JPH09295927A - Tyrosinase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a natural tyrosinase inhibitor effective for beautifying, whitening, etc., a skin. SOLUTION: This tyrosinase inhibitor contains a sugar ester of 2'-(S)-8-C- glycosyl-7-methylaloesol, e.g. 2"-p-cumaroyl ester of 2'-(S)-8-C- glycosyl-7-methylaloesol as an active ingredient. Theses compounds are obtained from an aloe vera juice of pharmaceutical plant belonging to genus Aloe such as Aloe perryi Baker, Aloe arborescens Mill. var. natalensis Berger, etc. The above active ingredient is obtained by removing fibers of the aloe vera juice, adsorbing the juice with an activated carbon, extracting the obtained adsorbed material to the activated carbon with ethanol and then purifying the extract by a high speed liquid chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a natural tyrosinase inhibitor effective for whitening skin, removing spots, freckles, etc. and improving spots on the skin due to liver damage.

[0002]

2. Description of the Related Art Hyperpigmentati on the skin
The melanin pigment that causes (on) coloring is produced in the melanogenic granules (melanosomes) in the melanocytes (melanocytes) between the epidermis and the dermis, and the produced melanin diffuses to adjacent cells by the osmotic action. . The biochemical reaction in this melanocyte is currently estimated to be of the following reaction formula (Fragrance Journal No. 63, p. 24 (1983) et al.).

[0003]

Embedded image

As described above, tyrosine, which is an essential amino acid, becomes dopaquinone by the action of the enzyme tyrosinase,
The process in which this changes to black melanin through red and colorless pigments by the enzymatic or non-enzymatic oxidation action is the melanin pigment formation process.

[0005]

Therefore, it has been imagined that suppressing the action of tyrosinase, which is the first step of the reaction, or reducing quinones in the intermediate step, can suppress the production of melanin. , For example, a substance that binds to copper that is the active center of tyrosinase (for example, thiourea, cystine), a substance that can be a competitive substrate with tyrosine that is a substrate for tyrosinase (for example, N-acetyltyrosine, γ-pyrone, hinokitiol), A substance that prolongs the induction period of the enzymatic reaction between tyrosinase and a substrate (for example, Tween 20), a substance that selectively competes with an o-dihydroxy group such as dopa (for example, aniline), a reducing agent for o-quinones (for example, , Ascorbic acid, hydroquinone and its derivatives) were proposed, but their toxicity to humans, preservability, Considering the ability effects like, but not enough satisfactory. For example, hydroquinone has been used for treatment of melasma in many countries for many years, but it is ineffective unless it is administered in a large amount, and moreover, an effective concentration causes side effects such as erythema and pain. In addition, although it is certain that cystine suppresses the formation of dopa pigment, there is a problem in using it as a cosmetic because of its sulfur odor. In addition, ascorbic acid is vulnerable to oxidation and browns the formulation, which is also a practical problem.

Furthermore, in recent years, attention has been paid to flavonoids in an aqueous drug solution for controlling plant growth, and in the context of the present invention, the anti-thyrodinase action of an anthraquinone glycoside, aloin (barvaloin), has been announced. (Japanese Patent Publication No. 53-45376). However, its anti-tyrosinase action is not so remarkable as described later.

The present invention has been made in view of such circumstances, and an object thereof is to provide a safe and reliable tyrosinase inhibitor using aloe, which has been famous as a folk medicinal plant since ancient times.

[0008]

In order to achieve the above object, the tyrosinase inhibitor of the present invention contains 2 '-(S)-.
A sugar ester of 8-C-glucosyl-7-methylaloesol is used as an active ingredient.

That is, the inventor of the present invention conducted an inhibition test using mushroom tyrosinase to obtain 2 '-(S) -8-C-glucosyl-7-methylaloesol [below] contained in fresh leaves of aloe. It was found that the sugar ester of the formula (1)] (in particular, the ester having an acyl group bonded to the sugar 2 ″ position of the following formula (1)) has a remarkable tyrosinase inhibitory activity comparable to that of ascorbic acid. Then, the sugar ester of the following formula (1) was obtained by adsorbing the freeze-dried product of fresh aloe leaf foliage onto activated carbon and eluting it with ethanol. It was found that it can be obtained by performing the image purification.

[0010]

Embedded image

The present inventor has also found that the sugar 2 ″ hydroxyl group of 2 ′-(S) -8-C-glucosyl-7-methylaloesol represented by the above formula (1) is p-coumaric acid. Ester-bonded 2 '-(S) -8-C-glucosyl-7-methylaloesol 2 "-p-coumaroyl ester [the following formula (2)] and cinnamic acid ester-bonded 2'-
It has been found that the 2 ″ -cinnamoyl ester of (S) -8-C-glucosyl-7-methylaloesol [the following formula (3)] has a particularly excellent tyrosinase inhibitory action among the above sugar esters. .

[0012]

Embedded image

[0013]

Embedded image

[0014]

Next, embodiments of the present invention will be described in detail.

The tyrosinase inhibitor of the present invention is 2'-
(S) -8-C-Glucosyl-7-methylaloesol (8-C-β-D-glucopyranosyl-2-[(S)-
2′-hydroxy] propyl-7-methoxy-5-methylchromone) is used as an active ingredient.

Aloe, which is a raw material of the above-mentioned tyrosinase inhibitor, is known as Aloe perryi B since ancient times.
aker), as well as Kidachi aloe (A. arborescens Mill.var.na
talensis Berger), Curacao aloe (also known as aloe vera) (A. barbadensis Miller), Cape aloe (A. ferox M)
iller or this with A.afiricana Miller. and A.spic
hybrid with ata Baker), Natal aloe (A. candelabr
um Berger) and other medicinal aloe plants can be used.

2'in the tyrosinase inhibitor of the present invention
As the sugar ester of-(S) -8-C-glucosyl-7-methylaloesol, as described above, 2 "of sugar can be used.
Particularly preferred is an ester in which the position hydroxyl group is acylated,
In addition to this, sugar 2 ", 3", 4 "and 6" positions
Examples thereof include an ester or an enol ester in which all or part of the hydroxyl groups at positions are acylated.

Next, the experimental facts on which the present invention is based will be described.

[Extraction Method] Fresh leaves of Aloe vera are shredded and lyophilized, and then the powder is suspended in water, and Aloe vera juice is prepared using a juicer. Next, fibers and the like present in Aloe vera juice were removed by filtration using a 250 μm mesh sieve, and the filtrate was passed through an activated carbon layer. After thoroughly washing this activated carbon layer with water, the adsorbed substance was eluted from the activated carbon layer with ethanol. The yield of yellow ethanol eluate from fresh Aloe vera leaves is 0.5-1.0%.
Met.

Next, the yellow ethanol elution part (10
0 g) was subjected to Sephadex LH-20 column chromatography and eluted with 50% methanol to give fraction 1 (1
9.2 g) and fraction 2 (12.5 g) were separated. The Fraction 1 was further subjected to Diaion CHP-20P column chromatography to obtain Compound 1 (74 mg) from the 15% methanol eluate and Compound 3 (51 mg) from the 80% methanol eluate. On the other hand, after fractionating the above fraction 2 by Sephadex LH-20 column chromatography (50% methanol elution), Diaion CHP-20P column chromatography (60%)
Fractionation by elution with methanol) gave compound 2 (151 mg).

[High Performance Liquid Chromatography (HPL
C) -1] Column: Wakosil-II 5C18HG (5 μm,
Inner diameter 4.6 × 150 mm, manufactured by Wako Pure Chemical Industries, Ltd.). Eluent: acetonitrile / water (0-19 minutes, 12-23%; 19-
24 minutes, 23-28%; 24-39 minutes, 28-46
%). Flow rate: 1 ml / min. Monitor: 290 mm. Column temperature: 45 ° C. Measuring instrument type: UV-8000 type UV-visible detector (manufactured by Tosoh Corporation), pump: CCPD type (manufactured by Tosoh Corporation), Waters 990J type photodiode array detector, data processing: Chromatocorder-II (system instrument).

[High Performance Liquid Chromatography (HPL
C) -2] Column: Wakosil-II 5C18HG (5 μm,
Inner diameter 4.6 × 150 mm, manufactured by Wako Pure Chemical Industries, Ltd.). Eluent: acetonitrile /0.1%H ₃ PO ₄ (0~9 minutes, 15
%; 9 to 14 minutes, 15 to 35%; 14 to 20 minutes, 35
%). Flow rate: 1 ml / min. Monitor: 290 mm. Column temperature: 45 ° C. Measuring instrument type: UV-8000 type UV-visible detector (manufactured by Tosoh Corporation), pump: CCPD type (manufactured by Tosoh Corporation), Waters 990J type photodiode array detector, data processing: Chromatocorder-II (system instrument).

[Compound 1] Yellow amorphous, mp135-1
36 ° C., [α] ³⁰ _D + 9.1 ° (MeOH, C = 1.0
0), UVλ ^MeOH _max nm (log ε): 214 (4.
16), 226 (4.17), 243 (4.07), 2
52 (4.05), 293 (3.91), HR-positi
ve FAB-MS m / z: 411.1651 ([M +
H] _{^{+, Calcd for C 20 H 27}} O 9: 411.165
4).

¹ H of compound 1 obtained by the above extraction method
And ¹³ C-NMR spectrum data are used as a standard for 2 ′-(R) -8-C-glucosyl-7-O-methylaloesol [mp 141-143 ° C., [α] ³⁰ _D -3.
9.5 ° (MeOH, C = 0.23)] ¹ H and ¹³
It was compared with C-NMR spectrum data.

Further, the HPLC of the compound 1 was compared with the standard HPLC by using the HPLC shown above. As a result, the retention time of the standard under the condition of HPLC-1 was 5.03.
The retention time of Compound 1 was 5.58 minutes, while the time was minutes. Literature [G.Speranza, G.Dada, L.Lanazzi, P.Gram
Atica, P. Manitto (1986) Phytochemistry, 2219-2222], the two were compared under the HPLC conditions, and both were 2 ′-(R) -8-C-glucosyl-7-O- which was a standard product.
Methyl aloesol (5.77 minutes) and compound 1 (6.
29 minutes).

From the above facts, the above compound 1 is 2 '-(S) -8-C-glucosyl-represented by the above formula (1).
It was determined to be 7-O-methyl aloesol.

[Compound 2] colorless amorphous form, mp156-1
60 ° C., [α] ²⁷ _D −156.8 ° (MeOH, C =
0.25), UVλ ^MeOH _max nm (log ε): 213
(4.52), 228 (4.58), 242 sh, 2
52 (4.34), 300 (4.54).

¹ H of compound 2 obtained by the above extraction method
And ¹³ C-NMR spectrum data were obtained from alloresin D (8-C-β-D- [2'-O- (E) -p-coumaroyl] glucopyranosyl-2-[(R) -2-hydroxy] propyl-7. -Methoxy-5-methylchromone)
Compared with ¹ H and ¹³ C-NMR spectral data.
As a result, it was confirmed that the ¹ H- and ¹³ C-NMR spectral data of Compound 2 closely resembled that of alloresin D [G. Speranza, G. Dada, L. Lanazzi, P. Gramat.
ica, P. Manitto (1986) Phytochemistry, 2219-2222].

Compound 2 was added to 0.2 M sodium hydroxide or pancreatin (pH 8.0, 37 ° C., 2-7).
Compound 1 was confirmed by HPLC-1 and p-coumaric acid by HPLC-2.

From the above facts, Compound 2 was determined to be the p-coumaric acid ester of Compound 1. And p-
Coumaric acid is bound to ¹ H and ¹³ C of alloresin D.
-By comparison with the NMR spectrum data, it was determined to be at the 2 "-position of the sugar, and Compound 2 represented by the above formula (2) was named isoalloresin D.

[Compound 3] Light yellow amorphous form, mp 86-9
2 ° C., [α] _{^{26 D -112.6 ° (MeOH, C}} = 0.
55), UVλ ^MeOH _max nm (log ε): 205 s
h, 217 (4.44), 223 (4.43), 244
(4.26), 252 (4.30), 281 (4.3)
9), HR-positive FAB-MS m / z: 54
1.2073 ([M + H] ⁺ , Calcd for C ₂₉ H
₃₃ O ₁₀ : 541.2073).

UV of compound 3 obtained by the above extraction method
The spectral data was compared to the UV spectral data for compound 2. As a result, it was confirmed that the UV spectrum data of compound 3 was very similar to that of compound 2.

¹ H and ¹³ C-NMR of compound 3
The spectral data are shown as ¹ H and ¹³ C-NM of compound 2.
R spectrum data was compared. As a result, compound 3
Was suggested to have a structure in which cinnamic acid was bonded in place of p-coumaric acid bonded to the 2 ″ position of the sugar of Compound 2.

Compound 3 was added to 0.2 M sodium hydroxide or pancreatin (pH 8.0, 37 ° C., 2-
Compound 1 was confirmed by HPLC-1 and cinnamic acid was confirmed by HPLC-2 by hydrolysis for 7 days).

From the above facts, the compound 3 has a structure in which the cinnamic acid is ester-bonded to the 2 "-position of the sugar of the compound 1 and is represented by the above formula (3) 8-C-β-D- [2. '-O-
(E) -Cinnamoyl] glucopyranosyl-2-
[(S) -2-hydroxy] propyl-7-methoxy-
It was determined to be 5-methylchromone. Then, this compound 3 was named as aloeresin E.

[Measurement of Tyrosinase] Tyrosinase was measured by the Pomerantz method (Pomerantz, SH, (1963) J. Biol. C.
hem., 238, 2351) with some modifications. That is, 0.4 μM of a sample was added to 1 ml of 10% dimethyl sulfoxide and dissolved by ultrasonic wave. 1/15 M for 1 ml of test solution
Phosphate buffer (pH 6.8) 1 ml, mushroom (mushroom) tyrosinase (96 U / ml, Sigma Chemical Co.) 0.5 ml and L-dopa (3,4-dihydroxyphenylalanine) (1 μM / ml) 0.5 m
1 was added and kept at 25 ° C. for 2 minutes. Then, the amount of dopachrome (red color) in the reaction mixture was measured by the absorption amount at 475 nm. The change in absorption at 475 nm in the presence or absence of sample was linear within 2 minutes. FIG. 1 is a graph showing the relationship between the time course and reaction rate of dopa-tyrosinase and tyrosin-tyrosinase reactions.

The inhibition rate (%) of the tyrosinase reaction is calculated according to the following formula.

[0038]

[Equation 1] A: Absorption after incubation at 475 nm in the absence of test sample. B: Absorption before incubation at 475 nm in the absence of test sample. C: Absorption after incubation at 475 nm in the presence of test sample. D: Absorption before incubation at 475 nm in the presence of test sample.

The molecular absorption coefficient of dopachrome at 475 nm is 3.7 × 10 ³ .

[Measurement of Tyrosinase Inhibition Rate] Table 1 below
The relationship between the concentration and the inhibition rate was measured using various compounds having the molecular weights shown in 1 and the structure shown in FIG. The results are shown in FIGS. 3 and 4.

[0041]

[Table 1]

As is clear from FIG. 3, Compound 1 does not show inhibitory activity, but Compound 2 (isoalloresin D) and Compound 3 (Alloresin E), which are sugar esters of Compound 1, show high inhibitory activity. confirmed. In particular, it was confirmed that the compound 3 exhibits substantially the same high inhibitory activity as that of the ascorbic acid and the alloresin A (p-coumaroyl aloesin) as controls.

As is clear from FIG. 4, p-coumaric acid and cinnamic acid have lower inhibitory activities than ascorbic acid as a control but higher inhibitory activities than aloesin and barvaloin widely distributed in aloe. confirmed.

From these facts, Compound 1 does not show inhibitory activity as it is, but p-coumaric acid and cinnamic acid which show high inhibitory activity are ester-bonded to the 2 "-position of sugar of Compound 1 to give It seems that the inhibitory activity is higher as in Compound 2 and Compound 3.

Thus, the novel compound of Aloe vera is H
It is widely distributed in plants of the genus Aloe by searching with PLC, and is expected to have a whitening effect as an effective and safe natural tyrosinase inhibitor.

[Formulation] The tyrosinase inhibitor of the present invention is
For example, it can be used for external medicines, cosmetics and the like. The dosage forms include ointments, tinctures, soaps,
Examples include cleansing cream, cold cream, vanishing cream, foundation cream, lotion, astringent, emulsion, foundation lotion, cleansing lotion, white powder, pack and the like.
When preparing these, known external preparations, bases for skin cosmetics, additives and the like can be added.
For example, isopropyl myristate, isopropylmitate, liquid paraffin, nujol, vaseline, solid paraffin, ceresin, microcrystal wax, lanolin, acylated lanolin, carbowax, carboxyvinyl polymer, various surfactants, beeswax, vegetable oil, borax, Bleaching beeswax, vegetable volatile oil, sorbitol, cetanol,
Spermaceti, stearic acid, propylene glycol, glycerin, methyl cellulose, triethanolamine, silicone oil, ozokerite, titanium dioxide, lambritol
Wax, ethanol, lemon juice, pectin, tracant gum, loofah water, citric acid, alum, zinc sulfate,
Other ingredients such as polyethylene glycol fatty acid ester, talc, kaolin, precipitated calcium carbonate, zinc stearate, magnesium carbonate, zinc dioxide, sulfur flower, pigments, fragrances and preservatives, ascorbic acid or its sodium salt, hinokitiol, cystine, koji Acids, cyclic α-ketols such as pyromeconic acid and maltol, and known tyrosinase inhibitory compounds such as hydroquinone and catechol can be added.

[Formulation Examples] Next, several types of formulation examples containing the tyrosinase inhibitor of the present invention will be shown.

Formulation Example 1 (Cold cream type nutrition cream) ○ Oil phase (%) Lanolin alcohol 5.0 Acetylated lanolin 1.0 Whale wax 4.0 Bleached beeswax 10.0 Liquid paraffin 20.0 White petrolatum 20.0 Sorbitan fatty acid ester 3.0 Tween 60 1.0 Polyethylene glycol 400 2.0 Methylcellulose 0.1 2 ′-(S) -8-C-Glucosyl-7-methylaloesol 2 ″ -p-coumaroyl ester freezing Dried product (Compound 2) 0.5 ○ Aqueous phase Propylene glycol 5.0 Methyl paraoxybenzoate 0.1 Add fragrance and water 100.0

Formulation Example 2 (Hand cream) ○ Oil phase (%) Isopropyl myristate 3.0 Microcrystal wax 2.0 Cetanol 6.0 Lanolin 1.0 Sorbitan monolaurate 0.8 2 '-(S)- Lyophilized product of 2 ″ -cinnamoyl ester of 8-C-glucosyl-7-methylaloesol (Compound 3) 0.5 ○ Water phase Sorbitol (70%) 5.0 Sodium sorbate 0.1 Water In addition 100.0

Formulation Example 3 (vanishing cream) ○ Oil phase (%) Wool wax sterol 5.0 Acetylated lanolin alcohol 2.0 Acetylated and ethoxylated lanolin 3.0 Stearic acid monmoglyceride 5.0 Stearic acid 15.0 Lyophilized product of 2 ″ -p-coumaroyl ester of 2 ′-(S) -8-C-glucosyl-7-methylaloesol (Compound 2) 0.5 ○ Water phase Propylene glycol 5.0 Fragrance and water In addition 100.0

Formulation Example 4 (milk lotion) (%) Liquid paraffin 20.0 Polyethylene glycol 600 monostearate 5.0 Cetyl trimethyl ammonium chloride 3.0 Isopropyl myristate 2.0 Propylene glycol 4.0 2 '-(S ) -8-C-Glucosyl-7-methylaloesol 2 ″ -Cinnamoyl ester lyophilizate (Compound 3) 0.5 Add perfume, preservative and water 100.0

[0052]

INDUSTRIAL APPLICABILITY As described above, the tyrosinase inhibitor of the present invention is effective in removing skin whitening, spots, freckles, and improving spots on the skin due to liver damage, and is safe.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the time course and reaction rate of dopa-tyrosinase and tyrosin-tyrosinase reactions.

FIG. 2 is a diagram showing structures of compounds 1 to 3, aloeresin A, aloesin and barvaloin.

FIG. 3 is a graph showing the relationship between the concentration and the inhibition rate when compounds 1 to 3 and other compounds (aloresin A, ascorbic acid) were used.

FIG. 4 compounds (barvaloin, aloesin, cinnamic acid,
It is a graph which shows the relationship between the concentration and the inhibition rate when using p-coumaric acid and ascorbic acid.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display area A61K 31/70 ADA A61K 31/70 ADA AED AED // C07H 17/07 C07H 17/07

Claims (2)

[Claims] 1. 2 '-(S) -8-C-glucosyl-7
-A tyrosinase inhibitor comprising a sugar ester of methylaloesol as an active ingredient. 2. The sugar ester of 2 '-(S) -8-C-glucosyl-7-methylaloesol is 2'-
2'-p-coumaroyl ester of (S) -8-C-glucosyl-7-methylaloesol, 2 '-(S) -8-
The tyrosinase inhibitor according to claim 1, which is a 2 ″ -cinnamoyl ester of C-glucosyl-7-methylaloesol.