WO2011074758A1 - Process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids - Google Patents

Process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids Download PDF

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WO2011074758A1
WO2011074758A1 PCT/KR2010/005033 KR2010005033W WO2011074758A1 WO 2011074758 A1 WO2011074758 A1 WO 2011074758A1 KR 2010005033 W KR2010005033 W KR 2010005033W WO 2011074758 A1 WO2011074758 A1 WO 2011074758A1
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process according
polyunsaturated fatty
ethyl ester
oil
fatty acid
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PCT/KR2010/005033
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French (fr)
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Yong Wook Lee
Jung Nam Kim
Moon Soo Park
Dong Hyeon Ko
Jae-Taek Han
Eun-Jeong Nam
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Chemport Inc.
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/06—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils with glycerol
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/003—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with alcohols
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/005—Splitting up mixtures of fatty acids into their constituents
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/02—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with glycerol
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids, and a use of the triglyceride oil prepared by the process. More specifically, the present invention relates to a process for easily and efficiently preparing triglyceride oil containing high concentration of polyunsaturated fatty acids by reacting highly purified polyunsaturated fatty acid ethyl ester oil with glycerol in the presence of an enzyme, and a composition for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing high concentration of eicosapentaenoic acids (EPA) prepared by the process.
  • EPA eicosapentaenoic acids
  • Polyunsaturated fatty acids are fatty acids which have eighteen or more carbon atoms and two or more double bonds. Polyunsaturated fatty acids have been used to increase functionality of food products and animal feedstuffs, since they have several special biological activities. Examples of polyunsaturated fatty acids include linoleic acid (LA), ⁇ -linolenic acid (ALA), ⁇ -linolenic acid (GLA), dihomo- ⁇ -linolenic acid (DGLA), mead acid (MA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc. Polyunsaturated fatty acids are used primarily in the form of triglycerides, although they may be used in the form of free fatty acids or phospholipids.
  • sources for polyunsaturated fatty acids are limited to extracts from biological resources, since it is difficult and expensive to synthesize polyunsaturated fatty acids chemically. They are generally extracted from oil plants, seafood, microorganisms, seaweed and the like.
  • Polyunsaturated fatty acids usually exist in the form of triglycerides in these organisms.
  • the triglycerides have low concentration of polyunsaturated fatty acids and do not exhibit desired biological activities sufficiently, or if it is taken in large amount to provide desired biological functions, calorie intake is increased.
  • conventional processes have difficulties in applying to commercial mass production since they need to use analytical instruments such as high performance liquid chromatography.
  • triglyceride oil containing high concentration of polyunsaturated fatty acids can be easily and economically prepared by reacting highly purified polyunsaturated fatty acid ethyl ester oil with glycerol in the presence of an enzyme to complete the present invention.
  • An object of the present invention is, therefore, to provide a process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids easily and efficiently.
  • Another object of the present invention is to provide a composition for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing high concentration of eicosapentaenoic acid (EPA) prepared by the process.
  • EPA eicosapentaenoic acid
  • One aspect of the present invention relates to a process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids, comprising:
  • step (i) when naturally occurring triglyceride oil is reacted with ethanol, fatty acids in the form of triglycerides are converted to ethyl esters by ester exchange reaction to yield fatty acid ethyl ester oil.
  • the ethanol is preferably used in the amount of 30 to 100 wt% based on 100 wt% of the naturally occurring triglyceride oil.
  • the ester exchange reaction is preferably performed in the presence of a base, more preferably sodium hydroxide.
  • the amount of the base is preferably 0.5 to 2 wt% based on 100 wt% of the naturally occurring triglyceride oil.
  • the reaction temperature is preferably 40 °C to 70 °C.
  • the naturally occurring triglyceride oil is extracted from animals or plants. Generally, it is extracted from oil plants, seafood, microorganisms, seaweed and the like. It is preferably extracted from fishes, more preferably anchovy, sardine, herring, menhaden or tuna.
  • step (ii) the fatty acid ethyl ester oil containing various types of fatty acid ethyl esters is separated and purified to provide polyunsaturated fatty acid ethyl ester oil containing high concentration, preferably 70% or more, of a desired specific polyunsaturated fatty acid ethyl ester.
  • the separation and purification can be performed by molecular distillation, vacuum fractional distillation, recrystallization and the like.
  • the molecular distillation is preferably performed by first distillation at 0.1 torr or less and 100 °C to 150 °C, followed by second distillation at 0.1 torr or less and 140 °C to 170 °C, using a conventional molecular distillation apparatus.
  • the vacuum fractional distillation is preferably performed at 0.5 torr or less and 120 °C to 200 °C using a conventional vacuum fractional distillation apparatus.
  • first molecular distillation with a molecular distillation apparatus then vacuum fractional distillation, and finally second molecular distillation with a molecular distillation apparatus may be performed.
  • the recrystallization is preferably performed with urea and an alcohol, particularly, methanol by a conventional method.
  • polyunsaturated fatty acids include, but not limited to, linoleic acid (LA), ⁇ -linolenic acid (ALA), ⁇ -linolenic acid (GLA), dihomo- ⁇ -linolenic acid (DGLA), mead acid (MA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Eicosapentaenoic acid, or docosahexaenoic acid is particularly preferable.
  • LA linoleic acid
  • ALA ⁇ -linolenic acid
  • GLA ⁇ -linolenic acid
  • DGLA dihomo- ⁇ -linolenic acid
  • MA mead acid
  • AA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • step (iii) when the polyunsaturated fatty acid ethyl ester oil obtained in step (ii) is reacted with glycerol in the presence of an enzyme, polyunsaturated fatty acids bind to glycerol by ester exchange reaction to generate triglyceride oil.
  • the content of polyunsaturated fatty acids in the produced triglyceride oil varies depending on the separation and purification level of the polyunsaturated fatty acid ethyl ester oil, and is preferably 70 wt% or more.
  • the separated and purified polyunsaturated fatty acid ethyl ester oil may be used alone or in a mixture of two or more.
  • the enzyme may be a lipase originated from microorganisms such as Rhizopus sp., Aspergillus sp., and Mucor sp., or a non-site specific lipase such as a lipase originated from Candida cylindracea or Candida antartica.
  • Novozyme 435 originated from Candida antartica or a similar lipase is preferable.
  • the amount of the enzyme used is preferably from 1 to 7 wt% based on the polyunsaturated fatty acid ethyl ester oil. If the amount of the enzyme is less than 1 wt%, the reaction does not proceed. If the amount of the enzyme is more than 7 wt%, the reaction rate does not increase any more, which is not economical.
  • the optimum reaction temperature depends on the type of the enzyme, but it is important to maintain activity of the enzyme in consideration of reuse of the enzyme.
  • 50 to 70 °C is preferable. While the reaction hardly proceed at less than 50 °C, the enzyme activity decreases at more than 70 °C and thus there may be a problem in the reuse of the enzyme.
  • the reaction is preferably performed under vacuum in order to rapidly remove ethanol generated during the reaction.
  • the level of vacuum is preferably 10 to 0.05 torr. High level of vacuum is desired, but too high level of vacuum increases possibility of loss of glycerol and needs expensive apparatus resulting in economic loss.
  • the amount of glycerol used is preferably 5 to 13 wt%, more preferably 7 to 9 wt% based on the polyunsaturated fatty acid ethyl ester oil.
  • a triglyceride (TG) which is glycerol bonded to 3 fatty acids is more slowly generated than a monoglyceride (MG) or a diglyceride (DG) which is glycerol bonded to one fatty acid or two fatty acid, respectively.
  • MG monoglyceride
  • DG diglyceride
  • the content of triglyceride in the produced triglyceride oil is preferably 60% or more.
  • the enzyme catalyst is expensive, it is preferable to recover and reuse it after completion of the reaction. Thus, it is preferable to use the enzyme adsorbed on a solid support to facilitate recovery.
  • Another aspect of the present invention relates to a composition for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing 70% or more of eicosapentaenoic acid (EPA) prepared by the process according to the present invention.
  • EPA eicosapentaenoic acid
  • the composition according to the present invention can be used as a pharmaceutical composition for ameliorating or alleviating symptoms of rheumatoid arthritis.
  • the pharmaceutical composition according to the present invention can be administered orally, e.g., ingestion or inhalation; or parenterally, e.g., injection, deposition, implantation or suppositories.
  • the injection can be, for example, intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal.
  • the pharmaceutical composition of the present invention may be formulated as tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injection solutions, emulsions, suspensions, syrups, aerosols, etc.
  • composition of the present invention can be prepared in a manner well known in the art using a pharmaceutically acceptable carrier(s) which are usually used for each form.
  • pharmaceutically acceptable carriers include excipient, filler, expander, binder, disintegrator, lubricant, preservative, antioxidant, isotonic agent, buffer, coating agent, sweetening agent, dissolvent, base, dispersing agent, wetting agent, suspending agent, stabilizer, colorant, etc.
  • the pharmaceutical composition of the present invention contains 0.01 to 100 wt% of triglyceride oil containing 70% or more of EPA depending on the form thereof.
  • the specific dosage of the present pharmaceutical composition can be varied with species of mammals including a human-being, route of administration, body weight, gender, age, severity of disease, judgment of doctor, etc. It is preferable that 0.01 to 50 mg of the active ingredient is administered per kg of body weight a day for oral use, while 0.01 to 10 mg of the active ingredient is administered per kg of body weight a day for parenteral use.
  • the total daily dosage can be administered once or over several times depending on the severity of disease, judgment of doctor, etc.
  • the composition according to the present invention can be used as a functional food for ameliorating or alleviating symptoms of rheumatoid arthritis.
  • the functional food according to the present invention can be oral preparation such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., or ordinary food such as candies, snacks, gums, ice creams, noodles, breads, drinks, etc. to which the active ingredient is added.
  • the functional food according to the present invention can be prepared in a manner well known in the art using a sitologically acceptable carrier(s) such as filler, extender, binder, wetting agent, disintegrator, sweetening agent, flavoring agent, preservative, surfactant, lubricant, excipient, etc. depending on the form thereof,
  • a sitologically acceptable carrier(s) such as filler, extender, binder, wetting agent, disintegrator, sweetening agent, flavoring agent, preservative, surfactant, lubricant, excipient, etc. depending on the form thereof,
  • the functional food of the present invention contains 0.01 to 100 wt% of the triglyceride oil containing 70% or more of EPA depending on the form thereof.
  • triglyceride oil containing high concentration of polyunsaturated fatty acids with desired biological activities can be easily and efficiently prepared using an enzyme.
  • concentrations and types of the polyunsaturated fatty acids contained in the triglyceride oil can be controlled by adjusting level of purification and type of polyunsaturated fatty acid ethyl ester oil which is reacted with glycerol.
  • triglycerides oil containing 70% or more of eicosapentaenoic acid (EPA) prepared by the present process can be used for preparing a composition for ameliorating or alleviating symptoms of rheumatoid arthritis.
  • EPA eicosapentaenoic acid
  • Figure 1 is a graph illustrating increase rates of edema in the normal control group, arthritis control group, 500, 1000 and 2000 mg/kg of triglyceride administration groups, and positive control group.
  • Figure 2 is a graph showing inhibition rates of edema in the 500, 1000 and 2000 mg/kg of triglyceride administration groups and positive control group.
  • triglyceride oil originated from anchovy oil
  • 50 wt% of ethanol and 1 wt% of sodium hydroxide were added, and the mixture was stirred for 2 hrs while maintaining its temperature at 70 °C. And then, the reaction solution was washed with water 5 times, and degassed to give eicosapentaenoic acid ethyl ester oil containing 17% of eicosapentaenoic acid.
  • the obtained oil was distilled firstly at 0.1 torr or less and 120 °C, and then distilled secondly at 0.1 torr or less and 160 °C in a molecular distillation apparatus to provide eicosapentaenoic acid ethyl ester oil containing 24% of eicosapentaenoic acid.
  • the obtained oil was distilled at 0.5 torr or less and 120 °C in a vacuum fractional distillation apparatus to provide eicosapentaenoic acid ethyl ester oil containing 70% of EPA.
  • triglyceride oil originated from tuna oil
  • 50 wt% of ethanol and 1 wt% of sodium hydroxide were added, and the mixture was stirred for 2 hrs while maintaining its temperature at 70 °C. And then, the reaction solution was washed with water 5 times, and degassed to give docosahexaenoic acid ethyl ester oil containing 24% of docosahexaenoic acid (DHA).
  • DHA docosahexaenoic acid
  • the obtained oil was distilled firstly at 0.1 torr or less and 125 °C, and then distilled secondly at 0.1 torr or less and 165 °C in a molecular distillation apparatus to provide docosahexaenoic acid ethyl ester oil containing 45% of DHA.
  • the obtained oil was distilled at 0.5 torr or less and 170 °C in a vacuum fractional distillation apparatus to provide docosahexaenoic acid ethyl ester (DHA 70EE) oil containing 70% of DHA.
  • DHA 70EE docosahexaenoic acid ethyl ester
  • DHA 70EE oil obtained in Preparative Example 2 urea and methanol were mixed in the weight ratio of 100:200:800. After they were dissolved completely at the temperature of 60 °C or more, they were recrystallyzed by cooling at a rate of 1.0 °C/min. The urea crystal was filtered and removed, and the filtrate was washed with water 4 times and degassed to give docosahexaenoic acid ethyl ester (DHA 80EE) oil containing 80% of DHA.
  • DHA 80EE docosahexaenoic acid ethyl ester
  • EPA 70EE oil obtained in Preparative Example 1 urea and methanol were mixed in the weight ratio of 100:200:800. After they were dissolved completely at the temperature of 60 °C or more, they were recrystallyzed by cooling at a rate of 1.0 °C/min. The urea crystal was filtered and removed, and the filtrate was washed with water 4 times and degassed to give eicosapentaenoic acid ethyl ester (EPA 90EE) oil containing 90% of EPA.
  • EPA 90EE eicosapentaenoic acid ethyl ester
  • Eicosapentaenoic acid ethyl ester oil containing 24% of EPA obtained through two molecular distillations as described in Preparative Example 1 and EPA 90EE oil obtained in Preparative Example 4 were mixed in the ratio of 1:6 to give eicosapentaenoic acid ethyl ester (EPA 80EE) oil containing 80% of EPA.
  • Example 1 Preparation of triglyceride oil containing 70% eicosapentaenoic acid
  • the prepared triglyceride oil was methyl esterified by treatment with BF 3 for gas chromatography (GC).
  • GC gas chromatography
  • the content of EPA was 70% by GC.
  • HPLC high performance liquid chromatography analysis showed 65% of triglycerides, 20% of diglycerides (DG) and 15% of ethyl esters (EE), and 94% of recovery rate.
  • Example 2 Preparation of triglyceride oil containing 70% docosahexaenoic acid
  • the prepared triglyceride oil was methyl esterified by treatment with BF 3 for gas chromatography.
  • the content of DHA was 70% by GC.
  • HPLC analysis showed 64% of TG, 22% of DG and 14% of EE, and 93% of recovery rate.
  • Example 3 Preparation of triglyceride oil containing 80% docosahexaenoic acid
  • the prepared triglyceride oil was methyl esterified by treatment with BF 3 for gas chromatography.
  • the content of DHA was 80% by GC.
  • HPLC analysis showed 62% of TG, 22% of DG and 16% of EE, and 93% of recovery rate.
  • Example 4 Preparation of triglyceride oil containing 90% eicosapentaenoic acid
  • the prepared triglyceride oil was methyl esterified by treatment with BF 3 for gas chromatography.
  • the content of EPA was 90% by GC.
  • HPLC analysis showed 90% of TG, 6% of DG and 4% of EE, and 92% of recovery rate.
  • Example 5 Preparation of triglyceride oil containing 80% eicosapentaenoic acid
  • the prepared triglyceride oil was methyl esterified by treatment with BF 3 for gas chromatography.
  • the content of EPA was 80% by GC.
  • HPLC analysis showed 65% of TG, 20% of DG and 15% of EE, and 94% of recovery rate.
  • Example 6 Preparation of triglyceride oil containing 53% eicosapentaenoic acid and 25% docosahexaenoic acid
  • reaction solution was cooled to room temperature, filtered with filter paper to separate Novozyme 435 and oil, and 100 g of water was added to the oil to remove residual glycerol. Then, the oil was degassed under vacuum to remove moisture.
  • the prepared triglyceride oil was methyl esterified by treatment with BF 3 for gas chromatography.
  • the contents of EPA and DHA were 53% and 25% by GC, respectively.
  • HPLC analysis showed 63% of TG, 24% of DG and 13% of EE, and 91% of recovery rate.
  • FCA Freund's complete adjuvant
  • Triglyceride administration group arthritis was induced by administering FCA subcutaneously to hind legs of the test animals, and triglyceride oil obtained in Example 1 was administered.
  • the amount of administered solution for each group was 5 mL/kg body weight, dosages of the triglyceride oil were 500, 1000 and 2000 mg/kg body weight, and dosage of phenylbutazone was 50 mg/kg body weight.
  • the triglyceride oil and phenylbutazone were orally administered once daily for 21 days after administration of FCA.
  • Increase rate of edema [(edema of paw before FCA administration edema of paw after FCA administration)/ edema of paw before FCA administration] X 100
  • Inhibition rate of edema [(average edema after specified time in the arthritis control group - average edema after specified time in the triglyceride administration group)/ average edema after specified time in the arthritis control group] X 100
  • the increase rate of edema was 65.92 ⁇ 6.24 % at 5 days after administration of FCA in the arthritis control group.
  • the increase rates of edema were 55.23 ⁇ 4.22 %, 47.71 ⁇ 8.50 % and 54.55 ⁇ 4.90 % for 500 mg/kg, 1000 mg/kg and 2000 mg/kg of triglyceride administration groups, respectively.
  • the increase rate of edema was 49.65 ⁇ 5.40 % in the positive control group. Therefore, there was no statistical significance compared to the arthritis control group, but there was a tendency of inhibition of edema increase rate.
  • the increase rates of edema for the arthritis control group and the positive control group at 13 days after administration of FCA were 106.44 ⁇ 7.78 % and 89.19 ⁇ 6.89 %, respectively.
  • the increase rates of edema for 500, 1000 and 2000 mg/kg of triglyceride administration groups were 96.03 ⁇ 4.69 %, 95.95 ⁇ 4.94 % and 93.53 ⁇ 7.67 %, respectively, and were lower than that of the arthritis control group.
  • the increase rates of edema were 111.44 ⁇ 5.42 %, 89.78 ⁇ 3.65 %, and 88.93 ⁇ 3.46 % for the arthritis control group, 500 mg/kg of triglyceride administration group and 1000 mg/kg of triglyceride administration group, respectively, and statistically significant (p ⁇ 0.01) inhibition of edema increase rate was observed.
  • the increase rates of edema for 2000 mg/kg of triglyceride administration group and the positive control group were 76.84 ⁇ 3.03 % and 72.51 ⁇ 3.97 %, respectively, and statistically very significant (p ⁇ 0.001) inhibition of edema increase rate was observed.
  • the inhibition rates of edema for 500 mg/kg, 1000 mg/kg and 2000 mg/kg of triglyceride administration groups and the positive control group were 3.3%, 12.1%, 6.7%, and 9.8%, respectively.
  • the inhibition rates of edema for 500 mg/kg, 1000 mg/kg and 2000 mg/kg of triglyceride administration groups and the positive control group were 2.5%, 0.8%, 2.0%, and 4.2%, respectively.
  • the inhibition rates of edema for 500 mg/kg, 1000 mg/kg and 2000 mg/kg of triglyceride administration groups and the positive control group were 7.1%, 7.8%, 14.8%, and 16.4%, respectively.
  • tendency of inhibition of edema was observed compared to the arthritis control group.
  • the administration of the triglyceride oil containing 70% of EPA according to the present invention showed decrease of paw edema and thus increase of edema inhibition rate compared to the arthritis control group in FCA induced arthritis model. Accordingly, the triglyceride oil according to the present invention has effect of ameliorating symptoms of rheumatoid arthritis.

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Abstract

The present invention relates to a process for easily and efficiently preparing triglyceride oil containing high concentration of polyunsaturated fatty acids by reacting highly purified polyunsaturated fatty acid ethyl ester oil with glycerol in the presence of an enzyme, and a composition for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing high concentration of eicosapentaenoic acid (EPA) prepared by the process.

Description

PROCESS FOR PREPARING TRIGLYCERIDE OIL CONTAINING HIGH CONCENTRATION OF POLYUNSATURATED FATTY ACIDS
The present invention relates to a process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids, and a use of the triglyceride oil prepared by the process. More specifically, the present invention relates to a process for easily and efficiently preparing triglyceride oil containing high concentration of polyunsaturated fatty acids by reacting highly purified polyunsaturated fatty acid ethyl ester oil with glycerol in the presence of an enzyme, and a composition for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing high concentration of eicosapentaenoic acids (EPA) prepared by the process.
Polyunsaturated fatty acids (PUFA) are fatty acids which have eighteen or more carbon atoms and two or more double bonds. Polyunsaturated fatty acids have been used to increase functionality of food products and animal feedstuffs, since they have several special biological activities. Examples of polyunsaturated fatty acids include linoleic acid (LA), α-linolenic acid (ALA), γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA), mead acid (MA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc. Polyunsaturated fatty acids are used primarily in the form of triglycerides, although they may be used in the form of free fatty acids or phospholipids.
In general, sources for polyunsaturated fatty acids are limited to extracts from biological resources, since it is difficult and expensive to synthesize polyunsaturated fatty acids chemically. They are generally extracted from oil plants, seafood, microorganisms, seaweed and the like. Polyunsaturated fatty acids usually exist in the form of triglycerides in these organisms. However, the triglycerides have low concentration of polyunsaturated fatty acids and do not exhibit desired biological activities sufficiently, or if it is taken in large amount to provide desired biological functions, calorie intake is increased. Thus, it is necessary to separate and purify specific triglyceride species from triglyceride mixtures. However, conventional processes have difficulties in applying to commercial mass production since they need to use analytical instruments such as high performance liquid chromatography.
The present inventors discovered that triglyceride oil containing high concentration of polyunsaturated fatty acids can be easily and economically prepared by reacting highly purified polyunsaturated fatty acid ethyl ester oil with glycerol in the presence of an enzyme to complete the present invention.
An object of the present invention is, therefore, to provide a process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids easily and efficiently.
Another object of the present invention is to provide a composition for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing high concentration of eicosapentaenoic acid (EPA) prepared by the process.
One aspect of the present invention relates to a process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids, comprising:
(i) reacting naturally occurring triglyceride oil with ethanol to give fatty acid ethyl ester oil;
(ii) separating and purifying the fatty acid ethyl ester oil to give polyunsaturated fatty acid ethyl ester oil; and
(iii) reacting the polyunsaturated fatty acid ethyl ester oil with glycerol in the presence of an enzyme.
In step (i), when naturally occurring triglyceride oil is reacted with ethanol, fatty acids in the form of triglycerides are converted to ethyl esters by ester exchange reaction to yield fatty acid ethyl ester oil.
The ethanol is preferably used in the amount of 30 to 100 wt% based on 100 wt% of the naturally occurring triglyceride oil.
The ester exchange reaction is preferably performed in the presence of a base, more preferably sodium hydroxide. The amount of the base is preferably 0.5 to 2 wt% based on 100 wt% of the naturally occurring triglyceride oil.
The reaction temperature is preferably 40 ℃ to 70 ℃.
The naturally occurring triglyceride oil is extracted from animals or plants. Generally, it is extracted from oil plants, seafood, microorganisms, seaweed and the like. It is preferably extracted from fishes, more preferably anchovy, sardine, herring, menhaden or tuna.
In step (ii), the fatty acid ethyl ester oil containing various types of fatty acid ethyl esters is separated and purified to provide polyunsaturated fatty acid ethyl ester oil containing high concentration, preferably 70% or more, of a desired specific polyunsaturated fatty acid ethyl ester.
The separation and purification can be performed by molecular distillation, vacuum fractional distillation, recrystallization and the like.
The molecular distillation is preferably performed by first distillation at 0.1 torr or less and 100 ℃ to 150 ℃, followed by second distillation at 0.1 torr or less and 140 ℃ to 170 ℃, using a conventional molecular distillation apparatus.
The vacuum fractional distillation is preferably performed at 0.5 torr or less and 120 ℃ to 200 ℃ using a conventional vacuum fractional distillation apparatus.
Also, first molecular distillation with a molecular distillation apparatus, then vacuum fractional distillation, and finally second molecular distillation with a molecular distillation apparatus may be performed.
The recrystallization is preferably performed with urea and an alcohol, particularly, methanol by a conventional method.
In order to obtain highly purified polyunsaturated fatty acid ethyl ester oil, it is preferable to perform the molecular distillation, vacuum fractional distillation and/or recrystallization successively.
Examples of polyunsaturated fatty acids include, but not limited to, linoleic acid (LA), α-linolenic acid (ALA), γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA), mead acid (MA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Eicosapentaenoic acid, or docosahexaenoic acid is particularly preferable.
In step (iii), when the polyunsaturated fatty acid ethyl ester oil obtained in step (ii) is reacted with glycerol in the presence of an enzyme, polyunsaturated fatty acids bind to glycerol by ester exchange reaction to generate triglyceride oil.
The content of polyunsaturated fatty acids in the produced triglyceride oil varies depending on the separation and purification level of the polyunsaturated fatty acid ethyl ester oil, and is preferably 70 wt% or more.
The separated and purified polyunsaturated fatty acid ethyl ester oil may be used alone or in a mixture of two or more.
The enzyme may be a lipase originated from microorganisms such as Rhizopus sp., Aspergillus sp., and Mucor sp., or a non-site specific lipase such as a lipase originated from Candida cylindracea or Candida antartica. In particular, Novozyme 435 originated from Candida antartica or a similar lipase is preferable.
The amount of the enzyme used is preferably from 1 to 7 wt% based on the polyunsaturated fatty acid ethyl ester oil. If the amount of the enzyme is less than 1 wt%, the reaction does not proceed. If the amount of the enzyme is more than 7 wt%, the reaction rate does not increase any more, which is not economical.
The optimum reaction temperature depends on the type of the enzyme, but it is important to maintain activity of the enzyme in consideration of reuse of the enzyme. When the lipase from Candida antartica is used, 50 to 70 ℃ is preferable. While the reaction hardly proceed at less than 50 ℃, the enzyme activity decreases at more than 70 ℃ and thus there may be a problem in the reuse of the enzyme.
The reaction is preferably performed under vacuum in order to rapidly remove ethanol generated during the reaction. The level of vacuum is preferably 10 to 0.05 torr. High level of vacuum is desired, but too high level of vacuum increases possibility of loss of glycerol and needs expensive apparatus resulting in economic loss.
The amount of glycerol used is preferably 5 to 13 wt%, more preferably 7 to 9 wt% based on the polyunsaturated fatty acid ethyl ester oil. A triglyceride (TG) which is glycerol bonded to 3 fatty acids is more slowly generated than a monoglyceride (MG) or a diglyceride (DG) which is glycerol bonded to one fatty acid or two fatty acid, respectively. Thus, if too much glycerol is used, lots of MG and DG are generated and desired level of TG may not be generated. The content of triglyceride in the produced triglyceride oil is preferably 60% or more.
Since the enzyme catalyst is expensive, it is preferable to recover and reuse it after completion of the reaction. Thus, it is preferable to use the enzyme adsorbed on a solid support to facilitate recovery.
Another aspect of the present invention relates to a composition for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing 70% or more of eicosapentaenoic acid (EPA) prepared by the process according to the present invention.
The composition according to the present invention can be used as a pharmaceutical composition for ameliorating or alleviating symptoms of rheumatoid arthritis. The pharmaceutical composition according to the present invention can be administered orally, e.g., ingestion or inhalation; or parenterally, e.g., injection, deposition, implantation or suppositories. The injection can be, for example, intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal. Depending on the route of administration, the pharmaceutical composition of the present invention may be formulated as tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injection solutions, emulsions, suspensions, syrups, aerosols, etc. The above various forms of the pharmaceutical composition of the present invention can be prepared in a manner well known in the art using a pharmaceutically acceptable carrier(s) which are usually used for each form. Examples of the pharmaceutically acceptable carriers include excipient, filler, expander, binder, disintegrator, lubricant, preservative, antioxidant, isotonic agent, buffer, coating agent, sweetening agent, dissolvent, base, dispersing agent, wetting agent, suspending agent, stabilizer, colorant, etc.
The pharmaceutical composition of the present invention contains 0.01 to 100 wt% of triglyceride oil containing 70% or more of EPA depending on the form thereof.
The specific dosage of the present pharmaceutical composition can be varied with species of mammals including a human-being, route of administration, body weight, gender, age, severity of disease, judgment of doctor, etc. It is preferable that 0.01 to 50 mg of the active ingredient is administered per kg of body weight a day for oral use, while 0.01 to 10 mg of the active ingredient is administered per kg of body weight a day for parenteral use. The total daily dosage can be administered once or over several times depending on the severity of disease, judgment of doctor, etc.
Meanwhile, the composition according to the present invention can be used as a functional food for ameliorating or alleviating symptoms of rheumatoid arthritis. There is no particular limit to the kinds of the functional food according to the present invention. It can be oral preparation such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., or ordinary food such as candies, snacks, gums, ice creams, noodles, breads, drinks, etc. to which the active ingredient is added.
The functional food according to the present invention can be prepared in a manner well known in the art using a sitologically acceptable carrier(s) such as filler, extender, binder, wetting agent, disintegrator, sweetening agent, flavoring agent, preservative, surfactant, lubricant, excipient, etc. depending on the form thereof,
The functional food of the present invention contains 0.01 to 100 wt% of the triglyceride oil containing 70% or more of EPA depending on the form thereof.
According to the present process, triglyceride oil containing high concentration of polyunsaturated fatty acids with desired biological activities can be easily and efficiently prepared using an enzyme. In particular, the concentrations and types of the polyunsaturated fatty acids contained in the triglyceride oil can be controlled by adjusting level of purification and type of polyunsaturated fatty acid ethyl ester oil which is reacted with glycerol.
Further, the triglycerides oil containing 70% or more of eicosapentaenoic acid (EPA) prepared by the present process can be used for preparing a composition for ameliorating or alleviating symptoms of rheumatoid arthritis.
Figure 1 is a graph illustrating increase rates of edema in the normal control group, arthritis control group, 500, 1000 and 2000 mg/kg of triglyceride administration groups, and positive control group.
Figure 2 is a graph showing inhibition rates of edema in the 500, 1000 and 2000 mg/kg of triglyceride administration groups and positive control group.
The present invention is further illustrated by the following examples, which are not to be construed to limit the scope of the invention.
Preparative Example 1: Preparation of 70% eicosapentaenoic acid ethyl ester (EPA 70EE) oil
To 100 wt% of triglyceride oil originated from anchovy oil, 50 wt% of ethanol and 1 wt% of sodium hydroxide were added, and the mixture was stirred for 2 hrs while maintaining its temperature at 70 ℃. And then, the reaction solution was washed with water 5 times, and degassed to give eicosapentaenoic acid ethyl ester oil containing 17% of eicosapentaenoic acid. The obtained oil was distilled firstly at 0.1 torr or less and 120 ℃, and then distilled secondly at 0.1 torr or less and 160 ℃ in a molecular distillation apparatus to provide eicosapentaenoic acid ethyl ester oil containing 24% of eicosapentaenoic acid. The obtained oil was distilled at 0.5 torr or less and 120 ℃ in a vacuum fractional distillation apparatus to provide eicosapentaenoic acid ethyl ester oil containing 70% of EPA.
Preparative Example 2: Preparation of 70% docosahexaenoic acid ethyl ester (DHA 70EE) oil
To 100 wt% of triglyceride oil originated from tuna oil, 50 wt% of ethanol and 1 wt% of sodium hydroxide were added, and the mixture was stirred for 2 hrs while maintaining its temperature at 70 ℃. And then, the reaction solution was washed with water 5 times, and degassed to give docosahexaenoic acid ethyl ester oil containing 24% of docosahexaenoic acid (DHA). The obtained oil was distilled firstly at 0.1 torr or less and 125 ℃, and then distilled secondly at 0.1 torr or less and 165 ℃ in a molecular distillation apparatus to provide docosahexaenoic acid ethyl ester oil containing 45% of DHA. The obtained oil was distilled at 0.5 torr or less and 170 ℃ in a vacuum fractional distillation apparatus to provide docosahexaenoic acid ethyl ester (DHA 70EE) oil containing 70% of DHA.
Preparative Example 3: Preparation of 80% docosahexaenoic acid ethyl ester (DHA 80EE) oil
DHA 70EE oil obtained in Preparative Example 2, urea and methanol were mixed in the weight ratio of 100:200:800. After they were dissolved completely at the temperature of 60 ℃ or more, they were recrystallyzed by cooling at a rate of 1.0 ℃/min. The urea crystal was filtered and removed, and the filtrate was washed with water 4 times and degassed to give docosahexaenoic acid ethyl ester (DHA 80EE) oil containing 80% of DHA.
Preparative Example 4: Preparation of 90% eicosapentaenoic acid ethyl ester (EPA 90EE) oil
EPA 70EE oil obtained in Preparative Example 1, urea and methanol were mixed in the weight ratio of 100:200:800. After they were dissolved completely at the temperature of 60 ℃ or more, they were recrystallyzed by cooling at a rate of 1.0 ℃/min. The urea crystal was filtered and removed, and the filtrate was washed with water 4 times and degassed to give eicosapentaenoic acid ethyl ester (EPA 90EE) oil containing 90% of EPA.
Preparative Example 5: Preparation of 80% eicosapentaenoic acid ethyl ester (EPA 80EE) oil
Eicosapentaenoic acid ethyl ester oil containing 24% of EPA obtained through two molecular distillations as described in Preparative Example 1 and EPA 90EE oil obtained in Preparative Example 4 were mixed in the ratio of 1:6 to give eicosapentaenoic acid ethyl ester (EPA 80EE) oil containing 80% of EPA.
Example 1: Preparation of triglyceride oil containing 70% eicosapentaenoic acid
100 g of EPA 70EE oil obtained in Preparative Example 1 and 8.7 g of glycerol were mixed, and then degassed at 45 ℃ for 30 min. 5 g of Novozyme 435 was added to the mixture, and the mixture was stirred at 300 rpm at 60 ℃ and 1 torr. After 27 hrs, the reaction was terminated when 65% of triglycerides were formed. And then, the reaction solution was cooled to room temperature, filtered with filter paper to separate Novozyme 435 and oil, and 100 g of water was added to the oil to remove residual glycerol. Then, the oil was degassed under vacuum to remove moisture.
The prepared triglyceride oil was methyl esterified by treatment with BF3 for gas chromatography (GC). The content of EPA was 70% by GC. Also, high performance liquid chromatography (HPLC) analysis showed 65% of triglycerides, 20% of diglycerides (DG) and 15% of ethyl esters (EE), and 94% of recovery rate.
Example 2: Preparation of triglyceride oil containing 70% docosahexaenoic acid
100 g of DHA 70EE oil obtained in Preparative Example 2 and 7.9 g of glycerol were mixed, and then degassed at 45 ℃ for 30 min. 5 g of Novozyme 435 was added to the mixture, and the mixture was stirred at 300 rpm at 60 ℃ and 1 torr. After 30 hrs, the reaction was terminated when 64% of triglycerides were formed. And then, the reaction solution was cooled to room temperature, filtered with filter paper to separate Novozyme 435 and oil, and 100 g of water was added to the oil to remove residual glycerol. Then, the oil was degassed under vacuum to remove moisture.
The prepared triglyceride oil was methyl esterified by treatment with BF3 for gas chromatography. The content of DHA was 70% by GC. Also, HPLC analysis showed 64% of TG, 22% of DG and 14% of EE, and 93% of recovery rate.
Example 3: Preparation of triglyceride oil containing 80% docosahexaenoic acid
100 g of DHA 80EE oil obtained in Preparative Example 3 and 8.09 g of glycerol were mixed, and then degassed at 45 ℃ for 30 min. 5 g of Novozyme 435 was added to the mixture, and the mixture was stirred at 300 rpm at 60 ℃ and 1 torr. After 30 hrs, the reaction was terminated when 62% of triglycerides were formed. And then, the reaction solution was cooled to room temperature, filtered with filter paper to separate Novozyme 435 and oil, and 100 g of water was added to the oil to remove residual glycerol. Then, the oil was degassed under vacuum to remove moisture.
The prepared triglyceride oil was methyl esterified by treatment with BF3 for gas chromatography. The content of DHA was 80% by GC. Also, HPLC analysis showed 62% of TG, 22% of DG and 16% of EE, and 93% of recovery rate.
Example 4: Preparation of triglyceride oil containing 90% eicosapentaenoic acid
100 g of EPA 90EE oil obtained in Preparative Example 4 and 8.65 g of glycerol were mixed, and then degassed at 45 ℃ for 30 min. 5 g of Novozyme 435 was added to the mixture, and the mixture was stirred at 300 rpm at 60 ℃ and 0.2 torr. After 48 hrs, the reaction was terminated when 90% of triglycerides were formed. And then, the reaction solution was cooled to room temperature, filtered with filter paper to separate Novozyme 435 and oil, and 100 g of water was added to the oil to remove residual glycerol. Then, the oil was degassed under vacuum to remove moisture.
The prepared triglyceride oil was methyl esterified by treatment with BF3 for gas chromatography. The content of EPA was 90% by GC. Also, HPLC analysis showed 90% of TG, 6% of DG and 4% of EE, and 92% of recovery rate.
Example 5: Preparation of triglyceride oil containing 80% eicosapentaenoic acid
100 g of EPA 80EE oil obtained in Preparative Example 5 and 8.84 g of glycerol were mixed, and then degassed at 45 ℃ for 30 min. 5 g of Novozyme 435 was added to the mixture, and the mixture was stirred at 300 rpm at 60 ℃ and 1 torr. After 27 hrs, the reaction was terminated when 65% of triglycerides were formed. And then, the reaction solution was cooled to room temperature, filtered with filter paper to separate Novozyme 435 and oil, and 100 g of water was added to the oil to remove residual glycerol. Then, the oil was degassed under vacuum to remove moisture.
The prepared triglyceride oil was methyl esterified by treatment with BF3 for gas chromatography. The content of EPA was 80% by GC. Also, HPLC analysis showed 65% of TG, 20% of DG and 15% of EE, and 94% of recovery rate.
Example 6: Preparation of triglyceride oil containing 53% eicosapentaenoic acid and 25% docosahexaenoic acid
94.6 g of EPA 80EE oil obtained in Preparative Example 5 and 5.4 g of DHA 70EE oil obtained in Preparative Example 2 were mixed to prepare mixed oil containing 53% EPA and 25% DHA. 100 g of the mixed oil and 8.55 g of glycerol were mixed, and then degassed at 45 ℃ for 30 min. 5 g of Novozyme 435 was added to the mixture, and the mixture was stirred at 300 rpm at 60 ℃ and 1 torr. After 27 hrs, the reaction was terminated when 63% of triglycerides were formed. And then, the reaction solution was cooled to room temperature, filtered with filter paper to separate Novozyme 435 and oil, and 100 g of water was added to the oil to remove residual glycerol. Then, the oil was degassed under vacuum to remove moisture.
The prepared triglyceride oil was methyl esterified by treatment with BF3 for gas chromatography. The contents of EPA and DHA were 53% and 25% by GC, respectively. Also, HPLC analysis showed 63% of TG, 24% of DG and 13% of EE, and 91% of recovery rate.
Experimental Example 1: Anti-rheumatoid arthritis efficacy test
1. Test method
1.1 Test groups
Male Wister rats were used as test animals, and each group consisted of 8 rats.
Test groups were as follows:
i) Normal control group: arthritis was not induced, and only corn oil as excipient was administered.
ii) Arthritis control group: arthritis was induced by administering Freund's complete adjuvant (FCA) subcutaneously to hind legs of the test animals, and corn oil was administered.
iii) Triglyceride administration group: arthritis was induced by administering FCA subcutaneously to hind legs of the test animals, and triglyceride oil obtained in Example 1 was administered.
iv) Positive control group: arthritis was induced by administering FCA subcutaneously to hind legs of the test animals, and phenylbutazone as a positive control material was administered.
The amount of administered solution for each group was 5 mL/kg body weight, dosages of the triglyceride oil were 500, 1000 and 2000 mg/kg body weight, and dosage of phenylbutazone was 50 mg/kg body weight. The triglyceride oil and phenylbutazone were orally administered once daily for 21 days after administration of FCA.
1.2 Measurement of edema
After the joint region of hind ankle of rat was marked with oily pen, the volume of paw was measured prior to administration, and at 5, 13, and 21 days after administration using plethysmometer. Level of edema was measured, and increase rate of edema and inhibition rate of edema were calculated according to the equations below, and compared to the arthritis control group.
Increase rate of edema (%) = [(edema of paw before FCA administration edema of paw after FCA administration)/ edema of paw before FCA administration] X 100
Inhibition rate of edema (%) = [(average edema after specified time in the arthritis control group - average edema after specified time in the triglyceride administration group)/ average edema after specified time in the arthritis control group] X 100
1.3 Statistical treatment of data
All data were compared to the arthritis control group, and determined to be statistically significant when p < 0.05 using Student's t-test.
2. Results
2.1 Increase rate of edema (Fig. 1) and inhibition rate of edema (Fig. 2)
As shown in Fig. 1, the increase rate of edema was 65.92±6.24 % at 5 days after administration of FCA in the arthritis control group. The increase rates of edema were 55.23±4.22 %, 47.71±8.50 % and 54.55±4.90 % for 500 mg/kg, 1000 mg/kg and 2000 mg/kg of triglyceride administration groups, respectively. Also, the increase rate of edema was 49.65±5.40 % in the positive control group. Therefore, there was no statistical significance compared to the arthritis control group, but there was a tendency of inhibition of edema increase rate. The increase rates of edema for the arthritis control group and the positive control group at 13 days after administration of FCA were 106.44±7.78 % and 89.19±6.89 %, respectively. On the other hand, the increase rates of edema for 500, 1000 and 2000 mg/kg of triglyceride administration groups were 96.03±4.69 %, 95.95±4.94 % and 93.53±7.67 %, respectively, and were lower than that of the arthritis control group. At 21 days, the increase rates of edema were 111.44±5.42 %, 89.78±3.65 %, and 88.93±3.46 % for the arthritis control group, 500 mg/kg of triglyceride administration group and 1000 mg/kg of triglyceride administration group, respectively, and statistically significant (p < 0.01) inhibition of edema increase rate was observed. The increase rates of edema for 2000 mg/kg of triglyceride administration group and the positive control group were 76.84±3.03 % and 72.51±3.97 %, respectively, and statistically very significant (p < 0.001) inhibition of edema increase rate was observed.
Referring to Fig. 2, at 5 days after administration of FCA, the inhibition rates of edema for 500 mg/kg, 1000 mg/kg and 2000 mg/kg of triglyceride administration groups and the positive control group were 3.3%, 12.1%, 6.7%, and 9.8%, respectively. At 13 days after administration of FCA, the inhibition rates of edema for 500 mg/kg, 1000 mg/kg and 2000 mg/kg of triglyceride administration groups and the positive control group were 2.5%, 0.8%, 2.0%, and 4.2%, respectively. At 21 days after administration of FCA, the inhibition rates of edema for 500 mg/kg, 1000 mg/kg and 2000 mg/kg of triglyceride administration groups and the positive control group were 7.1%, 7.8%, 14.8%, and 16.4%, respectively. As the period of administration increases, tendency of inhibition of edema was observed compared to the arthritis control group.
As shown in the above results, the administration of the triglyceride oil containing 70% of EPA according to the present invention showed decrease of paw edema and thus increase of edema inhibition rate compared to the arthritis control group in FCA induced arthritis model. Accordingly, the triglyceride oil according to the present invention has effect of ameliorating symptoms of rheumatoid arthritis.

Claims (19)

  1. A process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids, comprising
    (i) reacting naturally occurring triglyceride oil with ethanol to give fatty acid ethyl ester oil;
    (ii) separating and purifying the fatty acid ethyl ester oil to give polyunsaturated fatty acid ethyl ester oil; and
    (iii) reacting the polyunsaturated fatty acid ethyl ester oil with glycerol in the presence of an enzyme.
  2. The process according to claim 1, wherein the reaction of step (i) is performed in the presence of a base.
  3. The process according to claim 2, wherein the base is sodium hydroxide.
  4. The process according to claim 1, wherein the naturally occurring triglyceride oil is extracted from fishes.
  5. The process according to claim 4, wherein the fishes are anchovy, sardine, herring, menhaden or tuna.
  6. The process according to claim 1, wherein the separation and purification in step (ii) is performed by molecular distillation, vacuum fractional distillation, recrystallization, or a combination thereof.
  7. The process according to claim 1, wherein the polyunsaturated fatty acid is linoleic acid (LA), α-linolenic acid (ALA), γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA), mead acid (MA), arachidonic acid (AA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA).
  8. The process according to claim 7, wherein the polyunsaturated fatty acid is eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA).
  9. The process according to claim 1, wherein the triglyceride oil containing high concentration of polyunsaturated fatty acid comprises 70% or more of polyunsaturated fatty acid.
  10. The process according to claim 1, wherein the triglyceride oil containing high concentration of polyunsaturated fatty acids comprises 60% or more of triglyceride.
  11. The process according to claim 1, wherein the enzyme in step (iii) is a lipase originated from microorganisms such as Rhizopus sp., Aspergillus sp., and Mucor sp., or a non-site specific lipase such as a lipase originated from Candida cylindracea or Candida antartica.
  12. The process according to claim 11, wherein the enzyme is a lipase originated from Candida antartica.
  13. The process according to any one of claims 1, 11 and 12, wherein the amount of the enzyme used is 1 to 7 wt% based on the polyunsaturated fatty acid ethyl ester oil.
  14. The process according to claim 1, wherein the reaction temperature in step (iii) is 50 to 70 ℃.
  15. The process according to claim 1, wherein the reaction of step (iii) is performed under vacuum of 10 to 0.05 torr.
  16. The process according to claim 1, wherein the amount of glycerol used is 5 to 13 wt% based on the polyunsaturated fatty acid ethyl ester oil.
  17. A process for preparing triglyceride oil containing 70% or more of eicosapentaenoic acid, comprising
    (i) reacting triglyceride oil originated from anchovy with ethanol to give fatty acid ethyl ester oil;
    (ii) separating and purifying the fatty acid ethyl ester oil to give eicosapentaenoic acid ethyl ester oil containing 70% or more of eicosapentaenoic acid; and
    (iii) reacting the eicosapentaenoic acid ethyl ester oil with glycerol in the presence of an enzyme.
  18. A pharmaceutical composition for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing 70% or more of eicosapentaenoic acid prepared by the process according to claim 17.
  19. A functional food for ameliorating or alleviating symptoms of rheumatoid arthritis comprising triglyceride oil containing 70% or more of eicosapentaenoic acid prepared by the process according to claim 17.
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Publication number Priority date Publication date Assignee Title
FR3103355A1 (en) * 2019-11-26 2021-05-28 Polaris Oil composition of microorganisms enriched with polyunsaturated fatty acids
WO2021105267A1 (en) * 2019-11-26 2021-06-03 Polaris Microorganism oil composition enriched in polyunsaturated fatty acids

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