KR20030041239A - Novel antioxidative compounds from Daldinia concentrica and antioxidative composition comprising it as an active material - Google Patents

Abstract

PURPOSE: A novel antioxidizing compound and an antioxidizing composition containing the compound as an effective component are provided, wherein the compound protects the nerve cell from the oxidative toxicity due to iron and shows no cell cytotoxicity. CONSTITUTION: The antioxidizing compound is represented by the formula 1 or 2, and is obtained from the Daldinia extract or synthesized by a chemical method. The compound of the formula 1 or 2 is obtained by extracting ground Daldinia with methanol and separating and purifying the compound by chromatography.

Description

Novel antioxidative compounds from Daldinia concentrica and antioxidative composition comprising it as an active material

The present invention relates to a novel antioxidant compound derived from soybean mushroom and an antioxidant composition comprising the same as an active ingredient. More specifically, the present invention relates to a novel antioxidant compound having an antioxidant activity obtained by purely separating and extracting from soy mushrooms, and to an antioxidant composition comprising the same as an active ingredient.

Free radicals are a direct or indirect cause of various diseases, including stroke, myocardial infarction, diabetic vascular disorders, hyperlipidemia, acute inflammation, rheumatic diseases, alcoholic hepatitis, arteriosclerosis, carcinogenesis, and aging due to cerebrovascular disorders. have. Active oxygen scavengers have been reported to reduce or prevent these diseases, and various active oxygen scavengers have been found in microorganisms and plants and are used as pharmaceuticals or cosmetics and food additives.

Iron produces hydroxyl radicals (OH) through the Fentone reaction, which in turn causes peroxidation of lipids and proteins and crosslinkage of DNA, leading to Cause death.

Highly reactive activity such as superoxide (OH ₂ ), hydroxyl radical (OH) and hydrogen peroxide (H ₂ O ₂ ) due to various oxidation reactions, chemicals, food, human diseases and radiation When oxygen and free radicals are generated in vivo, the phospholipids of the cell membrane containing a large amount of unsaturated fatty acids are oxidized, thereby producing lipid peroxides on the cell membrane. Accumulation of lipid peroxides in the cell membrane reduces the fluidity and functionality of the cell membrane, thereby inhibiting the overall function of the cell and changing the structure of the cell. Therefore, if excessive reactive oxygen and free radicals accumulate in the human body, various human diseases such as stroke, myocardial infarction, diabetic vascular disorders, hyperlipidemia, acute inflammation, rheumatic diseases, alcoholic hepatitis, and the like are caused. Therefore, the active oxygen scavenger that is powerful but not toxic will be very useful as a therapeutic agent for various diseases or as an anti-aging cosmetic additive and food additive. Until now, synthetic antioxidants such as butylated hydroxy toluene (BHT) and butylated hydroxyanisol (BHA) have been used as antioxidant compounds. However, these BHT and BHA have excellent antioxidant activity but are highly likely to cause cancer or malformation. There was a disadvantage. Therefore, it is required to develop an antioxidant that has no toxicity or side effects by creating a new active oxygen scavenger from natural products.

In view of the above, the present inventors collected and investigated various mushrooms and medicinal plants, and extracted and purified soybean mushrooms using methanol, and purified by chromatography to obtain novel antioxidant compounds daldine A and daldine B. After the chemical structure and physicochemical properties were identified, the present invention was completed by examining neuronal protective activity.

Accordingly, an object of the present invention is to provide a novel antioxidant compound having neuronal protective activity obtained by purely purified from soybean mushroom. Another object of the present invention to provide an antioxidant composition containing a novel antioxidant compound having a neuronal protective activity obtained by purely purified from soybean mushroom as an active ingredient.

The object of the present invention was achieved by methanol extraction of soy mushrooms, purified by chromatography to obtain a novel antioxidant compound, the chemical structure and physicochemical properties of the soy mushroom, and then investigated the neuronal protective activity.

Hereinafter, the configuration of the present invention.

The present invention comprises the steps of pulverizing the soy mushroom, extracted with methanol and purified purely the new antioxidant compounds daldine A and B using chromatography; Examining the chemical structures and physicochemical properties of the compounds daldine A and daldine B obtained in the step; Investigating the neuronal protective activity of the compounds daldine A and B of the above step.

The novel neuronal protective antioxidant Daldine A according to the present invention has the following formula (1).

Physicochemical properties of the novel neuronal protective antioxidant Daldine A according to the present invention are as follows.

Appearance: Colorless latex

Molecular Weight: 294

Molecular Formula: C ₁₅ H ₁₈ O ₆

Radiance: [α] _D -8.9 ( c 0.9, MeOH)

Mass Spectrum Spectrum: HREIMS m / z 294.1099 [M] ⁺

EIMS m / z 294 [M] ⁺

UV absorption spectrum: UV λ max (MeOH) (log ε )

210 (4.3) 245 (sh. 3.68), 270 (sh. 3.03) nm

Infrared spectrum: IR (KBr) ν max

2942, 2842, 1770, 1592, 1463, 1238, 1127 cm ^-1

Hydrogen Magnetic Resonance Spectrum: ppm (500 MHz)

¹ H NMR (CDCl ₃ ): δ 6.56 (2H, s, H-2 ′, 6 ′), 4.61 (1H, d, J = 6.6 Hz, H-2), 4.52 (1H, dd, J = 6.6, 9.6 Hz, H-8), 4.39 (1H, t, J = 9.0 Hz, H-4), 4.36 (1H, dd, J = 2.1, 9.6 Hz, H-8), 4.21 (1H, dd, J = 3.6, 9.0 Hz, H-4), 3.87 (6H, s, 3 ′, 5′-OCH ₃ ), 3.84 (3H, s, 4′-OCH ₃ ), 3.45 (1H, dt, J = 3.6, 9.0 Hz, H-5), 3.14 (1H, m, H-1)

Carbon Magnetic Resonance Spectrum: ppm (125 MHz)

¹³ C NMR (CDCl ₃ ): δ 177.9 (C-6), 153.6 (C-3 ′, 5 ′), 138.0 (C-4 ′), 134.5 (C-1 ′), 102.8 (C-2 ′ , 6 ′), 86.1 (C-2), 70.2 (C-4), 69.8 (C-8), 60.7 (4′-OCH ₃ ), 56.1 (3 ′, 5′-OCH ₃ ), 48.4 (C -1), 46.0 (C-5)

The novel neuronal protective antioxidant Daldine B according to the present invention has the following formula (2).

Physicochemical properties of the novel neuronal protective antioxidant Daldine B according to the present invention are as follows.

Appearance: Colorless latex

Molecular Weight: 212

Molecular Formula: C ₁₁ H ₁₆ O ₄

Radiance: [α] _D -20 ( c 0.2, MeOH)

Mass Segmentation Spectrum: HREIMS m / z 212.1040 [M] ⁺

EIMS m / z 212 [M] ⁺

UV absorption spectrum: UV λ max (MeOH) (log ε )

210 (4.53) 245 (sh. 3.82), 268 (3.37) nm

Infrared spectrum: IR (KBr) ν max

3437, 2930, 1593, 1462, 1235, 1128 cm ^-1

Hydrogen Magnetic Resonance Spectrum: ppm (500 MHz)

¹ H NMR (CDCl ₃ ): δ 6.61 (2H, s, H-2 ′, 6 ′), 4.83 (1H, d, J = 6.36 Hz, H-1), 3.87 (6H, s, 3 ′, 5 ′ -OCH ₃ ), 3.84 (3H, s, 4′-OCH ₃ ), 1.45 (3H, d, J = 6.36 Hz, H-2)

Carbon Magnetic Resonance Spectrum: ppm (125 MHz)

¹³ C NMR (CDCl ₃ ): δ 153.2 (C-3 ′, 5 ′), 141.7 (C-1 ′), 137.0 (C-4 ′), 102.2 (C-2 ′, 6 ′), 70.5 ( C-1), 60.7 (4′-OCH ₃ ), 56.1 (3 ′, 5′-OCH ₃ ), 25.2 (C-2)

From the interpretation of the above physicochemical properties and nuclear magnetic resonance spectra, compounds Daldin A and B according to the present invention have been identified as novel neuronal protective antioxidant compounds.

The novel antioxidant compound according to the present invention is extracted from organic mushrooms (alcohol, ether, acetone, etc.) from mushrooms belonging to the genus Daldinia , separation of ethyl acetate and water, column chromatography, etc. The well-known method used for these can be obtained easily or in combination suitably. The crude extract can be further purified according to a commercial method as needed.

The chromatography used in the present invention includes silica gel column chromatography, L-20 column chromatography, thin layer chromatography and high performance liquid chromatography. (high performance liquid chromatography) and the like can be used.

The novel compounds daldine A and B according to the present invention have the effect of inhibiting the oxidative toxicity of brain-induced neuronal cells and can be advantageously used as an antioxidant in vivo because it is lower in cytotoxicity than other antioxidants.

The novel compounds daldine A and B according to the present invention can be used as additives such as food, beverages and cosmetics as antioxidants as well as having excellent stability to protect nerve cells from oxidative toxicity and high stability.

When applied to pharmaceuticals, the compounds Daldine A and Daldine B according to the present invention can be formulated as oral or parenteral administration in the form of solid, semi-solid or liquid by adding a commercially available inorganic or organic carrier. .

Examples of preparations for oral administration include tablets, pills, granules, soft and hard capsules, powders, fine granules, powders, emulsions, syrups and pellets. have. Preparations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. In order to formulate the active ingredient of the present invention, it can be easily formulated according to the conventional method, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspensions, and other commonly used auxiliaries can be suitably used.

Hereinafter, the specific method of the present invention will be described step by step with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Isolation and Purification of Compounds Daldine A and Daldine B

460 g of soybean fruiting bodies were finely crushed and then immersed in 70% methanol for 2 days and extracted at room temperature. The methanol extract was concentrated under reduced pressure, and the remaining residue was extracted with ethyl acetate, and the extract was concentrated under reduced pressure, and subjected to silica gel column chromatography using CHCl ₃ : MeOH as a developing solvent. After concentrating the active fractions obtained above, Sepadex LH-20 column chromatography was performed using methanol as a developing solvent, and ODS gel column chromatography was performed using 60% methanol. 5 mg of the compound Daldine A and 3 mg of Daldine B were isolated.

Example 2 Physicochemical Properties and Chemical Structure Analysis of Compounds Daldine A and Daldine B

Compounds Daldine A and Daldine B obtained as described above were obtained as a colorless emulsion. As a result of EI-mass measurement, the molecular weight of Daldine A was 294 and Daldine B was 212. As a result of the high resolution EI-mass measurement, it was determined that the molecular formula of Daldine A was C ₁₅ H ₁₈ O ₆ and that of Daldine B was C ₁₁ H ₁₆ O ₄ . As a result of measuring a UV spectrum daldin A and B are both 210, 245, exhibited a maximum absorption peak at about 270 nm from the result of measuring the IR spectrum daldin A at 1770 cm ^-1 is a carbonyl group (carbonyl group), B is daldin At 3437 cm ⁻¹ , a band attributable to hydroxyl was observed. Daldin A and Daldine B's luminescence was -8.9 ° and -20 °, respectively.

In order to elucidate the chemical structures of Dardin A and B, one-dimensional NMR spectra including ¹ H, ¹³ C and DEPT and two-dimensional NMR spectra including COSY and HMBC were measured. Based on the interpretation of these NMR data and the above physicochemical properties, the chemical structures of the novel antioxidant compounds daldines A and B were identified.

Example 3 Investigation of Biological Activities of Compounds Daldine A and B

Cortical neurons isolated from fetuses of the 15th day of gestation were isolated and cultured for 12-14 days and treated with FeSO ₄ (II), which induces neuronal death by oxidative toxicity. At the same time, treatment with the neuronal cell protective active substances Daldine A and B and 24 hours after the death of neurons were observed by light microscopy.

The primary mouse cortical neuron was treated with 50 μM of FeSO ₄ (II) and treated with Dardin A and B to examine the neuroprotective activity of the neurons. Showed a 50% neuronal cell death inhibitory effect at 5μM. Neuronal cell death was completely inhibited by 10 μM of neuronal cell protective material. The compound of the present invention was significantly lower in cytotoxicity than other antioxidants reported so far in primary mouse cortical neuronal culture of primary cultured rats.

As described above, the compounds Daldine A and Daldine B of the present invention have antioxidant activity to eliminate free radicals caused by iron, and thus protect neurons from oxidative toxicity as well as natural soybean mushrooms. It is a very useful invention for the pharmaceutical, cosmetic and food industries because there is no cytotoxicity by extracting from.

Claims (3)

Antioxidant compound, characterized in that represented by the following formula (I) prepared by soybean mushroom extract or chemical synthesis. [Formula 1] Antioxidant compound, characterized in that represented by the formula (II) prepared by soybean extract or chemical synthesis. [Formula 2] An antioxidant composition comprising at least one or more selected from the group consisting of compounds of claims 1 and 2 as an active ingredient.