# Patent

## Title
Agent for preventing or ameliorating obesity

## Abstract

The present invention provides a method for preventing or ameliorating obesity, for inhibiting accumulation of visceral fat, for inhibiting accumulation of liver lipid, for preventing or ameliorating fatty liver, including using a material for a pharmaceutical product or food, which has inhibitory actions on body weight increase, accumulation of visceral fat and accumulation of liver lipid. The present invention is also directed to a method for preventing or ameliorating obesity, a method for inhibiting accumulation of visceral fat, a method for inhibiting accumulation of liver lipid, and a method for preventing or ameliorating fatty liver, which methods include administering potassium alginate to a subject in need thereof or having the subject take potassium alginate.

## Background
FIELD OF THE INVENTION
The present invention relates to an agent for preventing or ameliorating obesity, to an agent for inhibiting accumulation of visceral fat, to an agent for inhibiting accumulation of liver lipid, and to an agent for preventing or ameliorating fatty liver.
BACKGROUND OF THE INVENTION
In recent years, Japanese have consumed more of a Western diet, resulting in taking in excessive energy (increase in ingestion of fat and sucrose), and have had fewer chances for physical exercise. Therefore, the number of patients of lifestyle-related diseases such as obesity and diabetes continuously has increased. Under such social circumstances, prevention and amelioration of obesity is a key issue.
One of the methods for the preventing or ameliorating of obesity, that dieticians generally propose is to consume low-calorie or low-fat food. In recent years, it was reported that each of water-insoluble dietary fibers such as wheat bran, water-soluble dietary fibers such as indigestible dextrin, and resistant starches such as high amylose starch has an action of promoting lipid excretion (Non-Patent Document 1), an inhibitory action of sugar absorption (Non-Patent Document 2), and an action of reducing blood neutral fat (Non-Patent Document 3), respectively and these materials have an action of ameliorating glucose tolerance (Non-Patent Documents 3, 4 and 5). Thus, these materials are thought to be effective for the preventing or ameliorating of obesity.
Since a rapid rise in postprandial blood lipid level is thought to promote accumulation of fat, an approach of suppressing postprandial hyperlipidemia (increase in blood triglyceride level) is also a key issue for the prevention or amelioration of obesity. In recent years, there have been reported some safe and effective lipid-absorption inhibitors such as xanthan gum and propylene glycol alginate ester (Patent Document 1), chitosan (Patent Document 2) and a composition for ameliorating obesity containing an extract from animal meat and/or fish meat and a substance capable of suppressing fat accumulation, in combination (Patent Document 3).
It is also reported that sodium alginate, which is a high-molecular acidic polysaccharide found in brown algae, has an inhibitory action on increase in blood glucose level and thus is useful for the prevention of obesity and diabetes (Patent Document 4 and Non-Patent Document 7).
The aforementioned low-calorie food or low-fat food can exhibit a temporary effect of reducing body weight. However, since a food composing of the low-calorie food or low-fat food is generally not tasty (i.e., monotonous), such food becomes unacceptable by those who consume it over a long period, making long-term ingestion thereof difficult. Also, in order to attain the aforementioned physiological effect, the aforementioned conventional food materials such as water-insoluble dietary fiber, water-soluble dietary fiber, and resistant starch must be continuously ingested in a large amount over a long period of time. Although the physiological effect can be attained, suppression of obesity has not yet been confirmed. When foods and beverages are produced by using such food materials, their intrinsic appearance, taste, sensations such as touch to the teeth and smoothness are generally impaired. Therefore, the food materials cannot be easily incorporated into foods and beverages in a satisfactory amount, and a limitation is imposed on the type of the foods and beverages into which the food materials are added. In addition, difficulty is encountered in long-term ingestion of such foods and beverages.
Due to insolubility, making beverages containing water-insoluble dietary fiber is difficult. Resistant starch or water-soluble dietary fiber can be dissolved or dispersed in water. However, difficulty is also encountered in producing beverages containing resistant starch or water-soluble dietary fiber in a sufficient amount to expect anti-obesity effect, since the viscosity of the beverages increases.
Meanwhile, potassium alginate is widely used as a thickener for food and as a gelling agent incorporated into a dental impression material, and it is reported that a hypertension inhibitory action of potassium alginate based on active excretion of sodium in the body (Non-Patent Document 8).
However, potassium alginate has never been known to have an action of preventing or ameliorating obesity.
Patent Document


    Patent Document 1: JP-A-H05-186356
    Patent Document 2: JP-A-H03-290170
    Patent Document 3: WO 2006/103750 Pamphlet
    Patent Document 4: Japanese Patent No. 2643669


Non-Patent Document


    Non-Patent Document 1: Am. J. Clin. Nutr., 1978, 31(10 Suppl): S21-S29
    Non-Patent Document 2: Folia Endocrinologica Japonica, 1992, 68(6): 623-35
    Non-Patent Document 3: Am. J. Clin. Nutr., 1989, 49(2): 337-44
    Non-Patent Document 4: Acta Paediatr Hung., 1985, 26(1):75-7
    Non-Patent Document 5: J. Endcrinol., 1995, 144(3): 533-8
    Non-Patent Document 6: Y. Kimura et al., Journal of Ethnopharmacology, 54, (1996) 47-54
    Non-Patent Document 7: Keisuke Tsuji et al, Journal of Home Economics of Japan, Vol. 39, No. 3, pages 187-195 (1988)


SUMMARY OF THE INVENTION
The present invention is directed to a method for preventing or ameliorating obesity, a method for inhibiting accumulation of visceral fat, a method for inhibiting accumulation of liver lipid, and a method for preventing or ameliorating fatty liver, which methods include administering potassium alginate to a subject in need thereof or having the subject take potassium alginate.
The present invention is also directed to a method for treating or remedying obesity or for reducing the likelihood of developing obesity and a method for treating or remedying fatty liver and reducing the likelihood of developing fatty liver.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for preventing or ameliorating obesity, a method for inhibiting accumulation of visceral fat, a method for inhibiting accumulation of liver lipid, and a method for preventing or ameliorating fatty liver, including using a material for a pharmaceutical product or a food, which has an inhibitory action on body weight increase, accumulation of visceral fat and/or accumulation of liver lipid.
The present inventors have investigated a material which is applied to the above product or food and is also effective in a beverage. As a result, they have found that potassium alginate has an inhibitory action on body weight increase, accumulation of visceral fat, and/or accumulation of liver lipid and exhibits the effect of preventing or ameliorating obesity or fatty liver, in the case where potassium alginate is ingested as aqueous solution or as an ingredient incorporated into a meal.
According to the present invention, in fields of pharmaceutical and food, a variety of lifestyle-related diseases such as obesity and fatty liver can be prevented, treated, or ameliorated.
Alginic acid is a high-molecular acidic polysaccharide (molecular weight: several tens thousands to several hundreds of thousands) which is mainly formed from uronic acid (D-mannuronic acid and L-guluronic acid) as a constituent saccharide and which is found in all brown algae as a cell wall substance. One constituent unit thereof has one carboxyl group. Potassium alginate is a salt formed through bonding potassium ions to the carboxyl groups of alginic acid.
The potassium alginate employed in the present invention is a low-molecule potassium alginate which has a weight average molecular weight, as determined through HPLC, of 60,000 or less, preferably 10,000 to 60,000, more preferably 20,000 to 60,000, even more preferably 20,000 to 50,000. Particularly when the potassium alginate is used in the form of a peroral liquid preparation, the viscosity of the liquid is preferably low, from the viewpoints of production thereof and favorable touch to the throat, sensation of sliminess, swallowability, and other considerations during drinking. Based on these viewpoints, the potassium alginate employed preferably has a low weight average molecular weight of 10,000 to 50,000, more preferably 10,000 to 40,000, even more preferably 10,000 to 30,000.
The potassium alginate of the present invention may be produced through a known method such as degradation under pressure and heat (JP-A-H06-7093) or enzymatic degradation (JP-A-H02-303468, JP-A-H03-94675, JP-A-H04-169189, JP-A-H06-245767, or JP-A-H06-217774). In one specific procedure, high-molecular-weight potassium alginate or alginic acid, serving as a raw material, is degraded under the conditions of, for example, pressure and heat, heating at ambient pressure, and in the presence of an enzyme, to thereby attain a molecular weight of interest. The thus-formed lower molecule product is optionally neutralized, dehydrated, and freeze-dried, to thereby yield potassium alginate. In the case of thermal degradation, molecular weight may be tuned by regulating pH at reaction, reaction temperature, reaction time, etc.
As described in the Examples hereinbelow, the thus-obtained potassium alginate of the present invention exhibits inhibitory effects on body weight increase, accumulation of visceral fat, and accumulation of liver lipid, which are associated with obesity promoted by intake of high-fat food, in the case where potassium alginate is ingested as aqueous solution (i.e., beverage) or as an ingredient incorporated into a meal.
Thus, the potassium alginate of the present invention can be employed in methods for preventing or ameliorating obesity, for inhibiting increase in body weight, for inhibiting accumulation of visceral fat, and for inhibiting accumulation of liver lipid, which are associated with obesity promoted by intake of high-fat food, which methods include administering potassium alginate to a subject (human or animal) in need thereof or having the subject take potassium alginate. Namely, the potassium alginate may serve as an agent for preventing or ameliorating obesity, an agent for inhibiting accumulation of visceral fat, an agent for inhibiting accumulation of liver lipid, or an agent for preventing or ameliorating fatty liver (hereinafter collectively referred to as “agent for preventing or ameliorating obesity, etc.”), which exhibits an inhibitory effects of body weight increase, accumulation of visceral fat and accumulation of liver lipid, which are associated with obesity promoted by intake of high-fat food.
The agent for preventing or ameliorating obesity may be pharmaceuticals, foods, feeds or the like to exhibit, per se, the effect for preventing or ameliorating obesity, for inhibiting increase of body weight, for inhibiting accumulation of visceral fat and for inhibiting accumulation of liver fat; or materials and preparations to be incorporated into the pharmaceuticals or the like. The foods may be a food such as beauty food, functional food, patient food, or food for specified health uses, with an optional label indicating a concept of prevention or amelioration of obesity, inhibition of accumulation of visceral fat, inhibition of accumulation of liver lipid, or prevention or amelioration of fatty liver.
As used herein, the term “high-fat food” refers to a food containing fat in a large amount, as defined by “The Lipid Reference Intakes for Japanese” (see “Dietary Reference Total Lipid Intakes,” “Dietary Reference Saturated Fatty Acid Intakes,” “Dietary Reference n-6 Fatty Acid Intakes,” and “Dietary Reference Cholesterol Intakes,” attached) as described in “The Dietary Reference Intakes for Japanese (2005 edition)” (Comments on “The Dietary Reference Intakes for Japanese (2005 edition)” on the homepage of the Ministry of Health, Labour and Welfare, issued on Nov. 22, 2004 by Health Policy Bureau, General Affairs Division, Office of Lifestyle-Related Disease Control, Nutrition Guidance Group <URL: http://www.mhlw.go.jp/houdou/2004/11/h1122-2.html#betu>). For example, the “high-fat food” has an energy ratio of total lipid to total intake energy of 30% or higher, preferably 40% or higher, more preferably 50% or higher, even more preferably 50 to 90%.
The total lipid energy ratio of the “high-fat food” may be calculated on the basis of one food, one meal, or meals consumed during the course of a day.
The pharmaceutical products containing potassium alginate may be, for example, peroral solid preparations such as tablet and granules, and peroral liquid preparations such as oral liquid and syrup.
A peroral solid preparation may be produced by mixing the potassium alginate of the present invention with a pharmaceutically acceptable carrier such as a diluent and, if required, a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, a corrigent, etc., and forming the mixture into tablets, coated tablets, granules, powders, capsules, etc., through a conventional method. A peroral liquid preparation may be produced by mixing the potassium alginate with a flavoring agent, a buffer, a stabilizer, a corrigent, etc. and forming the mixture into peroral liquid, syrup, elixir, etc. through a routine method.
The amount of potassium alginate incorporated into each preparation is generally 0.01 to 100 wt. %, preferably 0.1 to 80 wt. %. More preferably, the amount is 1 to 50 wt. % for solid preparations, and 0.1 to 20 wt. % for liquid preparations.
A food and feed containing therein the potassium alginate of the present invention includes foods such as breads, confectionaries, jellied foods, dairy products, frozen foods, instant foods, starch-processing products, processed meat products and other processing products, drinks, soups, seasonings and nutritional supplement; feeds such as feeds for small animals including rabbits, rats and mice and pet foods for dogs, cats, birds, squirrels and the like.
The food or feed, if necessary, may optionally contain other component combined with the potassium alginate of the present invention. The other component preferably includes carriers acceptable in a food or feed field. Examples of the acceptable carries includes solvents, softeners, fats and oils, emulsifiers, preservatives, flavors, stabilizers, colorants, antioxidants, humectants, thickeners, gelatinizers, shape retaining agents, pH adjusters, seasonings and nutritional supplement products.
No particular limitation is imposed on a form of foods or feed. In addition to liquid, semi-solid and solid form, tablets, pills, capsules, liquids, syrups, powders, granules, or the like may be included, similar to a form of the peroral preparations.
The food or feed contains therein the potassium alginate of the present invention, depending on a form thereof, in an amount of 0.001 to 100 mass %, preferably 0.01 to 80 mass %, more preferably 0.1 to 50 mass % in term of dry weight. The drink preferably contains therein the potassium alginate of the present invention in an amount of 0.001 to 20 mass %, more preferably 0.1 to 10 mass %.
When the potassium alginate of the present invention is used for pharmaceuticals or foods, no particular limitation is imposed on the dose (effective amount taken by subject) thereof, so long as the target effect(s) can be attained. The daily dose (intake), which may vary depending on the condition, body weight, sex, or other factors of the subject, is preferably ≧0.001 g/kg-body weight in terms of potassium alginate, more preferably 0.01 to 15.0 g/kg-body weight.
No particular limitation is imposed on the timing of administration of a variety of the preparation to a subject or having the subject take the preparation, and a timing of “during or before meals” is preferred. In the case where administration of the aforementioned preparation to a subject (or having the subject take the preparation) is carried out “between meals,” the timing is preferably “before or while having a snack.” Alternatively, the preparation may be administered or have taken as a snack.
No particular limitation is imposed on the frequency of ingestion or administration of a variety of the preparation per day, since it is highly safe. Administration or ingestion of one or more times per day is preferred. The frequency of ingestion or administration of the preparation per week is preferably 3 days or more, more preferably 5 days or more, even more preferably daily. The period of ingestion or administration of the preparation is preferably 2 weeks or longer, more preferably 4 weeks or longer.
No particular limitation is imposed on the subject to which the pharmaceutical products or foods of the present invention is administered or consumed, so long as the subject is in need of the agent. However, preferred target subjects include patients of nonalcoholic steatohepatitis (NASH); patients of obesity (BMI≧25, and/or waist measurement of ≧85 cm (male) or ≧90 cm (female), and/or visceral fat area of ≧100 cm2); patients of diabetes or impaired glucose tolerance (fasting blood glucose level of ≧110 mg/dL and/or OGTT 2-hour level of ≧140 mg/dL (WHO standards, 1998)); and patients of hyperlipidemea or impaired lipid metabolism (fasting blood neutral fat level of ≧150 mg/dL and/or HDL cholesterol level of <40 mg/dL).
Meanwhile, when a subject who is not at present in the state of obesity, diabetes or impaired glucose tolerance, or hyperlipidemia or impaired lipid metabolism (i.e., a healthy subject or a candidate) takes potassium alginate with a meal, particularly a high-fat food, the time of onset of obesity, diabetes, impaired glucose tolerance, hyperlipidemia, or impaired lipid metabolism, which will possibly occur in the future, is thought to be prolonged. Thus, healthy people may take potassium alginate for the purpose of prevention of such diseases or conditions.
EXAMPLES
Production Example (1)
Preparation of Potassium Alginate having a Weight Average Molecular Weight of about 50,000
A 5% solution of alginic acid (Duck Acid A (Lot. X-2702): product of Kibun Food Chemifa Co., Ltd.) was prepared, and the solution was heated at 100° C. for 45 minutes for degradation. The degradation product was neutralized with potassium hydroxide to a pH of 7. Subsequently, ethanol was added to the neutralization product so as to give 80% of ethanol content, and the formed precipitate was recovered through centrifugation (3000 rpm, 10 min) and dried, to thereby prepare a potassium alginate of interest.
The weight average molecular weight of the obtained potassium alginate, as measured through the below-described method, was found to be 52,163.
Production Example (2)
Preparation of Potassium Alginate having a Weight Average Molecular Weight of about 25,000
A 5% solution of alginic acid (Duck Acid A (Lot. X-2702): product of Kibun Food Chemifa Co., Ltd.) was prepared, and the solution was heated at 100° C. for 120 minutes for degradation. The degradation product was neutralized with potassium hydroxide to a pH of V. Subsequently, ethanol was added to the neutralization product so as to give 80% of ethanol content, and the formed precipitate was recovered through centrifugation (3000 rpm, 10 min) and dried, to thereby prepare a potassium alginate of interest.
The weight average molecular weight of the obtained potassium alginate, as measured through the below-described method, was found to be 25,801.
Determination of Average Molecular Weight of Alginic Acid or Salt thereof (Weight Average Molecular Weight Determination Method)
The weight average molecular weight of alginic acid (or a salt thereof) was determined through high-performance liquid chromatography (HPLC).
A portion (0.1 g) of alginic acid (or a salt thereof) was quantitatively dissolved in distilled water, to thereby provide a 0.1% solution thereof. The solution was employed as an HPLC analytical sample.
HPLC was performed under the following conditions. A calibration curve for the calculation of molecular weight was drawn by use of standard pullulan (Shodex STANDARD P-82, product of Showa Denko K.K.). An HPLC analytical sample (100 μL) was injected to an HPLC, and the weight average molecular weight of alginic acid (or a salt thereof) contained in the analytical sample was calculated from the obtained chromatographic chart.
<HPLC Operational Conditions>
Columns:


    (1) Super AW-L (guard column) (product of Tosoh Corporation)
    (2) TSK-GEL Super AW4000 (GPC column, exclusion limit of molecular weight: 4×105 PEO/DMF, length: 15 cm, inner diameter: 6 mm) (product of Tosoh Corporation)
    (3) TSK-GEL Super AW2500 (GPC column, exclusion limit of molecular weight: 2×103 PEO/DMF, length: 15 cm, inner diameter: 6 mm) (product of Tosoh Corporation)


The aforementioned columns AW-L, AW4000, and AW2500 are linked in that order.
Column temperature: 40° C.
Detector: refractive index detector
Mobile phase: 0.2 mol/L aqueous sodium nitrate
Flow rate: 0.6 mL/min
Injection amount: 100 μL
Test Example 1
Effect of Potassium Alginate on Anti-Obesity
1-1 Test Samples
The potassium alginate produced in Production Example 1 (hereinafter referred to as “K alginate (mol. wt. 50,000)”) and the potassium alginate produced in Production Example 2 (hereinafter referred to as “K alginate (mol. wt. 25,000)”) were employed as potassium alginate samples.
1-2 Test Groups
K alginate intake was realized through the following two methods: (1) dissolving K alginate in purified water and feeding the aqueous solution (feeding water), and (2) incorporating K alginate into a meal (feeding meal).
The following six groups were subjected to a breed test (N=10 in each group).
In the Test Examples, the term “high-fat food” refers to a food containing fat in a large amount, with reference to the aforementioned “The Dietary Reference Intakes for Japanese (2005 edition), Dietary Reference Total Lipid Intakes” (lipid energy ratio: 52 to 53%), and the term “low-fat food” refers to a food containing fat in a small amount, with reference to the aforementioned “The Dietary Reference Intakes for Japanese (2005 edition), Dietary Reference Total Lipid Intakes” (lipid energy ratio: 12%).










TABLE 1







Meal feeding
Water feeding (test



Test groups
(test food)
water)


















1
low-fat food
low-fat food
purified water


2
high-fat food
high-fat food
purified water


3
K alginate (mol.
high-fat food
K alginate (mol.



wt.: 25,000) in

wt.: 25,000) 5% aq.



water (5%)

solution


4
K alginate (mol.
high-fat food + K
purified water



wt.: 25,000) in
alginate




meal (5%)
(mol. wt.: 25,000)





(5%)



5
K alginate (mol.
high-fat food
K alginate (mol.



wt.: 50,000) in

wt.: 50,000) 5% aq.



water (5%)

solution


6
K alginate (mol.
high-fat food + K
purified water



wt.: 50,000) in
alginate (mol. wt.:




meal (5%)
50,000)(5%)









1-3 Feeding Meal (Test Food) Composition

Table 2 shows the compositions of the test foods given to each of the Test groups.







TABLE 2







Compositions of test foods (content: %)














Test food for
Test food for



Test food
Test food for
Test group 4
Test group 6



for Test
Test groups
high-fat food +
high-fat food +



group 1
2, 3, and 5
K alginate
K alginate



low-fat
high-fat
(mol. wt.:
(mol. wt.:


Ingredients
food
food
25,000) (5%)
50,000) (5%)














Corn oil
5
25
25
25


Lard
—
5
5
5


K alginate
—
—
5
—


(mol. wt.:






25,000)






K alginate
—
—
—
5


(mol. wt.:






50,000)






Sucrose
—
13
13
13


Casein
20
20
20
20


Cellulose
4
4
4
4


AIN76
3.5
3.5
3.5
3.5


mineral






mixture






AIN76
1
1
1
1


vitamin






mixture






α-Potato
66.5
28.5
23.5
23.5


starch






Total
100
100
100
100


Lipid energy
11.5
52.3
53.4
53.4


ratio (%)









In the test foods, corn oil, lard, casein, cellulose, AIN76 mineral mixture, AIN76 vitamin mixture, and α-potato starch were products of Oriental Yeast Co., Ltd., and sucrose was fine granules of sucrose (special grade) and a product of Wako Pure Chemical Industries, Ltd.
1-4 Fed Water (Test Water) Composition
A 5 w/w % aqueous solution of K alginate (mol. wt.: 25,000) and that of K alginate (mol. wt.: 50,000) were prepared on the day when the test water was applied. These two aqueous solutions have so low viscosity that they can be fed by means of a water feed bottle generally employed in breeding animals. Thus, test animals can readily take the test water as tap water.
1-5 Test Animal and Breeding Conditions
7-Week-old male C57BL/6J Jcl mice (purchased from Clea Japan) were bred for one week, while maintained on a common food (CE-2: moisture 9.3%, crude protein 25.1%, crude fat 4.8%, crude fiber 4.2%, crude ash 6.7%, and NFE 50.0%, product of Clea Japan). After breeding, at the age of 8 weeks, the mice were weighed and classified into six Test groups so that the initial body weights of these groups were substantially equal.
After classification, feeding of meal and water (shown in Table 1) was carried out for the test to week 18. The mice were maintained on food and water ad libitum, where Roden Cafe was used as a base of the food, and water was supplied by means of a water feed bottle.
Energy intake was calculated from the following unit values: lipid (9.0 kcal/g), sugar (4.0 kcal/g), protein (4.0 kcal/g), and potassium alginate (2 kcal/g) (see Note 1). (Note 1) Unit calorie value of potassium alginate
In the notifications of the Ministry of Health, Labour and Welfare (Feb. 17, 2003); “Partial Revision of ‘Practice of Nutrition Labeling Standards’” (Issue No. 0217001) and “Partial Revision of ‘Method for Analysis of nutrients in Nutrition Labeling Standards’ (Issue No. 0217002), energy conversion factors of various dietary fibers are described. However, potassium alginate, which was employed in the test of the invention, is not described in these notifications. According to the above notifications of the Ministry of Health, Labour and Welfare, the energy conversion factor for a dietary fiber which is not described in these notifications is provisionally determined as 2 kcal/g. Thus, in the present invention, energy calculation was carried out with an energy conversion factor of potassium alginate of 2 kcal/g.
1-6 Removal of the Visceral Fat and Livers for Measuring Visceral Fat Weight and Liver Lipid
On the final day of the test, mice were maintained on food and water ad libitum just before anatomization. Under anesthetization with ether, the mice were immediately subjected to peritoneotomy and sacrificed through exsanguination via the abdominal vena cava. Immediately thereafter, the liver was removed from each mouse, and the weight of the liver was measured. The thus-obtained liver was quickly frozen in liquid nitrogen for the measurement of liver lipid. Furthermore, visceral fat (periepididymal, perinephric, retroperitoneal, and mesenteric) was removed and weighed. The sum of the fat weights was employed as the amount of visceral fat.
The obtained liver was stored in a freezer at −80° C. from completion of anatomization to measurement of liver lipid.
1-7 Data Analysis and Statistical Test
During breeding, two mice were severely and widely injured by fighting, and the injuries were observed until the final test day (injury of about 1⁄5 or more of the body surface visually observed, presence of muscle exposure, continuous from breeding week 10 or 11 to breeding week 18 (final test day)). Thus, the two mice were excluded from data analysis.
The body weight, and the amounts of visceral fat and liver lipid were measured, and average± standard error (S.E.) values were calculated. Statistical testing was performed through Fisher's PLSD test.
1-8 Results
1-8 (1) Body Weight
Tables 3 and 4 show the body weights of mice before the test, after breeding week 4, breeding week 8, breeding week 12, and breeding week 18.
Test group 3 (K alginate, mol. wt.: 25,000, 5% in water) and Test group 5 (K alginate, mol. wt.: 50,000, 5% in water) exhibited significantly lower body weights after breeding week 4, as compared with Test group 2 (high-fat food), indicating suppression of increase in body weight. During the test period, Test groups 3 and 5 exhibited body weights not lower than the body weight of Test group 1 (low-fat food). Therefore, growth of mice in Test groups 3 and 5 was not thought to be inhibited.
Test group 4 (K alginate, mol. wt.: 25,000, 5% in meal) and Test group 6 (K alginate, mol. wt.: 50,000, 5% in meal) exhibited significantly lower body weights after breeding week 12, as compared with Test group 2 (high-fat food), indicating suppression of increase in body weight. During the test period, Test groups 4 and 6 exhibited body weights not lower than the body weight of Test group 1 (low-fat food). Therefore, growth of mice in Test groups 4 and 6 was not thought to be inhibited.
Through the above test, potassium alginate is thought to exhibit excellent inhibitory effect on body weight increase, which would otherwise be promoted by intake of high-fat food, when potassium alginate is in the form of aqueous solution or has been incorporated into a meal.







TABLE 3







Body weights (before test, week 4, and week 8)












Test groups
Before test (g)
Week 4 (g)
Week 8 (g)
















1
low-fat food
21.12 ± 0.12
26.79 ± 0.33
***
31.33 ± 0.43
***


2
high-fat food
21.21 ± 0.09
30.07 ± 0.46
—
37.61 ± 0.72
—


3
K alginate (mol.
21.15 ± 0.10
28.48 ± 0.24
**
32.78 ± 0.66
***



wt.: 25,000) in








water (5%)







4
K alginate (mol.
21.27 ± 0.13
29.45 ± 0.34
N.S.
36.62 ± 0.41
N.S.



wt.: 25,000) in








meal (5%)







5
K alginate (mol.
21.13 ± 0.12
28.35 ± 0.38
**
33.84 ± 0.83
***



wt.: 50,000) in








water (5%)







6
K alginate (mol.
21.43 ± 0.13
29.56 ± 0.40
N.S
36.76 ± 0.98
N.S



wt.: 50,000) in








meal (5%)





Each of values indicates Av. ± S.E., Fisher's PLSD test was performed with respect to Test group 2 (high-fat food)


(** P < 0.01, *** P < 0.001)













TABLE 4







Body weights (week 12 and week 18)











Test groups
Week 12 (g)
Week 18 (g)















1
low-fat food
33.63 ± 0.46
***
36.10 ± 0.60
***


2
high-fat food
42.28 ± 1.10
—
46.66 ± 0.98
—


3
K alginate (mol.
35.45 ± 0.68
***
39.34 ± 0.77
***



wt.: 25,000) in







water (5%)






4
K alginate (mol.
39.69 ± 0.58
*
42.26 ± 0.87
**



wt.: 25,000) in







meal (5%)






5
K alginate (mol.
36.85 ± 1.14
***
39.66 ± 1.53
***



wt.: 50,000) in







water (5%)






6
K alginate (mol.
39.44 ± 1.09
*
42.35 ± 1.33
**



wt.: 50,000) in







meal (5%)





Each of values indicates Av. ± S.E., Fisher's PLSD test was performed with respect to Test group 2 (high-fat food)


(* P < 0.05, ** P < 0.01, *** P < 0.001)






Table 5 shows the total calorie intake (during 18 test weeks) and feed efficiency (body weight increase (18 weeks)/total calorie intake). The amounts of test food intake and test water intake were measured cage by cage. Thus, the total calorie intake was calculated through averaging two cages of each group.
While the high-fat food group exhibited a total calorie intake of 1,534 kcal/mouse, the potassium alginate intake groups exhibited a total calorie intake of 1,404 to 1,528 kcal/mouse; i.e., potassium alginate intake groups exhibited a cumulative calorie intake lower than that of the high-fat food group. However, although the high-fat food group exhibited a feed efficiency of 16.6 mg/kcal/mouse, the potassium alginate intake groups exhibited a low feed efficiency of 12.7 to 14.6 mg/kcal/mouse. Thus, when the same amount calories is taken in, the potassium alginate intake groups are thought to exhibit a smaller increase in body weight, as compared with the high-fat food group, and intake of potassium alginate is thought to inhibit body weight increase.







TABLE 5







Total calorie intake of test food and water and feed efficiency












Total calorie
Feed efficiency;




intake of test
18-weeks bodyweight




food and water
increase (mg)/total calorie



Test groups
(kcal/mouse)
intake (kcal/mouse)













1
low-fat food
1,605
9.3


2
high-fat food
1,534
16.6


3
K alginate (mol.
1,404
13.0



wt.: 25,000) in





water (5%)




4
K alginate (mol.
1,436
14.6



wt.: 25,000) in





meal (5%)




5
K alginate (mol.
1,456
12.7



wt.: 50,000) in





water (5%)




6
K alginate (mol.
1,528
13.7



wt.: 50,000) in





meal (5%)









1-8 (2) Visceral Fat Amount

Table 6 shows visceral fat amounts of the test groups.
Test group 2 (high-fat food) exhibited a significantly larger visceral fat amount, as compared with Test group 1 (low-fat food), indicating accumulation of visceral fat.
Test group 3 (K alginate, mol. wt.: 25,000, 5% in water), Test group 5 (K alginate, mol. wt.: 50,000, 5% in water), and Test group 6 (K alginate, mol. wt.: 50,000, 5% in meal) exhibited a significantly smaller visceral fat amount, as compared with Test group 2 (high-fat food), indicating inhibition of accumulation of visceral fat.
Test group 4 (K alginate, mol. wt.: 25,000, 5% in meal) exhibited a slightly smaller visceral fat (P=0.0695), as compared with Test group 2 (high-fat food), indicating inhibition of accumulation of visceral fat.
Through the above test, potassium alginate is thought to exhibit excellent inhibitory effect on accumulation of visceral fat, which would otherwise be promoted by intake of high-fat food, when potassium alginate is in the form of aqueous solution or has been incorporated into a meal.







TABLE 6







Visceral fat amount










Test groups
Visceral fat amount (g)













1
low-fat food
2.8599 ± 0.1259
***


2
high-fat food
4.9539 ± 0.1386
—


3
K alginate (mol.
3.4313 ± 0.2084
***



wt.: 25,000) in





water (5%)




4
K alginate (mol.
4.2154 ± 0.2144
P = 0.0695



wt.: 25,000) in





meal (5%)




5
K alginate (mol.
3.5511 ± 0.4010
***



wt.: 50,000) in





water (5%)




6
K alginate (mol.
3.8556 ± 0.3561
**



wt.: 50,000) in





meal (5%)





Each of values indicates Av. ± S.E., Fisher's PLSD test was performed with respect to Test group 2 (high-fat food)


(** P < 0.01, *** P < 0.001)






Test Example 2
Inhibitory Effect of Potassium Alginate on Accumulation of Liver Lipid
The livers obtained in Test Example 1 were used. Specifically, each liver which had been frozen at −80° C. was thawed at room temperature, and a portion (0.25 g) taken from the liver was subjected to liver lipid extraction through the Folch method. The amount of lipid in 1 g of liver was calculated. The results are shown in Table 7.
Test group 2 (high-fat food) exhibited a significantly larger liver lipid amount, as compared with Test group 1 (low-fat food), indicating accumulation of fat in the liver (fatty liver).
In contrast, Test group 3 (K alginate, mol. wt.: 25,000, 5% in water), Test group 4 (K alginate, mol. wt.: 25,000, 5% in meal), Test group 5 (K alginate, mol. wt.: 50,000, 5% in water), and Test group 6 (K alginate, mol. wt.: 50,000, 5% in meal) exhibited a significantly smaller liver lipid amount, as compared with Test group 2 (high-fat food), indicating inhibition of accumulation of lipid in the liver.
Through the above test, potassium alginate is thought to exhibit excellent inhibitory effect on accumulation of liver lipid, which would otherwise be promoted by intake of high-fat food, when potassium alginate is in the form of aqueous solution or has been incorporated into a meal.







TABLE 7







Liver lipid amount










Test groups
Liver lipid amount (g/1 g-liver)













1
low-fat food
0.0892 ± 0.0085
***


2
high-fat food
0.1451 ± 0.0096
—


3
K alginate (mol.
0.0971 ± 0.0054
***



wt.: 25,000) in





water (5%)




4
K alginate (mol.
0.1021 ± 0.0066
***



wt.: 25,000) in





meal (5%)




5
K alginate (mol.
0.1060 ± 0.0077
**



wt.: 50,000) in





water (5%)




6
K alginate (mol.
0.0916 ± 0.0080
***



wt.: 50,000) in





meal (5%)





Each of values indicates Av. ± S.E., Fisher's PLSD test was performed with respect to Test group 2 (high-fat food)


(** P < 0.01, *** P < 0.001)

## Claims

1. A method for ameliorating obesity, the method comprising administering potassium alginate to a subject in need thereof or having the subject take potassium alginate for a period of at least 4 weeks.

2. The method of claim 1, wherein ameliorating obesity comprises reducing the accumulation of visceral fat.

3. The method of claim 1, wherein ameliorating obesity comprises reducing the accumulation of liver lipids.

4. The method of claim 1, wherein ameliorating obesity comprises ameliorating fatty liver.

5. The method for ameliorating obesity according to claim 1, wherein potassium alginate has a weight average molecular weight of 10,000 to 60,000.

6. The method of ameliorating obesity according to claim 1, wherein potassium alginate has a weight average molecular weight of 20,000 to 50,000.

7. The method of ameliorating obesity according to claim 6, comprising administering potassium alginate to a subject in need thereof or having the subject take potassium alginate at least once per day.

8. The method of ameliorating obesity according to claim 1, comprising administering potassium alginate to a subject in need thereof or having the subject take potassium alginate for a period of at least 8 weeks.

9. The method of ameliorating obesity according to claim 8, wherein potassium alginate has a weight average molecular weight of 20,000 to 50,000.

10. The method of ameliorating obesity according to claim 9, comprising administering potassium alginate to a subject in need thereof or having the subject take potassium alginate at least once per day.

11. The method of ameliorating obesity according to claim 1, comprising administering potassium alginate to a subject in need thereof or having the subject take potassium alginate for a period of at least 12 weeks.

12. The method of ameliorating obesity according to claim 11, wherein potassium alginate has a weight average molecular weight of 20,000 to 50,000.

13. The method of ameliorating obesity according to claim 12, comprising administering potassium alginate to a subject in need thereof or having the subject take potassium alginate at least once per day.