JP2805260B2 - Glycerophosphate dehydrogenase inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a glycerophosphate dehydrogenase inhibitor containing 5-alkylresorcinol (1,3-dihydroxy-5-alkylbenzene) as an active ingredient.

[0002]

2. Description of the Related Art In recent years, the problem of obesity has come to the forefront with the westernization of eating habits. At the same time, various slimming methods have been devised one after another for obese people, but most of them have disappeared without being put to practical use. The reason for this is that triglycerides, the main cause of obesity, have the property that once accumulated, they do not easily decrease, so that extraordinary efforts are required to reduce the fat. Was. Once these properties became known, there was a movement to prevent obesity by reducing caloric intake, assuming that the main cause of the accumulation of neutral fat was due to excessive calorie intake. . The so-called diet, which has caused a new problem of anorexia nervous caused by extremely reducing calorie intake for dieting.

[0003] Therefore, it has been desired to develop a substance capable of preventing obesity without becoming nervous in calorie intake.

On the other hand, 5-alkylresorcinol having 14 to 16 carbon atoms in the alkyl group (hereinafter referred to simply as 5-alkylresorcinol) is disclosed in The Journal of Antibioti.
cs, 870-875 (December 1971) and JP-A-47-31951.
As natural products, those isolated from cashew nut juice (oil) and wheat husks are known. However, although the action of this alkylresorcinol as an antioxidant or antibacterial substance is known, there is no mention of any inhibitory activity on glycerophosphate dehydrogenase.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a glycerophosphate dehydrogenase inhibitor which can be used as an antiobesity agent.

[0006]

Means for Solving the Problems The present inventors inhibited glycerophosphate dehydrogenase, which acts at the previous stage, in the metabolic process from the production of glucose to the production of triglyceride, a neutral fat, in animal cells. As a result of intensive search for a substance having an activity, it was found that 5-alkylresorcinol had such an activity, and the present invention was completed.

[0007] That is, the present invention provides a glycerophosphate dehydrogenase inhibitor comprising, as an active ingredient, a 5-alkylresorcinol having an alkyl group having 14 to 16 carbon atoms.

The alkyl group used in the present invention has 1 carbon atom.
5-alkylresorcinols in the range of 4-16 are
The Journal of Antibiotics, 870-875 (Dec. 1971
) Or JP-A-47-31951, but it is preferable to use those extracted from a culture of actinomycetes.

[0009] Such actinomycetes include streptoma
Preferably used Isis Shianeusu (Streptomycescyaneus). More specifically, Streptomyces
Cyaneus ( Streptomyces cyaneus ) 2299-SV1 strain ((F
ERM BP-3607). This bacterium is 1% soluble starch, 1% polypeptone, 1% molasses, 1% Beefex
tract, pH 7.2, and 2% glycerin, 1% molasses, 0.5% casein, 0.1% polypeptone, 0.4% calcium carbonate, pH 7.0, respectively. After culturing at 27 ° C., the obtained culture solution is subjected to centrifugation or the like to remove solid substances such as cells, and then the target substance is extracted with an appropriate solvent. The obtained active substance can be used as it is, but it is optional to further purify and isolate it by column chromatography or the like before use.

The above strain 2299-SV1 is an actinomycete isolated from a soil sample collected at the riverside of Azusa River, Azumi Village, Nagano Prefecture. "Characteristics" of this strain was determined according to the method described in "Japan Patent Office Industrial Examination Standards". The basal hypha of this strain is not broken. Aerial hyphae form a long main axis, branch irregularly, and at the tip form a compact spiral spore chain. Spores are non-motile, cylindrical to oblong, 0.4-0.5 width
μm, 0.8-1.2 μm in length, and the spore surface is smooth.
The spore chain was tightly wound and a spore-like morphology was observed.
No special morphology such as sclerotia is observed. The cells are wall type I.

Table 1 shows the culture characteristics. The flora color of the colony surface was a gray series and the back color was unclear, pH-insensitive, and the diffusible pigment was slightly white-brown only on tyrosine agar. The physiological properties are shown in Table-2. This strain was mesophilic and used all the diagnostic sugars for its carbon source assimilation ability. Based on the morphological properties and cell wall chemical type of this strain, this strain was Streptomyces
(Hereinafter referred to as S.). Based on the above properties, a search was made for known species of the genus S, and S. colinus was selected as the closest relative. A recently published book “Mr. Barjay's Recent Phylogenetic Handbook, Volume 4” (Berge
According to the description of Williams, et. al. in the S genus of the y's Manual of Systematic Bacteriology Vol. 4), S. colinas is a synonym (synonym) of S. cyaneus. Therefore, the present strain 2299
-SV1 is contained in S. cyaneus-strain and identified as Streptomyces. Cyaneus (Streptomyces cyaneus)
Designated 2299-SV1 strain.

Table 1 Culture characteristics culture medium Microflora color of colony surface Backside color of colony Diffusible pigment sucrose ・ No growth Light yellow (2ca) None Nitrate agar Glucose / As Gray series Light yellow (2ca) None Paragine agar Light brown taste gray (3gc) Glycerin / As Gray series Light brown taste gray (3gc) None Paragine agar Inorganic salt / starch Gray series From pale yellow brown (3ie) None Agar Grayish yellow brown (3lg) Tyrosine agar No growth Light brown taste gray White brown (3ec) (3cb) Nutrient agar No growth Light brown taste gray None (3ec) Yeast / malt Gray series Brown (5lg) None Agar Oatmeal Gray series Light brown taste gray (3gc) None Agar to light yellow brown (3ie)・ () indicates the color code of the Color Harmony Manual (Container Corporation of America, 1950).

Table 2 Physiological properties Growth temperature range 20-45 ° C Optimum temperature 20-37 ° C Melanin-like pigment Tyrosine agar + Peptone yeast iron agar + Trypton yeast broth + Hydrolysis of starch + Liquefaction of gelatin + Degreasing Peptonization of milk powder + coagulation of skim milk powder-reduction of nitrate + assimilation of carbon source D-glucose + L-arabinose + D-xylose + D-fructose + sucrose + L-rhamnose + raffinose + i -inositol + D-man Knit +

The measurement of the glycerophosphate dehydrogenase inhibitory activity in the present invention is carried out according to the method described in The Journal of Biological Chemist.
Wise and Gree as described in ry 254, 273-275, 1979
According to the method of n, the procedure was as follows. 100 mM triethanol / HCl containing 2.5 mM EDTA
2.25 ml of buffer (pH 7.5) is placed in a 1 cm square cell, and 0.3 ml of 6 mM dihydroxyacetone phosphate, 0.1 ml of 3 mM β-mercaptoethanol and 0.1 ml of 3.6 mM NADH are placed therein. And the active substance (substance to be tested) is 0.5 to 3.0.
0.1 ml of methanol solution containing μg / ml
And mix well. To this, 0.15 ml of a 2 unit / ml glycerophosphate dehydrogenase solution is added, and after mixing, immediately, the absorbance at 340 nm is time-scanned. The initial rate of decrease in the absorbance at 340 nm at this time is defined as the enzyme activity, and the enzyme activity when the active substance is added is calculated, where the enzyme activity when the methanol solution containing no active substance is added is defined as 100.

Inhibition rate (%) = 100− (activity of sample / activity of control) × 100 Confirmation of the effect of preventing fat accumulation in the present invention was carried out using Comp. Bioc.
Ka described in hem. Physiol. 96A, 323-326, 1990.
According to the method of wada et al., the procedure was as follows.

0.5 ml of Dulbecco's modified Eagle's medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum was placed in a 24-well multi-dish, and 1000 mouse fibroblast 3T3-L1 cells / The cells are inoculated at a ratio of 1 well, and cultured at 37 ° C., 5% CO _2, pH 7.2 for 6 days. Next, the cultured cells are added to the above medium for 1 to 3.
The cells were transferred to a medium supplemented with 7 μg / ml of the active substance, and the medium was further added to 0.25 mM dexamethasone, 0.5
mM 1-methyl-3-isobutylxanthine and 10 mM
0.05 ml with μg / ml insulin added
, And cultured for 7 days at 37 ° C., 5% CO _2, pH 7.2. After the culture, the medium was removed, the cells were washed with a phosphate buffer-saline, and then 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA.
Is added to the cells to scrape the cells, and the suspension is sonicated to destroy the cells. The amount of triglyceride in the obtained liquid was determined by an enzyme method (triglyceride G-test).
It is measured with a Wako kit (manufactured by Wako Pure Chemical Industries, Ltd.).

The glycerophosphate dehydrogenase inhibitor of the present invention may be a 5-alkylresorcinol itself or a composition mixed with a physiologically acceptable carrier and / or diluent, for example, powders, granules, tablets, capsules , Dry syrup, injections and the like can be administered orally or parenterally. For example, when administered orally, weight 1
The dose is preferably 10 to 100 mg per kg, and 2 to 20 mg per kg for parenteral administration.

[0018]

EFFECTS OF THE INVENTION The active substance of the present invention has a glycerophosphate dehydrogenase inhibitory activity which is at least 10 times stronger than that of stearic acid, which is considered to be the strongest inhibitory activity of existing active substances. Promising. Next, the present invention will be described with reference to examples.

[0019]

Example 1 (Production of active substance) In a large test tube, a medium (1% soluble starch, 1% polypeptone, 1% molasses, 1% Beef extrac) was added.
(t, pH 7.2), 15 ml, into which streptomyces cyaneus 2299
One loopful of -SV1 strain (FERM BP-3607) was inoculated and cultured with shaking at 27 ° C for 72 hours. Then, 1 ml of this culture was added to a medium (2% glycerin, 1% molasses, 0.1%
5% casein, 0.1% polypeptone, 0.4% calcium carbonate, pH 7.0)
The mixture was transferred to an Erlenmeyer flask and shake cultured at 27 ° C. for 4 days.

After the completion of the culture, 8.2 l of a culture liquid from which about 10 l of the culture liquid was removed by centrifugation to remove solid substances such as bacterial cells was added, and ethyl acetate was added thereto to extract the product in the culture liquid. Next, ethyl acetate was separated, and ethyl acetate was distilled off under reduced pressure to obtain 830 mg of a crude extract. Next, the crude extract was subjected to column chromatography (developing solvent; chloroform: methanol = 200: 1) using silica gel (Wakogel C-300; manufactured by Wako Pure Chemical Industries, Ltd.) as a carrier, and Toyopearl HW-40 (manufactured by Tosoh Corporation). Column chromatography (developing solvent: methanol) using a white powder 3
0 mg was obtained.

The result of GC / MS analysis of this substance revealed that it was a mixture of about 7 components (FIG. 1). The EI-MS spectra of these substances all had a base peak at m / z 124, suggesting analogs. Furthermore, each substance was obtained by preparative liquid chromatography (developing solvent; 95% methanol) using an ODS column.

Among them, ¹ H-NMR, ¹³
From each spectrum of C-NMR, IR and FAB-MS, it was confirmed that the substance was 5-n-pentadecylresorcinol. Measurement results: ¹ H-NMR solvent; heavy chloroform, see FIG. 2 ¹³ C-NMR solvent; heavy chloroform, see FIG. 3 IR film method, see FIG. 4 High resolution FAB-MS: m / z = 321.2870 (M
+ H) 321.22849 (m + H) For other substances, the chain length and terminal of the side chain were determined from the molecular ion peak by EI-MS and the coupling of alkyl side chain terminal methyl (δ 0.87) by ¹ H-NMR analysis. These substances have 14 to 16 carbon atoms.
It was confirmed to be 5-alkylresorcinols having the following alkyl group. The results are shown in Table-3.

Table 3 No. GC / MS Rete EI-MS analysis (m / z) ¹ H-NMR Compound name Absorber base Molecular ion Cup Im (min) Peak Peak ring 1 20.8 124 306 d 5-Isotetra Decyl resorcinol 2 21.1 124 306 t 5-n-tetradecyl resorcinol 3 21.6 124 320 d 5-Isopentadecyl resorcinol 4 21.8 124 320 t 5-n-pentadecyl resorcinol 5 22.3 124 334 d 5- Isohexadecyl resorcinol 6 22.4 124 334 7 22.6 124 334 t 5-n-hexadecyl resorcinol d and t in the table represent terminal methyl (δ in ¹ H-NMR).
D in the coupling of 0.87); doublet
t: triplet.

[0024]

Example 2 (Inhibition activity) 100 mM containing 2.5 mM EDTA
Triethanol / HCl buffer (pH 7.5)
2.25 ml was put into a 1 cm square cell, and 6 mM
0.3 ml of dihydroxyacetone phosphoric acid, 0.1 ml of 3 mM β-mercaptoethanol and 0.1 ml of 3.6 mM NADH, and 0.5 to 3.0 μg / ml of the active ingredient alone or as a mixture. Was added and mixed well. To this, 0.15 ml of a 2 unit / ml glycerophosphate dehydrogenase solution was added, and after mixing, 3
The absorbance at 40 nm was time scanned.

The initial rate of decrease in the absorbance at 340 nm is defined as the enzyme activity, and the relationship between the enzyme activity when the active substance is added and the concentration of the active substance relative to the enzyme activity when a methanol solution containing no active ingredient is added. Is shown in FIG.

[0026]

Example 3 (Prevention of Fat Accumulation in Animal Cells) 0.5 ml of Dulbecco's modified Eagle's medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum was placed in a 24-well multi-dish, and mice were Fibroblast 3T3-L1 cells at 1000 cells / 1
Inoculated at the ratio of the holes, 37 ° C, 5% CO _2, pH 7.2
For 6 days. Next, the cells after the culture are transferred to a medium obtained by adding an active substance of 1 to 3.7 μg / ml to the above-mentioned medium, and further, 0.25 mM dexamethasone and 0.5 mM 1-methyl-3-isobutyl are added to the medium. Xanthine and 0.05 ml to which 10 μg / ml of insulin was added were added, and the mixture was added at 37 ° C., 5% CO _{2 for} 7 days _.
The cells were cultured under the condition of pH 7.2. After the culture, remove the medium,
After washing the cells with phosphate-bapper saline, 0.3 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA was added, and the cells were scraped off.
The suspension was sonicated to destroy the cells. The amount of triglyceride in the obtained liquid was measured by an enzyme method (triglyceride G-test Wako kit (manufactured by Wako Pure Chemical Industries, Ltd.)).

FIG. 6 shows the relationship between the concentration of the active ingredient and the amount of accumulated triglyceride in the mouse fibroblast 3T3-L1 cells.

[0028]

[Brief description of the drawings]

FIG. 1 is a total ion chromatogram of a 5-alkylresorcinol as an active ingredient in GC / MS analysis.

FIG. 2 is a diagram showing a ¹ H-NMR spectrum chart of 5-n-pentadecylresorcinol using deuterated chloroform as a solvent.

FIG. 3 is a chart showing a ¹³ C-NMR spectrum chart of 5-n-pentadecylresorcinol using deuterated chloroform as a solvent.

FIG. 4 is a view showing an IR spectrum chart of 5-n-pentadecylresorcinol measured by a film method.

FIG. 5 is a graph showing the relationship between the concentrations of 5-n-pentadecyl resorcinol and 5-isopentadecyl resorcinol and the glycerophosphate dehydrogenase inhibitory rate.

FIG. 6. In mouse fibroblast 3T3-L1 cells,
It is a figure showing the relation between the concentration of 5-n-pentadecyl resorcinol and 5-isopentadecyl resorcinol, and the amount of triglyceride accumulation.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 465) (72) Inventor Makiko Mizogami 1-5-7 Mikitei Sakaecho, Higashi Osaka City, Osaka Inside House Foods Industry Co., Ltd. 56) References. ANTIBIOT. VOL. 45, NO. 6, (1992) p. 886-891 (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 31/05 CA (STN) MEDLINE (STN)

Claims (1)

(57) [Claims] 1. A glycerophosphate dehydrogenase inhibitor comprising, as an active ingredient, a 5-alkylresorcinol having an alkyl group having 14 to 16 carbon atoms.