WO2013018779A1 - CRISTAL DE α-GLUCOSYL-HESPÉRIDINE ET UTILISATION DE CELUI-CI - Google Patents

CRISTAL DE α-GLUCOSYL-HESPÉRIDINE ET UTILISATION DE CELUI-CI Download PDF

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WO2013018779A1
WO2013018779A1 PCT/JP2012/069378 JP2012069378W WO2013018779A1 WO 2013018779 A1 WO2013018779 A1 WO 2013018779A1 JP 2012069378 W JP2012069378 W JP 2012069378W WO 2013018779 A1 WO2013018779 A1 WO 2013018779A1
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crystal
glucosyl hesperidin
hesperidin
glucosyl
crystals
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Japanese (ja)
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渋谷　孝
精祐 伊澤
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株式会社林原
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/07—Benzo[b]pyran-4-ones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a novel crystal of ⁇ -glucosyl hesperidin and a powder consisting essentially of the crystal, and its use, and more specifically, ⁇ -glucosyl hesperidin whose crystal structure has been determined by X-ray crystal structure analysis.
  • the present invention relates to a novel crystal, a powder substantially consisting of the crystal, and its use as a pharmaceutical material.
  • ⁇ -Glucosyl hesperidin is an ⁇ -glucosyl derivative of hesperidin in which one molecule of glucose is bonded via an ⁇ -glucoside bond to the hydroxyl group at the 4-position of glucose constituting the rutinose structure of hesperidin, which is a kind of vitamin P (patent no. No. 3060227, Japanese Patent No. 2805273, Japanese Patent No. 3989561, Y. Suzuki et al., The First International Congress on “Vitamins and Biofactors in Life Science” "(The First International Congress on” Vitamins and Biofactors in Life Science "(ICVB)), Kobe, 1991, See Abstract 4-IV-5. .
  • ⁇ -Glucosyl hesperidin has extremely high water solubility while hesperidin is hardly soluble in water, and also has excellent properties such that it is easily hydrolyzed in vivo and exhibits the physiological activity inherent in hesperidin. Yes. Therefore, ⁇ -glucosyl hesperidin is more convenient than hesperidin and acts as hesperidin, mainly in the food and cosmetics fields, such as yellow colorants, antioxidants, quality improvers, skin beautifiers, fairing agents, etc. It is used as.
  • ⁇ -glucosyl hesperidin 75-85% by mass of ⁇ -glucosyl hesperidin produced by spray-drying method in terms of anhydride (hereinafter, unless otherwise specified, ⁇ -glucosyl hesperidin-containing amorphous powder mainly for foods (for example, trade name “Hayashibara Hesperidin S”, sold by Hayashibara Corporation; trade name “ ⁇ G Hesperidin PA”) -T ", sold by Toyo Seika Co., Ltd., etc.), and by reacting an enzyme having ⁇ -L-rhamnosidase activity (eg, hesperidinase), the remaining sparingly water-soluble hesperidin is decomposed to become water-soluble.
  • an enzyme having ⁇ -L-rhamnosidase activity eg, hesperidinase
  • an ⁇ -glucosyl hesperidin-containing amorphous mainly for cosmetics Powder for example, trade name “alpha glucosyl hesperidin”, sold by Hayashibara Biochemical Laboratories Co., Ltd .; trade name “ ⁇ G hesperidin PS”, sold by Toyo Seika Co., Ltd. Contains 75% or more ⁇ -glucosyl hesperidin in terms of anhydride.
  • the present applicant as one of the joint applicants in Japanese Patent No. 3833811, precipitated ⁇ -glucosyl hesperidin crystals from a 99% (v / v) methanol solution in which ⁇ -glucosyl hesperidin was dissolved.
  • Disclosed is a method for producing a high content of ⁇ -glucosyl hesperidin to be separated and collected.
  • the crystals obtained at this time are massive crystals in which a large number of minute crystals that can barely be confirmed as crystals are fixed to each other. It was impossible to identify even a single crystal form.
  • the present invention was made to eliminate the above-mentioned obstacles when ⁇ -glucosyl hesperidin was used as a pharmaceutical material, and to pioneer the use of ⁇ -glucosyl hesperidin as a pharmaceutical material. It is an object of the present invention to provide a novel crystal of glucosyl hesperidin, a powder substantially composed of the crystal, and its use as a pharmaceutical material.
  • the ⁇ -glucosyl hesperidin crystal having at least such a crystal structure has not been known before the filing of the present application, and the ⁇ -glucosyl hesperidin crystal is a novel crystal.
  • oxygen atoms and carbon atoms constituting the ⁇ -glucosyl hesperidin molecule have atomic coordinates shown in Tables 3 and 4 of the present specification.
  • the ⁇ -glucosyl hesperidin crystal of the present invention is a crystal substantially in the form of a single crystal in a preferred embodiment thereof.
  • the present invention also solves the above problem by providing a powder substantially consisting of the ⁇ -glucosyl hesperidin crystals.
  • the ⁇ -glucosyl hesperidin crystal of the present invention or a powder consisting essentially of the crystal is ⁇ -glucosyl as an optical isomer when subjected to high performance liquid chromatography (HPLC) analysis using an optical resolution column. Contains 70% or more of-(R) -hesperidin.
  • the present invention solves the above problems by providing a crystal of ⁇ -glucosyl hesperidin of the present invention as a pharmaceutical material or a powder consisting essentially of the crystal. That is, the ⁇ -glucosyl hesperidin crystal of the present invention whose crystal structure has been elucidated or a powder consisting essentially of the crystal can be easily improved in purity and stability as compared with a conventional amorphous powder. In addition, since the effectiveness and safety can be easily confirmed, it is extremely useful as a pharmaceutical material.
  • hesperidin has pharmacological effects such as blood vessel strengthening action, lipid metabolism improving action, carcinogenesis inhibiting action, UV protection action, antioxidant action, wound treatment action (Kyoichi Yoshioka et al., “Bioindustry”, Vol. 20, No. 5, pages 19 to 29 (2003)), the ⁇ -glucosyl hesperidin crystal of the present invention or a powder substantially consisting of the crystal is expected to have the same pharmacological action. It can be used as a pharmaceutical material that becomes an active ingredient such as a therapeutic agent for system diseases, a therapeutic agent for joint diseases, a therapeutic agent for vitamin C deficiency.
  • the crystal structure of the ⁇ -glucosyl hesperidin crystal of the present invention is clear, the physical and chemical properties of ⁇ -glucosyl hesperidin necessary for use as a pharmaceutical material are elucidated, and the presence or absence of crystal polymorphism is examined.
  • the advantage is that it is very easy to clarify the polymorphic phenomena.
  • the ⁇ -glucosyl hesperidin crystal of the present invention or a powder consisting essentially of the crystal is used as a pharmaceutical material, its effectiveness and safety can be confirmed more easily, and hesperidin is inherently present. With the expectation of the same pharmacological effect as the pharmacological effect, it is possible to obtain an advantage that it can be used more advantageously as a pharmaceutical material having extremely high water solubility compared to hesperidin.
  • 3 is an example of a micrograph of ⁇ -glucosyl hesperidin crystals obtained by crystallization using a 30% (v / v) aqueous methanol solution (magnification: 250 times). 3 is a stereomicrograph of ⁇ -glucosyl hesperidin crystals (0.1 ⁇ 0.1 ⁇ 0.03 mm) used for X-ray crystal structure analysis. 2 is an example of an X-ray diffraction pattern of a crystal of ⁇ -glucosyl hesperidin. It is an ORTEP diagram of ⁇ -glucosyl hesperidin excluding a hydrogen atom.
  • 3 is a crystal structure diagram showing packing of ⁇ -glucosyl hesperidin molecules in a crystal unit cell (b-axis direction). It is a chromatogram when the crystal of ⁇ -glucosyl hesperidin used for X-ray crystal structure analysis is subjected to optical resolution HPLC.
  • 2 is an example of a micrograph of ⁇ -glucosyl hesperidin crystals obtained by crystallization using a 99% (v / v) methanol solution (magnification: 500 times).
  • the present invention relates to a novel crystal of ⁇ -glucosyl hesperidin, specifically, a space group, a lattice constant, and a crystal system, which will be described later.
  • This relates to crystals of ⁇ -glucosyl hesperidin having the atomic coordinates shown in Table 4.
  • the novel crystal of ⁇ -glucosyl hesperidin of the present invention comprises ⁇ -glucosyl hesperidin having sufficient properties to analyze the crystal structure by X-ray crystal structure analysis, preferably in the form of a single crystal It is in.
  • a crystal having sufficient properties to analyze a crystal structure means, for example, (1) The same molecules in a solid (crystal) are arranged three-dimensionally with a certain regularity, and twins or fine single crystals in which two or more single crystals are bonded together are fixed to each other. Substantially free of polycrystals of (2) Appropriate shape and size of the crystal; for example, a prismatic columnar, plate-like, or needle-like crystal with at least one side grown to about 0.1 to about 0.5 mm, preferably Is preferably a rectangular columnar or plate-like crystal grown on all three sides, or a crystal having a width and thickness of about 0.02 mm or more.
  • a crystal having a size of about 0.1 ⁇ 0.1 ⁇ 0.1 mm to 0.3 ⁇ 0.3 ⁇ 0.3 mm is preferable.
  • (3) have chemical stability, mechanical stability, physical stability; It means a crystal with the characteristics of
  • the ⁇ -glucosyl hesperidin crystal of the present invention is not limited by the purity of the entire ⁇ -glucosyl hesperidin as long as the crystal has sufficient properties to analyze the crystal structure by X-ray crystal structure analysis. However, those having an ⁇ -glucosyl hesperidin purity of usually 95% or more, desirably 98% or more, and more desirably 99% or more are preferable.
  • purity of ⁇ -glucosyl hesperidin refers to 0.1% (w / w) of purified ⁇ -glucosyl hesperidin sample in the form of a solution, amorphous powder, crystal-containing powder, crystal or the like.
  • HPLC device “LC-20AD” (manufactured by Shimadzu Corporation)
  • Degasser “DGU-20A3” (manufactured by Shimadzu Corporation)
  • Eluent water / acetonitrile / acetic acid (80/20 / 0.01 (volume ratio))
  • Flow rate 0.7 ml / min
  • Temperature 40 ° C
  • Detection UV detector “SPD-20A” (manufactured by Shimadzu Corporation)
  • Data processor “Chromatopack C-R7A” (manufactured by Shimadzu Corporation)
  • ⁇ -Glucosyl hesperidin content Analyzed by HPLC, quantified based on the peak area using the reagent hesperidin (sold by Wako Pure Chemical Industries, Ltd.) as a standard substance, and calculated in terms of molecular weight.
  • Hesperidin content Analyzed by HPLC, and quantified based on the peak area using the reagent hesperidin (Wako Pure Chemical Industries, Ltd.) as a standard substance.
  • 7-Glucosyl hesperetin Analyzed by HPLC, quantified based on the peak area using the reagent hesperidin (sold by Wako Pure Chemical Industries, Ltd.) as a standard substance, and calculated in terms of molecular weight.
  • Other glycosides Analyzed by HPLC and quantified based on the peak area using the reagent hesperidin (sold by Wako Pure Chemical Industries, Ltd.) as a standard substance.
  • single crystals and other crystals can be selected by observing ⁇ -glucosyl hesperidin crystals under a polarizing microscope in advance.
  • ⁇ -glucosyl hesperidin crystals are directly analyzed by X-ray crystal structure analysis, that is, single crystal structure analysis by X-ray diffraction known to those skilled in the art (for example, Toshio Sakurai, “Guide for X-ray structure analysis”, (See, for example, Bunch issue (1983)) and determining whether or not an X-ray diffraction pattern (diffraction spot) as illustrated in FIG.
  • a commercially available single crystal X-ray diffractometer for example, an imaging plate single crystal automatic X-ray structure analyzer “R-AXIS RAPID” manufactured by Rigaku Corporation may be used.
  • the X-ray diffractometer is preinstalled with computer software for structural analysis.
  • the analysis determines crystallographic parameters in the single crystal,
  • the atomic coordinates of the ⁇ -glucosyl hesperidin molecule (a value indicating the spatial positional relationship of each atom) and a three-dimensional structural model can be obtained.
  • the atomic coordinates of ⁇ -glucosyl hesperidin are (1) A step of irradiating a monochromatized X-ray on the ⁇ -glucosyl hesperidin crystal of the present invention to obtain an X-ray diffraction pattern; (2) obtaining X-ray diffraction intensity data from the X-ray diffraction pattern; (3) Direct method (program “SIR92”, A. Altomare et al., Journal of Applied Crystallography (J. Appl.
  • the atomic coordinates may not be determined when the size of the single crystal is relatively small and the X-ray diffraction intensity is weak. Even in such cases, the basic three-dimensional structure of the ⁇ -glucosyl hesperidin molecule can be obtained by molecular modeling based on the atomic coordinates of only non-hydrogen atoms (in the case of ⁇ -glucosyl hesperidin, oxygen and carbon atoms). The structure can be revealed.
  • This crystal is a single crystal having sufficient properties to analyze the crystal structure by X-ray crystal structure analysis, but the crystal of the present invention is a crystal having the above space group, lattice constant, and crystal system. As long as it is not necessarily limited to a single crystal form as a whole.
  • the crystal of ⁇ -glucosyl hesperidin according to the present invention is more specifically the atomic coordinates shown in Tables 3 and 4 (continuation of Table 3) described below for each oxygen atom and each carbon atom of the ⁇ -glucosyl hesperidin molecule in the crystal. And gives the ORTEP diagram shown in FIG.
  • the “powder substantially consisting of ⁇ -glucosyl hesperidin crystals” as used in the present specification means almost the entire amount except for impurities that cannot be completely excluded due to the production process or technical reasons. It means a powder composed of ⁇ -glucosyl hesperidin crystals of the present invention having the crystallographic parameters described above, and the purity of ⁇ -glucosyl hesperidin in such a powder is usually 95.0% or more, preferably 98 0.0% or more, more preferably 99.0% or more.
  • the term “powder” as used herein means not only powder but also an aggregate of solid particles including, for example, granules, granules, and molded bodies.
  • hesperidin has two types of optical isomers, R-form and S-form, due to the difference in configuration of 3′-hydroxy-4′-methoxyphenyl group, and S-form exists in nature.
  • R-form optical isomers
  • S-form exists in nature.
  • the configuration is easily reversed by heating in an aqueous solution or the like, and becomes a mixture with the R form. Therefore, ⁇ -glucosyl hesperidin produced from hesperidin by a transglycosylation reaction usually contains a mixture of R and S isomers as optical isomers.
  • the R and S forms of ⁇ -glucosyl hesperidin are shown in the following chemical formulas 1 and 2.
  • the R and S isomers which are optical isomers of hesperidin, can be separated and quantified by HPLC using an optical resolution column, and the R and S isomers of ⁇ -glucosyl hesperidin can also be analyzed by the same method.
  • the ⁇ -glucosyl hesperidin crystal of the present invention or a powder consisting essentially of the crystal has an R-isomer as an optical isomer of 70% or more, specifically 70% or more, as shown in an experiment described later. Contains less than 80%.
  • the origin of ⁇ -glucosyl hesperidin as a raw material for producing the ⁇ -glucosyl hesperidin crystal of the present invention is not particularly limited, and it may be obtained by an organic synthesis method or an enzyme synthesis method. Good. Usually, ⁇ -form obtained by allowing cyclomaltodextrin / glucanotransferase (hereinafter abbreviated as “CGTase”) to act on a partially decomposed starch in the presence of hesperidin, followed by glucoamylase and further purification. Glucosyl hesperidin is preferred.
  • the ⁇ -glycosyl hesperidin-containing powder obtained by this enzyme synthesis method contains impurities derived from the production method.
  • the “contaminant derived from the production method” as used in the present specification refers to a hesperidin-related glycoside (hereinafter referred to simply as “other glycosides”, which is inherently mixed in a slight amount in the production raw material hesperidin. ), Unreacted hesperidin remaining in the transglycosylation reaction, and 7-glucosyl hesperetin, which is a degradation product of the unreacted hesperidin when degraded by the action of an enzyme having ⁇ -L-rhamnosidase activity Means.
  • the ⁇ -glucosyl hesperidin used for the preparation of the crystal of the present invention may be of a purity sufficiently high to form a crystal.
  • the purity of ⁇ -glucosyl hesperidin can be confirmed by a conventional purity confirmation means (for example, the HPLC analysis described above).
  • the content of ⁇ -glucosyl hesperidin as a raw material for producing crystals is usually 90% or more, desirably 95% or more, more desirably 98% or more, and even more desirably 99% or more ⁇ -glucosyl hesperidin purity. Those having the following are preferred.
  • Crystallization of an organic compound is usually performed based on the property that, when a solution containing the organic compound is brought into a supersaturated state, the solution state is changed to an insoluble state, and when a specific condition is satisfied, the organic compound is precipitated as a crystal. .
  • crystallization of ⁇ -glucosyl hesperidin (1) Add an aqueous alcohol solution to ⁇ -glucosyl hesperidin and dissolve it by heating; (2) The temperature of the solution in which ⁇ -glucosyl hesperidin is dissolved is lowered to a supersaturated state; (3) The supersaturated ⁇ -glucosyl hesperidin solution is maintained at a constant temperature to precipitate crystals; It can be done by the operation. Moreover, crystallization can also be accelerated
  • an aqueous alcohol solution obtained by mixing water with a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, and t-butanol is used.
  • a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, and t-butanol.
  • the methanol concentration is usually 80% (v / v) or less, preferably 60% (v / v) or less, more preferably 30 to 40% (v / v). Is preferred.
  • the methanol concentration is more than 80% (v / v), although depending on the concentration of ⁇ -glucosyl hesperidin, it is not preferable because crystals rapidly precipitate and a massive crystal is formed in which fine crystals are fixed to each other. . Further, if the alcohol concentration is too low, there is a disadvantage that crystals do not precipitate or even if they are precipitated, it takes a long time for growth.
  • the concentration of ⁇ -glucosyl hesperidin dissolved in the lower alcohol aqueous solution may be a concentration suitable for crystallization, and is usually 0.1 to 20% (w / v), preferably 0.5 to 10% ( w / v), more desirably 1 to 5% (w / v).
  • concentration of ⁇ -glucosyl hesperidin during crystallization is higher than 20% (w / v)
  • crystals are precipitated rapidly, and the bulk crystals in which minute crystals are fixed to each other Is not preferable.
  • the ⁇ -glucosyl hesperidin concentration is lower than 0.1% (w / v), there is a disadvantage that a crystal does not precipitate or a long time is required for growth even if it is precipitated.
  • the temperature condition depends on the concentration of ⁇ -glucosyl hesperidin and the alcohol concentration of the solvent, it is usually 0 to 50 ° C., preferably 3 to 30 ° C., more preferably 4 to 10 ° C.
  • crystals having a size suitable for X-ray crystal structure analysis are selected from the crystals crystallized using a stereomicroscope. Avoid as much as possible those with multiple crystals attached, those with cracks, those with white turbidity, and those with microcrystals fixed on the surface. In addition, in order to avoid further precipitation of fine crystals from the mother liquor on the surface of the crystals, it is possible to advantageously carry out coating of the target crystals with Palaton oil. The crystals thus obtained can be used for X-ray crystal structure analysis.
  • the ⁇ -glucosyl hesperidin crystal of the present invention or a powder consisting essentially of the crystal is highly purified and crystallized to a purity that can analyze its crystal structure, and is a high-quality ⁇ -glucosyl hesperidin prepared. It is.
  • the ⁇ -glucosyl hesperidin crystal of the present invention and the powder consisting essentially of the crystal are themselves effective, safe and stable pharmaceutical materials as well as hesperidin for viral diseases, bacterial diseases, cardiovascular diseases.
  • the dosage form when the ⁇ -glucosyl hesperidin crystal of the present invention and a powder consisting essentially of the crystal are used as a pharmaceutical material, and it may be used in the form of solid, powder, granule, tablet or the like. It can also be used in the form of a composition with other pharmaceutical materials.
  • ⁇ Reference Example Preparation of amorphous ⁇ -glucosyl hesperidin amorphous powder> An ⁇ -glucosyl hesperidin-containing amorphous powder for cosmetics (trade name “alpha glucosyl hesperidin”, ⁇ -glucosyl hesperidin content 83.8%, sold by Hayashibara Biochemical Laboratories Co., Ltd.) (hereinafter referred to as “Sample 1”). It was used as a raw material for preparing high purity ⁇ -glucosyl hesperidin amorphous powder. That is, 200 g of Sample 1 was suspended in 134 ml of 80% (v / v) ethanol aqueous solution, stirred and heated to 80 ° C.
  • Example 2 The purity of ⁇ -glucosyl hesperidin in Sample 2 was 96.3%.
  • Example 3 The purity of ⁇ -glucosyl hesperidin in each fraction obtained by ODS column chromatography was measured by HPLC analysis, and fractions with a purity of 98% or more were collected, freeze-dried by a conventional method, and containing ⁇ -glucosyl hesperidin with a purity of 99.0% 13.2 g of an amorphous lyophilized powder (hereinafter referred to as “Sample 3”) was obtained.
  • Table 1 summarizes the compositions of Samples 1 to 3 described above, that is, ⁇ -glucosyl hesperidin-containing amorphous powder, ⁇ -glucosyl hesperidin-containing amorphous powder, and ⁇ -glucosyl hesperidin-containing amorphous lyophilized powder for cosmetics. .
  • the remaining crystal suspension from which crystals for X-ray crystal structure analysis were collected was collected by filtering with a Kiriyama funnel using filter paper (No. 4, manufactured by Advantech Toyo Co., Ltd.), and then dried. About 1.2 g of ⁇ -glucosyl hesperidin crystals were obtained. The purity of ⁇ -glucosyl hesperidin of this crystal product was 99.3%.
  • ⁇ X-ray crystal structure analysis of ⁇ -glucosyl hesperidin single crystal The ⁇ -glucosyl hesperidin single crystal mounted on the sample holder in Example 1 was set in a single crystal X-ray diffractometer (“R-AXIS RAPID-R”, manufactured by Rigaku Corporation), and nitrogen gas ( ⁇ 170 ° C.) Under an atmosphere, an X-ray diffraction pattern was measured by vibration photography under the following conditions.
  • FIG. 2 shows a stereomicrograph of ⁇ -glucosyl hesperidin crystal used for single crystal structure analysis by X-ray diffraction
  • FIG. 3 shows an example of the X-ray diffraction pattern.
  • the X-ray diffraction pattern many diffraction spots (spots) were confirmed, and it was confirmed that the crystal was a single crystal.
  • the shape of the X-ray diffraction peak was relatively good, the peak intensity was weak. Of the 39,960 reflections (diffraction) observed, 7,806 were unique reflections.
  • the initial structure is obtained by a direct method, and a structural model is created with reference to the structural formula of ⁇ -glucosyl hesperidin. Refined by.
  • “Crystal Structure Ver. 3.8.2” manufactured by Rigaku Corporation was used as analysis software. Table 2 summarizes the crystallographic data of ⁇ -glucosyl hesperidin obtained by X-ray crystal structure analysis.
  • Table 3 shows the distance between each atom except the hydrogen atom in the ⁇ -glucosyl hesperidin molecule
  • Table 6 and Table 7 show the bond angle between each atom except the hydrogen atom. It was.
  • FIG. 4 shows an ORTEP diagram of ⁇ -glucosyl hesperidin calculated from the refined coordinate data and displayed. Further, the packing structure of ⁇ -glucosyl hesperidin molecules per unit cell of the crystal is shown in FIG. 5 as a crystal structure diagram when viewed from the b-axis direction.
  • the numbers of oxygen atoms and carbon atoms in Tables 3 to 7 correspond to the numbers of oxygen atoms and carbon atoms described in the ORTEP diagram of ⁇ -glucosyl hesperidin in FIG. 4, respectively.
  • or 7 means a standard deviation.
  • carbon numbers C1 to C16 represent the structure of hesperetin in the ⁇ -glucosyl hesperidin molecule
  • carbon numbers C17 to C28 represent the structure of rutinose (L-rhamnosyl ( ⁇ 1 ⁇ 6) glucose)
  • Carbon numbers C29 to C34 represent the structures of glucose bonded via an ⁇ -glucoside bond to the 4-position hydroxyl group of glucose constituting the rutinose structure, respectively.
  • FIG. 5 it can be well understood that in this crystal, two molecules of ⁇ -glucosyl hesperidin are packed per unit cell.
  • This crystal can also be used for X-ray crystal structure analysis in the same manner as the crystal obtained in Example 1.
  • the crystal suspension was recovered by filtration through a Kiriyama funnel using filter paper (No. 4, manufactured by Advantech Toyo Co., Ltd.) to obtain about 0.9 g of ⁇ -glucosyl hesperidin crystals. The purity of ⁇ -glucosyl hesperidin of this crystal product was 99.1%.
  • each crystal product was dissolved in 10 ml of an eluent (20 mM aqueous potassium phosphate solution (pH 3.0) / acetonitrile (86/14)) and subjected to optical resolution HPLC under the conditions shown below. The abundance ratio of was measured.
  • An example of the chromatogram of the crystal of Example 1 is shown in FIG.
  • Table 8 shows the analysis results of each crystal product.
  • HPLC apparatus Column: “SUMICHIRAL OA-7000” (4.6 ⁇ 250 mm) (manufactured by Sumika Chemical Analysis Co., Ltd.) Sample injection volume: 10 ⁇ l Eluent: 20 mM monopotassium phosphate aqueous solution (pH 3.0) / acetonitrile (86/14) Flow rate: 0.4 ml / min Column temperature: 35 ° C Detection: UV280nm Data processor: “Chromatopack CR-4A” (manufactured by Shimadzu Corporation) The abundance ratio was the peak area ratio of R-form and S-form of ⁇ -glucosyl hesperidin in the chromatogram.
  • the R-form and S-form peaks of ⁇ -glucosyl hesperidin were completely separated by an optical resolution column under the present analysis conditions, and retention times (Rt) were 31.4 minutes and 34.9 minutes, respectively. Eluted.
  • the crystal product of ⁇ -glucosyl hesperidin obtained in Example 1 has a ratio of R to S as an optical isomer of about 8: 2, and the same ratio of the crystal product of Example 3 was about 7: 3, and the abundance ratio of the R form was high.
  • ⁇ Method for producing powder substantially consisting of crystals of ⁇ -glucosyl hesperidin> Completely by adding 100 parts by weight of 35% (v / v) aqueous methanol solution to 4 parts by weight of the ⁇ -glucosyl hesperidin-containing amorphous lyophilized powder obtained by the method of Reference Example and heating to 80 ° C. with stirring. Then, the obtained solution was crystallized by maintaining at 10 ° C. for 14 days. As a result, colorless and plate-like crystals equivalent to those obtained in Example 1 were obtained. The crystal suspension was recovered by filtration and then dried to obtain about 2 parts by mass of a powder substantially consisting of ⁇ -glucosyl hesperidin crystals. The purity of ⁇ -glucosyl hesperidin of this product was 99.2%. This product can be suitably used as a pharmaceutical material.
  • ⁇ Tablets> A powder obtained by uniformly mixing 20 parts by mass of powder substantially consisting of ⁇ -glucosyl hesperidin crystals, 30 parts by mass of maltose, and 4 parts by mass of corn starch obtained by the method of Example 5 was applied to a tableting machine by a conventional method. One tablet of 0.5 g was produced.
  • This product can be advantageously used not only as a vitamin P supplement, but also as a vascular disease treatment agent, a joint disease treatment agent, and the like.
  • ⁇ Dissolving injection for use> Completely by adding 150 parts by weight of 35% (v / v) aqueous methanol solution to 3 parts by weight of the ⁇ -glucosyl hesperidin-containing amorphous lyophilized powder obtained by the method of Reference Example and heating to 80 ° C. with stirring. Then, the pyrogen was removed by microfiltration according to a conventional method, and ⁇ -glucosyl hesperidin was crystallized in the same manner as in Example 1 except that all the operations were performed in consideration of the absence of pyrogen contamination. After drying, it was pulverized to obtain about 1 part by mass of pyrogen-free powder substantially consisting of ⁇ -glucosyl hesperidin crystals.
  • the purity of ⁇ -glucosyl hesperidin of this product was 99.3%. 50 mg of this powder was aseptically placed in a 20 ml ampoule, purged with nitrogen, sealed, and used as an ampule-injectable injection containing an ampoule.
  • This product can be administered intramuscularly or intravenously alone or mixed with other vitamins, minerals, etc., and is advantageous not only as a vitamin P supplement, but also as a therapeutic agent for vascular diseases, therapeutic agents for joint diseases, etc. Available.
  • ⁇ Effect of ⁇ -glucosyl hesperidin on cell autophagy Using the crystalline ⁇ -glucosyl hesperidin sample with a purity of 99% or more obtained by the method of Example 3, ⁇ -glucosyl hesperidin is a cell autophagy, that is, unnecessary protein accumulated in the cell, damage We investigated the effect on the phenomenon of preventing disease by actively eliminating mitochondria.
  • HOZOT cells that are immune system cells (see re-published patent WO2007 / 105797) or SH-SY-5 strain that is a neuronal cell line are tested, ⁇ -glucosyl hesperidin is a final concentration of 1 or 10 ⁇ M in the case of HOZOT cells,
  • SH-SY-5 strain it was added to the culture solution so as to have a final concentration of 10 or 25 ⁇ M, treated for 24 hours, and then cells were collected, and a cell extract was prepared for each. Next, the cell extract was subjected to SDS-PAGE, and then the expression level of LC-3B, which is a marker protein for autophagy, was measured by Western blotting.
  • the results when HOZOT cells are used as test cells are shown in Table 9, and the results when SH-SY-5 strain is used are shown in Table 10, respectively.
  • ⁇ -glucosyl hesperidin is useful as a cell autophagy enhancer.
  • Autophagy is known to be associated with neurodegenerative diseases such as Huntington's disease and type 2 diabetes, and ⁇ -glucosyl hesperidin, which has an autophagy enhancing effect on cells, can be applied to treat these diseases. .
  • the present invention provides a novel crystal of ⁇ -glucosyl hesperidin whose crystal structure has been elucidated, a powder substantially consisting of the crystal, and its use as a pharmaceutical material.
  • the ⁇ -glucosyl hesperidin crystal and the powder substantially consisting of the crystal according to the present invention are themselves effective, safe and stable pharmaceutical materials, like hesperidin, viral diseases, bacterial diseases, circulation Not only can it be used as a preventive or therapeutic agent for various diseases for which hesperidin is considered to be effective, such as organ diseases and malignant tumors, but also solid physical properties such as solubility and stability of ⁇ -glucosyl hesperidin, and crystal polymorphism It is also extremely useful as a reagent for elucidating the presence or absence of metastasis and the transfer phenomenon.
  • the present invention greatly opens up the use of ⁇ -glucosyl hesperidin as a pharmaceutical material, and its industrial utility is extremely large.

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Abstract

La présente invention concerne : un nouveau cristal de α-glucosyl-hespéridine, dont la structure cristalline est élucidée; une poudre comprenant sensiblement le cristal; et l'utilisation de la poudre en tant que matériau pour un agent médicinal. L'objet peut être réalisé en fournissant : un cristal de α-glucosyl-hespéridine, qui a un groupe spatial cristallographique de P21, et dont le réseau unitaire a une constante de réseau a de 13,983 Å, une constante de réseau b de 7,620 Å et une constante de réseau c de 20,065 Å, et qui est un cristal monoclinique dans lequel α = γ = 90° et β = 93,475°; une poudre comprenant substantiellement le cristal; et l'utilisation de la poudre en tant que matériau pour un agent médicinal.
PCT/JP2012/069378 2011-08-01 2012-07-30 CRISTAL DE α-GLUCOSYL-HESPÉRIDINE ET UTILISATION DE CELUI-CI WO2013018779A1 (fr)

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Cited By (3)

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WO2015133483A1 (fr) * 2014-03-03 2015-09-11 株式会社林原 Hespéridine glucosyle, son procédé de fabrication, et application associée
WO2016027837A1 (fr) * 2014-08-20 2016-02-25 株式会社林原 Agent de soins de beauté d'administration par voie orale
WO2017038951A1 (fr) * 2015-09-02 2017-03-09 株式会社林原 Préparation externe dermatologique pour réduire le jaunissement cutané

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CN116437912A (zh) 2020-09-17 2023-07-14 株式会社力森诺科 自噬激活剂
WO2022059776A1 (fr) 2020-09-17 2022-03-24 昭和電工株式会社 Activateur d'autophagie
WO2022059775A1 (fr) 2020-09-17 2022-03-24 昭和電工株式会社 Activateur d'autophagie
WO2022059767A1 (fr) 2020-09-17 2022-03-24 昭和電工株式会社 Activateur d'autophagie

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WO1998042859A1 (fr) * 1997-03-24 1998-10-01 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo PROCEDE DE PRODUCTION D'UNE SUBSTANCE RICHE EN α-MONOGLUCOSYLHESPERIDINE
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US5627157A (en) * 1989-06-03 1997-05-06 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo α-glycosyl hesperidin and its uses
US5885969A (en) * 1996-06-26 1999-03-23 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Enzyme-treated hesperidin, process for producing the same and method of using enzyme-treated hesperidin
WO1998042859A1 (fr) * 1997-03-24 1998-10-01 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo PROCEDE DE PRODUCTION D'UNE SUBSTANCE RICHE EN α-MONOGLUCOSYLHESPERIDINE

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015133483A1 (fr) * 2014-03-03 2015-09-11 株式会社林原 Hespéridine glucosyle, son procédé de fabrication, et application associée
KR20160127763A (ko) * 2014-03-03 2016-11-04 가부시기가이샤하야시바라 글리코실헤스페레틴과 그 제조방법 및 용도
JPWO2015133483A1 (ja) * 2014-03-03 2017-04-06 株式会社林原 グリコシルヘスペレチンとその製造方法並びに用途
US10703773B2 (en) 2014-03-03 2020-07-07 Hayashibara Co. Ltd. Glycosyl hesperetin and process for producing the same and uses thereof
KR102172553B1 (ko) 2014-03-03 2020-11-03 가부시기가이샤하야시바라 글리코실헤스페레틴과 그 제조방법 및 용도
US11453694B2 (en) 2014-03-03 2022-09-27 Hayashibara Co. Ltd. Glycosyl hesperetin and process for producing the same and uses thereof
WO2016027837A1 (fr) * 2014-08-20 2016-02-25 株式会社林原 Agent de soins de beauté d'administration par voie orale
JPWO2016027837A1 (ja) * 2014-08-20 2017-07-20 株式会社林原 経口美容剤
WO2017038951A1 (fr) * 2015-09-02 2017-03-09 株式会社林原 Préparation externe dermatologique pour réduire le jaunissement cutané
CN107920981A (zh) * 2015-09-02 2018-04-17 株式会社林原 暗黄降低用皮肤外用剂
US10568827B2 (en) 2015-09-02 2020-02-25 Hayashibara Co., Ltd. External dermal agent for reducing yellowish dullness

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