JP2000143525A - Active oxygen eliminating action agent and aldose reductase inhibitory action agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an active oxygen eliminating action agent or an aldose reductase inhibitory action agent having no problems such as tachyphylaxis and various adverse effects shown in the case of a medicine synthesized by a conventional chemical means. SOLUTION: This active oxygen eliminating action agent comprises a component derived from Syzygium jambos as an active ingredient. This aldose reductase inhibitory action agent comprises also the component derived from Syzygium jambos as an active ingredient. These agents are effective for preventing and treating various diseases induced by oxidative damage in vivo and also effective for preventing and treating diabetic complication such as diabetic cataract, renal insufficiency, nervous disorder, arteriosclerosis, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an active oxygen scavenging agent and an aldose reductase inhibitor. More specifically, the present invention relates to an active oxygen-scavenging agent and an aldose reductase inhibitor which comprise an extract from a plant as an active ingredient.

[0002]

2. Description of the Related Art In normal living organisms, when bacteria or viruses, which are foreign substances to the body, invade, the phagocytes dissolve and remove the foreign substances taken in by phagocytic cells. And dissolve foreign matter. However, during abnormal times, excess active oxygen is produced, which flows out of the phagocytic cells, dissolves in the tissues of the body, and destroys normal cells. Or cause serious obstacles. In particular, polyunsaturated fatty acids, from the structural point of view,
It is very susceptible to the reaction of active oxygen generated in the living body, and has the property of easily becoming a chemically active alkyl radical. Further, the alkyl radical reacts with oxygen to form a peroxide called a hydroperoxide, that is, a lipid peroxide via a peroxy radical. It is further decomposed and polymerized to produce various aldehydes,
This produces ketones, polymers and the like. Further, it is said that the indiscriminate and harmful oxidation reaction in vivo caused by active oxygen directly causes various disease states such as cancer and aging.

[0003] As described above, the reaction in the living body due to active oxygen is a cause of diseases such as cancer, arteriosclerosis, high blood pressure, and senile dementia. In connection with aging, the relationship with lipid peroxidation has received particular attention.
This is because free radicals derived from active oxygen induce peroxidation of unsaturated fatty acids, which are constituents of biological membranes, and the resulting cell and tissue damage is thought to lead to aging. It has already been proved by animal experiments that lipid peroxide increases with aging. That is, it can be said that the living body has a reduced ability to remove active oxygen, free radicals, lipid peroxides, and the like with aging. These cause cell deterioration, metabolic abnormalities, etc., and cause various diseases as described above. Therefore, from the viewpoint of prevention of diseases such as cancer, arteriosclerosis, hypertension, senile dementia and the like, aging, etc., it is necessary to suppress and eliminate the in vivo effects of active oxygen, which is a cause of these diseases. Is desired.

[0004] The function of aldose reductase in vivo is still largely unknown, but it is considered at present that it works to regulate intracellular osmotic pressure. This aldose reductase is an enzyme involved in producing sorbitol from glucose in a polyol metabolism system. The importance of polyol metabolism was revealed
It begins with the discovery of the fructose production pathway, the energy source of sperm. Around the same time, it has become clear that aldose reductase, the rate-limiting enzyme of this metabolic pathway, is present in various tissues such as capillaries, peripheral nerves, lenses, and retina where diabetes complications frequently occur. It has been recognized that this enzyme is important in the context of diabetes.

[0005] Normally, most of glucose taken into cells is metabolized to glycolysis by hexokinase, which has a stronger affinity for glucose than aldose reductase. However, in a hyperglycemic state such as diabetes, in an insulin-independent tissue, the increased aldose reductase activity facilitates the promotion of polyol metabolism, and as a result, glucose alcohol (polyol) is converted from glucose. Will be produced. However, since the product here is relatively stable, once produced, it will accumulate in cells, thereby causing an increase in osmotic pressure, water retention and cell damage, resulting in cataract, Various diabetic complications such as retinopathy will be caused. In addition, administration of an aldose reductase inhibitor to rat or dog diabetic animal models actually inhibits the production of polyol in tissues, inhibits the progression of cataracts and vascular lesions of the retina, and decreases the peripheral nerve conduction velocity. Has also been reported to be improved. Therefore, it is expected to prevent and treat various diabetic complications by inhibiting aldose reductase activity that causes the above-mentioned symptoms.

To date, a large number of compounds exhibiting an active oxygen scavenging action and an aldose reductase inhibitory action have been reported, but many of them have been synthesized by chemical methods and have a continuous nature. At present, tachyphylaxis and various side effects due to its use cannot be avoided. Therefore, there is a strong demand for a drug exhibiting an active oxygen scavenging action and an aldose reductase inhibitory action, which hardly causes such side effects. On the other hand, Futomomo ( Syzygium jambos ) is called incense fruit in Taiwan, and its fruit is edible. However, it has not been known at all that the component derived from Futomomo has both the active oxygen scavenging action and the aldose reductase inhibitory action.

[0007]

SUMMARY OF THE INVENTION As described above, the present invention provides an active oxygen scavenging agent and an aldose reductase inhibitor which hardly cause various side effects such as those observed in a drug synthesized by a conventional chemical method. It is intended to provide an agent.

[0008]

Means for Solving the Problems [Summary of the Invention] <Summary> The present invention is based on the fact that the components contained in the branches and leaves of the Japanese peach, which has conventionally consumed fruit, have both an active oxygen scavenging action and an aldose reductase inhibitory action. While having an effect, it is based on the discovery that the component has almost no side effects as seen with conventional similar drugs.

That is, the active oxygen scavenging agent according to the present invention is characterized by comprising a component derived from Futomomo as an active ingredient. On the other hand, the aldose reductase inhibitor according to the present invention is characterized in that a component derived from Futomomo is used as an active ingredient. In each of these cases, a representative of the fuming peach-derived component is an organic solvent or water extract of the fuming peach.

[0010] Here, the term "futomomo-derived component" refers to an extract of the branches and leaves of the peach. And this "extract"
The term includes both the state including the extractant and the state from which the extractant has been removed, and also includes the state after the extractant has been removed and further subjected to a purification treatment.

<Effects> The active oxygen-scavenging agent derived from Futomomo provided by the present invention exhibits a remarkable lipid peroxide-suppressing action, free radical-scavenging action and superoxide-scavenging action. Various diseases caused by reactions in a living body can be effectively prevented and treated. That is, the active oxygen scavenging agent of the present invention has a remarkable superoxide scavenging effect in addition to a lipid peroxide suppressing effect and a free radical scavenging effect, and is considered to be directly caused by active oxygen in the living body. It can act more effectively on cancer and aging. In addition, the aldose reductase inhibitor provided by the present invention exhibits a remarkable aldose reductase inhibitory effect, and therefore, is effective for diabetic complications such as diabetic cataract, retinopathy, renal failure, neuropathy, and arteriosclerosis. Can be prevented and treated. Furthermore, the active oxygen scavenging agent and the aldose reductase inhibitor of the present invention do not cause problems such as tachyphylaxis and various side effects which are observed in the case of conventional drugs.

[0012]

DESCRIPTION OF THE INVENTION [Detailed Description of the Invention] <Futomomo> Futomomo ( Syzygium jambos ) is an evergreen tree that belongs to the genus Futomomo and belongs to the genus Futomomo, and is native to India. It is native to Southeast Asia and Japan. It is widely distributed in East Asia, including. Futomomo is also called incense fruit in Taiwan, and its fruit is edible. However, it has not been known until now that this Futomomo has both an active oxygen scavenging action and an aldose reductase inhibitory action. The present invention is based on the discovery that the components of this peach have an active oxygen scavenging activity and an aldose reductase inhibitory activity, and utilizes the discovered activity.

In the present invention, as long as the components having the above-mentioned active oxygen scavenging action and aldose reductase inhibitory action can be obtained, either whole or part of the Futomomo plant can be used. However, in the present invention, it is typical to use the branch and foliage of Futomomo. When used in the present invention, the Futomomo plant is used as it is or in a processed state. Preferably, the branches and leaves are dried and then pulverized to be used as a pulverized material. More preferably, an extract from Futomomo is used, as described in the next section.

<Extraction of Futomomo> Extraction of the Futomomo plant, typically its branches and leaves, is carried out by pulverizing the plant of the Futomomo either raw or by drying and then extracting it with an extraction solvent (extractant). ) Is obtained by performing extraction using water, preferably hot water, or an organic solvent. Extraction can be performed by combining organic solvent extraction and water extraction, if necessary. When the organic solvent is water-soluble, a mixture with water, that is, an aqueous solution, can be used as the extractant. In the case of hot water extraction, typically, a ground pulverized plant extract is extracted under heating and reflux. Examples of the organic solvent used when extracting with an organic solvent include methanol, n-butanol, and ethyl acetate. Preferably, methanol is used. For example, in the case of extracting with methanol, it is typical to extract the ground plant of Futomomo under heating and reflux.

<Fumomo extract> The fumomo extract obtained as described above usually gives a tar-like black-brown extract when the extractant is distilled off under reduced pressure. When the extractant to be used is non-toxic, for example, water, it can be used in the preparation while containing the extractant, but it is usual to apply the extract separated from the extractant to the preparation. The extract after separation of the extractant can be used again for preparation by dissolving it in a suitable solvent, for example, water. The extract thus obtained is purified or improved in titer by a suitable purification means, for example, fractional extraction, partitioning between two solvents, fractional adsorption / elution with an appropriate adsorbent or chromatography. Can also be performed.

When the Futomomo extract obtained as described above is purified as it is or by solvent partitioning, column chromatography or a combination thereof, each of the obtained fractions has an active oxygen scavenging action and / or It has been found that there is an aldose reductase inhibitory effect.

For example, as described in the section of [Examples] below, in various fractions (samples 1 to 17) obtained from the Futomomo extract, the active oxygen scavenging action and / or ) It has been found to have an aldose reductase inhibitory effect. That is, in the active oxygen scavenging action,
The free radical scavenging action was described in Samples 1, 4, 6, 7,
8, 9, 11, 12, 13, 14, and 15, the superoxide scavenging action was confirmed by Samples 1, 2, 3, 4, 5, 6,
7, 8, 9, 11, 12, 13, 14, 15, and the lipid peroxide-suppressing effect was shown in Samples 1, 2, 3, 4, 5, 6,
7, 8, 9, 11, 12, 13, 14, 15, 17,
In each case a strong activity has been found. In addition, the aldose reductase inhibitory activity was determined by the following samples 1, 2, 3, 4,
7, 8, 9, 10, 12, and 13 have found strong activities, respectively.

In the present specification, when evaluating the active oxygen scavenging action, the free radical scavenging action,
Superoxide scavenging action and lipid peroxide suppression action are considered. Considering these effects, superoxidants serve as a source of active oxygen, and free radicals function to generate lipid peroxide derived from active oxygen. Is caused by active oxygen and induces various diseases, and thus suppressing the elimination thereof has a very close relationship in evaluating the effect of the active oxygen elimination effect required in the present invention. Because.

<Active Oxygen Scavenger and Aldose Reductase Inhibitor> The active oxygen scavenger and aldose reductase inhibitor according to the present invention contain the above-mentioned components from Futomomo as an active ingredient. The term "futomomo-derived component" as used herein has the broadest meaning in the expression of "derived component", and a typical example of "extract" is also one containing no extractant. As described above, not only good but also those purified after removing the extractant are included. Further, “being an active ingredient” naturally includes the case where a carrier corresponding to the dosage form is included, and also means that other agents that can be used in combination may be contained. Such concomitant drugs include, for example, excipients, preservatives, binders, stabilizers, flavors, and the like.

The active oxygen scavenging agent and the aldose reductase inhibitor according to the present invention are provided in the form of a drug or food. When used as pharmaceuticals, dry powders, powders, granules, tablets, dragees, capsules,
It is provided in the form of a liquid or the like. When used as a food, it is provided in the form of gum, candy, jelly, tablet, beverage, and the like. When administered as a pharmaceutical, various administration forms such as oral administration, parenteral administration, inhalation, rectal administration, and topical administration can be adopted. Among these, parenteral administration includes subcutaneous injection, intravenous administration, intramuscular administration, intranasal administration or infusion. The dose is generally about 0.1-200 mg / dose.
It is in the range of kg body weight and is usually administered 1 to 5 times a day. However, the exact dose depends on the patient's age, weight,
It is determined from the above range in consideration of the administration route and the like.

[0021]

The following examples illustrate the invention. However, the present invention is not limited to these.

[Example 1] Dried product of foliage of Futomomo 4.6
After pulverizing the kg, methanol extraction was performed at room temperature, and the solvent was distilled off under reduced pressure. As a result, 280 g of a black-brown tar (sample 1) was obtained.

Example 2 The methanol extract of Example 1 was partitioned between ethyl acetate and water.
Was obtained by evaporation of the solvent.

Example 3 When the aqueous layer of Example 2 was partitioned between n-butanol and water, 36 g of a black-brown tar (sample 3) was obtained from the n-butanol layer by distilling off the solvent. Further, 11 g of a light brown amorphous substance (sample 4) was obtained from the aqueous layer by distilling off the solvent.

Example 4 The extract (sample 4) from the aqueous layer of Example 3 was subjected to column chromatography using polystyrene gel (Diaion CHP20P manufactured by Mitsubishi Chemical Corporation) as a carrier, and the fraction eluted with water. Min to 150 mg of a light brown amorphous substance (sample 5), a fraction eluted with 10% methanol, 85 mg of a light brown powder (sample 6),
160 mg light brown powder from the fraction eluted with 30% methanol (sample 7), 180 mg light brown powder from the fraction eluted with 50% methanol (sample 8), 70%
20 mg of an almost transparent amorphous substance (sample 9) from the fraction eluted with methanol, and 5 mg of an almost transparent powder (sample 1) from the fraction eluted with 100% methanol.
0).

Example 5 Sample 7 was further subjected to column chromatography using dextran gel (Sephadex LH-20 manufactured by Pharmacia) as a carrier and 100% methanol as a developing solvent. 15 was obtained. (Sample 11) Almost transparent powder 21mg (Sample 12) Light brown powder 25mg (Sample 13) Light brown powder 28mg (Sample 14) Almost transparent powder 20mg (Sample 15) Almost transparent powder 21mg

Example 6 Sample 12 was further subjected to preparative TLC (Me
Using RP- _8F254S manufactured by _rck , methanol: water =
Thin layer chromatography was carried out using 7: 1 as a developing solvent, and samples 16 and 17 were obtained from Rf values of 7.5 and 5.0, respectively. At the time of fractionation, the dissolved silica gel was removed by column chromatography using dextran gel (Sephadex LH-20 manufactured by Pharmacia) as a carrier (elution solvent: methanol: water = 1: 1). (Sample 16) Almost transparent amorphous 2mg (Sample 17) Almost transparent amorphous 3mg

(1) Reactive oxygen scavenging action test Using these samples 1 to 17, active oxygen scavenging action tests (free radical scavenging action test, superoxide scavenging action test and lipid peroxide inhibitory action test) were performed. .

<< Free radical scavenging action test >> 100 m
M 1,1-diphenyl-2-picrylhydrazyl in ethanol (900 μl) at various concentrations (10 or 2.5
(μg / ml), and the amount of decrease in absorbance at a wavelength of 517 nm after 20 minutes was measured with a spectrophotometer. In addition, 100 μg of ascorbic acid
The inhibition rate (unit:%) of each sample was determined with the amount of decrease in absorbance when using / ml as 100. The higher the inhibition rate, the stronger the activity of the free radical scavenging action.

<< Superoxide scavenging action test >> 300
mM calcium phosphate buffer solution (pH 7.8, 600 m
M disodium ethylenediaminetetraacetic acid)
250 μl, cytochrome C (0.06 mM) aqueous solution 25
A solution containing 0 µl, 250 µl of 0.3 mM xanthine aqueous solution and 500 µl of water was added to various concentrations (10 or 1 µm).
g / ml) or 150 μl of the prepared water,
There, xanthine oxidase (7.5-15 × 10 ⁻⁵⁾
mM) at 25 ° C. after addition of 100 μl.
The increase in absorbance at nm was recorded continuously for 2 minutes, and the change value was determined from the linear portion for 1 minute, which was defined as the inhibition rate (unit:%). The higher the inhibition rate, the stronger the activity of superoxide scavenging action.

<< Lipid peroxide inhibitory effect test >> 7 (w / v)
% Rat brain homogenate in 40 mM phosphate buffer (pH
7.4, containing 14.2 mM NaCl) in 1.5 ml,
15 μl of a sample or water prepared at various concentrations (10 or 1 μg / ml) is added and shaken at 37 ° C. for 1 hour. Then, after adding 600 μl of a 28% aqueous trichloroacetic acid solution, the mixture was centrifuged at 3000 rpm for 10 minutes, 1.2 ml of the supernatant was collected, 300 μl of a 1% aqueous thiobarbituric acid solution was added thereto, and the mixture was placed on a boiling water bath. For 15 minutes to react. The concentration of the resulting red solution was measured with a spectrophotometer (wavelength 532 nm), and the inhibition rate (unit:%) was determined. As a control, 2 hours before shaking at 37 ° C.
An 8% aqueous solution of trichloroacetic acid was added, and the mixture was reacted and treated in the same manner. The higher the inhibition rate, the stronger the activity of the lipid peroxide-suppressing action. Table 1 shows the test results.

Table 1 Sample No. Free radical superoxide Lipid peroxide suppression Elimination effect test Elimination effect test Effect test (inhibition rate (%)) (inhibition rate (%)) (inhibition rate (%)) Sample concentration (μg / ml) 10 2.5 10 1 10 1 1 14.8-90.0 3.4 98.8 35.8 2 17.6-95.3 12.5 98.8 10.2 3 28.8-100 17.0 98.8 26.5 4 95.7 27.0 97.8 23.6 98.8 60.0 5 27.4-66.0 7.5 73.3 2.8 6 96.4 44.5 100 90.9 97.1 42.9 7 96.5 43.5 100 90.9 96.7 40.6 8 94.8 32.9 100 86.3 95.2 12.2 9 83.9 26.3 100 46.5 92.4 3.4 10 5.7-21.3-3.9-11 86.2 22.1 100 16.3 100 18.4 12 94.2 33.4 100 37.0 100 48.2 13 94.5 35.1 100 35.0 100 49.4 14 94.8 37.4 100 23.7 100 50.9 15 95.3 40.7 100 28.7 100 49.8 16 3.0 0.8 0.0 − 0.0 − 17 36.0 7.0 44.1 − 60.1 −

As is evident from the results obtained (Table 1 above), the free radical scavenging action was the same for Samples 1, 4, 6, 7,
At 8, 9, 11, 12, 13, 14 and 15, the superoxide scavenging effect was similar to samples 1, 2, 3, 4, 5, 6,
7, 8, 9, 11, 12, 13, 14 and 15, and the lipid peroxide inhibitory effect was shown in samples 1, 2, 3, 4, 5, 6,
7, 8, 9, 11, 12, 13, 14, 15, and 17
In each case, a strong activity was found. From these results, the active oxygen scavenging agent extracted from Futomomo shows an excellent effect on the lipid peroxide suppressing action, free radical scavenging action and superoxide scavenging action. It is found to be effective for prevention and treatment of various diseases caused by the reaction.

(2) Assay for aldose reductase inhibition
Using the test samples 1 to 15, an aldose reductase inhibitory test was performed.

<< Preparation of Enzyme Solution >> The lens extracted from rat eyes was homogenized in 5 mM phosphate buffer (pH 7.4), and the supernatant after centrifugation was salted out with a 40-75% aqueous ammonium sulfate solution. did. The resulting oxygen solution is
It was adjusted to a concentration of 0.015 to 0.020 U / ml. At this time, 1 U indicates the activity of oxidizing 1 μM NADPH per minute.

<< Activity Test >> 0.2 M phosphate buffer (pH
6.2) 2M ammonium sulfate aqueous solution 2
00 μl, 16 mM NADPH 10 μl, the above oxygen solution 20 μl, water 250 μl and test substance (final concentration 10 μl)
g / ml) or 10 μl of water (blank), 10 μl of 1.0 M DL-glyceraldehyde was added thereto, followed by an enzyme reaction at 30 ° C., and NADP after 10 minutes.
The H decrease was determined from the decrease in absorbance at a wavelength of 340 nm, and the inhibition rate (unit:%) was calculated. Under the conditions of this experiment, ONO-223 used as a comparative control drug was used.
5 showed an inhibitory activity of 50.3% at 5 × 10 ⁻⁸ M.
The higher the inhibition rate, the stronger the activity of the aldose reductase inhibitory action. Table 2 shows the test results.

[0037]

As is evident from the results obtained (Table 2 above), the aldose reductase inhibitory effect was as shown in Sample 1,
At 2, 3, 4, 7, 8, 9, 10, 12 and 13, strong activity was found, respectively. From these results, this aldose reductase inhibitor extracted from Futomomo has an extremely excellent effect on aldose reductase inhibitory action, such as diabetic cataract, retinopathy, renal failure, neuropathy, arteriosclerosis, etc. Is effective for the prevention and treatment of diabetic complications.

[0039]

The active oxygen-scavenging agent obtained according to the present invention exhibits a remarkable lipid peroxide-suppressing effect, free radical-scavenging effect, superoxide-scavenging effect and the like without side effects. Is effective for the prevention and treatment of various diseases caused by a reaction in a living body to an excess of active oxygen, and the aldose reductase inhibitor according to the present invention has remarkable aldose reductase without side effects. It shows an inhibitory effect, and it is effective for the prevention and treatment of diabetic complications such as diabetic cataract, retinopathy, renal failure, neuropathy, and arteriosclerosis. Has been described above.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 39/06 A61K 31/00 639C 43/00 643D (72) Inventor Toyoyoshi Yoshizawa Kitamachi, Otsumachi, Kitaibaraki City, Ibaraki Prefecture 1-1-8 F term (reference) 4C088 AB57 AC05 AC06 BA08 BA09 BA10 MA17 NA14 ZA01 ZA33 ZA45 ZA81 ZC20 ZC21 ZC35

Claims (4)

[Claims] (1) a fumomo-derived component as an active ingredient;
Active oxygen scavenger. 2. The active oxygen scavenging agent according to claim 1, wherein the component derived from Futomomo is an organic solvent extract or a water extract. 3. A composition comprising a fumomo-derived component as an active ingredient.
Aldose reductase inhibitor. 4. The aldose reductase inhibitor according to claim 3, wherein the component derived from Futomomo is an organic solvent extract or an aqueous extract.