JP6101888B2 - Fatty liver suppressant - Google Patents

Description

  The present invention relates to a drug capable of effectively suppressing fatty liver.

  In general, hepatitis is classified into viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, drug hepatitis, autoimmune hepatitis, primary biliary cirrhosis, and the like depending on the cause.

  Among these hepatitis, nonalcoholic steatohepatitis is called fatty liver and has been conventionally considered to be a benign liver disease. In recent years, however, dietary habits have changed in Japan, and lifestyle-related diseases have become a problem as they become overnourished. Along with this, it is becoming clear that fatty liver is directly related to the onset and progression of metabolic syndrome and arteriosclerosis, and it has also been found that hepatitis and cirrhosis may progress to liver cancer. It was.

  On the other hand, as the liver is said to be a silent organ, there is almost no subjective symptom, especially at the beginning of each disease. In the case of fatty liver as well, there is no subjective symptom at the beginning, and general symptoms of liver disease such as tiring, dullness, and loss of appetite appear as the patient progresses. It is often made worse by leaving it alone.

  Therefore, it is important to try to prevent fatty liver from the usual lifestyle, and it is preferable to improve it at an early stage.

  Drugs for treating insulin resistance and diabetes are often used as therapeutic agents for fatty liver. For example, Patent Document 1 discloses a PPARα (peroxisome proliferator-activated receptor α) activator as a therapeutic agent for fatty liver. However, such a drug is a synthetic drug used for the treatment of diabetes itself and is considered to have strong side effects and is not suitable for preventive use. For example, fenofibrate, an agonist of PPARα, is known to have side effects such as liver hypertrophy.

  On the other hand, herbal medicines that are said to be effective for fatty liver, not synthetic drugs, include Daihuang, Garlic, Hawthorn, Sadako, Dansang, Licorice, Hull, Toki, Tochu, Chen, Hakone, Shihu, Jinji , Huang Huang, gold and silver flowers, Huang Huang, Mulberry leaves, Inchinkou, Sawa Hou, and lewd sheep are known.

  By the way, Patent Document 2 describes that an extract of a plant belonging to the genus Salacia shows an effect for the treatment of diabetes and the like, and a method for judging the activity (quality) of the extract based on the content of mangiferin Is disclosed.

JP 2002-220345 A JP 2002-267655 A

  As described above, various drugs that are said to have an effect on fatty liver have been known so far.

  However, synthetic drugs are difficult to use for preventive purposes due to problems such as side effects. On the other hand, fatty liver often progresses with no subjective symptoms, or even if there is a subjective symptom, if it is left confused with a cold or the like, prevention is very important.

  In addition, the present inventors selected animal drugs that are considered to be effective for fatty liver from herbal medicines that are considered to be relatively safe, and conducted animal experiments. That is, a hot water extract was obtained from Daiki and was orally administered to experimental animals at a high dose of 500 mg / kg for 5 weeks, but no effect was found.

  Furthermore, although Patent Document 2 describes that a plant extract of Salacia is effective for diabetes and the like, there is no description or suggestion of an effect on fatty liver. Moreover, in the quality determination method of Patent Document 2, mangiferin is adopted as a reference substance, and the correlation between the inhibitory activity of α-glucosidase that degrades starch and the like into glucose and the content of mangiferin has been experimentally shown. . However, in Patent Document 2, not only fatty liver is not described, but mangiferin is not a direct component of the blood glucose level increase inhibitory effect, and the active ingredient remains unknown.

  Under such circumstances, the problem to be solved by the present invention is to provide a drug for suppressing fatty liver that is safe and can be taken every day.

  The inventors of the present invention have made extensive studies to solve the above problems. As a result, some of the plants belonging to the genus Salacia that were originally grown in India and Sri Lanka and used as folklore medicines were first introduced in Japan in 1995 by the present inventors. The present invention was completed by finding that it was extremely effective for suppression.

  The fatty liver suppressant according to the present invention is characterized by containing, as an active ingredient, an extract of at least one root selected from Salacia reticulata and Salacia chinensis.

As the above-mentioned extract which is an active ingredient of the fatty liver inhibitor according to the present invention, an extract containing at least one selected from aqueous solvents, particularly water and C _1-4 alcohols is preferable. Moreover, as the extract, among the extracts extracted with an aqueous solvent, an extract that is further soluble in methanol is preferable. Furthermore, as said extract, what contains 0.5 mass% or more of mangiferins is also preferable. The excellent effect of improving fatty liver of these extracts has been confirmed by experiments by the present inventors.

  Fatty liver is also closely related to alcohol. That is, if a large amount of alcohol is ingested, it becomes easy for the liver to get fat. Recently, however, fatty liver caused by excessive intake of nutrients such as overeating has become a serious problem because it can cause other lifestyle-related diseases. It has been confirmed by experiments by the inventor that the fatty liver inhibitor according to the present invention exhibits a particularly excellent improvement effect on non-alcoholic fatty liver.

  The fatty liver suppressant according to the present invention is very safe because it contains an extract of a plant of the genus Salacia, which is also used as a tea, and can be taken or taken daily. Moreover, the fatty liver inhibitor which concerns on this invention is very excellent in the preventive action and therapeutic action of a fatty liver. Therefore, this invention is useful as what can suppress the fatty liver which became a problem by the westernization of the eating habits in recent years.

FIG. 1 is a photograph of a hepatoxylin-eosin stained liver tissue piece from a control rat fed with free intake of water and normal feed. FIG. 2 is a photograph of hepatoxylin-eosin stained liver tissue pieces from control rats fed with fructose loading. FIG. 3 is a photograph of hepatoxylin-eosin stained liver tissue pieces of control rats bred while administering fenofibrate, a lipid lowering drug, in addition to fructose load. FIG. 4 is a photograph of hematoxylin and eosin stained liver tissue pieces of control rats bred with administration of a relatively low dose of mangiferin in addition to fructose load. FIG. 5 is a photograph of hematoxylin and eosin-stained liver tissue pieces from control rats fed with a relatively high dose of mangiferin in addition to fructose load. FIG. 6 is a photograph of a liver tissue piece of a control rat bred with free intake of water and normal feed stained with oil red O. FIG. 7 is a photograph of a liver tissue piece of a control rat bred with fructose loading and stained with oil red O. FIG. 8 is a photograph of oil red O-stained liver tissue pieces of control rats bred while administering a relatively low dose of mangiferin in addition to fructose load. FIG. 9 is a graph for comparing the ratio of oil droplet portions in the photographs of FIGS.

  The fatty liver suppressant according to the present invention is characterized by containing, as an active ingredient, an extract of at least one root selected from Salacia reticulata and Salacia chinensis.

  Salacia reticulate and Salacia chinensis are plants of the asterae family widely distributed in subtropical regions such as India, Sri Lanka, and Southeast Asia. Therefore, it can be obtained from these countries.

  In the present invention, either Salacia reticulata or Salacia chinensis may be used as a raw material, or a combination thereof may be used.

  The fatty liver inhibitor which concerns on this invention contains the extract of the root part of the said plant as an active ingredient. The root portion is a portion of the plant that exists in the ground, and it is preferable to use a portion that can be clearly distinguished from the stem of the ground portion in terms of color and the like.

  In the extraction, the root of the plant may be dried after washing. As a drying method, natural drying, heat drying, reduced pressure drying, or the like can be used in appropriate combination. In extraction, the root may be coarsely cut and pulverized.

As the extraction solvent for obtaining the extract according to the present invention, an aqueous solvent is preferably used. In the present invention, the aqueous solvent refers to water and a water-miscible organic solvent that is liquid at 30 ° C. Examples of water-miscible organic solvents include methanol, ethanol, 1-propanol, 2-propanol, n-butanol, isobutanol, s-butanol, t-butanol C _1-4 alcohols; ethers such as diethyl ether and tetrahydrofuran Examples thereof include amide solvents such as dimethylformamide and dimethylacetamide; sulfoxide solvents such as dimethyl sulfoxide.

As the solvent used in the present invention, a solvent selected from the above may be used alone, or two or more may be selected and mixed. For example, a C _1-4 alcohol aqueous solution (simply referred to as “C _1-4 alcohol water” in some cases) of 10 v / v% or more and 90 v / v% or less can be used.

  The amount of the extraction solvent used is not particularly limited and may be adjusted as appropriate. For example, it can be about 1 L / kg or more and 50 L / kg or less with respect to the root mass.

  The extraction temperature can also be adjusted as appropriate, for example, 60 ° C. or higher and 120 ° C. or lower. In addition, when extracting above the boiling point of a solvent, it is preferable to seal or pressurize an extraction container.

  The extraction time can also be adjusted as appropriate, for example, about 30 minutes or more and 10 hours or less.

  Moreover, in order to improve extraction efficiency, you may repeat extraction operation 2 times or more. That is, after normal extraction, the root and the solvent are separated by filtration, centrifugation, or the like, and the operation of adding fresh solvent to the root again and extracting is repeated to combine the extracts. The number of extractions in this case is preferably 2 or more, 5 or less, more preferably 2 or 3, and most preferably 2.

  The extracted liquid mixture can be used as it is, but normal post-treatment may be performed. For example, the root and the solvent may be separated by filtration or centrifugation, or the separated extract may be dried by appropriately combining heating drying, reduced pressure drying, freeze drying, and the like.

  The extract according to the present invention may be fractionated according to the solubility in a solvent. For example, the present inventor has experimentally found that a water extract is fractionated in accordance with the concentration of methanol water, and in particular, a methanol-soluble component exhibits an extremely excellent effect of improving fatty liver.

  The dosage form of the fatty liver inhibitor according to the present invention is not particularly limited. For example, as described above, it may be a mixed solution containing the root and the extract, or the extract itself from which the root is removed after extraction. In addition, it is also conceivable that the extract once dried is made into a liquid preparation such as a solution or an emulsion preparation for administration as an injection. However, as shown in the examples below, the fatty liver inhibitor according to the present invention is highly effective in oral administration. Therefore, it is preferable to use an oral preparation from the viewpoint of ease of ingestion and administration.

  Although it does not restrict | limit especially as an oral agent, For example, a tablet, a powder, a capsule, a sugar-coated tablet, a granule etc. can be mentioned. For the fatty liver inhibitor according to the present invention, a pharmaceutically acceptable additive may be used in accordance with the dosage form. Examples of such additives include excipients, bases, preservatives, auxiliaries, stabilizers, wetting agents, pH adjusting agents, antioxidants, colorants, sweeteners and the like. Moreover, you may make a solution and suspension into a drink, and may add to a general diet.

  The administration frequency and dosage of the fatty liver inhibitor according to the present invention may be adjusted as appropriate depending on whether it is prophylactic or therapeutic use, the severity of fatty liver, the age, sex, condition, etc. of the patient. . According to experimental data to be described later, a significant fatty liver inhibitory effect was confirmed by daily administration of 500 mg of the dry extract per rat body weight (kg). From these results, it is preferable that the dosage for humans is about 1 mg / kg body weight or more and about 1 g / kg body weight or less per day for the dried extract. The number of administrations per day is preferably 1 or more and 5 or less, more preferably 1 or more and 3 or less, and even more preferably 1 or more and 2 or less.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Example 1 Preparation of SR-Derived Extract of the Invention In 2010, Salacia reticulata (SR) native to Tamil Nada on the eastern southernmost side of India was collected and allowed to dry naturally. The root portion of 1.0 kg was roughly cut, put into 10 L of hot water, and heated for 3 hours. Subsequently, it filtered and isolate | separated the rough cut root part, and it applied to the same extraction operation again, and obtained the extract. The extracts were combined and concentrated under reduced pressure at 45 ° C. or lower to obtain 69 g of SR root extract. The extraction yield was about 7%.

Example 2 Preparation of SC-derived extract of the present invention The root of SC was obtained in the same manner as in Example 1 except that Salacia chinensis (SC) native to Khon Kaen in northeastern Thailand was collected and used in 2010. 70 g (extraction yield: about 7%) of the extract was obtained.

Example 3 Fatty Liver Improvement Action Test Male Sprague-Dawley rats (hereinafter referred to as “SD rats”) having a body weight of 210 to 230 g were placed in a light and dark breeding room every 12 hours at room temperature 21 ± 1 ° C. and humidity 55 ± 5%. Feed and water were freely taken and pre-bred for 1 week.

  28 rats were randomly divided into 4 groups, 7) 1) water administration group, 2) fructose administration group, 3) fructose + SR root extract administration group, 4) fructose + SC root extract administration group, The animals were raised for 9 weeks. Fructose was given using a 10 mass% aqueous solution instead of water. Each extract was suspended in a 5% aqueous gum arabic solution and forcibly orally administered at a rate of 500 mg / kg over 10 weeks between 9 am and 10 am daily using a rat sonde. During the breeding, water or fructose solution and feed intake were measured. Fasted from the night before the 9th week (63rd day), the body weight was measured after the 10th week and sacrificed, and the liver was taken out and weighed. The liver was frozen with liquid nitrogen and stored at -80 ° C until measurement.

  Neutral fat and cholesterol in the liver were measured as follows. 100 mg of liver tissue was collected, homogenized, and extracted with 2 ml of isopropyl alcohol. The obtained extract was centrifuged at 3,000 rpm to obtain a supernatant. The amount of neutral fat and cholesterol in the supernatant were measured using a commercially available kit.

  First, Table 1 shows the total fructose intake, total feed intake, and changes in body weight of each group. In Table 1, “*” indicates a case where there is a significant difference at p <0.05 with respect to the fructose administration group in the t test.

  As shown in Table 1, no difference was found in the total intake of fructose in any group. Regarding feed, a decrease in intake was observed in the group ingested fructose. There was no difference between groups in terms of body weight.

  Table 2 shows the liver weight, liver weight / body weight ratio, liver neutral fat amount, and liver total cholesterol amount with respect to the liver removed at 10 weeks. In Table 2, “*” indicates a case where there is a significant difference at p <0.05 with respect to the fructose administration group in the t test.

  As shown in the results of Table 2, the amount of triglyceride in the liver tissue significantly increased with the intake of fructose relative to the control group (water only intake). However, administration of SR and SC root extracts significantly decreased the amount closer to the control group. Therefore, it was demonstrated that the extract according to the present invention can effectively treat fatty liver due to overnutrition.

Example 4 Fatty liver improving effect test As shown in the results of Example 3 above, SR and SC root extracts were found to be effective against fructose-induced fatty liver, and thus the SC root extract was fractionated. Measurements were made. Specifically, the SC root extract (50 g) obtained in Example 2 was subjected to column chromatography (GE Healthcare, Sephadex LH-20), 100% water elution part, water: methanol = 50. : 50 fractions and methanol 100% fraction were separated into three fractions. Hereinafter, each fraction is referred to as SC fractions 1 to 3, respectively. The yield of each fraction was 32 g, 6.5 g, and 6.8 g, respectively, with respect to 50 g of SC root extract.

  Based on the above yield, the SC fraction 1 dose was 200 mg / kg, the SC fraction 2 and SC fraction 3 doses were 50 mg / kg, and fenofibrate, a lipid-lowering drug (manufactured by Sigma), was used as a positive control. The experiment was conducted in the same manner as in Example 3 except that was orally administered at a dose of 20 mg / kg. First, Table 3 shows the total fructose intake, the total feed intake and the body weight after 10 weeks of breeding in each group. In Table 3, “*” indicates a case where there is a significant difference at p <0.05 with respect to the fructose administration group in the t test.

  As shown in the results of Table 3, there was a decrease in intake in the group ingested fructose, and there was an inhibitory effect on body weight and feed intake by fenofibrate, as well as total fructose intake, total feed intake and body weight. There was no difference.

  Next, regarding the liver excised at 10 weeks, the liver weight, liver weight / body weight ratio, liver neutral fat amount and liver total cholesterol amount are shown in Table 4. In Table 4, “*” indicates a case where there is a significant difference at p <0.05 with respect to the fructose administration group in the t test.

  As shown in Table 4, when fenofibrate, a lipid lowering drug, was administered in addition to fructose load, the amount of triglyceride in the liver was significantly decreased while the ratio of liver weight to body weight was significantly increased. Hepatic hypertrophy was observed. On the other hand, when SC fraction 3 was administered in addition to the fructose load, the amount of neutral fat in the liver could be significantly reduced without significantly increasing the liver weight / body weight ratio. Thus, it was clarified that the methanol-solubilized portion of the SC root aqueous extract has an even more excellent effect of improving fatty liver.

Example 5 Fatty liver improving effect test As shown in Example 4 above, since the effect of improving fatty liver was clearly observed in the SC extract fraction 3, the components of the SC extract fraction 3 were analyzed.

  First, the SC extract fraction 3 solution was separated by thin layer chromatography (Silicagel 60F254, manufactured by Merck & Co., Inc.), and then a 10% cerium sulfate aqueous solution was sprayed on it and heated, and there was a spot that strongly colored in red. . From this result, it was estimated that SC extract fraction 3 contained a phenolic compound.

  Next, the SC extract fraction 3 was fractionated by chromatography or the like, the light intensity was measured, and analysis by NMR strongly suggested the presence of mangiferin, a polyphenol compound. Therefore, an experiment was conducted in accordance with Example 3 using mangiferin (hereinafter referred to as “MG”) alone. That is, 7 rats each of 35 rats: 1) Water administration group (control group), 2) Fructose administration group, 3) Fructose + fenofibrate administration group, 4) Fructose + low MG administration group, 5) Fructose + high The experiment was conducted by arbitrarily dividing the MG administration group into 5 groups. It should be noted that the levels of the low MG administration group and the high MG administration group are merely relative and do not indicate that the dose is lower or higher than the standard. Mangiferin was orally administered at a rate of 5 mg / kg to the low MG administration group, and orally administered at a rate of 10 mg / kg to the high MG administration group.

  Table 5 shows the total fructose intake, total feed intake, and body weight after 10 weeks of breeding for each group. In Table 5, “*” indicates a case where there is a significant difference at p <0.05 with respect to the fructose administration group in the t test.

  As shown in the results of Table 5, a decrease in intake was observed in the group ingested fructose, and a suppressive effect was observed on body weight and feed intake by fenofibrate, except for the total fructose intake, total feed intake and body weight. There was no difference.

  Next, regarding the liver excised at 10 weeks, the liver weight, the liver weight / body weight ratio and the amount of neutral fat in the liver are shown in Table 6. In Table 6, “*” indicates a case where there is a significant difference at p <0.05 with respect to the fructose administration group in the t test.

  As shown in Table 6, when fenofibrate, a lipid-lowering drug, was administered in addition to fructose load, the amount of triglyceride in the liver was significantly decreased while the ratio of liver weight to body weight was significantly increased. The tendency of liver enlargement was observed. In contrast, when mangiferin was administered in addition to the fructose load, the amount of triglycerides in the liver could be significantly reduced without significantly increasing the liver weight / body weight ratio.

  In addition, we made a pathological section of the liver this time, and observed the repair of the cavity due to fatty liver. Specifically, liver tissue pieces stored at −80 ° C. were fixed with 10% formalin, embedded in paraffin, cut to a size of 4 μm, stained with hematoxylin and eosin, and then a research system microscope (Olympus). Observation was performed using BX-51). The results are shown in FIGS.

  As shown in FIG. 2, when fructose was loaded as compared with the control group (FIG. 1) in which only water was administered, neutral fat was accumulated between hepatocytes and cavitation was observed. On the other hand, when fenofibrate or mangiferin, which is a lipid lowering drug, is administered (FIGS. 3 to 5), such cavitation is clearly reduced and is close to the liver slice image of the control group.

  Further, in order to quantify the amount of oil droplets in the liver slice, the liver slices of the water administration group (control group), the fructose administration group and the fructose + low MG administration group were stained with oil red O, and image analysis software (manufactured by NIH) , Image J 1.43), the ratio of the oil droplet portion was quantified. Liver slice images stained with oil red O are shown in FIGS. 6 to 8, and a graph of quantitative values is shown in FIG. In FIG. 9, “*” indicates a case where there is a significant difference at p <0.05 in the t test. As shown in FIGS. 6 to 9, neutral fat is significantly accumulated in the liver tissue when fructose is loaded as compared with the case where only water is administered. On the other hand, when mangiferin is administered, the amount of neutral fat is increased. Proved to be significantly reduced.

  Here, SR and SC leaves (about 5 to 8 cm), branches (about 1 cm in diameter), stems (about 5 cm in diameter), and roots of mangiferin (MG) contained in Sri Lanka, India, and Thailand. ) Is disclosed. Quantification was performed by drying a sample collected in 2010 until the loss on drying reached 5 to 6%, and extracting the sample with 50 v / v% methanol water by HPLC. In Examples 1 and 2, water is used as the extraction solvent. In this case, the amount of MG extraction is 1/5 to 1/10 as compared with the case where 50 v / v% methanol water is used.

  As shown in Table 7, the amount of mangiferin contained in the root of SR or SC is about 10% by mass at most. On the other hand, as for the liver fat reduction effect, as shown in Table 4 and Table 6, there is no significant difference between mangiferin at 5 mg / kg and 10 mg / kg, and the SC root extract before water-methanol fractionation Is more effective when 50 mg / kg is administered. From these results, mangiferin itself has an effect of improving fatty liver, but it is extremely excellent due to a component other than mangiferin contained in the SR root extract or SC root extract or due to the synergistic effect of the component and mangiferin. It is presumed that the effect of improving fatty liver is exerted.

Example 6 Toxicity Test An SC root extract was obtained in the same manner as in Example 2 except that the extraction solvent was changed from water to 50 v / v% methanol water, and its acute toxicity was examined. Specifically, the extract is administered once to DD8-Y male mice weighing about 20 g at a rate of 2,000 mg / kg that can be physically orally administered, and water and normal feed are freely ingested. Raised for 1 week.

  As a result, no deaths and abnormalities were observed at all during the 1-week observation period. Therefore, it was concluded that the extract according to the present invention is very safe.

Claims (5)

An inhibitor of non-alcoholic fatty liver induced by fructose, comprising an extract of at least one root selected from Salacia reticulata and Salacia chinensis as an active ingredient. The inhibitor of non-alcoholic fatty liver induced by fructose according to claim 1, wherein the extract is an extract with an aqueous solvent. The inhibitor of nonalcoholic fatty liver induced by fructose according to claim 2, wherein the aqueous solvent is at least one selected from water and C _1-4 alcohol. The inhibitor of non-alcoholic fatty liver induced by fructose according to claim 2 or 3, wherein the extract is a methanol-soluble component of an aqueous solvent extract. The inhibitor of nonalcoholic fatty liver induced by fructose according to any one of claims 1 to 4, wherein the extract contains 0.5% by mass or more of mangiferin.