JPH10158185A - Anti-oxidizing action agent, xanthin oxidase inhibitory action agent and aldose reductase inhibitory action agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anti-oxidizing action agent, a xanthin oxidase inhibitory action agent and an aldose reductase inhibitory action agent having strong activity, comprising an extract of Bauhinia splendens as an active ingredient. SOLUTION: This anti-oxidizing agent comprises an extract of Bauhinia splendens, a plant of the genus Bauhinia of the family Leguminosae in an organic solvent or water as an active ingredient. For example, Bauhinia splendens is ground in a raw state as it is or dried and ground and extracted with an organic solvent such as methanol or water. A dose is about 10-200mg/kg weight and administered once to 5 times daily.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antioxidant, a xanthine oxidase inhibitor, and an aldose reductase inhibitor. More specifically, the present invention relates to an antioxidant, a xanthine oxidase inhibitor, and an aldose reductase inhibitor comprising an extract from a plant as an active ingredient.

[0002]

2. Description of the Related Art Oxidative damage to the human body leads to denaturation and oxidation of biological constituents such as proteins, lipids and carbohydrates, thereby causing damage to biological membranes and genes, and finally irreversible cell damage. And it is the cause of the development and progression of various diseases. In particular, as the relationship between lipid peroxidation and aging becomes clear, it is becoming increasingly thought that oxidative damage leads to aging.

On the other hand, it is also apparent that during ischemia-reperfusion typified by stroke, a large amount of reactive oxygen is explosively produced under xanthine oxidase, causing abnormal lipid peroxidation and brain damage. It became. Due to the existence of such a mechanism, brain damage caused by ischemia-reperfusion is considered to be significantly suppressed by a xanthine oxidase inhibitor.

[0004] Aldose reductase is also known as NAD.
It is an enzyme that converts glucose into a sugar alcohol (polyol) using PH as a coenzyme, and has attracted considerable attention in recent years as an enzyme causing diabetic complications. With the progression of diabetes, the development of various complications seen in organs,
It is thought that aldose reductase-mediated mechanisms are involved in the early cell damage.

In fact, when an aldose reductase inhibitor is administered to a rat or dog diabetic animal model, the production of polyol in tissues is inhibited, the progression of cataracts and vascular lesions of the retina are inhibited, and the peripheral nerve conduction velocity is decreased. It is reported that the point will also be improved. Therefore, the development of aldose reductase inhibitors as preventive and therapeutic agents for diabetic complications is considered to be an important urgent need at present.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide an antioxidant agent, xanthine oxidase, comprising as an active ingredient an extract from a plant which has almost no side effect on such various diseases. An object of the present invention is to provide an inhibitor and an aldose reductase inhibitor.

[0007]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
This can be achieved by an antioxidant, a xanthine oxidase inhibitor or an aldose reductase inhibitor having an organic solvent extract or water extract of Sipo escarda as an active ingredient.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Bauhinia sp.
lendens) is a plant belonging to the genus Japonica in Brazil, and is consumed as tea in Brazil.It is known to be effective in treating urological diseases, but it has an antioxidant effect, It is not known at all to have a xanthine oxidase inhibitory action or an aldose reductase inhibitory action.

[0009] Extraction from Sipo Escada is carried out using an organic solvent or water (including hot water) as a solvent, after neat or drying and pulverizing them. Are used in combination. As the organic solvent, methanol, ethanol, acetone or the like is used, and preferably, methanol is used.

[0010] Each fraction obtained by purifying these extracts as they are or by solvent partitioning, column chromatography or a combination thereof may have an antioxidant effect, a xanthine oxidase inhibitory effect or an aldose reductase inhibitory effect. Was found.

Among the antioxidant effects, the free radical scavenging effect is described in Samples 3, 4 and 11 below, the superoxide scavenging effect is described in Samples 4, 5, 10 and 11 described below, and the lipid peroxide suppressing effect is described in Samples 2 and 3 below. Strong activity was found in 3, 4, 6, 10, and 11, respectively. Further, the xanthine oxidase inhibitory effect was found in Samples 7 and 8 described below, and the aldose reductase inhibitory effect was found in Samples 5 and 10 described later.

These extracts are provided in the form of a medicine or food. When used as a medicament, it is provided in the form of powders, granules, tablets, dragees, capsules, liquids, and the like, and when used as a food, it is provided in the form of gum, candy, jellies, tablet confections, beverages, etc. You. When used as a medicament, it is administered by oral administration, parenteral administration, inhalation, rectal administration, topical administration and the like. Parenteral administration includes subcutaneous injection, intravenous administration, intramuscular administration, nasal administration or infusion. The amounts used will generally be within the range of about 10-200 mg / kg body weight per dose, usually administered 1-5 times daily. However, the exact dose is determined from the above range in consideration of the age, weight, symptoms, administration route and the like of the patient.

[0013]

According to the present invention, there are provided an antioxidant, a xanthine oxidase inhibitor, and an aldose reductase inhibitor having a strong activity, which are extracted from Sipo escarda.

This antioxidant has excellent effects on free radical scavenging, superoxide scavenging and lipid peroxide suppression, and is useful for various diseases caused by oxidative damage in living organisms. Effective for prevention and treatment. In addition, this xanthine oxidase inhibitor suppresses the generation of a large amount of reactive oxygen that is explosively produced during ischemia-reperfusion, and is effective for ameliorating cerebral ischemic disease. Furthermore, this aldose reductase inhibitor has an excellent effect on aldose reductase inhibitory activity, and is effective in preventing and treating diabetic complications such as diabetic cataract, renal failure, neuropathy, and arteriosclerosis. It is.

[0015]

Next, the present invention will be described by way of examples.

Example (1) Dried material of Sipo Escada collected in Brazil
After crushing 1.8 kg, methanol was extracted at room temperature, the solvent was distilled off under reduced pressure, the residue was partitioned with chloroform and water, and 12.8 g of the extract (sample 1) from the chloroform layer was distilled off. Obtained.

(2) The extract obtained by distilling off the solvent from the aqueous layer of the above (1) is subjected to column chromatography using a polystyrene gel (Diaion CHP20P manufactured by Mitsubishi Chemical Corporation) as a carrier, and Brown powder from the eluted fraction (Sample 2)
86.8 g was obtained.

Samples 3 to 6 were obtained from fractions eluted with methanol of various concentrations by column chromatography. (Sample 3) 17.16 g of brown powder from the fraction eluted with 10% methanol (Sample 4) 3.45 g of yellow powder from the fraction eluted with 30% methanol (Sample 5) 0.71 g of brown powder from the fraction eluted with 60% methanol (Sample 6) 80 3.44 g of brown powder from the fraction eluted with% methanol

Finally, the fraction eluted with 100% methanol was subjected to Cosmosil 75 C ₁₈ -OPN column chromatography.
I got (Sample 7) 0.3 g of brown powder from 85% methanol-eluting fraction (Sample 8) 0.2 g of brown powder from 90% methanol-eluting fraction (Sample 9) 1.41 g of brown powder from 98% methanol-eluting fraction

(3) Cosmosil 75 C ₁₈ -OP was further added to the sample 4.
Samples 10 to 12 were obtained from N-column chromatography and fractions eluted with methanol at various concentrations. (Sample 10) Brown powder from 10% methanol elution fraction 0.82 g (Sample 11) Brown powder from 30% methanol elution fraction 0.46 g (Sample 12) Yellow powder from 50% methanol elution fraction 1.93 g

This sample 12 was further recrystallized with methanol to obtain 1.72 g of yellow crystals (sample 13).

(4) The methanol extraction residue of (1) was extracted with hot water, and the solvent was removed from the extract under reduced pressure to obtain 27 g of a brown powder (sample 14).

Using these samples 1 to 14, an antioxidant activity test (free radical scavenging activity, superoxide scavenging activity and lipid peroxide inhibitory activity), a xanthine oxidase inhibitory activity test and an aldose reductase inhibitory activity test were conducted. .

Free radical scavenging test: 100 mM 1,1-
Diphenyl-2-picrylhydrazyl in ethanol 900
100 μl of the sample prepared at various concentrations was added to μl, and the amount of decrease in absorbance at a wavelength of 517 nm after 20 minutes was measured with a spectrophotometer, and the inhibition rate with the amount of decrease in absorbance at a concentration of ascorbic acid of 100 μg / ml being 100 ( (Unit:%) was determined.

Table 1 Sample concentration 100 μg / ml 10 μg / ml 1 μg / ml sample 1 68.2 11.6 sample 2 96.1 74.0 14.5 sample 3 97.8 94.9 14.6 sample 4 96.5 90.9 15.0 sample 5 97.9 62.3 10.4 sample 6 99.0 69.0 7.8 sample 7 91.8 19.1 sample 8 96.4 16.7 Sample 9 98.5 40.6 Sample 10 97.1 81.6 14.0 Sample 11 99.0 91.0 12.8 Sample 12 96.1 57.1 9.0 Sample 13 98.4 64.1 9.6 Sample 14 96.4 68.9 11.5

Superoxide scavenging action test: 250 μl of 300 mM calcium phosphate buffer solution (pH 7.8; containing 600 mM disodium ethylenediaminetetraacetate), 250 μl of aqueous solution of cytochrome C (0.06 mM), 250 μm of 0.3 mM xanthine aqueous solution
To a solution containing μl and 500 μl of water, add 150 μl of sample or water prepared at various concentrations, add 100 μl of xanthine oxidase (7.5 to 15 × 10 to ⁵ mM), and then increase the absorbance at 550 nm at 25 ° C. Was continuously recorded for 2 minutes, and the change value was obtained from the linear portion for 1 minute, and the change was defined as the inhibition rate (unit:%).

Table 2 Sample concentration 150 μg / ml 15 μg / ml 1.5 μg / ml 0.75 μg / ml sample 1 95.2 28.6 sample 2 100 95.2 9.5 sample 3 100 95.2 23.8 sample 4 100 95.2 33.3 sample 5 100 95.2 52.4 9.5 sample 6 95.2 95.2 23.8 Sample 7 100 57.1 9.5 Sample 8 95.2 42.9 Sample 9 100 38.1 Sample 10 100 100 47.6 Sample 11 100 95.2 38.1 Sample 12 100 47.6 Sample 13 95.2 38.1 Sample 14 100 85.7 9.5

Lipid peroxide inhibitory activity test: 40 mM phosphate buffer (pH 7.4; 14.2 mM Na) of 7 (w / v)% rat brain homogenate
(Containing Cl) 5ml, sample or water 50μl prepared at various concentrations
And shake at 37 ° C for 1 hour. Next, after adding 2 ml of a 28% aqueous trichloroacetic acid solution, centrifugation was performed at 3000 rpm for 10 minutes, 4 ml of the supernatant was collected, 1 ml of a 1% aqueous thiobarbituric acid solution was added, and 15 ml of a boiling water bath was added. Reaction was carried out by heating for minutes. Determine the concentration of the resulting red solution with a spectrophotometer
(Wavelength 532 nm), and the inhibition rate (unit:%) was determined. At that time, as a control, a 28% aqueous solution of trichloroacetic acid was added before shaking at 37 ° C., and the mixture was similarly reacted and treated.

Table 3 Sample concentration 10 μg / ml 5 μg / ml 1 μg / ml sample 1 32.3 sample 2 98.0 74.1 18.4 sample 3 99.6 95.0 26.4 sample 4 100 93.8 18.6 sample 5 94.3 42.3 sample 6 96.9 93.2 14.1 sample 7 45.2 sample 8 27.5 sample 9 59.0 42.7 Sample 10 98.3 80.2 21.2 Sample 11 99.0 56.6 56.7 Sample 12 28.3 Sample 13 13.2 Sample 14 99.8 31.5

Xanthine oxidase inhibitory activity test: 30
250 μl of 0 mM calcium phosphate buffer solution (pH 7.8; containing 600 mM disodium ethylenediaminetetraacetic acid), 0.3 mM
In a solution containing 250 μl of xanthine aqueous solution and 750 μl of water,
After adding 150 μl of sample or water prepared at various concentrations and adding 100 μl of xanthine oxidase (7.5 to 15 × 10 to ⁵ mM), the absorbance at 550 nm at 25 ° C. was increased by 2 μm.
The change was recorded continuously for 1 minute, and the change value was determined from the linear portion for 1 minute, and the change was defined as the inhibition rate (unit:%).

Table 4 Sample concentration 150 μg / ml 15 μg / ml 1.5 μg / ml sample 1 100 51.9 sample 2 51.9 48.1 sample 3 92.6 48.1 sample 4 88.9 55.6 14.8 sample 5 59.3 18.5 sample 6 85.2 59.3 11.1 sample 7 100 92.6 48.1 sample 8 100 77.8 Sample 9 66.7 33.3 Sample 10 81.5 51.9 18.5 Sample 11 70.4 44.4 Sample 12 55.6 25.9 Sample 13 37.0 Sample 14 77.8 40.7

Aldose reductase inhibitory test: The lens isolated from the rat eye was treated with 5 mM phosphate buffer (pH 7.4).
After centrifugation, the supernatant was salted out with a 40 to 75% aqueous ammonium sulfate solution. The obtained enzyme solution was adjusted to a concentration of 0.015 to 0.020 U / ml. 1 U at this time indicates an activity in which 1 μmol of NADPH is oxidized per minute.

In 500 μl of 0.2 mM phosphate buffer (pH 6.2), 200 μl of 2 M ammonium sulfate aqueous solution, 10 μl of 16 mM NADPH,
20 μl of the above enzyme solution, 250 μl of water and a sample (final concentration 10 μg /
or 10 μl of water, 10 μl of 1.0 M DL-glyceraldehyde was added thereto, and the enzyme reaction was carried out at 30 ° C..The amount of decrease in NADPH after 10 minutes was determined from the amount of decrease in absorbance at a wavelength of 340 nm. (Unit:%) was calculated. Under the conditions of this experiment, ONO-2235 was used as a control,
It showed 50.3% inhibitory activity at a concentration of 5 × 10 to ⁸ mol / l.

Table 5 Sample concentration 10 μg / ml Sample 1 31.7 Sample 2 37.5 Sample 3 38.9 Sample 4 51.0 Sample 5 88.4 Sample 6 1.6 Sample 7 53.6 Sample 9 3.4 Sample 10 76.4 Sample 13 17.6 Sample 14 37.5

Claims (3)

[Claims] 1. An antioxidant comprising, as an active ingredient, an organic solvent or an aqueous extract of Sipo Escada. 2. A xanthine oxidase inhibitor comprising, as an active ingredient, an organic solvent or an aqueous extract of Sipo Escada. 3. An aldose reductase inhibitor comprising, as an active ingredient, an organic solvent or an aqueous extract of Sipo Escada.