JPH11349436A - Collagenase inhibitor and skin agent containing the same and used for external use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a collagenase inhibitor having a specific inhibiting action against intersitial collagen produced by eosinophile leukocytes, mesenchymal cells such as fibroblasts, etc., and useful for improving and preventing disease states related to the collagen, such as inflammation, wounds, cancer metastasis, tumorigenesis and skin aging, and to obtain a skin agent used for external use and effective for preventing and improving skin aging symptoms. SOLUTION: This collagenase inhibitor is obtained by subjecting the 50 vol.% ethanol extract of Eucalyptus globulus Labillardiere or its relative plant to an adsorption styrene polymer gel chromatography using an ethanol-water mixture solvent, dividing a 50 vol.% ethanol elusion fraction, subjecting the fraction to a silica gel chromatography using a chloroform-methanol-water mixture solvent, dividing a 50 vol.% ethanol elusion fraction, and subsequently using the fraction as such or after dissolved in a solvent, a base agent, etc. A skin agent for external use is obtained by adding the collagenase inhibitor to a base for the agent for external use.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a collagenase inhibitor which acts specifically on stromal collagenase produced mainly by mesenchymal cells, and a skin aging condition comprising the same, which comprises the same. It relates to an external preparation for skin that is effective for prevention and improvement. More specifically, a collagenase inhibitor comprising a fraction obtained by purifying and fractionating from a 50% by volume ethanol extract of Eucalyptus or a related plant thereof,
Further, the present invention relates to a skin external preparation containing the same.

[0002]

2. Description of the Related Art In recent years, it has become clear that matrix metalloprotease (MMP), which promotes degradation of matrix fibers, is involved in various disease states. MMP
Among them, mesenchymal cells such as fibroblasts, and stromal collagenase produced by eosinophils and the like present at the site of inflammation are involved in metastasis and ulceration of cancer cells, periodontitis and caries formation, etc. Has been reported to be involved. In addition, the collagenase degrades collagen fibers in the dermis of the skin, and is also involved in reducing skin elasticity and generating wrinkles. It is also known that production of these collagenases is promoted by cytokines induced by external stimuli such as ultraviolet rays and allergens.

[0003] Therefore, screening for a collagenase inhibitor that inhibits the activity of collagenase as described above has been actively conducted. For example, acylphenylglycine derivatives (JP-A-7-101925), plant extracts such as pomegranate seeds, lemon balm seeds and leaves, guava and hamamelis (JP-A-7-195526, and 7-291873).
No. 8-283133), polypolenic acid and holo mushroom extract containing the same (JP-A-9-40552), dicarboxylic acid (JP-A-9-124472), substances produced by microorganisms belonging to the genus Aspergillus (JP-A-9-241287), and the like. Have been.

[0004] However, among the collagenase inhibitors obtained so far, those having a strong inhibitory activity against bacterial collagenase, having low specificity for stromal collagenase, or those having insufficient inhibitory activity. Some lacked stability.

[0005]

Accordingly, in the present invention, the present invention specifically exhibits an inhibitory activity against stromal collagenase produced by mesenchymal cells such as fibroblasts, eosinophils, etc., and exhibits inflammation and Obtain collagenase inhibitors that can improve or prevent collagenase-related pathologies such as wounds, cancer metastasis, ulceration, and skin aging, and prevent skin aging symptoms such as reduced skin elasticity and wrinkles And to obtain a skin external preparation effective for improvement.

[0006]

Means for Solving the Problems To solve the above-mentioned problems, the present inventors screened a collagenase inhibitor having high stability and safety and high activity, and have already screened Eucalyptus, Salvia, Dokudami, An excellent collagenase inhibitory activity was found in extracts of buttons and peonies or their closely related plants, and a collagenase inhibitor containing the same was disclosed (Japanese Patent Application No. 9-33498).
1). This time, among the plant extracts, a fraction exhibiting particularly high collagenase inhibitory activity can be obtained by further fractionating an extract of Eucalyptus or a closely related plant thereof with 50% by volume of ethanol. As a result, the present invention has been completed.

That is, in the present invention, Eucalyptus globulus Labillardiere or a closely related plant thereof ( Eucalyptus polybractea RTBaker, Eucalyptus)
dives Schauer) was fractionated with 50% ethanol by a mixed solvent of ethanol and water by adsorption gel chromatography made of styrene polymer, and the fraction eluted with 50% ethanol by ethanol was then subjected to silica gel chromatography. After elution with a mixed solvent of chloroform-methanol-water, a fraction eluted with 50% by volume of ethanol is collected and used as it is or as a collagenase inhibitor as it is contained in a solvent, a base or the like. Further, this collagenase inhibitor is contained in an external preparation base to prepare a skin external preparation.

[0008] Note that eucalyptus extract is formulated in cosmetics and skin external agents conventionally, Porphyromonas ging
effect of inhibiting collagenase produced is known by Ivalis (JP 8-109118). However, there has been no report that a fraction having a specific and high inhibitory effect on stromal collagenase was obtained from the plant extract.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In obtaining the collagenase inhibitor of the present invention, Eucalyptus used as a starting material
globulus Labillardiere) is an evergreen tree belonging to the family Myrtaceae native to Australia and used as a base plant of crude drug eucalyptus oil. Related plants ( Eucalyptus p
olybractea RTBaker, Eucalyptus dives Schauer) can also be used. For extraction, flowers, stems,
Leaves, roots and whole trees can be used. In the present invention, an extract of these plants with 50% by volume of ethanol is used.

The above plants may be subjected to an extraction operation as they are, but in consideration of the extraction efficiency, it is preferable to carry out the extraction after performing a treatment such as shredding, drying and pulverization. The extraction is performed by immersing in 50% by volume ethanol as an extraction solvent. Stirring may be performed to increase the extraction efficiency, or homogenization may be performed in an extraction solvent. 5 ℃ -50 ℃ as extraction temperature
The degree is appropriate. The extraction time is about 4 hours to 10 days.

A 50% by volume ethanol extract of Eucalyptus or a closely related plant thereof is first fractionated by an adsorption gel chromatography made of styrene polymer with a mixed solvent of ethanol and water, and a fraction eluted with 50% by volume of ethanol Get. As an adsorption gel made of styrene polymer, MCI
GEL (manufactured by Mitsubishi Chemical Corporation) or the like can be preferably used. The fraction is further subjected to silica gel chromatography, and eluted with a mixed solvent of chloroform-methanol-water. A silica gel adsorbed substance that is not eluted with this mixed solvent is eluted with 50% by volume of ethanol to obtain a desired fraction. The adsorption gel chromatography and the silica gel chromatography are suitably performed as column chromatography.

The fraction obtained from the 50% by volume ethanol extract of Eucalyptus or the like can be used as it is as a collagenase inhibitor, but it can be dissolved or suspended in a polar solvent such as water or ethanol, or can be used as an emulsion, cream,
Collagenase inhibitors can also be dispersed in a base such as gel or ointment.

The collagenase inhibitor according to the present invention includes physiologically active components such as active oxygen scavengers, anti-inflammatory agents, anticancer agents, whitening agents, skin cell activators, bactericides, oils, and interface agents. Raw materials for general pharmaceuticals and cosmetics, such as activators, humectants, ultraviolet absorbers, powders, fragrances, preservatives, etc., can also be included.

In the present invention, the collagenase inhibitor obtained as described above is contained in an external preparation base to prepare an external preparation for skin. The amount added to the skin external preparation was 0.0
About 001 to 5.0% by weight is appropriate.

The external preparation for skin according to the present invention can be provided in the form of lotions, emulsions, gels, creams, ointments and the like, and further, skin cosmetics such as lotions, emulsions, creams, packs, etc. , Makeup base lotion, makeup base cream, emulsion or creamy or ointment type foundation, makeup cosmetics such as eye color, cheek color, body cosmetics such as hand cream, leg cream, body lotion, etc. Can be provided. Further, in addition to the collagenase inhibitor according to the present invention, general pharmaceutical and cosmetic raw materials such as oils, surfactants, humectants, ultraviolet absorbers, pigments, fragrances, and preservatives, active oxygen scavengers, Anti-inflammatory, whitening,
Physiologically active ingredients such as skin cell activators can also be included.

[0016]

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

Example 1 Eucalyptus g
lobulus Labillardiere) 500 g of dry powder of leaves
Immerse in 1,000 ml of 50% by volume ethanol aqueous solution,
It was allowed to stand at 25 ° C. for 7 days. Thereafter, the plant powder was removed by filtration, and the filtrate was subjected to MCI GEL (manufactured by Mitsubishi Chemical Corporation) column chromatography, fractionated with a mixed solvent of ethanol and water, and fractions eluted with 50% by volume of ethanol were fractionated. . Next, this fraction was subjected to column chromatography on silica gel (manufactured by Wako Pure Chemical Industries, Ltd.), and a chloroform-methanol-water mixed solvent (volume ratio = 7: 3:
A fraction obtained by eluting with 50% by volume of the column adsorbed material eluted in 0.5) and not eluted with this mixed solvent was used as a collagenase inhibitor.

When the collagenase inhibitor of Example 1 was hydrolyzed by refluxing in 3% by volume hydrochloric acid-methanol,
A solid containing a water-soluble saccharide and a solid soluble in methanol were obtained, indicating that the main component of Example 1 was a glycoside. In addition, qualitative analysis was performed by silica gel thin layer chromatography, and it was confirmed that the main component of Example 1 was a polyphenol compound, but did not have a flavonoid skeleton. Among the hydrolysates, a high collagenase inhibitory activity was also observed in the methanol-soluble fraction, indicating that the aglycone as the main component of Example 1 also has a collagenase inhibitory activity.

For Example 1 described above, a 50% ethanol extract as a starting material and the methanol-soluble hydrolyzate of Example 1 were used to compare and evaluate the inhibitory effect on collagenase. Purified I as collagenase
Type collagenase (0.25 units / ml) and human fibroblast-derived collagenase were used. Collagenase activity was determined by incubating collagenase with type I collagen labeled with 0.25 mg / ml fluorescein isothiocyanate (FITC) in a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 7.5) at 35 ° C. for 2 hours. Then, 5 μl of 40 mM o-phenanthroline was added to the reaction solution, and the mixture was treated at 35 ° C. for 30 minutes to obtain a collagen-containing supernatant decomposed by an ethanol precipitation method.
The fluorescence intensity of the collagen contained in the supernatant was measured with a fluorescence spectrophotometer at an excitation wavelength of 495 nm and a fluorescence wavelength of 520.
It was determined by measuring in nm. The collagenase inhibitory activity of Example 1 and the like according to the present invention is determined by measuring the enzyme activity by allowing each of the above collagenases to act on the above-mentioned collagen in the presence of 25 μg / ml of each sample. Calculated and expressed. The results are shown in Table 1.

(Equation 1)

As the human fibroblast-derived collagenase, human fibroblasts were used at 5 × 10 ⁴ cells / well.
Seeded into a 48-well microplate so that
After time, the medium was replaced with Dulbecco's modified basal nutrient medium (DMEM) supplemented with 10 ng / ml of each of interleukin-1α (IL-1α) and epidermal growth factor (EGF).
After culturing at 48 ° C. for 48 hours, the medium was collected, trypsin was added to the collected medium to a concentration of 0.04 mg / ml, and the mixture was reacted at 37 ° C. for 30 minutes to activate collagenase in the supernatant. Was used.

[0021]

[Table 1] As is evident from Table 1, Example 1 of the present invention
Both types of collagenase and human fibroblast-derived collagenase almost completely inhibited the activity, at 99%. On the other hand, in the 50% by volume ethanol extract as a starting material, a 90% inhibitory effect on each collagenase was observed. In addition, the methanol-soluble hydrolyzate of Example 1 also showed a high inhibitory effect of 99% on each collagenase.

Subsequently, the stability to heat and light of Example 1 of the present invention was evaluated. Example 10 at 100 ° C.
The collagenase inhibitory rate when 25 μg / ml was added was measured for each of the cases of heat treatment for 3 minutes and exposure and storage for 3 months, and the results are shown in Table 2 in comparison with the collagenase inhibitory rates of the untreated cases. As collagenase, a type I collagenase preparation was used.

[0023]

[Table 2] As is clear from Table 2, Example 1 of the present invention shows very good stability to heat and light, and 98% when heat-treated at 100 ° C. for 10 minutes, and exposure for 3 months. When stored, it retained 95% collagenase inhibitory activity.

Further, Example 1 of the present invention was evaluated for cytotoxicity to cultured human fibroblasts. Human-derived fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells / well, and 24 hours later,
Example 1 at 1.0 mg / ml to 100.0 mg / ml in DMEM supplemented with 1.0% by volume fetal calf serum at 37 ° C.
The cells were further cultured for 24 hours, and the number of viable cells was counted to determine the cell viability, and the 50% lethal concentration (LD ₅₀ ) was calculated. As a result, the LD ₅₀ value of Example 1 was greater than 100.0 mg / ml, and no cytotoxicity was observed at the concentrations tested.

Next, the formulations of the examples of the external preparation for skin according to the present invention are shown.

[Example 2] Lotion for skin (1) Ethanol 10.0 (wt%) (2) Hydroxyethylcellulose 1.0 (3) Collagenase inhibitor (Example 1) 0.1 (4) Paraoxybenzoate Methyl acid 0.1 (5) Purified water 88.8 Production method: (1) to (4) are sequentially added to (5) and uniformly dissolved.

Example 3 Skin Emulsion (1) Stearic acid 0.2 (% by weight) (2) Cetanol 1.5 (3) Vaseline 3.0 (4) Liquid paraffin 7.0 (5) Polyoxy Ethylene (10E.O.) monooleic acid 1.5 ester (6) Tocopherol acetate 1.0 (7) Glycerin 5.0 (8) Methyl parahydroxybenzoate 0.1 (9) Triethanolamine 1.0 (10 ) Purified water 79.5 (11) Collagenase inhibitor (Example 1) 0.2 Production method: The oil phase components (1) to (6) are mixed, heated and uniformly dissolved, and kept at 70 ° C. On the other hand, the aqueous phase components (7) to (10) are mixed and heated to be uniform, and the temperature is set to 70 ° C. The oil phase component is gradually added to the aqueous phase component while stirring to emulsify, and after cooling, (11) is added and mixed at 40 ° C.

Example 4 Skin Gel (1) Dipropylene glycol 10.0 (% by weight) (2) Carboxyvinyl polymer 0.5 (3) Potassium hydroxide 0.1 (4) Methyl paraoxybenzoate 0.1 (5) Purified water 88.8 (6) Collagenase inhibitor (Example 1) 0.5 Production method: After uniformly dissolving (2) in (5), dissolving (4) in (1) Then, (3) is added to increase the viscosity, and (6) is added and mixed.

Example 5 Skin Cream (1) Beeswax 6.0 (% by weight) (2) Cetanol 5.0 (3) Reduced Lanolin 8.0 (4) Squalane 27.5 (5) Glyceryl fatty acid ester 4.0 (6) Lipophilic glyceryl monostearate 2.0 (7) Polyoxyethylene (20E.O.) sorbitan 5.0 Monolaurate (8) Propylene glycol 5.0 (9) Paraoxybenzoic acid Methyl 0.1 (10) Purified water 36.4 (11) Collagenase inhibitor (Example 1) 1.0 Production method: Mix and dissolve oil phase components (1) to (7) and heat to 75 ° C . On the other hand, the aqueous phase components (8) to (10) are mixed and dissolved, and heated to 75 ° C. Next, the oil phase component is added to the water phase component and pre-emulsified, and then uniformly emulsified by a homomixer. After cooling, (11) is added and mixed at 40 ° C.

Example 6 Oil-in-water emulsion ointment (1) White petrolatum 25.0 (% by weight) (2) Stearyl alcohol 25.0 (3) Glycerin 12.0 (4) Sodium lauryl sulfate 1.0 (5) Methyl paraoxybenzoate 0.1 (6) Purified water 36.3 (7) Collagenase inhibitor (Example 1) 0.6 Production method: Mix and dissolve oil phase components (1) to (4) And uniform
Heat to 75 ° C. On the other hand, (5) is dissolved in (6) and heated to 75 ° C., the oil phase component is added thereto to emulsify, and after cooling, (7) is added and mixed at 40 ° C.

Example 7 Toner (1) Ethanol 10.00 (% by weight) (2) 1,3-butylene glycol 5.00 (3) Collagenase inhibitor (Example 1) 0.01 (4) Fragrance 0.10 (5) Purified water 84.89 Production method: (1) to (4) are sequentially added to (5), and uniformly mixed and dissolved.

Example 8 Serum (1) Glycerin 2.00 (% by weight) (2) 1,3-butylene glycol 3.00 (3) Polyoxyethylene (25E.O.) oleyl ether 0.50 (4) Collagenase inhibitor (Example 1) 0.05 (5) Ethanol 15.00 (6) Methyl paraoxybenzoate 0.10 (7) Fragrance 0.10 (8) Purified water 79.25 Production method: (6) ) And (7) are dissolved in (5), and this is added to (8) together with (1) to (4) and uniformly mixed and dissolved.

[Example 9] Emollient cream (water-in-oil type) (1) Liquid paraffin 30.00 (wt%) (2) Microcrystalline wax 2.00 (3) Vaseline 5.00 (4) Diglyceryl geo Leic acid ester 5.00 (5) Sodium L-glutamate 1.60 (6) L-serine 0.40 (7) Propylene glycol 3.00 (8) Methyl parahydroxybenzoate 0.10 (9) Purified water 52. 75 (10) Fragrance 0.10 (11) Collagenase inhibitor (Example 1) 0.05 Production method: (5) and (6) are dissolved in a part of (9) to 50 ° C.
Add slowly to (4) heated to 0 ° C. with stirring. This was previously mixed and uniformly dispersed in (1) to (3), which were heated and dissolved at 70 ° C., and (7) and (8) were dissolved in the remainder of (9) and heated to 70 ° C. Add while stirring and emulsify with a homomixer. After cooling, (10) and (11) are added and mixed at 40 ° C.

Example 10 Makeup Base Cream (1) Stearic acid 12.00 (% by weight) (2) Cetanol 2.00 (3) Glyceryl tri-2-ethylhexanoate 2.50 (4) Self-emulsification Type glyceryl monostearate 2.00 (5) Propylene glycol 10.00 (6) Potassium hydroxide 0.30 (7) Purified water 69.58 (8) Titanium oxide 1.00 (9) Bengala 0.10 ( 10) Yellow iron oxide 0.40 (11) Fragrance 0.10 (12) Collagenase inhibitor (Example 1) 0.02 Production method: Mix oil phase components (1) to (4) and heat to 75 ° C And make it uniform. On the other hand, mix the aqueous phase components (5) to (7)
The mixture is heated and dissolved to make the mixture uniform, and the pigments (8) to (10) are added to the mixture and uniformly dispersed by a homomixer. The oil phase component is added to the water phase component, emulsified by a homomixer, cooled, and (11) and (12) are added and mixed at 40 ° C.

Example 11 Emulsion Foundation (1) Stearic acid 2.00 (% by weight) (2) Squalane 5.00 (3) Octyldodecyl myristate 5.00 (4) Cetanol 1.00 (5) Decaglyceryl monoisopalmitate 9.00 (6) 1,3-butylene glycol 6.00 (7) Potassium hydroxide 0.10 (8) Methyl parahydroxybenzoate 0.10 (9) Purified water 53.40 ( 10) Titanium oxide 9.00 (11) Talc 7.40 (12) Bengala 0.50 (13) Yellow iron oxide 1.10 (14) Black iron oxide 0.10 (15) Fragrance 0.15 (16) Collagenase Inhibitor (Example 1) 0.15 Production method: The oil phase components (1) to (5) are mixed and heated to 75 ° C. to make uniform. On the other hand, the aqueous phase components of (6) to (9) were mixed and
The mixture is heated and dissolved to make the mixture uniform, and the pigments (10) to (14) are added to the mixture and uniformly dispersed by a homomixer. The oil phase component is added to the water phase component, and the mixture is uniformly emulsified by a homomixer, then cooled, and (15) and (16) are added and mixed at 40 ° C.

Example 12 Hand Cream (1) Cetanol 4.0 (% by weight) (2) Vaseline 2.0 (3) Liquid paraffin 10.0 (4) Glyceryl monostearate 1.5 (5) Polyoxyethylene (60E.O.) glyceryl 2.5 isostearate (6) Tocopherol acetate 0.5 (7) Glycerin 20.0 (8) Methyl paraoxybenzoate 0.1 (9) Purified water 59.2 ( 10) Collagenase inhibitor (Example 1) 0.2 Production method: Mix and dissolve the oil phase components (1) to (6) and heat to 75 ° C. On the other hand, the aqueous phase components (7) to (9)
Heat to 5 ° C. Next, the oil phase component is added to the water phase component and pre-emulsified, then uniformly emulsified and cooled with a homomixer, and (10) is added and mixed at 40 ° C.

With respect to Examples 2 to 6 among the above Examples of the present invention, the effect of preventing wrinkles caused by ultraviolet rays was evaluated. In each of the Examples, Comparative Example 2 to Comparative Example 6 were obtained by replacing the collagenase inhibitor of Example 1 with 50% by volume of ethanol. The effect of preventing wrinkles was determined by applying 5 hairless mice to one group, applying Examples and Comparative Examples to the back once a day for each group, and applying 1 J / cm ²
/ Week long-wave ultraviolet light (UVA) was applied for 50 weeks, the occurrence of wrinkles in hairless mice was observed, and scored according to the criteria shown in Table 3. At this time, a group to which only purified water was applied was used as a control. The results were calculated as the average value of each group, and are shown in Table 4 in relation to the number of UVA irradiation days.

[Table 3]

[0038]

[Table 4] As shown in Table 4, in the control group, the depth of wrinkles formed reached a moderate level when the number of UVA irradiation days exceeded 40 weeks, and the occurrence of deep wrinkles was observed after 50 weeks. Was. On the other hand, in each of the groups to which the examples of the present invention were applied, the occurrence of wrinkles was remarkably suppressed to the extent that slight to slight wrinkles were observed after 50 weeks in each case. On the other hand, in the group to which the comparative example was applied, in the group to which the comparative example 3 containing tocopherol acetate was applied, the occurrence or occurrence of wrinkles was not significantly prevented or reduced except that the degree of wrinkles was slightly reduced.

Subsequently, with respect to Examples 2 to 6 of the present invention, the anti-inflammatory effect and the effect of promoting wound healing were evaluated. Using a group of 5 mice with artificial inflammation or wound on the back skin, in each group, apply 0.5 g of each of Examples and Comparative Examples twice a day to an inflamed or wounded site for 7 days. Then, on day 7, the condition of each part was observed. "Effective", "Slightly effective", "Ineffective" for anti-inflammatory effect, "Complete healing", "Almost healing" for wound healing promoting effect,
The results were evaluated in three stages of “incomplete healing”, and the results are shown in Table 5 in terms of the number of mice that obtained each evaluation.

[0040]

[Table 5] As apparent from Table 5, no anti-inflammatory effect was observed in any of the mice to which the present invention was applied in the group to which the present invention was applied, and an effective anti-inflammatory effect was observed in two or more mice. I was Regarding the effect of promoting wound healing, none of the mice to which the wound healing was incomplete were observed in the group to which the Examples of the present invention were applied, and complete healing was recognized in two or more mice. On the other hand, in the group to which the comparative example was applied, one mouse each having a slightly effective anti-inflammatory effect was observed in each of the groups to which the comparative example 3 and the comparative example 5 were applied. I couldn't see it. In addition, the wound healing of the mice was incomplete in any of the application groups, except for one mouse in which the wound was almost healed in the group to which Comparative Example 3 was applied.

Next, a practical use test for 6 months was performed on Examples 2 to 12 of the present invention. As the panelists, women in their 40s to 60s who exhibited remarkable skin aging symptoms such as wrinkles and decreased skin elasticity were used, and one group consisted of 20 women. In this use test, the collagenase inhibitor of Example 1 blended in each Example was replaced with 50% by volume of ethanol in Comparative Examples 2 to 3 in the same manner as described above.
Comparative Example 12 was made. The use test was conducted by blindly using each of the examples and comparative examples for each group of panelists exhibiting skin aging symptoms. Before the start of the use test and after the end of the use test, the condition of the skin was observed, and the wrinkle and skin elasticity were evaluated in three stages of "improvement", "slight improvement", and "no change". The degree of wrinkles was evaluated by photographing and replica sampling, and the skin elasticity was measured and evaluated by a cute meter. Result is,
Table 6 shows the number of panelists who obtained each evaluation.

[0042]

[Table 6] As shown in Table 6, the improvement of the wrinkles was observed in all the groups using the examples of the present invention. In particular, Examples 2 to 6, and Example 1
In the two-use group, a clear improvement was recognized in 50% or more of the panelists. Regarding the state of improvement of skin elasticity, the improvement tendency was observed in all the groups using the examples, and 35%
The above panelists have clearly seen improvement. On the other hand, in the group using the comparative example, no panelists who recognized a clear improvement in both wrinkles and skin elasticity were observed, and the wrinkles were 70%.
As described above, regarding skin elasticity, 65% or more of panelists did not improve symptoms.

In Examples 2 to 12 of the present invention, no change in state such as precipitation, separation, agglomeration, discoloration, or odor of the contained components was observed at all during the above use test period. In addition, in each group used in Examples, there was no paneler showing a skin irritating reaction or a skin sensitizing reaction.

[0044]

As described in detail above, the present invention exhibits a specific and high inhibitory activity against stromal collagenase produced by mesenchymal cells such as fibroblasts and eosinophils. A collagenase inhibitor could be obtained. Collagenase inhibitors according to the present invention, inflammation and wounds, cancer metastasis,
It is useful for improving or preventing pathologies involving collagenase such as ulceration and skin aging.

According to the present invention, wrinkles and skin elasticity are prevented by suppressing the progress of collagen degradation in dermal fibroblasts due to various external stimuli such as ultraviolet rays and aging, and preventing the destruction of matrix fibers. A skin external preparation which effectively prevents or ameliorates the aging symptoms of the skin, such as a decrease in skin loss, and which is also excellent in the anti-inflammatory action and the wound healing promoting action, was obtained.

Claims (2)

[Claims] [Claim 1] Eucalyptus globulus La
billardiere) or its closely related plant ( Eucalyptus polybrac)
tea RTBaker, Eucalyptus dives Schauer) with 50% by volume of ethanol was fractionated with an ethanol-water mixed solvent by styrene polymer adsorption gel chromatography, and a 50% by volume ethanol eluted fraction was collected. Then, the mixture was subjected to silica gel chromatography, eluted with a mixed solvent of chloroform-methanol-water, and
A collagenase inhibitor comprising a fraction obtained by elution with 0% by volume of ethanol. 2. An external preparation for skin comprising the collagenase inhibitor according to claim 1.