阅读说明：本技术 一种n-乙酰神经氨酸钙盐晶体及其制备方法和应用 (N-acetylneuraminic acid calcium salt crystal and preparation method and application thereof ) 是由 朱薇薇 袁丽霞 陈祥松 吴金勇 王煜 王刚 费贤春 李翔宇 孙立洁 王纪 姚建铭 于 2019-10-08 设计创作，主要内容包括：本发明提供一种N-乙酰神经氨酸钙盐晶体及其制备方法和应用,所述N-乙酰神经氨酸钙盐晶体的分子式为(C<Sub>11</Sub>H<Sub>18</Sub>NO<Sub>9</Sub>)<Sub>2</Sub>Ca,在粉末X射线衍射谱图中有明显的衍射峰,具有优异的溶解性和高温稳定性,184℃高温处理下不会变色。本发明所述的N-乙酰神经氨酸钙盐晶体可以通过N-乙酰神经氨酸和钙源在水相环境中反应、进而结晶析出得到,其制备方法简单,产物的收率和纯度高,适宜规模化生产。本发明提供的高稳定性、高溶解度的N-乙酰神经氨酸钙盐晶体吸收率高,能够同时满足人体对钙和N-乙酰神经氨酸的补充需求,具有广阔的应用前景。(The invention provides an N-acetylneuraminic acid calcium salt crystal and a preparation method and application thereof, wherein the molecular formula of the N-acetylneuraminic acid calcium salt crystal is (C) 11 H 18 NO 9 ) 2 Ca has obvious diffraction peak in powder X-ray diffraction spectrum, excellent solubility and high temperature stability, and does not generate reaction at 184 deg.CAnd (5) changing color. The N-acetylneuraminic acid calcium salt crystal can be obtained by reacting N-acetylneuraminic acid and a calcium source in an aqueous environment and then crystallizing and separating out, and the preparation method is simple, high in product yield and purity and suitable for large-scale production. The N-acetylneuraminic acid calcium salt crystal with high stability and high solubility provided by the invention has high absorptivity, can simultaneously meet the supplement requirements of human bodies on calcium and N-acetylneuraminic acid, and has wide application prospects.)

1. The N-acetylneuraminic acid calcium salt crystal is characterized in that the molecular formula of the N-acetylneuraminic acid calcium salt crystal is (C)₁₁H₁₈NO₉)₂Ca。

2. The crystal of the calcium salt of N-acetylneuraminic acid according to claim 1, which has a powder X-ray diffraction pattern having diffraction peaks at diffraction angles of 2 θ of 15.98 ± 0.2 °, 17.70 ± 0.2 °, 22.46 ± 0.2 °, 25.16 ± 0.2 °, 26.86 ± 0.2 °, 28.62 ± 0.2 °, 29.34 ± 0.2 °, 32.46 ± 0.2 °, 33.48 ± 0.2 °, 35.68 ± 0.2 °, 38.12 ± 0.2 °, 42.98 ± 0.2 °, 44.10 ± 0.2 °, 46.88 ± 0.2 °, 51.80 ± 0.2 °, 61.96 ± 0.2 °, 65.82 ± 0.2 °.

3. The calcium N-acetylneuraminic acid salt crystals according to claim 1 or 2, wherein the melting point of the calcium N-acetylneuraminic acid salt crystals is 232 to 235 ℃;

preferably, the melting point of the N-acetylneuraminic acid calcium salt crystals is 233.35 ℃.

4. A method for producing the crystal of N-acetylneuraminic acid calcium salt according to any one of claims 1 to 3, which comprises the steps of:

(1) mixing N-acetylneuraminic acid, a calcium source and water, and reacting to obtain a reaction solution;

(2) adding a crystallization solvent into the reaction solution obtained in the step (1), standing for crystallization, and carrying out solid-liquid separation to obtain a solid phase which is the N-acetylneuraminic acid calcium salt crystal.

5. The method according to claim 4, wherein the calcium source in the step (1) is calcium hydroxide or calcium carbonate.

6. The method according to claim 4 or 5, wherein the molar ratio of N-acetylneuraminic acid to the calcium source in the step (1) is (1.9-2.1): 1, preferably 2: 1;

preferably, the mass volume ratio of the N-acetylneuraminic acid to the water in the step (1) is 10-200 g/L;

preferably, the temperature of the reaction in the step (1) is 20-60 ℃;

preferably, the reaction time in the step (1) is 2-8 h;

preferably, the reaction of step (1) is carried out under stirring conditions.

7. The method according to any one of claims 4 to 6, wherein the step (1) further comprises a decolorization treatment of the reaction solution;

preferably, the decolorant for the decoloration treatment is activated carbon;

preferably, the adding amount of the activated carbon is 0.1-1.0% of the mass of the reaction liquid;

preferably, the decoloring treatment method comprises the following steps: mixing the reaction solution with activated carbon, decoloring, and performing solid-liquid separation to obtain a liquid phase which is the decolored reaction solution;

preferably, the decolorization is performed under stirring or shaking conditions;

preferably, the temperature of the decolorization is 30-70 ℃;

preferably, the decoloring time is 10-60 min;

preferably, the solid-liquid separation method is membrane separation;

preferably, the pore size of the membrane in the membrane separation is 0.22 μm;

preferably, the step (1) further comprises a concentration treatment of the reaction solution;

preferably, the concentration treatment method comprises the following steps: heating the reaction solution to volatilize the solvent until the concentration of the calcium N-acetylneuraminic acid in the reaction solution is 0.15-2.5 mol/L.

8. The preparation method according to any one of claims 4 to 7, wherein the crystallization solvent in step (2) is selected from any one of an alcohol solvent, a ketone solvent, acetonitrile or tetrahydrofuran, or a combination of at least two thereof;

preferably, the alcohol solvent is a C1-C5 alcohol solvent;

preferably, the crystallization solvent is methanol and/or ethanol;

preferably, the volume ratio of the crystallization solvent to the reaction solution is (2.5-5): 1, and more preferably (3-5): 1;

preferably, the temperature of the standing crystallization in the step (2) is 0-10 ℃, and further preferably 3-5 ℃;

preferably, the standing crystallization time in the step (2) is 8-24 h;

preferably, the solid-liquid separation method in the step (2) is membrane separation;

preferably, the pore size of the membrane in the membrane separation is 0.22 μm;

preferably, the step (2) further comprises a post-treatment step;

preferably, the post-treatment step comprises washing and drying;

preferably, the washing solvent is an aqueous solution of C1-C5 alcohol solvent or C1-C5 alcohol solvent;

preferably, the drying temperature is 50-80 ℃;

preferably, the drying is reduced pressure drying or normal pressure drying;

preferably, the drying time is 5-12 h.

9. The method according to any one of claims 4 to 8, wherein the method comprises the steps of:

(1) mixing N-acetylneuraminic acid, a calcium source and water, and stirring at 20-60 ℃ for reacting for 2-8 hours to obtain a reaction solution; wherein the molar ratio of the N-acetylneuraminic acid to the calcium source is (1.9-2.1): 1, the mass volume ratio of the N-acetylneuraminic acid to the water is 10-200 g/L, and the calcium source is calcium hydroxide or calcium carbonate;

(2) adding a crystallization solvent with the volume of 2.5-5 times that of the reaction liquid obtained in the step (1), wherein the crystallization solvent is selected from any one or a combination of at least two of an alcohol solvent, a ketone solvent, acetonitrile or tetrahydrofuran; standing and crystallizing for 8-24 h at 0-10 ℃, separating solid from liquid, collecting solid phase, washing and drying to obtain the N-acetylneuraminic acid calcium salt crystal.

10. Use of the crystal of calcium N-acetylneuraminic acid salt according to any one of claims 1 to 3 in a medicament, a health product, a food or a skin care product.

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to an N-acetylneuraminic acid calcium salt crystal and a preparation method and application thereof.

Background

N-acetylneuraminic acid (Neu 5Ac) is the most predominant of the fifty sialic acids found to date, and accounts for over 99% of all sialic acids. Neu5Ac has a pyranose structure with 9 carbon atoms in the molecule, is an acidic amino sugar mainly in the form of alpha-glycoside at the end of non-reducing oligosaccharides such as glycoproteins or glycolipids, is easily soluble in water, and can be stably stored in aqueous solution for a long period of time. Neu5Ac is the first contact site for cell information transmission, has diverse molecular structures, can participate in multiple physiological processes such as cell recognition, signal transduction, tumorigenesis and fertilization, and has wide application in the fields of food, medicine and rapid disease detection. In addition, Neu5Ac can regulate IgG anti-inflammatory activity, influence nerve cell integrity, permeability and activity, enhance infant immunity, and promote infant brain development.

In recent years, the research on the medical value and the nutritional value of N-acetylneuraminic acid has led to the development of the preparation process of the compound, and the feasible preparation methods of N-acetylneuraminic acid comprise a natural extraction method, a microbial fermentation method, an enzyme catalysis method and a chemical synthesis method, and the synthesized N-acetylneuraminic acid crystals comprise dihydrate crystals and anhydrous crystals. However, in the application process of the N-acetylneuraminic acid crystal, researchers find that the N-acetylneuraminic acid aqueous solution shows strong acidity and generally small solubility which is about 100g/L at normal temperature, and the N-acetylneuraminic acid crystal and the N-acetylneuraminic acid aqueous solution have poor stability and gradually turn into dark brown in the storage process, so that the application of Neu5Ac is greatly limited. Therefore, the method improves the water solubility and the stability of the N-acetylneuraminic acid, and has important significance for further expanding the application range of the N-acetylneuraminic acid.

For example, CN107207552A discloses a crystal of N-acetylneuraminic acid ammonium salt anhydride and a method for producing the same, wherein the method specifically comprises: adding or dropwise adding a solvent consisting of alcohols and ketones into an aqueous solution containing N-acetylneuraminic acid containing an ammonium compound with the pH of 3.0-9.0 to separate out crystals of an N-acetylneuraminic acid ammonium salt anhydrous substance to obtain a product; the crystal melting point of the N-acetylneuraminic acid ammonium salt anhydrate is about 167 ℃, and the solubility in water and the stability at a high temperature of 60 ℃ are remarkably improved. CN106573950A discloses crystals of an alkali metal salt anhydride of N-acetylneuraminic acid and a process for producing the same, which comprises adding or dropping a solvent comprising an alcohol and a ketone to an aqueous solution of N-acetylneuraminic acid containing an alkali metal compound at a pH of 3.0 to 9.0 to precipitate crystals of the alkali metal salt anhydride of N-acetylneuraminic acid, and collecting the crystals of the alkali metal salt anhydride of N-acetylneuraminic acid from the aqueous solution.

Calcium is a blood coagulation factor, can reduce excitability of nerves and muscles, is a basic stone forming human skeleton, and has important significance for the growth of infants and the physical health of adults. The calcium agent for supplementing calcium to human body is divided into inorganic calcium and organic calcium, the inorganic calcium is represented by calcium carbonate, and the calcium products have the characteristics of general solubility and general absorptivity; the organic calcium is represented by calcium gluconate, and the calcium product has the characteristics of better dissolution and higher absorption rate.

Because the calcium element and the N-acetylneuraminic acid are both beneficial to the health of human bodies and are particularly beneficial to the growth and development of infants, the development of a substance simultaneously containing the calcium element and the N-acetylneuraminic acid has very important significance in the aspects of biological medicines, health-care foods and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the N-acetylneuraminic acid calcium salt crystal, the preparation method and the application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a crystal of N-acetylneuraminic acid calcium salt having the formula (C)₁₁H₁₈NO₉)₂Ca。

Preferably, in the powder X-ray diffraction spectrum of the crystal of the N-acetylneuraminic acid calcium salt, diffraction peaks are found at the 2 theta diffraction angles of 15.98 +/-0.2 degrees, 17.70 +/-0.2 degrees, 22.46 +/-0.2 degrees, 25.16 +/-0.2 degrees, 26.86 +/-0.2 degrees, 28.62 +/-0.2 degrees, 29.34 +/-0.2 degrees, 32.46 +/-0.2 degrees, 33.48 +/-0.2 degrees, 35.68 +/-0.2 degrees, 38.12 +/-0.2 degrees, 42.98 +/-0.2 degrees, 44.10 +/-0.2 degrees, 46.88 +/-0.2 degrees, 51.80 +/-0.2 degrees, 61.96 +/-0.2 degrees and 65.82 +/-0.2 degrees.

In the powder X-ray diffraction spectrogram of the N-acetylneuraminic acid calcium salt crystal, +/-0.2 is the error in the test.

Preferably, the melting point of the N-acetylneuraminic acid calcium salt crystal is 232 to 235 ℃, for example, 232.1 ℃, 232.3 ℃, 232.5 ℃, 232.7 ℃, 232.9 ℃, 233 ℃, 233.2 ℃, 233.4 ℃, 233.6 ℃, 233.8 ℃, 234 ℃, 234.2 ℃, 234.4 ℃, 234.6 ℃, 234.8 ℃ or 234.9 ℃.

Preferably, the melting point of the N-acetylneuraminic acid calcium salt crystals is 233.35 ℃.

In another aspect, the present invention provides a method for preparing the crystal of calcium N-acetylneuraminic acid salt according to the first aspect, which comprises the steps of:

(1) mixing N-acetylneuraminic acid, a calcium source and water, and reacting to obtain a reaction solution;

(2) adding a crystallization solvent into the reaction solution obtained in the step (1), standing for crystallization, and carrying out solid-liquid separation to obtain a solid phase which is the N-acetylneuraminic acid calcium salt crystal.

Preferably, the calcium source in step (1) is calcium hydroxide or calcium carbonate.

Preferably, the molar ratio of N-acetylneuraminic acid to the calcium source in step (1) is (1.9-2.1): 1, for example, 1.91:1, 1.93:1, 1.94:1, 1.95:1, 1.97:1, 1.99:1, 2:1, 2.02:1, 2.04:1, 2.05:1, 2.07:1 or 2.09:1, and more preferably 2: 1.

Preferably, the mass-to-volume ratio of N-acetylneuraminic acid to water in the step (1) is 10-200 g/L, such as 12g/L, 15g/L, 17g/L, 20g/L, 23g/L, 25g/L, 28g/L, 30g/L, 33g/L, 35g/L, 38g/L, 40g/L, 45g/L, 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, 75g/L, 80g/L, 85g/L, 90g/L, 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, 150g/L, 160g/L, 170g/L, 180g/L, 190g/L or 195g/L, as well as the specific points between those points, are not exhaustive for the invention and for brevity.

Preferably, the temperature of the reaction in the step (1) is 20 to 60 ℃, for example, 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃, 36 ℃, 38 ℃, 40 ℃, 42 ℃, 45 ℃, 48 ℃, 50 ℃, 52 ℃, 55 ℃, 57 ℃ or 59 ℃, and the specific values therebetween are limited by space and for the sake of brevity, and the invention is not exhaustive.

Preferably, the reaction time in step (1) is 2-8 h, such as 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h or 7.5h, and the specific values therebetween are not exhaustive, and for brevity and clarity, the invention is not intended to be exhaustive.

Preferably, the reaction of step (1) is carried out under stirring conditions.

Preferably, the step (1) further comprises a decolorization treatment of the reaction solution.

Preferably, the decoloring agent for decoloring treatment is activated carbon.

The amount of the activated carbon added is preferably 0.1 to 1.0% by mass of the reaction solution, for example, 0.15%, 0.18%, 0.2%, 0.23%, 0.25%, 0.28%, 0.3%, 0.33%, 0.35%, 0.37%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, or 0.95%.

Preferably, the decoloring treatment method comprises the following steps: mixing the reaction solution with activated carbon, decoloring, and performing solid-liquid separation to obtain a liquid phase which is the decolored reaction solution.

Preferably, the decolorization is performed under stirring or shaking conditions.

Preferably, the temperature of the decolorization is 30 to 70 ℃, such as 32 ℃, 35 ℃, 38 ℃, 40 ℃, 42 ℃, 44 ℃, 46 ℃, 48 ℃, 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, 66 ℃, 68 ℃ or 69 ℃.

Preferably, the time for decoloring is 10-60 min, such as 12min, 15min, 18min, 20min, 23min, 25min, 28min, 30min, 33min, 35min, 37min, 40min, 45min, 50min or 55 min.

Preferably, the solid-liquid separation method is membrane separation.

Preferably, the pore size of the membrane in the membrane separation is 0.22 μm.

Preferably, the step (1) further comprises a concentration treatment of the reaction solution.

Preferably, the concentration treatment method comprises the following steps: the reaction solution is heated to volatilize the solvent until the concentration of calcium N-acetylneuraminic acid in the reaction solution is 0.15 to 2.5mol/L, for example, 0.17mol/L, 0.19mol/L, 0.2mol/L, 0.25mol/L, 0.3mol/L, 0.35mol/L, 0.4mol/L, 0.5mol/L, 0.6mol/L, 0.8mol/L, 1mol/L, 1.2mol/L, 1.4mol/L, 1.6mol/L, 1.8mol/L, 2mol/L, 2.2mol/L or 2.4 mol/L.

Preferably, the crystallization solvent in step (2) is selected from any one of an alcohol solvent, a ketone solvent, acetonitrile or tetrahydrofuran, or a combination of at least two thereof.

Preferably, the alcohol solvent is a C1 to C5 (e.g., C1, C2, C3, C4, or C5) alcohol solvent.

Preferably, the crystallization solvent is methanol and/or ethanol.

The volume ratio of the crystallization solvent to the reaction solution is preferably (2.5 to 5):1, for example, 2.6:1, 2.8:1, 3:1, 3.2:1, 3.5:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1, or 4.9:1, and more preferably (3 to 5): 1.

As a preferred embodiment of the present invention, the crystallization solvent may be added all at once, or the crystallization solvent may be added in an amount of 2 times the volume of the reaction solution and then the crystallization solvent may be continuously added dropwise under heating (60 to 80 ℃ C., e.g., 62 ℃ C., 65 ℃ C., 68 ℃ C., 70 ℃ C., 73 ℃ C., 75 ℃ C., 78 ℃ C., etc.) until turbidity appears in the system.

In the preparation method of the N-acetylneuraminic acid calcium salt crystal, the addition amount of the crystallization solvent in the step (2) is preferably 2.5-5 times of the volume of the reaction liquid. If the addition amount of the crystallization solvent is too small, the precipitation amount of crystals is extremely small, the precipitation speed is slow, and even no crystallization occurs; if the addition amount of the crystallization solvent is too much, the waste of the organic solvent is caused, and the post-treatment cost of the industrial three wastes is increased. If the preparation method does not have the crystallization process described in the step (2) and directly dries the reaction solution obtained in the step (1), the product is amorphous calcium salt instead of high-stability N-acetylneuraminic acid calcium salt crystal.

Preferably, the temperature of the standing crystallization in the step (2) is 0 to 10 ℃, for example, 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 9.5 ℃, and more preferably 3 to 5 ℃.

Preferably, the standing crystallization time in the step (2) is 8-24 h, such as 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 23.5h, and the specific values therebetween are not exhaustive, and for the sake of brevity, the invention is not exhaustive.

Preferably, the solid-liquid separation method in the step (2) is membrane separation.

Preferably, the pore size of the membrane in the membrane separation is 0.22 μm.

Preferably, the step (2) further comprises a post-treatment step.

Preferably, the post-treatment step comprises washing and drying.

Preferably, the solvent for washing is an aqueous solution of a C1 to C5 (e.g., C1, C2, C3, C4, or C5) alcohol solvent or a C1 to C5 (e.g., C1, C2, C3, C4, or C5) alcohol solvent.

Exemplary, the washed solvents include, but are not limited to: absolute ethyl alcohol, absolute methyl alcohol, 75% ethanol aqueous solution or 75% methanol aqueous solution, etc.

Preferably, the drying temperature is 50-80 deg.C, such as 52 deg.C, 54 deg.C, 56 deg.C, 58 deg.C, 60 deg.C, 62 deg.C, 64 deg.C, 66 deg.C, 68 deg.C, 70 deg.C, 72 deg.C, 74 deg.C, 76 deg.C, 78 deg.C or 79 deg.C.

Preferably, the drying is drying under reduced pressure or drying under normal pressure.

Preferably, the drying time is 5-12 h, such as 5.5h, 6h, 7h, 8h, 9h, 10h, 11h or 11.5 h.

Preferably, the preparation method comprises the following steps:

(1) mixing N-acetylneuraminic acid, a calcium source and water, and stirring at 20-60 ℃ for reacting for 2-8 hours to obtain a reaction solution; wherein the molar ratio of the N-acetylneuraminic acid to the calcium source is (1.9-2.1): 1, the mass volume ratio of the N-acetylneuraminic acid to the water is 10-200 g/L, and the calcium source is calcium hydroxide or calcium carbonate.

(2) Adding a crystallization solvent with the volume of 2.5-5 times that of the reaction liquid obtained in the step (1), wherein the crystallization solvent is selected from any one or a combination of at least two of an alcohol solvent, a ketone solvent, acetonitrile or tetrahydrofuran; standing and crystallizing for 8-24 h at 0-10 ℃, separating solid from liquid, collecting solid phase, washing and drying to obtain the N-acetylneuraminic acid calcium salt crystal.

In another aspect, the present invention provides a use of the crystal of calcium N-acetylneuraminic acid salt according to the first aspect in a medicament, a health product, a food or a skin care product.

Compared with the prior art, the invention has the following beneficial effects:

the molecular formula of the N-acetylneuraminic acid calcium salt crystal provided by the invention is (C)₁₁H₁₈NO₉)₂Ca, which has obvious diffraction peak in powder X-ray diffraction spectrum; the solubility in water is good, and the solubility at room temperature can reach more than 400 g/L; has excellent stability, is still white solid after being treated at the high temperature of 184 ℃ for 20min, and the water solution of the white solid is colorless, clear and transparent at the high temperature of 120 ℃ without color change. The N-acetylneuraminic acid calcium salt crystal can be obtained by reacting N-acetylneuraminic acid and a calcium source in an aqueous environment and then crystallizing and separating out, the preparation method is simple, the yield of the product is high and can reach 70-85%, and the method is suitable for large-scale production. The N-acetylneuraminic acid calcium salt crystal with high stability and high solubility provided by the invention has high absorptivity, can simultaneously meet the supplement requirements of human bodies on calcium and N-acetylneuraminic acid, and has wide application prospects in health care products, foods, biological medicines and cosmetics.

Drawings

FIG. 1 is an infrared spectrum of the crystal of calcium N-acetylneuraminic acid salt provided in example 1;

FIG. 2 is an infrared spectrum of N-acetylneuraminic acid as a raw material;

FIG. 3 is a polycrystalline X-ray diffraction pattern of the crystal of calcium N-acetylneuraminic acid salt provided in example 1;

FIG. 4 is a polycrystalline X-ray diffraction pattern of the calcium salt of N-acetylneuraminic acid provided by comparative example 2;

FIG. 5 is a graph showing the comparison of the stability of the aqueous solution of the crystal of calcium salt of N-acetylneuraminic acid provided in example 1 and the raw material N-acetylneuraminic acid;

FIG. 6 is a graph showing a comparison of the solubility of the crystal of calcium salt of N-acetylneuraminic acid provided in example 1 and the starting material N-acetylneuraminic acid.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In the following examples of the present invention, the raw material N-acetylneuraminic acid (anhydrate) is commercially available, and may be N-acetylneuraminic acid (anhydrate) prepared by natural extraction, chemical synthesis, enzymatic catalysis or microbial fermentation, and the source thereof is not particularly limited in the present invention.