JPH0643329B2 - Aldo-reductase inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an aldose reductase inhibitor containing as an active ingredient one or more kinds of water or an aqueous organic solvent extract selected from cinnamon bark, peony root, oju, ginger, licorice, soy sauce, and Fuzi. It is about.

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 first step in this pathway, the conversion between aldose and polyol, is called aldose reductase,
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, as a result of conducting research on aldose reductase inhibitory action for various Chinese herbal formulas and their constituent crude drugs, one or more kinds selected from cinnamon bark, peony root, oju, ginger, licorice, soy sauce, and Fuzi It was found that water or an aqueous organic solvent extract has an aldose reductase inhibitory action. The present invention is based on this finding, effective in the treatment of complications in diabetic patients, cinnamon, peony, ozouraku, ginger, licorice, soy sauce, one or more kinds of water or water organic selected from Fuzi The present invention provides an aldose reductase inhibitor comprising a solvent extract.

Water or an aqueous organic solvent extract of crude drug in the present invention means one or more kinds of water or an aqueous organic solvent selected from cinnamon bark, peony root, large jujube, ginger, licorice, soy sauce, and Fuzi (for example, an aqueous solvent such as ethanol. It means an extract obtained by extraction with (alcohol) or a dried extract obtained by drying the obtained extract by a usual method such as spray drying, freeze drying or concentration to dryness. In order to obtain the target extract, one or more of the above crude drugs may be mixed and extracted, or each crude drug may be extracted and then the respective extracts may be mixed. The extraction conditions may be room temperature or heating (heating to about 80 to 100 ° C.), but heating is preferably performed. Although this extract can be used as it is, powders, granules, tablets, capsules, etc. can be prepared according to a conventional method of manufacturing a formulation by adding excipients, adjuvants and the like used in usual formulations. It can also be used in the form of a preparation. If desired, this extract may be further purified by a conventional method such as dialysis or various chromatographies and used. Of these, cinnamon 3 ~
4 parts by weight, 3-4 parts by weight of peony, 3-4 parts by weight of oju, 3-4 parts by weight of ginger, 2 parts by weight of licorice, 3-4 parts by weight of soy sauce, 0.5-1 part by weight of sardine The Kampo prescription is generally called Keishin Kashutsu-to, and its medicinal effect is described in the Kampo book (medical practice). It is used.
However, it has never been known that it has an aldose reductase inhibitory action.

A specific example of the production of the crude drug extract is as follows.

Example 1 4 g of cinnamon bark, 4 g of peony, 4 g of large jujube, 4 g of ginger, 2 g of licorice,
10 times the amount of mixed crude drug of Sogaku 4g and Fuzi 1g, ie 2
30 ml of water was added and the mixture was heated and extracted at about 100 ° C. for 1 hour, and the extract obtained was filtered and spray-dried to obtain 4.5 g of dry extract powder.

Example 2 15 g of cinnamon bark was added with a 10-fold amount, that is, 150 ml of water, and heat-extracted at about 100 ° C. for 1 hour. The obtained extract was filtered and concentrated to dryness to obtain 1.4 g of dried extract.

Example 3 To 15 g of soy sauce, 10 times amount, that is, 150 ml of water was added, and the mixture was heated and extracted at about 100 ° C. for 1 hour. The obtained extract was filtered and concentrated to dryness to obtain 1.8 g of dried extract.

Example 4 15 g of peony root was added with a 10-fold amount, that is, 150 ml of water, and heat-extracted at about 100 ° C. for 1 hour. The obtained extract was filtered and concentrated to dryness to obtain 1.7 g of dried extract.

Specific Example 5 15 g of licorice was added with 10 times amount, that is, 150 ml of water, and the mixture was heated and extracted at about 100 ° C. for 1 hour. The obtained extract was filtered and concentrated to dryness to obtain 1.6 g of dried extract.

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 homogenized with a 135 mM sodium-potassium-phosphate buffer solution (pH 7.0) containing 0.5 mM phenylmethylsulfonyl fluoride and 30,000 rpm.
It was centrifuged for 30 minutes. The supernatant was used as a sample for aldose reductase activity measurement. Also, all the above operations are 4
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)]. That is, 100
mM lithium sulfate, 0.03mM NADPH (reduced nicotinam
ide 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.
A drug solution 1 in which 00 μl and the drug obtained in each of the above specific examples 1 to 5 were dissolved in distilled water to a concentration of 1 mg / ml
00 μl was added to each and reacted at 0 ° C. for 30 minutes. Then, the reaction was stopped by adding 0.3 ml of 0.5N hydrochloric acid, 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.

The results are shown in Table 1.

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
Krebs-Ringer, which was prepared by dissolving glucose and the drugs obtained in the above-mentioned specific examples 1, 4 and 5 with distilled water so as to have a concentration of 2.5 mg / ml and equilibrated with 95% oxygen and 5% carbon dioxide, respectively. Bicarbonate buffer
1 ml of the erythrocyte suspension was added to 4 ml of ffer) and incubated at 37 ° C. After 60 minutes, 6% cold perchloric acid was added to stop the reaction, and the protein was removed by centrifugation at 3,000 rpm for 10 minutes at 4 ° C. 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 for 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 described above was used as a control value except that the chemical agents obtained in Specific Examples 1, 4 and 5 were not added at the time of reaction and were added after the reaction was completed.

This result was used as the control sorbitol content of 10
It is shown in Table 2 as 0%.

From the above results, the aldose reductase inhibitor of the present invention was observed to inhibit the activity of aldose reductase, reduce the accumulation of sorbitol in erythrocytes, and is expected to be effective in the prevention or treatment of diabetic complications. To be done.

Next, an acute toxicity test of the drugs obtained in Examples 1 to 5 by oral administration was carried out using ddY mouse and Wistar rat, and the drugs obtained in Examples 1 to 5 were 15
No death occurred even by oral administration of g / kg (dose 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 produced according to Example 1 above was added to lactose 95
g and 5 g of magnesium stearate, and the mixture is compression molded, crushed, sized and sieved to 2
Good granules with 0-50 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 produced according to Example 5 above was added to lactose 95
g and 5 g of magnesium stearate, and the mixture is compression molded, crushed, sized and sieved to 2
Good granules with 0-50 mesh particles were obtained.

This granule is taken at a daily dose of 1.5 to 5 g (corresponding to 1 to 10 g as the dry extract powder weight of the aldose reductase inhibitor of the present invention) in three divided doses depending on the symptoms.

Example 3 200 g of the drug produced according to Example 3 above was added to lactose 95
g and 5 g of magnesium stearate, and the mixture is compression molded, crushed, sized and sieved to 2
Good granules with 0-50 mesh particles were obtained.

This granule is taken at a daily dose of 1.5 to 5 g (corresponding to 1 to 10 g as the dry extract powder weight of the aldose reductase inhibitor of the present invention) in three divided doses depending on the symptoms.

Example 4 200 g of the drug prepared according to Example 2 above was mixed with 45 g of microcrystalline cellulose and 5 g of magnesium stearate, and the mixture was tabletted with a single-shot tableting machine to produce a tablet having a diameter of 9 mm and a weight of 250 mg. did. 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 5 200 g of the drug prepared according to Example 4 above 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 produce a tablet having a diameter of 9 mm and a weight of 250 mg. did. 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 6 200 g of the drug prepared according to Example 5 above 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 produce a tablet having a diameter of 9 mm and a weight of 250 mg. did. 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 7 A hard capsule was filled with 250 g of the drug produced according to Example 1 above. This capsule is 4 times daily depending on the symptoms.
Take ~ 40 capsules 3-6 times daily.

Example 8 A hard capsule was filled with 250 g of the drug prepared according to Example 3 above. This capsule is 4 times daily depending on the symptoms.
Take ~ 40 capsules 3-6 times daily.

Example 9 A hard capsule was filled with 250 g of the drug produced according to Example 4 above. This capsule is 4 times daily depending on the symptoms.
Take ~ 40 capsules 3-6 times daily.

Claims (1)

[Claims] 1. Cinnamomi cortex and peony
ae Radix), large jujube (Zizyphi Fructus), ginger (Zingibe
ris Rhizoma), licorice (Glycyrrhizae Radix), blue (A)
tractylodis Lanceae Rhizoma), Fuzi (Aconiti Tube)
An aldose reductase inhibitor containing, as an active ingredient, water or an aqueous organic solvent extract of one or more crude drugs selected from r).