Patent Description:
Vitamin D3 itself has no physiological effect (H. De Luca et al. The H<NUM> labeled vitamin D3 is used to perform study, vitamin D3 that is absorbed in the intestine first undergoes hydroxylation of <NUM>-carbon atom in the liver to produce <NUM>-hydroxycholecalciferol, the <NUM>-hydroxycholecalciferol is converted into <NUM>,<NUM>-dihydroxy D3 in the kidney mitochondria and then delivered to the small intestine mucosa and the kidneys to promote absorption and reabsorption of two minerals, calcium and phosphorus, and meanwhile produce other functions required for animal body metabolism. Studies have shown that the <NUM>-hydroxycholecalciferol has a different absorption principle from ester-soluble vitamins, and has a higher utilization efficiency.

<NUM>-hydroxycholecalciferol, also known as <NUM>-hydroxyvitamin D3, is an expression type of activity of vitamin D3 in the body, with a structural formula as shown in formula <NUM>. <CHM>
<CHM>.

The <NUM>-hydroxycholecalciferol may be obtained by any method. For example, <CIT> reported a method of isolation and purification of <NUM>-hydroxycholecalciferol, <CIT> reported for a method of preparation of <NUM>-hydroxycholecalciferol by a photochemical reaction, <CIT> reported a method of preparation of <NUM>-hydroxycholecalciferol by fermentation, and <CIT> reported a method of production of <NUM>-hydroxycholecalciferol. Furthermore, it was also reported in related research that <NUM>-hydroxycholecalciferol may be utilized as an active ingredient in a feed. However, a stability test of <NUM>-hydroxycholecalciferol showed that <NUM>-hydroxycholecalciferol has a low stability, and after storage at <NUM> for one month, a content of <NUM>-hydroxycholecalciferol is reduced from <NUM>% to <NUM>%. Therefore, how to enhance the stability of <NUM>-hydroxycholecalciferol, so as to facilitate production, storage and use of relevant preparations, and thus make use of biological efficacy thereof, is an urgent problem in this field.

<NPL>) discloses the isolation of a monohydrate by slow evaporation from methanol.

In view of the abovementioned problem, the present invention provides a <NUM>-hydroxycholecalciferol monohydrate crystal, which can effectively enhance the stability of <NUM>-hydroxycholecalciferol, and is more beneficial to production and storage of related preparations, and thus biological properties of <NUM>-hydroxycholecalciferol can be effectively utilized, and a preparation method thereof.

The present invention provides a <NUM>-hydroxycholecalciferol monohydrate crystal, an X-ray powder diffraction spectrum of <NUM>-hydroxycholecalciferol monohydrate crystal includes the following peaks:.

and wherein the <NUM>-hydroxycholecalciferol monohydrate crystal is obtained via a preparation method comprising the following steps: <NUM>) heating and refluxing <NUM>-hydroxycholecalciferol in an organic solvent until <NUM>-hydroxycholecalciferol is dissolved to obtain a <NUM>-hydroxycholecalciferol solution; and <NUM>) adding water to the <NUM>-hydroxycholecalciferol solution while stirring until a solid is precipitated out, continuing to stir, and after being cooled to room temperature, filtering to obtain a filter cake, and drying the filter cake until moisture content in the filter cake is less than 20wt%, to prepare the <NUM>-hydroxycholecalciferol monohydrate crystal.

It should be understood that, in general, when measuring a crystal by X-ray powder diffraction, there may be some small errors in peaks in the spectrogram due to differences in measuring instruments, measuring conditions and the like, and thus, other peak may also be included in the spectrogram of a crystal of the present invention in addition to the abovementioned peaks. The measuring conditions of the invention are as follows: Cu Kα line, tube voltage <NUM> kV, tube current <NUM> mA. <FIG> is an XRPD spectrogram of a <NUM>-hydroxycholecalciferol monohydrate prepared in an embodiment of the present invention. The X-ray diffractometer (Bruker D8 advance) of <FIG> has working parameters as follows:
Cu Kα line; tube voltage: 40kV; tube current: 40mA; scanning type: fixed coupling; increase amount: <NUM>°; scanning range: <NUM>°-<NUM>°; scanning speed: <NUM>/step.

A stability test shows that after the <NUM>-hydroxycholecalciferol monohydrate crystal of the present invention is left open for six months at <NUM>, a mass content of <NUM>-hydroxycholecalciferol in the <NUM>-hydroxycholecalciferol monohydrate crystal is <NUM>%. Therefore, the <NUM>-hydroxycholecalciferol monohydrate crystal of the present invention has a stable crystal property, which can effectively improve stability of <NUM>-hydroxycholecalciferol.

The <NUM>-hydroxycholecalciferol solution is obtained by dissolving <NUM>-hydroxycholecalciferol in an organic solvent. The present invention has no limitation on a source of <NUM>-hydroxycholecalciferol raw material, which may be commercially available or prepared in-house.

The abovementioned organic solvent for preparation of the <NUM>-hydroxycholecalciferol solution may be selected from one or more of an ether solvent, an alcohol solvent, a ketone solvent, and a chlorohydrocarbon solvent. Where the ether solvent includes, but is not limited to, tetrahydrofuran and <NUM>-methyltetrahydrofuran; the alcohol solvent includes, but is not limited to, methanol, alcohol and isopropanol; the ketone solvent includes, but is not limited to, acetone and butanone; the chlorinated hydrocarbon solvent includes, but is not limited to, dichloromethane, chloroform and the like. When the organic solvent is a mixture of the above solvents, the present invention has no limitation on a ratio between various solvents. At the same time, when preparing the <NUM>-hydroxycholecalciferol solution, a mass volume ratio of <NUM>-hydroxycholecalciferol to the organic solvent is <NUM>: <NUM>, and preferably <NUM>: <NUM>-<NUM>, and more preferably <NUM>: <NUM>-<NUM>.

When preparing the <NUM>-hydroxycholecalciferol monohydrate crystal via a reaction of the <NUM>-hydroxycholecalciferol solution with water, the present invention does not limit a reaction molar ratio of <NUM>-hydroxycholecalciferol to water, and the reaction molar ratio may be <NUM>: (<NUM>-<NUM>), and preferably <NUM>: (<NUM>-<NUM>), and more preferably <NUM>: (<NUM>-<NUM>). When a molar amount of <NUM>-hydroxycholecalciferol is greater than a molar amount of water, the <NUM>-hydroxycholecalciferol monohydrate crystal has a too low yield; when a molar amount of <NUM>-hydroxycholecalciferol is less than a molar amount of water, the <NUM>-hydroxycholecalciferol monohydrate crystal will have a significantly increased yield. It is worth noting that, since <NUM>-hydroxycholecalciferol has only one site for binding to water, even a molar amount of <NUM>-hydroxycholecalciferol is less than a molar amount of water, the product is still a <NUM>-hydroxycholecalciferol monohydrate crystal, and it is impossible to obtain a <NUM>-hydroxycholecalciferol polyhydrate.

The present invention also provides a preparation method of the <NUM>-hydroxycholecalciferol monohydrate crystal any one of embodiments above, including the following steps:.

Specifically, after adding <NUM>-hydroxycholecalciferol into the organic solvent, this reaction system is heated, so that the organic solvent is refluxed to dissolve <NUM>-hydroxycholecalciferol. The organic solvent may be selected from one or more of the ether solvent, the alcohol solvent, the ketone solvent and the chlorohydrocarbon solvent mentioned above. After <NUM>-hydroxycholecalciferol is dissolved, stirring is continued while heating is stopped, and water is slowly added to the <NUM>-hydroxycholecalciferol solution until a white solid is precipitated out, and after addition of water is completed and a temperature of the system is reduced to room temperature, stirring is continued for <NUM>-<NUM> hours.

After the completion of stirring, a solid-liquid separation is performed on the system. Specifically, it is possible to collect a filter cake by means of centrifugation or filtration and dry the filter cake. In order to avoid detachment of crystal water in the <NUM>-hydroxycholecalciferol monohydrate crystal, a drying temperature may be set less than <NUM>, and preferably is set to <NUM>-<NUM>, and more preferably <NUM>-<NUM>; the drying method may be an atmospheric pressure drying, and when there is a low drying temperature, drying under a reduced pressure may be selected in order to enhance drying effect. The present invention has no excessive requirement on the degree of drying, generally, moisture content is controlled to be less than 20wt%, and preferably less than 10wt%, and more preferably less than 5wt%.

The preparation method for the <NUM>-hydroxycholecalciferol monohydrate crystal of the present invention is simple and highly operable, and the <NUM>-hydroxycholecalciferol monohydrate crystal obtained by the method has a high stability. A stability test shows that after the <NUM>-hydroxycholecalciferol monohydrate crystal prepared by the method of the present invention is left open for six months at <NUM>, a mass content of <NUM>-hydroxycholecalciferol in the <NUM>-hydroxycholecalciferol monohydrate crystal is <NUM>%.

Also disclosed is a microemulsion, the microemulsion includes of the <NUM>-hydroxycholecalciferol monohydrate crystal any one of embodiments above. Since <NUM>-hydroxycholecalciferol is easily soluble in methanol, ethanol and dimethyl sulfoxide, slightly soluble in diethyl ether, and almost insoluble in water, a microemulsion containing <NUM>-hydroxycholecalciferol has a very low solubility in water. Therefore, the solubility of microemulsion needs to be improved, so that it can be effectively absorbed in the body. Accordingly, the present disclosure provides a microemulsion containing a <NUM>-hydroxycholecalciferol monohydrate crystal, the microemulsion having good stability and good water solubility.

Further, the microemulsion includes the following components by weight percentage: <NUM>-<NUM>% of <NUM>-hydroxycholecalciferol monohydrate crystal according to any one of embodiments above, <NUM>-<NUM>% of Tween-<NUM>, <NUM>-<NUM>% of sodium stearoyl lactate, <NUM>-<NUM>% of Span-<NUM>, <NUM>-<NUM>% of <NUM>,<NUM>-di-tert-butyl-<NUM>-methylphenol, <NUM>-<NUM>% of sorbic acid, <NUM>-<NUM>% of propylene glycol and <NUM>-<NUM>% of water.

Further, the microemulsion includes the following components by weight percentage: <NUM>% of the <NUM>-hydroxycholecalciferol monohydrate crystal according to any one of embidiments above, <NUM>% of Tween-<NUM>, <NUM>% of sodium stearoyl lactate, <NUM>% of Span-<NUM>, <NUM>% of <NUM>,<NUM>-di-tert-butyl-<NUM>-methylphenol, <NUM>% of sorbic acid, <NUM>% of propylene glycol and <NUM>% of water.

The microemulsion provided in the present disclosure may reach nanometer scale, and specifically, it may have an average particle size of up to <NUM>-<NUM>, and an encapsulation rate of up to <NUM>-<NUM>%. The encapsulation rate refers to a percentage of an encapsulated substance (for example, a certain free drug) in a total amount of the drug in a liposome suspension. The encapsulation rate is an important indicator of mass control of liposomes and nanoparticles and reflects a degree to which a drug is encapsulated by a carrier. The encapsulation rate includes determinations of an encapsulation percentage and an encapsulation volume, and the encapsulation rate (EN%) is generally calculated by Equation <NUM> below:<MAT> in Equation <NUM>, Cf is an amount of an encapsulated substance (a free drug); Ct is a total amount of drug in a nanoparticle or liposome suspension.

The present disclosure also provides a preparation method of the microemulsion according to any one of the embodiments above, including the following steps:.

Generally, oscillating by the vortex oscillator for <NUM>-<NUM> is sufficient to ensure homogeneous mixing of various components.

The present disclosure also provides a use of the microemulsion according to any one of the embodiments above in a feed additive, the microemulsion as the feed additive can shorten metabolic time of vitamin D3 in bodies of livestock and poultry, promote absorption and reabsorption of calcium and phosphorus elements, and fill a blank of using <NUM>-hydroxycholecalciferol as the feed additive in domestic.

The present disclosure also provides a feed, including the microemulsion according to any one of the embodiments above, the feed can effectively promote the absorption of calcium and phosphorus elements in bodies of livestock and poultry, and lay a foundation for healthy growth of livestock and poultry.

Also disclosed are at least the following advantages:.

In order to make the purposes, technical solutions and advantages of the present invention more clear, technical solutions of the embodiments of the present invention will be described clearly and completely below in combination with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are merely part, but not all, of the embodiments of the present invention.

A specific preparation method includes the following steps:
adding <NUM> of <NUM>-hydroxycholecalciferol into <NUM> of acetone, stirring, heating and refluxing until the <NUM>-hydroxycholecalciferol was fully dissolved; adding slowly <NUM> of water dropwise until a white solid was precipitated out, and then stopping heating and continuing stirring for <NUM> hours; filtering under an atmospheric pressure to collect a filter cake, and drying the filter cake under <NUM> for <NUM> to obtain a <NUM>-hydroxycholecalciferol monohydrate crystal. The inventor performed the following tests on the <NUM>-hydroxycholecalciferol monohydrate crystal prepared in this embodiment.

<FIG> and Table <NUM> verified that compositions of the <NUM>-hydroxycholecalciferol monohydrate crystal prepared in the embodiment of the present invention, and a crystal form of <NUM>-hydroxycholecalciferol is clearly characterized.

The content of <NUM>-hydroxycholecalciferol was measured by the following method: taking a sample to prepare a solution of about <NUM>µg/mL, injecting <NUM>µL for a HPLC analysis, and calculating the content thereof according to an external standard method.

Test conditions of the HPLC were as follows:.

It can be known from table <NUM> that the <NUM>-hydroxycholecalciferol monohydrate crystal prepared in the embodiment has a good stability, and there is almost no loss of <NUM>-hydroxycholecalciferol therein after six months of being left open at <NUM>.

A specific preparation method includes the following steps.

It was detected that the <NUM>-hydroxycholecalciferol monohydrate microemulsion prepared in the embodiment has a particle size of <NUM>, and an encapsulation rate of <NUM>%.

The microemulsion prepared in the embodiment was sealed and stored at <NUM>, and the content of the <NUM>-hydroxycholecalciferol contained therein was tested at one month, two months, three months and six months, and meanwhile, a microemulsion prepared from water-free <NUM>-hydroxycholecalciferol by the same operation as the present embodiment was taken as a control example, and specific results were shown in <FIG> is graphs of stability tests of the microemulsion prepared in Embodiment <NUM> and the microemulsion of the control example. As shown in <FIG>, a stability of the microemulsion prepared in the present embodiment is obviously better than that of the microemulsion prepared according to the prior art.

The content of <NUM>-hydroxycholecalciferol was tested in the following method: when a date for stability test was due, taking a sample of about <NUM>µg/mL solution, injecting <NUM>µL for an HPLC analysis, and calculating the content thereof according to an external standard method.

Claim 1:
A <NUM>-hydroxycholecalciferol monohydrate crystal, wherein an X-ray powder diffraction spectrum of <NUM>-hydroxycholecalciferol monohydrate crystal comprises the following peaks:

<TAB>

and wherein the <NUM>-hydroxycholecalciferol monohydrate crystal is obtained via a preparation method comprising the following steps:
<NUM>) heating and refluxing <NUM>-hydroxycholecalciferol in an organic solvent until <NUM>-hydroxycholecalciferol is dissolved to obtain a <NUM>-hydroxycholecalciferol solution; and
<NUM>) adding water to the <NUM>-hydroxycholecalciferol solution while stirring until a solid is precipitated out, continuing to stir, and after being cooled to room temperature, filtering to obtain a filter cake, and drying the filter cake until moisture content in the filter cake is less than 20wt%, to prepare the <NUM>-hydroxycholecalciferol monohydrate crystal.