JPH08143457A - Antienzyme and inhibitor for hyperlipemia - Google Patents

Abstract

PURPOSE: To obtain an antienzyme against esterase and lipase, containing ebelactone B and/or ebelactone A as an active ingredient and to provide an inhibitor for hyperlipemia having high inhibitory activity against hyperlipemia. CONSTITUTION: This antienzyme contains ebelactone B or ebelactone A or both as active ingredients. Ebelactone B and ebelactone A have inhibitory activity against lipase and esterase enzyme, suppresses absorption of fats and represses an amount of triglyceride in serum. The antienzyme is useful for studying the role of lipase or esterase in hyperlipemia. An inhibitor for hyperlipemia is obtained by properly mixing ebelactone B and/or ebelactone A with a conventional preparation auxiliary and pharmaceutically manufacturing the mixture by a conventional method into a preparation for oral administration. A dose is 0.1-2.5g per adult and is properly administered one hour before each meal. Ebelactone B shows stronger inhibitory activity against esterase and lipase than ebelactone A.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel inhibitor of the enzymatic activity of lipase or esterase containing ebelactone B and / or ebelactone A as an active ingredient, and ebelactone B and / or ebelactone A as an active ingredient. The present invention relates to a novel hyperlipidemia inhibitor.

[0002]

2. Description of the Related Art In Japan, the proportion of deaths due to hyperlipidemia among the deaths due to adult diseases tends to increase year by year. Although researches on therapeutic agents for arteriosclerosis due to hyperlipidemia and high cholesterol have made rapid progress in recent years, researches on therapeutic agents mainly on substances having inhibitory activity of cholesterol biosynthesis have been conducted.

It is known that the majority of fats in foods are neutral fats (triglycerides, TG) consisting of long-chain fatty acids, which are emulsified by bile and hydrolyzed into fatty acids and monoglycerides by the action of pancreatic juice lipase. There is. The resulting fatty acid and monoglyceride are associated with cholesterol by the action of bile acid to form micelles, which are absorbed by diffusion from the jejunal epithelial cell membrane. It is known that the absorbed long-chain fatty acid and monoglyceride enter the mucosal epithelial cell and are then resynthesized into triglyceride, and together with cholesterol, apoprotein synthesized intracellularly constitutes lipoprotein. Therefore, it is considered that the absorption of fat can be suppressed by inhibiting the pancreatic juice lipase.

It is considered that by inhibiting lipolytic enzymes in the gastrointestinal tract lumen and thereby suppressing the production of monoglyceride, lymphatic absorption of neutral fat and cholesterol can be inhibited. Reducing the gastrointestinal absorption of dietary fat is considered a good strategy for preventing or treating hyperlipidemia.

On the other hand, Ebelactone A and Ebelactone B are known to be physiologically active substances having anti-esterase activity and anti-formylmethionine aminopeptidase activity, and Ebelactone A and Ebelactone B have the following structural formulas. It is a compound
-10953 and U.S. Pat. No. 4,358,602).

Ebelactone A

Ebelactone B

The present inventors have an extremely unique mechanism of inhibiting lipolytic absorption of neutral fat and cholesterol by inhibiting lipolytic enzyme activity in the gastrointestinal tract lumen and suppressing monoglyceride production. The research was repeated for the purpose of providing a novel prophylactic or therapeutic drug for hyperlipidemia.

[0009]

Means for Solving the Problems As a result of intensive studies to develop a new hyperlipidemia inhibitor, the present inventors have found that ebelactone B and ebelactone A have activity of inhibiting lipase and esterase enzymes. It was found that the absorption of fat was prevented and the amount of TG in serum was suppressed, and the present invention was completed.

That is, the first aspect of the present invention provides a lipase or esterase enzyme activity inhibitor containing ebelactone B or ebelactone A or both as an active ingredient.

The second aspect of the present invention provides a hyperlipidemia inhibitor containing ebelactone B, ebelactone A, or both as active ingredients.

The toxicity of Ebelactone B and Ebelactone A was low, and no signs of toxicity were observed, for example, when 100 mg / kg of mice was intraperitoneally injected.

The enzyme inhibitor according to the first aspect of the present invention is useful for studying the role of lipase or esterase in hyperlipidemia.

Further, ebelactone A and ebelactone B, which are used as active ingredients in the hyperlipidemia inhibitor according to the second aspect of the present invention, produce hyperlipidemia in various fat loading models including hypercholesterolemia in rabbits and rats. It has been confirmed to be effective in suppressing dysfunction and is expected to be useful as a preventive or therapeutic agent for hyperlipidemia in clinical situations.

Ebelactone B and Ebelactone A used as active ingredients of the hyperlipidemia inhibitor according to the second aspect of the present invention.
Although the dose varies depending on the type of target disease, age, sex, body weight, symptoms of patient or administration form, administration for 1 hour before meal is preferable. Generally, one hour before each meal for adults
A dose of 0.1 g-2.5 g is suitable.

The second drug of the present invention is preferably administered orally for the prevention or treatment of hyperlipidemia. The active ingredient, ebelactone, can be formulated in the form of a composition mixed with a pharmaceutically acceptable carrier. As a formulation for oral administration, a powder,
Solid preparations such as granules, capsules and tablets, or liquid preparations such as syrups and elixirs can be prepared.

These preparations are produced according to a conventional method by adding a pharmacologically or pharmaceutically acceptable carrier, ie, a formulation auxiliary, to the active ingredient ebelactone and mixing them. Further, it is also possible to prepare a sustained-release preparation by a known technique.

The above-mentioned carrier may be a carrier commonly used in oral preparations (oral preparations). For example, in the case of oral preparations and mucosal administration preparations, the carrier may be starch or lactose, for example. , Crystalline cellulose, calcium lactose, magnesium aluminometasilicate, anhydrous silicic acid. As disintegrants, for example, carboxymethyl cellulose, carboxymethyl cellulose calcium; as lubricants, such as magnesium stearate, talc; as coating agents, such as hydroxyethyl cellulose, sucrose, hydroxypropyl cellulose, polyvinylpyrrolidone, corn protein; and for flavoring agents Additives can be added.

In order to produce granules, a powdery composition containing an active ingredient, a carrier and other additive ingredients can be wet or dry granulated. Also, to make tablets,
These powders and granules may be tableted as they are or by adding a lubricant such as magnesium stearate and talc. For example, a mixture of 1 part of Ebelactone B or A and 5 parts of lactose can be tableted by a conventional method to give tablets. Furthermore,
These granules or tablets can be coated with an enteric base such as hydroxypropylmethyl cellulose phthalate, methacrylic acid, and methyl methacrylate copolymer to prepare an enteric preparation, or coated with ethyl cellulose, carnauba wax, hardened oil, etc. It can also be made into a sustained-release preparation. In order to produce capsules, a hard capsule is filled with a powder or granules containing the active ingredient, or an oily solution of the active ingredient dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. is coated with a gelatin film. It can be made into a soft capsule. In the case of a capsule, the content of the capsule may be 100 wt% of ebelactone B or ebelactone A.

To prepare a liquid preparation for oral administration, the active ingredient and a sweetener such as sucrose, sorbitol and glycerin are dissolved in water to give a transparent syrup, or essential oil, ethanol and the like are added. It may be an elixir or may be an emulsion or suspension prepared by adding gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose or the like. If desired, flavoring agents, coloring agents, preservatives and the like may be added to these liquid preparations.

In addition to the above, the hyperlipidemia-suppressing agent of the second aspect of the present invention having the above-described structure has been subjected to a known production method, for example, the method described in the Japanese Pharmacopoeia 10th Edition General Rules for Preparations or appropriate improvements. It can also be formulated by a method.

The present invention will be described in detail below with reference to test examples.

Test Example 1 In this example, the enzyme inhibitory activity of Ebelactone A or B is tested. Pig liver esterase (Sigma Chemical Co., U.
SA) and porcine pancreatic lipase (Nutritional Biochem. Co.,
USA) and each contains 0.2% Triton X-100 0.05
Store in a cool dark place as a 1 mg / ml enzyme solution by dissolving in M phosphate buffer (pH 7.0). At the time of use, it is diluted with the above buffer solution and adjusted so that 0.03 ml of the esterase or lipase enzyme solution releases about 50 nmol of p-nitrophenol in a reaction for 15 minutes (room temperature). The enzyme inhibitory activity was measured by using a 96-well plate (nunclon, E96) containing 50 μl of a substrate solution [containing 0.1 M p-nitrophenylacetic acid as a substrate for esterase or 0.02 M p-nitrophenyllauric acid as a substrate for lipase]. Buffer solution (100 μl), ebelactone A or B sample solution (25 μl) and esterase or lipase enzyme solution (25 μl) were added and reacted at room temperature for 15 minutes, and then 4 with Microplate Reader Model 3550 (Bio-Rad).
The absorbance at 05 nm was measured. As a control test, a blank test was conducted without using Ebelactone A or B. By comparing the measured absorbance values, Ebelactone A
Alternatively, the concentration at which Ebelactone B inhibits the enzyme activity of esterase or lipase by 50% (IC ₅₀ value, μg / ml) was calculated. The test results are shown in Table 1 below.

[0024]

Ebelactone B showed a stronger inhibitory activity than Ebelactone A against both esterase and lipase.

Test Example 2 In this example, hyperlipidemia was induced in a fat-loaded rat after oral administration of olive oil and cholesterol,
The effect of ebelactone B was examined by the single oral administration of ebelactone B to reduce or suppress the lipid level in blood.

That is, 5 ml / kg of olive oil containing 10% cholesterol was orally administered, and the effect of ebelactone B on hyperlipidemia induced 6 hours later was examined. Ebelactone B is 10% CHO-60 (Nippon Chemicals, FFgrade)
Was dissolved in a solution of 10 mg / 5 ml, and 1.25 ml of the solution was orally administered 60 minutes before fat loading. The dose of Ebelactone B was equivalent to 10 mg / kg per rat. Blood was collected 6 hours after fat loading, and total lipid (TL), triglyceride (TG), total cholesterol (TC) and phospholipid (P) in serum were collected.
The concentration of L) was measured. As a control test, instead of Ebelactone B, 1.25 ml of physiological saline was administered 60 minutes before fat loading, and blood was collected 6 hours after fat loading, and the same test was performed.

The results are shown in Table 2 below.

[0029]

As is clear from the results shown in Table 2, when Ebelactone B was administered 60 minutes before fat loading, the serum lipid level 6 hours after fat loading was significantly reduced as compared with the control test. It has been found. Therefore, it was confirmed that Ebelactone B has an action of significantly suppressing the increase in the amount of lipid in blood.

[0031]

INDUSTRIAL APPLICABILITY Ebelactone B and Ebelactone A inhibited lipase and esterase, and significantly lowered the lipid in serum as compared with the fat loading model. Therefore, Ebelactone B and Ebelactone A can be effectively used as enzyme inhibitors and as hyperlipidemia inhibitors.

Claims (2)

[Claims] 1. An enzyme activity inhibitor of lipase or esterase, which comprises Ebelactone B, Ebelactone A, or both as an active ingredient. 2. An agent for suppressing hyperlipidemia, which comprises Ebelactone B, Ebelactone A, or both as an active ingredient.