JP4051482B2 - Pharmaceutical composition comprising α-glycosyl hesperidin - Google Patents

Description

  The present invention relates to a novel α-glycosyl hesperidin, a method for producing the same, and uses, and more specifically, α-glycosyl hesperidin in which D-glucose residue is α-bonded to equimolar or more in hesperidin, and hesperidin and α -A method for producing α-glycosyl hesperidin, characterized in that a glycosyltransferase is allowed to act on a solution containing a glucosyl sugar compound to produce novel α-glycosyl hesperidin, and this is collected. Various compositions such as beverages characterized by containing α-glycosyl hesperidin obtained, foods and beverages such as processed foods, prophylactic agents for sensitive diseases, anti-sensitive disease agents such as therapeutic agents, cosmetics such as skin beautifying agents, fair skin agents, etc. It relates to the use for things.

  Hesperidin has the chemical structure shown below, and has been known for a long time as vitamin P, which has physiological functions such as strengthening of capillaries, prevention of bleeding and blood pressure adjustment, and as a yellow pigment, and is used in foods, pharmaceuticals, cosmetics, etc. Has been.

Vitamin P is involved in the physiological activity of vitamin C in vivo, for example, the hydroxylation reaction of proline and lysine necessary for the synthesis of collagen which is the main component of biological connective tissue, and for example, Fe ^{++ of} cytochrome C. It is known to be involved in redox reactions such as reducing Fe to Fe ⁺⁺ , and further to enhance the immune response by increasing leukocytes, and plays an important role in maintaining and promoting the health of the body. is doing.

  The use of hesperidin is not limited to vitamin P fortifier as a nutrient, but due to its chemical structure and physiological function, it can be used alone or in combination with other vitamins, for example, yellow colorant, antioxidant, stabilizer. , For food and drink as a quality improver, UV absorber, etc., for prophylactic and therapeutic agents for sensitive diseases such as viral diseases, bacterial diseases, cardiovascular diseases, malignant tumors, ie anti-sensitive disease agents, It extends to cosmetics such as skin-beautifying agents and whitening agents as yellow colorants, stabilizers, antioxidants, ultraviolet absorbers, melanin production inhibitors, etc., and its application range is extremely wide.

  However, hesperidin is hardly soluble in water, and only 1 g (about 0.002 w / v%) is dissolved in 50 L of water at room temperature, making it extremely difficult to use.

  As a method for improving this, a method is known in which dimethyl sulfate is allowed to act on hesperidin to convert hesperidin into a methyl derivative to increase water solubility.

  However, this method is performed by an organic chemical method and uses dimethylsulfuric acid which is toxic to the reaction. Therefore, there is considerable difficulty in purifying the resulting derivative, and its nontoxicity, ensuring safety, economic efficiency, etc. It was not satisfactory in terms of. There is also a drawback that the resulting methyl derivative has a bitter taste.

  Realization of a hesperidin derivative that eliminates the disadvantages of conventional hesperidin or its derivatives, has excellent water solubility, is substantially tasteless, odorless, has no fear of toxicity, and can sufficiently exhibit physiological activity in vivo. It is strongly desired.

  The present invention is for overcoming the above-mentioned drawbacks, and is based on the results of intensive research aimed at a novel hesperidin derivative using a biochemical technique, among others.

  As a result, by allowing glycosyltransferase to act on a solution containing hesperidin and an α-glucosyl sugar compound, it is excellent in water solubility, substantially tasteless, odorless, without concern for toxicity, and easily in vivo. Found to produce a novel α-glycosyl hesperidin that is hydrolyzed and exhibits the physiological activity inherent in hesperidin, its production method, and its use in various compositions such as foods and drinks, prophylactic agents, therapeutic agents, and cosmetics for sensitive diseases To complete the present invention.

  In addition, when purifying α-glycosyl hesperidin produced by this transglycosylation reaction, it was also found that it can be easily purified by contacting the reaction solution with a porous synthetic adsorbent and utilizing the difference in adsorbability. The present invention has been completed.

  Therefore, the production method of α-glycosyl hesperidin according to the present invention eliminates the disadvantages of the prior art at once, and makes it very easy to realize its industrialization.

  As described in the text, α-glycosyl hesperidin in which D-glucose residue is α-bonded to equimolar or more to hesperidin, which is a novel substance of the present invention, is excellent in water solubility, substantially tasteless, odorless and toxic. Without concern, it is easily hydrolyzed into hesperidin and D-glucose in vivo, and exhibits the physiological activity inherent in hesperidin.

  In addition, since this α-glycosyl hesperidin can be easily produced by a biochemical method in which a glycosyltransferase is allowed to act on a solution containing hesperidin and an α-glucosyl sugar compound, it is excellent in economic efficiency and its industrial implementation. Easy.

  Furthermore, it was found that the reaction concentration of hesperidin can be increased and the concentration of α-glycosyl hesperidin can be easily generated at a high concentration. At the same time, when purifying the reaction solution, the reaction solution is made porous. It has also been found that α-glycosyl hesperidin can be purified by contact with a synthetic adsorbent, which greatly facilitates the mass production of α-glycosyl hesperidin.

  In addition, α-glycosyl hesperidin thus obtained has characteristics such as good water solubility, good light resistance and stability, and sufficient physiological activity, and is a highly safe natural vitamin P. Not only as a toughening agent, but also as a yellow colorant, antioxidant, stabilizer, quality improver, preventive agent, therapeutic agent, ultraviolet absorber, anti-degradation agent, etc., food and drink, favorite food, feed, feed, anti-sensitivity It is advantageously used for cosmetics such as disease agents, skin-beautifying agents, whitening agents, and plastic products.

  Therefore, the industrial production method of α-glycosyl hesperidin according to the present invention and the establishment of its use have great industrial significance in the food, beverage, cosmetic, pharmaceutical and plastic industries.

  Hereinafter, the present invention will be described in detail.

  Hesperidin used in the present invention is not limited to highly purified hesperidin, but a mixture of hesperidin and a flavonoid glycoside such as citronin, naringin, rutin, and various kinds containing hesperidin. Plant-derived extracts, juices, or partially purified products thereof can be used as appropriate.

  As plant tissue, for example, citrus fruits, pericarps, immature fruits and the like can be advantageously used.

  The α-glucosyl sugar compound used in the present invention may be any compound that can generate α-glycosyl hesperidin from hesperidin by a glycosyltransferase used at the same time. For example, starch such as amylose, dextrin, cyclodextrin, maltooligosaccharide A partially hydrolyzed product, further liquefied starch, gelatinized starch, and the like are appropriately selected.

  Therefore, in order to facilitate the production of α-glycosyl hesperidin, an α-glucosyl sugar compound suitable for glycosyltransferase is selected.

  For example, when α-glucosidase (EC 3.2.1.20) is used as a glycosyltransferase, malto-oligosaccharides such as maltose, maltotriose, maltotetraose, or starch having a partial hydrolysis of DE of about 10 to 70 are used. When a cyclomaltodextrin glucanotransferase (EC 2.4.1.19) is used, a starch partially hydrolyzed product having a DE of about 60 from cyclodextrin or starch gelatinized product having a DE of 1 or less is suitable. When α-amylase (EC 3.2.1.1) is used, a starch gelatinized product of DE1 or less to a dextrin of about DE 30 or a partially hydrolyzed starch is preferable.

  The α-glucosyl sugar compound concentration during the reaction is preferably in the range of about 0.5 to 50 times that of hesperidin.

  The hesperidin-containing liquid during the reaction is preferably one containing hesperidin as high as possible. For example, hesperidin is suspended or dissolved at a high temperature, or dissolved at an alkaline pH exceeding 7.0. A solution containing a high concentration of hesperidin is suitable, and the concentration of hesperidin is a high concentration of about 0.005 w / v% or more, preferably about 0.01 to 10.0 w / v%. It means the solution that is.

  The glycosyltransferase used in the present invention produces α-glycosyl hesperidin without decomposing hesperidin when acting on a solution containing hesperidin and an α-glucosyl sugar compound having properties suitable for the enzyme. I just need it.

  For example, α-glucosidase is used in pig liver, buckwheat seeds, etc., plant tissue-derived enzymes, fungi belonging to the genus Mucor, Penicillium, or the genus Saccharomyces. An enzyme derived from a culture obtained by culturing a microorganism such as yeast belonging to a nutrient medium, cyclomaltodextrin glucanotransferase is an enzyme derived from a bacterial culture belonging to the genus Bacillus, Klebsiella, etc. As the α-amylase, a bacterium belonging to the genus Bacillus or the like, an enzyme derived from a mold culture belonging to the genus Aspergillus or the like can be appropriately selected. These glycosyltransferases do not necessarily need to be purified and used as long as the above conditions are satisfied. Usually, the object of the present invention can be achieved with a crude enzyme.

  If necessary, it may be purified by various known methods. Commercially available glycosyltransferases can also be used. The amount of enzyme used and the reaction time are closely related, and the enzyme amount is usually selected so that the reaction is completed in about 5 to 80 hours from the viewpoint of economy. Further, it is also possible to appropriately select to use the immobilized glycosyltransferase repeatedly in a batch system and in a reaction or in a continuous system in a continuous reaction.

  In the reaction method of the present invention, it is desirable to allow glycosyltransferase to act in a state where the concentration of hesperidin is increased. For example, when reacting hesperidin in a suspended state, a hesperidin-rich solution containing about 0.1 to 2.0 w / v% suspended hesperidin and an appropriate amount of an α-glucosyl sugar compound is added at a pH of about 4.5 to 6.5, and maintained at a temperature as high as possible at which glycosyltransferase can act, specifically about 70 to 90 ° C. When glycosyltransferase is allowed to act on this, hesperidin is converted into α-glycosyl hesperidin. Suspended hesperidin gradually dissolves as it is converted, and at the same time α-glycosyl hesperidin is readily produced in high concentrations.

  Further, for example, when hesperidin is reacted on the alkali side exceeding pH 7.0, about 0.2 to 5.0 w / v% hesperidin is dissolved by heating in water having a pH of about 7.5 to 10.0, A solution containing a high amount of hesperidin obtained by dissolving an appropriate amount of an α-glucosyl sugar compound in this solution is as high as possible at the pH and high temperature at which glycosyltransferase can act. Specifically, the pH is about 7.5 to 10.0, and the temperature. When maintained at about 50 to 80 ° C. and allowed to act on glycosyltransferase, α-glycosyl hesperidin is easily produced at a high concentration. At this time, since hesperidin in the alkaline solution is liable to be decomposed, it is desirable to keep it as light-shielded and anaerobic as possible in order to prevent this. If necessary, an antioxidant such as L-ascorbic acid or erythorbic acid may coexist.

  Furthermore, a method of combining the above conditions, for example, a hesperidin-rich solution containing about 0.5 to 10.0 w / v% hesperidin and an appropriate amount of α-glucosyl sugar compound is adjusted to a pH of about 7.5 to 10.0. When the temperature is maintained at about 50 to 80 ° C. and a glycosyltransferase is allowed to act thereon, α-glycosyl hesperidin is easily produced at a high concentration.

  Further, as hesperidin, for example, about 0.1 to 1.0 N caustic soda aqueous solution, caustic potash aqueous solution, sodium carbonate aqueous solution, calcium hydroxide water, aqueous ammonia, etc., about 0.5 to 10.0 w / A solution dissolved at a high concentration of v% is added, and an aqueous acid solution such as hydrochloric acid or sulfuric acid is added thereto to adjust to a pH at which the enzyme can act, preferably a pH exceeding 7.0, and α-glucosyl sugar It is very convenient to add a compound and immediately act on glycosyltransferase, since α-glycosyl hesperidin can be easily produced at a high concentration.

  At this time, even if the hesperidin solution dissolved at high concentration is adjusted with an acidic aqueous solution, hesperidin is likely to precipitate. Therefore, before adjusting the pH, α-glucosyl sugar compound or a small amount of α-glycosyl hesperidin may be added. It is also possible to advantageously carry out the transglycosylation reaction while suppressing the precipitation of hesperidin together.

  In addition, if necessary, in order to increase the solubility of hesperidin before the reaction and facilitate the transglycosylation reaction to hesperidin, an organic solvent that can be dissolved in water with hesperidin-rich solution such as methanol, ethanol, n The coexistence of lower alcohols such as -propanol, iso-propanol, n-butanol, acetol, and acetone, and lower ketones can also be appropriately selected.

  Moreover, the relatively high molecular weight α-glycosyl hesperidin produced by the transglycosylation reaction may be used as it is or after purification with a porous synthetic adsorption resin, after which glucoamylase (EC 3.2.1.3) is used. Or by partial hydrolysis with an amylase such as β-amylase (EC 3.2.1.2) to reduce the number of α-D-glucosyl groups of α-glycosyl hesperidin. For example, when glucoamylase is allowed to act, a high-molecular substance higher than α-maltosyl hesperidin can be hydrolyzed to produce glucose and to accumulate and produce α-glucosyl hesperidin, and also to act on β-amylase. In some cases, α-maltotriosyl hesperidin or higher polymer can be hydrolyzed to produce maltose, and a mixture of α-glucosyl hesperidin and α-maltosyl hesperidin can be accumulated and produced.

  The reaction solution in which α-glycosyl hesperidin is produced as described above can be directly used as an α-glycosyl hesperidin product. Usually, the reaction solution is filtered and concentrated to form a syrup, and further dried and powdered to a powdered α-glycosyl hesperidin product.

  This product is not only used as a vitamin P fortifier, but also as a highly safe natural yellow colorant, antioxidant, stabilizer, quality improver, preventive agent, therapeutic agent, ultraviolet absorber, etc. It can be advantageously used for various compositions such as taste products, feeds, feeds, anti-sensitive diseases, cosmetics, and plastic products.

  Furthermore, when producing a purified α-glycosyl hesperidin product, the α-glycosyl hesperidin is separated from impurities such as an α-glucosyl sugar compound by utilizing the difference in adsorptivity by the porous synthetic adsorbent. What is necessary is just to refine.

  The porous synthetic resin referred to in the present invention is a synthetic resin such as a porous, wide adsorption surface area, and nonionic styrene-divinylbenzene polymer, phenol-formalin resin, acrylate resin, and methacrylate resin. For example, commercially available brand names manufactured by Rohm & Haas, Amberlite XAD-1, Amberlite XAD-2, Amberlite XAD-4, Amberlite XAD-7, Amberlite XAD-8, Amberlite XAD-11 , Amberlite XAD-12, trade name manufactured by Mitsubishi Chemical Industries, Ltd. Diaion HP-10, Diaion HP-20, Diaion HP-30, Diaion HP-40, Diaion HP-50, manufactured by IMACTI Product name Imakti Syn-42, Imakti Syn-44 And Immacy Syn-46.

  The method for purifying a reaction solution in which α-glycosyl hesperidin according to the present invention is produced is as follows. When the reaction solution is passed through, for example, a column packed with a porous synthetic adsorbent, α-glycosyl hesperidin and a relatively small amount of unreacted hesperidin. Adsorbs to the porous synthetic adsorbent, whereas the α-glucosyl sugar compound and water-soluble saccharide that coexist in large quantities flow out as they are without being adsorbed.

  If necessary, after completion of the reaction of glycosyltransferase and before contacting with the porous synthetic adsorbent, for example, the reaction solution is heated to remove insoluble matter, or magnesium aluminate silicate, Treat with magnesium aluminate to adsorb and remove proteinaceous substances in the reaction solution, or treat with strongly acidic ion exchange resin (H type), medium basic or weakly basic ion exchange resin (OH type), etc. It is also optional to use a combination of purification methods such as desalting.

  As described above, the α-glycosyl hesperidin selectively adsorbed on the porous synthetic adsorbent column and the relatively small amount of unreacted hesperidin are washed with a dilute alkali, water, etc., and then a relatively small amount of organic solvent or If a mixed liquid of an organic solvent and water, for example, methanol water, ethanol water, or the like is passed, α-glycosyl hesperidin is eluted first, and if the amount of liquid passed is increased or the concentration of the organic solvent is increased, unreacted hesperidin is It will elute.

  This eluate containing a large amount of α-glycosyl hesperidin is distilled, and after the organic solvent is distilled off, it is concentrated to an appropriate concentration to obtain a syrup-like product containing α-glycosyl hesperidin as a main component. Further, by drying and pulverizing this, a powdered product mainly composed of α-glycosyl hesperidin can be obtained.

  The elution operation of α-glycosyl hesperidin and unreacted hesperidin with the organic solvent also serves as a regeneration operation of the porous synthetic adsorbent, thereby enabling repeated use of the porous synthetic adsorbent.

  Further, the purification by the porous synthetic adsorbent of the present invention has a feature that not only α-glucosyl sugar compounds and water-soluble saccharides but also impurities such as water-soluble salts can be removed at the same time. The α-glycosyl hesperidin thus obtained has the following features.

(1) Compared with hesperidin, α-glycosyl hesperidin is extremely water-soluble. Moreover, (alpha) -glycosyl hesperidin suppresses precipitation of hesperidin from hesperidin containing aqueous solution, and prevents the cloudiness of this solution. Accordingly, α-glycosyl hesperidin can be advantageously used as a stabilizer that can suppress precipitation of hesperidin contained in citrus fruit juice and prevent white turbidity.
(2) α-Glycosyl hesperidin has higher light resistance and stability than hesperidin.
(3) α-glycosyl hesperidin is hydrolyzed to hesperidin and glucose by an enzyme in the body, and exhibits the physiological activity (vitamin P) inherent to hesperidin. Unlike other vitamins and antioxidants such as thiamine, riboflavin, vitamin C, and vitamin E, α-glycosyl hesperidin is substantially tasteless and odorless, and there are concerns about discoloration, browning, and off-flavor generation. It is also convenient to use α-glycosyl hesperidin in combination with one or more of vitamins and antioxidants. Moreover, the combined use with vitamin C can enhance their physiological activity.
(4) In the case of a product containing an α-glucosyl sugar compound, not only the effect of α-glycosyl hesperidin is exhibited, but the α-glucosyl sugar compound may exhibit shaping, a bulking effect and a sweetening effect. In the case of a purified product from which the α-glucosyl sugar compound has been removed, the effect of α-glycosyl hesperidin can be exerted with almost no shaping or increase, and it is virtually tasteless and odorless, so it is free. Can be seasoned and scented.

  From these features, α-glycosyl hesperidin is not only a highly safe natural vitamin P enhancer, but also a yellow colorant, antioxidant, stabilizer, quality improver, viral disease, bacterial disease, circulation As preventives, treatments, UV absorbers, etc. for sensitive diseases such as organ diseases and malignant tumors, foods and beverages, foods, feeds, feeds, anti-sensitive disease agents, skin beautifying agents, whitening agents, etc., and plastic products Etc., and can be advantageously used for various compositions.

  In addition, α-glycosyl hesperidin is well harmonized with various substances having tastes such as acidity, salt to taste, astringency, umami, and bitterness, and has high acid resistance and heat resistance. For example, seasonings, Japanese confectionery, Western confectionery, ice confectionery, beverages, spreads, pastes, pickles, canned bottles, processed meat products, processed fish / fishery products, processed milk / eggs, processed vegetables, processed fruits, processed cereals, etc. Can be used in a wide range. In addition, it is also convenient to mix and use feeds, feeds, and the like for domestic animals such as domestic animals, poultry, bees, cormorants, and fish for the purpose of enhancing vitamin P and improving palatability.

  In addition, various solids and pastes such as tobacco, troches, liver oil drops, complex vitamins, mouth fresheners, mouth-flavored tablets, mouthwashes, tube nutrients, oral medicines, injections, toothpaste, lipstick, lip balm, sunscreen It can also be advantageously used in combination with cosmetics such as anti-sensitive disease agents, skin-beautifying agents, skin-whitening agents, and the like. It can also be advantageously carried out by blending it into a plastic product or the like as an absorbent or a deterioration inhibitor.

  In addition, the susceptibility disease referred to in the present invention is a disease that is prevented or treated by α-glycosyl hesperidin, and examples thereof include viral diseases, bacterial diseases, traumatic diseases, immune diseases, rheumatism, diabetes. It may be a cardiovascular disease, a malignant tumor or the like. The preventive agent and therapeutic agent for α-glycosyl hesperidin can be freely selected depending on the purpose. For example, liquids such as sprays, eye drops, nasal drops, gargles, injections, ointments, patches, pastes such as creams, solids such as powders, granules, capsules and tablets. In the preparation, if necessary, it is used in combination with one or more other ingredients such as therapeutic agents, physiologically active substances, antibiotics, adjuvants, bulking agents, stabilizers, coloring agents, flavoring agents, etc. It is also optional to do.

  The dose can be appropriately adjusted depending on the content, administration route, administration frequency and the like. Usually, α-glycosyl hesperidin is preferably in the range of about 0.001 to 10.0 grams per adult day.

  In the case of cosmetics, they can be used according to the aforementioned preventive and therapeutic agents.

  As a method of using α-glycosyl hesperidin, known methods such as mixing, kneading, dissolution, dipping, infiltration, spraying, coating, spraying, pouring, etc. are required until the products of these various compositions are completed. Is appropriately selected.

  Hereinafter, an example of α-glycosyl hesperidin according to the present invention will be described by experiments.

(Experiment 1)
<Preparation of α-glycosyl hesperidin>
(1) Transglycosylation reaction 5,000 parts by weight of water is added to 1 part by weight of hesperidin and 6 parts by weight of dextrin (DE20), heated and dissolved, and then cyclomaltodextrin derived from Bacillus stearothermophilus Glucanotransferase (sales by Hayashibara Biochemical Laboratories Co., Ltd.) was added at 20 units per gram of dextrin and reacted at pH 6.0 and 70 ° C. for 18 hours to convert about 70% of hesperidin to α-glycosyl hesperidin. Thereafter, the mixture was inactivated by heating to obtain a reaction solution containing unreacted hesperidin together with α-glycosyl hesperidin.

(2) Purification The reaction solution obtained by the method of (1) is filtered, and the filtrate is passed through a column filled with a porous synthetic adsorbent and trade name Diaion HP-10 (Mitsubishi Kasei Kogyo Co., Ltd.) with SV2. did. After washing this column with water, 50 v / v% ethanol was passed through, the eluate was concentrated to distill off the solvent, and powdered to obtain a pale yellow α-glycosyl hesperidin sample [I] as a starting material. The yield was about 130% based on the weight of hesperidin.

(3) Hydrolysis by amylase The α-glycosyl hesperidin sample [I] obtained by the method of (2) was dissolved in water at 1 w / v%, and this was dissolved in glucoamylase (EC 3.2.1.3, raw Chemical Industry Co., Ltd.) was added 100 units per gram of the sample, and the reaction was continued for 5 hours while maintaining the pH at 5.0 and 55 ° C. The reaction solution was heated to inactivate the enzyme, filtered, and the filtrate was passed through a column of a porous synthetic adsorbent, trade name Diaion HP-10 (sold by Mitsubishi Kasei Kogyo Co., Ltd.) with SV2. As a result, α-glycosyl hesperidin and unreacted hesperidin in the solution were adsorbed on the porous synthetic adsorbent, and glucose, salts, and the like flowed out without being adsorbed. Next, the column was flushed with water, washed, and then passed while gradually increasing the concentration of the aqueous ethanol solution. The α-glycosyl hesperidin fraction was collected, concentrated under reduced pressure, powdered, and light yellow α-glycosyl. A hesperidin sample [II] was obtained in a yield of about 70% based on the weight of the raw material hesperidin per solid.

  Further, in place of glucoamylase, β-amylase (EC 3.2.1.2, sold by Seikagaku Corporation) was used to add α-glycosyl hesperidin sample [I] according to the above method. By decomposing, purifying, concentrating and pulverizing, a pale yellow α-glycosyl hesperidin sample [III] was obtained in a yield of about 70% based on the weight of the raw material hesperidin.

(Experiment 2)
<Physicochemical properties of α-glycosyl hesperidin>
(1) Improvement of water solubility The α-glycosyl hesperidin-containing reaction solution prepared by the transglycosylation reaction by the method of Experiment 1 (1) and the enzyme used in this method are heated and deactivated in advance to obtain the method of Experiment 1 (1). The control solution treated in the same manner was allowed to stand in a cold room at 4 ° C. for 2 days. In the control solution, hesperidin precipitated and became cloudy, whereas the α-glycosyl hesperidin-containing reaction solution remained transparent. .

  Therefore, the α-glycosyl hesperidin produced by the transglycosylation reaction is remarkably improved in water solubility. In other words, α-glycosyl hesperidin suppresses the precipitation of unreacted hesperidin that coexists in the aqueous solution, and prevents the solution from becoming clouded.

(2) Solubility in solvents The α-glycosyl hesperidin sample is readily soluble in water, 0.1N caustic soda and 0.1 constant hydrochloric acid, slightly soluble in methanol and ethanol, and insoluble in ether, benzene and chloroform.

(3) Ultraviolet absorption spectrum α-glycosyl hesperidin sample was dissolved in 0.1N caustic soda solution and the ultraviolet absorption spectrum was examined, and either of sample [I], sample [II] or sample [III] Also had an absorption maximum in the vicinity of 286 nm as in the case of hesperidin.

(4) Infrared absorption spectrum The infrared absorption spectrum of the α-glycosyl hesperidin sample was examined by the KBr tablet method. The results of the α-glycosyl hesperidin sample [I] are shown in FIG. 1, and the results of the sample [II] are shown in FIG.

(5) Stability to hydrolysis (a) α-glycosyl hesperidin preparation is hydrolyzed by α-glucosidase (EC 3.2.1.20) derived from pig liver to produce hesperidin and D-glucos. To do.
(B) It is not hydrolyzed by β-glucosidase.

(6) Thin layer chromatography (a) Analytical method Plate: Merck
Product name Kieselgel 60F ₂₅₄
Developing solvent; n-butanol: acetic acid: water = 4: 2: 1
Color developing agent: 1 w / w% ceric sulfate 10 w / w% sulfuric acid aqueous solution (b) Analysis result When α-glycosyl hesperidin sample was analyzed, hesperidin spot of Rf 0.69 was obtained in the case of sample [I]. In addition, a new spot is recognized at Rf 0.48, 0.34, 0.22, 0.16, 0.10, 0.04 and the origin, and in the case of the standard [II], Rf 0.48. In the case of the sample [III], spots of Rf 0.48 and 0.34 were recognized.

  From the above physicochemical properties, the substance showing Rf0.48 contained in the specimen [I], the specimen [II] and the specimen [III] has 1 mole of D-glucose residue α-bonded to 1 mole of hesperidin. α-Glucosyl hesperidin (also known as hesperidin monoglucoside), a substance showing Rf0.34 contained in the sample [I] and the sample [III] is 2 mol of D-glucose in 1 mol of hesperidin Α-diglucosyl hesperidin (also known as hesperidin diglucoside), and similarly, a substance showing a plurality of spots of Rf 0.22 or less contained in the sample [I] has a D-glucose residue in 1 mol of hesperidin. Is α-oligoglucosyl hesperidin that is α-linked by 3 mol or more.

  As described above, α-glycosyl hesperidin of the present invention is a novel hesperidin sugar derivative with good water solubility in which D-glucose residue is α-bonded to hesperidin in an equimolar amount, and when ingested in vivo, α-glucosidase It is easily hydrolyzed by, and exhibits the physiological activity inherent in hesperidin.

(Experiment 3)
<Acute toxicity>
The α-glycosyl speridine preparation [I] prepared by the method of Experiment 1 (2) was orally administered using a 7-week-old dd mouse, and an acute toxicity test was conducted. There were no deaths. Therefore, the toxicity of this substance is extremely low. In addition, when this test was conducted in the same manner using the α-glycosyl hesperidin sample [II] prepared by the method of Experiment 1 (3), it was found that the same results as described above were obtained and the toxicity was extremely low. did.

  As examples of the present invention, examples of production of α-glycosyl hesperidin are described in Example A, and examples of use of α-glycosyl hesperidin in various compositions are described in Example B.

(Example A-1)
<Α-glycosyl hesperidin>
1 part by weight of hesperidin is dissolved in 4 parts by weight of 1N caustic soda solution, neutralized with 0.01N hydrochloric acid solution and 4 parts by weight of dextrin (DE10) is added, and immediately derived from Bacillus stearothermophilus Cyclomaltodextrin glucanotransferase (available from Hayashibara Biochemical Laboratories Co., Ltd.) was added at 20 units per gram of dextrin, maintained at pH 6.0 and 75 ° C., and reacted for 24 hours with stirring. When the reaction solution was analyzed by thin layer chromatography, about 70% of hesperidin was found to be α-glucosyl hesperidin, α-diglucosyl hesperidin, α-triglucosyl hesperidin, α-tetraglucosyl hesperidin, α-pentaglucosyl hesperidin, etc. It was converted to glycosyl hesperidin. The reaction solution is heated to inactivate the enzyme, filtered, and the filtrate is desalted and purified with an ion exchange resin (H-type and OH-type) according to a conventional method, concentrated, and syrup-like α-glucosyl sugar compound An α-glycosyl hesperidin product containing was obtained in a yield of about 90% based on raw material weight per solid.

  This product is not only used as a vitamin P fortifier, but also as a highly safe natural yellow colorant, antioxidant, stabilizer, quality improver, preventive agent, therapeutic agent, UV absorber, etc. It can be advantageously used for various compositions such as taste products, feeds, feeds, anti-sensitive disease agents, cosmetics, and plastic products.

(Example A-2)
<Α-Glucosyl hesperidin>
1 part by weight of an α-glycosyl hesperidin product having a syrup-like α-glucosyl sugar compound prepared according to the method of Example A-1 was dissolved in 4 parts by weight of water, and glucoamylase (EC 3.2.1) was dissolved therein. .3, sold by Seikagaku Corporation) was added at 100 units per gram of α-glycosyl hesperidin product solid, and reacted at 50 ° C. for 5 hours. When the reaction solution was analyzed by thin layer chromatography, α-glycosyl hesperidin was converted to α-glucosyl hesperidin.

  The reaction solution was heated to inactivate the enzyme, filtered, and the filtrate was passed through a column of a porous synthetic adsorbent, trade name Diaion HP-10 (sold by Mitsubishi Kasei Kogyo Co., Ltd.) with SV2. As a result, α-glucosyl hesperidin and unreacted hesperidin in the solution were adsorbed on the porous synthetic adsorbent, and glucose, salts, and the like flowed out without being adsorbed. Next, the column was passed with water and washed, and then passed while gradually increasing the concentration of the aqueous ethanol solution. The α-glucosyl hesperidin fraction was collected, concentrated under reduced pressure, powdered, and powdered α-glucosyl. Hesperidin was obtained in a yield of about 60% based on the weight of the raw material hesperidin per solid.

  When α-glucosyl hesperidin was hydrolyzed with acid, 1 mol of hesperidin produced 1 mol of L-rhamnose and 2 mol of D-glucose, and α-glucosyl hesperidin was extracted from pig liver and partially purified. It was found that when α-glucosidase was allowed to act, it was hydrolyzed to hesperidin and D-glucose.

  This α-glucosyl hesperidin is a highly purified highly water-soluble vitamin P fortifier, and as a yellow colorant, antioxidant, stabilizer, quality improver, preventive agent, therapeutic agent, ultraviolet absorber, etc. It can be advantageously used for various compositions such as foods and drinks, foods, anti-sensitive diseases, and cosmetics.

(Example A-3)
<Α-glycosyl hesperidin>
1 part by weight of hesperidin is dissolved in 500 parts by weight of water at a pH of 9.5, and 10 parts by weight of dextrin (DE8) is dissolved in 10 parts by weight of water. Then, these solutions are mixed and mixed with cyclomaltodextrin gluca Notransferase was added at 30 units per gram of dextrin and maintained at pH 8.2, 65 ° C., and reacted for 40 hours with stirring. When the reaction solution was analyzed by thin layer chromatography, about 80% of hesperidin was converted to α-glycosyl hesperidin. The reaction solution was heated to inactivate the enzyme and then filtered. The filtrate was passed through a column of a porous synthetic adsorbent, trade name Amberlite XAD-7 (manufactured by Rohm & Haas) at SV1.5.

  As a result, α-glycosyl hesperidin and unreacted hesperidin in the solution were adsorbed on the porous synthetic adsorbent, and dextrin, oligosaccharide, salts, and the like flowed out without being adsorbed. The column was flushed with water, washed, and then passed through 50 v / v methanol to elute α-glycosyl hesperidin and hesperidin, which was concentrated and powdered to obtain a powdered α-glycosyl hesperidin product. The yield was about 120% based on the weight of the raw material hesperidin.

  This product is not only used as a highly water-soluble vitamin P fortifier, but also as a highly safe natural yellow colorant, antioxidant, stabilizer, quality improver, preventive agent, therapeutic agent, ultraviolet absorber, etc. It can be advantageously used for various compositions such as foods and drinks, foods, feeds, feeds, anti-sensitive diseases, cosmetics, and plastic products.

(Example A-4)
<Α-glycosyl hesperidin>
(1) Preparation of α-glucosidase preparation Maltose 4 w / v%, potassium phosphate 0.1 w / v%, ammonium nitrate 0.1 w / v%, magnesium sulfate 0.05 w / v%, potassium chloride 0.05 w / v Into 500 parts by weight of a liquid medium consisting of v%, polypeptone 0.2 w / v%, calcium carbonate 1 w / v% (separately sterilized by dry heat and added aseptically at the time of inoculation) and water, Mucor Javanicus (Mucor javanicus) IFO 4570 was cultured with shaking at a temperature of 30 ° C. for 44 hours. After completion of the culture, the mycelium was collected, 500 parts by weight of 4 M urea solution dissolved in 0.5 M acetate buffer (pH 5.3) was added to 48 parts by weight of the wet mycelium, and the mixture was allowed to stand at 30 ° C. for 40 hours. And then centrifuged. The supernatant was dialyzed overnight in running water, and then saturated with ammonium sulfate, left to stand overnight at 4 ° C., and the produced salted-out product was collected by filtration, and 50 parts by weight of 0.01 M acetate buffer (pH 5.3). After suspending and dissolving, the supernatant was collected by centrifugation and used as an α-glucosidase preparation.

(2) Preparation of α-glycosyl hesperidin 5 parts by weight of hesperidin was dissolved in 100 parts by weight of 0.5N caustic soda solution, and 0.01N hydrochloric acid solution was added thereto to adjust the pH to 9.5, and dextrin (DE30) 20 parts by weight was added, and immediately 10 parts by weight of the α-glucosidase preparation prepared by the method (1) was added, and the mixture was reacted at 55 ° C. for 40 hours while maintaining the pH at 8.5. When the reaction solution was analyzed by thin layer paper chromatography, about 0% of hesperidin was converted to α-glycosyl hesperidin. The reaction solution was purified in the same manner as in Example 3, concentrated and pulverized to obtain a powdered α-glycosyl hesperidin product at a yield of about 110% based on the weight of the raw material hesperidin.

  As in Example A-3, this product is not only a highly water-soluble vitamin P fortifier, but also a highly safe natural yellow colorant, antioxidant, stabilizer, quality improver, It can be used for various compositions as a prophylactic agent, therapeutic agent, ultraviolet absorber, and the like.

(Example B-1)
<Hard candy>
Reduced maltose syrup (Hayashibara Shoji Co., Ltd., registered trademark Mabit) 1,500 parts by weight, concentrated under reduced pressure to a water content of about 2% or less, to which 15 parts by weight of citric acid and Example A-3 1 part by weight of powdered α-glycosyl hesperidin obtained by the method and a small amount of lemon flavor were mixed, and then molded and packaged according to a conventional method to obtain a hard candy.

  This product is a yellow lemon candy with fortified vitamin P, which has low caries and low calories.

(Example B-2)
<Boiled buffalo>
Peel off the cypress, cut it to an appropriate length, immerse it in a thin saline solution for several hours, and mix it with syrup-like α-glycosyl hesperidin obtained by the method of Example A-1 and Blue No. 1. It was boiled with a liquid containing the prepared green colorant to obtain a boiled green buffalo.

  This product adds color as a material for various Japanese-style dishes and also exhibits physiological effects as dietary fiber.

(Example B-3)
<Fertilization>
1.2 parts by weight of water is mixed with 1 part by weight of mochi seed starch, and gelatinized by heating, 1.5 parts by weight of sugar and 0.7 parts by weight of crystalline β-maltose (manufactured by Hayashibara Co., Ltd., registered trademark Sun Malt) Then, 0.3 part by weight of chickenpox and 0.2 part by weight of syrup-like α-glycosyl hesperidin obtained by the method of Example A-1 were mixed and thereafter shaped and packaged in accordance with a conventional method to produce a fertilizer.

  This product is a kiwi-dango-style Japanese confectionery with good fertilization in both flavor and mouthfeel.

(Example B-4)
<Mixed sweetener>
100 parts by weight of honey, 50 parts by weight of isomerized sugar, 2 parts by weight of brown sugar and 1 part by weight of powdered α-glycosyl hesperidin obtained by the method of Example A-4 were mixed to obtain a mixed sweetener.

  This product is a sweetener enriched with vitamin P and is suitable as a health food.

(Example B-5)
<Sand cream>
1,200 parts by weight of crystalline α-maltose (produced by Hayashibara Co., Ltd., registered finetose), 1,000 parts by weight of shortening, 10 parts by weight of powdered α-glycosyl hesperidin obtained by the method of Example A-3, lecithin 1 part by weight, 1 part by weight of lemon oil and 1 part by weight of vanilla oil were mixed by a conventional method to produce a sand cream.

  This product is a vitamin P-enriched, yellow-colored sand cream that is good in mouthfeel, melting, and flavor.

(Example B-6)
<Tablets>
After uniformly mixing 20 parts by weight of ascorbic acid with 13 parts by weight of crystalline β-maltose, 4 parts by weight of corn starch and 3 parts by weight of powdered α-glucosyl hesperidin obtained by the method of Example A-2, the diameter was 12 mm, 20R. Tableting was performed using a scissors.

  This product is a complex vitamin preparation of ascorbic acid and α-glucosyl hesperidin, which has good ascorbic acid stability and is easy to drink.

(Example B-7)
<Capsule>
10 parts by weight of calcium acetate monohydrate, 50 parts by weight of L-magnesium lactate trihydrate, 57 parts by weight of maltose, 20 parts by weight of α-glucosyl hesperidin obtained by the method of Example A-2 and 20% of eicosapentaenoic acid After 12 parts by weight of the contained γ-cyclodextrin inclusion compound was uniformly mixed and granulated by a granulating machine, it was encapsulated in a gelatin capsule according to a conventional method to produce a capsule containing 150 mg of one capsule.

  This product can be advantageously used as a blood cholesterol lowering agent, an immunostimulant, a skin beautifying agent, etc., as a prophylactic agent, a therapeutic agent, a health promotion food, etc. for sensitive diseases.

(Example B-8)
<Ointment>
1 part by weight of sodium acetate / trihydrate and 4 parts by weight of DL-calcium lactate are uniformly mixed with 10 parts by weight of glycerin, and this mixture is mixed with 50 parts by weight of petroleum jelly, 10 parts by weight of wax, 10 parts by weight of lanolin, and sesame oil 14 In addition to a mixture of 5 parts by weight, 1 part by weight of α-glycosyl hesperidin obtained by the method of Example A-4 and 0.5 part by weight of mint oil, the mixture was further uniformly mixed to prepare an ointment.

  This product can be advantageously used as a sunscreen, skin beautifying agent, whitening agent, etc., and further as a healing accelerator for wounds and burns.

(Example B-9)
<Injection>
The α-glucosyl hesperidin obtained by the method of Example A-2 was dissolved in water, purified and filtered by conventional methods to make pyrogen free, and this solution was added to a 20 mL ampoule so that α-glucosyl hesperidin was 50 mg. This was dispensed in vacuo, dried under reduced pressure and sealed to produce an injection.

  This injection can be administered alone or mixed with other vitamins, minerals and the like intramuscularly or intravenously. In addition, this product does not need to be stored at a low temperature and has extremely good solubility in physiological saline and the like when used.

(Example B-10)
<Injection>
6 parts by weight of sodium chloride, 0.3 parts by weight of potassium chloride, 0.2 parts by weight of calcium chloride, 3.1 parts by weight of milk sodium, 45 parts by weight of maltose and α-glucosyl hesperidin 1 obtained by the method of Example A-2 Part by weight was dissolved in 1,000 parts by weight of water, purified and filtered according to a conventional method to make pyrogen-free, and this solution was filled in 250 mL sterilized plastic containers to produce an injection.

  This product is not only supplemented with vitamin P, but also an injection for caloric supplementation and mineral supplementation, and can be advantageously used to promote treatment and recovery during and after illness.

(Example B-11)
<Tube nutrients>
Crystalline α-maltose 20 parts by weight, glycine 1.1 parts by weight, sodium glutamate 0.1 part by weight, sodium chloride 1.2 parts by weight, citric acid 1 part by weight, calcium lactate 0.4 part by weight, magnesium carbonate 0.1 A formulation consisting of 0.1 parts by weight of α-glycosyl hesperidin obtained by the method of Example A-3, 0.01 parts by weight of thiamine and 0.01 parts by weight of riboflavin is prepared. 24 g of this blend was filled in a laminated aluminum sachet and heat sealed to prepare a tube nutrient.

  The tube feeding agent can be advantageously used as an oral or parenteral nutritional solution for the nasal cavity, stomach, intestine, etc. by dissolving one bag in about 300 to 500 mL of water.

(Example B-12)
<Bath agent>
21 parts by weight of DL-sodium lactate, 8 parts by weight of sodium pyruvate, 5 parts by weight of α-glycosyl hesperidin obtained by the method of Example A-1 and 40 parts by weight of ethanol were mixed with 26 parts by weight of purified water and coloring and flavoring agents. Mixed with appropriate amount, bath preparation was produced.

  This product is suitable as a skin-beautifying agent and a light-whitening agent, and it may be used by diluting 100 to 10,000 times in hot water for bathing. As in the case of bathing hot water, this product can be advantageously used by diluting it into facial cleansing water, lotion, or the like.

(Example B-13)
<Emulsion>
0.5 parts by weight of polyoxyethylene behenyl ether, 1 part by weight of polyoxyethylene sorbitol tetraoleate, 1 part by weight of glyceryl monostearate, 0.5 parts by weight of pyruvic acid, 0.5 parts by weight of behenyl alcohol, avocado oil 1 part by weight, 1 part by weight of α-glycosyl hesperidin obtained by the method of Example A-3, appropriate amounts of vitamin E and preservatives were dissolved by heating according to a conventional method, and 1 part by weight of L-sodium lactate, 1, An emulsion was prepared by adding 5 parts by weight of 3-butylene glycol, 0.1 part by weight of carboxyvinyl ponmer and 85.3 parts by weight of purified water, emulsifying with a homogenizer, adding an appropriate amount of a fragrance, stirring and mixing.

  This product can be advantageously used as a sunscreen, skin cleansing agent, whitening agent, and the like.

(Example B-14)
<Cream>
2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of glyceryl monostearate, 2 parts by weight of α-glucosyl hesperidin obtained by the method of Example A-2, 1 part by weight of liquid paraffin, glyceryl trioctanoate 10 An appropriate amount of parts by weight and preservative is dissolved by heating in accordance with a conventional method, and 2 parts by weight of L-lactic acid, 5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water are added thereto, emulsified through a homogenizer, and further flavored. An appropriate amount of was added and stirred to prepare a cream.

  This product can be advantageously used as a sunscreen, skin cleansing agent, whitening agent, and the like.

It is an infrared absorption spectrum of (alpha) -glycosyl hesperidin sample [I] as an example of this invention.

It is an infrared absorption spectrum of (alpha) -glycosyl hesperidin sample [II] as an example of this invention.

Claims (3)

Excellent water solubility as hesperidin in preventive or therapeutic agents for viral diseases, bacterial diseases, traumatic diseases, immune diseases, rheumatism, diabetes , cardiovascular diseases, or malignant tumors containing hesperidin as an active ingredient A prophylactic or therapeutic agent characterized by using α-glycosyl hesperidin, which is hydrolyzed into hesperidin and D-glucose in vivo, wherein hesperidin has α-bonded D-glucose residues in an equimolar amount or more.   Furthermore, thiamine is contained, The preventive agent or therapeutic agent of Claim 1 characterized by the above-mentioned.   The prophylactic or therapeutic agent according to claim 1 or 2, further comprising riboflavin.

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