KR20030082275A - Novel antioxidant from Salicornia herbacea and its manufacture and use - Google Patents

Abstract

PURPOSE: A novel compound having an antioxidizing activity separated from Salicornia herbacea, its preparation method and an antioxidant composition containing the compound are provided, to apply the compound as an antioxidant and a health food. CONSTITUTION: The compound is represented by the formula 1. The method comprises the steps of drying Salicornia herbacea in a shady place, extracting the dried one with methanol, filtering it and concentrating the filtrate under reduced pressure to obtain a methanol extract; suspending the methanol extract in distilled water, extracting the suspension in shake with ethyl acetate, and obtaining the water layer; obtaining an active fraction by performing the adsorption chromatography with a methanol aqueous solution; and drying the active fraction under reduced pressure and performing HPLC with an acetonitrile aqueous solution at an acidic condition to obtain the compound of the formula 1.

Description

Novel antioxidant from Salicornia herbarium and its manufacture and use

The invention tungtung novel compounds having the antioxidant activity away from the node, relates to the preparation methods and uses, more particularly novel represented by the following formula (1) isolated from a type of Halophytes tungtung node (Salicornia herbacea) compound The present invention relates to a method for preparing the compound and to the use of a novel compound which can be used in various therapeutic agents and health foods related to the antioxidant action.

Aerobic organisms gain energy by breathing oxygen, the most abundant element on Earth, as their final electron acceptor. However, the basic triplet oxygen, which is oxygen essential for this life phenomenon but stable molecular state, produces free radicals of oxygen toxicity which are fatal to the living body due to physicochemical and environmental factors. Representative free radicals, which are the biggest problem in vivo, are generated from enzymes, reduction metabolism, chemicals, pollutants and photochemical reactions from the base triplet oxygen, a stable molecular state widely available in nature as active oxygen. As a product are O ₂ ^- (superoxide), H ₂ O ₂ (dihydro genpa oxide), OH and (hydroxyl radical), ¹ O ₂ (singlet oxy gen) etc., and these producing cells in the granular and cytoplasmic do. Free radicals are indispensable substances used in the sterilization of macrophages in vivo, removal of old proteins, etc., but they can exhibit harmful effects in vivo due to their high reactivity.

However, in the case of abnormality in the active oxygen defense mechanism or when the active oxygen is excessively increased by physical or chemical factors, the active oxygen may cause fatal damage to each part of the body. Because of this, it is known to cause various diseases such as brain diseases such as stroke, Parkinson's disease, heart disease, ischemia, arteriosclerosis, skin disease, digestive diseases, inflammation, rheumatism, autoimmune diseases, and other cancers. Antioxidants that remove free radicals or inhibit free radical production may be used for the prevention and treatment of various diseases caused by excessively produced free radicals and to inhibit skin aging.

Recently, the development of antioxidants as food additives, health foods for the prevention of various diseases, and the pharmaceutical and cosmetics industry for the purpose of inhibiting aging is diversified. Currently used antioxidants include synthetic antioxidants such as tert-butylhydroxytoluene (BHT) and tert-butylhydroxyaniso (BHA), and natural antioxidants such as tocopherol, carotenoids, flavonoids, tannins, and antioxidant enzymes such as SOD. Etc. are used. Synthetic antioxidants are limited to use due to the toxicity and the expensive price to the human body, it is necessary to develop a natural antioxidant that is harmless to the human body and shows a stronger antioxidant effect.

Among the natural antioxidants, the extracts of various spices, such as speamint, marjoram, sweet basil, and gagyo, have reported similar antioxidant effects to tocopherol and BHA, and sesameol (sesaminol) as an antioxidant component. , γ-tocopherol, trans-ferulic acid, and bisepoxyligan homologues were reported. Studies on the antioxidant effects from edible plants have shown that there is an antioxidant effect from the ethanol extract of Deodeok. Flavonoid puerarin was found to be an antioxidant from the roots, benzene-based antioxidants were isolated from edible seaweeds, salicylic acid, ρ-cumaric acid, Phenolic antioxidants such as gentisic acid and caffeic acid were isolated. In the methanol extract of rice hulls, flavonoid-based substances are present and they exhibit an antioxidant effect. The active substance was found to be C-glycosyl flavonoid. The ethanol extract of Rhus japonica, which is used as an herbal medicine, also showed a strong antioxidant effect. When used with tocopherol, synergistic effect was observed. In addition, various edible plants have been reported to have antioxidant activity in Schizandra chinensis, Kernel and mustard, Sansa.

Accordingly, the present inventors found an antioxidant activity in the plump node while searching for an antioxidant substance from the medicinal plant material and analyzed the structure by separating the antioxidant substance, thereby preparing a new compound represented by the formula (1). To complete.

An object of the present invention is to provide a novel compound exhibiting antioxidant activity isolated from Salicornia herbacea and a method for producing the same.

In addition, the present invention has another object to provide an antioxidant composition containing a novel compound showing an excellent antioxidant activity effect as an active ingredient, and a pharmaceutical or health food comprising the same.

Figure 1 shows the hydrogen nuclear magnetic resonance spectrum of the compound represented by the formula (1).

Figure 2 shows the carbon nuclear magnetic resonance spectrum of the compound represented by formula (1).

Figure 3 shows the two-dimensional nuclear magnetic resonance spectrum of the compound represented by the formula (1).

The present invention is characterized by a novel compound represented by the following formula (1) having an antioxidant activity isolated from spurs and a method of preparing the same.

In addition, it includes an antioxidant composition containing the compound as an active ingredient, and pharmaceuticals or health foods containing the composition.

Formula 1

Referring to the present invention in more detail as follows.

The present invention is isolated from Salicornia herbacea , which is a kind of salt plant, and then a new compound represented by the following formula (1), a method for preparing the compound, and the compound may be used in various therapeutic agents and health foods related to antioxidant activity. It relates to the use of the novel compounds.

The preparation method of the novel compound according to the present invention

1) extracting Salicornia herbacea , dried in the shade, extracted with methanol, filtered and the filtrate was concentrated under reduced pressure to obtain a methanol extract;

2) suspending the methanol extract in distilled water and shaking off with ethyl acetate to obtain a water layer;

3) desorption of the water layer with an aqueous methanol solution to obtain an active fraction;

4) drying the active fraction under reduced pressure, and performing high-speed liquid chromatography with acetonitrile aqueous solution under acidic conditions.

As a result of purifying the separated active fraction and analyzing the structure of the new compound by NMR, caffeic acid and 7,8-dihydrocaffeic acid form ester bonds among two hydroxyl groups among three hydroxyl groups of quinic acid. The molecular formula is C ₂₅ H ₂₆ O _{12 The} molecular weight is 518, it is represented by the formula (1).

Antioxidant activity of this compound was confirmed by performing a PDH reduction method. Therefore, the compound represented by Chemical Formula 1 may be usefully used to treat various diseases caused by harmful oxygen or free radicals as an active ingredient having an antioxidant effect.

Antioxidant composition comprising the compound as an active ingredient can be administered orally or parenterally, for example, intravenous, subcutaneous, intraperitoneal or topical in clinical administration, can be used in the form of general medicines and functional foods .

The antioxidant composition may be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Is formulated. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation into tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above. In addition, the pharmaceutical composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. To formulate into a parenteral formulation, the composition is prepared in solution by mixing in water with stabilizers or buffers and formulated into unit dosage forms of ampoules or vials.

Effective dosages of the compounds may vary depending on the age, physical condition, weight, etc. of the patient, but are generally administered within the range of 1-100 mg / kg body weight per day. In addition, within a daily effective dosage range is divided into once a day or several times a day.

Antioxidant composition comprising such a compound represented by the formula (1) is useful for preventing or treating diseases related to the antioxidant action.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Isolation of Antioxidant Active Substances in the Rite

The inventors used extraction and chromatography to separate antioxidants with strong antioxidant activity from the stalks.

First, 1.5 kg of the dried bark taken from the west coast tidal flats were crushed, extracted with 100% methanol for 3 days, and filtered under reduced pressure. Suspended. The methanol extract suspended in distilled water was shaken and extracted with 2 L of ethyl acetate in a separatory filter to obtain a water layer, and the water layer was concentrated under reduced pressure in a 60 ° C. water bath to remove ethyl acetate in the water layer. The above water layer, from which ethyl acetate was removed, was adsorbed on Diion HP-20 column chromatography equilibrated in distilled water, washed with water, and then extracted with active substance with 30% methanol to obtain an active fraction. . The obtained active fraction was concentrated under reduced pressure, dissolved in a small amount of methanol, and developed on a Sephadex LH-20 gel column chromatography equilibrated with methanol to obtain an active fraction, and the fraction was subjected to YMC gel ODS column chromatography. In column chromatography, high-performance liquid chromatography was performed using 20% acetonitrile aqueous solution as a solvent under acidic conditions.

Example 2 Structure Analysis of Antioxidants

The purified material was dissolved in methanol (CD ₃ OD) to determine the chemical shift of the proton and carbon of the following Table 1 through ¹ H-NMR, ¹³ C-NMR, two-dimensional NMR. In addition, ¹ H-NMR, ¹³ C-NMR, two-dimensional NMR spectrum results are shown in the accompanying drawings.

Chemical shifts of protons and carbon from isolated and refined materials Carbon number Chemical shift of proton NMR (ppm) Chemical shift of carbon NMR (ppm) Quinic acid One - 75.1 2 2.08,2.30 36.9 3 5.57 69.9 4 4.88 76.6 5 4.28 65.7 6 2.06,2.17 41.9 7 - 177.8 Caffeic acid One' - 127.0 2' 7.04 115.1 3 ' - 149.5 4' - 146.7 5 ' 6.77 116.5 6 ' 6.94 123.0 7 ' 6.26 115.2 8' 7.50 147.3 9 ' - 168.6 Dihydrocaffeic acid One" - 133.5 2" 6.65 116.3 3 " - 146.1 4" - 144.5 5 " 6.64 116.3 6 " 6.48 120.4 7 " 2.70 31.2 8" 2.55 37.2 9 " - 174.0

The separated and purified material is a compound in which caffeic acid and 7,8-dihydrocaffeic acid form ester bonds in two hydroxyl groups among three hydroxyl groups of quinic acid. The molecular formula is C ₂₅ H ₂₆ O ₁₂ and has a molecular weight of 518. . This compound is referred to as toongtoongmadic acid, 3- [3- (3,4-dihydroxy-phenyl0-acryloyloxy] -4 [3- (3,4-dihydroxy-phenyl) -propionyloxy] -1,5-dihydroxy- cyclohexanecarboxylic

acid), and its structure is represented by the following Chemical Formula 1.

Formula 1

Example 3: Confirmation of Antioxidant Activity of Purified Compounds

1,1-diphenyl-2-picrylhydrazyl, which contains free radicals in the molecule, readily accepts electrons or hydrogen radicals and becomes a stable molecule, thereby measuring free radical scavenging ability. The hydrogen donating ability of the sample was measured as follows. After dissolving 12 mg of DPI in 100 ml of ethanol, 100 ml of distilled water was added to make 0.5 mM DPI solution, and 200 µL of DPI solution was added to 2 µl of the sample in each separated tablet. After 30 minutes, the absorbance was measured at 517 nm using a spectrophotometer, and the absorbance decrease of the control group was expressed as antioxidant activity as a PDH radical scavenging activity. In this case, when the compound of Formula 1 was reacted with 2 μl of 0.5 mM DPI at a concentration of 50 μg / ml, the absorbance was reduced by 79% at 517 nm, which is 10 times better than that of alpha-tocopherol. It indicates activity.

Example 4: Toxicity Test

Toxicity experiments were carried out on the novel compounds according to the invention as follows.

The compound represented by Chemical Formula 1 was dissolved in dimethylsulfoxide (dimethylsulfoxide, DMSO), diluted with water, and then administered to the mice (10 mice per group) at 100 mg / kg, respectively, and observed for 7 days, but no mice died.

Formulation Example 1 Preparation of Syrup

Syrup containing the new compound according to the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) was prepared by the following method.

Acid addition salts, saccharin and sugars of the novel compounds were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

Formulation Example 2: Preparation of Tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

250 g of the novel compound according to the invention were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

Formulation Example 3: Preparation of Capsule

500 mg of a novel compound according to the present invention was filled into a hard gelatin capsule to prepare a capsule.

Formulation Example 4 Preparation of Injection

200 mg of the novel compound according to the present invention was heat-dissolved in 200 mg of physiological saline containing polyoxyethylene hydrogenated Castro oil to prepare an injection containing the mixed extract at a concentration of 0.1.

Formulation Example 5: Beverage Preparation

After dissolving 500 mg of the novel compound according to the present invention in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharides as an auxiliary component were added, and an appropriate amount of sodium benzoate was added as a preservative, followed by addition of water. 100 ml was prepared to prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

As can be seen from the above description, the novel compound according to the present invention is excellent in antioxidant activity, and thus it is useful to use as a therapeutic or health food for diseases related to antioxidant activity.

Claims (6)

A compound characterized by the following formula (1). Formula 1 1) extracting Salicornia herbacea , dried in the shade, extracted with methanol, filtered and the filtrate was concentrated under reduced pressure to obtain a methanol extract; 2) suspending the methanol extract in distilled water and shaking off with ethyl acetate to obtain a water layer; 3) desorption of the water layer with an aqueous methanol solution to obtain an active fraction; 4) A method for preparing a compound represented by the following Chemical Formula 1, comprising drying the active fraction under reduced pressure and performing high-performance liquid chromatography with an acetonitrile aqueous solution under acidic conditions. Formula 1 An antioxidant composition containing the compound represented by the following formula (1) as an active ingredient. Formula 1 Antioxidant, characterized in that consisting of the composition of claim 3. The antioxidant according to claim 4, which is formulated with the addition of a pharmaceutically acceptable excipient in the form of tablets, capsules, syrups, injections, suspensions or edible beverages. Health food, characterized in that consisting of the composition of claim 3.