TW201319080A - Crystalline α -glucosyl hesperidin and uses thereof - Google Patents

Abstract

The purpose of the present invention is to provide: a novel crystal of a-glucosyl hesperidin, of which the crystal structure is elucidated; a powder substantially comprising the crystal; and use of the powder as a material for a medicinal agent. The purpose can be achieved by providing: a crystal of a-glucosyl hesperidin, which has a crystallographic space group of P21, and of which the unit lattice has a lattice constant a of 13.983 AA, a lattice constant b of 7.620 AA and a lattice constant c of 20.065 AA, and which is a monoclinic crystal wherein a = ? = 90 DEG and ss = 93.475 DEG ; a powder substantially comprising the crystal; and use of the powder as a material for a medicinal agent.

Description

Crystallization of α-glucosyl hesperidin and its use

The present invention relates to a novel crystal of α-glucosyl hesperidin and a powder substantially composed of the crystal, and a use thereof, and more specifically, an X-ray crystal structure is analyzed to determine a crystal structure thereof. The novel crystal of α-glucosyl hesperidin and the powder substantially composed of the crystal and the use as a raw material of the pharmaceutical.

Α-glucosyl hesperidin, which constitutes a rutinose structure of vitamin P, is a rutinose-structured glucose at the 4-position hydroxyl group, and one molecule of glucose is bound to hesperidin by α-glucoside binding. Alpha-glucosyl derivatives. (See Japanese Patent No. 3060227, Japanese Patent No. 2805273, Japanese Patent No. 3896561, (Y. Suzuki), etc. (The First International Congress on "Vitamins and Biofactors in Life Science" (ICVB)) In 1991, the keynote of the lecture was 4-IV-5). The α-glucosyl hesperidin has an extremely high water solubility with respect to water-insoluble hesperidin, and has excellent physiological properties which are inherently exerted by hesperidin which is easily hydrolyzed in a living body. Therefore, α-glucosyl hesperidin is easier to use than hesperidin, and as a substance acting like hesperidin, mainly in the field of food and cosmetics, as a yellow coloring agent, an antioxidant, a quality improving agent, a beauty agent, A whitening agent or the like is used.

By the way, as a commercially available α-glucosyl hesperidin, It is produced by a spray drying method and contains 75 to 85% by mass of α-glucosyl hesperidin in terms of an acid anhydride (hereinafter, unless otherwise indicated, the mass % of the specification is abbreviated as "%"), mainly as a food-oriented α-glucose. The stearyl glucoside contains an amorphous powder (for example, the trade name "Linhara orange saponin S", sold by Linyuan Commercial Co., Ltd.; the trade name "α G hesperidin PA-T", Toyo Fine Sugar Co., Ltd., etc.) Further, by the action of an enzyme having α-L-rhamnosidase activity (for example, hesperidinase), the residual water-soluble celloside is decomposed to improve its water solubility (refer to Japanese Patent No. 3833775). Bulletin) is mainly used as an amorphous powder containing α-glucosyl hesperidin for cosmetics (for example, the trade name "α-glucosyl hesperidin", sold by the Chemical Research Institute of Linyuan Biotechnology Co., Ltd.; The trade name "α G hesperidin PS", sold by Toyo Seiki Co., Ltd., etc., contains α-glucosyl hesperidin 75% or more in terms of acid anhydride.

However, these commercially available α-glucosyl hesperidin powders, all of which are amorphous forms of α-glucosyl hesperidin powder, can be used only for food or cosmetics, if used Pharmaceuticals, not purely in terms of purity or stability, or even effectiveness or safety.

On the other hand, the present applicant, Japanese Patent No. 3833811, as a co-applicant, discloses the precipitation of α-glucose from a 99% (v/v) methanol solution in which α-glucosyl hesperidin is dissolved. A method for producing a high content of α-glucosyl hesperidin by separating the crystal of quercetin. However, the crystals obtained at this time are as described below, and it is confirmed that the degree of crystallinity is a large crystal which is a plurality of block crystals which are fixed to each other, and it is not confirmed that the crystal is composed of a single crystal form. By. Now, as far as the Applicant is aware, there is a report on the crystal structure of hesperidin which is a glycosidic glycosidic group (see (W. Shin et al., (Acta Cryst.) C43, pp. 1946-1949 (1987). Years and (S. Fujii) et al. (Chem. Pharm. Bull.) Vol. 42, No. 5, pages 1143-1145 (1994), but for α-glucosyl hesperidin, the crystal There are no records in the structure.

As is well known, regarding an organic compound which is relatively small in molecular weight to be used as a pharmaceutical product, it is necessary to explain the physical understanding of the solid or the transfer phenomenon during preparation or storage. In the pharmaceutical industry, there is a mission to provide good medicine. As an organic compound of pharmaceuticals, if it is in the form of crystals, its crystal structure information, and when the organic compound exists as an optical isomer of natural substances, it is necessary for its optical purity information. of. However, α-glucosyl hesperidin has no information on its crystal structure or optical purity, and has become an obstacle to the use of α-glucosyl hesperidin as a raw material for pharmaceuticals.

An object of the present invention is to provide an α-glucosyl hesperidin which is used as a raw material for pharmaceuticals to remove the above-mentioned obstacles, and the use as a raw material of α-glucosyl hesperidin has been greatly developed and has been determined. A novel crystal of α-glucosyl hesperidin having a crystal structure, a powder substantially composed of the crystal, and a use as a raw material of the pharmaceutical.

In order to successfully solve the above-mentioned problems, as a result of repeated studies on the crystallization of α-glucosyl hesperidin, the present inventors, α-Port An amorphous powder containing α-glucosyl hesperidin having a purity of 95% or more of glycosylglycoside, dissolved in 30 to 40% (v/v) of methanol at a concentration of less than 5% (w/v) In the aqueous solution, it takes time to crystallize at a low temperature, and the crystal structure is analyzed by X-ray crystal structure analysis to obtain a single crystal of α-glucosyl hesperidin having sufficient properties and size, and the crystal structure is determined. The present invention has been completed.

That is, the technical means for solving the problem of the present invention is to provide a crystal of α-glucosyl hesperidin having a space group of P2 ₁ and a lattice constant of a unit lattice of a=13.983 Å and b=7.620 Å. , c = 20.065 Å, and is monoclinic with α = γ = 90 ° and β = 93.475 °. The α-glucosyl hesperidin crystal having at least such crystals is not known before the present application, and the crystal of the aforementioned α-glucosyl hesperidin is a novel crystal.

The crystal of α-glucosyl hesperidin of the present invention is more specifically described as having an oxygen atom and a carbon atom constituting the α-glucosyl hesperidin molecule having atomic coordinates as shown in Tables 3 and 4 in the present specification.

The α-glucosyl hesperidin crystal of the present invention is substantially a crystal of a single crystal form in this suitable aspect.

Further, the present invention solves the above problems by providing a powder substantially composed of the crystal of the above α-glucosyl hesperidin.

Further, the crystal of the α-glucosyl hesperidin of the present invention or the powder substantially composed of the crystal is contained in an optical analysis column for high-speed liquid chromatography, and contains 70% by mass or more as an optical isomer. α-Glucosyl-(R)-hesperidin.

According to the present invention, the above-mentioned problems are solved by providing a crystal of α-glucosyl hesperidin of the present invention as a raw material for pharmaceuticals or a powder substantially composed of the crystal. That is, it has been determined that the novel crystal of the α-glucosyl hesperidin crystal structure or the powder substantially composed of the crystal is easier to improve the purity or stability than the conventional amorphous powder. Since it is easy to confirm the validity or safety, it is extremely useful as a raw material for pharmaceuticals.

By the way, hesperidin, because of its vascular strengthening, lipid metabolism, cancer inhibition, UV defense, anti-oxidation, wound healing, etc. (Jigang Akira, "Bioindustry", 20, 5, 19 to 29 (2003)), it is expected that the crystal of α-glucosyl hesperidin of the present invention or the powder substantially composed of the crystal has the same pharmacological action and can be used as The pharmaceutical raw materials are used as active ingredients for therapeutic agents for vascular diseases, therapeutic agents for joint diseases, and therapeutic agents for vitamin C deficiency.

According to the α-glucosyl hesperidin crystal of the present invention, since the crystal structure is clear, the physical property of α-glucosyl hesperidin which is necessary as a raw material for pharmaceuticals is obtained. The explanation of the nature of the chemical, or the polymorphism of the presence or absence of the crystalline polymorphism becomes an extremely easy advantage. Further, when the crystal of the α-glucosyl hesperidin of the present invention is used or the powder composed of the crystal is substantially used, the effectiveness or safety of the crystal is more easily confirmed, and it is expected to have the original with hesperidin. The pharmacological effects of the same pharmacological effects are more advantageous as a raw material of a drug which is extremely water-soluble compared to hesperidin.

1. Crystallization of α-glucosyl hesperidin

The present invention is a novel crystal of α-glucosyl hesperidin, and more specifically, has a space group, a lattice constant, and a crystal system which will be described later, and further, the oxygen atom and the carbon atom thereof have the following Tables 3 and 4. Related to the atomic coordinates of α-glucosyl hesperidin crystals. The novel crystal of α-glucosyl hesperidin of the present invention is composed of an α-glucosyl hesperidin which is analyzed by an X-ray crystal structure and has a property of analyzing a crystal structure having a sufficient crystal structure, and is suitable for a single crystal form.

In the present specification, "analyzing a crystal having a sufficient crystal structure" means that, for example,

(1) The same molecule in a solid (crystal) which is a three-dimensional arrangement having a certain regularity, and substantially does not contain a polycrystal in which two or more single crystals of a single crystal are adhered to each other;

(2) Appropriate crystal shape and size; for example, at least one side grows into an angular columnar, plate-like, or needle-like crystal of about 0.1 to about 0.5 mm, and it is desired that the three sides are all grown in the same angular columnar shape. A plate-like crystal or a crystal having a width and a thickness of about 0.02 mm or more is preferred. Further, it is more preferable to crystallize to a size of about 0.1 × 0.1 × 0.1 mm to 0.3 × 0.3 × 0.3 mm.

(3) It has chemical stability, mechanical stability, physical stability, and crystallisation of characteristics.

The crystal of α-glucosyl hesperidin of the present invention is analyzed by an X-ray crystal structure as long as it has crystals having a sufficient crystal structure to be analyzed. The purity of the whole α-glucosyl hesperidin is not limited thereto, and is usually α-glucosyl hesperidin having a purity of 95% or more, preferably 98% or more, more preferably More than 99%.

In the present specification, the "purity of α-glucosyl hesperidin" means a solution of a solution, an amorphous powder, an α-glucosyl hesperidin containing crystal powder, crystals, or the like, which is diluted with purified water or Dissolved in 0.1% (w/v), filtered through a 0.45 μm membrane filter, and then subjected to HPLC analysis according to the following conditions, and used as a standard substance by the reagent drug hesperidin (sold by Wako Pure Chemical Industries Co., Ltd.). The peak area appearing on the UV280nm chromatogram is α-glucosyl hesperidin, 7-O-β-glucosyl hesperidin (hereinafter, only referred to as "7-glucosylhesin" The molecular weight of each component of hesperidin and other glycosides was calculated and converted into the content of α-glucosyl hesperidin in the composition of the acid anhydride mass.

<HPLC analysis conditions>

HPLC device: "LC-20AD" (made by Shimadzu Corporation)

Deaerator: "DGU-20A3" (made by Shimadzu Corporation)

Pipe column: "CAPCELL PAK C18 UG 120" (manufactured by Shiseido Co., Ltd.)

Sample injection amount: 10μl

Eluent: water / acetonitrile / acetic acid (80 / 20 / 0.01 (volume ratio))

Flow rate: 0.7ml/min

Temperature: 40 ° C

Check out: UV detector "SPD-20A" (Shimadzu Corporation stock Company system)

Measurement wavelength: 280 nm

Data processing device: "Chromatographic Pack C-R7A" (made by Shimadzu Corporation)

(1) The content of the α-glucosyl hesperidin was determined by HPLC analysis, and the amount of the converted molecular weight was calculated based on the peak area based on the test drug hesperidin (sold by Wako Pure Chemical Industries, Ltd.) as a standard substance.

(2) Hesperidin content: Quantitative analysis was carried out on the basis of the peak area by HPLC analysis using the test drug hesperidin (sold by Wako Pure Chemical Industries Co., Ltd.) as a standard substance.

(3) 7-glucosyl hesperidin: In the HPLC analysis, the reagent, hesperidin (sold by Wako Pure Chemical Industries, Ltd.) was used as a standard substance, and the molecular weight was calculated based on the peak area.

(4) Other glycosides: Quantitative analysis was carried out on the basis of the peak area in the HPLC analysis using the reagent drug hesperidin (sold by Wako Pure Chemical Industries Co., Ltd.) as a standard substance.

Generally, the single crystal can be confirmed to have a matte under a bias microscope, and if necessary, a single crystal and other crystals can be selected by observing the crystal of α-glucosyl hesperidin under a polarizing microscope in advance. Further, the crystal of α-glucosyl hesperidin is directly subjected to analysis of the X-ray crystal structure, that is, in the field, a single crystal structure is analyzed by a conventional technique by X-ray diffraction (for example, refer to Sakurai Sakurai) "X-ray structure analysis guide", published by Sang Wah House (1983), etc., by whether it can be obtained as shown in Figure 3 below. The X-ray diffraction pattern (diffraction spot) shown is judged to determine whether or not it has sufficient properties for analyzing the crystal structure. For the analysis of the single crystal structure, a commercially available single crystal X-ray diffraction apparatus, for example, an image forming plate single crystal automatic X-ray structure analyzer "R-AXIS RAPID" manufactured by Rigaku Corporation, and the like, such commercially available single crystals. The X-ray diffraction device has been equipped with a computer software for structural analysis.

The crystal of α-glucosyl hesperidin is analyzed by X-ray crystal structure. When it is a single crystal having a sufficient crystal structure to analyze its crystal structure, the crystallographic parameters are determined on the single crystal by the analysis. The atomic coordinates of the α-glucosyl hesperidin molecule (the value indicating the positional relationship of each atomic space) and the 3-dimensional structural model can be obtained. Specifically, the atomic coordinate of α-glucosyl hesperidin can be obtained by irradiating monochromated X-rays on (α) the α-glucosyl hesperidin crystal of the present invention to obtain an X-ray diffraction pattern. (2) a step of obtaining X-ray diffraction intensity data from the X-ray diffraction pattern; (3) via a direct method (Program "SIR92", A. Altomare et al., Journal of Applied Crystallography (J. Appl. Cryst.) , Volume 27, page 435, (1994)), the steps of obtaining the initial structure (electron density map); (4) A step of precisely constructing a carbon atom, an oxygen atom, and a hydrogen atom on the electron density map based on the chemical structure of α-glucosyl hesperidin to minimize the R value by a least square method The order is obtained as an atomic coordinate.

Incidentally, regarding the hydrogen atom, the specification of the single crystal is small, and the atomic coordinates may not be determined when the X-ray diffraction intensity is weak. Even in such a case, only the atomic coordinates of a non-hydrogen atom (in the case of α-glucosyl hesperidin is an oxygen atom and a carbon atom) can be understood as a basis for molecular modeling, and α-glucosyl hesperidin can be known. The basic 3 dimensional structure of the molecule.

Based on the X-ray diffraction intensity data, the crystallographic parameters of the crystal of the present invention can be determined. The crystal group of the present invention has a space group of P2 ₁ , and the lattice constant of the unit lattice is a=13.983 Å, b=7.620 Å, c=20.065 Å, and is monoclinic crystal with α=γ=90° and β=93.475°. Department (monoclinic). The crystal is analyzed by an X-ray crystal structure, and has a single crystal having a sufficient crystal structure. The crystal of the present invention is not limited to the crystal having the space group, the lattice constant, and the crystal system. The overall single crystal morphology.

The α-glucosyl hesperidin crystal of the present invention, more specifically, each of the oxygen atoms having an α-glucosyl hesperidin molecule and each carbon atom in the crystal are as shown in Tables 3 and 4 below (Continued Table 3) The atomic coordinates shown are provided in the ORTEP diagram shown in Figure 4.

In addition, in the present specification, the "powder consisting essentially of crystals of α-glucosyl hesperidin" means that it is almost impossible to completely exclude the presence of impurities in the technical or technical reasons. The powder of the α-glucosyl hesperidin crystal of the present invention having the above crystallographic parameters in its entirety, and the purity of the α-glucosyl hesperidin in the powder is usually 95.0% or more, preferably More than 98.0%, more preferably 99.0% or more. In addition, in the present specification, "powder" means a specific powder, for example, a package. An aggregate of solid particles containing granules, granules, and shaped bodies.

By the way, in the stereospecific configuration of the 3'-hydroxy-4'-methoxyphenyl group, there are two optical isomers of the R body and the S body, and the S body is naturally present. The aqueous solution is easily inverted into a mixture with the R body by a stereoscopic arrangement such as heating. Therefore, α-glucosyl hesperidin produced from glycosides by a sugar transfer reaction is usually mixed with the S body as an optical isomer. The R body and the S body of α-glucosyl hesperidin are represented by the following Chemical Formulas 1 and 2.

Chemical formula 1: α-glucosyl-(R)-hesperidin

Chemical formula 2: α-glucosyl-(S)-hesperidin

The R body and the S body of the optical isomer of hesperidin can be separated and quantified by HPLC using an optical analysis column, and the R body and the S body of α-glucosyl hesperidin can be analyzed by the same method. Further, the crystal of the α-glucosyl hesperidin of the present invention or the powder substantially composed of the crystal is as described in the experiment described later, and the R body as the optical isomer is 70% or more. In other words, it contains 70% or more and less than 80%.

2. Method for producing α-glucosyl hesperidin crystal

Hereinafter, a method for producing the α-glucosyl hesperidin crystal of the present invention will be described.

The α-glucosyl hesperidin which produces the raw material of the α-glucosyl hesperidin crystal of the present invention is not particularly limited, and may be obtained by an organic synthesis method or by an enzyme synthesis method. . Usually, in the presence of hesperidin, a cyclomaltodextran glucanotransferase acts on a partial decomposition product of starch (hereinafter, simply referred to as "CGTase"), followed by glucose conversion The α-glucosyl hesperidin obtained by purification is further suitable. The α-glucosyl hesperidin powder obtained by the enzyme synthesis method contains impurities derived from the method for producing the same. The term "miscellaneous material from the manufacturing method" as used in the present specification means that hesperidin which is a raw material is originally mixed in a trace amount in a glycoside similar to hesperidin (hereinafter, this specification is simply referred to as "other glycoside". ”), when the unreacted hesperidin and unreacted hesperidin remaining in the sugar transfer reaction decompose the enzyme having α-L-rhamnosidase activity, the decomposition product is 7-glucosylcholine Glycosides and the like.

The α-glucosyl hesperidin used for the crystallization of the present invention may be prepared so as to have a purity sufficiently high to form crystals. The purity of α-glucosyl hesperidin can be confirmed by a conventional purity confirmation means (for example, the aforementioned HPLC analysis). As the α-glucosyl hesperidin-containing substance for producing a raw material for crystallization, it is preferable that the α-glucosyl hesperidin having a purity of 90% or more is preferable, and it is preferably 95% or more, more preferably 98%. The above is more preferably 99% or more.

When the organic compound is crystallized, the solution containing the organic compound is usually changed from a solution state to a non-dissolved state in a supersaturated state, and when a specific condition is satisfied, it is carried out based on the property of precipitation as a crystal. Crystallization of α-glucosyl hesperidin, specifically, by (1) adding α-glucosyl hesperidin to an aqueous alcohol solution, heating to dissolve it; (2) reducing dissolved α-glucosyl orange peel The temperature of the glycoside solution becomes supersaturated; (3) The α-glucosyl hesperidin solution in a supersaturated state is maintained at a certain temperature to precipitate crystals; Further, crystallization can be promoted by adding seed crystals if necessary.

Incidentally, in the above-mentioned Japanese Patent No. 3833811, a freeze-dried solid (amorphous powder) purified to a purity of 81% or 85% of α-glucosyl hesperidin is added to a 99% (v/v) methanol solution. After heating and dissolving at 80 ° C, it was allowed to stand at room temperature to precipitate crystals, washed with 99% (v/v) methanol solution, and dried to produce crystals containing α-glucosyl hesperidin. Α-glucosyl hesperidin high content. However, when the crystallization method described in Japanese Patent No. 3833811 is reproduced, α-glucosyl hesperidin, as shown in Fig. 7, is a block crystal in which minute crystals are fixed to each other. With such crystallization, it is impossible to analyze the crystal structure of α-glucosyl hesperidin, and the crystal is not confirmed to be composed of a single crystal form.

As a solvent for preparing the crystal of the present invention, a lower alcohol having a carbon number of 1 to 4, such as methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol or t-butanol, is mixed with water. An aqueous alcohol solution is preferred. For example, when a methanol aqueous solution is used as the solvent, the methanol concentration is usually 80% (v/v) or less, preferably 60% (v/v) or less, more preferably 30 to 40% (v/v). When the methanol concentration exceeds 80% (v/v), the crystals are rapidly precipitated depending on the concentration of α-glucosyl hesperidin, and it is not preferable because the minute crystals are formed as block crystals which are fixed to each other. Further, when the concentration of the alcohol is too low, it is inconvenient that the crystal does not precipitate or precipitates and grows for a long time.

The concentration of α-glucosyl hesperidin dissolved in the above aqueous solution of the lower alcohol may be a concentration suitable for crystallization, and is usually 0.1 to 20% (w/v), preferably 0.5 to 10% (w/v). ), preferably 1 to 5% (w/v). When the concentration of α-glucosyl hesperidin is higher than 20% (w/v) in the crystallization, crystals are rapidly precipitated depending on the alcohol concentration of the solvent, and it is not preferable because the minute crystals are formed as block crystals which are fixed to each other. Further, when the concentration of α-glucosyl hesperidin is less than 0.1% (w/v), it is inconvenient that crystals do not precipitate or precipitate and grow for a long time.

The temperature condition, the concentration of α-glucosyl hesperidin, is also usually 0 to 50 ° C, preferably 3 to 30 ° C, more preferably 4 to 10 ° C, depending on the alcohol concentration of the solvent.

After the crystallization is obtained, a crystal suitable for the resolution of the X-ray crystal structure is selected from the crystallized crystal by a stereoscopic microscope. Try to avoid those who are covered by complex crystals, those who are cracked, those who are white, and those who have microcrystalline on the surface. Further, in order to avoid precipitation of microcrystals from the mother liquid on the surface of the crystal, it is advantageous to apply Paratone oil for the purpose of crystallization. The crystals thus obtained can be used for the analysis of the X-ray crystal structure.

The crystal of the α-glucosyl hesperidin of the present invention or the powder substantially composed of the crystal, the crystal structure is highly purified to a purity level that can be resolved, and is crystallized and prepared with high quality α- Glucosyl hesperidin. The crystal of α-glucosyl hesperidin of the present invention and the powder substantially composed of the crystal itself are effective and safe stable raw materials for medicine, and are also used as prevention or treatment of various diseases like hesperidin. Agent use is not just about Hesperidin such as viral diseases, bacterial diseases, cardiovascular diseases, and malignant tumors is effective, and is also used as a solid physical or crystal polymorphism for explaining the solubility, stability, and the like of α-glucosyl hesperidin. Tests with or without metastases are also extremely useful.

The crystal of the α-glucosyl hesperidin of the present invention and the powder substantially composed of the crystal are not particularly limited as long as they are used as a raw material for pharmaceuticals, and can be used according to solids, powders, granules, tablets, and the like. It can also be used in the form of a composition of other pharmaceutical raw materials.

Hereinafter, the present invention will be further described in detail based on the reference examples and examples. However, the invention is not limited thereto by these examples.

<Reference example: Preparation of high-purity α-glucosyl hesperidin amorphous powder>

Amorphous powder containing α-glucosyl hesperidin for cosmetics (trade name “α-glucosyl hesperidin”, α-glucosyl hesperidin content 83.8%, research on biochemistry of Linyuan Co., Ltd. The product (hereinafter referred to as "sample 1") is used as a raw material for preparing a high-purity α-glucosyl hesperidin amorphous powder. That is, 200 g of the sample 1 was suspended in 134 ml of an 80% (v/v) aqueous solution of ethanol, heated to 80 ° C while stirring, completely dissolved, cooled to room temperature, and allowed to stand at room temperature for 5 days, and then precipitated. Crystallization, the crystals become blocky pieces that are fixed to each other. The obtained crystal block was washed with about 200 ml of an 80% (v/v) aqueous ethanol solution, and the crystals were collected by filtration using a filter paper (No. 131, manufactured by Advantech Toyo Co., Ltd.), and dried at 45 ° C for 5 hours. The crucible was pulverized to obtain 15.3 g of a powder containing α-glucosyl hesperidin crystals (hereinafter referred to as "samples" 2"). The purity of α-glucosyl hesperidin on sample 2 was 96.3%.

15 g of the sample 2 obtained above was dissolved in purified water having a concentration of 12.5% (w/v), and the filtered product of a 0.22 μm membrane filter was divided into three times and about 5 g each was supplied to the ODS column layer under the following conditions. Analysis.

<ODS column chromatography conditions>

Device: "CCCP-D" (made by Tosoh Corporation)

Pipe column: "YMC-Pak ODS-AQ" (inner diameter 50mm × length 500mm) (manufactured by YMC Co., Ltd.)

Sample injection amount: 40ml

Eluent: 35% (v/v) aqueous methanol solution

Flow rate: 40ml/min

Temperature: 40 ° C

Detection: Differential refractometer "Shodex RI-102" (made by Showa Denko Co., Ltd.)

Data processing device: "Chromatographic Pack C-R7A" (made by Shimadzu Corporation)

Fraction: 50ml / fraction

The purity of α-glucosyl hesperidin was measured by HPLC analysis on each fraction obtained by ODS column chromatography, and a fraction having a purity of 98% or more was collected and freeze-dried by a usual method to obtain 13.2 g of purity 99.0. % amorphous freeze-dried powder containing α-glucosyl hesperidin (hereinafter referred to as "sample 3").

The above samples 1 to 3, that is, an amorphous powder containing α-glucosyl hesperidin for cosmetics, a powder containing α-glucosyl hesperidin crystals, and an α-glucosyl orange peel The composition of the amorphous freeze-dried powder of glycosides is summarized in Table 1.

In sample 1, the purity of α-glucosyl hesperidin was increased from 83.8% to 96.3% by crystallization with 80% (v/v) ethanol, and the content of 7-glucosylhesin in the impurity was 12.3. % decreased to 0.5%, and the content of other glycosides decreased only slightly. Next, the purity of α-glucosyl hesperidin was increased from 96.3% to 99.0% by ODS column chromatography, and the content of other glycoside was decreased to 0.9%. Finally, an amorphous freeze-dried powder containing α-glucosyl hesperidin having a purity of 99.0% used for the preparation of crystals was obtained (Sample 3).

[Example 1] <Preparation of α-glucosyl hesperidin single crystal>

3 g of an amorphous freeze-dried powder containing α-glucosyl hesperidin (sample 3) was added to 150 ml of a 30% (v/v) aqueous methanol solution, and the mixture was stirred and heated to 80 ° C to complete the reaction. Dissolved, and the resulting solution was crystallized by maintaining at 4 ° C for 14 days. A micrograph of the obtained crystal suspension is exemplified in Fig. 1 . As seen in Fig. 1, a relatively large colorless plate-like crystal precipitation was observed. From the crystals of the crystallized α-glucosyl hesperidin, the appropriate size was collected under a stereo microscope of a serial digital microscope ("MX-1200II/NDL", manufactured by Nakaden Co., Ltd.), immediately at Baladong. After coating with oil (Paratone oil), it was mounted on a sample holder and used as a sample for X-ray crystal structure analysis.

The crystal suspension remaining in the crystal for the analysis of the X-ray crystal structure was obtained, and the filter paper (No. 4, manufactured by Advantech Toyo Co., Ltd.) was used. The crystals were collected by filtration from a Kiriyama funnel, and dried to obtain about 1.2 g of crystals of α-glucosyl hesperidin. Further, the α-glucosyl hesperidin of the present crystal product had a purity of 99.3%.

[Embodiment 2] <Analysis of single crystal X-ray crystal structure of α-glucosyl hesperidin>

In the single crystal X-ray diffractometer ("R-AXIS RAPID-R", manufactured by Rigaku Corporation), the single crystal of the α-glucosyloside was placed in the sample holder in Example 1. The X-ray diffraction pattern was measured by a vibrating photo method under the following conditions in an environment of 170 ° C.

<X-ray diffraction pattern measurement conditions>

X light source: Cu

Output: 50kV, 100mA

Incident X-ray: CuK α line (λ=1.54187Å)

Incident X-ray size: about 0.5mm

Crystal size: 0.10 × 0.10 × 0.03mm

Detector: imaging plate

Measuring temperature: about -170 ° C (nitrogen spraying method)

A photomicrograph of α-glucosyl hesperidin crystals analyzed by X-ray diffraction for single crystal structure is shown in Fig. 2, and one of the X-ray diffraction patterns is shown in Fig. 3, respectively. In the X-ray diffraction pattern, a large number of diffraction spots (Spot) were confirmed, and it was confirmed that the crystal was a single crystal. Also, X-ray The shape of the diffraction peak is relatively good, and the peak intensity is weakened. It was observed that within 39,960 reflections (diffraction), the intrinsic reflection was 7,806.

Based on the X-ray diffraction intensity of the X-ray diffraction pattern, the initial structure was obtained by the direct method, and the structural model was prepared with the structural formula of α-glucosyl hesperidin as a reference, and then based on the minimum of 4,739 reflections. The two-time method makes it precise. In addition, as the analysis software, "Crystal Structure Ver. 3.8.2" manufactured by Rigaku Corporation was used. The crystallographic data of α-glucosyl hesperidin obtained by X-ray crystal structure analysis are summarized in Table 2.

As shown in Table 2, from the obtained X-ray diffraction intensity data, the crystal system to which the crystal belongs is determined to be monoclinic, the space group is P2 ₁ , and the lattice constant is a=13.983 Å, b=7.620 Å. , c = 20.065 Å, α = γ = 90 °, β = 93.475 °, V = 213395 Å ³ . Further, the Z value of the number of α-glucosyl hesperidin molecules per unit lattice of the crystal was 2, and in the present crystal, it was judged that two molecules of α-glucosyl hesperidin were contained per unit lattice of the crystal.

<Analysis of molecular structure and crystal structure>

According to the results of the direct method analysis, the hydrogen atom on the α-glucosyl hesperidin molecule is unidentifiable, and the basic skeleton of the non-hydrogen atom (carbon atom and oxygen atom) can be based on α-glucosyl orange. The structure information of dermatan is to arrange elements at each atomic position. The atomic coordinates (x, y, z) of each oxygen atom and carbon atom on the α-glucosyl hesperidin molecule and their respective isotropic (Isotropy) temperature factors (B _eq ) are obtained by precision of coordinate data. The values are shown in Tables 3 and 4 (continued Table 3). Further, the distance between the atoms other than the hydrogen atom on the α-glucosyl hesperidin molecule is shown in Table 5, and each shows the bonding angle between the atoms except the hydrogen atom in Table 6 and Table 7. (Continued from Table 6).

Further, an ORTEP diagram showing the expression of α-glucosyl hesperidin from the refined coordinate data is shown in Fig. 4 . Further, the packaging structure of the α-glucosyl hesperidin molecule per crystal unit lattice is shown in Fig. 5 as a crystal structure diagram seen from the b-axis direction. Further, in Tables 3 to 7, the numbers of oxygen atoms and carbon atoms correspond to the numbers of oxygen atoms and carbon atoms described in the ORTEP diagram of α-glucosyl hesperidin of Fig. 4 . Further, the numerical values in parentheses in the numerical values in Tables 3 to 7 mean the standard deviation.

Further, on the ORTEP diagram shown in Fig. 4, each shows the celloside structure of the carbon number C1 to C16 on the α-glucosyl hesperidin molecule, and the carbon number C17 to C28 Rutinose (L) - the structure of rhamnopyranosyl (α 1 → 6) glucose), and the carbon number C29 to C34 constitute a rutinose structure of the 4th hydroxyl group of glucose combined by α-glucoside combination Glucose structure. On the other hand, from the crystal structure diagram shown in Fig. 5, in the present crystal, it is understood that each unit lattice is packaged by two molecules of α-glucosyl hesperidin.

[Example 3] <Modulation of α-glucosyl hesperidin crystals>

2 g of the amorphous freeze-dried powder containing α-glucosyl hesperidin (sample 3) was added to 50 ml of a 40% (v/v) aqueous methanol solution, and the mixture was stirred and heated to 80 ° C to complete the reaction. After dissolution, the resulting solution was crystallized by maintaining at 10 ° C for 10 days. The same colorless, plate-like crystals as those obtained in Example 1 were obtained. This crystal was also used for the analysis of the X-ray crystal structure in the same manner as the crystal obtained in Example 1. Crystalline suspension It was collected by filtration through a Kiriyama funnel (No. 4, manufactured by Advantech Toyo Co., Ltd.) to obtain a crystal of α-glucosyl hesperidin of about 0.9 g. Further, the purity of α-glucosyl hesperidin of the crystal product was 99.1%.

[Example 4] <Measurement of the existence ratio of optical isomers on α-glucosyl hesperidin crystals>

The optical isomer ratio of α-glucosyl hesperidin on the α-glucosyl hesperidin crystals obtained in Examples 1 and 3 was investigated by an optical analysis column.

Each of the crystals was dissolved in about 10 mg, and each was dissolved in 10 ml of an eluent (20 mm aqueous potassium phosphate solution (pH 3.0) / acetonitrile (86/14)), and subjected to optical analysis HPLC under the conditions shown below to measure optical isomers. There is a ratio. An example of the crystal chromatogram of Example 1 is shown in Fig. 6. Further, the analysis results of the respective crystal products are shown in Table 8.

<HPLC analysis conditions>

HPLC device:

Pipe column: "SUMICHIRAL OA-7000" (4.6×250mm) (manufacturing center of Sumitomo Co., Ltd.)

Sample injection amount: 10μl

Eluent: 20mm aqueous solution of potassium phosphate (pH 3.0) / acetonitrile (86/14)

Flow rate: 0.4ml/min

Column temperature: 35 ° C

Detection: UV280nm

Data processing device: "Chromatographic Pack CR-4A" (made by Shimadzu Corporation)

The peak area ratio of the R body and the S body of α-glucosyl hesperidin was used as a ratio of existence on the chromatogram.

As shown in Fig. 6, under the present analysis conditions, the R body and the S body peak of α-glucosyl hesperidin were completely separated by an optical analysis column, and each was eluted at a holding time (Rt) of 31.4 minutes and 34.9 minutes. As is clear from Table 8, the ratio of the R body to the S body of the crystalline product of α-glucosyl hesperidin obtained in Example 1 as an optical isomer was about 8:2, and the crystal product of Example 3 The year-on-year rate is about 7:3, so the existence of the R body is relatively high.

[Example 5] <Method for Producing Powder Consisting of Crystal of α-Glucosylhesin

4 parts by mass of an amorphous freeze-dried powder containing α-glucosyl hesperidin obtained by the method of Reference Example was added to 100 parts by mass of a 35% (v/v) aqueous methanol solution, and heated to 80 ° C while stirring. It was completely dissolved, and then the resulting solution was crystallized by maintaining at 10 ° C for 14 days. The same colorless, plate-like crystals as those obtained in Example 1 were obtained. After the crystal suspension is recovered by filtration, it is dried to obtain about 2 parts by mass of a powder substantially composed of crystals of α-glucosyl hesperidin. Further, the α-glucosyl hesperidin of the product has a purity of 99.2%. This product is suitable for use as a raw material for pharmaceuticals.

[Embodiment 6] <Lings>

A powder obtained by the method of Example 5, which is substantially composed of 20 parts by mass of a crystal of α-glucosyl hesperidin, 30 parts by mass of maltose, and 4 parts by mass of corn starch, is generally mixed. One lozenge of 0.5 g of lozenge was produced on a tablet press. This product can be advantageously used not only as a vitamin P supplement, as a therapeutic agent for vascular diseases, as a therapeutic agent for joint diseases, and the like.

[Embodiment 7] <multivitamin agent>

10 parts by mass of sodium L-ascorbate (test grade, sold by Wako Pure Chemical Industries Co., Ltd.), and the crystal obtained by the method of Example 5 consisting essentially of crystals of α-glucosyl hesperidin Powder 15 quality The mixture was mixed with a general-purpose mixer, left overnight, and pulverized to prepare a powdery multivitamin. This product stabilizes L-ascorbic acid via α-glucosyl hesperidin, not only for the prevention or treatment of vitamin C deficiency and vitamin P deficiency, but also for the purpose of strengthening microvessels and improving gastrointestinal diseases. on.

[Embodiment 8] <Dissolved injection before use>

The α-glucosyl hesperidin obtained by the method of the reference example contained 3 parts by mass of an amorphous freeze-dried powder in 150 parts by mass of a 35% (v/v) aqueous methanol solution, and was heated to 80 ° C while stirring. After it was completely dissolved, it was subjected to precision filtration in accordance with an ordinary method to remove the pyrogenic property, and in the same manner as in Example 1, the α-glucosyl hesperidin was crystallized and dried in the same manner as in Example 1. Thereafter, the powder was pulverized to obtain about 1 part by mass of the pyrogen-free powder substantially composed of the crystal of α-glucosyl hesperidin. Further, the α-glucosyl hesperidin of the product has a purity of 99.3%. This 50 mg of the powder was placed in a sterile 20 ml jar and replaced with nitrogen, and then sealed to form a pre-use dissolved injection containing an ampoule. This product can be used as a vitamin P supplement, as a therapeutic agent for vascular diseases, a therapeutic agent for joint diseases, etc., by using a monomer or a mixture of other vitamins and minerals.

[Embodiment 9] Effect of <α-glucosyl hesperidin on autophagy of cells>

The α-glucosyl hesperidin obtained by the method of Example 3 was used as a crystalline α-glucosyl hesperidin standard having a purity of 99% or more, and the α-glucosyl hesperidin was investigated for the self of the cells. The phagocytosis, that is, the effect of preventing the disease phenomenon by actively excluding the mitochondria which are accumulated in the cells, such as proteins and damage. The HOZOT cells of the immune system cells (refer to the re-patented WO2007/105797) or the SH-SY-5 strain of the neural cell strain were used as test subjects, and α-glucosyl hesperidin was added to the HOZOT cells. The culture solution having a concentration of 1 or 10 μM and a final concentration of 10 or 25 μM in the SH-SY-5 strain was recovered after 24 hours, and the cell extract was prepared. Next, after the cell extract was subjected to SDS-PAGE, the expression amount of LC-3B which is an autophagy marker protein was determined by Western blotting. The results when HOZOT cells were used as test cells are shown in Table 9, and the results when using SH-SY-5 strain are shown in Table 10, respectively.

As is clear from Table 9, in the case of HOZOT cells, treatment with 1 μM of α-glucosyl hesperidin increased the expression amount of LC-3B to 4.6 times that of no treatment (Control 1). The effect of this α-glucosyl hesperidin was stronger than that of the drug MG-132 (Control 2) which is known to promote autophagy, and to the same extent as the thiazolidinedione (Control 3). On the other hand, it is clearly understood from Table 10 that in the case of the SH-SY-5 strain, treatment with 10 μM or 25 μM of α-glucosyl hesperidin increased the expression amount of LC-3B to no treatment (Control 1). It is about 6.3 times or about 7.5 times. This effect was stronger than in the case of treatment with 1 μM thiazolidinedione (Control 3). From the results of these, it can be judged that α-glucosyl hesperidin is useful as an autophagy promoter for cells. Autophagy, known to be associated with neurodegenerative diseases represented by Huntington's disease or type 2 diabetes, alpha-glucosyl hesperidin with cellular autophagy promotion can be used for the treatment of these diseases .

[Industry use possibility]

The present invention provides a novel crystal of α-glucosyl hesperidin having a crystal structure and a powder substantially composed of the crystal, and a use as a raw material of the pharmaceutical. The crystal of α-glucosyl hesperidin associated with the present invention and the powder substantially composed of the crystal itself are effective and safe stable raw materials for medicine, and are not only usable as orange peel, like hesperidin. The use of a prophylactic or therapeutic agent for various diseases such as a viral disease, a bacterial disease, a cardiovascular disease, and a malignant tumor, and the solid physical property as a solution for the solubility, stability, and the like of α-glucosyl hesperidin A reagent for the presence or absence of a crystalline polymorphism or a transfer phenomenon is also extremely useful. The use of the present invention as a raw material for pharmaceuticals of α-glucosyl hesperidin has been greatly expanded, and its industrial usefulness is extremely large.

Fig. 1 is a micrograph of a crystal of α-glucosyl hesperidin crystal obtained by crystallization from a 30% (v/v) aqueous methanol solution (magnification: 250 times).

Fig. 2 is a solid-state micrograph of α-glucosyl hesperidin crystal (0.1 × 0.1 × 0.03 mm) used for analysis of an X-ray crystal structure.

Fig. 3 is an example of an X-ray diffraction pattern of α-glucosyl hesperidin crystals.

Fig. 4 is an ORTEP diagram of α-glucosyl hesperidin which removes a hydrogen atom.

Fig. 5 is a crystal structure diagram (b-axis direction) showing a molecular package of α-glucosyl hesperidin on a crystal unit lattice.

Fig. 6 is a chromatogram of an α-glucosyl hesperidin crystal used for analysis of an X-ray crystal structure for optical analysis HPLC.

Fig. 7 is a micrograph of a microscope photograph of α-glucosyl hesperidin crystal obtained by crystallization using a 99% (v/v) methanol solution (magnification: 500 times).

Claims (6)

A crystal of α-glucosyl hesperidin having a space group of P2 ₁ and a lattice constant of a unit lattice of a=13.983 Å, b=7.620 Å, c=20.065 Å, and α=γ=90 °, β = 93.475 ° monoclinic system (monoclinic). The crystal of the first aspect of the patent application, wherein the oxygen atom and the carbon atom constituting the α-glucosyl hesperidin molecule have atomic coordinates as shown in Tables 3 and 4. For example, the crystal of item 1 or item 2 of the patent application is in the form of a single crystal. A powder consisting essentially of the crystals described in any one of the first to third aspects of the patent application. A crystal according to the first to third aspects of the patent application or a powder according to the fourth aspect of the patent application, wherein, in the case of high-speed liquid chromatography using an optical analysis column, 70% by mass or more is contained as an optical difference. The structure of α-glucosyl-(R)-hesperidin. The crystallization described in the first to third patent applications or the powder according to item 4 of the patent application is used as a raw material for pharmaceuticals.