KR20010008772A - Hispidin-series chemical compound capable of removing active oxygen and process for prepration thereof - Google Patents

Abstract

PURPOSE: Provided are a hispidin-based active oxygen scavenger and its preparation method thereof. The compound is a free radical scavenger wherein free radicals are as follows: a superoxide radical, tertiary butyl-oxy-radical, 1,1-diphenyl-2-picrylhydrazyl(DPPH), and has an anti-oxidation activity. CONSTITUTION: A hispidin-based active oxygen scavenger that has an anti-oxidation activity is characterized by formula(I). Its preparation method is characterized by comprising the following steps: (i)extracting ground Inonotus xeranticus fruit body with ethanol, performing vacuum concentration; (ii)extracting it with ethyl acetate again to separate ethyl-acetate layer and water layer; (iii)obtaining an active fraction by performing concentration gradient silica gel column chromatography of ethyl-acetate layer through using chloroform-methanol mixed solvent; (iv)purifying the active fraction through sephadex LH-20 column chromatography, ODS column chromatography sequentially; (v)after obtaining the active fraction from performing ODS TLC with methanol, separating and purifying the compound by performing HPLC with 60% methanol.

Description

Hispidin-based active oxygen scavenging substance and method for preparing the same {Hispidin-series chemical compound capable of removing active oxygen and process for prepration

The present invention relates to a novel hispidin-based active oxygen scavenging substance and a method for producing the same. More specifically, the present invention relates to a novel hispidine-based spiro type compound for strongly scavenging the active oxygen isolated from the extract of Inoletus xeranticus and a method for producing the same.

Free radicals and free radicals are produced by various oxidation reactions, causing oxidative damage to cells and causing various diseases. That is, highly reactive free radicals such as superoxide (O ₂ ), hydroxyl radical (OH) and hydrogen peroxide (H ₂ O ₂ ) due to various oxidation reactions, chemicals, food, human diseases and radiation When free radicals and the like are generated in vivo, lipids of cell membranes containing a large amount of unsaturated fatty acids are oxidized, thereby producing lipid peroxides on the cell membranes. 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. In addition, free radicals and free radicals modify or destroy cell constituents such as nucleic acids and sugars, resulting in cancer, liver disease such as alcoholic hepatitis, stroke due to cerebrovascular disorders, myocardial infarction, diabetic vascular disorders, hyperlipidemia, and acute inflammation. And various human diseases such as rheumatoid disease. Therefore, the discovery of a powerful but non-toxic reactive oxygen scavenger will be very useful as a therapeutic agent for various diseases, anti-aging cosmetic additives and food additives. Until now, synthetic antioxidants such as butylated hydroxy toluene (BHT) and butylated hydroxy anisol (BHA) have been used as lipid peroxidant inhibitors, but these BHT and BHA have excellent antioxidant activity. The possibility of causing cancer or malformation was very high, there was a disadvantage that can not be used continuously. Therefore, it is necessary to create an antioxidant that has no toxicity or side effects by creating a new active oxygen scavenger from natural products.

The search for physiologically active substances exhibiting new active oxygen scavenging activity from microorganisms and natural products has been relatively recent. New Flavonoid Glycosides Available in Cosmetics and Medicines from Eucalyptus Extracts (Japanese Patent Laid-Open No. Hei 6-65278, 1994.3.8), Antioxidants Showing Active Oxygen Scavenging Activity (JP 05186768, July 27, 1993) A number of known antioxidants (JP 05186769, July 27, 1993) are known, but newer substances that have more powerful and non-toxic free radical scavenging activity are required.If such substances are found, they are used to develop treatments for various diseases or to prevent aging. It will be very useful as a cosmetic additive and food additive.

The inventors of the present invention, while searching for new active oxygen scavengers from various natural products such as microorganisms, mushrooms or medicinal plants, found a spirotype compound having a new hispidine skeleton from the extract of the brier mushroom (Inonotus xeranticus) and having excellent active oxygen scavenging ability The present invention was completed by identifying the physicochemical properties of the substance, establishing the preparation method and confirming the pharmacological activity.

Accordingly, an object of the present invention is to isolate and provide a hispidine-based compound having excellent active oxygen scavenging action from an extract of Inortus xeranticus. Another object of the present invention is to provide a method for extracting and separating the hispidine-based compound.

The object of the present invention is to identify the pure compound obtained by purifying the extract extracted from the staple fruiting fruit body by mass spectrum, ultraviolet absorption spectrum, infrared absorption spectrum, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum to identify as hispidine-based compound After determining the structure thereof, the biofilm peroxidation inhibitory activity, superoxide radical scavenging activity, tertiary butylperoxy radical scavenging activity and 1-1-diphenyl-2-picrylhydrazyl (1,1-Diphenyl- 2-picrylhydrazyl: DPPH) radical scavenging ability was measured to determine the excellent active oxygen scavenging activity and safety of the hispidine-based compound of the present invention.

Hereinafter, the configuration and operation of the present invention.

Figure 1 shows the high-speed atomic impact mass spectrum of the hispidine-based active oxygen scavenging material of the present invention.

Figure 2 shows the ultraviolet absorption spectrum of the hispidine-based active oxygen scavenger of the present invention

Figure 3 shows the infrared spectrum of the hispidine-based active oxygen scavenger of the present invention.

Figure 4 shows the hydrogen nuclear magnetic resonance spectrum of the histidine-based active oxygen scavenger of the present invention.

Figure 5 shows the carbon nuclear magnetic resonance spectrum of the hispidine-based active oxygen scavenging material of the present invention.

The present invention is to extract the methanol extract and ethyl acetate extract from the staple mushroom fruiting body sequentially silica gel column chromatography, Sephadex LH-20 column chromatography, reverse phase column chromatography to prepare the histidine-based active oxygen scavenger step; Investigation of physicochemical properties and determination of chemical structural formulas by investigating the mass spectrum, ultraviolet absorption spectrum, infrared absorption spectrum, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum of the extracted histidine-based active oxygen scavenger step; Using the microsomes isolated from the liver of rats as a biofilm lipid source, the reaction solution of the present invention hispidine-based active oxygen scavenger and FeSO ₄ solution was terminated with trichloroacetic acid and the supernatant obtained by centrifugation of the reaction solution. After reacting with thiobarbituric acid, the reaction is measured using a spectrophotometer, the control material is treated in the same manner as above, and the amount of the reaction positive material is measured in the same manner. Comparing and investigating the biological membrane lipid peroxidation inhibitory ability; Superoxide radical scavenging ability was measured by measuring absorbance while reacting a phosphate-buffered solution in which xanthine and nitroblueterazolium were dissolved, the present hisidine-based active oxygen scavenger, and xanthine oxidase. Measuring; Staphylococcus aureus 209 (Staphylococcus aureus 209) fungus suspension and hemoglobin were mixed with the histidine-based active oxygen scavenger and the control of the present invention, respectively, and reacted with the addition of tertiary butyl peroxide, followed by mannitol. Investigating tertiary butylperoxy radical scavenging ability by measuring the minimum concentration of the sample to disinfect the phenol red medium and completely inhibit the sterilization of radicals while overnight culture; After mixing the DPPH solution and the present invention hispidine-based active oxygen scavenger dissolved in methanol, the light absorbance is measured and the light absorbance of the control compound is measured in the same manner to compare the DPPH radical scavenging ability.

The hispidine compound of the present invention contains a spirotype skeleton and is extracted by water or an organic solvent (alcohol, ether, acetone, etc.) from mushrooms belonging to basidiomycete as shown by the following formula (I): A well-known method which can be used for isolation | extraction extraction of mushroom components, such as ethyl and other organic solvents and various column chromatography, can be obtained individually or in mixture suitably, and crude extract can be refine | purified more purely by a conventional method as needed.

Hispidin-based compounds containing the spirotype skeleton of the present invention have the action of scavenging free radicals and free radicals and exhibit stronger antioxidant activity than other known compounds similar to the compounds of the present invention. In addition to the first field can be very useful in the field of physiological activity associated with the peroxidation reaction of biological membrane lipids.

The hispidine-based compound containing the spirotype skeleton of the present invention exhibits excellent active oxygen scavenging activity and is also very safe, and can be used as various medicines using excellent in vivo antioxidant activity in addition to food or cosmetics as an antioxidant. Do. When used as a medicament, each compound specified in the present invention is used as an active ingredient, and the corresponding inorganic or organic carrier is added to form a solid, semi-solid or liquid form, and to be formulated not only for oral administration but also for parenteral administration such as trauma. Can be. Formulations for oral administration may include tablets, pills, granules, capsules, pellets, powders, granules, powders, emulsions, suspensions, syrups, and the like. Parenteral dosages include injections, drops, sap, Ointments, sprays, suspensions, emulsions, suppositories, and the like.

In order to formulate the active ingredient of the present invention can be formulated relatively easily according to the conventional method, surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents and the like can also be appropriately selected according to the conventional method.

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

Example 1 Preparation of the Hispidine Compound of the Invention

500 g of the dried brier (Inonotus xeranticus) fruiting body was finely pulverized with a grinder, extracted with 8 L of 80% methanol, concentrated under reduced pressure, and extracted again with ethyl acetate, and then divided into an ethyl acetate layer and a water layer. The ethyl acetate layer was mixed with chromoform-methanol 60: 1, 20: 1, 10: 1, and subjected to concentration gradient silica gel column chromatography, followed by active fractionation. Sephadex LH-20 column chromatography, reversed phase (ODS) ) Column chromatography was sequentially performed, followed by ODS TLC using 67% methanol as a solvent, and active fractions were HPLC using 60% methanol to obtain 10.3 mg of purely purified yellow compound.

Example 2: Identification and Physicochemical Properties of Hispidine Compounds of the Invention

Characterization, molecular weight, molecular formula, mass spectrometry, photoluminescence, ultraviolet absorption spectrum, infrared absorption spectrum, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum of the yellow compound extracted and purified in Example 1 were identified and catabolized The chemical properties were investigated. Experimental results are as shown below, and from this result, the compound of the present invention was identified as a hispidine-based compound containing a spirotype skeleton.

Form of material: Yellow powder

Molecular Weight: 462

Molecular Formula: C ₂₅ H ₁₈ O ₉

Mass spectrometry (FIG. 1): HRFAB-MS ((M + H) ⁺ , m / z found 463.1045, calculated 463.1029]

Luminous intensity: [α] _D = 0 (c = 9.0, CH ₃ OH)

UV absorption spectrum (FIG. 2) (UVλmax nm (ε) in MeOH)

262 (11,822), 389 (11,568)

Infrared absorption spectrum (FIG. 3) (IRυmax in KBr)

3430, 1700, 1695, 1593, 1549, 1384, 1276, 1127 cm ^-1

Hydrogen Nuclear Magnetic Resonance Spectrum (Fig. 4): δ ppm (600 MHz, CD ₃ OD)

1.98 (s, Me-1 '), 5.57 (s, H-2'), 5.65 (s, H-5 '), 6.50 (s, H-4), 6.59 (dd, J = 8.1, 1.6, H -11 '), 6.71 (d, J = 1.6, H-7'), 6.72 (d, J = 15.9, H-6), 6.75 (d, J = 8.1, H-10 '), 6.79 (d, J = 8.2, H-12), 7.00 (dd, J = 8.2, 1.8, H-13), 7.08 (d, J = 1.8, H-9), 7.44 (d, J = 15.9, H-7)

Carbon nuclear magnetic resonance spectrum (FIG. 5): δ ppm (150 MHz, CD ₃ OD)

20.0 (Me-1 '), 94.5 (C-4'), 95.5 (C-4), 95.9 (C-5 '), 99.5 (C-2), 105.1 (C-2'), 115.0 (C- 9), 115.1 (C-7 '), 115.5 (C-10'), 115.8 (C-12), 116.6 (C-6), 120.2 (C-11 '), 122.7 (C-13), 123.2 ( C-6 '), 128.5 (C-8), 139.9 (C-7), 146.3 (C-8'), 147.0 (C-10), 147.8 (C-9 '), 149.5 (C-11), 160.6 (C-1), 167.0 (C-5), 176.8 (C-3), 192.9 (C-1 '), 203.1 (C-3')

Chemical structure

Example 3 Inhibition of Biofilm Lipid Peroxidation of the Hispidine Compound of the Present Invention

In this example, microsomes isolated from livers of rats were used as biological membranes in order to measure the lipid peroxidation inhibitory activity of the inventive hispidine-based compounds. Microsomes were separated by fractional centrifugation. That is, the livers of the rats were extracted, washed 2 to 3 times in a 0.25 M sucrose solution at 4 ° C, and then chopped. 0.1 mM EDTA mixed solution was added and ground using a tissue grinder. This was centrifuged at 7,000g for 10 minutes, the supernatant was taken, ultracentrifuged at 77,000g for 60 minutes, and the precipitate was taken out and washed three times with Tris-HCl buffer. This was suspended in tris-hydrochloric acid buffer and the protein was quantified by Lowry method to make 1 mg / mL. The liver microsomes of rats isolated as above were used as biological membrane lipid sources to measure lipid peroxidation inhibitory activity of the samples. The reaction was initiated by adding 10 μl of microsomal solution, 10 μl of sample in each concentration dissolved in methanol, 0.5 mL of 100 mM Tris-hydrochloric acid buffer (pH 7.5), 450 μl of distilled water, and 30 μl of 4mM FeSO ₄ solution. . The reaction was carried out for 30 minutes in a 37 DEG C water bath and the reaction was stopped by adding 0.3 mL of 3M trichloroacetic acid-2N hydrochloric acid 1: 1 mixture. After centrifugation at 3000g for 10 minutes, 1mL of the supernatant was added, and 0.3mL of 0.67% thiobarbituric acid (TBA) was added and reacted in a boiling water bath for 10 minutes. Absorbance at wavelength 530 nm was measured using a spectrophotometer to calculate lipid peroxidation inhibitory activity (%) according to the following formula. As a positive control, vitamin E and butylated hydroxy anisole (BHA) were used.

A: absorbance without the inhibitor

B: Absorbance of Without Inhibitors and Microsomes

C: absorbance of the inhibitor

As a result of the experiment, the lipid peroxidation inhibitory activity of the histodine compound of the present invention is shown in Table 1 below, with the concentration of 50% inhibiting lipid peroxidation (IC ₅₀ ) of the microsomes extracted from the rat liver. The compound of the present invention was slightly higher than the control compound 1 in biofilm lipid peroxidation inhibition and was found to be significantly higher than the control compound 2.

Example 4 Superoxide Radical Scavenging Activity of the Hispidine Compounds of the Present Invention

In order to investigate the superoxide radical scavenging ability of the hispidine-based compound of the present invention, xanthine (2 mM) dissolved in 50 mM, pH7.8 potassium phosphate buffer, per well of a 96-well microplate 100 μl of the mixed solution of nitroblueterazolium (NBT (0.1 mM)) and 5 μl of the sample of each concentration of the control substance including the invention hispidine dissolved in methanol, followed by 4 units / mL of xanthine oxidase (xanthine oxidase). ) 30 μl was added to initiate the reaction, and then immediately measured for absorbance at 570 nm with a microplate reader, and reacted for 20 minutes. Superoxide radical scavenging activity was calculated by calculating the absorbance change (A ₂₀ -A ₀ ) by subtracting the absorbance value of (A ₀ ) immediately after the start of the reaction from the absorbance (A ₂₀ ) after 20 minutes of reaction. The concentration of the sample for reducing the change in absorbance by 50% was set to IC ₅₀ as compared to the control without the addition. As a positive control, vitamin E and butylhydroxyanisole (BHA) were used.

A: Change in absorbance without the sample

B: Absorbance change of the sample

Result, the superoxide radical scavenging activity of the present invention Heath blood as to, in terms of the superoxide radical scavenging activity of dingye compound with IC ₅₀ shown in Table 2, the present invention Heath blood dingye compound showed a superior is superior compared to the control substance .

Example 5: tert-butylperoxy radical (t-butylperoxy radical, t-BOO.) Scavenging ability of the hispidine-based compound of the present invention

Staphylococcus aureus 209 strains were inoculated in 5 mL of brain-heart infusion medium and incubated overnight at 37 ° C., followed by centrifugation (1,000 × g). 3 times with PBS (pH 7.2). The bacteria were suspended in PBS to prepare 1 × 10 ⁷ CFU / mL. 70 μl of the microbial suspension for each well of the 96-well microplate, 10 μl of the sample of each concentration of the present invention and the control compound, and 30 μl of 1 mg / mL hemoglobin were mixed well, followed by 50 mM tertiary butyl peroxide (t -BOOH) was added and the mixture was immediately mixed to start the reaction, and the reaction solution was incubated at 37 ° C for 30 minutes. The reaction was stopped by adding 5 µl of mannitol phenol red medium (6 g of beep extract, 20 g of peptone, 10 g of salt, 20 g of mannitol, 0.07 g of phenol red, 1 L of water, pH 7.4). The mannitol phenol red medium was dispensed into 100 µl / well in a 96-well microplate, and then 50 µl of the above-described sterilization reaction solution was incubated at 37 ° C overnight. The medium in the well containing the viable bacteria by eliminating the tertiary butyl peroxy radical by the compound of the present invention on the plate, the pH was lowered by the growth of the bacteria, the color of the phenol red in the medium was changed from red to yellow. The tertiary butylperoxy radical scavenging ability of the compound was expressed as the minimum concentration (MIC) of the sample to completely inhibit the sterilization action of the radical, and vitamin E, BHA and probucol were used as a positive control. As a result, the tertiary butyl peroxy radical scavenging ability of the compound of the present invention was determined to be MIC, as shown in Table 3 below.

Example 6: 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of the hispidine-based compound of the present invention

1 mL of a solution of 0.15 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) dissolved in ethanol and the concentrations of the inventive hispidine-based compound and the reference compound dissolved in methanol After mixing 0.1 mL of the sample, the mixture was left for 30 minutes, and then the absorbance at 517 nm and the absorbance of the control group containing only methanol without the sample were measured to calculate DPPH radical scavenging ability according to the following formula. As a positive control, vitamin E and BHA were used.

A: absorbance without sample

B: absorbance of the sample

As a result, the DPPH radical scavenging ability of the histidine-based compound of the present invention showing the concentration of 50% scavenging DPPH radical (IC ₅₀ ) is shown in Table 4. It was found to be superior.

As described through the above embodiment, the present invention, hisspidin-based compound extracted from a staple mushroom (Inonotus xeranticus) has an antioxidant activity that strongly inhibits lipid peroxidation of microsomes extracted from rat liver and has a superoxide radical scavenging ability. , Tertiary butyloxy radical scavenging ability and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging effect has an excellent effect, and by changing the chemical formula of the present invention hispidine-based compound by changing the chemical formula Since one compound has the effect of obtaining a very useful invention in the food, cosmetics and pharmaceutical industries.

Claims (3)

A hispidine-based compound having an antioxidant activity that inhibits lipid peroxidation and whose chemical structure is represented by the following formula (I). Use of the hispidine-based compound according to claim 1 as an active oxygen scavenger in vivo. After crushing the fruiting body of Inertus mushroom (Inonotus xeranticus), methanol was extracted, concentrated under reduced pressure, and extracted again with ethyl acetate. The ethyl acetate layer was separated into an ethyl acetate layer and a water layer. The double ethyl acetate layer was concentrated on silica gel column using a chloroform-methanol mixed solvent. Chromatography was performed to obtain an active fraction, which was then purified sequentially using Sephadex LH-20 column chromatography and reverse phase (ODS) column chromatography, and then the active fraction obtained by performing ODS TLC with methanol as a solvent was recovered. A method for producing a hispidine compound according to claim 1, which is separated and purified by HPLC with 60% methanol.