A reduced coenzyme Q10 crystal and a preparation method thereof

Abstract

The present invention provides a reduced coenzyme Q10 crystal. When the temperature is raised at a rate of 10 k / min as measured by differential scanning calorimetry, an endothermic peak appears at 52 ± 2 °C. Compared with the prior art, the crystal form of the reduced coenzyme Q10 provided by the present invention is more stable than the reduced coenzyme Q10 crystal reported in the literature, and other physical properties such as water solubility and residual solvents are also more excellent; at the same time, the crystal form provided by the present invention also overcomes the disadvantages of the reduced coenzyme Q10 which is very easy to oxidize and has limitations in use; in addition, the reduced coenzyme Q10 crystal provided by the present invention and the crystalline solid containing the crystal not only have excellent physical properties of stability, but also are prominent in terms of high purity and low solvent residue.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound crystals, and particularly relates to a reduced coenzyme Q10 crystal and a preparation method thereof. Background Art

[0002] Coenzyme Q10, also known as ubiquinone 10, has the chemical name of 2,3-dimethoxy-5-methyl-6-decaprenylbenzoquinone, and its structure is similar to that of vitamin K. It was discovered by scientists in the mid-20th century and its chemical structure was confirmed. Coenzyme Q10 is a kind of vitamin-like substance, a fat-soluble organic quinone compound widely distributed in organisms, and has important physiological and pharmacological effects. In the early 1980s, Ernster in Sweden revealed the antioxidant effect and free radical scavenging effect of the vitamin-like substance coenzyme Q10. In 1972, Harman elaborated on the relationship between the function of mitochondria and aging. Coenzyme Q10 is mainly bound to the mitochondrial membrane to form a coenzyme in the respiratory chain link and participates in the transfer of hydrogen in the energy metabolism in organisms. Coenzyme Q10 is a metabolic activator that can activate cell respiration, generate the power of cells, and accelerate the production of adenosine triphosphate (ATP). At the same time, coenzyme Q10 itself is a natural antioxidant produced by cells themselves, which can inhibit mitochondrial peroxidation, protect the integrity of the biological membrane structure. At the same time, it is also a non-vitamin nutrient. After ingestion, it can activate cell respiration, accelerate the production of high-energy ATP, strengthen myocardial metabolism function, improve the heart beating efficiency, regulate the hypoxic state of cells and tissues, and has good protective and improvement effects on the liver, brain, heart and nervous system. Coenzyme Q10 also has a very specific enhancing effect on immunity, can increase the phagocytosis rate of phagocytes, and increase the production of antibodies. A large number of clinical application studies at home and abroad have shown that coenzyme Q10 has excellent curative effects on diseases such as heart disease, hypertension, cerebrovascular disorders, scurvy, viral hepatitis, etc., and can be used as a non-specific immune enhancer of the body, a cell metabolism and cell respiration activator. More recently, it has been found that it has a significant adjuvant curative effect on cancer and AIDS. Therefore, coenzyme Q10 plays an irreplaceable role and has broad application prospects in human health care, anti-aging and improving body immunity. At the same time, because coenzyme Q10 has no toxic and side effects and does not interact with any other drugs, it has become an important medicine and health product.

[0003] At present, most of the coenzyme Q10 sold on the market is oxidized coenzyme Q10. However, research has found that the two-electron reductant of oxidized coenzyme Q10, reduced coenzyme Q10, shows higher oral absorbability than oxidized coenzyme Q10, and the one that mainly plays a role in the body is also reduced coenzyme Q10. The only difference between the two is that one is in the benzoquinone form and the other is in the benzodiol form.

[0004] In addition, among the numerous publicly reported technical documents on the production of reduced coenzyme Q10, except for extraction from fermentation broth (most of which is reduced coenzyme Q10 during the fermentation process and gradually oxidizes to oxidized coenzyme Q10 during the extraction process), most use oxidized coenzyme Q10 as the raw material and reduce it to reduced coenzyme Q10 through conventional reducing agents. The reducing agents involved include sodium borohydride, sodium dithionite, ascorbic acid, and certain specific amino acids, etc. The solvents used mainly include aliphatic hydrocarbons, fatty acid esters, etc. In addition, several methods are also known for obtaining reduced coenzyme Q10 in crystalline form. For example, a method of precipitating reduced coenzyme Q10 in an alcohol solution and / or a ketone solution to produce crystals has been reported (WO2003 / 006409), and a method of crystallizing by adding a high-concentration liquid phase of reduced coenzyme Q10 to a poor solvent has been reported (Japanese Patent Laid-Open No. 2003-089669), etc.

[0005] On the other hand, the patent document (WO2012 / 176842) records that polymorphism is observed in reduced coenzyme Q10 and a new crystal form different from the above-mentioned documents is obtained. And it is reported that compared with the existing reduced coenzyme Q10, the newly discovered crystal form is very stable and other physical properties are also excellent, and its manufacturing method is also disclosed. Compared with the existing reduced coenzyme Q10 (hereinafter, this crystal is referred to as the reduced coenzyme Q10 crystal of Form I or Form I crystal), it is reported that the newly discovered crystal form (hereinafter, this crystal is referred to as the reduced coenzyme Q10 crystal of Form II or Form II crystal) is very stable and other physical properties are also excellent.

[0006] However, due to its structure, just like most of the fermentation broth is reduced coenzyme Q10 during the fermentation process and gradually becomes oxidized coenzyme Q10 during the extraction process, during the chemical production process, since reduced coenzyme Q10 is easily oxidized by oxygen in the air to oxidized coenzyme Q10, it is very difficult to completely eliminate molecular oxygen on a commercial production scale. Therefore, the residual oxygen in the production process has a greater adverse effect, generating almost ineliminable oxidized coenzyme Q10, which is then doped in the product, resulting in a decrease in product purity. Research has also found that in addition to being relatively stable in the solvent, the product is vulnerable to oxidation during filtration, drying, and storage. In order to obtain high-purity reduced coenzyme Q10 in crystal form, it is important to fully protect reduced coenzyme Q10 from the above oxidation.

[0007] In addition, the literature mentions that the crystal form has a relatively large impact on its oxidation rate. However, research shows that for the crystal forms obtained from different examples of the existing literature (WO2012 / 176842 Gazette, Form II), powder X-ray diffraction indicates that they are actually still of Form I, rather than Form II reported in the literature. There is also literature reporting that coating with oil or adding antioxidants will introduce new substances, which is unfavorable to the product. In addition, except for ethanol, when using aliphatic hydrocarbon solvents such as n-hexane and n-heptane for purification, there are situations where it is difficult to meet the requirements of solvent residues. Since the melting point of oxidized or reduced coenzyme Q10 is about 50 °C, if the drying temperature is increased, it is easily liquefied. And the crystallization with ethanol has the problems that the color of the obtained product is prone to discoloration, the appearance is yellow and in a slurry form, and filtration is difficult. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a reduced coenzyme Q10 crystal with high stability and purity and a preparation method thereof.

[0009] The present invention provides a reduced coenzyme Q10 crystal, which has an endothermic peak at 52 ± 2 °C when heated at a rate of 10 k / min as measured by differential scanning calorimetry.

[0010] Preferably, in the powder X-ray diffraction measured with Cu-Kα rays, characteristic peaks are shown at diffraction angles (2θ ± 0.2°) of 8.95°, 10.04°, 15.09°, 18.65°, 19.03°, 21.61° and 23.01°.

[0011] Preferably, the infrared absorption spectrum of the reduced coenzyme Q10 crystal measured by the KBr tablet pressing method has characteristic absorption peaks at wavenumbers 794 ± 1 cm -1 、877 ± 1 cm -1 、962 cm -1 and 1014 cm -1 .

[0012] Preferably, it has Figure 1 the powder X-ray diffraction pattern measured with Cu-Kα rays as shown;

[0013] and / or, has Figure 2 the infrared absorption spectrum diagram measured by the KBr tablet pressing method as shown;

[0014] and / or, has Figure 3 the differential scanning calorimetry curve diagram as shown.

[0015] The present invention also provides a preparation method of a reduced coenzyme Q10 crystal, comprising the following steps:

[0016] (S) Crystallize reduced coenzyme Q10 in the presence of a cyclic ether solvent, a fluorinated alcohol solvent and an organic base to obtain reduced coenzyme Q10 crystals.

[0017] Preferably, step (S) is specifically as follows:

[0018] Mix and dissolve reduced coenzyme Q10 with a cyclic ether solvent under heating conditions, then add a mixed solution of a fluorinated alcohol solvent and an organic base, and cool down for crystallization to obtain reduced coenzyme Q10 crystals;

[0019] The temperature of the crystallization is 0°C to 20°C.

[0020] Preferably, the cyclic ether solvent is selected from one or more of 2-methyltetrahydrofuran, 3-methyltetrahydrofuran, tetrahydrofuran and 2,5-dimethyltetrahydrofuran;

[0021] The fluorinated alcohol solvent is selected from one or more of trifluoromethanol, trifluoroethanol, trifluoropropanol and trifluorobutanol;

[0022] The organic base is selected from one or more of triethylamine, pyridine and piperidine.

[0023] Preferably, the ratio of reduced coenzyme Q10 to the cyclic ether solvent is 1 g:(1 - 10) mL;

[0024] The volume ratio of the cyclic ether solvent to the fluorinated alcohol solvent is 1:(1 - 10);

[0025] The mass of the organic base is 0.1% - 10% of the mass of reduced coenzyme Q10.

[0026] The present invention also provides a reduced coenzyme Q10 crystalline solid, comprising the above-mentioned reduced coenzyme Q10 crystals.

[0027] The present invention also provides a reduced coenzyme Q10 composition, comprising the above-mentioned reduced coenzyme Q10 crystals;

[0028] and / or, the above-mentioned reduced coenzyme Q10 crystalline solid.

[0029] The present invention provides a reduced coenzyme Q10 crystal. When the temperature is raised at a rate of 10 k / min by differential scanning calorimetry, an endothermic peak appears at 52 ± 2 °C. Compared with the prior art, the crystal form of the reduced coenzyme Q10 provided by the present invention is more stable than the reduced coenzyme Q10 crystal reported in the literature, and other physical properties such as water solubility and residual solvents are also more excellent. At the same time, the crystal form provided by the present invention also overcomes the disadvantages of the reduced coenzyme Q10, which is very easy to oxidize and has limitations in use. In addition, the reduced coenzyme Q10 crystal provided by the present invention and the crystalline solid containing the crystal not only have excellent physical properties of stability, but also are outstanding in terms of high purity and low solvent residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the powder X-ray diffraction pattern of the reduced coenzyme Q10 crystal obtained in Example 1 of the present invention;

[0031] Figure 2 It is the infrared absorption spectrum of the reduced coenzyme Q10 crystal obtained in Example 1 of the present invention;

[0032] Figure 3 It is the DSC spectrum of the reduced coenzyme Q10 crystal obtained in Example 1 of the present invention;

[0033] Figure 4 It is the powder X-ray diffraction pattern of the reduced coenzyme Q10 crystal obtained in Comparative Example 1 of the present invention;

[0034] Figure 5 It is the HPLC chart of the reduced coenzyme Q10 crystal obtained in Example 1 of the present invention after being stored for 360 days;

[0035] Figure 6 It is the HPLC chart of the reduced coenzyme Q10 crystal obtained in Comparative Example 1 of the present invention after being stored for 360 days;

[0036] Figure 7 It is the HPLC chart of the reduced coenzyme Q10 crystal obtained in Example 1 of the present invention;

[0037] Figure 8 It is the HPLC chart of the reduced coenzyme Q10 crystal obtained in Comparative Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0039] The present invention provides a reduced coenzyme Q10 crystal in a novel crystal form, whose physical and chemical properties and crystal structure are significantly different from those of the reduced coenzyme Q10 crystal reported in the literature.

[0040] Specifically, the present invention provides a reduced coenzyme Q10 crystal, which, when heated at a rate of 10 k / min by differential scanning calorimetry, has an endothermic peak at 52 ± 2 °C, and more specifically, has an endothermic peak indicating crystal melting at 52 ± 2 °C.

[0041] More specifically, the reduced coenzyme Q10 crystal has Figure 3 the differential scanning calorimetry curve shown.

[0042] According to the present invention, the reduced coenzyme Q10 crystal preferably shows characteristic peaks at diffraction angles (2θ ± 0.2°) of 8.95°, 10.04°, 15.09°, 18.65°, 19.03°, 21.61° and 23.01° in the powder X-ray diffraction measured with Cu-Kα rays; in particular, it shows characteristic strong diffraction peaks at diffraction angles (2θ ± 0.2°) of 8.95°, 19.03° and 23.01°.

[0043] More specifically, the reduced coenzyme Q10 crystal has Figure 1 the powder X-ray diffraction pattern measured with Cu-Kα rays shown. For Figure 1 the XRD diffraction pattern shown, the above characteristic peaks are completely different from the diffraction pattern of the crystal reported in the existing literature (CN103635452A), which exactly indicates that Figure 1 the reduced coenzyme Q10 crystal of the present invention shown is a novel polymorph of reduced coenzyme Q10.

[0044] And / or, more specifically, the reduced coenzyme Q10 crystal has characteristic absorption peaks in the infrared absorption spectrum measured by the KBr tablet method at wavenumbers 794 ± 1 cm -1 , 877 ± 1 cm -1 , 962 cm -1 and 1014 cm -1 ; among the above absorption peaks, the peaks near 794 ± 1 cm -1 and 877 ± 1 cm -1 are characteristic absorption peaks formed by two peaks of the same intensity; 962 cm -1 and 1014 cm -1The nearby peak is a characteristic absorption peak formed by two peaks of substantially the same intensity. Since the reduced coenzyme Q10 crystals reported in previous literature do not have an absorption peak of two peaks at this position, it is clearly indicated that the reduced coenzyme Q10 crystals provided by the present invention are a new crystal form of reduced coenzyme Q10 different from the crystal forms reported in previous literature.

[0045] More specifically, the reduced coenzyme Q10 crystal provided by the present invention has Figure 2 the infrared absorption spectrogram measured by the KBr tablet pressing method as shown.

[0046] According to the present invention, the reduced coenzyme Q10 crystal is the new reduced coenzyme Q10 crystal provided by the present invention as long as it contains one or more of the above DSC endothermic peaks, XRD diffraction patterns and IR absorption patterns.

[0047] According to the present invention, the reduced coenzyme Q10 crystal has good water solubility; the solubility of the reduced coenzyme Q10 crystal in purified water at a temperature of 25 °C is preferably at least 0.01 wt%, more preferably at least 0.1 wt%, and still more preferably at least 0.5 wt%; the solubility of the reduced coenzyme Q10 crystal provided by the present invention is significantly higher than the solubility (less than 0.001 wt%) shown by the reduced coenzyme Q10 crystals known in the past. As described above, since the reduced coenzyme Q10 crystal provided by the present invention shows a higher melting point and a higher water solubility property compared with the reduced coenzyme Q10 crystals reported in the literature, it is not only a crystal form with a different crystal structure from the reduced coenzyme Q10 crystals reported in the literature, that is, a new crystal form of reduced coenzyme Q10 (or a crystal containing this crystal form), but also can be said to be a more stable crystal. The stable form of the reduced coenzyme Q10 crystal provided by the present invention not only has high thermal stability but also has high water solubility.

[0048] The reduced coenzyme Q10 crystal provided by the present invention exhibits excellent stability to molecular oxygen. Previously, it was known that reduced coenzyme Q10 is easily oxidized by molecular oxygen in the air. However, as shown in the examples described later, the novel reduced coenzyme Q10 crystal form discovered in the present invention and the reduced coenzyme Q10 crystal mainly composed of the same exhibit higher stability than the crystal forms listed in the literature even in a state where no protective measures against molecular oxygen are taken in the air. In addition, even in the case where the reduced coenzyme Q10 crystals reported in previous literature and other non-crystalline components coexist, the high oxidation stability of the reduced coenzyme Q10 polymorph of the present invention can be exerted, and for the reduced coenzyme Q10 crystalline solid of the present invention, an oxidation stability that cannot be considered to be from the previous understanding is also shown. Due to the content of the novel reduced coenzyme Q10 crystal form in the crystal or crystalline solid, storage conditions, etc., the oxidation stability of the reduced coenzyme Q10 crystal and crystalline solid of the present invention cannot be generalized. For example, as the retention rate (%) of reduced coenzyme Q10 after storage for a specified period under the conditions of 25°C, in the air, and protected from light, it is generally about 80% or more, preferably about 85% or more, more preferably about 90% or more, and particularly preferably 95% or more. It should be noted that the so-called retention rate here means the value obtained by the ratio of the absolute content of reduced coenzyme Q10 (or the concentration in the crystalline solid) after storage for a specified period to the absolute content of reduced coenzyme Q10 in the composition before storage (or the concentration in the crystalline solid). In addition, the specified period is not particularly limited. For example, it is 6 months, preferably 12 months, and more preferably 36 months.

[0049] The crystal form of reduced coenzyme Q10 provided by the present invention is more stable than the reduced coenzyme Q10 crystal reported in the literature, and other physical properties including water solubility and residual solvents are also more excellent; at the same time, the crystal form provided by the present invention also overcomes the disadvantages of reduced coenzyme Q10, which was very easy to oxidize and had limitations in use in the past; in addition, the reduced coenzyme Q10 crystal provided by the present invention and the crystalline solid containing the crystal not only have excellent physical properties of stability, but also are prominent in terms of high purity and low solvent residue.

[0050] The present invention also provides a method for preparing a reduced coenzyme Q10 crystal, comprising the following step: S) Crystallizing reduced coenzyme Q10 in the presence of a cyclic ether solvent, a fluorinated alcohol solvent, and an organic base to obtain a reduced coenzyme Q10 crystal.

[0051] Among them, the present invention has no special restrictions on the sources of all raw materials, and they can be commercially available.

[0052] According to the present invention, it is preferred to first mix and dissolve reduced coenzyme Q10 with a cyclic ether solvent under heating conditions, and then add a mixed solution of a fluorinated alcohol solvent and an organic base, and cool down for crystallization to obtain reduced coenzyme Q10 crystals.

[0053] In the present invention, the reduced coenzyme Q10 can be the reduced coenzyme Q10 well-known to those skilled in the art, without any special limitation; the purity of the reduced coenzyme Q10 is preferably greater than or equal to 99%, more preferably greater than or equal to 99.5%.

[0054] As the solvent used in crystallization and subsequent processing, since reduced coenzyme Q10 is difficult to dissolve in alcohol solvents, it is not appropriate to perform recrystallization using the alcohols reported in the literature. However, as a poor solvent, the crystallization system is prone to slurry formation after addition, and filtration is difficult. The crystal form obtained is still the crystal form initially reported in the literature. When using a single fatty acid ester or ether as the solvent, due to the easy solubility of the sample, the crystallization yield is low. Unless the proportion of the solvent is reduced or the temperature is lower, it is possible to increase the yield. But this brings another problem. Due to the too low temperature or the low proportion of the solvent, the system is relatively viscous and difficult to filter, and the crystal form of the obtained crystals is still the crystal form reported in the literature. When using alkanes such as n-hexane, n-heptane, n-propane, and n-butane, with appropriate crystallization temperature, solvent and crude product ratio, a high-yield product can be obtained. However, the crystal form of the obtained crystals is confirmed to be the reduced coenzyme Q10 Form I reported in the literature, and there is still a situation where the solvent residue is difficult to meet the requirements after drying for 48 hours.

[0055] Through a large number of studies in the present invention, the use of a benign cyclic ether solvent, a poor fluorinated alcohol solvent, and a small amount of organic base not only results in a high yield, but also the crystal form precipitated has higher stability than that reported in the literature and better impurity removal effect.

[0056] Mix and dissolve reduced coenzyme Q10 with a cyclic ether solvent under heating conditions; the cyclic ether solvent is preferably one or more of 2-methyltetrahydrofuran, 3-methyltetrahydrofuran, tetrahydrofuran, and 2,5-dimethyltetrahydrofuran; the ratio of reduced coenzyme Q10 to the cyclic ether solvent is preferably 1 g:(1 - 10) mL, more preferably 1 g:(1 - 8) mL, still more preferably 1 g:(1 - 5) mL, and most preferably 1 g:(1 - 3) mL; in some embodiments provided by the present invention, the ratio of reduced coenzyme Q10 to the cyclic ether solvent is specifically 1 g:2 mL or 1 g:10 mL; the temperature of the mixing and dissolving is preferably 30°C - 50°C, more preferably 35°C - 45°C, and still more preferably 40°C.

[0057] Then, a mixed solution of a fluorinated alcohol solvent and an organic base is added, and the temperature is lowered for crystallization; the fluorinated alcohol solvent is preferably one or more of trifluoromethanol, trifluoroethanol, trifluoropropanol, and trifluorobutanol; the volume ratio of the cyclic ether solvent to the fluorinated alcohol solvent is preferably 1:(1-10), more preferably 1:(1-8), still more preferably 1:(1-7), and most preferably 1:(1-5); in some embodiments provided by the present invention, the volume ratio of the cyclic ether solvent to the fluorinated alcohol solvent is specifically 1:5 or 1:1; the mass of the organic base is preferably 0.1%-10% of the mass of reduced coenzyme Q10, more preferably 0.5%-10%, still more preferably 1%-10%, and most preferably 5%-10%; in some embodiments provided by the present invention, the mass of the organic base is preferably 5%, 7%, or 10% of the mass of reduced coenzyme Q10; the crystallization can be room temperature crystallization or cooling crystallization, preferably cooling crystallization or a combination of cooling crystallization and other crystallization methods; specifically, the temperature of the crystallization is preferably 0°C to 20°C; more specifically, in the present invention, the temperature of the crystallization is preferably 0°C to 10°C or 10°C to 20°C, more preferably 0°C to 5°C or 10°C to 15°C; the time of the crystallization is preferably more than 6 h, more preferably 6-24 h; specifically, in the present invention, when the crystallization temperature is 0°C to 10°C, the crystallization time is 6-12 h, and when the crystallization temperature is 10°C to 20°C, the crystallization time is 12-24 h; the crystallization can be carried out under static conditions or under stirring conditions, and there is no special limitation. In the present invention, it is preferably carried out under stirring conditions; the rotation speed of the stirring is preferably not less than 200 r / min. In the present invention, crystal seeds can also be added after the temperature is lowered; the crystal seeds are crystals of reduced coenzyme Q10; the present invention does not have any special limitation on the crystal form of the crystals of reduced coenzyme Q10 as crystal seeds, which can be the crystals provided by the present invention or Form I prepared in the existing literature, and there is no special limitation; by adding crystal seeds for induced crystallization, the addition amount of the crystal seeds has no special limitation. In the present invention, the mass of the crystal seeds is preferably more than 0.1% of the mass of reduced coenzyme Q10, more preferably 0.1%-5%, still more preferably 0.1%-3%, still more preferably 0.5%-3%, still more preferably 1%-2%, and most preferably 1%-1.5%.

[0058] Under the above crystallization conditions, in order to improve the conditions affecting crystallization such as the solubility, concentration, yield, slurry properties, or crystal properties of reduced coenzyme Q10, the above solvents can be mixed and used in a preferred ratio according to the characteristics of various solvents.

[0059] In a specific embodiment provided by the present invention, the crystallization concentration or the holding time after crystallization can be appropriately determined considering the solubility of reduced coenzyme Q10 in the solvent, etc., in order to obtain the reduced coenzyme Q10 crystals as the target. For example, when using 2-methyltetrahydrofuran as the solvent, a 2-methyltetrahydrofuran solution of reduced coenzyme Q10 with a concentration of 50% is prepared at a temperature below 40°C, and then a fluorinated alcohol and an organic base are added, followed by cooling and crystallization to 10°C. After the reduced coenzyme Q10 crystals are precipitated, the precipitated reduced coenzyme Q10 crystals can be directly held in the solvent at this temperature for more than 6 hours, preferably more than 8 hours, and further preferably more than 12 hours for production. In this holding step, the mixed solution of the precipitated reduced coenzyme Q10 crystals and the solvent can be stirred or can be in a static state. Stirring is preferred, and the stirring speed is not less than 200 r / min.

[0060] After crystallization, if necessary, it can be recovered through steps such as solid-liquid separation / drying by methods known in the prior art described in patent documents. For example, pressure filtration or centrifugal filtration can be used for solid-liquid separation, and generally, the method of pressure filtration with an inert gas is adopted. In addition, the drying temperature is confirmed according to the boiling point of the added solvent, and generally does not exceed 45°C. The dried crystalline solid can be pulverized or classified (screened) according to need and then recovered to obtain reduced coenzyme Q10 crystals.

[0061] It should be noted that in the above crystallization and post-treatment steps, it is preferably carried out in a deoxygenated environment. The deoxygenated environment can be replaced by using an inert gas, such as nitrogen, helium, argon, and carbon dioxide, and nitrogen or argon is preferred.

[0062] In addition, once the reduced coenzyme Q10 crystals can be produced or obtained, when performing the crystallization operation, by adding the reduced coenzyme Q10 crystals of the present invention as crystal seeds, under general conditions, the reduced coenzyme Q10 crystals of the present invention can be produced. At this time, a normal temperature or cooling crystallization method can be used for implementation. The preferred crystallization method is cooling crystallization, or a method in which cooling crystallization is combined with other crystallization methods.

[0063] The present invention also provides a reduced coenzyme Q10 crystalline solid, including the above-mentioned reduced coenzyme Q10 crystals.

[0064] Specifically, the reduced coenzyme Q10 crystalline solid can be obtained by adding, during the crystallization process, reduced coenzyme Q10 in a crystal form other than the crystal form of the reduced coenzyme Q10 crystals provided in the present application as crystal seeds.

[0065] More specifically, the content of the reduced coenzyme Q10 crystals in the reduced coenzyme Q10 crystalline solid is preferably 0.1 wt% or more, more preferably 1 wt% or more, still more preferably 10 wt% or more, still more preferably 30 wt% or more, still more preferably 50 wt% or more, still more preferably 70 wt% or more, and most preferably 85 wt% or more. When the lower limit value of the content of the reduced coenzyme Q10 crystals provided by the present invention is the above value, the upper limit value corresponding to each lower limit value is naturally 100% by weight. In the reduced coenzyme Q10 crystals and crystalline solids provided by the present invention, whether the reduced coenzyme Q10 crystals exist in a mixed state with the crystal forms reported in previous literatures and their proportions can be known by, for example, using DSC and measuring at a heating rate of 1 k / min. Under this condition, the respective endothermic peaks showing the melting of the reduced coenzyme Q10 crystals known in the prior art and the reduced coenzyme Q10 crystals provided by the present invention are clearly separated. Since the sizes of these peaks are related to the mixing ratio, even when the reduced coenzyme Q10 crystals reported in previous literatures are mixed in the reduced coenzyme Q10 crystals and crystalline solids of the present invention, the presence or content of the novel reduced coenzyme Q10 crystal form can be clearly determined.

[0066] The reduced coenzyme Q10 crystals provided by the present invention can coexist as a crystalline solid with the reduced coenzyme Q10 crystals reported in previous literatures as long as they contain the reduced coenzyme Q10 crystals having the above DSC endothermic peak, XRD diffraction pattern or IR absorption pattern. In addition, as long as it is a crystalline solid containing the reduced coenzyme Q10 crystals of the present invention, it is within the scope of the present invention regardless of whether it coexists with other solid forms of reduced coenzyme Q10. It should be noted that since the reduced coenzyme Q10 crystals contained in the reduced coenzyme Q10 crystalline solid of the present invention are more stable than the crystal forms reported in the literature, even if the reduced coenzyme Q10 crystals are only present in a small amount in the reduced coenzyme Q10 crystalline solid of the present invention, all crystal forms may transfer to the reduced coenzyme Q10 crystals provided by the present invention over time.

[0067] The present invention also provides a reduced coenzyme Q10 composition, comprising the above-mentioned reduced coenzyme Q10 crystals and / or the above-mentioned reduced coenzyme Q10 crystalline solid.

[0068] The reduced coenzyme Q10 crystals, reduced coenzyme Q10 crystalline solids and reduced coenzyme Q10 compositions provided by the present invention can be used in applications such as food, nutritional functional food, specific health food, nutritional supplement, animal medicine, beverage, feed, cosmetics, pharmaceuticals, therapeutic drugs, prophylactic drugs or pet food.

[0069] To further illustrate the present invention, a reduced coenzyme Q10 crystal and its preparation method provided by the present invention will be described in detail below in conjunction with embodiments.

[0070] It should be specifically pointed out that the measurement conditions for powder X-ray diffraction (XRD), differential scanning calorimetry (DSC), and infrared spectroscopy (IR) in the embodiments are as follows:

[0071] XRD measurement conditions:

[0072] Equipment model: X-ray powder diffractometer / SmartLab SE;

[0073] X-ray tube: Cu target;

[0074] Speed: 10° / min;

[0075] Collection angle: 3° - 60°;

[0076] Step size: 0.02°;

[0077] Slit width: 10mm;

[0078] Output voltage: 40kV;

[0079] Output current: 40mA.

[0080] DSC measurement conditions:

[0081] Equipment model: Differential scanning calorimeter / Netzsch DSC214

[0082] Sample crucible: Concavus Al;

[0083] Heating rate: 10K / min;

[0084] Test range: 30°C - 300°C;

[0085] Purge gas / protective gas: N2.

[0086] IR measurement conditions:

[0087] Equipment model: Shimadzu Fourier transform infrared spectrometer IRTracer-100;

[0088] Measurement method: KBr tablet method.

[0089] Example 1

[0090] Under nitrogen protection, 100 g of commercially available reduced coenzyme Q10 (purity 99.5%) and 200 mL of 2-methyltetrahydrofuran were added to a 3 L three-necked flask. The mixture was heated to 40 °C with stirring until completely dissolved, then cooled to 10 °C. 1000 mL of trifluoroethanol and 5 g of triethylamine were slowly added dropwise. After cooling to 2 °C, the mixture was kept warm and stirred for 12 hours, filtered, washed with 20 mL of trifluoroethanol, and dried under reduced pressure at 40 °C for 6 - 8 hours to obtain granular crystals, which were reduced coenzyme Q10 crystals.

[0091] The results of DSC analysis respectively confirmed that: when heating at a rate of 10 k / min, as Figure 3 shown, an endothermic peak of melting was shown at 52.2 °C.

[0092] The analysis results of powder X-ray diffraction, as Figure 1 shown, characteristic peaks were found at diffraction angles (2θ ± 0.2°) of 8.95°, 10.04°, 15.09°, 17.36°, 18.65°, 19.03°, 20.19°, 21.61° and 23.01°.

[0093] Furthermore, the results of IR analysis, as Figure 2 shown, were different from those of the reduced coenzyme Q10 crystals in the literature. Characteristic absorption peaks were present near 794 ± 1 cm -1 , 877 ± 1 cm -1 and 962 cm -1 , 1014 cm -1 .

[0094] Based on the above analysis results, it was confirmed that the reduced coenzyme Q10 crystals obtained in this example were in a different crystal form from those in the literature. When measuring the solubility of the obtained crystals in purified water, it was 0.5 wt% at 25 °C.

[0095] Example 2

[0096] Under nitrogen protection, 100 g of commercially available reduced coenzyme Q10 (purity 99.5%) and 1000 mL of 2,5-dimethyltetrahydrofuran were added to a 3 L three-necked flask. The mixture was heated to 40 °C with stirring until completely dissolved, and 1000 mL of trifluoropropanol and 7 g of triethylamine were slowly added dropwise. After cooling to 10 °C, 1 g of seed crystal (obtained in Example 1) was added. After stirring for 1 hour, the mixture was cooled to 2 °C, then kept warm and stirred for 12 hours, filtered, washed with 20 mL of trifluoropropanol, and dried under reduced pressure at 40 °C for 6 - 8 hours to obtain crystals.

[0097] The results of DSC analysis respectively confirmed that: when heating at a rate of 10 k / min, an endothermic peak of melting was shown at 50.2 °C.

[0098] The crystals obtained in Example 2 were analyzed and detected by powder X-ray diffraction and infrared spectroscopy. The detection results were similar to those of Example 1, and the error was within the range. It was determined that the crystal forms of the crystals obtained in Example 2 and Example 1 were the same.

[0099] Based on the above analysis results, it was confirmed that the reduced coenzyme Q10 crystals obtained in this example were different crystal forms from the reduced coenzyme Q10 in the literature. When measuring the solubility of the obtained crystals in purified water, it was 0.5 wt% at 25 °C.

[0100] Example 3

[0101] Under nitrogen protection, 100 g of commercially available or self-made reduced coenzyme Q10 and 1000 mL of tetrahydrofuran were added to a 3 L three-necked flask. The temperature was raised to 40 °C with stirring until completely dissolved. 1000 mL of trifluoroethanol and 10 g of triethylamine were added dropwise. After cooling to 10 °C, 1 g of seed crystal (obtained in Example 1) was added. After stirring for 1 hour, the temperature was cooled to 2 °C and kept warm while continuing to stir for 12 hours. Then it was filtered, washed with 20 mL of trifluoroethanol, and dried under reduced pressure at 40 °C for 6 - 8 hours to obtain crystals.

[0102] The results of DSC analysis confirmed that when heating at a rate of 10 °C / min, an endothermic peak of melting was shown at 52.2 °C. In addition, the analysis results of powder X-ray diffraction confirmed that the reduced coenzyme Q10 crystals obtained in this example were the same crystal form as the reduced coenzyme Q10 in Example 1.

[0103] Comparative Example 1 (Example 1 of CN103635452A)

[0104] After purging the inside of a 300 mL reaction flask (made of heat-resistant glass) with nitrogen, 40 g of commercially available reduced coenzyme Q10 (manufactured by Kaneka Corporation, a conventionally known reduced coenzyme Q10 crystal) and 60 g of n-hexane were added. The temperature was raised to 40 °C with stirring until completely dissolved. After cooling this solution to 25 °C at a cooling rate of 10 °C / hour, it was kept at 25 °C for 96 hours with continuous stirring. After filtration and drying (drying under reduced pressure, 20 °C - 40 °C), crystals were obtained.

[0105] The results of DSC analysis confirmed that when heating at a rate of 10 °C / min, an endothermic peak of melting was shown at 50.2 °C.

[0106] In addition, the analysis results of powder X-ray diffraction were as Figure 4As shown, it was confirmed that the reduced coenzyme Q10 crystals obtained in Comparative Example 1 were in the same crystal form as the reduced coenzyme Q10 Form Ⅰ reported in the literature, rather than Form Ⅱ reported in the literature. The test also confirmed that when using solvents reported in other patents, such as ethanol, heptane, etc., the crystal form obtained was also Figure 4 the Form Ⅰ shown, so it may be difficult to improve the stability of reduced coenzyme Q10 by conventional solvent crystallization.

[0107] Example 4

[0108] The crystals of reduced coenzyme Q10 obtained in Example 1 and Comparative Example 1 were respectively placed in a vacuum bag and stored at 25 °C in the dark. The weight ratio of reduced coenzyme Q10 to oxidized coenzyme Q10 was determined by the following HPLC analysis. The results are shown in Table 1. The HPLC chart of the crystals of reduced coenzyme Q10 obtained in Example 1 after 360 days of storage is as Figure 5 shown, and the HPLC chart of the crystals of reduced coenzyme Q10 obtained in Comparative Example 1 after 360 days of storage is as Figure 6 shown.

[0109] HPLC analysis conditions

[0110] Column: (Agilent) ZORBAX Extend C18 4.6×150mm, 5um;

[0111] Mobile phase: Mobile phase A: acetonitrile, Mobile phase B: isopropanol, Mobile phase C: methanol;

[0112] Detection wavelength: 290nm;

[0113] Flow rate: 1.5 mL / min;

[0114] Column temperature: 35 °C;

[0115] Injection concentration: 1 mg / mL;

[0116] Injection volume: 20 μL.

[0117] The elution program is shown in Table 2.

[0118] Table 1 Stability test results

[0119]

[0120] Table 2 Elution program

[0121] Time (min) Mobile Phase A (%) Mobile Phase B (%) Mobile Phase C (%) 0 43 17 40 40 43 17 40

[0122] As shown in the above results, it was confirmed that the reduced coenzyme Q10 crystals of the present invention have higher stability compared to the crystal forms in the literature, and higher purity can be obtained by using this crystallization method.

[0123] Comparative Example 3

[0124] The detection was carried out by the liquid phase method of Patent CN103635452, and the obtained results are as Figure 7 as that Figure 8 shown, where Figure 7 is the HPLC chromatogram of the crystal of reduced coenzyme Q10 obtained in Example 1, Figure 8 is the HPLC chromatogram of the crystal of reduced coenzyme Q10 obtained in Comparative Example 1, indicating that the results obtained by the existing method and the literature method are close.

[0125] Example 5

[0126] The solvent residues of the crystals of reduced coenzyme Q10 obtained in Example 1 and Comparative Example 1 were determined, and the results are shown in Table 3.

[0127] GC analysis conditions

[0128] Chromatographic column: PEG-20M 30m×0.53mm, 1.0μm or a capillary column with equivalent efficiency;

[0129] Temperature rising rate: The initial temperature is 35°C and is maintained for 10 minutes; the temperature is raised to 220°C at a rate of 20°C per minute;

[0130] Injection port temperature: 200°C;

[0131] Detector temperature: 250°C;

[0132] Headspace time:: 20 min;

[0133] Headspace temperature: 70°C

[0134] Injection volume: 1 mL.

[0135] Table 3 Solvent residue determination results

[0136]

[0137] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. However, the present invention is not limited thereto. Those skilled in the art can understand that within the scope of the technical concept of the present invention, the technical solutions of the present invention can be modified, or some technical features can be combined in any other way. These modifications or combinations do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of each embodiment of the present invention, and should be regarded as the content disclosed by the present invention and all fall within the protection scope of the present invention.

Claims

1. A method for preparing reduced coenzyme Q10 crystals, characterized in that, The reduced coenzyme Q10 crystal is determined by differential scanning calorimetry and has an endothermic peak at 52 ± 2 °C when heated at a rate of 10 k / min; moreover, it has a powder X-ray diffraction pattern measured with Cu-Kα radiation as shown in Figure 1; The preparation method includes the following steps: Mix and dissolve reduced coenzyme Q10 with a cyclic ether solvent under heating conditions, then add a mixed solution of a fluorinated alcohol solvent and an organic base, and cool down for crystallization to obtain reduced coenzyme Q10 crystals; The temperature for crystallization is 0 °C to 20 °C; The ratio of the reduced coenzyme Q10 to the cyclic ether solvent is 1 g:(2 - 10) mL; The volume ratio of the cyclic ether solvent to the fluorinated alcohol solvent is 1:(1 - 5); The mass of the organic base is 5% - 10% of the mass of the reduced coenzyme Q10; The cyclic ether solvent is selected from one or more of 2-methyltetrahydrofuran and tetrahydrofuran; The fluorinated alcohol solvent is selected from one or more of trifluoromethanol, trifluoroethanol, trifluoropropanol, and trifluorobutanol; The organic base is selected from triethylamine.

2. The preparation method according to claim 1, wherein The infrared absorption spectrum of the reduced coenzyme Q10 crystal measured by the KBr tablet pressing method has characteristic absorption peaks at wavenumbers 794±1 cm -1 , 877±1 cm -1 , 962 cm -1 and 1014 cm -1 .

3. The preparation method according to claim 1, characterized in that, It has an infrared absorption spectrum measured by the KBr tablet pressing method as shown in Figure 2; and / or, it has a differential scanning calorimetry curve as shown in Figure 3.

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