Oil composition, formulations comprising the oil composition, and the use thereof to reduce accumulation of visceral fat, improve glucose tolerance, and prevent or treat obesity related diseases and disorders

Abstract

This invention relates to an oil composition, preferably obtained from a copepod, and the use thereof to reduce accumulation of visceral fat and counteract impairment of heart function caused by obesity inducing Western diets. The oil composition of the present invention can thus be used to reduce abdominal obesity and improve glucose tolerance and thus to reduce the risk of obesity related diseases such as but not limited to type 2 diabetes or cardiovascular disease, or to prevent or treat such diseases.

Claims

1. A method for reducing the risk of contracting obesity and type-2 diabetes or treatment of abdominal obesity and type 2 diabetes comprising administering to a patient in need thereof a clinically effective dosage of a composition comprising 20-100% by weight of wax esters isolated from a copepod of the genus Calanus, wherein said wax esters consist of monoesters of mono- or polyunsaturated C16 to C22 fatty acids and monounsaturated C16 to C22 fatty alcohols, and wherein said fatty acids are comprised of 5-20% by weight of stearidonic acid (SDA).

2. A method according to claim 1, comprising administering to a patient in need thereof a clinically effective dosage of composition comprising 40-85% by weight of wax esters.

3. A method according to claim 1, comprising administering to a patient in need thereof a clinically effective dosage of composition wherein 70% or more of the monounsaturated fatty alcohols of the wax esters are C16:1, C20:1 and C22:1.

4. A method according to claim 1, comprising administering to a patient in need thereof a clinically effective dosage of composition wherein 70% or more of the fatty alcohols of the wax esters are monounsaturated, and wherein 50% or more of the fatty acids of the wax ester are either mono- or polyunsaturated n-3 fatty acids.

5. A method according to claim 1, comprising administering to a patient in need thereof a clinically effective dosage of a composition comprises 3-20% by weight of eicosapentaenoic acid (EPA) and 2-15% by weight of docosahexaenoic acid (DHA).

6. A method according to claim 5, comprising administering to a patient in need thereof a clinically effective dosage of a composition comprises fatty alcohols and SDA, DHA and EPA as monoesters.

7. A method according to claim 1, comprising administering to a patient in need thereof a clinically effective dosage of composition containing 500-4000 ppm of astaxanthin.

8. A method according to one of claims 1 to 7, wherein the clinically effective dosage is a daily amount of 0.5 to 5 g of the oil composition.

9. A method according to claim 8, comprising administering to a patient in need thereof a clinically effective dosage of a composition isolated from the copepod species Calanus finmarchicus.

10. A method according to one of claim 1 to 4 or 7, comprising administering to a patient in need thereof a clinically effective dosage of a composition, wherein the composition is provided as a pharmaceutical composition in capsules, tablets, emulsions or tonics.

11. A method according to claim 10, comprising administering to a patient in need thereof a clinically effective dosage of a composition further comprising one or more pharmaceutically acceptable additives selected from the group consisting of adjuvants, stabilizers, antioxidants, emulsifiers, surfactants and carriers.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows total perirenal fat as a function of body weight in rats fed low fat reference diet (Control), obesity inducing high fat diet (HF) and obesity inducing high fat diet containing 1.5% by weight of the oil composition of the present invention, CO (Copepod Oil) (HF+1.5% CO) (n=10).

(2) FIG. 2 shows glucose oxidation capacity (as mol/min/g dry weight) of heart muscle tissue of rats fed low fat reference diet (Control), obesity inducing high fat diet (HF) and obesity inducing high fat diet containing 1.5% by weight of the oil composition of the present invention (HF+1.5% CO). Values are mean95% C.I. (n=8, 6 and 6 for Control, HF and HF+1.5% CO, respectively).

(3) FIG. 3 (A) shows the blood glucose levels during intraperitoneal glucose tolerance test in mice given the control diet (Control), high fat diet (HF) and high fat diet containing 1.5% by weight of the oil composition of the present invention (HF+1.5% CO), respectively.

(4) FIG. 3 (B) shows the area under the curve of blood glucose levels during intraperitoneal glucose tolerance test in mice of the same groups shown in FIG. 3 (A). Data are expressed as meanS.E. *P<0.05 for HF vs. Control; #P<0.05 for HF+1.5% CO vs. HF.

DETAILED DESCRIPTION OF THE INVENTION

(5) The following term shall herein have the meaning as indicated below unless otherwise specifically stated:

(6) Wax esters: Wax esters are esters of long-chain or very long-chain acids (fatty acids) and long-chain or very long-chain alcohols (fatty alcohols). Long-chain in this context means from 14 to 22 carbons, whereas very long-chain refers to 24 or more carbon atoms. Wax esters are important component in waxes. Usually, the term wax refers to a wide class of lipids characterized by being solids at room temperature, e.g. when it looks like honeycomb material or bees-wax. However, waxes may be both solid and liquid. They are produced by animals (beeswax, wool wax (lanolin), sperm whale wax and orange roughy oil) and plants (candelilla, carnauba, rice-bran, sugar cane (policosanol) and jojoba. All leaf surfaces and many vegetables and fruits are covered by a microcrystalline layer of wax. Waxes are used in the food, pharmaceutical and cosmetic industries for the protection of surfaces.

(7) Wax esters of the present invention are monoesters of long-chain unsaturated fatty alcohols and long-chain unsaturated fatty acids, notably omega-3 fatty acids, and the oil composition of the present invention is completely soluble and free-flowing at room temperature.

(8) It has been shown that the oil composition of the present invention, used as a minor supplement in a high-fat, obesity inducing Western type of diet, specifically inhibits visceral fat accumulation induced by such a diet. Moreover, it has been shown that the oil composition of the present invention can counteract impairment of heart function induced by such a diet. The oil composition of the present invention can in other words be used to counteract the most harmful effects of obesity inducing Western diets.

(9) The oil composition of the present invention has been obtained from the marine copepod Calanus finmarchicus. The chemical composition of this oil differs markedly from that of other oils, and it was investigated if it would differ also regarding the possible biomedical responses it may elicit. However, there is nothing in the prior art that would lead a person skilled in the art to consider it likely that inclusion of as little as 1.5% (w/w) of the present oil composition in an obesity inducing high-fat (24% w/w) Western type of diet would result in a statistically highly significant reduction of visceral fat accumulation in rats, without affecting growth or deposition of fat in other adipose tissues. Moreover, and equally unexpected, the present oil composition reduced the visceral fat accumulation in rats fed on this high-fat diet to the same level as in rats fed on the low-fat (4% w/w) diet used as the reference diet, not inducing obesity. It is also evident that the present oil composition may counteract deterioration of healthy heart function typically seen in obesity induced type 2 diabetes, as demonstrated by high cardiac glucose metabolism even after long time feeding on an obesity inducing high-fat diet.

(10) The n-6/n-3 proportion of fatty acids in dietary lipids affects the pattern of fat accumulation in the body. Diets in which this proportion is high (e.g. >5/1), as in high-fat Western diets, have a stronger tendency to induce central obesity and accumulation of visceral fat than diets with a lower n-6/n-3 ratio. For comparison, since the oil composition of the present invention constituted only a minor fraction of an obesity inducing high-fat Western type of diet, the n-6/n-3 fatty acid ratio was still as high as 5/1 in our experimental diet.

(11) The oil composition of the present invention contains the n-3 fatty acids EPA (eicosapentaenoic acid=C20:5n-3) and DHA (docosahexaenoic acid=C22:6n-3), like other marine oils. However, the level of EPA and DHA is somewhat lower than in fish oil and krill oil (Table 1), whereas it is rich in stearidonic acid (SDA=C18:4n-3). Nevertheless, it might be argued that the observed inhibition of diet induced visceral obesity could be due to the presence of EPA and DHA. However, it should be borne in mind that wax esters, including marine wax esters, are digested very slowly compared to triglycerides and phospholipids and that EPA and DHA in the wax ester is not readily available for absorption as free acid. In fact, it is a common understanding among the skilled in the field that wax esters are not digestible, and that a marine wax ester rich oil like that from Calanus finmarchicus therefore is a poor source of EPA and DHA. Moreover, in the experiments of Belzung et al. (1993) described above, the amount of omega-3 consumed by the animals was in the range of 0.7-1.4 grams daily. By comparison, the oil composition of the present invention contributed with only 0.06 gram omega-3 daily to rats feeding on the obesity inducing high-fat diet. Accordingly, it can be concluded that the inhibition of visceral fat accumulation shown in the present invention neither can be ascribed to omega-3 as in the experiments of Belzung et al., nor can it be due to change in the ratio of n-6/n-3 fatty acids in a more healthy direction.

(12) The oil composition according to the present invention can be derived from marine copepods, preferably a copepod of the genus Calanus, such as Calanus finmarchicus. Freshly harvested, frozen/thawed or dehydrated raw material can be used as the raw material for obtaining the oil compositions, using any method known by the skilled in the art, such as but not limited to, conventional fish oil production technology, biotechnological methods, organic solvents or supercritical fluid extraction, and cold pressing. Independent of the procedure of obtaining the oil and the yield of oil, the typical gross composition will be as shown in Table 1.

(13) TABLE-US-00001 TABLE 1 Typical chemical composition of three different marine oils: (A) Copepod oil from Calanus finmarchicus caught in Norwegian waters, (B) cod liver oil from Atlantic cod Gadus morhua, and (C) krill oil from Euphausia superba caught in the Southern ocean, given in mg/g oil. Lipid classes and selected fatty acids A.sup.1 B.sup.2,4 C.sup.3,5 Triglycerides 10 955 260 Free fatty acids 90 14 13 Saturated fatty acids 160 160 300 Monounsaturated fatty aids 120 385 300 Polyunsaturated fatty acids 250 475 387 n-3 fatty acids 240 395 332 n-6 fatty acids 10 50 55 18:4 n-3 fatty acid (SDA) 86 21 51 20:5 n-3 fatty acid (EPA) 67 72 128 22:6 n-3 fatty acid (DHA) 49 188 101 Wax esters 670 0 0 Polar lipids <50 18 670 Neutral lipids >750 967 310 .sup.1Copepod oil produced by Calanus AS (www.calanus.no). .sup.2From Falch, et al., Process Biochemistry 2006; 41: 666-674 .sup.3From Phleger, et al. Comparative Biochemistry and Physiology Part B 2002; 131: 733-747 .sup.4From Standal et al., J. Am Oil Chem Soc 2008; 85: 105-112 .sup.5From Hagen et al., Marine Biology 2001; 139: 95-104

(14) Besides the notable difference in gross chemical composition, the three marine oils used here for illustration purposes, are different also in their content of n-3 fatty acids (Table 1).

(15) To illustrate the uniqueness of the oil composition according to the present invention, the corresponding compositions of conventional cod liver oil and hill oil are shown for comparison. It is evident from gross chemical analysis that these oils are highly different, in particular regarding their contents of wax esters, triglycerides, phospholipids, and of astaxanthin (not shown). It should be noted that wax esters constitute the major lipid component in the copepod oil of the present invention, unlike both cod liver oil and krill oil.

(16) The most noteworthy difference in fatty acid composition between the three oils is the higher stearidonic acid (SDA), but lower docosahexaenoic acid (DHA) content in the copepod oil.

(17) In the oil composition of the present invention, SDA, EPA and DHA exist to a large extent as esters with unsaturated long-chain fatty alcohols. In conclusion, the copepod oil of the present invention differs markedly from typical fish oil and krill oil in both gross chemical composition and fatty acid content. It is, however, similar to other marine oils in its overall level of n-3 fatty acids.

(18) A typical composition of wax esters and fatty alcohols/fatty acids combinations in an oil composition of the present invention is shown in Table 2.

(19) TABLE-US-00002 TABLE 2 Typical composition of wax esters and fatty alcohol/fatty acid combinations (% (w/w)) in copepod oil derived from Calanus finmarchicus..sup.1 Wax Major fatty Minor fatty ester alcohol/fatty acid alcohol/fatty acid % (w/w) 30:1 14:0/16:1 16:1/14:0 0.8 32:1 16:0/16:1 14:0/18:1 1.9 32:2 16:1/16:1 14:0/18:2 0.6 32:4 14:0/18:4 16:0/16:4 0.9 34:1 16:0/18:1 14:0/20:1 20:1/14:0 17.6 34:2 16:0/18:2 16:1/18:1 0.9 34:3 16:0/18:3 16:1/18:2 34:4 16:0/18:4 16:1/18:3 2.7 34:5 14:0/20:5 16:1/18:4 0.4 36:1 20:1/16:0 16:0/20:1 22:1/14:0 21.9 36:2 20:1/16:1 16:1/20:1 2.3 36:5 16:0/20:5 20:1/16:4 1.1 36:6 16:1/20:5 14:0/22:6 0.3 38:1 22:1/16:0 16:0/22:1 2.8 38:2 22:1/16:1 20:1/18:1 3.9 38:3 20:1/18:2 22:1/16:2 0.4 38:4 20:1/18:3 22:1/16:3 0.9 38:5 20:1/18:4 22:1/16:4 5.4 38:6 16:0/22:6 16:1/22:5 40:2 20:1/20:1 22:1/18:1 5.9 40:3 22:1/18:2 0.7 40:5 22:1/18:4 20:1/20:4 4.7 40:6 20:1/20:5 1.5 42:2 22:1/20:1 20:1/22:1 12.7 42.6 22:1/20:5 20:1/22:5 1.5 42:7 20:1/22:6 2.0 44:2 22:1/22:1 4.9 44:7 22:1/22:6 0.6 .sup.1Compiled from Graeve, M. and Kattner, G. Species-specific differences in intact wax esters of Calanus hyperboreus and C. finmarchicus from Fram Strait - Greenland Sea. Marine Chemistry 1992; 39: 269-281.

(20) The marine wax esters in the oil composition of the present invention are characterized by being either mono- or polyunsaturated, with long-chain monounsaturated fatty alcohols (dominated by C16:1, C20:1 and C22:1 fatty alcohols), and a high proportion of mono- and polyunsaturated fatty acids (predominantly C16 to C22) including n-3 fatty acids SDA (C18:4), EPA (C20:5) and DHA (C22:6). The oil composition of the present invention is therefore a low-viscous and completely free-flowing liquid at room temperature.

(21) Depending on the analytical methods used, the typical content of wax-ester of the oil composition of the present invention is 60-90%, whereas it contains 10-20% of other components such as free fatty acids, triacylglycerols, phospholipids and their residues, sterols and pigments. In certain applications, it may be advantageous or even desirable to remove free fatty acids and other components by suitable methods known to those skilled in the art. Thus, in one embodiment of the present invention the oil composition may contain up to 100% wax ester.

(22) It has been shown that the oil composition of the present invention may inhibit the accumulation of visceral fat and counteract deterioration of healthy heart function induced by a high-fat Western type diet, as shown in FIGS. 1-3. Thus, the composition according to the present invention may be useful in the prevention and treatment of diseases induced by obesity, such as but not limited to type 2 diabetes.

(23) The oil composition according to the present invention comprises from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, by weight up to 75%, 80%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% by weight of wax esters. Preferably the oil composition comprises from 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89% by weight up to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% by weight of wax esters.

(24) Further the oil composition of the present invention comprises from 5%, 6%, 7%, 8%, 9%, 10% by weight up to 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% by weight of SDA.

(25) The content of EPA in the oil composition may be 3%, 4%, 5%, 6%, 7%, by weight up to 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% by weight. The composition may comprise 2%, 3%, 4%, 5% by weight up to 6%, 7%, 8%, 9%, 10% by weight of DHA.

(26) In an embodiment of the present invention an oil composition, comprising 20-100% by weight of wax esters, preferably 40-85% by weight of wax esters, wherein said wax esters consist of monoesters of predominantly mono- or polyunsaturated C16 to C22 fatty acids and predominantly monounsaturated C16, C20 and C22 fatty alcohols for use as a medicament for the prevention or treatment of abdominal obesity and obesity related disease is provided.

(27) In one embodiment of the oil composition according to the invention 70% or more of the monounsaturated fatty alcohols of the wax esters are C16:1, C20:1 and C22:1.

(28) In another embodiment a composition is provided wherein the obesity related disease is type 2 diabetes.

(29) In a further embodiment the composition provided in addition comprises 5-20% by weight of stearidonic acid (SDA). An oil composition further comprising 3-20% by weight of eicosapentaenoic acid (EPA) and 2-15% by weight of docosahexaenoic acid (DHA) is provided in another embodiment of the invention.

(30) In another embodiment of the invention the oil composition comprises fatty alcohols and SDA, DHA and EPA as monoesters.

(31) In yet another embodiment of the present invention a composition further containing 500-4000 ppm of astaxanthin is provided.

(32) In an embodiment of the present invention an oil composition is provided wherein the oil composition is isolated from a copepod, and in a further embodiment the copepod is of the genus Calanus. In yet another embodiment the copepod is of the species Calanus finmarchicus.

(33) Dietary supplement formulation and functional food or feed formulation comprising an oil composition as defined according to the present invention are also provided as additional embodiments of the present invention.

(34) In an embodiment of the invention a pharmaceutical formulation comprising an oil composition as defined according to the present invention is provided. The pharmaceutical formulation may be provided in capsules, tablets, emulsions or tonics. Further the formulation comprises one or more pharmaceutically acceptable additives selected from the group consisting of adjuvants, stabilizers, antioxidants, emulsifiers, surfactants and carriers.

(35) In an embodiment of the present invention the use of an oil composition as defined above for the manufacture of medicaments for the prevention or treatment of abdominal obesity is provided. Further the use of the oil composition for the manufacture of medicaments for the prevention or treatment of a obesity related disease is provided, said obesity related disease is type 2 diabetes.

(36) In yet another embodiment of the present invention a method for the prevention or treatment of abdominal obesity is provided wherein a daily amount of 0.5-5 g of an oil composition as defined above is administered to a human subject in need of such treatment. Further the method for the prevention or treatment of an obesity related disease is provided; said obesity related disease is type 2 diabetes.

(37) In addition to its usefulness for mammals, particularly humans, it is also an embodiment of the present invention that it may used to prevent a major problem in aquacultured fish, including but not limited to salmon and trout. It is well-known among fish farmers and others who are familiar with fish farming, that fishes like salmon and trout accumulate high amounts of intestinal fat when they are feeding on compounded dry feed pellets, whereas this quality problem of farmed salmon is not evident in wild salmon feeding on naturally available food organisms in the ocean. Visceral fat accumulation may be so high that the intestine sometimes is merely discernible in gutted farmed fish. The reason for this quality difference between wild and farmed fishes such as salmon and trout, is not at all known by either international feed manufacturers or other people working within the aquaculture sector. It is nevertheless generally accepted that it has to do with some kind of imbalance in the artificial diet, or lack of one or more components the wild fish obtain from their natural food in the ocean. A further embodiment of the present invention is thus enrichment of fish feeds with this food source, either as a whole copepod preparation or as the oil composition of the present invention, to prevent visceral fat accumulation also in farmed fish.

(38) The following non-limiting experimental part and examples illustrate and document the present invention.

EXAMPLES

(39) Experimental

(40) Animal experiments were carried out at the Faculty of Medicine at the University of Troms, Norway. Six weeks old Wistar rats, with an average weight of 162 grams, were individually caged and housed in a room maintained at 21 C., 55% humidity and 12:12-h light-dark cycle (lights from 7 AM to 7 PM). Animals had free access to water and feed. All procedures were approved by the Norwegian Experimental Animal Board and were in accordance with the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (EST NR. 123, 1986). Rats were fed for 110 days on three different diets, which were produced by TestDiet (www.testdietcom): 1) a control diet containing 4% fat (2.37% soybean oil and 1.89% of lard, w/w), corresponding to 10% of the total dietary energy content (Control, 3.75 kcal/g); 2) an obesity-inducing high fat diet containing 24% fat (2.89% soybean oil and 20.68% of lard, w/w) corresponding to 45% of the total dietary energy content (HF, 4.65 kcal/g); 3) an experimental high fat diet containing 24% fat (2.91% of soybean oil, 19.15% lard and 1.5% of the oil formulation of the present invention), corresponding to 45% of the total dietary energy (HF+1.5% CO, 4.47 kcal/g feed). The diets were prepared without antioxidants or preservatives and stored frozen prior to feeding.

(41) At the day of sacrifice, after administration of heparin and anesthesia, total body weight was recorded. The weight of liver and hearth were recorded after being removed from the body, and visceral (perirenal) fat was dissected out and weighed. The data are shown in Table 3 and FIG. 1. Cardiac metabolism was measured as glucose oxidation in isolated perfused hearts according to standard methodology (E. Aasum, A. M. Khalid, O. A. Gudbrandsen, O.-J. How, R. K. Berge, T. Larsen. Fenofibrate modulates cardiac and hepatic metabolism and increases ischemic tolerance in diet-induced obese mice. Journal of Molecular and Cellular Cardiology 2008; 44:201-209). The results are shown in FIG. 2.

(42) In another set of experiments mice were fed for 50 days with the same diets as described for rats. For glucose tolerance test, mice were fasted for 5 h and given an intraperitoneal injection of glucose (1.3 g/kg). Blood samples were taken from the saphenous vein before injection and at 15, 30, 60, and 120 min after the injection. Blood glucose levels were determined using a glucometer (Bayer Ascensia Contour Glucose Meter). The results are shown in FIG. 3.

(43) Biological Effects

(44) TABLE-US-00003 TABLE 3 Biometrical data for rats fed low fat reference diet (Control), the obesity inducing high fat diet (HF) and the experimental high fat diet containing 1.5% by weight of the oil formulation of the present invention (HF + 1.5% CO). Values given for body weight at start of the feeding trials (body wt start), body weight at the end of the feeding trials (body wt sacrifice), liver weight at the end of the feeding trials (liver wt), heart weight at the end of the feeding trials (hearth wt), and the ratio between hearth weight and body weight, and tibia (shinbone) length in mm, respectively, at the end of the feeding trials. Values are mean 95% C.I. (n = 10). Body wt Body wt start sacrifice Liver wt Heart wt Heart wt Heart wwt/ Heart dwt/ (g) (g) (g) (g wet) (g dry) body wt* tibia length* Control 162 5 457 12 13.51 0.75 1.20 0.03 0.228 7 2.63 0.05 7.15 0.24 HF 162 3 483 16 12.27 0.44 1.21 0.03 0.232 7 2.54 0.10 7.31 0.19 HF + 1.5% 162 4 484 9 11.85 0.37 1.29 0.08 0.249 14 2.69 0.20 7.67 0.44 CO *wwt = wet weight in gram, dwt = dry weight in gram. Values have been multiplied by 1000.

(45) As shown in Table 3, the average total body weight (and thus weight gain) after 110 days was the same of rats fed the two high fat (high energy) diets, whereas the rats fed on the lower energy reference diet had a lower weight gain, as expected.

(46) Other biometrical data show that there are no notable differences between the groups. Rats in the two high fat dietary groups had the same daily feed intake and their water intake was also the same (data not shown). The reference group consumed somewhat more feed than rats in the two high fat groups, but their daily energy intake was lower, explaining their lower weight gain. In conclusion, the experiment showed that the oil composition of the present invention does not affect performance of rats fed a high fat diet and there were no signs of adverse effects on the animals.

(47) When the animals were sacrificed after 110 days of feeding and the bellies opened up, a notable difference between the two high-fat groups could be observed with the naked eye: Rats on the experimental high-fat diet supplemented with the oil composition of the present invention, had visibly less visceral fat than the corresponding high-fat group, resembling more the visceral fat accumulation in rats that had been fed on the low fat reference diet, when comparing rats of about same size. Statistical analysis of the weights of visceral fat in the three groups confirmed this direct visual observation. Since the amount of visceral fat increases with body size, independent on feeding regime, the primary data on perirenal fat in the three dietary groups have been plotted as a function of body weight at sacrifice, as shown in FIG. 1.

(48) Statistical treatment of these weight data revealed the following: There was a clear linear correlation between visceral fat weight and body weight in all three groups, with a level of statistical significance of p=0.034 for the reference group (Control), p=0.003 for the high-fat group (HF) and p=0.018 for the experimental group containing the oil composition of the present invention (HF+1.5% CO). Based on the fact that a statistically significant correlation exists between visceral fat weight and body weight, the statistical difference between the groups could be calculated by ANCOVA analysis, using weight as a covariate. The analysis showed that the difference between the group containing the experimental oil and the corresponding high-fat diet was statistically significant (p=0.00241), whereas there was no statistically significant difference between the experimental oil group and the low fat group. The experimental oil group and the low fat reference group, taken together, were significantly different from the high-fat group (p=0.0084). In conclusion therefore, the oil of the present invention, given as a minor supplement to high fat obesity inducing diet, inhibits the accumulation of visceral fat.

(49) FIG. 2 shows the glucose oxidation capacity of heart muscle tissue of rats which had been feeding on the three diets. Rats on the high-fat diet (HF) had reduced glucose oxidation capacity compared to rats on the low-fat control diet (Control), whereas in rats that had been fed on the high-fat diet containing 1.5% of the present oil composition (HF+1.5% CO), glucose oxidation capacity was comparable to that of the low-fat control rats. It is evident therefore that the present oil composition may counteract deterioration of healthy heart function even after long time feeding on an obesity inducing high-fat diet.

(50) FIG. 3(A) shows the blood glucose levels during intraperitoneal glucose tolerance test in mice given the control diet (Control), high fat diet (HF) and high fat diet with oil composition (HF+1.5% CO), respectively. FIG. 3(B) shows the area under the glucose tolerance curves shown in panel A. Data are expressed as meanSE. *P<0.05 for HF vs Control; #P<0.05 for HF+1.5% CO vs HF.

(51) Blood glucose levels were significantly increased during the intraperitoneal glucose tolerance test in the HF mice compared with the Control mice. These results show that feeding with a high fat diet for 50 days induced insulin resistance characteristic of type 2 diabetes. Importantly, inclusion of 1.5% of the oil composition of the present invention to the high fat diet counteracted the development of insulin resistance.

(52) Mice on the high fat diet (HF) had reduced myocardial glucose oxidation rates compared to mice on the control diet (Control) (p<0.05). Glucose oxidation was partly restored, however, in mice that had been fed the high fat diet containing 1.5% of the present oil composition (HF+1.5% CO) (p<0.05). It is evident, therefore, that the oil composition of the present invention may counteract the reduction in myocardial glucose oxidation capacity which occur after feeding with a high fat diet.