JP3010215B1 - Lipase inhibitor - Google Patents

Abstract

Abstract: PROBLEM TO BE SOLVED: To provide a lipase inhibitor and a pharmaceutical and food additive based on the same. SOLUTION: A lipase inhibitor, an antiobesity agent, an acne treatment agent and a food deterioration inhibitor containing sclerothioline as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a lipase inhibitor, an antiobesity agent and an acne treatment agent, and a food deterioration inhibitor.

[0002]

2. Description of the Related Art Lipase is an enzyme that hydrolyzes glyceride and releases fatty acids, and is widely distributed in mammalian body fluids, organs, plants, microorganisms, and the like. Among them, pancreatic lipase is secreted into the duodenum by lipase contained in pancreatic juice, and decomposes glyceride in the intestinal tract to contribute to lipid absorption. Therefore, since the absorption of lipids from the intestinal tract is inhibited by inhibiting the pancreatic lipase activity, it is known that pancreatic lipase inhibitors are useful as anti-obesity drugs with few side effects (International Journal of Obesity ( 199
5) 19, 221-226, laboratory test vol. 42, No. 4, 440-443).

It is said that the main causes of acne are increased sebum secretion, accelerated keratinization, and proliferation of propionobacterium acnes (bacillus acne) resident in the skin. Of these, lipase produced by Bacillus acne is known to be a component that inhibits free fatty acids produced by decomposing triglycerides in sebum, as it acts as an irritant, and that components that inhibit this are useful for acne treatment. (JP-A-2-121)
No. 908).

[0004] Fats contained in foods are hydrolyzed by lipases contained in foods such as plants or lipases derived from microorganisms mixed in foods.
Decomposed into glycerol and free fatty acids. This free fatty acid causes deterioration and foul odor, and it is important to suppress the generation of free fatty acid from the viewpoint of maintaining the quality of food, and the development of lipase inhibitors as food deterioration inhibitors has been developed. Various methods have been proposed (Japanese Patent No. 2598873, Japanese Patent Application Laid-Open No. 9-40689).

[0005]

However, these conventional anti-obesity agents, acne treatment agents and food deterioration inhibitors based on lipase inhibitory activity are not yet fully satisfactory, and new drugs are required. I have.

[0006]

The present inventors have searched for substances having a lipase inhibitory activity from cultures of various microorganisms, and found that sclerothioline produced by a microorganism belonging to Penicillium sclerothiorum is
It has an inhibitory effect on pancreatic lipase, lipase derived from acne bacillus, lipase derived from microorganisms, and lipase derived from plants, and is found to be useful as an antiobesity drug, a therapeutic agent for acne, and a food deteriorating inhibitor. The invention has been completed.

[0007] That is, the present invention provides a lipase inhibitor, an antiobesity agent, an acne treatment agent and a food deterioration inhibitor containing sclerothioline as an active ingredient.

[0008]

DETAILED DESCRIPTION OF THE INVENTION Sclerothioline used in the present invention has the following formula (1)

[0009]

Embedded image

A known compound represented by the following formula: Biochemical Journal (Biochem. J.), 34 , 1419 (1940),
There are many reports such as Chemical and Pharmaceutical Bulletin (Chem. Pharm. Bull.), 11 , 366 (1963). gram-positive bacteria action, Arutsunaimiteru - (. Arzneimittel-Forsh) Forushunku 12,
1143 (1962) reported an antitumor effect. Recently, Agriculture and Biological Chemistry (Agric. Biol. Chem.), 52 ,
In 307 (1988), the fungus-inducing activity of chlamydospore-like cells was reported in the Journal of Antibiotics (J. Antibiotics).
t.), 48, 913 ( 1995) endothelin antagonist activity are, in JP-A-2-255615 is phospholipase A ₂ inhibitory activity have been reported. However, no report has been made on the lipase inhibitory effect of sclerothioline.

[0011] Sclerothioline is produced, for example, by culturing a sclerothioline-producing bacterium belonging to the genus Penicillium, and collecting from the culture. According to the study of the present inventors, in the production of sclerothioline by the present method, if a medium containing ethanol as a carbon source is used,
It has been found that productivity is dramatically improved as compared with the case where another carbon source is used. Therefore, in the present method, it is preferable to use a medium containing ethanol as a carbon source.

[0012] Sclerothioline-producing bacteria include Penicillium sclerotioru (Penicillium sclerotioru).
m) The EF-1646 strain is preferred. This EF-1646
The strain was isolated from a soil sample collected at Shinonoi Shiozaki, Nagano City, Nagano Prefecture, and has the following bacteriological properties.

<Characteristics on Various Media> Teleomorphs are not formed in various media such as potato dextrose agar plate media and Zapec yeast extract agar plate media, and anamorphs consisting of broom-shaped conidiophores and conidia are formed. It was abundantly formed, and its conidial formation was phyaroal.

Next, Table 1 shows the characteristics of the growth state when cultured on a malt extract agar plate medium and a Zapek yeast extract agar plate medium at 25 ° C. for 7 to 9 days.

[0015]

[Table 1]

The anamorphs observed on these media consisted of broom-shaped conidiophores erecting from the aerial or vegetative mycelium and conidia chains. Conidiophores are colorless, smooth, unbranched, 3-4 μm in diameter, 28-120 μ in length
m, which is a single-wheeled living body in which 5 to 10 phialides are formed on a vesicle having a diameter of 4 to 5 μm. The phialide was ampoule-shaped and had a size of 7 to 11 μm × 2 to 3 μm. The conidium formed is pale green, smooth surface, spherical, diameter 2
３3 μm.

<Physiological characteristics> The EF-1646 strain contains 10
It can grow in the range of ℃ ~ 34 ℃, the optimal growth temperature is 2
4-25 ° C.

[0018] The present strain, from the features, Penicillium genus of imperfect fungi Aspergillus Giroi death subgenus of (Penicillium Link) (Subgenus Aspergilloides Dierckx ), seemed to belong to the Aspergillus Giroi death clause (Section Aspergilloides Pitt). Penicillium sclerotiorum van beyma ( Penicillium sclerotior) is characterized by the fact that the backside of the colony is orange in the species belonging to the same subgenus.
um van Beyma). Therefore, this strain was used for Penicillium sclerothiorum EF-1646 ( Penicillium sc
lerotiorum EF-1646). This penicillium
Sclerothiolm EF-1646 strain was granted to FERM P-1711 by the Institute of Biotechnology and Industrial Technology, MITI.
No. 7 was deposited on December 22, 1998.

Culture of the sclerothioline-producing bacterium may be carried out according to a conventional method using a medium in which a genus Penicillium grows, but the use of a medium containing ethanol as described above is preferred in terms of productivity. In particular, EF-1
The 646 strain grows well in a medium containing ethanol as a carbon source, and produces sclerothioline at a much higher rate than when other carbon sources are used. preferable. The concentration of ethanol in the medium is not particularly limited, but may be 0.01 to 5%.
(V / v)% is preferred. As a particularly preferred medium, a medium (pH 5) consisting of ethanol 3% (v / v), defatted soybean flour 1.2%, peptone 0.32%, sodium nitrate 0.08%, and sodium sulfate heptahydrate 0.1% .6).

The thus obtained sclerothioline has an excellent inhibitory effect on pancreatic lipase, acne bacillus lipase, plant-derived lipase and microorganism-derived lipase.
And the toxicity of sclerothioline is very low, for example, 100 mg / kg body weight of 10-week-old (weight 35-40 g) I
Even if it is intraperitoneally administered to a CR male mouse as a 0.5% methylcellulose suspension, it shows no toxicity and is highly safe. Therefore, sclerothioline is based on lipase inhibitory action,
It is useful as an anti-obesity drug, a treatment for acne, and a food deterioration inhibitor.

The antiobesity agent of the present invention is used orally or parenterally in mammals including humans. As the form of the preparation, various forms can be selected according to the purpose of treatment, and typical examples are oral preparations such as tablets, pills, granules, powders, capsules, suspensions, emulsions, and syrups. And parenteral administration agents such as injections (subcutaneous, intravenous, intramuscular injection, etc.).

The acne treating agent of the present invention is used orally or parenterally. As the form of the preparation, various forms can be selected according to the purpose of treatment, and typical examples are oral preparations such as tablets, pills, granules, powders, capsules, suspensions, emulsions, and syrups. And parenteral administration agents such as injections (subcutaneous, intravenous, intramuscular injection, etc.) and external administration agents in the form of solid preparations such as ointments. In addition, in the case of an acne therapeutic agent, it can be a cosmetic for acne treatment such as lotions, emulsions, creams, gels, sticks and the like.

The food deterioration inhibitor of the present invention is used as a food additive and applied to foods containing the same. The food additive can be used as a powder or as a solution or suspension diluted with an appropriate solvent. Furthermore, it can be used as a powder or paste by blending with other additives by ordinary operations such as mixing and drying.

The dosage of sclerothioline as an antiobesity agent or an acne therapeutic agent depends on its usage, patient weight, age,
The dose is appropriately selected depending on gender and other conditions, the degree of the disease, and the like. Usually, the dose is 0.5 to 2000 mg, preferably 1 to 1000 mg per day. Effective single doses range from 0.01 to sclerothioline per kg of patient weight.
It is selected in the range of 50 mg, preferably in the range of 0.05 to 20 mg. The amount of sclerothioline in the external preparation or acne cosmetic is preferably 0.001 to 10% by weight (hereinafter abbreviated as wt%), more preferably 0.05 to 5% by weight, based on the total amount of the composition. It is. Also,
When blended in food as a food deteriorating inhibitor, 0.0001 to 1 wt% of sclerothioline is suitable for food, and more preferably 0.001 to 0.1 wt%.

[0025]

EXAMPLES The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the present invention.

Production Example 1 500 ml of 100 ml of a medium (pH 5.6) consisting of 1.2% defatted soy flour, 0.32% peptone, 0.08% sodium nitrate and 0.1% sodium sulfate heptahydrate was used.
Was charged into three Erlenmeyer flasks and sterilized at 121 ° C. for 15 minutes. Then, filter sterilized ethanol
% (V / v) each to obtain a liquid medium. In each flask, add Penicillium
sclerotiorum ) EF-1646 strain was inoculated one loop at a time, and cultured with stirring on a rotary shaker at 25 ° C for 4 days. Next, 15 l of the same medium was charged into a 30-liter jar fermenter, sterilized at 121 ° C. for 15 minutes, inoculated with the entire amount of the culture obtained above, and cultured at 25 ° C. for 6 days.
The resulting culture was extracted twice with ethyl acetate. After the extract was washed with a 5% sodium hydrogen carbonate solution, the ethyl acetate layer was dehydrated, filtered, concentrated and dried to dryness, and subjected to silica gel column chromatography (1 l of silica gel) to elute with benzene: ethyl acetate = 5: 1. The eluate was fractionated 1 l each, fractions showing lipase inhibitory activity were collected, the solvent was distilled off under reduced pressure, and the residue was crystallized from methanol to obtain 2 g of crude crystals. This was further recrystallized from methanol to obtain 1.2 g of yellow needle-like crystals of sclerothioline.

Melting point: 205-208 ° C. Specific rotation: [α] _D = + 463 ° (c 0.2, EtOH) IR (KBr): 1740, 1720, 1636, 1528 cm ^-1 UV λ _max nm (ε): 220 ( 13000), 270 (13100), 370 (302
00) ¹ H-NMR (CDCl ₃ , δ): 7.94 (1 H, s), 7.06 (1 H, d, J = 16 Hz),
6.65 (1H, s), 6.08 (1H, d, J = 16Hz), 5.71 (1H, d, J = 10Hz),
2.49 (1H, m), 2.17 (3H, s), 1.85 (3H, s), 1.57 (3H, s), 1.2
5-1.50 (2H, m), 1.00 (3H, d, J = 6 Hz), 0.87 (3H, t, J = 7 Hz) ¹³ C-NMR (CDCl ₃ , δ): 191.7, 186.0, 170.1, 158.1, 15
2.7, 148.8, 142.9, 138.7, 132.0, 115.7, 114.6, 106.
4, 84.6, 35.1, 30.0, 22.5, 20.2, 20.1, 12.4, 12.0 Rf = 0.40 (silica gel plate, hexane: ethyl acetate = 3: 1) Solubility: soluble; methanol, ethanol, acetone, dichloromethane, chloroform, acetic acid Ethyl, benzene insoluble; water FAB-MS, M / Z (%): 391 (M ⁺ +1, 100), 393 (M ⁺ +3, 37) EI-MS, M / Z (%): 390 (M ⁺ , 100), 392 (M ⁺ +2, 34)

Production Example 2 (Productivity depending on the difference in carbon source) A medium comprising 1.2% defatted soy flour, 0.32% peptone, 0.08% sodium nitrate, and 0.1% sodium sulfate heptahydrate ( (pH 5.6) 100 ml was charged into a 500 ml Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes.
Thereafter, 3% (v / v) of ethanol sterilized by filtration was added to obtain a liquid medium. Similarly, ethanol 3% (v / v) is replaced with glucose 3% (w / v) and glycerin 3% (w / v).
Was prepared and sterilized at 121 ° C. for 15 minutes. Hereinafter, each medium is called an ethanol medium, a glucose medium, and a glycerin medium. In the flask in which the ethanol medium was prepared, add Penicillium sclerothiolm ( Peni
Cillium sclerotiorum ) EF-1646 strain was inoculated one platinum loop at a time, and cultured with stirring on a rotary shaker at 25 ° C. for 4 days. Next, each of the flasks containing the ethanol, glucose, and glycerin
% (V / v) was inoculated and cultured with stirring on a rotary shaker at 25 ° C. for 7 days. The culture solution was extracted twice with ethyl acetate, dehydrated, filtered, and dried, then dissolved in a fixed amount of methanol, and the pancreatic lipase inhibitory activity was examined in the same manner as in Example 1 described later to calculate sclerothioline productivity. did. The result is shown in FIG. As a result, it was confirmed that sclerothioline, which is an active ingredient of the present invention, was effective as a carbon source in culturing using the EF-1646 strain.

Example 1 (pancreatic lipase inhibitory activity) An inhibitory activity test of sclerothioline on porcine pancreatic lipase was conducted. To 100 μl of 0.1 mM 4-methylumbelliferyl oleate solution, 10 μl of methanol solution of sclerothioline at various concentrations, 40 μl of 0.1 M Mcllvaine buffer (pH 7.4), 5 μg / ml of porcine pancreatic lipase (Sigma) ) Add 50 μl of solution and add 2
Enzymatic reaction was performed for 0 minutes. Porcine pancreatic lipase solution is 0.0
It was dissolved in the same buffer containing 04% bovine serum albumin. After the reaction, 1 ml of 0.1N hydrochloric acid was added to the reaction solution to stop the enzymatic reaction. Then, 2 ml of a 0.2 M sodium citrate solution was added, and the fluorescence of 4-methylumbelliferone generated was excited at an excitation wavelength of 320 nm. Quantification was performed at a wavelength of 450 nm. A sample to which 10 μl of methanol was added was used as a control. The inhibition rate (%) at each concentration is
[(Control fluorescence intensity-Fluorescence intensity of each sample) / Control fluorescence intensity] × 100. The results are shown in Table 2 with IC ₅₀ values Sukurerochiorin. Hesperidin, apigenin, whose pancreatic lipase inhibitory activity has already been reported,
Quercetin was used as a positive control.

[0030]

[Table 2]

Example 2 (Inhibition of lipase derived from Propionibacterium acnes) An inhibitory activity test of sclerothioline on lipase derived from Propionibacterium acne was performed. Propionibacterium acne (Prop)
For ionibacterium acnes), 50% saturated ammonium sulfate was added from a culture solution cultured in a commercial GAM medium (manufactured by Nissui) at 37 ° C. for 24 hours using JCM6425, and salted out at 4 ° C. overnight. Collect the precipitate by centrifugation,
The enzyme solution was dissolved in Mcllvaine buffer (pH 6.8). The lipase inhibitory activity was determined according to Example 1, using the enzyme solution prepared with the enzyme, and the pH of the Mcllvaine buffer used for the reaction.
Tested at 6.8. The results are shown in Table 3 below as IC ₅₀ values of sclerothioline.

[0032]

[Table 3]

Example 3 (Inhibitory activity on lipase derived from plants and microorganisms) The inhibitory activity of sclerothioline on lipase derived from plants and microorganisms was tested. Lipases derived from wheat (Wheat Germ) as plant-derived lipases (manufactured by Sigma), lipases derived from Pseudomonas sp. Lipase (manufactured by Sigma) was used. Wheat-derived lipase is Mcllvaine containing 0.004% bovine serum albumin.
It was used after being dissolved in a buffer solution (pH 7.4). Mcllvaine buffer (pH 7.4) was used for the buffer solution used for the reaction.
0.004 lipase from Pseudomonas sp.
Mcllvaine buffer containing 7% bovine serum albumin (pH 7.
0). The buffer solution used for the reaction was Mcll
A vaine buffer (pH 7.0) was used. 0.004% lipase from Candida rugosa
Mcllvaine buffer containing bovine serum albumin (pH 7.
Used after dissolving in 2). The buffer solution used for the reaction was Mcll
A vaine buffer (pH 7.2) was used. Other reaction conditions were the same as in Example 1. The results are shown an IC ₅₀ value of Sukurerochiorin in Table 4 below.

[0034]

[Table 4]

Example 4 Production of Capsules According to the formulation shown in Table 5, each component was mixed well and filled in No. 1 capsule, and the capsule of the lipase inhibitor of the present invention was added to 300 capsules.
Individually manufactured.

[0036]

Table 5 Sclerothioline 5 g Lactose 40 g Potato starch 50 g Hydroxypropyl methylcellulose 3.5 g Magnesium stearate 1.5 g Total 100 g

Example 5 Preparation of Ointment The ingredients shown in Table 6 were mixed well to prepare the lipase inhibitor ointment of the present invention.

[0038]

[Table 6] Sclerothioline 5g Glycerin 12g Stearyl alcohol 25g White petrolatum 25g Methyl p-oxybenzoate 0.025g P-propyl oxybenzoate 0.025g Sterilized purified water Total 100g

Example 6 Preparation of Skin Cream A mixture of the components A and B in Table 7 heated and dissolved at 80 ° C. was mixed, and then cooled to 30 ° C. while stirring to prepare a skin cream.

[0040]

Table 7 A Sclerothioline 5 g Stearic acid 7 g Cetanol 3 g Beeswax 2 g Olive oil 15 g Polyoxyethylene sorbitan monooleate 2 g B Propylene glycol 5 g Methyl paraben 0.1 g Purified water 100 g

Example 7 Manufacture of biscuit A dough was prepared by a conventional method with the composition shown in Table 8, and molded and roasted to manufacture a biscuit.

[0042]

[Table 8] Sclerothioline 0.1 g Flour 120 g Sugar 35 g Shortening 15 g Whole egg powder 1.5 g Salt 1 g Sodium bicarbonate 0.6 g Ammonium carbonate 0.75 g Water 20 g

[0043]

EFFECTS OF THE INVENTION Sclerothioline is highly safe and has a high pancreatic lipase inhibitory activity. Therefore, it suppresses lipid degradation in the intestinal tract, thereby suppressing lipid absorption and contributing to suppression, prevention, weight loss and the like of obesity. obtain. In addition, Propionibacterium acne, the causative agent of acne (Propioniba
It also has an inhibitory activity against lipase produced by cterium acnes), which is useful for the prevention and treatment of acne. Furthermore, the decomposition of fats and oils in foods by lipase derived from food materials or microorganisms can be suppressed, and as a result, deterioration of foods and generation of bad smell caused by free fatty acids can be prevented. Therefore, a food that can be stored for a long time can be provided. In addition, the calorie of food can be reduced due to its pancreatic lipase inhibitory action.

[Brief description of the drawings]

FIG. 1 is a graph showing differences in sclerothioline productivity due to differences in carbon sources in culturing sclerothioline-producing bacteria.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // C07D 311/76 C07D 311/76 C12P 17/06 C12P 17/06 (C12P 17/06 C12R 1:80) (58) Survey Field (Int.Cl. ⁷ , DB name) A61K 31/35 CAPLUS (STN)

Claims (4)

(57) [Claims] 1. A lipase inhibitor comprising sclerothioline as an active ingredient. 2. An anti-obesity drug containing sclerothioline as an active ingredient. 3. A treatment for acne comprising sclerothioline as an active ingredient. 4. A food deterioration inhibitor comprising sclerothioline as an active ingredient.