 7 18 Water fraction 37.4 11

As shown in the above Table 1, the extract of R. squid has a strong antioxidative activity even in the form of crude extract. Especially, the extract of hexane and chloroform has excellent antioxidative effect.

≪ Example 3 > Separation and structure determination of antioxidants from the above-ground part

The active substance was isolated from the solvent fraction obtained from the above-mentioned ground of Example 1 in a butanol fraction having a strong antioxidative activity and relatively easy separation and purification, according to the following procedure.

The butanol fraction (7 g) was eluted and separated on a glass column (5 x 70 cm) filled with silica gel (70-230 mesh, Merck) suspended in chloroform. At this time, a solvent system of chloroform: methanol was used as an eluting solvent, and 5 g of fraction showing antioxidative activity was obtained. A portion (2 g) of the active fraction was eluted with a solvent system of methanol: distilled water using a glass column (5 x 70 cm) packed with ODS gel (70-230 mesh, YMC) to obtain 1.1 g of the active fraction. This was eluted with a glass column packed with Sephadex LH-20 gel (100 g, Pharmacia) in chloroform: methanol to obtain 35.7 mg of the active fraction as a single substance.

The separated compounds were identified by a signal (2H) of 7.00 ppm shown in the hydrogen nuclear magnetic resonance spectrum (see Fig. 2), a signal of benzene ring of 108.9-145.5 ppm of the carbon nuclear magnetic resonance spectrum (see Fig. 3) and a ketone of 164.6 ppm , And six signals from sugar at 60.5-94.4 ppm were observed to be glycoside compounds in which sugar was ester bonded to garlic acid. The signal of the anomeric proton at 5.50 ppm of J = 7.8 Hz of the hydrogen nuclear magnetic resonance spectrum is β-D-glucos, and in the HMBC spectrum, 1 'of sugar and aglycon 3, Cross peaks were observed in the first ketone carbon from the fourth protons and the structure was determined to be β-glucogallin. Saijo et al., Phytochemistry 29, 267 , 1990).

The physico-chemical properties of the beta-glucogalline compound of the present invention are as follows.

(1) Common name: beta-glucogallin

Chemical name: Beta-D-Glucopyranos 1- (3,4,5-trihydroxybenzoate)

(? -D-glucopyranose 1- (3,4,5-trihydroxybenzoate)

(2) Appearance of substance: tan yellow oil phase

(3) Molecular weight: 332

(4) Molecular formula: C ₁₃ H ₁₆ O ₁₀

(5) Mass spectrum (FAB-MS, m / z):

1, and the mass spectrometric value ([M + H] ⁺ ) is 333.

(6) Hydrogen nuclear magnetic resonance spectrum:

Dimethylsulfoxide (DMSO) was used as a solvent and tetramethylsilane (TMS) was used as a reference material. The results are shown in Fig.

(7) Carbon Nuclear Magnetic Resonance Spectrum:

Dimethylsulfoxide (DMSO) was used as a solvent and tetramethylsilane (TMS) was used as a reference material. The results are shown in Fig.

(8) Solubility: It dissolves well in hot water and Dimethylsulfoxide.

(9) Chemical formula:

Formula 1

Thus, the main antioxidant in the plant was found to be beta glucogalline. The separation of the beta glucogallgine compounds from the corresponding plants was first carried out by the present inventors.

The active ingredient of the fructose acetate fraction, which showed high antioxidant activity in the aerial part of the plant, was identified as isoquercitrin, which is known as an antioxidant.

Example 4: Antioxidant activity of beta glucogallin compound

The antioxidative activity of the beta-glucogalline compound was examined by the method of Example 2 above. RC ₅₀ (μg / ml) used for the antioxidant activity was determined by measuring the amount (μg) of sample required for reducing the absorbance value of the control by 50% using 1,1-diphenyl-2-picrylhydrazide (DPPH) to be.

Antioxidant Activity of Beta Glucoglin Compounds compound Antioxidant activity RC ₅₀ ^a) (μg) Beta Grokogallin 8.5 BHA 14 Tocopherol 12

The antioxidant activity of beta - glucogalline was about 1.65 times higher than that of BHA, which is a synthetic antioxidant, and about 1.41 times higher than that of tocopherol, a natural antioxidant. Therefore, beta-glucogallin is expected to be used as an antioxidant.

As described above, the extract of the present invention and the beta glucogallgyline compound exhibit excellent antioxidative activity and can be widely used as safe and effective antioxidants derived from plants in the future.

In addition, the extracts obtained by the method of the present invention can be widely applied not only to medicines but also to foodstuffs.

Claims (10)

An antioxidant comprising a beta glucogallin compound of formula (1) as an active ingredient. Formula 1 Extract of the extracts with the antioxidant activity obtained by extracting the corresponding alcohols with lower alcohol. 3. The extract according to claim 2, characterized in that the leaf is a dry leaf of the leaf or the leaf, and the lower alcohol is methanol. 3. The extract according to claim 2, wherein the extract is further extracted with an organic solvent containing hexane, chloroform, ethyl acetate and butanol. The extract according to any one of claims 2 to 4, which comprises beta glucogallin as an active ingredient. The methanol extracts were obtained by immersing the above-ground portions in methanol and repeatedly extracting them. The methanol extracts were suspended in distilled water and an organic solvent including hexane, chloroform, ethyl acetate and butanol was added in order to obtain the corresponding solvent-extracted fraction. ≪ / RTI > A purification method for obtaining a beta glucogallgyline compound having an antioxidative activity by performing silica gel chromatography, ODS column chromatography, and GPC column chromatography on a corresponding extract prepared by the method of claim 6. 8. The method according to claim 7, wherein the extract is a butanol extract. 2. A pharmaceutical composition having an antioxidative activity using the extract of claim 2 as an active ingredient. Functional food, food additive, functional beverage or beverage additive containing the extract of claim 2 as an active ingredient.