WO1999043316A1

WO1999043316A1 - Production of coenzyme q

Info

Publication number: WO1999043316A1
Application number: PCT/US1999/004202
Authority: WO
Inventors: Richard A. Willis
Original assignee: The Board Of Regents, The University Of Texas System
Priority date: 1998-02-26
Filing date: 1999-02-25
Publication date: 1999-09-02
Also published as: AU2791999A

Abstract

Coenzyme Q is obtained by subjecting a mixture from soybeans, particularly a deodorizer distillate, and the like to molecular distillation, supercritical extraction or column chromatography.

Description

-1- PRODUCTION OF COENZYME Q

BACKGROUND OF INVENTION

This invention pertains to the production of coenzyme Q, particularly coenzyme Q₁₀, from naturally occurring oils.

Coenzyme Q is available commercially. However, the cost of coenzyme Q is quite high, in part due to the costly methods used for production. In particular, it is known to make coenzyme Q through either a multi-step synthesis or by genetically engineered microorganisms. A new and inexpensive process would be highly desirable. There are currently no methods available for obtaining coenzyme Q from natural sources on an industrial scale. A need thus exists for obtaining coenzyme Q from natural sources on a large scale.

SUMMARY OF INVENTION

The present invention provides a solution to one or more of the disadvantages and deficiencies described above.

A process has now been discovered which obtains coenzyme Q through use of existing, relatively uncomplicated, separation substeps. Advantageously, the coenzyme Q is obtained from inexpensive sources such as oils from a variety of legumes and vegetables, as well as from fish oils presently used to obtain EPA. Likewise, the legume oils, such as peanut and soybean oil, may be the same oil used to obtain tocopherol (vitamin E). Tocopherol and coenzyme Q can thus be obtained in the same process.

In one broad respect, this invention is a process for the production of coenzyme Q, comprising obtaining a deodorizer distillate containing tocopherol and coenzyme Q; -2-

separating any sterols, phospholipids, and fatty acids from the distillate to form a mixture; subjecting the mixture to molecular distillation to separate tocopherol and form a second mixture; and subjecting the second mixture to molecular distillation to provide concentrated coenzyme Q fraction.

In another broad respect, this invention is a process for the production of a coenzyme Q concentrate, comprising obtaining a mixture from a deodorizer distillate wherein the mixture comprises tocopherol and coenzyme Q; and subjecting the mixture to molecular distillation to provide a coenzyme Q concentrate.

In yet another broad respect, this invention is a process for the production of a coenzyme Q concentrate, comprising obtaining a mixture from a deodorizer distillate wherein the mixture comprises tocopherol and coenzyme Q; subjecting the mixture to molecular distillation to provide a tocopherol concentrate and a residue mixture; and subjecting the residue mixture to molecular distillation to provide a coenzyme Q concentrate.

As used herein, substantially free of sterols, phospholipids and fatty acids refers to compositions that contain small, but detectable amount of these materials, for instance less than about 1 microgram/mg of these substances.

Uses for coenzyme Q are numerous, including medicinal use for congestive heart failure and periodontal disease. Coenzyme Q is also know as ubiquinone.

DETAILED DESCRIPTION OF THE INVENTION

Treatment of vegetable oils with steam and vacuum produces a byproduct referred to as deodorizer distillate, or deodorizer sludge. This deodorizer distillate is rich in tocopherols and sterols, but also contains a multitude of other compounds. Methods for the producton of deodorizer distillate are well known as discussed in U. S. Patent No. 5,424,457, incorporated herein by reference. In general, deodorizer distillates are obtained by conventional steam distillation of the crude oils from peanuts, soybean, kinako, azuki beans, almonds, pistachios, -3-

walnuts, chestnuts, cereal grains, a variety of vegetables oils from fishes such as mackerel, sardines, tuna, eel and cuttlefish, and oils from various seeds such as corn, cottonseed, safflower, sunflower, grapeseed, olive, sesame and rice. The deodorizer distillates may contain tocopherols, sterols, fatty acids, glycerides, a variety of hydrocarbons and coenzyme Q components.

Separation of fatty acids and sterols from the deodorizer distillate may be performed using the techniques described, for instance, in U. S. Patent No. 5,242,457 to thereby provide the mixture comprising coenzyme Q and tocopherol. For example, tocopherols can be recovered from vegetable oils and fats by reacting the fatty acid glycerides with an aliphatic monohydric alcohol in the presence of an alkaline alcoholysis catalyst, followed by flash distillation of residual alcohol, glycerol and fatty acid esters, such as is described in U.S. Patent 2,432, 181. The present inventor has found that this procedure is preferably conducted in the presence of pyrogallol to prevent loss of coenzyme Q. U.S. Patent 2,729,655 discloses that sterols can be recovered from distillate by saponification and acidulation to convert glycerides and sterol esters to free fatty acids and free alcohols (glycerol, sterols respectively). The free fatty acids are esterified with a monohydric lower alcohol. The sterols are crystallized by the addition of a hydrocarbon/water solvent to the mixture. U.S. Patent 3,153,055 discloses a process for the isolation of sterols and tocopherols from deodorizer distillate by esterification of higher fatty acids into lower monohydric alcohol esters under strongly acidic conditions. The sterols and tocopherols are fractionally extracted from the esterification product with a combination of polar and nonpolar solvents. U.S. Patent 3,335,154 discloses that the distillate is saponified and acidulated to convert glycerides and sterol esters to free fatty acids and free alcohols (glycerol, sterols respectively). The free fatty acids are esterified with a monohydric lower alcohol and mineral acid catalyst. The sterols are precipitated by the addition of water to the mixture, and the tocopherols are concentrated by removal of the fatty esters by molecular distillation. U.S. Patent 3,840,570 discloses that sterols can be concentrated from a plant-derived source-by phase separation caused by the addition of a water-alcohol solvent. Sterol esters are saponified with an alkali metal base and free sterols are crystallized from an aprotic solvent. U.S. Patent 4,148,810 discloses that sterols can be isolated by transesterification of distillate with methanol, catalyzed by alkali -4-

metal alcoholates or alkali metal hydroxides. The sterols are isolated from the transesterification mixture by adduct formation with calcium chloride in an aprotic solvent. U.S. Patents 4,374,776 and 4,451 ,564 disclose a method for the concentration of sterols by base-catalyzed transesterification of distillate residues with a lower monohydric alcohol, followed by molecular distillation. The base catalyst is an alkali metal alcoholate or alkali metal hydroxide.

In U.S. Patent 5,424,457, the deodorizer distillate is treated by a process comprising the steps of a) reacting the deodorizer distillate mixture with a C₁-C₈ alcohol in the presence of a monoalkyl or dialkyltin or phenyl phosphonic acid catalyst at a temperature of about 150°-240° C; b) removing C-.--C₈ alcohols by distillation; c) crystallization of sterols and isolation of said sterols; followed by d) removing C,-C₈ esters of fatty acids by distillation; and e) molecular distillation of the resulting mixture to provide a concentrated tocopherol mixture. As an alternative process there is provided a process for recovering sterols and tocopherols from a deodorizer distillate mixture, said mixture comprised of sterols, tocopherols, free fatty acids, mono, di-, and tri-glycerides, said process comprising the steps a) reacting the deodorizer distillate mixture with a -C*-* alcohol in the presence of a monoalkyl or dialkyltin or phenylphosphonic acid catalyst at a temperature of about 150°- 240° C; b) removing C--C₈ esters of fatty acids and C,-C₈ alcohols by distillation; c) crystallization of sterols and isolation of said sterols; d) molecular distillation of the resulting mixture to provide a concentrated tocopherol mixture. The present inventor has recognized that the residue mixture left after separation of the tocopherol mixture contains coenzyme Q and, importantly, may be further purified to yield coenzyme Q product.

In the practice of this invention, the residue mixture that is separated from the tocopherol mixture may be subjected to molecular distillation to provide a coenzyme Q concentrate product. Molecular distillation is typically carried out at temperatures of about 140°C to about 250°C. Temperatures outside this range may be employed. The pressure will vary depending on temperature, equipment, composition of the material and so forth. In general, pressures are sub-atmospheric, and are typically less than about 100 mm Hg. Ultralow pressures such as less than 1 mm Hg (or, e.g., in the range from 50 to 1000 microns) -5-

may often be desirable to delimit the amount of coenzyme Q degradation from heat. Apparatus which are commonly used for molecular distillation include a centrifugal molecular still, thin film evaporation, a falling film evaporator, a rotary film centrifugal type molecular distillation apparatus, a wiped film evaporator as well as other short path distillation apparatus. In this invention, tocopherol is typically collected overhead first, with distillation conditions being adjusted to distill off the coenzyme Q. Since distillation is typically cost effective on a commercial scale, molecular distillation is typically preferred in the practice of this invention relative to chromatography and supercritical extraction.

Molecular distillation is preferred in the practice of this invention. However, as an alternative to molecular distillation, it is contemplated that chromatography may be used for the tocopherol and coenzyme Q separation. Coenzyme Q is readily identified in the column owing to its distinctive orange color. In general, coenzyme Q may be separated using column chromatography such as liquid/liquid chromatography, liquid/solid chromatography, and high performance liquid chromatography (HPLC). Common solvents may be used for the chromatography such as hydrocarbons such as octane, hexane and toluene, alcohols such as ethanol, methanol and isopropanol, ketones such as acetone and methyl ethyl ketone, chlorinated hydrocarbons such as chloroform, ethers such as ethyl ether and tetrahydrofuran, and mixtures thereof. When a solid phase is employed, conventional packing materials may be used such as silica, alumina, glass beads and ion-exchange resins. For example, coenzyme Q can be extracted from soybean oil by the following representative, non-limiting procedure. First, the oil is saponified with 10% alcoholic KOH and 5% pyrogallol and extracted several times with hexane. The hexane fraction is then placed on a silica column. Plastoquionone is eluted from the column with 3:1 hexane:dichloromethane. Coenzyme Q is then eluted from the column with dichloromethane. The sample is evaporated under nitrogen to dryness. The sample is then re-dissolved in isopropanol and quantified using high-performance liquid chromatography. Samples are run for 25 minutes at a rate of 1.2 mL/minute in 7% hexane/93% methanol and read at 275 nanometers. Coenzyme Q₁₀ has a retention time under these conditions of 13 minutes. Alternatively, the sample obtained after the evaporation step can be dissolved in warm ethanol and crystallized from the solution which has been chilled. -6-

Using these procedures and HPLC, several oils were treated to form a coenzyme Q concentrate. The amount of coenzyme Q in the oil was then calculated. The results are shown in Table 1 , which also provides the commercial sources of such oil.

TABLE 1

Oil Type CoQ₁₀ CoQ₉ Source of Oil microgram/mg oil microgram/mg oil soybean oil 0.12 Hain canola oil 0.09 Loriva canola oil 0.12 Whole Foods corn oil 0.46 Spectrum rice bran oil 0.03 0.12 Loriva safflower oil 0.04 Spectrum peanut oil 0.04 Spectrum sesame oil 0.02 Spectrum olive oil 0.004 0.11 Bella extra virgin shark liver oil 0.1 Sea-West

Laboratories,

Canada

In another alternative embodiment, supercritical carbon dioxide extraction is employed to purify coenzyme Q. Supercritical carbon dioxide extraction, optionally using a co-solvent such as ethanol or isopropanol, is described in Kuk, JAOCS. Volume 71, Number 12, pages 1353-1356 (December 1994). In general, the supercritical carbon dioxide extraction may be accomplished by introducing a mixture of tocopherol and coenzyme Q into a cylindrical stainless steel vessel with glasswool placed at both ends, placing the vessel in an isothermal chamber at the extraction temperature of about 80 degrees Centigrade, charging the vessel with carbon dioxide using a positive displacement pump to bring the pressure to about 7000 pounds per square inch, allowing the tocopherol or coenzyme Q to be extracted into the supercritical carbon dioxide, with or without a co-solvent. -7-

The types and quantities of coenzyme Q obtained may vary depending on the starting material. See in this regard, Kamei et al., "The Distribution Content of Ubiquinone in Foods," International Journal for Vitamins and Nutrition Research, volume 56, pages 57-63 (1986). In one embodiment of this invention, coenzymes Q₈, Q₉, Q,₀ and mixtures thereof may be obtained.

It should be appreciated that the types and amounts of coenzyme Q varies depending on the type of food. A study of coenzyme Q in various foods is described in M. Kamei et al; InternationalJournal for Vitamins and Nutrition Research, Vol. 56, pages 57-63 (1986).

Further modifications and alternative embodiments of this invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the manner of carrying out the invention. Equivalent elements may be substituted for those illustrated and described herein, and certain features of the invention may be utilized independently of the use of other features, all as would be apparent to one skilled in the art after having the benefit of this description of the invention.

Claims

-8- WHAT IS CLAIMED IS : 1. A process for the production of coenzyme Q, comprising: obtaining a deodorizer distillate containing tocopherol and coenzyme Q; separating any sterols, phospholipids, and fatty acids from the deodorizer distillate to form a mixture; subjecting the mixture to a first separation step to separate tocopherol and form a second mixture; subjecting the second mixture to a second separation step to provide a concentrated coenzyme Q fraction.

2. The process of claim 1 wherein the deodorizer distillate is obtained from steam distillation of oil removed from an oil source.

3. The process of claim 2 wherein the oil source is peanuts or soybeans.

4. The process of claim 1 wherein the coenzyme Q is coenzyme Q₈, Q₉, Q₁₀ or mixtures thereof.

5. The process of claim 1 wherein the first and second separation steps are by molecular distillation.

6. The process of claim 5 wherein the molecular distillation occurs at a pressure of less than about 100 mm Hg and a temperature in the range from about 140 degrees Centigrade to about 250 degrees Centigrade.

7. The process of claim 6 wherein the pressure is less than about 1 mm Hg.

8. A process for the production of a coenzyme Q concentrate, comprising: obtaining a mixture from a deodorizer distillate wherein the mixture comprises tocopherol and coenzyme Q; and subjecting the mixture to molecular distillation to provide a coenzyme Q concentrate. -9-

9. A process for the production of a coenzyme Q concentrate, comprising: obtaining a mixture from a deodorizer distillate wherein the mixture comprises tocopherol and coenzyme Q; subjecting the mixture to molecular distillation to provide a tocopherol concentrate and a residue mixture; and subjecting the residue mixture to molecular distillation to provide a coenzyme Q concentrate.

10. The process of claim 9 wherein the deodorizer distillate is obtained from steam distillation of oil removed from an oil source.

11. The process of claim 10 wherein the oil source is peanuts or soybeans.

12. The process of claim 9 wherein the coenzyme Q is coenzyme Q₈, Q₉, Q₁₀ or mixtures thereof.

13. The process of claim 9 wherein the molecular distillation occurs at a pressure of less than about 100 mm Hg and a temperature in the range from about 140 degrees Centigrade to about 250 degrees Centigrade.

14. The process of claim 13 wherein the pressure is less than about 1 mm Hg.

15. A process for manufacture of a coenzyme Q concentrate, comprising: obtaining a mixture comprising tocopherol and coenzyme Q; and subjecting the mixture to column chromatography to obtain coenzyme Q concentrate.

16. The process of claim 15 wherein a tocopherol concentrate is also obtained. -10-

17. The process of claim 1 wherein the mixture is obtained from a deodorizer distillate that is obtained from steam distillation of oil removed from an oil source.

18. The process of claim 17 wherein the oil source is peanuts or soybeans.

19. The process of claim 16 wherein the mixture is saponified prior to the column chromotography.