JP5951512B2 - Composition for improving lipid metabolism comprising cepatiolanes as active ingredient, and food additive - Google Patents

Description

The present invention relates to a pharmaceutical composition and a food additive for inhibiting fat accumulation in the liver, which are effective for preventing, treating and / or improving diseases caused by fat accumulation in the liver such as fatty liver.

Fatty liver is a symptom caused by accumulation of neutral fat or the like in the liver due to excessive eating or drinking. Leaving fatty liver can lead to cirrhosis and liver cancer, as well as increased risk of developing diabetes and the promotion of arteriosclerosis. So far, several substances having an effect of suppressing fat accumulation in the liver have been found, and examples of such substances include processed products of blueberry leaves, seaweed fermented foods, and soy protein ( Patent Documents 1 and 2, Non-Patent Document 5). In addition, it has been reported in the paper that tea, fish oil, unsaturated fatty acids, etc. also have an effect of suppressing the accumulation of fat in the liver. It has also been shown that onion or onion extract has a fatty liver inhibitory action and a body fat accumulation inhibitory action (Patent Documents 4 and 5). However, it has not been specified which component in the onion contributes to its action.

An onion is one of the vegetables widely eaten all over the world since ancient times, and there are many reports regarding showing an effective physiological action on humans (Non-patent Document 1). In 2002, a lacrimatory factor synthase (hereinafter referred to as LFS) was discovered, and an onion lacrimatory factor (hereinafter referred to as LF), propanthial S-oxide, is the precursor S-. The enzyme alliinase acts on 1-propenyl-cysteine sulfoxide (hereinafter referred to as PRENCSO) to become 1-propenylsulfenic acid, and 1-propenylsulfenic acid is isomerized by LFS. The resulting mechanism became clear. For this reason, if the expression of LFS could be suppressed, it was expected that the amount of useful components in the onion would increase and the effect of useful physiological actions would become stronger (Non-patent Document 2).

Based on the results of component analysis of LFS-suppressed onions produced using RNAi technology, LFS-suppressed onions have an increased amount of thiosulfinate compared to normal onions and an increased component called Zwiebelene isomer. (Non-patent Document 3). However, the usefulness of the increased compounds has not been clarified due to the high reactivity of sulfur-containing compounds.

Non-Patent Document 4 clarifies that the planar structure of the component that increases in the LFS-suppressed onion is Formula 1. It was also clarified that the compound has an activity of inhibiting cyclooxygenase-1 (COX-1) and an activity of inhibiting α-glucosidase. However, the fat accumulation inhibitory action of the component of Formula 1 (hereinafter referred to as Cepathiolanes) has not been studied. Moreover, about the optical isomer of the compound shown by Formula 1, it has been reported as an onion component which has a leek flavor (patent document 3). However, no mention was made of health functionality.

JP 2008-189931 A JP 2003-02001 A JP 2010-143866 A JP 2009-089703 A JP 2007-326800 A

Griffiths G et al .; Onions-A Global Benefitto Health. Phytother. Res., 16, 603-615 (2002) Imai S et al .; An onion enzyme that makes theeyes water.Nature, 419, 685 (2002) Eady C. C. et al .; Silencing OnionLachrymatory Factor Synthase Causes a Significant Change in the SulfurSecondary Metabolite Profile.Plant Physiology, 147, 2096-2106 (2008) Aoyagi M et al .; Structure and Bioactivity of Thiosulfinates Resulting from Suppression of Lachrymatory Factor Synthase in Onion.J.Agric.Food Chem, 59, 10893 – 10900, (2011) Ascencio C et al .; Soy Protein Affects SerumInsulin and Hepatic SREBP-1 mRNA and Reduces Fatty Liver in Rats.J. Nutr, 134,522 – 529, (2004)

  An object of the present invention is to provide a novel means capable of suppressing the accumulation of fat in the liver and suppressing the onset of fatty liver.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the planar structure obtained by decomposing the substrate PRENCSO with the enzyme alliinase increases in the LFS-suppressed onion or decomposes with the enzyme alliinase in the absence of LFS. The present inventors have found that a component, that is, Cepathiolanes has an effect of suppressing fat accumulation in the liver, and completed the present invention.

The present invention includes the following inventions.
[1] A pharmaceutical composition for inhibiting fat accumulation in the liver, comprising Cepathiolanes.
[2] An expression inhibitor of a gene involved in synthesis of triglyceride in the liver, characterized by containing Cepathiolanes.
[3] A food additive having an action of suppressing accumulation of fat in the liver, comprising Cepathiolanes.
[4] A food or drink comprising the food additive of [3].
[5] A method for producing a food / beverage composition having an action of inhibiting fat accumulation in the liver, comprising blending Cepathiolanes into a material of the food / beverage product composition.

  ADVANTAGE OF THE INVENTION According to this invention, the novel pharmaceutical composition etc. which can suppress the fat accumulation to a liver are provided. By ingesting the pharmaceutical composition of the present invention, it is possible to prevent, treat and / or improve diseases caused by fat accumulation in the liver such as obesity, hyperlipidemia or impaired glucose tolerance.

FIG. 1 is a graph showing changes in body weight of each group of test mice (control group, Cepathiolanes administration group, PRENCSO administration group). (Example 2) FIG. 2 is a graph showing changes in food intake of each group of test mice (control group, Cepathiolanes administration group, PRENCSO administration group). (Example 2) FIG. 3 is a graph showing changes in water intake in each group of test mice (control group, Cepathiolanes administration group, PRENCSO administration group). (Example 2) FIG. 4 shows the LDL-cholesterol concentration in plasma of each group of test mice (control group, Cepathiolanes administration group, PRENCSO administration group). (Example 2) FIG. 5 is a graph showing plasma HDL-cholesterol concentrations in each group of test mice (control group, Cepathiolanes administration group, PRENCSO administration group). (Example 2) FIG. 6 is a graph showing the total triglyceride concentration in plasma of each group of test mice (control group, Cepathiolanes administration group, PRENCSO administration group). (Example 2) FIG. 7 is a graph showing the total cholesterol concentration in the liver of each group of test mice (control group, Cepathiolanes administration group, PRENCSO administration group). (Example 2) FIG. 8 shows the total triglyceride concentration in the liver of each group of test mice (control group, Cepathiolanes administration group, PRENCSO administration group). (Example 3) FIG. 9 is a diagram showing the expression level of each gene involved in triglyceride synthesis. (Example 3) FIG. 10 is a graph showing changes in body weight of each group of test rats (control group, Cepathiolanes administration group). Example 4 FIG. 11 is a graph showing changes in food intake of each group of test rats (control group, Cepathiolanes administration group). Example 4 FIG. 12 is a graph showing changes in water intake in each group of test rats (control group, Cepathiolanes administration group). Example 4 FIG. 13 is a view showing the total triglyceride concentration in the liver of each group of test rats (control group, Cepathiolanes administration group). Example 4

1. Preparation of components represented by Formula 1 The compound represented by Formula 1 is obtained by treating PRENCSO represented by Formula 2 with alliinase and decomposing it. Although this decomposition reaction proceeds by grinding onion, LFS is contained in ordinary onion grinds, so the degradation product of PRENCSO by alliinase is used for the synthesis of LF (tearing component) by LFS and quickly. Consumed and hard to accumulate. However, the compound represented by Formula 1 can be prepared by the following two methods. Specifically, the first production method is a method of pulverizing a low LFS onion with reduced LFS activity. The second production method is a method of degrading PRENCSO with alliinase in the absence of LFS.

The first method will be described. A low LFS onion is an onion with reduced LFS activity compared to a normal onion and can be obtained by genetic recombination or mutation treatment. A preferred low LFS onion is an LFS activity that is 20 times or less, preferably 40 times or less, more preferably 1/160 or less, particularly preferably 1/400 or less of the LFS activity of the parent strain (normal onion). Have

Next, the second manufacturing method will be described in detail. S-1-propenyl-cysteine sulfoxide (PRENCSO) used as a raw material has a structure represented by Formula 2 when it is naturally derived. However, a stereoisomer of PRENCSO different from the natural type (for example, an isomer having a different configuration of sulfoxide) can also be used as the raw material PRENCSO.

PRENCSO used as a raw material may be synthesized or may be derived from onion or the like. Furthermore, a composition containing PRENCSO can be used as a PRENCSO raw material. For example, juice and paste (heated onion juice, heated onion paste) prepared by heat-treating onion to inactivate the enzyme and then crushing it is a composition containing PRENCSO, and it is to be subjected to alliinase treatment as a PRENCSO raw material Can do.

Alliinase (EC4.4.1.4, S-alk (en) yl-L-cysteine sulfoxide
lyase) is an enzyme that breaks down alkyl cysteine sulfoxide into dehydroalanine and sulfenic acid, and it was named because it breaks down alliin. It is now also called CS lyase (Source: Food function of spice ingredients, edited by Kazuo Iwai, Nobuji Nakatani, Mitsuyokan P174-175 (1989)). The origin of alliinase that can be used in the second production method is not particularly limited. The source is the same as above).

Conditions for treating PRENCSO with alliinase are not particularly limited. However, since the properties of alliinase vary depending on the origin, the degradation of PRENCSO can be made more efficient by adapting the characteristics of individual alliinase using the reaction conditions. For example, when alliinase is derived from garlic, since the optimum pH range is wide, the pH may be in the range of 5 to 8, and the temperature is optimally 37 ° C. On the other hand, when using an onion-derived alliinase, the enzyme activity is optimized when the pH is adjusted to 7.5 to 8.6, and when using an alliinase derived from the leaf of Welsh onion, the pH is optimized at 7.0 (source: spices) Ingredient food function Kazuo Iwai, Enji Nakatani, edited by Koseikan Co., Ltd. P174-175 (1989)). Since alliinase uses pyridoxal phosphate as a coenzyme, use of a buffer to which pyridoxal phosphate is added as in Example 1 is also useful for optimizing the activity of alliinase.

Cepathiolanes are produced in a short time after treating PRENCSO with alliinase, and thereafter decomposed over time. Therefore, it is preferable to extract the compound immediately after the reaction solution, preferably within 2 hours. The product obtained by decomposing PRENCSO with alliinase removes solids by an operation such as filtration or centrifugation, if necessary. Depending on the operation performed in the next step, the extract may be used as it is, or the solvent may be distilled off and partially concentrated or dried. The obtained extract can be used after being concentrated or dried and then washed and purified with an appropriate solvent, for example, an insoluble solvent, or further dissolved or suspended in an appropriate solvent. . Furthermore, in the present invention, for example, the obtained solvent extract can be used as a dry extract of onion-derived components by subjecting it to ordinary means such as drying under reduced pressure or freeze drying.

2． Applications The active ingredient Cepathiolanes according to the present invention has an action of suppressing the accumulation of liver cholesterol and triglycerides, as shown in the examples described later. Specifically, as shown in FIGS. 7 and 8, the liver triglyceride concentration is lower in the mouse / rat test group administered with the treatment compared to the mouse / rat test group not administered with Cepathiolanes. doing.

In addition, as shown in FIG. 9, the mechanism of action of the liver is by suppressing the expression of LXRα, which is a gene involved in triglyceride synthesis in the liver, and downstream genes (eg, SREBP1c, FAS, DGAT2, etc.). It is considered that the accumulation of medium triglyceride is suppressed and the onset of fatty liver is suppressed.
Therefore, onion-derived components including Cepathiolanes and Cepathiolanes can treat, prevent, or improve diseases associated with these genes by suppressing the expression of genes related to triglyceride synthesis in the liver, such as LXRα and SREBP1c.
Diseases or symptoms that can be treated, prevented, or ameliorated by suppression of LXRα expression include abnormal lipid metabolism (eg, hyperlipidemia, arteriosclerosis, and fatty liver), particularly hyperlipidemia (eg, hypercholesterolemia) Disease, low HDL-cholesterolemia, hypertriglyceridemia); obesity; and weight gain.

In the present invention, the above-mentioned cepathiolanes can be used alone or in solid or liquid form, but a pharmaceutically acceptable carrier or additive is added to the above-mentioned cepathiolanes. It can also be prepared as a solid or liquid pharmaceutical composition.
The form of the pharmaceutical composition of the present invention is not particularly limited, but is preferably a form suitable for oral administration. For example, solid compositions for oral administration (solid pharmaceutical preparations) include tablets (including sugar-coated tablets), pills, capsules, fine granules, granules and the like, and liquid compositions for oral administration (liquid pharmaceuticals). Formulations may take the form of emulsions, solutions, suspensions, syrups and the like. In these preparations, in addition to the active ingredients, excipients, binders, disintegrants, lubricants, colorants, flavoring agents, pH adjusting agents, and the like can be appropriately blended depending on the dosage form. And can be formulated according to conventional methods.

The pharmaceutical composition of the present invention may contain Cepathiolanes which are active ingredients and other functional ingredients such as vitamins in addition to the carrier or additive. The composition ratio of the active ingredients of the pharmaceutical composition cannot be specified because it varies depending on the purpose of use and the like, but can be appropriately adjusted using an effective dose described later as a reference value.

The food additive according to the present invention is a food additive containing an effective amount of the active ingredient according to the present invention. Here, “containing an effective amount of an active ingredient” refers to such a content that an active ingredient is ingested within a range as described later when an amount normally consumed in each food or drink is ingested. In the food according to the present invention, the active ingredient according to the present invention can be blended in the food as it is or in the form of the composition as described above.
In the present invention, “food” is used in the meaning including health food, functional food, food for specified health use, and food for the sick.

Since the composition and food additive according to the present invention are onion-derived components or derivatives that humans have taken as food and drink for many years, they have low toxicity and mammals (eg, humans, mice, rats, rabbits, etc.) It is used safely. The dose or intake of the active ingredient according to the present invention can be determined depending on the recipient, the age and weight of the recipient, symptoms, administration time, dosage form, administration method, drug combination, and the like. For example, when the active ingredient according to the present invention is taken orally, it can be taken at 0.3 to 4 mg / kg body weight per adult in terms of the substrate PRENCSO.

Example 1-1.
Preparation of purified PRENCSO solution (1) Extraction of PRENCSO from heated onion The peel of 3 balls (1Kg) of raw onion was peeled off, wrapped and heated in a microwave oven at 600W for 12 minutes. The heated onion was put in a mixer, and after adding an equal amount of distilled water, it was roughly crushed, and the crushed liquid was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected, cation exchange resin IR120B (H type) was added and stirred, and then the resin was collected by suction filtration. Distilled water (1 L) was added to the recovered resin, and concentrated aqueous ammonia was added to adjust the pH to 8.5. The supernatant was recovered by suction filtration, and ammonia water having a pH of 8.5 was added to the remaining resin again. Again, the supernatant was collected by suction filtration. The resulting solution was neutralized to pH 7.0 by adding 1N hydrochloric acid. The solution was concentrated to dryness using an evaporator.

(2) Purification of PRENCSO 50 ml of distilled water was added to the residue and dissolved. The resulting crude PRENCSO solution was purified by medium pressure reverse phase chromatography. If necessary, further purification was performed by HPLC (column: ODS, mobile phase: acidic water pH 3.3, temperature: 35 ° C., UV: 230 nm). The eluate obtained from the column was dried using an evaporator and a freeze dryer to obtain purified PRENCSO powder (about 100 mg).
Repeat steps (1) and (2) above to make the amount required for the following test, dissolve in distilled water to a concentration of 20 mg / ml or 40 mg / ml, and keep it at -20 ° C or lower until use. Saved with.

Example 1-2.
Preparation of purified alliinase solution (1) Grinding and acid precipitation of garlic First, a mixer mug was placed in a refrigerator and allowed to cool. In addition, the rotor was set in a low-temperature centrifuge, and the temperature was set at 4 ° C. to be cooled. An equal amount of buffer A (described later) was added to garlic pieces (100 g) and pulverized with a mixer. Fasten the double gauze with a rubber band to the mouth of a mug placed on ice. The ground material was poured over the gauze and filtered. After a certain amount of filtrate was obtained, the ground material on the gauze was wrapped with gauze and squeezed. The obtained filtrate was centrifuged at 12000 rpm for 10 minutes at 4 ° C., and the supernatant was recovered. While stirring the collected centrifugal supernatant on ice, acetic acid was added while monitoring the pH, and the pH was adjusted to 4.0. When the pH reached 4.0, it was allowed to stand for 5 minutes. The sample from which the precipitate appeared was centrifuged at 12000 rpm for 10 minutes at 4 ° C., and the centrifuged pellet was recovered (buffer A was used).
The collected pellets were made 50 ml to 100 ml (buffer A) and allowed to stand at 5 ° C. for 30 minutes. Samples were centrifuged for 10 minutes at 12000 rpm at 4 ° C. The pH of the collected centrifugal supernatant was adjusted to 6.5 using 1N aqueous sodium hydroxide solution. This was used as the crude alliinase extract.

(2) Hydroxyapatite column treatment The above alliinase crude extract (centrifugal supernatant) was applied to a hydroxyapatite column equilibrated with buffer A. A yellow band was adsorbed on the hydroxyapatite column. The hydroxyapatite column to which the sample was applied was washed with 300 ml of buffer A. 300 ml of buffer C (described later) was passed through the hydroxyapatite column after washing to elute it. The eluate was fractionated by 10 ml using a fraction collector, and the fractions in which the yellow eluate was fractionated were collected.

(3) Purification of alliinase by ConA column The concentration of calcium ions and magnesium ions in the sample solution was increased by adding 20 mM calcium chloride / magnesium chloride solution in a volume 1/20 times the collected fraction (20 ml). After that, it was applied to a ConA column equilibrated with starting buffer (recommended buffer described in the ConA Sepharose 4B manual). The ConA column after applying the sample was washed with 50 ml of starting buffer. 50 ml of ConA elution buffer (recommended buffer described in the ConA Sepharose 4B manual) was passed through the ConA column after washing to elute. The yellow eluate was collected.

The buffer A (pH 7.0) (50 mM buffer A for purifying alliinase) was prepared as follows. A 50 mM dipotassium hydrogen phosphate solution (5.22 g dissolved in 600 ml) and a 50 mM potassium dihydrogen phosphate solution (3.4 g dissolved in 500 ml) were prepared. While monitoring the pH, both were mixed to adjust the pH to 7.0. Glycerol was added 1x vol to 9x vol of the finished buffer and mixed well. 5.3 mg of pyridoxal phosphate was added to and mixed with 1 L of the glycerol-containing buffer. The completed buffer was stored at 10 ° C.

Buffer C (pH 7.0) (500 mM buffer C for purifying alliinase) was prepared as follows. A 500 mM dipotassium hydrogen phosphate solution (43.6 g dissolved in 500 ml) and a 500 mM potassium dihydrogen phosphate solution (34.0 g dissolved in 500 ml) were prepared. While monitoring the pH, both were mixed to adjust the pH to 7.0. Glycerol was added 1x vol to 9x vol of the finished buffer and mixed well. 5.3 mg of pyridoxal phosphate was added to and mixed with 1 L of the glycerol-containing buffer. The finished buffer was kept at 10 ° C.
The above operations (1), (2), and (3) were repeated to produce the amount necessary for the following test. The prepared alliinase was appropriately diluted with 100 mM potassium phosphate buffer pH 6.5 so as to be 500 U / ml, and stored at −80 ° C. or less until use.

The lipid metabolism improvement effect was evaluated using C57BL / 6N mice (female). In other words, 5 to 7 5-week-old C57BL / 6NCrlCrlj females (Nippon Charles River Co., Ltd.) per group were bred for one week with free intake of commercial powder feed MF (Oriental Yeast Co., Ltd.) and water. After that, the group was divided into three groups: a control group, a Cepathiolanes administration group, and a PRENCSO administration group so that the average body weight of each group was equalized by a computer-based complete random sampling method, and the diet was switched from MF to a high fat, high cholesterol diet . Distilled water was used as the control group, and 40 mg / ml purified PRENCSO prepared in Examples 1-1 and 1-2 and 500 U / ml purified alliinase were mixed at a ratio of 1: 1 for the Cepathiolanes administration group and reacted at room temperature for 1 minute. The reaction product and the PRENCSO administration group were prepared by mixing 40 mg / ml purified PRENCSO prepared in Example 1-1 and distilled water in a ratio of 1: 1 to 200 μL / 20 g (mouse body weight), respectively. As described above, forced oral administration was performed using a disposable syringe and an oral sonde. The administration was performed from 9 am to 12 am, and the administration period was 42 days.

By the way, the high fat and high cholesterol diet was mixed in the ratio of powdered general feed: lard: cholesterol: cholic acid = 78.7: 20: 1: 0.3. The preparation method is to first weigh the required amount of powdered feed, cholesterol and cholic acid, transfer to Kenmix mixer (Aiko Seisakusho Co., Ltd.), add the required amount of lard little by little while stirring well with Kenmix mixer, and more fully Mixed. The preparation was performed once a week, and after preparation, it was put in a container and stored at room temperature.

During the test period, general conditions and life and death were observed once a day for all cases, and body weight, food intake, and water intake were measured once a week (FIGS. 1-3). In addition, fasting was performed from 16:00 on the 42nd day of administration, and the next day, whole blood was collected from the abdominal aorta under ether anesthesia. The collected blood was placed in a Nunk tube containing EDTA 2K and centrifuged at 1700 × g for 15 minutes at 4 ° C. to collect plasma. After all blood collection, the livers of all cases were removed. The collected plasma and liver were stored frozen at −80 ° C. until used for various measurements. Plasma determined LDL-cholesterol, HDL-cholesterol, and total triglyceride, and liver determined total cholesterol and total triglyceride.

LDL-cholesterol, HDL-cholesterol, and total triglycerides in plasma are separated according to particle size by HPLC with two gel filtration columns connected in series, and the system is used to quantify the concentration by online enzymatic reaction. (Skylight Biotech, Inc., Akita, Japan). Total cholesterol and total triglycerides in the liver are determined by the Folch method (Folch J et al., J Biol Chem
226: 497-509 (1957)), after extracting total lipids from the liver, total cholesterol
(Sekisui Medical Co., Ltd.) and total triglycerides were quantified using Collestest TG (Sekisui Medical Co., Ltd.). The respective results are shown in FIGS. The measured value was expressed as mean value ± standard error, and the statistical analysis used Dunnett's test because uniformity was recognized by the Bartlett test. Significance levels were expressed as 5% (*) and 1% (**).

During the test period, there was no difference in food intake and water intake between the control group, Cepathiolanes administration group, and PRENCSO administration group (Figs. 2 and 3). In the Cepathiolanes administration group, the total cholesterol and triglyceride concentrations in the liver were significantly reduced compared to the control. However, such an effect was not confirmed in the PRENCSO administration group (FIGS. 7 and 8). In addition, body weight and total triglyceride in plasma tended to decrease in the Cepathiolanes administration group compared with the control group, although not significantly decreased (FIGS. 1 and 6).

The action mechanism of the Cepathiolanes group that had an effect of suppressing fat accumulation in the liver in Example 2 was elucidated. In Example 2, 200 mg of the control group and the Cepathiolanes administration group among the liver tissues cryopreserved at −80 ° C. were immersed in RNAlater ® -ICE (manufactured by Life Technologies Corporation) and stored at −20 ° C. RNA was collected from about 50 mg of liver tissue stored in RNAlater ® -ICE using Isogen (Nippon Gene) according to the attached manual. Collected RNA is NanoDrop
After measuring the concentration using 1000 (Thermo Fisher Scientific), reverse transcription was performed using ReverTra Ace ® qPCR RT Master Mix with gDNA Remover (TOYOBO) according to the attached manual to synthesize cDNA. Quantification of each gene was performed using the 7900 HT Fast Real-Time PCR System (Applied) using the primer set shown in the table below and THUNDERBIRD ® SYBR ® qPCR Mix (TOYOBO).
Biosystems). The results were corrected by the expression level of 36B4, which is one of the housekeeping genes, and the expression level of each gene was shown with the expression level of the control group being 100% (FIG. 9). The measured values are expressed as mean ± standard error, and statistical analysis is performed using the Student t test when uniformity is found by F test, and Mann- when the uniformity is not found by variance. Whitney test was used. Significance levels were expressed as 5% (*) and 1% (**). Table 1 shows the primer sequences used for qRT-PCR.

As a result of statistical analysis, the expression of LXRα, SREBP1c, FAS, GPAT, and DGAT2 was significantly suppressed. SCD1, DGAT1, PPARγ, and CD36 also showed a tendency to be suppressed, although not significantly different. All of these genes have been reported to be involved in triglyceride synthesis in the liver. From the above results, it was suggested that the lipid metabolism improvement effect by ingestion of Cepathiolanes was caused by the suppression of triglyceride synthesis in the liver.

  Evaluation was performed using SD rats (female) for the purpose of confirming that Cepathiolanes showed an effect of improving lipid metabolism at a lower concentration than in Example 2. That is, six 5-week-old Slc: SD rat females (Japan SLC Co., Ltd.) per group were bred for 8 days with free intake of commercial powder feed MF (Oriental Yeast Co., Ltd.) and water. After that, divided into two groups, Cepathiolanes administration group and control group, so that the average body weight of each group becomes equal by a complete random sampling method using a computer, the feed is changed from MF to high fat and high cholesterol diet described in Example 2 Switched. In the Cepathiolanes administration group, 20 mg / ml purified PRENCSO prepared in Examples 1-1 and 1-2 and 500 U / ml purified alliinase were mixed at a ratio of 2: 1 and reacted at room temperature for 1 minute. Thereafter, the reaction product was placed on ice and administered by gavage using a disposable syringe and an oral sonde so that the reaction product was 150 μl / 200 g (rat body weight). In the control group, distilled water was forcibly orally administered using a disposable syringe and an oral sonde so as to be 150 μL / 200 g (rat body weight). Each administration was performed between 9:00 am and 12:00 am, and the administration period was 56 days.

During the test period, general conditions and life and death were observed once a day, body weight was measured twice a week, and food intake and water intake were measured once a week. Fasting was performed from 16:00 on the 55th day of administration, and 2 hours after the completion of administration on the 56th day (24 hours after administration on the 55th day), whole blood was collected from the rat abdominal aorta under ether anesthesia.

After all blood collections were completed, the livers of all cases were removed and weighed. The collected liver was stored frozen at −80 ° C. until used for various measurements. Total triglyceride in the liver was quantified.

Total triglyceride in the liver is determined by the Folch method (Folch J
et al., J Biol Chem 226: 497-509 (1957)), total lipids were extracted from the liver, and then quantified using Cholestest TG (manufactured by Sekisui Medical). The results are shown in FIGS. The measured values are expressed as mean ± standard error, and statistical analysis is performed using the Student t test when uniformity is found by F test, and Mann- when the uniformity is not found by variance. Whitney test was used. Significance levels were expressed as 5% (*) and 1% (**).
During the test period, there was no difference in food intake and water intake between the control group and the Cepathiolanes administration group (Figs. 11 and 12). In the Cepathiolanes administration group, the body weight and the triglyceride concentration in the liver tended to decrease compared to the control. (Figures 10 and 13). From the above results and the results of Example 2, it was confirmed that Cepathiolanes showed an effect of reducing triglyceride in the liver in the range of 15 mg / kg to 200 mg / kg in rodents such as rats and mice. When this concentration was converted into a human dose in consideration of sensitivity to humans, it was estimated that the effect was approximately 0.3-4 mg / kg.

Claims (5)

A pharmaceutical composition for inhibiting fat accumulation in the liver, comprising a component of Formula 1 (hereinafter referred to as Cepathiolanes).   An expression inhibitor of a gene involved in synthesis of triglyceride in the liver, characterized by containing Cepathiolanes. A food additive for suppressing fat accumulation in the liver, comprising Cepathiolanes. A food or drink composition for suppressing fat accumulation in the liver, comprising the food additive according to claim 3. The manufacturing method of the food-drinks composition for fat accumulation suppression to a liver including mix | blending Cepathiolanes with the material of food-drinks composition.