WO2015027463A1

WO2015027463A1 - Agents for preventing or treating alcoholic fatty liver

Info

Publication number: WO2015027463A1
Application number: PCT/CN2013/082664
Authority: WO
Inventors: Li Han; Tomoko Tsuji; Conglin Zuo; Maowu Guo; Hongling Wang; Qin LIAO
Original assignee: Nippon Suisan Kaisha, Ltd.
Priority date: 2013-08-30
Filing date: 2013-08-30
Publication date: 2015-03-05

Abstract

Compositions that have efficacy for preventing and treating alcoholic fatty liver and which are highly safe to use are provided. The compositions can be used as agents for preventing or treating alcoholic fatty liver in which phospholipids that contain highly unsaturated fatty acids as constituent fatty acids, in particular krill oil, are contained as an active ingredient. The daily intake of krill oil is preferably in the range of 1 to 100 mg/kg body weight.

Description

AGENTS FOR PREVENTING OR TREATING ALCOHOLIC FATTY LIVER

TECHNICAL FIELD

[0001]

The present invention relates to compositions that are effective in preventing or treating alcoholic fatty liver, More specifically, the invention relates to those compositions in which phospholipids that contain highly unsaturated fatty acids as constituent fatty acids, in particular krill oil, are contained as active ingredients, said compositions being derived from natural products and hence featuring high enough safety to be suitable for long-term intake.

BACKGROUND ART

[0002]

A very high proportion of people who are suffering from or highly likely to develop metabolic syndrome have been diagnosed as fatty liver. Fatty liver refers to excessive fat accumulation in the liver, at levels higher than the normal (3-5%). The incidence of fatty liver is relatively high among people in their thirties to seventies and peaks in middle-aged and older people, somewhere around forty in males and older than forty in females; in terms of gender, ten out twenty-five males and one out of five females have fatty liver. Pathogenesis of fatty liver involves excessive calorie intake, dietary imbalance, and inadequate exercise (in the case of asymptomatic fatty liver or non-alcoholic steatohepatitis, NASH) and prolonged, continuous alcohol intake is another possible cause (alcoholic steatohepatitis, ASH). Middle-aged and older males in particular have a significantly high risk for having alcoholic fatty liver since they often drink alcohol while eating too much or ingesting a high fat content. Alcoholic fatty liver is such a disease that continued heavy drinking of alcohol over a prolonged period will not only damage liver ceils to cause various disorders in the functions of the liver; if left untreated, the disease is gradually aggravated and will progress with high likelihood to a more serious disease such as cirrhosis or hepatitis. Thus, alcoholic fatty liver is a "disease" (may be presymptomatic) that is associated with the lifestyle of an individual, so it would be dealt with more effectively by the food- or health food-based approach rather than by the medication-based approach. As yet, however, there have been developed or distributed no medications or health foods that are targeted to alcoholic fatty liver.

[0003]

Commercial turmeric-containing drinks, which have high visibility as products that can mitigate uncomfortable symptoms due to alcohol intake, are presumed to work effectively through detoxification due to their high antioxidative action; however, in the absence of reports that describe their contribution to an improved lipid metabolism, it is impossible to expect that commercial turnieric-coiitaining drinks will suppress alcohol-induced lipid accumulation in the liver. Alcoholic fatty liver would be more effectively suppressed by functional materials that work holistically from various aspects including alcohol absorption suppression, lipid metabolism enhancement, and liver ceil protection.

Patent Document 1 discloses that soy lecithin has an action of suppressing the synthesis of triglycerides in the liver. Non-patent Document 1 discloses that n-3 fatty acids contribute to the regeneration of liver cells. Patent Document 2 discloses that krill oil is effective in suppressing the elevation of blood alcohol levels. Non-patent Document 2 discloses that krill oil can be used in patients with non-alcoholic steatohepatitis. Non-patent Document 3 discloses that soy lecithin, phosphatidylcholine with polyunsaturated fatty acid, suppresses alcohol-induced fatty liver.

CITATION LIST PATENT DOCUMENT

[0004] Patent Document 1 : WO 2012/029898

Patent Document 2: Japanese Patent No, 5271454

NON-PATENT DOCUMENT

[0005]

Non-patent Document 1 : V. irimlioglu, et a!, , Effect of Fish Oil, Olive oil, and Vitamin E on Liver Pathology, Cell Proliferation, and Antioxidant Defense System in Rats Subjected to Partial Hepatectomy. Transplantation Proceeds ngs , 38, 564-567 (2006)

Non-patent Document 2: S, Tandy, et ai,, Dietary Krill Oil Supplementation Reduces HepaticSteatosis, Glycemia, and Hypercholesterolemia in High-Fat-Fed Mice. Journal of Agricultural and Food Chemistry, 57, 9339-9345 (2009)

Non-patent Document 3: K. P. Navder, et aL Polyenylphosphatidylcholine Attenuates Alcohol-Induced Fatty Liver and Hyperlipemia in Rats. Journal of Nutrition, 127, 1800-1806 (1997)

SUMMARY OF THE INVENTION TECHNICAL PROBLEM

[0006]

An object of the present in vention is to provide compositions that have efficacy for preventing or treating alcoholic fatty liver and which are highly safe to use.

SOLUTION TO PROBLEM

[0007]

Research reports on animal models with alcoholic fatty liver have so far been published occasionally but no reports have been made on animal test systems that provide consistently reproducible results. The present inventors successfully prepared an alcoholic fatty liver model. Using this model, the inventors confirmed the efficacy of krill oil in suppressing the formation of alcoholic fatty liver, thereby accomplished the present invention.

[0008] An object, therefore, of the present invention is to provide agents for preventing or treating alcoholic fatty liver as recited below under (1) to (15), as well as methods for preventing or treating alcoholic fatty liver as recited under (16) to (20).

(1) An agent for preventing or treating alcoholic fatty liver which contains a lipid as an active ingredient, the lipid comprising a highly unsaturated fatty acid as a constituent fatty acid.

(2) An agent for preventing or treating alcoholic fatty liver as recited in (1) which contains a phospholipid as the lipid.

(3) An agent for preventing or treating alcoholic fatty liver as recited in (1) or (2), wherein the lipid consists of a phospholipid comprising a highly unsaturated fatty acid as a constituent fatty acid.

(4) An agent for preventing or treating alcoholic fatty liver as recited in any one of (1 ) to (3), wherein the highly unsaturated fatty acid is an n-3 highly unsaturated fatty acid.

(5) An agent for preventing or treating alcoholic fatty liver as recited in (4), wherein the n-3 highly unsaturated fatty acid is eicosapentaenoic acid, docosapentaenoic acid or docosahexenoic acid.

(6) An agent for preventing or treating alcoholic fatty liver as recited in any one of (2) to

(5) , wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolaniiiie, phosphatidic acid, phosphatidylglycerol and phosphati dy 1 inositol .

(7) An agent for preventing or treating alcoholic fatty liver as recited in any one of (1) to

(6) , wherein the lipid is krill oil.

(8) An agent for preventing or treating alcoholic fatty liver as recited in (7), wherein the krill oil is refined krill oil.

(9) An agent for preventing or treating alcoholic fatty liver as recited in (7) or (8), wherein the krill oil is produced via a thermal coagulum of krill. (10) An agent for preventing or treating alcoholic fatty liver as recited in any one of (7) to

(9) , wherein the krill oil contains a t least 10 wt% of phospholipid.

(11) An agent for preventing or treating alcoholic fatty liver as recited in any one of (7) to

(10) , wherein at least 5 wt% of the constituent fatty acids in the krill oil is an n-3 highly unsaturated fatty acid.

( 12) An agent for preventing or treating alcoholic fatty liver as recited in any one of (7) to

(11) , wherein at least 2 wt% of the constituent fatty acids in the krill oil is eicosapentaenoic acid

(13) An agent for preventing or treating alcoholic fatty liver as recited in any one of (7) to

(12) , wherein at least 1 wt% of the constituent fatty acids in the krill oil is docosahexenoic acid.

(14) An agent for preventing or treating alcoholic fatty liver as recited in any one of (1 ) to

(13) , which is for administering the lipid to a subject at a dosage of 1 to 100 mg kg body weight/day.

(15) An agent for preventing or treating alcoholic fatty liver as recited in any one of (7) to (13), which is for administering krill oil to a subject at a dosage of 1 to 100 mg kg body weight/day.

[0009]

(16) A method for preventing or treating alcoholic fatty liver, which comprises administering a human or a non-human animal with an agent for preventing or treating alcoholic fatty liver as recited in any one of (1) to (15).

(17) Use of a phospholipid comprising a highly unsaturated fatty acid as a constituent fatty acid in the manufacture of a medicine for treating alcoholic fatty liver as recited in any one of (l) to (15).

(18) A food or beverage, a feed or a medication containing an agent for preventing or treating alcoholic fatty liver as recited in any one of (1 ) to (15). (19) A food or beverage or a feed as recited in (18) for permitting ingestion of the lipid in an amount of 100 to 4500 mg per application,

(20) A food or beverage or a feed as recited in (18) for permitting ingestion of krill oil in an amount of 100 to 4500 mg per application.

ADVANTAGEOUS EFFECTS OF INVENTION

[0010]

If, for example, krill oil is administered orally according to the present invention, fatty liver, in particular, alcoholic fatty liver which can become serious enough as the result of alcohol intake can be prevented or treated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]

Fig. 1 is a graph showing changes in body weight for the groups tested in Example 1 .

Fig. 2 is a graph comparing the kidney and testis peripheral fat tissue to body weight ratios among the groups tested in Example 1.

Fig, 3 is a graph comparing the liver to body weight ratios among the groups tested in Example I ,

Fig. 4 is a graph comparing the T. CHO levels in the liver among the groups tested in Example 1.

Fig. 5 is a graph comparing the TG levels in the liver among the groups tested in Example 1 .

Fig, 6 is a graph comparing the severity of liver steatosis among the groups tested in Example 1.

Fig. 7 is a set of photos showing pathological sections (representative samples) depicting the severity of liver steatosis for the groups tested in Example 1.

Fig. 8 is a graph comparing the blood ALT concentrations among the groups tested in Example 1 . Fig. 9 is a graph comparing the blood AST^' concentrations in the groups tested in Example 1.

Fig. 10 is a graph comparing the T. CHO levels in the liver among the groups tested in Example 2.

Fig. 11 is a graph comparing the TG levels in the liver among the groups tested in Example 2.

Fig. 12 is a graph comparing the severity of liver steatosis among the groups tested in Example 2.

[0012]

The data for the Example 1 are shown graphically as MeaniSE. Significant differences were all calculated with the T test and indicated as follows:

$) p < 0.05, $$) p < 0.01, $$$) p < 0.001 : vs. ND group

#) p < 0.05, ##) p < 0.01, ###) p < 0.001 : vs. HFD group

*) p < 0.05, **) < 0.01 , ***) p < 0.001 : vs. Cont group

&) p < 0.05: vs. KO-M group

¥) p < 0.05, ¥¥) p < 0.01 : vs. F+S-M group

The data for the Example 2 are also shown graphically as Mean±SE. Significant differences were all calculated with the T test and indicated as follows:

) p < 0.05, $$) p < 0.01, $$$) p < 0.001 : vs. ND group

#) p < 0.05, ##) p < 0.01 , ###) p < 0.001 : vs. Cont. l group

ρ < 0.1 , *) p < 0.05, **) p < 0,01 : vs. Cont,2 group

6) p < 0.05: vs. Cont. 3 group

DESCRIPTION OF EMBODIMENTS

[0013]

The krill oil to he used in the present invention is a lipid derived from krill. Krill is an arthropod belonging to the phylum Arthropoda, subphylum Crutacea, class Maiacostraca, and examples include arthropods belonging to the phylum Arthropoda, subphylum Crutacea, class Malacostraca, order Eucarida, family Euphausiacea, such as antarctic krill (Euphausiasuperb), and arthropods belonging to the phylum Arthropoda, subphylum Crutacea, class Malacostraca, order Peracarida, family Mysidae, such as mysids caught in the seas around Japan. It should, however, be noted that antarctic krill is particularly preferred from the viewpoints of stability in catch and uniformity in the qual ity of the lipid component.

[0014]

The krill-derived lipid to be used in the present invention can be obtained by known production methods. For example, it can be produced by referring to the known methods described in WO2000/023546, WO2009/027692, WO2010/035749, WO2010/035750, etc,

Krill oil can be obtained by extraction with suitable organic solvents from krill either as such or in the form of a dried product or thermal coagulum, typically in accordance with the methods described in the international publications identified above. Suitable organic solvents include, for example, alcohols such as methanol, ethanol, propanol, isopropanol, hutanol, propylene glycol, and butylenes glycol, as well as methyl acetate, ethyl acetate, acetone, chloroform, toluene, pentane, hexane, and cyclohexane, which may be used either alone or in combination of two or more species.

The krill-derived thermal coagulum mentioned above can be obtained in the following manner: dried krill, milled krill, raw krill or frozen krill, either in whole or in part, is pressed into a liquid form and the resulting pressed liquid is heated to separate a solid content from water-soluble components. The pressing operation can be performed with commonly employed apparatuses, including a hydraulic press, a screw press, a meat and bone separator, a press dehydrator, a centrifuge, and combinations thereof.

[0015]

The pressed liquid may be heated under an atmospheric, superatmospheric, or subatmospheric pressure at 50 °C or higher, preferably at 70-150 °C, more preferably at 85-110 °C. Upon this heating, a solid content (thermal coagulum) is separated from water-soluble components and filtered, centrifuged or otherwise treated to give a thermal coagulum. The coagulum may be dried as appropriate before use. If the drying temperature is unduly high, the lipid is oxidized to give off a malodor; hence, drying is typically performed at 90 °C or below, preferably at 75 °C or below, more preferably at 55 °C or below. Drying is a preferred process because it allows removal of volatile impurities.

The thermal coagulum of the pressed liquid from krill or the dried product thereof may be washed with water to remove water-soluble impurities. Washing with water can be performed using freshwater or seavvater in a volume at least four times, preferably at least ten times, the weight of the dr content in the thermal coagulum or the dried product thereof. Preferably, washing is done at least twice, more preferably at least three times.

A phospholipid fraction can be obtained from the above-described thermal coagulum or its dried product or the washings thereof. For example, these materials may be treated with a solvent such as ethanol, hexane, chloroform or acetone to extract an oil, which is then subjected to chromatography using silica gel or the like to thereby obtain a phospholipid fraction. Phosphatidylcholine is abundant in this fraction.

In krill oil, a phospholipid containing a polyvalent unsaturated fatty acid as a constituent fatty acid is contained as an active ingredient.

[0016]

Phospholipids are known as a major component of ceil membranes and have a hydrophilic phosphate part and a hydrophobic fatty acid part. Phospholipids are divided into two types: diacylgiycerophospholipids and lysoacylglycerophospholipids. Diacylglycerophospholipids have the fatty acid part at 1- and 2-positioins on the glycerol backbone. Lysoacylglycerophospholipids are divided into 1 -acy lglycerolysophospholipids having the fatty acid part, only at 1 -position on the glycerol backbone and 2-acylglycerolysophospholipids having the fatty acid part only at 2 -position on the glycerol backbone. The phospholipids as referred to herein embrace all of these substances, with diacylgiycerophospholipids being particularly preferred. The diacylgiyicerophospholipids contained in krill oil are mainly composed of phosphatidylcholine (PC). The content of phospholipids in krill oil is typically 5-80 wt%, with the range of 30-60 wt% being particularly preferred. The krill oil to be used in the present invention preferably contains at least 10 wt%, more preferably at least 25 wt%, and even more preferably at least 35 wt%, of phospholipids.

[0017]

Krill oil has highly unsaturated fatty acids as the fatty acid part. The highly unsaturated fatty acids as referred to herein are fatty acids having at least three double bonds and containing at least 18 carbon atoms, preferably having at least four double bonds and containing at least 20 carbon atoms. Preferred highly unsaturated fatty acids are n-3 highly unsaturated fatty acids and specific examples include a-linolenic acid (18:3, ALA), eicosapeniaenoic acid (20:5, EPA), docosapentaenoic acid (22:5, DPA), and docosahexaenoic acid (22:6, DHA), with EPA, DPA and DHA being preferred.

The n-3 highly unsaturated fatty acids typically account for 5-60 wt%, preferably 10-50 wt%, more preferably 10-30 wt%, of the constituent fatty acids in krill oil. For the purpose of the present invention, a preferred value is at least 5 wt%, more preferably at least 10 wt%, and even more preferably at least 15 wt%. in particular, krill oil containing at least 2 wt%, preferably at least 10 wt%, of eicosapentaenoic acid and/or at least 1 wt%, preferably at least 3 wt%, of docosahexaenoic acid is desirably used. In order to increase the concentration of n-3 highly unsaturated fatty acids in kril l oil, concentrating and refining may be performed by known methods. The concentration and purity of lipids can be adjusted to any values by using techniques such as concentrating, extracting and/or refining and blending that are commonly known in the art concerned.

The composition to be used in the present invention may contain other components of krill oil, as exemplified by astaxanthin and sterols,

[0018] The agent for preventing or treating alcoholic fatty liver according to the present invention contains an effective amount of a lipid or krill oil. The term "effective amount" as used herein refers to the amount required for preventing or treating alcoholic fatty liver and, in the case of krill oil, may he illustrated by 1-500 mg/kg, preferably 1-100 mg, and even more preferably 5-100 mg/kg per day. In the case of human subjects, the effective amount of krill oil is preferably 1 -4,500 mg per day. Specifically, the agent may be formulated as a capsule or supplement that enables 100-4,500 mg, preferably 200-3,000 mg, and more preferably 300-2,000 mg, of krill oil to be ingested per day. This also applies to lipids other than krill oil. Since krill oil features high safety, it may be ingested in large amounts but if its intake is excessive, undesirable effects will result, such as retarded absorption, dyspepsia, indigestion, and loss of appetite. The amounts of ingestion indicated above may refer to a single intake or multiple, for example, two or three intakes.

Administration of the agent for preventing or treating alcoholic fatty liver according to the present invention contributes to improving symptoms of fatty liver due to alcohol intake, such as an increased liver weight, elevated total cholesterol and triglyceride levels in the liver, and fat accumulation in the liver.

The present invention can be used for human and other animals. Animals, other than "humans" as referred to in this specification, are such as cows, horses, pigs, sheep, goats, mules, camels, llamas, Asian elephants, alpacas, reindeer (caribou), zebras (zebu), water buffalo, yaks, guinea pigs, hares (rabbits), minks, chicken, ducks, geese, turkeys, Muscovy ducks, quails, ostriches, feral pigeons, pheasants, cormorants, dogs, cats, hamsters, guinea pigs, ferrets, squirrels, monkeys and the like; fish: sea bream, tuna, rudderfish, goldstriped amberjack, jack mackerel, chub mackerel, Japanese seaperch, eel, halibut, olive flounder, globefish, salmon, trout, catfish, sea bass, barramimdi, cobia, tilapia, and the like.

[0019]

The agent for preventing or treating alcoholic fatty liver according to the present invention may optionally contain known other ingredients capable of improving functions of the liver, as exemplified by antioxidants, vitamins, and amino acids. Exemplary antioxidants include tocopherol, dried yeasts, glutathione, lipoic acid, quercetin, catechin, coenzyme Q10, enzogenol, proanthocyanidins, anthocyanidin, anthocyanin, carotenes, lycopene, flavonoid, resveratrol, isoflavones, zinc, melatonin, ginkgo leaf, Alpiniazerumbet leaf, hibiscus, and extracts thereof.

The agent for preventing or treating alcoholic fatty liver according to the present invention may be prepared in forms suitable as pharmaceutical compositions, functional foods, health foods, supplements and the like; examples include various solid formulations such as granules (including dry syrup), capsules (soft and hard capsules), tablets (including chewable tablets and the like), powders (dusts), and pills; liquid formulations such as liquids for internal use (including liquids, suspensions, and syrups.)

Examples of additives that may be used to prepare formulations include excipients, lubricants, binders, disintegrants, fluidizatioii agents, dispersaiits, wetting agents, antiseptics, viscous agents, pH adjusting agents, colorants, corrigents, surfactants, and solvent promoters. To prepare liquids, thickening agents such as pectin, xanthan gum, and guar gum may be incorporated. If desired, coating agents may be used to formulate coated tablets or gelatin pastes. Even other forms of preparation may be produced in accordance with conventional methods. If desired, the agent for preventing or treating alcoholic fatty liver according to the present invention may also be ingested together with various foods and beverages (feed in case of animals).

[0020]

On the following pages, examples of the present invention are described but should in no ways be taken as limitings.

EXAMPLES EXAMPLE 1

[0021]

Effect against Alcohol-Induced Fatty Liver Formation (Preventive Effect) SD rats (male, 6-7 weeks old at the first dose) were given a high fat diet and alcohol to induce alcoholic fatty liver, which were subjected to tests for comparing the effects of krill oil, refined fish oil, and soy lecithin on fatty liver formation.

Test animal: SD rats (male, 6-7 weeks old, 12/group). Animals were purchased, acclimated, and randomized into different groups according to body weights prior to administration of test articles,

Diets: Two kinds of diets were used, a normal diet (ND) and a high fat diet (HFD), ND was of a SPF grade; HFD was a modified ND with no vitamin B₁₂ and methionine supplemented and with additional lard supplemented in amounts that the total lipid content was in the range of 20 to 25 wt%. The ingredients of the respective diets are shown in Table 1. The test animals were allowed to ingest the diets ad libitum.

Test articles: krill oil (manufactured by Nippon Suisan Kaisha, Ltd. under trade name Nissui Krill Oil); refined fish oil (commercially available under trade name Fish Oil); soy lecithin (commercially available under trade name Lecithin, from soybean); placebo/vehicle (commercial product, MCT). The ingredients of the respective test articles are shown in Table 2.

Dosage and duration: There were nine test groups as shown in Table 3 and the test articles were orally administered via a gastric tube once daily for 6 weeks at the dosages indicated in Table 3 after being formulated with MCT to reach a dose volume of 1 ml/kg. Starting on the first day of the administration of each test article, alcohol and glucose in water (dose volume: 10 ml/kg) were administered via a gastric tube in two divided doses per day at the dosages indicated in Table 4, The first dose on each day was given at least one hour after the administration of each test article, and the second dose was given at least four hours apart from the first dose.

[0022]

Evaluations:

* General observations: twice daily « Detailed observations: once weekly

* Body weights and food consumption: 3 times weekly

* Serum chemistry data of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) were collected prior to dosing initiation and 6 weeks post-dose.

* On the second day of the final dose, the rats were necropsied, liver weights and kidney/testis peripheral fat weights were collected, and liver tissues were collected for liver total cholesterol (T. CHO) and triglyceride (TG) measurements and histopathoiogical evaluation of fatty liver.

The severity of fatty liver was determined according to the following criteria:

-) the hepatocytes with fat vesicles were only occasional observed

+) the hepatocytes with fat vesicles were below 1/3

++) the hepatocytes with fat vesicles were between 1/3 to 2/3

+++) the hepatocytes with fat vesicles were above 2/3

[0023]

Table 1

Ingredients Normal Diet High Fat Diet

(wt%) (ND) (HFD)

Water 9.8 6.82

Protein 25.1 18.2

Lipid 6.6 22.52

Fiber 3.5 -

Ash 7.15 3.48

Calcium 1.29 0.55

Phosphorus 0.85 0.4 [0024]

Table 2

N.B.) As calculated

[0025]

Table 3

Groups Diet and Alcohol Test/Control article treatment

Diet Alcohol Test/Control article Dose

(mg/kg)

ND ND MCT 0 HFD HFD MCT 0

Contl HFD MCT 0

KO-L1 HFD - - Krill oil 10

KO-M 1 HFD Kril! oil 100

KO-H l HFD + Krill oil 1 ,000

FO-M HFD + F-Oil 100

S-M HFD + Soy Lecithin 100

F+S-M HFD F-Oil + Soy Lecithin 50 + 50

[0026]

Table 4

[0027]

Results:

The administration of alcohol caused a temporary decrease in the food consumption during 4 to 5 weeks after the first dose but thereafter all groups acclimated to alcohol administration well enough, with no apparent effect on their body weight increase (Fig. 1 ,)

As shown in Table 1, the high fat diet (HFD) used in the experiment contained higher level of fat as compared to the normal diet (22.52% vs. 6.6%). This high fat diet intake induced a significant increase in the kidney/testis peripheral fat tissue weights but had no effect on the fat content in the liver (Figs. 2 and 3.) In contrast, the administration of alcohol was found to increase the liver to body weight ratio significantly. This increase in the liver to body weight ratio could be significantly suppressed by administration of krill oil (Fig. 3) but not by fish oil, soy lecithin or their mixture.

The use of the high fat diet elevated the T. CHO level in the liver significantly. This increase could be significantly suppressed only in the krill oil treated groups (Fig. 4.)

The elevated TG level in the liver due to the intake of the high fat diet was further increased significantly after administration of alcohol. This increase could be significantly suppressed only in the krill oil treated groups, with complete elimination of the effect of alcohol in the high dose group (Fig. 5.)

Tissue sections of the test animals were histopathologically evaluated for fat vesicles in the liver (i.e., excessive fat accumulation generally seen as a sign of fatty liver). The severity of fatty liver increased significantly after high fat diet intake and alcohol administration, and the increased severity reduced significantly after administration of krill oil (Figs. 6 and 7), Fatty liver, if not treated appropriate, will progressed gradually to alcoholic hepatitis, liver fibrosis and cirrhosis. As shown in the experiment, fat accumulation in the liver was confirmed in the Cont. l group while no sign of fibrosis was noted. Thus, the fatty liver induced in the rats in this experiment represented the early stage of alcoholic liver disease.

[0028]

The ALT level, as a blood biomarker for liver function, increased significantly after alcohol administration for the Cont. l group. There were no apparent differences among the Cont. l group and the test article treated groups (Fig. 8). There was no apparent change in the AST level after alcohol administration for the Cont.l group. There were no apparent differences among the Cont.l group and the test article treated groups (Fig. 9). These findings showed: (1 ) the administration of alcohol and high fat diet intake for 6 continuous weeks caused characteristic fatty liver related changes including significant increase in liver to body weight ratio, T. CHO and TG levels in the liver, as well as the severity of fatty liver in histopathologica! evaluation; (2) the administration of foil! oil had a suppressive effect on the fatty liver development characterized as reversing the fatty liver related changes listed above; (3) however this suppressive effect was not noted in the groups treated with either fish oil or soy lecithin, indicating the preventive effect on alcoholic fatty liver is attributed to the ingredient of phospholipid-boimd n-3 HUFA contained in krill oil. EXAMPLE 2

[0029]

Therapeutic Effect on Alcohol -Induced Fatty Liver

SD rats (male, 7-8 weeks old at modeling initiation) were given a high fat diet and alcohol to induce alcoholic fatty liver, which were subjected to tests for verifying die therapeutic effect of krill oil on the fatty liver.

Test animal: SD rats (male, 7-8 weeks old, 10/group). Animals were purchased, acclimatized, and randomized into different groups according to body weights prior to administration of test articles.

Diets: As in Example 1 , two kinds of diets with the ingredients shown in Table 1 were used. The test animals were allowed to ingest the diets ad libitum.

Test articles: krill oil and placebo/vehicle (MCT), which were the same as in Example I . Dosage and duration: There were eight test groups as shown in Table 5 and the test article were orally administered via a gastric tube once daily for 4 weeks (from 7^th to 10*" week) at the dosages indicated in Table 5 after being formulated with MCT to reach a dose volume of 1 ml/kg. From 1 to 6 weeks or from 1 to 10 weeks, alcohol and glucose in water (dose volume: 10 ml/kg) were administered via a gastric tube in two divided doses per day at the dosages indicated in Table 6. The first dose on each day was given at least one hour after the administration of test article, and the second administration was given at least four hours apart from the first dose.

Evaluations:

* General observations: twice daily

* Detailed observations: once weekly

* Body weights and food consumption: 3 times weekly

* On the second day of the final dose, the rats were necropsied, liver weights and kidney/testis peripheral fat weights were collected, and liver tissues were collected for liver total cholesterol (T. CHO) and triglyceride (TG) measurements and histopathoiogical evaluation of fatty li ver.

[0030]

Table 5

Notes:

Cont.1 : Same as Cont, 1 in Example 1 , test was completed at 6 weeks after the addition of HFD and alcohol. Cont.2: Same as Cont. l from 1 to 6 weeks, no alcohol administration in the subsequent

7- 10. weeks,

[0031]

Table 6

Weeks ND Cont. l Cont.2 to KO-H2 Cont.3 to KO-H3

1 30% Glu 8% Ale + 30% Glu 8% Ale + 30% Glu 8% Ale + 30% Glu

9 20% Glu 12% Ale + 20%) Glu 12% Ale + 20% Glu 12%) Ale + 20% Glu

3 15% Glu 20% Ale + 15% Glu 20% Ale + 15% Glu 20% Ale + 15% Glu

4 10% Glu 30% Ale + 10% Glu 30% Ale + 10% Glu 30% Ale + 10% Glu

5-6 10% Glu 40% Ale + 10% Glu 40% Ale + 10%, Glu 40%) Ale + 10% Glu

7-10 10%, Glu 10%) Glu 40% Ale + 10% Glu

[0032]

Results:

The intake of the high fat diet elevated the T. CHO level in the liver significantly. In contrast, the degree of alcohol administration did not exert any further effect on the accumulation of cholesterols. The group administered with the higher dose of krill oil had a significant decrease in the elevated cholesterol level (Fig. 10.)

The high fat diet intake and the 6-week administration of alcohol increased the TG level in the liver significantly but no further elevation was noted upon continued alcohol administration up to lO"¹ week. When the alcohol administration was stopped, the TG level in the liver spontaneously returned to the normal and this process was accelerated by krill oil treatment. In the group continuously administered with alcohol, the use of krill oil caused significant decrease in the TG level in the liver (Fig. 11). liistopathol ogi cal e valuation of tissue sections of the liver showed t he simil ar results as in Example 1; the administration of krill oil decreased the severity of fatty liver (Fig. 12.)

These findings showed that krill oil not only prevents the formation of alcoholic fatty liver but also had a therapeutic effect on the fatty liver.

INDUSTRIAL APPLICABILITY

[0033]

The present invention can provide compositions that have efficacy for preventing and ameliorating alcoholic fatty liver and which are safe to be ingested for an extended period of time.

Claims

1. An agent for preventing or treating alcoholic fatty liver which contains a lipid as an active ingredient, the lipid comprising a highly unsaturated fatty acid as a constituent fatty acid.

2. An agent for preventing or treating alcoholic fatty liver according to claim 1 which contains a phospholipid as the lipid.

3. An agent for preventing or treating alcoholic fatty liver according to claim 1 or 2, wherein the lipid consists of a phospholipid comprising a highly unsaturated fatty acid as a constituent fatty acid.

4. An agent for preventing or treating alcoholic fatty liver according to any one of claims 1 to 3, wherein the highly unsaturated fatty acid is an n-3 highly unsaturated fatty acid.

5. An agent for preventing or treating alcoholic fatty liver according to claim 4, wherein the n-3 highly unsaturated fatty acid is eicosapentaenoic acid, docosapentaenoic acid or docosahexenoic acid.

6. An agent for preventing or treating alcoholic fatty liver according to any one of claims 2 to 5, wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatide acid, phosphatidylglyceroi, and phosphatidylinositol.

7. An agent for preventing or treating alcoholic fatty liver according to any one of claims 1 to 6, wherein the lipid is krill oil.

8. An agent for preventing or treating alcoholic fatty liver according to claim 7, wherein the krill oil is refined krill oil.

9. An agent for preventing or treating alcoholic fatty liver according to claim 7 or 8, wherein the krill oil is produced via a thermal coagulum of krill.

10. An agent for preventing or treating alcoholic fatty liver according to any one of claims 7 to 9, wherein the krill oil contains at least 10 wt% of phospholipid.

1 1 . An agent for preventing or treating alcoholic fatty liver according to any one of claims 7 to 10, wherein at least 5 wt% of the constituent fatty acids in the krill oil is an n-3 highly unsaturated fatty acid.

12. An agent for preventing or treating alcoholic fatty liver according to any one of claims 7 to 11, wherein at least 2 wt% of the constituent fatty acids in the krill oil is eicosapentaenoic acid.

13. An agent for preventing or treating alcoholic fatty liver according to any one of claims 7 to 12, wherein at least 1 wt% of the constituent fatty acids in the krill oil is docosahexenoic acid.

14. An agent for preventing or treating alcoholic fatty liver according to any one of claims 1 to 13, which is for administering the lipid to a subject at a dosage of 1 to 100 mg/kg body weight/day.

15. An agent for preventing or treating alcoholic fatty liver according to any one of claims 7 to 13, which is for administering krili oil to a subject at a dosage of 1 to 100 mg/kg body weight/day.

16. A method for preventing or treating alcoholic fatty liver, which comprises administering a human or a non-human animal with an agent for preventing or treating alcoholic fatty liver according to any one of claims 1 to 15.

17. Use of a phospholipid comprising a highly unsaturated fatty acid as a constituent fatty acid in the manufacture of a medicine for treating alcoholic fatty liver according to any one of claims 1 to 15.

18. A food or beverage, a feed or a medication containing an agent for preventing or treatmg alcoholic fatty liver according to any one of claims 1 to 15.

19. A food or beverage or a feed according to claim 18 for permitting ingestion of the lipid in an amount of 100 to 4500 mg per application.

20. A. food or beverage or a feed according to claim 18 for permitting ingestion of krill oil in an amount of 100 to 4500 mg per application.