RU2535928C1 - Rapid catalytic method for preparing recovered form of coenzyme q10 to be used in pharmaceutical and food compositions - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the chemical-pharmaceutical and food industries. What is presented is a catalytic method for synthesis of a recovered form of coenzyme Q₁₀ (ubiquinol) which can be used for the rapid preparation of ubiquinol and various based pharmaceutical compositions and biologically active food additives possessing antioxidant and antihypoxant activity. The method for preparing ubiquinol of an oxidised form of coenzyme Q₁₀ is based on the catalytic recovery of Q₁₀ in the presence of a chemical reducing agent, a solvent and trace amounts of a Cu²⁺ copper catalyst for the pharmaceutical and food applications. In addition to the substantial reduction of the time for preparing ubiquinol, using the catalyst enables reducing the consumption of the chemical reducing agent and energy. Prepared ubiquinol can be used, e.g. for preparing various compositions, including ones in the liquid form and in the form of water-soluble powders containing inclusion complexes.

EFFECT: method for the synthesis of the recovered form of coenzyme Q₁₀ (ubiquinol).

10 cl, 9 ex

Description

Coenzyme Q ₁₀ (KoQ) in two forms - oxidized (ubiquinone) and reduced (ubiquinol) is a coenzyme of the mitochondrial tissue respiration system, which is involved in the processes of biological oxidation and ATP production. The most studied use of coenzyme Q ₁₀ with the so-called mitochondrial diseases - heart failure, diabetes, metabolic syndrome, neurodegenerative disorders - Alzheimer's, Parkinson's disease, Haitington's syndrome, and also as a geroprotector. The use of the oxidized form of KoQ is associated with a number of difficulties associated with a) its low bioavailability, which is only 3–7%; b) with a decrease in the activity of enzymes I-III of the molecular complexes of mitochondria due to gene mutations, oxidative stress, the inhibitory effect of xenobiotics and drugs, and aging. This blocks the formation of the reduced form of KoQH ₂ and causes a decrease in the level of ATP synthesis, and in general depletion of the energy potential of the cell.

Meanwhile, normally 95% of the blood plasma KoQ should be represented by the restored form, which is unattainable in diabetes, cardiovascular pathology, etc., as well as in people over 40 years of age, i.e. when the ability of mitochondrial enzymes to reduce ubiquinone to ubiquinol is significantly reduced (S. Pere et al., 2007; Shults CW et al., 2002). Clinical trials have shown that the use of the reduced form of coenzyme Q ₁₀ increases its level in the blood several times more effectively than the use of its oxidized form (Yamamoto Y., Yamashita S., 1997). Therefore, the use of ready-made reconstituted KoQH ₂ (ubiquinol) allows not only to remove these problems, but, given its high bioavailability (8 times higher than KoQ) (Hosoe K., 2007; Takahashi T., Okamoto T., Mori K. et al., 1993), in a short time it is easy to create the necessary concentration of the product in the blood and other tissues. In addition, only the reduced form of KoQ has antiradical and antioxidant activity (Bentinger M. et al., 2007) and is able to maintain an effective concentration of the active reduced form of vitamin E (Thomas SR et al., 1997), preventing its oxidation. Therefore, for preventive and therapeutic purposes, the use of precisely the restored form of coenzyme Q ₁₀ , ubiquinol, is recognized as promising.

In connection with the foregoing, obtaining ubiquinol is the first step towards creating effective dosage forms of drugs and dietary supplements. However, the rapid preparative preparation of reduced coenzyme Q _{10 is} difficult due to the low rates of non-catalytic reduction of ubiquinone.

It is proposed to use the catalytic effect of the reduction of ubiquinone in an environment suitable for biological pharmaceutical and food use of an ethanol solvent in the presence of trace amounts of copper Cu ²⁺ and a natural reducing agent, ascorbic acid, as a method for accelerated production of the substance of the reduced form of coenzyme Q ₁₀ . Moreover, the catalytic effect in the same system, but with the participation of reduced iron, Fe ^{2+ is} not observed.

The present invention relates to the development of a technologically simple and low-cost method for producing the reduced coenzyme Q ₁₀ - ubiquinol using a catalyst.

Patent information on the catalytic production of the reduced form of coenzyme Q _{10 is} practically absent. However, there is patent data on non-catalytic methods for producing ubiquinol for cosmetic purposes by restoring ubiquinone with various natural plant extracts in ethanol (US patent appl. 20120201802 - Takao Yamaguchi, Takaaki Jikihara, Shiro Kitamura, Yasuyoshi Ueda. - Method for producing reduced coenzyme Q ₁₀ , method for stabilizing the same, and composition composition same, Kaneka Corp., pablished on august, 2012).

The disadvantage of this method is the use of significant amounts of expensive hydrophobic plant extracts for preparative KoQ recovery and, as a result, the difficulty of separating the recovered product obtained in the final mixture from extracts (US patent appl. 20120201802, august, 2012).

A known method of reducing coenzyme Q10 using basic synthetic reducing agents, such as sodium borohydride, sodium dithionite, sodium hydrosulfite, etc., in particular, described in US patent appl. 20120201802, august, 2012, as well as in non-patent literature (e.g., Mukai K. et al., 1990). The disadvantage of this method is the use to restore coenzyme Q ₁₀ not natural compounds, but synthetic chemical reducing agents.

An enzymatic method for reducing ubiquinone using one or more reductases extracted from biological material of bacteria, fungi and yeast is also described (WIPO Patent appl. WO / 2011/078648 - Tan Bok Hooi, Xiong Jun. - Method of producing reduced coenzyme Q10 by enzymatic method , pablished on July, 2011). The disadvantage of this method, as in the US patent appl. 20120201802, is the high cost of the biotechnological process and the complexity of separating reductases from the resulting product.

In contrast to the listed methods for restoring ubiquinone in US patent appl. 20110123505 proposed a preparative method for producing ubiquinol (US patent appl. 20110123505 - Tarahiro Ueda, Yozo Nagira, Shiro Kitamura. - Method of producing reduced coenzyme Q10, and method of stabilizing the same, Kaneka Corp., pablished on may 2011) by non-catalytic coenzyme recovery Q10 with ascorbic acid in boiling ethanol (78 ° C) for 30 hours.

The disadvantages of this method are: the duration of the non-catalytic reduction Q ₁₀ - 16 hours (US patent appl. 20120201802, august, 2012) or 30 hours (US patent appl. 20110123505, may, 2011) and, as a result, the possibility of oxidation of the final product in conditions of high temperature and a long time interval. The disadvantage is the complexity of the process associated with compliance with safety precautions and the difficulty of avoiding the loss of boiling solvent and the high energy consumption of the process.

The objective of the present invention was to develop a technologically simple and low-cost method for producing reduced coenzyme Q ₁₀ without the use of commonly used chemical reducing agents, for its further stabilization in liquid form of aqueous, alcoholic and oil solutions. This problem is solved in that in the described method, the quantitative reduction of coenzyme Q ₁₀ proceeds at 25 ° C without heating under the action of pharmaceutical L-ascorbic acid and in the presence of trace amounts of Cu ²⁺ ion catalyst in the form of copper pentahydrate (CuSO ₄ · 5H ₂ O) . Under these conditions, the formation of ubiquinol with a yield close to 95% can occur even at room temperature in just a few minutes. In the described method for dissolving the reagents, as well as in the above methods, only acceptable in the food industry and pharmaceutically approved 95% ethanol is used.

A feature of the present invention is a significant simplification of the process using simple equipment, a significant reduction in the recovery period of coenzyme Q ₁₀ from several hours to several minutes using the proposed catalyst, obtaining a reduced form of coenzyme Q ₁₀ with a quantitative yield (> 95%) at 25 ° C ( without heating, solvent loss and additional energy consumption). An optimal ratio of starting ubiquinone to reducing agent was also obtained, which is different from that described in US patent appl. 20120201802 and US patent appl. 20110123505 and providing a process acceleration of ~ 6000 times. The content of 2.2 mg / ml of reduced coenzyme Q ₁₀ (ubiquinol) (equivalent to solubility in ethanol at 25 ° C) corresponds to the bioavailability of 16 mg of ubiquinone, since ubiquinol has 8 times higher bioavailability (Hosoe K., 2007; Takahashi T., Okamoto, T., Mori, K. et al., 1993). Thus, 2.2 mg of reduced coenzyme Q ₁₀ in 1 ml is equivalent to 16 mg - a half daily prophylactic dose of the oxidized form of coenzyme Q ₁₀ .

The described method includes: (i) preliminary dissolution of the oxidized form of coenzyme Q ₁₀ and ascorbic acid, taken in a molar ratio of at least 1: 5; (ii) adding trace amounts of catalyst and catalytic reduction of coenzyme Q ₁₀ to form its reduced form (ubiquinol); (iii) preventing the reverse reaction from occurring by adding a complexing compound with Cu ²⁺ to achieve a quantitative yield of the target product; (iv) crystallization and isolation of the target product — the reduced form of coenzyme Q ₁₀ , followed by washing with water to remove traces of the catalyst. This ensures almost quantitative yield of the recovered synthesis product and the efficiency of the process.

1. Preparation and yield of reduced coenzyme Q ₁₀ with the participation of a catalyst

Under standard conditions, the full recovery of ubiquinone with a large excess of ascorbic acid (1:50) is at least a day, and under conditions of elevated temperature (60 ° C) with the same excess of a reducing agent, up to 6 hours. In the present method, the time period for the complete reduction of ubiquinone at a temperature of 25 ° C is sharply reduced to 4-7 minutes with a smaller excess of the reducing agent 1: 5. When the molar excess of AK is not less than 1: 5, the yield of reduced coenzyme Q ₁₀ is about 95%. A decrease in the ratio of Q ₁₀ : AK leads to a decrease in the yield of the product. The time to reach a residual concentration of ubiquinone (about 5%) with an increase in the excess of ascorbic acid from 1: 5 to 1:45 practically does not change and is 4-5 minutes. The time to reach a residual concentration of ubiquinone with an increase in the catalyst content (from 0.6 mmol / L to 2.4 mmol / L) sharply decreases from 7 to 1 minute.

1.1. The preparation of the desired reduced form of coenzyme Q ₁₀

A suspension of coenzyme Q ₁₀ in ethanol with a concentration 2 times higher than the limit for dissolution of 2.18 mg / ml (2.53 mmol / L) is mixed with the same volume of an ethanol solution of ascorbic acid taken in excess, achieving complete dissolution of ubiquinone. Then add the catalyst in the form of a solution of copper sulfate pentavodnoy (CuSO ₄ · 5H ₂ O) of the selected concentration. A kinetic analysis of the content and expenditure in the ubiquinone reaction is carried out at 405 nm by a spectrophotometric method.

Example 1. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 2.68 mmol / L and 113.6 mmol / L of ascorbic acid taken in a 45-fold excess (45: 1), 0.6 mmol / L of catalyst solution was added. The time period for complete (95%) recovery of ubiquinone is 4 minutes.

Example 2. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 2.68 mmol / L and 56.8 mmol / L of ascorbic acid taken in a 22.7-fold excess (22.7: 1), 0.6 mmol / L of catalyst solution was added. The period of complete (95%) recovery of ubiquinone is 4 - 4.5 minutes.

Example 3. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 2.71 mmol / L and 28.4 mmol / L of ascorbic acid taken in an 11.3-fold excess (11.3: 1), 0.6 mmol / L of catalyst solution was added. The time period for complete (95%) recovery of ubiquinone is 5 minutes.

Example 4. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 2.84 mmol / L and 14.2 mmol / L of ascorbic acid taken in a 5-fold excess (5.1: 1), 0.6 mmol / L of catalyst solution was added. The time period for complete (95%) recovery of ubiquinone is 4.5 minutes.

Example 5. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 2.80 mmol / L and 5.68 mmol / L of ascorbic acid taken in a 2-fold excess (2: 1), 0.6 mmol / L of catalyst solution was added. A decrease in the yield of the reduction product to 84% is observed due to an increase in the rate of the reverse reaction — the oxidation of ubiquinol.

Example 6. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 2.71 mmol / L and 5.68 mmol / L of ascorbic acid taken in a 2-fold excess (2.1: 1), 1.5 mmol / L of catalyst solution was added. There is an increase in product yield to 97.5%, speed, and a reduction in recovery period to 3 minutes.

Example 7. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 2.71 mmol / L and 5.68 mmol / L of ascorbic acid taken in a 2-fold excess (2.1: 1), 2.4 mmol / L of catalyst solution was added. The time period for complete (98%) recovery of ubiquinone is 3.5 minutes.

Example 8. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 2.71 mmol / L and 5.68 mmol / L of ascorbic acid taken in a 2-fold excess (2.1: 1), 3.0 mmol / L of catalyst solution was added. The time period for complete (98%) recovery of ubiquinone is 3 minutes.

Example 9. In an ethanol solution of coenzyme Q _{10 with an} initial concentration of 1.36 mmol / L and 41.9 mmol / L of ascorbic acid taken in a 31-fold excess (30.8: 1), 2.4 mmol / L of catalyst solution was added. The 90% recovery period of ubiquinone is about 1 minute.

A further increase in the excess of ascorbic acid to 250: 1 did not lead to an increase in the reaction rate and a decrease in the period of complete recovery of ubiquinone. On the contrary, the period for reaching the residual concentration increases from 3 min for AK: Q ₁₀ 250: 1 and 3 mmol / l of catalyst to 10 or more minutes, while decreasing to 0.6 mmol / l of catalyst and the same ratio AK: Q ₁₀ 250: 1.

Thus, the optimal mode of obtaining the reduced form of coenzyme Q ₁₀ at a temperature of 25 ° C from the point of view of the recovery period, product yield and consumption of the reducing agent is the mode in which the molar ratio AK / Q ₁₀ is at least 5: 1, and the catalyst content does not exceed 6.0 · 10 ^-4 mol / L.

Raising the temperature to 60 ° C allows you to increase the amount of restored coenzyme Q ₁₀ 4-5 times only by increasing its solubility in ethanol. The recovery rate corresponds to a recovery period of 1 to 3 minutes, with a product yield close to 95%. This ensures that the desired amount of the reduced form of coenzyme Q _{10 is} acceptable for preparative production. After synthesis was carried out at elevated temperature, the resulting solution with reduced coenzyme Q ₁₀ was cooled, and the precipitate formed upon cooling was either left in ethanol or filtered off, washed with an ice-cold ascorbic acid solution and dried for subsequent use.

2. The removal of the catalyst from the reaction system

To prevent the reverse oxidation reaction from occurring when the reduced coenzyme Q ₁₀ reaches a quantitative yield, sodium ethylene diamine tetraacetate (EDTA) is introduced into the reaction system as an effective complexing compound in an optimally selected ratio of EDTA: Cu ²⁺ close to 1.7: 1. Complete inhibition of the reverse oxidation reaction is observed both at the moment of achieving a quantitative yield of the reduced coenzyme Q ₁₀ , and at any other moment of the reaction of its reduction.

2.1. Ensuring the stability of ubiquinol

Example 1. The stabilization of the reduced coenzyme Q ₁₀ and its shelf life in an alcohol solution is determined by adding an excess of reducing agent and its concentration: 1.0% ensures the safety of the restored coenzyme Q _{10 for} at least 3 months.

Example 2. To the solution obtained after synthesis was complete, the reduced coenzyme Q _{10 was} added with 10 vol.% Distillate, the precipitate formed was filtered on a glass filter, washed with an ice-cold aqueous solution of ascorbic acid, and dried. The resulting powder was immediately used to obtain the liquid form of the reduced coenzyme Q ₁₀ .

Example 3. The solution obtained at the end of the synthesis was evaporated on a rotary evaporator, after which ubiquinol was removed with methylene chloride and the solvent was completely removed. The obtained powder was used to obtain finished forms of reduced coenzyme Q ₁₀ (ubiquinol).

Example 4. Obtained in example 2 in the form of a precipitate, the reconstituted form of coenzyme Q _{10 was} solubilized for subsequent introduction into a 20% aqueous solution of ascorbic acid (mass ratio KoQ: AK 1: 4). The resulting solution contains 5% reduced coenzyme Q ₁₀ .

Example 5. Obtained in example 2 as a precipitate, the reconstituted form of coenzyme Q _{10 was} solubilized for subsequent introduction into a 10% aqueous solution of ascorbic acid (mass ratio KoQ: AK 1: 1). The resulting gel contains 10% reduced coenzyme Q ₁₀ .

Example 6. The reconstituted form of coenzyme Q ₁₀ obtained in Example 2 as a precipitate was solubilized for subsequent introduction into a 20% aqueous solution of ascorbic acid (mass ratio KoQ: AK 1: 4) or mixed with a 4% solution of ascorbyl palmitate in a solubilizate . Obtained using thickeners (fructose, sucrose, pectin, etc.), the syrup contains 5% reduced coenzyme Q ₁₀ .

Example 7. The reduced form of coenzyme Q ₁₀ obtained in Example 2 as a precipitate was dissolved in essential oils (for example, grapefruit, orange, lemon) containing up to 90 - 95% limonene. The solution was mixed with a 4% solution of ascorbyl palmitate or propyl gallate in a solubilizate. The content of KoQ is 10%, and the stabilization of the reduced coenzyme Q _{10 is} provided up to 2 years.

Example 8. The reduced form of coenzyme Q ₁₀ obtained in Example 2 as a precipitate was dissolved in essential oils (for example, grapefruit, orange, lemon) containing 85-95% limonene, an excess of a reducing agent (for example, 2-4% ascorbyl palmitate, propyl gallate) was added . The content of KoQ is 11%, and the stabilization of the reduced coenzyme Q _{10 is} provided up to 2 years.

Example 9. Carotenoids — astaxanthin, lycopene or carotenoid bixin (permitted and available food supplement E150b — Annato) were introduced into the solutions of the reduced form of coenzyme KoQ ₁₀ obtained in examples 7 and 8 to inhibit photooxidation. This encapsulation, which limits the access of atmospheric oxygen, contributes to an increase in the shelf life of the reduced form of coenzyme Q _{10 for} more than 2 years. The content of reduced KoQ in the capsule is 8%.

Literature

1. Thomas S.R., Jifi N, Stocker R. Inhibition of LDL oxidation by ubiquinol-10. A protective mechanism for coenzyme Q10 in atherogenesis. - Mol. Aspects Med., 1997, 18, 85-103.

2. Bentinger M., Brismar K., Dallner G. The antioxidant role of coenzyme Q. // Mitochondrion. - 2007. - Vol.7S. - P.41 - 50.

3. Mukai K., Kikuchi S., Urano S. Stopped-flow kinetic study of the regeneration reaction of tocopheroxyl radicals by reduced ubiquinone-10 in solution. // Biochim. Biophys. Acta. - 1990. - Vol. 1035. - P.77 - 82.

4. Pepe S., Marasco S.F., Haas S.J. ety al. Coenzyme Q10 in cardiovascular disease. // Mitochondrion. - 2007. - Vol.7S. - P.154 - 167.

5. Shults C.W., Oakes D., Kieburtz K., et al. Effects of coenzyme Q10 in early Parkinson disease: evidence of slowing of the functional decline. // Arch. Neurol. - 2002. - Vol. 59, No. 10. - P. 1541 - 1550.

6. Hosoe K., Kitano M., Kishida H., et al. Study on safety and bioavailability of ubiquinol after single and 4-week multiple oral administration to healthy volunteers. // Regul. Toxicol. Pharmacol - 2007. - Vol. 47, No. 1. - P.19-28.

7. Yamamoto Y., Yamashita S. Plasma ratio of ubiquinol and ubiquinone as a marker of oxidative stress. // Mol. Aspects Med., 1997, Vol. 18S, 79-84.

8. Takahashi T., Okamoto T., Mori K. et al. Distribution of ubiquinone and ubiquinol homologues in rat tissues and subcellular fractions.// Lipids. - 1993. - Vol. 28. - P.803 - 809.

Claims (10)

1. A method of producing a reduced coenzyme Q ₁₀ by reducing the oxidized form of coenzyme Q _{10 with} ascorbic acid in ethanol, characterized in that it comprises the following steps: (i) pre-dissolving the oxidized form of coenzyme Q ₁₀ and ascorbic acid in a molar ratio of at least 1: 5, respectively; (ii) adding trace amounts of a catalyst for Cu ^{2+ ions} and catalytic reduction of coenzyme Q ₁₀ to form its reduced form (ubiquinol); (iii) adding a complexing agent with Cu ²⁺ compound to achieve a quantitative yield of the target product; (iv) crystallization and isolation of the target product is a reduced form of coenzyme Q ₁₀ followed by washing with water. 2. The method according to p. 1, characterized in that at the stage (i) use a solvent of 95% ethanol. 3. The method according to p. 1, characterized in that the ethanol after distillation can be regenerated for subsequent use. 4. The method according to p. 1, characterized in that at the stage (iii) there is a catalytic reduction of coenzyme Q ₁₀ (ubiquinone) quantitatively at 25 ° C. 5. The method according to p. 1, characterized in that the amount of the final product increases 4-5 times when the catalytic reduction of coenzyme Q _{10 is} carried out at a temperature of 60 ° C. 6. The method according to p. 1, characterized in that in stage (iv) in the resulting solution with reduced coenzyme Q ₁₀ add 10 vol.% Water. 7. The method according to p. 1, characterized in that the recovered coenzyme Q ₁₀ isolated in stage (iv) can be stabilized with ascorbic acid (mass ratio KoQ: AK 1: 1) in an aqueous solution by introducing into a solubilizer and then obtaining a gel containing 10% reduced coenzyme KoQ. 8. The method according to p. 1, characterized in that the recovered coenzyme Q ₁₀ isolated in stage (iv) can be stabilized with ascorbic acid or ascorbyl palmitate in an aqueous solution with a solubilizer and thickeners (for example, fructose, pectins, etc.) for the subsequent creating a syrup containing up to 10% of the recovered KoQ enzyme. 9. The method according to p. 1, characterized in that the recovered coenzyme Q ₁₀ isolated in stage (iv) can be stabilized by an excess of ascorbyl palmitate in essential oil (for example, grapefruit, orange, etc.) and used to create a capsule form of the drug with the content of reduced KoQ up to 10%. 10. The method according to p. 1, characterized in that the recovered coenzyme Q ₁₀ isolated in stage (iv) can be stabilized in a mixture of essential oil (grapefruit, etc.) and a solubilizer containing an excess of ascorbyl palmitate, which inhibits carotenoid photooxidation, and is used to create a capsule form of the drug with a reduced KoQ content of up to 8%.