JPS63104920A - Aldose reactase inhibitor - Google Patents

Abstract

PURPOSE:To obtain an aldose reactase inhibitor containing a specific compound such as liquiritin, liquiritigenin, etc., as an active ingredient and useful in treatment for various complications of diabetes such as cataract, retinopathy, nervous disorder, kidney disease, etc. CONSTITUTION:A compound expressed by the formula (R is H or glucose; R' is H or OH), namely liquirithin (R=glucose, R'=H), liquiritigenin (R=H, R'=H), choerospondin (R=glucose, R'=OH) or naringenin (R=H, R'=OH) is contained as an active ingredient. Said compound is an active ingredient contained in Licorice. The Licorice is extracted with water or/and alcohols and the extract solution is then subjected to column chromatography containing Sephadex, etc., used as a carrier to provide each fraction and water-methanol (1:1) elution part and methanol elution part are subjected to column chromatography and thin layer chromatography to afford each compound.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to an aldose reductase which has an aldose reductase inhibitory effect and is useful for the treatment of various complications of diabetes such as cataracts, retinopathy, neuropathy, and nephropathy. It concerns inhibitors.

[Prior Art and Problems] In recent years, the accumulation of intracellular sorbitol via the polyol pathway, which is a glucose metabolic pathway, has attracted attention as a cause of various complications in diabetes such as cataracts, retinopathy, and nephropathy. The polyol pathway is a metabolic pathway in which aldoses such as glucose and galactose are converted to ketoses such as fructose via polyols such as sorbitol and galactitol, and immunohistochemical techniques have revealed that they are widely present in all organs throughout the body. It has become.

The first step in this pathway, the enzyme that catalyzes 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, it has been desired to develop a drug that has an aldose reductase inhibitory effect.

[Means for Solving the Problems] As a result of research on the aldose reductase inhibitory effect of various herbal medicines, the present inventors found that licorice (Gly
cyrrhiza uralensis FISIIE
R.

Glycyrrhiza glabra LINNE
var, glanduliferaREGEL et
We discovered that the roots and stolons of IIERDER and other congenerous plants have a strong aldose reductase inhibitory effect, and as a result of conducting research on the active ingredients of licorice, we found that the compound represented by the general formula has an extremely strong aldose reductase inhibitory effect. The present invention was completed based on the discovery that the present invention has the following properties. That is, the present invention is based on the general formula [where R represents a hydrogen atom or glucose, and R'' represents a hydrogen atom or a hydroxyl group]. This is an aldose reductase inhibitor containing a compound represented by (hereinafter referred to as a compound of the general formula) as an active ingredient.

Compounds of the general formula include the compounds shown below.

For example, the following methods can be used to obtain these compounds.

Licorice is extracted with water, alcohol, or a mixed solvent of water and alcohol, and the residue removed from the extract is sequentially extracted.
Water, water-methanol (1:, 1), methanol as an elution solvent, subjected to column chromatography using Sephadex such as Sephadex LH-20, porous polymer such as Diaion IP-20, etc. as a carrier, Obtain each fraction. Next, the water-methanol (1:l) eluted portion and the methanol eluted portion were treated with Sephasics using at least one selected from water, methanol, ethanol, ether, acetone, chloroform, benzene, and a water-acetic acid-methanol mixture as an elution solvent. Dex LH-20
Column chromatography using Sephadex, M(, I gel CHP20P, etc.), cellulose, silica gel, or reversed-phase silica gel as a carrier was performed several times, and the target components were confirmed using thin layer chromatography. It can be obtained by fractionation. Optionally, it may be purified by recrystallization using an appropriate solvent such as methanol, ethanol, or water.

A specific example of the production of the compound of the general formula is as follows.

Specific Example 1 Licorice 1.4 to 9 was extracted with 10&ml of water, and the water was removed from the extract to obtain 400 g of Suiko Kiss. After dissolving this Suiko Kiss again in Water IQ, Sephadex LH-20 (
Subjected to column chromatography (manufactured by Pharmacia),
Elute sequentially with water, water-methanol (1:1), and methanol. By concentrating the water-methanol (III) eluate under reduced pressure and crystallizing the precipitated white powder from water-ethanol, an Rf value of 0.30 [thin layer plate: Kieselgel 60Fzs], developing solvent: chloroform-methanol (3 :l), coloring reagent: 10% sulfuric acid (orange color)] was obtained. This compound matched the standard liquiritin in the mixing test and infrared absorption spectrum.

Specific Example 2 The methanol eluate obtained by column chromatography on Sephadex LH-20 in Specific Example 1 was again subjected to Sephadex L11-20 to increase the concentration from water-methanol (1:0) to (0:1). By applying a gradient elution and crystallizing both eluted fractions from water-ethanol (4:6), the Rf value was 0.45 [thin layer plate: Kiegelgel 60F0].Developing solvent: benzene-ether (
Colorless needle-shaped crystals of 1:1), coloring reagent: 10% sulfuric acid (orange) were obtained. The physicochemical properties of this compound are described in the literature [C4a
n Hulle.

Braeckman, P., Jandewalle, P.
lanta Med, 20, 278 (1971)].

Specific Example 3 Both fractions eluted with water-methanol (4:6) in the MCI Gel C11P20P column chromatography of Specific Example 2 were treated with Fujigel (ODSG a type,
The mixture was subjected to column chromatography (Mito Kagaku Gijutsu Kenkyushin) to obtain both fractions, which were eluted with water-methanol (1:l). This fraction was concentrated under reduced pressure and the precipitated crystals were recrystallized from water-methanol to obtain an Rf value of 0.40 [thin layer plate: Kiegelgel 60 F25 developing solvent:
Chloroform-methanol (3:1), color reagent: 10
% sulfuric acid (yellow)] was obtained. The physical and chemical properties of this compound are described in the literature [Lu Yongzhen, Wang Yulan, Lou Zhuoxiong, Zu Jingyu, Lianguanqing 2 Zhou Zhuolun, Kuxuexuepo, +8. +99 (1983
)] was consistent with the properties of kerosbondein.

Specific Example 4 The fraction eluted with water-methanol (3N) in Sephadex LH-20 column chromatography in Specific Example 1 was then subjected to MCI gel CHP20P column chromatography, and a 5-degree gradient was applied with water-methanol. Both fractions were obtained, eluting with water-methanol (3N).

This was subjected to cellulose (Avicel, manufactured by Fukaisei) column chromatography, and 2% acetic acid-methanol (4:1) was used.
It was eluted. Both fractions eluted with the first 112 were concentrated under reduced pressure, and the precipitated crystals were recrystallized with water-methanol to obtain an Rf value of 0.50 [thin layer plate: Kiegelgel 6].
0F25<, developing solvent: benzene-acetone (2:1)
, chromogenic reagent.

Colorless needle-shaped crystals of 10% sulfuric acid (yellow) were obtained. The physical and chemical properties of this compound are described in the literature [Lu Yongzhen, Wang Yulan.

Lou Zhu Xiong, Zu Jing, Yu, Liang Kuanqing, 2 Zhou Zhuolun, and Ping Xuebao.

LL, 199 (1983)].

Next, the fact that the compound of the general formula has an aldose reductase inhibitory effect will be explained with reference to experimental examples.

Experimental Example 1 Measurement of aldose reductase activity> Stored at -20°C.

The crystalline lens was homogenized in 135mM sodium-potassium-phosphate buffer (pH 7.0) containing 0.5mM phenylmethylsulfonyl fluoride.
Centrifuged at pm for 30 minutes. The supernatant was used as a sample for aldose reductase activity measurement.

Furthermore, all of the above operations were performed at 4°C, and the specimens were stored at 0°C.

Aldose reductase activity was measured using Dufuran (Dufuran).
frane) et al. [Biochea+1cal M
edicine.

LL, 99-105 (1984)].

i.e. 100mM lithium sulfate, 0.03mMN
ADP 11 (reduced nicotinamide
adenine nucleotide phos
phate), and 135 mM sodium-potassium-phosphate buffer (p[!7.0) prepared to contain 0.1 mM DL-glyceraldehyde or 20 mM glucose as a substrate.
A 100 degree drug solution prepared by dissolving each of the compounds obtained in Specific Examples 1 to 4 above in ethanol to a final concentration of 1 x 10-11!9/d was added to each, and reacted at 30°C for 30 minutes. I let it happen. Next, 0.5N hydrochloric acid 0.377
was added to stop the reaction, and 1 d of 6N sodium hydroxide containing 10 mM imidazole was added to remove NADP (oxidized nicoti) produced by the above reaction.
namide adenine dinucleoti
de phosphate) into a fluorescent substance, and 6
The fluorescence intensity was measured 0 minutes later. The fluorescence intensity was measured at room temperature using a spectrophotometer RF-510 (manufactured by Shimadzu Corporation) under conditions of an excitation wavelength of 360 nm and a fluorescence wavelength of 460 nm.

Further, the fluorescence intensity measured by reacting in the same manner as above except that ethanol was added instead of adding the drug solution was used as a control value.

Aldose reductase uses NADPH as a coenzyme,
DL-An enzyme that converts glyceraldehyde or glucose into polyol, and along with this reaction, NADP
H changes to NADP. Therefore, if NADP is low, aldose reductase is inhibited.

The results are expressed as the degree of inhibition (%) and the 50% inhibitory concentration (lC
5o) shown in Table 1.

Table 1 Rat Lens Aldose Reductase From the above results, it was confirmed that the compound of the general formula inhibits the activity of aldose reductase, and is expected to be effective in preventing or treating diabetic complications.

Next, acute toxicity tests were conducted on oral administration of the compounds obtained in Examples 1 to 4 using ddY mice and Wistar (llista) mice.
When experiments were conducted using rats of the r) type, there were no deaths when the compounds obtained in Specific Examples 1 to 4 were orally administered at a dose of 1 g/kg.

As described above, the compound of the general formula has extremely low toxicity and high safety.

Considering the experimental data and the results of acute toxicity tests in the present invention, the effective dosage of the compound of the general formula is based on the patient's age,
Although it varies depending on the body weight and severity of the disease, it is generally considered appropriate for adults to take the general formula compound in doses of 1120 to 600 Q per day, divided into three doses per day depending on the symptoms.

Next, the present invention will be specifically explained using an example, but the present invention is not limited thereto.

Example 1 100g of the compound obtained in Example 1 above was mixed with 20g of silicic anhydride.
75 g of corn starch was added thereto and further mixed. A 10% ethanol solution of 10% hydroxypropyl cellulose was added to this mixture, and the mixture was wetted in a conventional manner, extruded, dried and sieved to obtain granules of 20 to 50 mesh particles.

This granule has a dosage of 80 to 40,014 depending on the symptoms.
9 (40 to 200 as the weight of the compound obtained in Specific Example 1)
xw) 3 times a day.

Example 2 40 g of the compound obtained in Example 2 was mixed with 20 g of silicic anhydride, 50 g of microcrystalline cellulose IOf, magnesium stearate, and lactose were added and mixed, and the mixture was compressed into tablets using a single-shot tablet machine. Tablets with a diameter of 7 mm and a weight of 12,019 were produced.

One tablet of the present invention contains 40m9 of the compound obtained in Specific Example 2. This tablet is taken 1 to 5 tablets at a time, three times a day.

Example 3 Compound 401!9 obtained in Example 3 was converted into lactose 1001! r9
and filled into gelatin capsules No. and O to obtain capsules.

This capsule preparation is taken 1 to 5 capsules at a time, three times a day, depending on the symptoms.

Example 4 100 g of the compound obtained in Example 4 was mixed with 20 g of silicic anhydride, and 75 g of corn starch was added thereto and further mixed. To this mixture, 1001 d of 10% hydroxypropylcellulose ethanol solution was added, and the mixture was stirred in a conventional manner, extruded, dried, and sieved.
Granules with particles of 0 to '50 mesh were obtained.

This granule has a dosage of 80 to 400 tons depending on the symptoms.
ttg (40 to 20 as the weight of the compound obtained in Specific Example 4)
(equivalent to 0■) and should be taken three times a day.

Claims (1)

[Claims] General formula▲ Numerical formulas, chemical formulas, tables, etc.▼ [In the formula, R represents a hydrogen atom or glucose, and R' represents a hydrogen atom or a hydroxyl group. ] An aldose reductase inhibitor containing a compound represented by the following as an active ingredient.