JPH0930984A - Aldose reductase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inhibitor comprising a specific natural substance selected from astilbin, taxifolin, etc., as an active ingredient, effective for preventing and treating various diabetic complication, having high safety free from an unpleasant smell. SOLUTION: This aldose reductase inhibitor comprises an active ingredient selected from astilbin, neoastilbin, isoastilbin, neoisoastilbin and taxifolin (for short astilbins). A leaf of Enge lhardiachrysolepis providing having an especially high astilbins content is preferable as a raw material for the objective inhibitor. Astilbins are preferably extracted from the leaf of Enge lhardiachrysolepis by immersing the dried leaf in an extracting solvent such as a 1-4C alcohol, etc., in the weight ratio of 5-15 times the amount of the leaf and eluting a soluble component at a normal temperature or under reflux by heating. The extract is preferably made more active and much more readily usable by purification. When the extract or the purified substance is hydrolyzed, a substance having high taxifolin content is obtained. A dose of the objective inhibitor is preferably 5-500mg/day as astilbins.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the prevention of diabetic complications, which has an action of inhibiting the activity of aldose reductase which is a causative enzyme of diabetic complications such as neuropathy, cataract, retinopathy and renal disorder. It relates to a therapeutically effective aldose reductase inhibitor.

[0002]

2. Description of the Related Art Diabetic complications such as neuropathy, cataracts, retinopathy, and renal disorders are alleged to be caused by polyol metabolism abnormalized by hyperglycemia, that is, activation of the sorbitol pathway.

The sorbitol pathway is considered to be an interconversion pathway between glucose and fructose, is found in systemic tissues, and is rapidly enhanced in hyperglycemic conditions. The rate-limiting enzyme is aldose reductase. In this pathway, sorbitol is produced as an intermediate metabolite, and the produced sorbitol is difficult to be metabolized and does not pass through the cell wall, so that intracellular accumulation of sorbitol progresses with the activation of the sorbitol pathway. As a result, increase in intracellular osmotic pressure →
It is thought that cell damage occurs by the mechanism of cell swelling → oxygen deficiency. In fact, it has been reported that sorbitol in red blood cells of diabetic patients has a high value and is closely associated with complications.

Therefore, the most effective measure against diabetic complications is to inhibit the intracellular accumulation of sorbitol by inhibiting the action of aldose reductase, which is the rate-limiting enzyme of the sorbitol pathway.

From these viewpoints, in recent years, the search for a substance having an aldose reductase inhibitory action has been advanced, a synthetic substance, eparelstat has been developed, and extracts such as cinnamon, peony, mulberry and licorice are effective. It is reported that there is.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel aldose reductase inhibitor which is made from a highly safe natural product as a raw material.

[0007]

The aldose reductase inhibitors successfully provided by the present invention are based on the novel finding that astilbin, neoastilbin, isoastilbin, neoisoastilbin and taxifolin show a strong aldose reductase inhibitory action. It is based on one or more of these compounds as an active ingredient.

[0008] Astilbin, neoastilbin, isoastilbin and neoisoastilbin are dihydroflavonol glycosides having taxifolin as an aglycone and having stereoisomeric relationships with each other (hereinafter, these stereoisomeric groups are referred to as astilbins). ).

Since astilbins are contained in large amounts in the yellow and yellow leaves as described later, the yellow and yellow leaf extract containing astilbins can also be a constituent component of the aldose reductase inhibitor of the present invention.

It has been confirmed that the active ingredient of the above-mentioned aldose reductase inhibitor of the present invention exhibits a non-competitive inhibition mode for aldose reductase activity.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Astilbins are evergreen trees of the walnut family, yellow-brown (Koki, Engelhardtiachrysolepi).
s) is included in the leaf part, and Nejiki (Lyonia ov
alifolia), Senryo (Chloranthus glaber), Kenashisarutoibara (Smilax glabra), Weeping beetle (Ast
ilbe microphylla), Trish gram (Astilbe odonto)
It has been confirmed that it is also contained in phylla) and the like, and the easiest way to obtain it is to extract it from these plants.

Yellow mullet leaves give an extract with a particularly high content of astilbins. Moreover, since it has been used as a kind of sweet tea or has been blended with ordinary tea since ancient times in southern China, there is no problem in safety. Therefore, it is extremely advantageous as a raw material for the aldose reductase inhibitor of the present invention.

Hereinafter, the method for producing the aldose reductase inhibitor of the present invention from yellow trefoil will be described in detail.

Examples of the solvent that can be used to extract astilbins from yellow mulberry leaves are methanol, ethanol, isopropanol, butanol, etc.
4 lower alcohols; lower fatty acid esters such as ethyl acetate; propylene glycol, 1,3-butylene glycol,
There are polyhydric alcohols such as glycerin; water and the like, and a mixture thereof can also be used.

An extract containing astilbins can be obtained by immersing the dried yellow musk leaf in an extraction solvent having a weight ratio of about 5 to 15 times and eluting the soluble component under heating at room temperature or under reflux. The extract obtained by distilling off the solvent can be used as an aldose reductase inhibitor as it is or by simply performing a simple deodorizing treatment, a decolorizing treatment and the like.

If this extract is subjected to any purification treatment such as liquid-liquid partition extraction, chromatography and ion exchange resin treatment to increase the content of astilbins, the aldose reductase inhibitory activity will be stronger and it will be easier to use. Become.

When the above extract or purified product is hydrolyzed with a mineral acid such as hydrochloric acid or sulfuric acid or a hydrolase such as hesperidinase, naringinase or anthocyanase, all or part of the astilbins are hydrolyzed. As a result, taxifolin is obtained, and a high taxifolin content can be obtained.

The yellow and yellow leaf extract containing astilbins and the astilbins and taxifolin which are advantageously obtained from them can be used alone or in combination of two or more, and further in combination with other aldose reductase inhibitors. It can be an aldose reductase inhibitor of the invention. Examples of aldose reductase inhibitors that can be used in combination include mulberry, licorice, epimedium, Gymnema,
There are extracts such as cinnamon bark, peony root, large jujube, ginger, soy sauce, and fusi.

The aldose reductase inhibitor of the present invention can be used in any dosage form such as tablets, capsules, troches, powders, solutions, syrups, etc. , Disintegrant, lubricant, stabilizer,
Auxiliary agents such as flavoring agents, flavoring agents, and fragrances can be appropriately blended as necessary.

An acute toxicity test using mice was carried out on the safety of the product of the present invention. The yellow-leaf extract was 1 g / kg,
No deaths were observed even after oral administration of 5 g / kg of astilbin. The mutagenicity was tested by a test method using the expression of the umu-gene directly involved in the mutation as an index.
In the range of g / ml, astilbins are 20-0.03 mg /
In the range of ml, no mutagenicity was observed regardless of the presence or absence of the metabolic activator S-9 mix.

From the above, the safety of the aldose reductase inhibitor of the present invention is considered to have no problem at all.

The preferred dose of the aldose reductase inhibitor of the present invention is about 5-500 mg / astilbins /
Day.

The aldose reductase inhibitor of the present invention may be mixed or attached to any food or drink, cosmetics, etc., and may be taken into the body through daily eating and drinking. Astilbins, which are the active ingredient of the aldose reductase inhibitor of the present invention, are easily soluble in water and have only a weak sweetness, and thus can be incorporated in foods and drinks without adversely affecting the flavor.

[0024]

【Example】

Example 1 200 g of the dried pulverized product of yellow and yellow leaves was extracted with 2 liters of hot water for 2 hours, and the obtained extract was concentrated under reduced pressure to obtain 32 g of hot water extract. Further, 34 g of an ethanol extract was obtained in the same manner as above except that 90% by volume ethanol was used instead of hot water. The results of quantifying astilbins in the hot water extract and the ethanol extract are shown in Table 1.
Shown in

[0025]

[Table 1] Content of astilbins (%) Compound Hot water extract Ethanol extract Astilbin 3.87 13.11 Neoastilbin 2.28 1.20 Isoastilbin 0.81 0.96 Neoisoastilbin 2.71 1 .01

Next, the above ethanol extract was suspended in 200 ml of water and extracted three times with 200 ml of chloroform,
It was subsequently extracted 3 times with 200 ml of ethyl acetate. The obtained ethyl acetate extract was further subjected to high performance liquid chromatography (column: TSKgel ODS-120T, 21.5 mm × 30 cm; elution solvent:
Separation with acetonitrile / 0.05% trifluoroacetic acid = 20: 80) gave 2.29 g of astilbin, 0.22 g of neoastilbin, 0.21 g of isoastilbin, and 0.18 g of neoisoastilbin.

Also, 0.5 g of the obtained astilbin was added to 5
Dissolve in 0 ml of water, add 50 ml of 4N hydrochloric acid, heat on a boiling water bath for 2 hours, neutralize, and add 3 with 50 ml of ethyl acetate.
By extracting twice, 0.21 g of taxifolin was obtained.

The activity inhibition rate of aldose reductase was determined by the following method for the hot water extract, ethanol extract, astilbin, neoastilbin, isoastilbin, neoisoastilbin and taxifolin obtained as described above.

[Measurement method of aldose reductase activity inhibition rate] Preparation of aldose reductase: After pork lens was homogenized in 135 mM phosphate buffer (pH 7.0) containing 10 mM β-mercaptoethanol, the mixture was homogenized at 30 × 10,000 × g.
Centrifuged for minutes. Ammonium sulphate was added to the resulting supernatant to 75% saturation, and then 10 × 10,000 × g.
Centrifuged for minutes. The obtained precipitate was dissolved in the above buffer solution and further centrifuged at 10,000 xg for 10 minutes, and the supernatant was used as a crude aldose reductase solution.

Measurement of aldose reductase activity inhibition rate: The following enzyme reaction solution is prepared. 3 mM glyceraldehyde solution (using 50 mM phosphate buffer of pH 6.2 containing 400 mM lithium sulfate) 900 μl crude aldose reductase solution 50 μl sample solution (concentration 0.1%) 50 μl

This enzyme reaction solution (sample concentration becomes 50 μg / ml) was heated at 37 ° C. for 2 minutes, and then 2.5 mM NADP was added.
Add 50 μl of H (reduced nicotinamide adenine dinucleotide phosphate) and absorb NADPH at a wavelength of 340 nm
The absorbance is measured (aldose reductase is NAD
It is an enzyme that reduces glyceraldehyde to a polyol by using PH as a coenzyme, and NADPH changes to NADP along with this reaction. Therefore, the generated NADPH
Is small and the measured change in absorbance is small, it means that the activity of aldose reductase is inhibited). As a control, the same operation is performed when water is added instead of the sample solution. In the above operation, the absorbance is also measured before the start of the enzymatic reaction of heating at 37 ° C. for 2 minutes.

From the measurement results, the inhibition rate of aldose reductase activity is calculated by the following formula. Inhibition rate (%) = [1- (A ₀ −A ₁ ) / (A ₂ −A ₃ )] ×
100 However, A ₀ : Absorbance before initiation of enzymatic reaction A ₁ : Absorbance after enzymatic reaction A ₂ : Control; Before initiation of enzymatic reaction A ₃ : Control; After enzymatic reaction The results are shown in Table 2.

[0033]

Table 2 specimen inhibition rate (%) Yellow杞葉hot water extract 56 yellow杞葉ethanol extract 51 astilbin 56 Neoasuchirubin 58 Isoasuchirubin 59 Neo iso A still bottle 55 taxifolin 84

Example 2 The effect of the aldose reductase inhibitor component of the present invention on sorbitol accumulation in erythrocytes was examined by the following method.

First, blood collected from a vein of a healthy person is treated with heparin, and washed with physiological saline three times by centrifugation. The obtained red blood cells are suspended in physiological saline so that the hematocrit value is around 30%. 1 ml of this red blood cell suspension, 28
3.9 ml of mM glucose (using Krebs-Ringer buffer) and 100 μl of sample solution (concentration 0.25%) are mixed (sample concentration is 50 μg / ml), and reacted at 37 ° C. for 60 minutes under 5% carbon dioxide. The reaction was stopped by adding 3 ml of 6% perchloric acid, and the protein was removed by centrifugation at 2000 xg for 10 minutes at 4 ° C. Then, 2.5 M potassium carbonate is added to the supernatant for neutralization, and the amount of sorbitol in the red blood cells is quantified using the sorbitol dehydrogenase method.

Sorbitol dehydrogenase method: 50 mM glycine buffer (pH 9.4) and 0.2 mM NAD in 1 ml.
(Oxidized nicotinamide adenine dinucleotide) and 0.64 units of sorbitol dehydrogenase were added to an enzyme reaction solution, and the deproteinized supernatant solution was added to the enzyme reaction solution.
Add 5 ml and react. NA generated by this reaction
DH (reduced nicotinamide adenine dinucleotide)
Is detected at an excitation wavelength of 366 nm and a fluorescence wavelength of 452 nm (This reaction detects intracellular sorbitol and NAD by fructose and NAD by sorbitol dehydrogenase.
Since it is a reaction for converting into H, the higher the amount of NADH after the reaction, the higher the content of sorbitol. ). As a control, the same operation is performed when water is added instead of the sample solution.

A control sorbitol content of 10
The result calculated as 0% is shown in Table 3. It can be seen that astilbins and the like inhibited the accumulation of sorbitol in red blood cells.

[0038]

TABLE 3 specimen erythrocyte sorbitol content (%) Control 100 Yellow杞葉hot water extract 67 yellow杞葉ethanol extract 68 astilbin 69 Neoasuchirubin 65 Isoasuchirubin 67 Neo iso A still bottle 69 taxifolin 32

[0039]

EFFECTS OF THE INVENTION According to the present invention, a novel aldose reductase inhibitor, which has been used as a raw material for drinks for a long time, is provided. Since this inhibitor is highly safe and does not have an unpleasant taste or odor, it becomes possible to provide a food or drink containing the inhibitor which has a preventive / therapeutic effect on diabetic complications and facilitate daily intake.

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Claims (2)

[Claims] 1. An aldose reductase inhibitor, which comprises, as an active ingredient, one or more compounds selected from the group consisting of astilbin, neoastilbin, isoastilbin, neoisoastilbin and taxifolin. 2. An aldose reductase containing as an active ingredient a yellow-fruited leaf extract containing one or more compounds selected from the group consisting of astilbin, neoastilbin, isoastilbin and neoisoastilbin. Inhibitor.