JPH11147834A - Serine protease inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inhibitor comprising an extract of sage or a plant of the genus Morus as an active ingredient, useful as a therapeutic agent for pancreatitis, acute arteritis, pulmonary emphysema, arteriosclerosis, arthrorheumatism, metastasis of cancer, infiltration, etc., capable of preventing aging of skin by recovering and maintaining tension and elasticity of skin. SOLUTION: This serine protease inhibitor comprises an extract of one or more plants selected from, among Salvia officinalis L. and plants of Morus (e.g. Morus bombycis Koidz. Morus alba L. var. stylosa Bur. Morus albaL., Morus atropurpure Roxb., etc.), as an active ingredient. One or a mixed solvent of two or more selected from among methanol, ethanol, 1,3-butylene glycol, propylene glycol and purified water is preferable as the extracting solvent in obtaining the extract. A method for immersing the plant in the solvent at room temperature in a cooled or heated state, etc., may be cited as the extraction method. Preferably the extraction is preferably carried out by uisng the solvent in an amount of 0.5-100 times as much as the plant under normal pressure at room temperature to the boiling point of the solvent for 2 hours to 2 weeks.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sage ( Salvia of Salvia).
ficinalis L.) and a serine protease inhibitor comprising an extract of one or more plants selected from the genus Morus as an active ingredient, and more specifically, a therapeutic agent for pancreatitis, acute arteritis, emphysema, It is expected to be effective as a therapeutic agent for arteriosclerosis, rheumatoid arthritis, metastasis and invasion of cancer, and to prevent skin aging by restoring and maintaining skin elasticity and elasticity, and to maintain a youthful skin condition. The present invention relates to a serine protease inhibitor and an inhibitor of elastase, trypsin, chymotrypsin and plasmin, which are a kind of serine protease.

[0002]

2. Description of the Related Art Various serine proteases such as trypsin, chymotrypsin, thrombin, plasmin, and elastase are present in a living body, and when these enzymes are abnormally activated by some factor, inflammation, pain, allergy, blood, etc. It is thought to cause diseases such as abnormalities and tissue destruction. For example, during acute pancreatitis or the acute lesion stage of chronic pancreatitis, destruction of pancreatic tissue by pancreatic protease activated by various factors, and systemic tissue and organ damage due to escape of the activated enzyme into the blood. It is known that severe symptoms develop. The involvement of trypsin as a pancreatic serine protease, particularly as a key enzyme, has been emphasized as the cause, and in recent years, several synthetic antitrypsin agents have been developed and marketed as therapeutic agents for improving the above-mentioned conditions.
In addition, due to UV exposure and aging, various inflammatory stimuli,
Due to overexpression of elastin, an elastin-disrupting enzyme, elastin is denatured and destroyed.
It is thought to lead to a decrease in the elasticity of the skin, and it is important to prevent elastin from degenerating and destroying, which gives the skin elasticity and firmness, by suppressing the action of elastase. In addition, neutrophil elastase inhibitors are used for acute arteritis, emphysema, arteriosclerosis, rheumatoid arthritis, metastasis of cancer,
It is expected to be used as a therapeutic agent for invasion and the like.

[0003] Many such serine protease inhibitors are known, but most of them are irreversible inhibitors that irreversibly bind to and deactivate the hydroxyl group of serine in the active center. Is not possible, so there are concerns about side effects. On the other hand, as reversible inhibitors, aldehydes, ketones, boronic acids and the like similar to substrates are known.

[0004] However, many of the conventional serine protease inhibitors are reversible, but their pharmacokinetics and side effects have not been solved.
In some cases, the action and effect were insufficient or the stability was poor, and a considerable amount had to be contained in order to obtain an effective effect.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a serine protease inhibitor which is free from problems such as side effects even when used continuously and has high safety.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have studied the serine protease inhibitory activity of a wide variety of natural products and found that sage ( Salvia officinalis L.) and mulberry ( Morus )
The present inventors have found that a plant extract has an excellent serine protease inhibitory action, has no side effects regardless of whether it is taken internally or is topically used, and has high safety, and has completed the present invention.

[0007] sage (Salvia officinalis L.) is a kind of plant of the mint family (Labiatae) salvia (Salvia), a height 30~70cm, evergreen perennial white fluff in all the grass is dense, in Europe, It has been used as a herb and folk medicine since ancient times. This sage ( Salvia officinalis
L.) extract, the antibacterial action (JP-A-7-267873, JP-A-8-119872, etc.), the antioxidant action (JP-A-3-9984), skin lipid peroxide Production inhibitory action (JP-A-61-24522), anti-inflammatory action (JP-A-1-83022, JP-A-60-156618, etc.), hyaluronidase inhibitory action (JP-A-1-128933), A protease inhibitory action derived from microorganisms (Japanese Patent Laid-Open No. 1-128934) is disclosed. However, it has not been known until now that sage extracts exhibit high inhibitory activity against serine proteases, especially elastase, trypsin, chymotrypsin and plasmin.

The genus Morus is a kind of plant of the mulberry family (Moraceae), and several species are widely cultivated in order to feed fruits and feed on leaves of silkworms. Also, the root bark of the mulberry plant is called mulberry bark, the leaves are mulberry leaves, and the fruit is mulberry,
Each has been used as a crude drug. For example, mulberry bark has been used as an anti-inflammatory diuretic and laxative,
The extract has a tyrosinase inhibitory action (JP-A-50-135236 and others), a microorganism-derived protease inhibitory action (JP-A-6-25000), and an antibacterial action (JP-A-8-151325 and others). ) Are disclosed. Mulberry leaves are pentosan, galactan, glucose,
Contains carotene, tannin, etc. as a carminative in China
In Japan, it is used as mulberry tea in the private sector, and the antioxidant action of mulberry leaf extract (JP-A-60-42485), the active oxygen scavenging action (JP-A-8-143466) and the like are disclosed. . Furthermore, mulberry contains organic acids, mucus, pigments, sugars, etc., and is said to have an effect on diuresis and antitussive, and is also used for raw eating and sake brewing. However, it has not been known that mulberry plants exhibit a high inhibitory activity against serine proteases, particularly elastase, trypsin, chymotrypsin and plasmin.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, sage ( Salvia officinali) is used.
s L.), the site to be subjected to extraction is not particularly limited, and leaves, flowers, and whole plants can be used as they are or dried.

The mulberry ( Morus ) genus plant used in the present invention includes mulberry (Yamaguwa, Nogwa) ( Morus bomby ).
cis Koidz., Morus japonica LHBailey non Sieb.,
Morus alba L.var. Stylosa Bur.) , Morus alba (Karayamaguwa, white mulberry) (Morus alba L., Morus atropurpure
Roxb.), Rosou (Logwa, Marbagwa, Mochiwa) ( Mor
us multicaulis Perr., Morus latifolia (Bur.) Poir.
, Morus alba L.var. Latifolia Bur. , Morus alba L.
var. multicaulis Loud.), Ogasawara Harrow (Morus bonin
ensis Koidz.), Ichibei (Morus argutidens Koidz.),
Shimaguwa ( Morusaustralis Poir., Morus acidosa Grif
f), Hamaguwa ( Morus bombycis Koidz.var. maritima Ko
idz.), Hachijogwa ( Morus kagayamae Koidz.), Mokogwa ( Morus mongolica (Bur.) Schneid, Morus alba
L.var. Mongolica Bur.), Kuromiguwa (Morus nigra
L.), Akamigua ( Morus rubra L.), Nogwa (Kegwa)
( Morustiliaefolia Makino) and the like, but are not particularly limited. The site to be subjected to extraction when obtaining the extract of the mulberry plant is not particularly limited.
It is preferable to obtain an extract of one or more sites selected from leaves, fruits, and flowers, and an extract from leaves is most preferable in terms of serine protease activity.

The extraction solvent used for obtaining the extract of the sage and mulberry plants used in the present invention includes purified water,
Ethanol, methanol, isopropanol, isobutanol, n-hexanol, methyl amyl alcohol, 2-
Alcohols such as ethylbutanol and n-octyl alcohol, glycerin, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, triethylene glycol, 1,3- Polyhydric alcohols such as butylene glycol and hexylene glycol or derivatives thereof, acetone, methyl ethyl ketone,
Ketones such as methyl isobutyl ketone and methyl-n-propyl ketone, esters such as ethyl acetate and isopropyl acetate, ethyl ether, isopropyl ether, n-
One or more mixed solvents selected from polar solvents such as ethers such as butyl ether can be suitably used,
In addition, a solvent to which an inorganic salt such as a phosphate buffered saline has been added can also be used, but is not particularly limited. For the purpose of the present invention, a polar solvent is preferred from the viewpoint of serine protease inhibitory action, and one or more kinds selected from methanol, ethanol, 1,3-butylene glycol, propylene glycol and purified water are further preferred. It is preferable to use a mixed solvent, particularly an aqueous ethanol solution, as the solvent.

[0013] The extraction method includes a method of immersion and extraction at room temperature, in a cooled or heated state, a method of extraction using a distillation method such as steam distillation, and a method of squeezing raw plants to obtain an extract. And the like, and these methods are used alone or in combination of two or more.

The ratio of the plant to the solvent at the time of extraction is not particularly limited.
000 weight times, especially 0.5 to 10 in terms of extraction operation and efficiency.
0 times by weight is preferred. The extraction temperature is conveniently in the range from room temperature to the boiling point of the solvent under normal pressure, and the extraction time varies depending on the extraction temperature and the like, but is preferably in the range of 2 hours to 2 weeks.

The extract of the sage and mulberry plants thus obtained can be used as it is, but it is purified by deodorization, decolorization, concentration, etc. within a range that does not lose the serine protease inhibitory action. It may be used as a fraction by performing additional operations or using column chromatography. These extracts, purified products, and fractionated products can be dried by removing the solvent from them, and can be further solubilized in a solvent such as alcohol.
Alternatively, it can be provided in the form of an emulsion.

The serine protease inhibitor of the present invention is mixed with a compound known in the art and used in the form of a drug, quasi-drug, cosmetic, or food suitable for parenteral administration, oral administration or external administration. be able to. In foods, it can be added to oils and fats products, emulsified products, soft drinks and the like.
Pharmaceuticals can be added to oral preparations, external preparations, injections, inhalants, nasal drops, eye drops, etc. Depending on the method of use, tablets, liquids, injections, ointments, creams, lotions, aerosols , Suppositories and the like. In addition, excipients, bases, emulsifiers, stabilizers, dissolution aids, flavoring agents, preservatives, fragrances, coloring agents, coating agents, and the like can be appropriately added as needed. As quasi-drugs / cosmetics, it can be added to lotions, emulsions, creams, etc. If necessary, oils, humectants, ultraviolet absorbers, water-soluble polymers, antioxidants, surfactants, metals An ion sequestering agent, an antibacterial preservative and the like can be blended.

The dosage of a plant extract when used as a medicament varies greatly depending on the type of plant used, the extraction solvent, the degree of purification, the age and symptoms of the patient, etc., but in general, the dosage is oral. 5 to 500 mg / dry weight
Range of days. When blended in foods and cosmetics, consider the effect, the scent when added, and the color tones.
It is desirable to set the concentration range of 1 to 5% by weight.

[0018]

EXAMPLES Further, the features of the present invention will be described in detail with reference to examples.

Examples 1 to 6 500 g of each part of the plant shown in Table 1 was immersed in 5000 ml of a 50% by volume aqueous ethanol solution, and extracted by leaving it to stand at room temperature for one week. Then, the plant body is removed by filtration,
After distilling off the solvent under reduced pressure, the obtained solid was redissolved in a 50% by volume aqueous ethanol solution to make up to 50 ml.
6 was obtained.

[0020]

[Table 1]

The serine protease inhibitory action of the Examples
Neutrophil elastase, trypsin, α-chymotrypsin, and plasmin were examined. The results are summarized in Table 2.

Inhibition of neutrophil elastase activity Using Examples 1 to 6, the inhibition rate of neutrophil elastase activity was measured. Neutrophil elastase activity was determined to be succinyl (O-methyl) -alanyl-alanyl-prolyl-
Human neutrophil-derived elastase (1 μg / ml, S) was used at 37 ° C. for 1 hour using valyl-4-methyl-cumaryl-7-amide (9 μM concentration, manufactured by Peptide Chemical Laboratories) as a substrate.
After the reaction with Sigma Co., Ltd.), the amount of 7-amino-4-methylcoumarin, which is a decomposition product, was measured at an excitation wavelength of 355 n.
The evaluation was performed by measuring the fluorescence intensity at a fluorescence wavelength of 460 nm. The enzymatic activity was measured for each of the examples where 0.1 mg / ml was added and for the case where no examples were added, and the neutrophil elastase activity inhibition rate was calculated using the following formula (1).

[0023]

(Equation 1)

Inhibition of trypsin activity Trypsin activity was measured using 0.1 M phosphate buffer (pH 8.
0) Porcine pancreatic trypsin (4000-4000) was added to 0.3 ml.
5000 USPunit / mg, Wako Pure Chemical Industries, Ltd.) solution (40 μg / m
l) Add 0.03 ml, incubate at 30 ° C for 5 minutes, and use BAPA (N-α-benzoyl-DL-
After adding 0.02 ml of arginine-p-nitroanilide hydrochloride) and further incubating at 30 ° C. for 30 minutes,
The reaction was stopped by adding 0.3 ml of a acetic acid solution at 405 nm.
Was evaluated by measuring the absorbance. The enzymatic activity was measured for each of the examples where 0.1 mg / ml was added and for the case where no examples were added, and the trypsin activity inhibition rate was calculated using equation (2).

[0025]

(Equation 2)

Inhibition of α-chymotrypsin activity α-chymotrypsin activity was measured using a 0.1 M phosphate buffer (p
H8.0) in 0.4 ml of an α-chymotrypsin type 7 solution (0.65 μg / ml 0.1 M phosphate buffer, Sigma)
0.05 ml), incubated at 37 ° C. for 5 minutes, and 0.02 ml of a succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide hydrochloride solution (3.0 mM 50% DMSO solution) as a substrate.
And further incubated at 37 ° C. for 30 minutes. Then, the reaction was stopped by adding 0.3 ml of a 20% acetic acid solution,
It was evaluated by measuring the absorbance at 405 nm. The enzymatic activity was measured for each of the examples where 0.1 mg / ml was added and for the case where no examples were added, and the α-chymotrypsin activity inhibition rate was calculated using equation (2).

Inhibition of plasmin activity Plasmin activity was determined by adding a 0.6% aqueous solution of plasminogen-removed fibrinogen type 2 to a Petri dish having a diameter of 9 cm.
Then, 4 ml of a 0.1 M phosphate buffer having a pH of 7.4 was added, and the mixture was stirred. 0.1 ml of thrombin (10 unit / ml) was added dropwise, mixed gently, and allowed to stand for 30 minutes to prepare a fibrin gel. . A plasmin solution (10 unit / ml) was added to the petri dish gel, incubated at 37 ° C. for 2 hours, and the dissolved area of the fibrin gel was measured. The enzymatic activity was measured when 0.1 mg / ml was added to each of the examples and when no example was added, and the plasmin activity inhibition rate was calculated using the formula (3).

[0028]

(Equation 3)

[0029]

[Table 2]

From the results shown in Table 2, Example 1 of the present invention was obtained.
~ 6 were shown to have a significant activity inhibitory effect on neutrophil elastase, trypsin, α-chymotrypsin, and plasmin at a 1% risk factor.

Subsequently, the stability to heat and light was evaluated for each example of the present invention. Each example was prepared at 100 ° C for 1
The neutrophil elastase activity inhibition rate at the time of addition of 0.1 mg / ml was measured for each of the case of heat treatment for 0 minute and the case of exposure and storage for 3 months. Table 3 shows a comparison. As is clear from Table 3, all the examples showed very good stability to heat and light, and the neutrophil elastase activity was inhibited by the heat treatment at 100 ° C. for 10 minutes and the exposure storage for 3 months. Little reduction in action was observed.

[0032]

[Table 3]

Further, for each of the examples of the present invention, cytotoxicity to cultured human fibroblasts was evaluated. Human-derived fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells / well, and 24 hours later,
Each of the examples was further cultured at 37 ° C. for 24 hours in Dulbecco's modified basal nutrient medium containing 1.0% by volume of fetal calf serum containing 1.0 mg / ml, and the number of viable cells was counted to determine the cell viability. The 50% lethal concentration (LD ₅₀ ) was calculated. As a result, as shown in Table 3, in all Examples, the LD ₅₀ was 100.0 mg / ml or more, and no cytotoxicity was observed at the tested concentrations.

[0034]

As described in detail above, sage ( Salvia of Salvia)
ficinalis L.) and the serine protease inhibitor of the present invention containing an extract of a mulberry plant ( Morus ) show an excellent action on inhibiting serine proteases such as elastase, trypsin, chymotrypsin, and plasmin, and are related to the present invention. Serine protease inhibitors are therapeutic agents for pancreatitis, acute arteritis, emphysema, arteriosclerosis, rheumatoid arthritis, metastasis and invasion of cancer, etc., and restore skin aging by restoring and maintaining skin elasticity and elasticity. The effect is expected to prevent and maintain youthful skin condition.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 35/78 ACJ A61K 35/78 ACJD ADA ADA ADU ADU AGU AGZ AGZ (72) Inventor Yuri Okano 112 Nogami, Okada-cho, Yokaichi, Shiga Prefecture -1 Within Noevir Basic Research Laboratory, Inc. (72) Inventor Hitoshi Masaki 112-1, Nogami, Okada-cho, Yokaichi, Shiga Prefecture Within Noevir Basic Research Laboratory, Inc.

Claims (5)

[Claims] 1. A serine protease inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants. 2. An elastase inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants. 3. A trypsin inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants. 4. A chymotrypsin inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants. 5. A plasmin inhibitor comprising as an active ingredient an extract of one or more plants selected from sage ( Salvia officinalis L.) and mulberry ( Morus ) plants.