JPH0643334B2 - Aldo-reductase inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an aldose reductase inhibitor consisting of Seokeiseito.

In recent years, accumulation of intracellular sorbitol through a polyol pathway, which is a glucose metabolism pathway, has been attracting attention as a cause of various complications in diabetes such as cataract, retinopathy, neuropathy, and nephropathy. The polyol pathway is a metabolic pathway in which aldoses such as glucose and galactose are converted into ketose such as fructose through polyols such as sorbitol and galactitol, and it is clear that they are widely present in various organs throughout the body by immunohistochemical techniques. It has become.

The enzyme that catalyzes the conversion between aldose polyols, which is the first step in this pathway, is called aldose reductase, and this enzyme is considered to be the rate-limiting enzyme in the polyol pathway.
It has been reported that inhibiting this aldose reductase and reducing the production and accumulation of sorbitol is effective in treating complications in diabetic patients. Therefore, the present inventors have conducted research on the aldose reductase inhibitory action of various Kampo prescriptions. It was found that Keiketsuto has an aldose reductase inhibitory action. The present invention is based on this finding, and provides an aldose reductase inhibitor consisting of Seokeiseito, which is effective in treating complications in diabetic patients.
Saikyoketsuto is described in the classical Chinese medicine formula (Menjujutsu) as to its constituent herbal medicines, doses, extraction methods, etc., and is used for various diseases such as neuralgia, arthralgia, back pain, and muscle pain. However, it has never been known that it has an aldose reductase inhibitory action.

According to the classic, Seokei-Ketsuto, 2.5g of peony medicine, ground 2g of peony, 1.5g of beef knees, 1.5g of leather, 1.5
g, It can be decocted with the above water to make 180 ml, and this can be taken as an aldose reductase inhibitor. However, in consideration of the ease of taking and the convenience of carrying, use the herbal medicine extract as the aldose reductase inhibitor. You can also For example, peony ~ 2.5 parts by weight, Soso 2-3 parts by weight, Toki 2-3.5 parts by weight, Tomonito 2-3 parts by weight, Furei 1-2 parts by weight, cow knee 1.
5 to 3 parts by weight, Chen skin 1.5 to 3 parts by weight, protected 1.5 to
2.5 parts by weight, windbreak 1.5-2.5 parts by weight, long gall 1.5
~ 2.5 parts by weight, licorice 1 part by weight, The extract obtained by hot extraction with 10 times the amount of water was dried and dried to obtain powder of dried extract of keisei-kaeto, which is an aldose reductase inhibitor, and an excipient used in ordinary preparations for this. It can also be used in the form of powders, granules, tablets, capsules and the like according to a conventional method for producing a preparation by adding an auxiliary agent and the like. If desired, this extract may be further purified by a conventional method such as dialysis or various chromatographies and used.

A specific example of the production of the aldose reductase inhibitor of the present invention is as follows.

Concrete example Ki 2g, Tomonito 2g, Furei 2g, Beef knee 1.5g, Chen skin 1.
5g, Protected 1.5g, Windproof 1.5g, Long gall 1.5g, Add 275 ml of water and heat extraction at about 100 ° C for 1 hour.
After the obtained extract was spray dried, 3.3 g
To obtain a dry extract powder.

Next, it will be described with reference to experimental examples that the aldose reductase inhibitor of the present invention has an aldose reductase inhibitory action.

Experimental Example 1 <Measurement of aldose reductase activity> A 6-week-old Wistar male rat was sacrificed under ether anesthesia, and the lens was immediately removed and stored at -20 ° C.

The lens is a 135 mM sodium-potassium-phosphate buffer solution (pH: 0.5 mM phenylmethylfonyl fluoride).
7.0) and homogenize at 30,000 rpm for 3
Centrifuge for 0 minutes. The supernatant was used as a sample for aldose reductase activity measurement. All the above operations were performed at 4 ° C, and the sample was stored at 0 ° C.

The measurement of aldose reductase activity was carried out using Dufram (Dufra).
ne) et al. [Biochemical Medicine, 32, 99-10]
5 (1984)]. Ie 100 mM
Lithium sulfate, 0.03mMNADPH (reduced nicotinamid
adenine dinucleotide phosphate) and 800 μl of 135 mM sodium-potassium-phosphate buffer (pH 7.0) prepared so as to contain 0.1 mM DL-glyceraldehyde or 20 mM glucose as a substrate, 100 μl of the above-mentioned sample and 100 μl of the drug solution prepared by dissolving the drug obtained in each example in distilled water to 1 mg / ml was added, and the mixture was reacted at 30 ° C. for 30 minutes. Next, 0.3 ml of 0.5N hydrochloric acid was added to stop the reaction, and 6N sodium hydroxide containing 10 mM imidazole was added.
NA was generated by the above reaction by adding ml.
DP (oxidized nicotinamide adenine dinucleotide phos
(phate) was converted into a fluorescent substance, and 60 minutes later, the fluorescence intensity was measured. Fluorescence intensity is spectrophotometer PF-51 at room temperature
0 (manufactured by Shimadzu Corporation) using an excitation wavelength of 360n
The measurement was performed under the conditions of m and fluorescence wavelength of 460 nm. Further, the fluorescence intensity measured by reacting in the same manner as above except that distilled water was added instead of the drug solution was used as a control value.

Aldose reductase is a DL-
It is an enzyme that converts glyceraldehyde or glucose into polyol, and NADPH changes into NADP along with this reaction. Therefore, if NADP is low, aldose reductase is inhibited.

As a result, the degree of inhibition of the drug obtained in the specific example on the aldose reductase activity of rat lens was 80.3% when the substrate was DL-glyceraldehyde, and 48.2% when the substrate was glucose. .

Experimental Example 2 <Quantification of sorbitol in erythrocytes> Erythrocytes obtained from blood of a forearm of a healthy person and treated with heparin were washed with cold saline three times, and further suspended so that the hematocrit value was around 30%. did. 28 mM
Glucose and the drug obtained in the above specific example were dissolved in distilled water to a concentration of 2.5 mg / ml, and oxygen was further adjusted to 95
%, Carbon dioxide 5% and Krebs-Ringer bicarbonate buffer 4 ml were added to the red blood cell suspension 1 ml and incubated at 37 ° C. After 60 minutes, 6% cold perchloric acid was added to stop the reaction.
It was deproteinized by centrifugation at 3,000 rpm for 10 minutes at ℃. After 2.5 M potassium carbonate was added to the supernatant for neutralization, this was used as a sample and the sorbitol concentration was measured by the method of Malone et al.

That is, 50 mM glycine buffer (pH = 1.0 ml)
9.4) and 0.2 mM NAD (oxidized nicotinamide)
adenine dinucleotide) and 0.64 unit of sorbitol dehydrogenase were prepared, and 0.5 ml of the sample deproteinized as described above was added and reacted. NADH produced by this reaction (reduced nico
tinamide adenine dinucleotide) was used as a fluorescent substance, and its fluorescence intensity was measured and used as an index of the amount of sorbitol. Since this reaction is a reaction of converting intracellular sorbitol and NAD into D-fructose and NADH by sorbitol dehydrogenase, the higher the amount of NADH after the reaction, the higher the content of sorbitol.

The fluorescence intensity measured by reacting in the same manner as above was used as a control value, except that the drug obtained in the specific example was not added during the reaction and was added after the reaction was completed.

As a result, the sorbitol content of the control was 100
%, The amount of sorbitol in red blood cells was reduced to 13.7% by the addition of the drug obtained in the specific example.

From the above results, it is observed that the aldose reductase inhibitor of the present invention inhibits the activity of aldose reductase, reduces the accumulation of sorbitol in erythrocytes, and is expected to be effective for complications or treatment of diabetes. .

Next, the acute toxicity test of the oral administration of the drug obtained in the specific example was conducted.
As a result of using Y mice and Wister rats, no drug died even when the drug obtained in the specific example was orally administered at 15 g / kg (administration limit).

Thus, the drug of the present invention has extremely low toxicity and high safety.

Judging from the experimental data and the results of the acute toxicity test in the present invention, the effective dose of the drug of the present invention varies depending on the age, body weight, and degree of disease of the patient. It is considered appropriate to administer the daily dose of 1 to 10 g divided into three times a day according to the symptoms.

Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 200 g of the drug manufactured according to the above specific example was added to 95 g of lactose.
And 5 g of magnesium stearate, and the mixture was compression molded, crushed, sized, and sieved to 20
Good granules with ˜50 mesh particles were obtained.

This granule has a daily dose of 1.5 to 15g depending on the symptoms.
(Corresponding to 1 to 10 g of the dry extract powder weight of the aldose reductase inhibitor of the present invention) is taken in 3 divided doses.

Example 2 200 g of the drug prepared according to the above specific example was mixed with 45 g of microcrystalline cellulose and 5 g of magnesium stearate, and this mixture was tabletted with a single-shot tableting machine to give a diameter of 9
mm tablets weighing 250 mg were produced. One tablet of the present invention contains 200 mg of the drug of the present invention.

The daily dose of this tablet is 5 to 50 tablets 3 to 6 times depending on the symptoms.
Take in divided doses.

Example 3 A hard capsule was filled with 250 g of the drug prepared according to the above specific example. This capsule is 4 to 4 times daily depending on the symptoms.
Take 40 capsules 3 to 6 times daily.

Claims (1)

[Claims] 1. An aldose reductase inhibitor consisting of Seokei-ekito.