JP2006240991A - Antioxidant using extract of green algae belonging to the genus monostroma as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-oxidant extractable from green algae belonging to the genus Monostroma with water, suitable as a food, etc., and having an antioxidant property and a high stability, and a method for evaluating the quality of the green algae. <P>SOLUTION: This extract of the green algae belonging to the genus Monostroma is characterized by being obtained as its dissolved fraction by subjecting the green algae belonging to the genus Monostroma of Monostromaceae under an extraction process with a polar solvent, and containing a substance showing a maximum absorption wavelength at 270 nm in an acidic condition. The purified substance is characterized by having the following (a) to (d) physicochemical properties. (a) A compound having ≤300 molecular weight. (b) Having 270 nm maximum absorption wavelength in an acidic condition. (c) Having a thermal stability at 100°C temperature. (d) Having a pH stability in an environment of pH 1-13. The beverages, foods, and medicinal and cosmetic compositions containing the above extract or purified substance are provided. The method for evaluating the quality of the green algae by measuring the color values of the green algae is also provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an extract of human alga green algae having an antioxidant activity obtained by extracting green alga belonging to the class of human exothera with a polar solvent such as water or alcohol, and a purification isolated as an active ingredient from the extract. And its use in the fields of food, medicine, cosmetics and the like. The present invention also relates to a simple method for evaluating the quality of green algae.

  Oxidation is said to be associated with deterioration of the quality of foods and with diseases such as aging due to active oxygen or cancer in vivo. Today, prevention against oxidation or active oxygen is attracting a great deal of attention in terms of various lifestyle-related diseases, aging prevention, health maintenance and preventive medicine, and the like. In addition, it is said that the main cause of diseases such as cancer and lifestyle-related diseases is oxidative stress (Nippon Food Science and Technology Journal Vol. 52, No. 1, 7-18, 2005) (Non-patent Document 1). Conventionally, various synthetic or natural antioxidants have been studied in order to prevent or suppress such oxidation or active oxygen, but they are obtained from nature for use in food or medicine taken by humans. That is particularly desirable.

  As a naturally-occurring antioxidant, Japanese Patent Application Laid-Open No. 2001-288078 (Patent Document 1) discloses that D-cystenol isolated from Aosa has a hydroxy radical generation inhibitory action and a singlet oxygen inhibitory action, It is disclosed that it is useful for health foods, pharmaceuticals and the like.

  Japanese Journal of Home Economics Vol.39, No.11, 1173-1178 (1988) (Non-Patent Document 2) extracts lipid categories from seven kinds of seaweeds of blue paste and contains lipids in these lipid categories. And that the main antioxidant is pheophytin.

  Japanese Patent Application Laid-Open No. 9-67266 (Patent Document 2) discloses a hyaluronidase inhibitor containing, as an active ingredient, an extract of seaweed belonging to the genus Human exosa, Aosa.

  Japanese Patent Application Laid-Open No. 2003-176213 (Patent Document 3) describes that extracts of human rush and bitter gourd are useful as hair restorers.

JP 2001-288078 A JP-A-9-67266 JP 2003-176213 A Japan Society for Food Science and Technology Vol.52, No.1, 7-18 (2005) Journal of Japanese Society for Home Economics Vol.39, No.11, 1173-1178 (1988)

  Further, as far as the present inventors know, there is still a report on the method for evaluating the quality of green algae by estimating the presence or degree of antioxidant activity of green algae or the content of proteins, amino acids, aroma components and pigment components. It has not been. Although the seaweed simmered is a traditional Japanese food, it is not known about the antioxidant properties of the human rush which is its raw material. An object of the present invention is to provide a highly stable antioxidant substance having an antioxidant action that can be extracted from green algae belonging to human rush with water suitable for foods and the like, and uses thereof. Another object of the present invention is to provide a method capable of evaluating the quality of green algae by a simple method.

  The present inventors examined the extract of a green algae belonging to the genus Aegyceae by subjecting it to an extraction step with a polar solvent such as water. As a result, a water-soluble fraction having a maximum absorption wavelength at 270 nm when acidic. Based on this finding, the present invention has been completed.

That is, the present invention provides a human exothera green algae extract having an antioxidant activity, a purified product isolated from the extract as an active ingredient, and an antioxidant (or oxidative stress inhibitor) having the following constitution. Is related to its use.
(1) It is obtained as a soluble fraction of an extraction solvent by subjecting green algae belonging to the genus Aegeanaceae to an extraction step with a polar solvent, and contains a substance having a maximum absorption wavelength at 270 nm when acidic. Characterized by a green extract of the genus Human exothera.
(2) The green algae extract according to (1) above, which is obtained by subjecting a green algae belonging to the genus Aurelia to an extraction step with water, a lower alcohol or a mixture thereof.
(3) The green algae extract according to (1) or (2) above, wherein the green algae belonging to the genus Aurelia is Monostroma nitidum .
(4) An isolated and purified product that can be obtained from the extract of the genus Human exothera described in any one of (1) to (3) above, wherein the following physicochemical properties (a) to (d) are obtained: A purified product characterized by comprising:
(A) A compound having a molecular weight of 300 or less.
(B) It has a maximum absorption wavelength at 270 nm when acidic.
(C) It has thermal stability to a temperature of 100 ° C.
(D) It has pH stability with respect to the environment of pH 1-13.
(5) An antioxidant comprising, as an active ingredient, the human exothera green algae extract described in any one of (1) to (3) above or the purified product described in (4) above.
(6) A food / beverage product having an antioxidative effect, comprising the human exothera green algae extract described in any one of (1) to (3) above or the purified product described in (4) above.
(7) A pharmaceutical or cosmetic composition comprising the human exothera green algae extract described in any one of (1) to (3) above or the purified product described in (4) above.
(8) The color value of the green algae is measured, and based on the obtained color value, the green algae is selected from the group consisting of antioxidant activity (or degree), protein, amino acid, aroma component, and pigment component (content) A method for evaluating green algae, wherein the degree (including presence or absence) of one or more types of index items is estimated.
(9) The evaluation method as described in (8) above, wherein the green algae is a green algae belonging to the genus Human exothera.

  The extract or purified product of the human exothera genus Green alga of the present invention has an excellent antioxidant action by various methods for measuring antioxidant activity, and has high thermal stability and wide-range pH stability. Therefore, the above-mentioned water-soluble extract or purified product of the present invention, which is derived from nature, is useful as an antioxidant suitable for use in humans, and can prevent oxidation of health food or food itself having an antioxidant action. It can be used for various foods such as, pharmaceuticals for prevention or treatment of diseases related to active oxygen, or cosmetics capable of suppressing the influence of active oxygen. In the present invention, the human exothera green algae extract which is a water-soluble fraction, that is, a water-soluble fraction having a maximum absorption wavelength at 270 nm when acidic has an excellent antioxidant action and is resistant to heat and a wide range of pH. It is understood that it was unexpected to have extremely high stability. Furthermore, according to the quality evaluation method of the present invention, it is possible to estimate the presence or absence or degree of antioxidant activity of green algae, or the degree of inclusion of proteins, amino acids, aroma components and pigment components only by measuring the color values of green algae. Therefore, it is useful as a simple method for quality control or evaluation of green algae raw materials in the production of foods using green algae.

  The following describes the present invention in detail. As described above, the extract of the present invention is characterized in that it is obtained as a dissolved fraction by subjecting green algae belonging to the genus Hemiptera to the extraction step with a polar solvent. And an antioxidant having a maximum absorption wavelength at 270 nm.

Green algae belonging to Hitoegusa family Hitoegusa genus as a raw material in the present invention is not particularly limited, for example Hitoegusa (Monostroma nitidum), Hiro Hanoi Hitoegusa (Monostroma latissimum), but Mo luck Hitoegusa (Monostroma zostericola) and the like, Hitoegusa are preferred. These green algae include natural or aquaculture collected from coastal areas of Japan, but are generally marketed in a wet or dry state and can be used. For example, human egusa has been popular for a long time as a boiled or sprinkled seaweed, and usually grown in the coastal area south of Fukushima is frozen or air-dried. In practice, commercially available products can be used.

  As for the above-mentioned raw material of the genus Green, which is collected from the place of production, it may be used as it is in the extraction process, but it may be used in the extraction process as it is, or it may be fragmented or refined, preferably a dried product. Is desirable in terms of extraction efficiency. As a dried product of green algae, a dried material obtained by drying a wet raw material containing moisture using a dryer or the like can be used. Examples of the method for fragmenting the dried material include a method using an impact pulverizer, a ball mill, a pin mill, a jet mill, a freeze pulverizer, and the like, preferably under conditions that destroy the cell wall. In the case of using a wet raw material, the human rusha raw material before drying can be fragmented in the same manner as in the case of the dry product using, for example, a colloid mill, a mill mill or the like. The size of the raw material that has been cut into small pieces is not particularly limited, but the extraction efficiency is improved as it is smaller, and for example, the particle size can be about 3 mm or less.

  In the present invention, the extraction of the raw material of the genus Human exothera is performed using a polar solvent. Examples of the polar solvent include water and alcohols (for example, methanol, ethanol, etc.), but water and lower alcohols (especially having 1 to 3 carbon atoms) are preferable, and water and ethanol are more preferable in view of appropriateness in food use. Water is particularly preferred. These solvents are usually used alone, but can also be used as a mixed solvent. Although the usage-amount of the polar solvent with respect to green algae raw material (preferably fragmented material) is not specifically limited, Usually, 3 to 200 weight part with respect to 1 weight part of seaweed raw material (as dry matter), Preferably about 10 to 100 weight part It is.

  The extraction operation is usually performed by adding a human exothera green algae raw material to a polar solvent or adding a polar solvent to a green algae raw material and mixing them for extraction. Although it may be in a stationary state during extraction, the extraction efficiency can be increased and the extraction time can be shortened by stirring with a mixer or a stirrer.

  Although the extraction temperature in an extraction process is not specifically limited, Usually, 5-50 degreeC, Preferably it extracts at 10-30 degreeC. The extraction time is not particularly limited as long as the components of the green algae raw material are sufficiently extracted.Although it cannot be generally defined by the size of the algae raw material, the presence or absence of the use of a stirrer, When the separated green algae raw material and the solvent are mixed and allowed to stand, it is usually about 15 to 60 minutes, and when extracting with stirring with a stirrer such as a mixer, it is usually about 10 to 30 minutes.

  After the extraction step with a polar solvent, a fraction dissolved in the solvent is collected. This dissolved fraction can be collected as a still, for example, by centrifugation (usually about 7000 × g, about 10 to 15 minutes) or filtration using a filter according to the size of the green algae raw material. The solubilized fraction obtained by extraction as described above contains a substance having a maximum absorption wavelength at 270 nm and an excellent antioxidant activity when acidic in the ultraviolet spectrum, as will be described later.

  When the polar solvent is water, the soluble fraction obtained by the polar solvent extraction obtained as described above can be used as the human exothera green algae extract of the present invention as it is, but by concentrating or removing the solvent, The human exothera green algae extract can be obtained as a concentrate or a dried product. In addition, when a polar solvent other than water is used, an extract obtained by removing the normal solvent and drying it can be used as a human exothera green alga extract. The human exothera green algae extract of the present invention finally obtained in this way contains an antioxidant having a maximum absorption wavelength at 270 nm when acidic, and is usually used when water is used as the extraction solvent. Further, rhamnan sulfate and D-cystenoic acid are included. When a polar solvent other than water such as ethanol is used, D-cystenoic acid, chlorophylls, and carotenoids are usually included in addition to the antioxidant.

  The human exothera green algae extract according to the present invention obtained as described above is isolated and purified from a substance having a maximum absorption wavelength at 270 nm when acidic by using a normal separation or purification means. Can be obtained as a purified product. As such isolation / purification means, purification can be carried out using ordinary purification means, for example, various chromatography such as column chromatography, high performance liquid chromatography, affinity chromatography, etc. alone or in combination. . In order to further increase the degree of purification, for example, the extracted fraction is extracted from an ion exchange resin column (DOWEX2 × 8 (anion exchange type (exchange group: dimethylhydroxyethylbenzylammonium)), DOWEX50W (cation exchange type). (Exchange group: sulfonic acid) etc.), or pretreatment such as precipitating with alcohol (for example, methanol, ethanol, isopropanol, etc.) may be performed in advance.

In the present invention, purification is performed using high performance liquid chromatography (HPLC), as shown in Examples below. The use conditions of high performance liquid chromatography at this time are as follows. Column: Shodex Asahipak NH ₂ P-50 2D (Amino column, Showa Denko KK), mobile phase: 85% acetonitrile aqueous solution (containing 2.25 mM KH ₂ PO ₄ ), flow rate: 0.3 ml / min, temperature: 40 ° C. Detection wavelength: 244 nm, retention time: 5-6 minutes. The purified product of the present invention isolated and purified under the above conditions is a substance having a maximum absorption wavelength at 270 μm and an excellent antioxidant activity when acidic in the ultraviolet absorption spectrum (for example, about pH 3). . Moreover, as a result of analyzing this substance by a commonly used electron impact ionization mass spectrometry (EI-MS: Hitachi, Ltd., double focusing type), the purified product of the present invention which is an antioxidant substance Is a compound having a molecular weight of 300 or less, has a wide range of pH from acidic to alkaline (pH 1 to 13), and has extremely high thermal stability and pH stability that are stable even at a temperature treatment of 100 ° C. It has characteristics suitable for use in foods or pharmaceuticals.

  In the present invention, the antioxidant activity of the extract of the human exothera green alga is measured by a well-known method in the art, that is, a 1,1-diphenyl-2-picrylhydrazyl (DPPH) -HPLC method (for example, Bioscience) for measuring radical scavenging activity. , Biotechnology and Biochemistry 62, 1201-1204 (1998)), β-carotene fading method for measuring the degree of inhibition of disappearance of β-carotene (see, for example, Journal of the Japan Food Industry Association Vol.41, 611-618 (1994)) The antioxidant activity is confirmed by the ORAC method (for example, Journal of Agricultural and Food Chemistry 51, 3273-3279 (2003)) for measuring the active oxygen absorption capacity. The ORAC (Oxygen Radical Absorbance Capacity) method is a well-known method called an active oxygen absorption capacity analysis method developed by the US Department of Agriculture, and to what extent the added test substance prevents the disappearance of the fluorescent substance by radicals. This is a method for measuring whether or not it can be performed. In the examples described later, a method for confirming the antioxidant activity according to this ORAC method is specifically shown.

The human exothera genus green algae extract or product of the present invention has an action of suppressing active oxygen such as the ability to absorb active oxygen as described above, so that it can be used as an antioxidant for foods, beverages, pharmaceuticals, cosmetics, etc. Can be used alone or as an additive. The antioxidant of the present invention has the ability to absorb or suppress active oxygen as described above, thereby preventing, treating or improving a disease or condition associated with active oxygen. Diseases related to active oxygen that are effective by antioxidant or active oxygen absorption are well known and include arteriosclerosis, ischemic disease, diabetes, hyperlipidemia, nephritis, aging, various cancers, eye diseases, skin diseases, etc. Illustrated. Regarding such well-known diseases, for example, for the arteriosclerosis, Journal of the American College of Nutrition 22, 258-268 (2003)), for diabetes, Journal of Nutrition 133, 2125-2130 (2003)), and for aging Current Medicinal Chemistry 8, 815-828 (2001), Carcinogenesis 21, 1836-1841 (2000) for various cancers, Journal of the European Academy of Dermatology and Venereology 17, 663-669 (2003) for skin diseases, etc. The literature is given.

  When using the human exothera genus green algae extract or purified product of the present invention in foods, the foods and drinks having an antioxidant action alone (which may contain other additives) or as additives in various foods and drinks It can be. The food and drink having an antioxidant action according to the present invention can take various food forms such as a health food such as a nutritional supplement and a nutrition-enriched food, or a functional food having an antioxidant action or an active oxygen suppressing action.

  The food and drink of the present invention include carbohydrates, lipids, proteins, vitamins, amino acids, pH regulators, surfactants, fragrances, pigments, thickeners, etc., in addition to the extract or purified product of human exothera. Additives generally used in foods and drinks such as sugars and starches as a form can be used. The food and drink of the present invention can be used in various product forms such as tablets, capsules, powders, pills, powders, granules, syrups, and troches. Moreover, it can also be set as the food / beverage products of the form which added the extract or purified product of this invention to various other food / beverage products, such as a solid food, a semi-fluid food, and a drink. In another embodiment of the present invention, a food product obtained by adding a human exothera green algal extract or purified product to various other food products may be a food product that prevents or suppresses oxidative alteration of the underlying food product itself. The food or drink to which the extract or purified product of the present invention is added is not particularly limited. Examples of the solid food include bread, noodles, confectionery, and sprinkles. Examples of the semi-liquid food include jelly and tofu. Yokan and the like are exemplified, and examples of the beverage include soups, tea, and green juice. The food having an antioxidant action according to the present invention is various diseases or pathologies or lifestyle-related diseases related to active oxygen, that is, arteriosclerosis, ischemic disease, diabetes, hyperlipidemia, nephritis, aging, It can be improved by acting gently against various cancers, eye diseases, skin diseases and the like. Moreover, the foodstuff of this invention can be made into the food-drinks (food for specific health use etc.) which attached | subjected the indication to be used for the improvement of the above diseases or pathologies as a product form. When ingesting the food or drink of the present invention, the oral intake as the purified product of the present invention is generally about 0.1 to 20 mg per meal, or about 0.5 to 50 mg per day.

  In the present invention, the amount of human exothera genus green algae extract or purified product used for health food or functional food can vary depending on the form of the food, but the content of the purified product of the present invention is, for example, It is usually about 0.05 to 5% by weight with respect to the total amount of the solid food, is usually about 0.05 to 5% by weight with respect to the total amount of the semi-liquid food, and is usually 0.05 to 5% with respect to the total amount of the beverage. About 5% by weight. Further, the blending amount in the case where the extract of human genus green algae or purified product of the present invention is used as an antioxidant for food itself is usually 0.01 to as a content in the product of the present invention, for example, relative to the total amount of solid food. It is about 1% by weight, usually about 0.01 to 1% by weight with respect to the total amount of the semi-fluid food, and usually about 0.01 to 1% by weight with respect to the total amount of the beverage.

  When the human exothera genus green algal extract or purified product of the present invention is used as a medicine or a pharmaceutical composition, the medicine is an antioxidant or an active oxygen absorber, and may be an oxidative stress inhibitor in another aspect. Therefore, well-known diseases or lifestyle-related diseases related to active oxygen that are effective by antioxidants or active oxygen absorbers, that is, ischemic diseases, arteriosclerosis, diabetes, hyperlipidemia, nephritis, etc. It can be used as a prophylactic or therapeutic agent for diseases such as cancer, skin diseases and eye diseases. The extract or purified product of the present invention as an antioxidant can be administered by various administration routes such as oral administration, subcutaneous administration, intravenous administration, intramuscular administration, rectal administration, intranasal administration and ocular administration. . When the agent of the present invention is used as a medicine, an appropriate dosage form determined by the method of administration, purpose of administration, etc. of the extract or purified product of the present invention, such as tablets, powders, granules, capsules, ointments, creams, liquids In various forms such as injections. In order to produce these preparations, pharmaceutically acceptable carriers or diluents, specifically, usual solvents, solubilizers, tonicity agents, excipients, binders, lubricants, etc. are added. can do. It goes without saying that the dose of the pharmaceutical of the present invention varies depending on the administration method, the patient's condition, etc., and the appropriate amount and the number of times of administration are determined by a specialist. Specifically, for example, the purified product of the present invention As an adult, it is about 0.1 to 100 mg per day.

  When using the human exothera green algae extract or purified product of the present invention for a cosmetic composition, it inhibits or suppresses the effect of active oxygen on the skin or hair or the like by an antioxidant action or an active oxygen absorption action. Can do. The cosmetic composition of the present invention may be in the form of a cosmetic or cosmetic raw material. The cosmetics can take various forms such as creams, lotions, packs, shampoos, rinses, etc., which are blended with the extract or purified product of the human exothera genus of the present invention. It can be a raw material for use in the above-mentioned cosmetics containing a human exothera green algae extract or purified product as an active ingredient, and these can be produced by using a method generally used in this field. The compounding amount of the extract or purified product of the human exothera genus Green algae of the present invention can be appropriately set in consideration of the cosmetic form, the strength of antioxidant activity, the dose as the above-mentioned pharmaceutical, and the like.

  The present invention also measures the color value of green algae, and based on the obtained color value, one or more indicators selected from the group consisting of antioxidant activity, protein, amino acid, aroma component and pigment component for the green algae There is also provided a method for evaluating the quality of green algae characterized by estimating the degree or content of an item. That is, in this method, based on the color value obtained by measuring the color value of green algae, the presence or absence of antioxidant activity and the protein, amino acid (D-cystenoic acid, glutamic acid, alanine, etc.), aroma By estimating the content of related components (dimethyl sulfide (DMS), precursor dimethyl-β-propiothetin (DMPT), etc.) and pigment components (chlorophyll a, chlorophyll b, β-carotene, luitein, etc.), It is to evaluate quality. Since the degree or content of the above-mentioned index items may vary depending on the type, production area, etc., the quality of green algae can be easily evaluated by the method of the present invention. The above items for quality assessment of green algae can be appropriately selected as a single or a combination of items depending on the type of food or drink using green algae.

Although Subject to the green algae evaluation is not particularly limited, for example, green algae belonging to Hitoegusa family Hitoegusa genus (Hitoegusa (Monostroma Nitidum), Hiro Hanoi Hitoegusa (Monostroma Latissimum), Mo luck Hitoegusa (Monostroma Zostericola) etc.) and the like, In particular, human rush is preferred. In the evaluation method of the present invention, the green algae can itself be a food, but it is usually used as a raw material for other foods and drinks. Examples of the other foods and drinks include various solid foods, semi-fluid foods and beverages exemplified above for the foods and drinks of the present invention.

  The above evaluation method according to the present invention is measured using a color difference meter as shown in Example 5 (including the antioxidant activity of human rush and the correlation between the content of various components and the color value). The color value of the green algae is highly correlated with index items such as antioxidant activity, protein, amino acid, aroma component and pigment component content, that is, the higher the color value, the greater the content of the index item. It was completed by finding In the correlation confirmation test (Example 5), the antioxidant activity was measured by the DPPH-HPLC method, the β-carotene fading method, and the ORAC method as described above. The protein was measured using the Kjeldahl method, the amino acid was measured using the HPLC method, the aromatic component was measured using the HPLC method, and the dye component was measured using the HPLC method.

The color value of green algae can be measured by any appropriate method, but a color difference meter is generally used. The color difference meter used is not particularly limited, and a normal reflection type or transmission type can be used. For example, as a reflective color difference meter, S and M Color Computer (SUGA TEST INSTRUMENTS) and the like are exemplified. Color measurement using a color difference meter is usually performed by irradiating the surface of green algae with a light source, or irradiating a light source on a thin sliced algae and reading the color values (coordinate values). As color values, L ^* values (lightness), a ^* values (chromaticity), b ^* values (chromaticity), C ^* values (saturation), and h ^* values (hue angle) are usually used. In the present invention, these color values can be used alone or in combination for quality assessment of green algae. However, in Table 1 (Example 5) described later, the h ^* value has a particularly high correlation with the above index items. Therefore, when used alone, it is preferable to use the h ^* value for the quality evaluation of green algae. Moreover, when using the said color value in combination, the combination of h ^* value and c ^* value is preferable from the result of Table 1.

  In actual evaluation, for example, the relationship between the measured values of various green algae colors and the activity or content of each of the above index items is obtained in advance (a calibration curve, etc.), and the standard value of these activities or content and the corresponding color value By setting the reference value of the desired value to the desired value, it is possible to evaluate the quality quality or the ranking (for example, the higher the reference value, the higher the quality), using the reference value of the color value as a guide. . Thus, the evaluation method according to the present invention is extremely useful in quality control or evaluation of raw materials in the production of foods using green algae.

  In the present invention or the specification, unless otherwise specified, the% display means weight%.

[Example 1]
A commercially available air-dried human egusa (produced by Isewan) was pulverized into a particle size of about 3 mm using a hammer mill (Atomizer: manufactured by Seishin Enterprise Co., Ltd.), immersed in a solvent of water or alcohol (methanol or ethanol), and centrifuged ( The supernatant was recovered by 7000 × g) (extract of the present invention). In addition, the usage-amount of a human rusha and a solvent was 1:20 (weight ratio), and extraction conditions were 30 minutes at the temperature of 25 degreeC. The collected supernatant was used as a column with Shodex Asahipak NH ₂ O-50 2D (manufactured by Showa Denko KK), the mobile phase with 85% acetonitrile aqueous solution (2.25 mM KH ₂ PO ₄ ), flow rate 0.3 ml / min, temperature 40 The extract was obtained by subjecting it to HPLC under the conditions of 0 ° C. and a detection wavelength of 244 nm and collecting the peak (FIG. 1) appearing after 5 to 6 minutes (purified product of the present invention). In FIG. 1, a small peak of vitamin C was detected after the peak.

[Example 2]
Antioxidant activity of the extract obtained by the method of Example 1 was measured by ORAC method (see Journal of Agricultural and Food Chemistry 51, 3273-3279 (2003)). The ORAC method is a method for measuring how much an added substance can prevent the disappearance of a fluorescent substance by radicals. 2,2'-azobis (2-amidino-propane) dihydrochloride (hereinafter referred to as AAPH) which is a radical generator is adjusted to 63.42 μM, and fluorescein sodium salt (hereinafter referred to as FL) which is a fluorescent substance. Was prepared using a phosphate buffer (0.075 M, pH 7.0) to a concentration of 0.1 μM. 240 μl of the FL solution and 10 μl of the extract obtained by the method of Example 1 were mixed, 50 μl of the AAPH solution was added thereto, and measurement of fluorescence (measurement wavelength 485 nm, excitation wavelength 535 nm) was started immediately. Fluorescence was measured every 2 minutes until the fluorescence was lost. The area (AUC) represented by taking the fluorescence intensity on the vertical axis and the measurement time on the horizontal axis was calculated by the following equation (1). As a result, the extract had a larger AUC representing the intensity of activity compared to the control water (FIG. 2). Therefore, it was revealed that this extract has antioxidant activity.
AUC = (f0 + f1 + f2 + f3 + f4... Fi) / f0 (1)
(AUC: area under curve, fi: fluorescence intensity measured for the i-th time)

[Example 3]
As active substances contained in blue paste, pheophytin (Japanese Journal of Home Economics Vol.39, 1173-1178 (1988)) and D-cystenoic acid obtained from Aosa (JP 2001-288078 A) have already been reported. The ultraviolet absorption spectrum (maximum absorption wavelength 270 nm in acidic (pH 3.5)) of this extract is clearly different from pheophytin (FIG. 3). D-cystenoic acid exhibits a ninhydrin reaction, whereas the extract does not exhibit a ninhydrin reaction. In this extract, the peak of HPLC is different from that of vitamin C (FIG. 1), and the elution time of HPLC is also different from that of β-carotene (not shown). From the above, it was found that the above extract (product of the present invention) is a substance different from pheophytin, D-cystenoic acid, vitamin C or β-carotene. Moreover, the molecular weight of this extract was confirmed to be 300 or less by analysis using electron impact ionization mass spectrometry (EI-MS) (double focusing type: manufactured by Hitachi, Ltd.).

[Example 4]
Whether this extract can be used as a food or a pharmaceutical product was confirmed for stability against heat and pH. For thermal stability, the extract was treated at 25 ° C. and 80 ° C., 100 ° C. for 30 minutes. For pH stability, the extract was treated with 75 mM phosphate buffer (pH 7.0), 50 mM aqueous HCl (pH 1.0), and 50 mM aqueous NaOH (pH 13.1) for 30 minutes. The antioxidant activity of these was measured by the ORAC method, and it was revealed that the activity was not reduced even after heat treatment and a wide range of pH treatment, and the stability was very high (FIG. 4). From this result, it was shown that this extract can be widely used as food and medicine.

[Example 5] (Quality evaluation of green algae by color value measurement)
Antioxidant activity, protein, amino acid (D-cystenolic acid), DMPT and pigment components (chlorophyll a, chlorophyll b, β-carotene, lutein) content of human rusha (10 to 32 samples) of different origin or harvest time are measured The correlation between these and color values was investigated.
The human egusa was pulverized to a particle size of about 3 mm using a hammer mill (Atomizer: manufactured by Seishin Enterprise Co., Ltd.) and used for each measurement. Extraction was performed using methanol for measurement of antioxidant activity and pigment component, and water for measurement of amino acid and DMPT. In the extraction method, 10 ml of water or methanol was added to 0.2 g of human rush and homogenized, and then the supernatant was collected by centrifugation (7000 × g, 10 minutes, 5 ° C.). Water or methanol was added again to the residue, and the same treatment was performed. This operation was repeated a total of 3 times, and the resulting supernatant was diluted to 50 ml. Antioxidant activity was measured by DPPH-HPLC method, β-carotene fading method and ORAC method. The protein was measured by the Kjeldahl method, and D-cystenolic acid, DMPT, and the dye component were measured by HPLC. Below are the HPLC conditions. The color value was measured with S and M Color Computer (manufactured by SUGA TEST INSTRUMENTS).
When the correlation between the measured antioxidant activity and the content of various components and the color value was examined, it was revealed that there was a high correlation between them (Table 1). From this, quality control or evaluation of a raw material can be performed simply by measuring a color value.

HPLC conditions:
・ D-cystenoic acid column Shim-pack Amino-Na type (manufactured by Shimadzu Corporation)
Mobile phase A: 0.2N sodium citrate solution
B: 0.6N sodium citrate solution
C: 0.2N sodium hydroxide solution, flow rate 0.5ml / min
Detection o-phthalaldehyde fluorescence method
(Measurement wavelength 348 nm, excitation wavelength 450 nm)

・ DMPT
Column Hitachi gel # 2618 (manufactured by Hitachi Chemical Co., Ltd.)
Mobile phase 0.05% phosphoric acid solution Flow rate 1ml / min
Detection 210nm
DMPT is decomposed by adding a sodium hydroxide solution to a sample, and acrylic acid which is a decomposition product is measured.

・ Dye component Column Waters Atlantis dC18 (manufactured by Waters)
Mobile phase A: 95% ethanol aqueous solution (5 mM NaCl)
B: 60% ethanol aqueous solution (5 mM NaCl)
Flow rate 1.0ml / min
Detection 410nm, 438nm

  INDUSTRIAL APPLICABILITY The present invention is useful for applications in the fields of anti-oxidants containing, as an active ingredient, extracts of human exothera green algae or isolated and purified products, and foods, medicines, cosmetics and the like having an antioxidant or active oxygen suppressing effect. In addition, it is useful for quality control or evaluation of raw materials in the production of foods using green algae.

The chromatogram which shows the separation pattern by the high performance liquid chromatography of the human eggplant extract obtained in Example 1. FIG. The graph which shows the antioxidant activity of the human eggplant extract obtained in Example 1. The ultraviolet absorption spectrum of the human rush extract obtained in Example 1 and pheophytin. The graph which shows the heat stability of the human rush extract (Example 1) with respect to various temperature. The graph which shows the pH stability of the human rush extract (Example 1) with respect to various pH.

Claims (9)

  It is obtained as a soluble fraction of the extraction solvent by subjecting green algae belonging to the genus Epusaceae to an extraction step with a polar solvent, and contains a substance having a maximum absorption wavelength at 270 nm when acidic. , Human exothera green algae extract.   2. The human exothera green alga extract according to claim 1, which is obtained by subjecting a green alga belonging to the genus Rhizomeaceae to an extraction step with water, a lower alcohol or a mixture thereof. The green alga extract according to claim 1 or 2, characterized in that the green alga belonging to the genus Human exothera is Monostroma nitidum . It is an isolated and purified product obtainable from the extract of the human alga green algae described in any one of claims 1 to 3, and has the following physicochemical properties (a) to (d): Purified product.
(A) A compound having a molecular weight of 300 or less.
(B) It has a maximum absorption wavelength at 270 nm when acidic.
(C) It has thermal stability to a temperature of 100 ° C.
(D) It has pH stability with respect to the environment of pH 1-13.   The antioxidant which uses the human Exa genus green algae extract described in any one of Claims 1-3, or the purified product described in Claim 4 as an active ingredient.   The food / beverage products which have the antioxidant effect | action containing the human Exa green algae extract described in any one of Claims 1-3, or the purified product described in Claim 4.   A pharmaceutical or cosmetic composition comprising the human exothera green algae extract according to any one of claims 1 to 3 or the purified product according to claim 4.   The color value of green algae is measured, and based on the obtained color value, the degree or content of one or more index items selected from the group consisting of antioxidant activity, protein, amino acid, aroma component and pigment component for the green algae A method for evaluating the quality of green algae, characterized by estimating the degree.   The quality evaluation method according to claim 8, wherein the green algae is a green algae belonging to the genus Human exothera.

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