JP3451114B2 - New glycoside and ultraviolet absorbing / scattering agent containing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel glycoside, and more specifically, it has excellent long-wavelength and short-wavelength ultraviolet absorption and scattering ability, and is used as a drug, quasi drug, cosmetic, etc. A novel glycoside useful as a compounding ingredient of

[0002]

2. Description of the Related Art It is said that external factors such as ultraviolet rays and dryness greatly affect the aging of the skin. Among them, with respect to ultraviolet rays, ultraviolet absorber-containing cosmetics and the like are widely used in order to prevent the generation of sunburn, spots, freckles and the like due to ultraviolet rays on the skin.

The ultraviolet absorber mainly absorbs long wavelength ultraviolet rays (about 320 to 400 nm, UV-A) called suntan wavelength and medium wavelength ultraviolet rays (about 280 to 320 nm, UV-B) called sunburn wavelength. Are mainly classified into those that mainly absorb and ultraviolet rays that absorb short wavelength ultraviolet rays (280 nm or less, UV-C). Many compounds have been known as UV-B absorbers for a long time, but UV-A reaches the dermis of the skin and has a blackening effect on the skin, and is a factor for the generation and exacerbation of spots, freckles, and the like. However, as a UV-A absorber, a sufficiently satisfactory compound has not yet been developed. Also, U
Most of V-C has not been paid much attention in the past because it is absorbed in the ozone layer, but since it has a carcinogenic effect, the development of its absorbent has been accompanied by the problem of ozone layer destruction by CFCs in recent years. Is desired.

On the other hand, conventional ultraviolet absorbers have an ultraviolet absorbing ability, but since they absorb the energy of ultraviolet rays, they are in a high energy state and are often unstable or have a bad influence on the living body. Therefore, it has been desired to develop a compound that can absorb ultraviolet rays that have a harmful effect on the human body and scatter the energy as safe visible light.

[0005]

SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide a component which can be incorporated into a skin external preparation as a UV absorber or a UV scatterer.

[0006]

Therefore, the present inventor pays attention to an eye that is constantly exposed to ultraviolet rays and the like, in particular, a crystalline lens, and diligently investigates to extract an ultraviolet absorbing substance and an ultraviolet scattering substance from a mammalian crystalline lens. As a result, the glycoside extracted from the protein fraction in the human lens has excellent UV-A absorption ability and UV.
-C absorption capacity, in addition to having an action of absorbing UV-A and UV-C and scattering into the visible region as fluorescence harmless to the human body, and having high stability, being useful as a component for external skin preparations The present invention has been completed and the present invention has been completed.

That is, the present invention provides 2-amino-3-hydroxy-acetophenone-O-β-D-glucoside. Further, the present invention provides an ultraviolet absorber containing the glucoside as an active ingredient. Furthermore, the present invention provides an ultraviolet scattering agent containing the glucoside as an active ingredient.

The 2-amino-3-hydroxy-acetophenone-O-β-D-glucoside of the present invention is a compound represented by the formula (1) and can be extracted from the lens of humans and primates. It can also be produced by a chemical or enzymatic synthesis method.

[0009]

[Chemical 1]

As a chemical synthesis method, α-glucopyranosyl bromide protected with a hydroxyl group is treated with 3-hydroxy-2-.
Reaction with nitroacetophenone to 3-acetyl-2-
Method for obtaining nitrophenoxy-hydroxyl group-protected-β-glucopyranoside, and then reducing the nitro group to an amino group by, for example, hydrogenation using a catalyst, and then removing the hydroxyl group to obtain the compound (1) of the present invention. Can be mentioned.

Further, as an enzymatic synthesis method, uridine-
A method for obtaining the compound (1) of the present invention can be mentioned by using an enzyme (UDPG transferase) obtained by extracting 5′-diphospho-α-glucoside (UDPG) and 2-amino-3-hydroxyacetophenone from wheat germ, for example.

To extract from the lens, for example, E
xp. Eye Res. , 14 , 53-57 (197
As described in 2), the homogenate obtained by grinding the lens with 10 times by weight of water is subjected to high-speed centrifugation to separate into a water-soluble protein fraction and a water-insoluble protein fraction. The obtained water-insoluble fraction is treated with an 80% ethanol solution containing 5% potassium hydroxide for 48 hours, followed by gel filtration and subsequent purification by reverse phase high performance liquid chromatography. Since the compound (1) of the present invention is present in the water-soluble protein fraction in addition to the water-insoluble protein fraction, it can be separated from the fraction.

The compound (1) of the present invention thus obtained has a wavelength of 260 nm, as is apparent from the ultraviolet absorption spectrum shown in FIG.
And having a maximum absorption peak near 370 nm, it is found to be useful as an ultraviolet absorber, particularly as a UV-A and UV-C absorber. Further, the compound (1) of the present invention emits fluorescence at 470 nm, which is harmless to the human body, when it absorbs light near 260 nm and 370 nm and is excited as shown in FIG.
It turns out that it is useful as an ultraviolet scattering agent. further,
The compound (1) of the present invention has a very short fluorescence lifetime of 1.5 × 10 ⁻⁹ seconds and is chemically stable, and therefore it is useful as a blending component for cosmetics and pharmaceuticals.

The compound (1) can be directly applied to the skin or the like to be used as an ultraviolet absorber or an ultraviolet scatterer, but it is used as a mixture with components or bases usually used in cosmetics, pharmaceuticals and the like. You can also do it. When blended into cosmetics, pharmaceuticals, etc., the blending amount is not particularly limited,
About 0.0001 to 50% by weight is preferable.

[0015]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Extraction of Compound (1): 50 human lenses obtained from a cataract patient were added with 10 times by weight of water and homogenized while cooling with water using a glass homogenizer. The homogenate was subjected to high-speed centrifugation under cooling at 27,000 xg for 60 minutes to obtain a precipitate (water-insoluble protein fraction). 2 g of the obtained water-insoluble fraction was added to 400 ml of an 80% ethanol solution of 5% potassium hydroxide to disperse the mixture, and the mixture was allowed to stand in a dark place at room temperature for 48 hours with gentle shaking. After the ethanol was distilled off, the mixture was neutralized with 1N hydrochloric acid, and the final concentration was 2.
Acetic acid was added to 8% and insoluble components were removed by centrifugation, and the supernatant was equilibrated with a 2.8% acetic acid solution. Toyopearl HW-40 Super Fine Column (2.2
Gel filtration was carried out at (× 40 cm). A flow rate of 30 ml / hour and a mobile phase of 2.8% acetic acid were used to collect 5 ml per fraction, and the fractions showing fluorescence of fractions No. 53 to 59 (fluorescence of 470 nm generated when excited at 370 nm) were collected. Collected (Fig. 1). The fraction showing the fluorescence was further purified by reverse phase high performance liquid chromatography (HPLC). H
PLC conditions are Senshu Pak ODS-115
1-sscolumn (4.6 x 150 mm), flow rate 1
ml / min, 0 to 19.6% linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid or 0.1% heptafluorobutyric acid as a counter ion, and treated for 24 minutes. Fractions having a retention time of about 20 minutes were repeatedly collected and purified to obtain the desired fluorescent substance (Fig. 2). The ultraviolet absorption spectrum of the obtained fluorescent substance is shown in FIG. 3, and the intensity of fluorescence generated when excited with light of 260 nm or 370 nm is shown in FIG. 4 (a, b). As a result, the compound of the present invention (1)
Has maximum absorption at 260 nm and 370 nm, 260 nm
It was found that when excited by light of 370 nm and 370 nm, fluorescence was generated at 470 nm.

(2) Structure Confirmation of Compound (1): (a) Nature, 230, 393-394 (197)
When the compound (1) was treated with β-glucosidase by the method described in 1) and its decomposed product was identified, the compound (1)
Was hydrolyzed by β-glucosidase to release D-glucose. On the other hand, the compound bound to D-glucose was a fluorophore, and it was confirmed that in Compound (1), the fluorophore and D-glucose were bound by an O-β-glucoside bond. In addition, the infrared absorption spectrum of the obtained fluorophore (JEOL JIR-6
300FT Infrared Spectropho
FIG. 5 shows the KBr method). as a result,
The medium sharp bands at 3481 and 3354 cm ^-1 in FIG. 5 indicate the NH stretch, and the sharp band at 1684 cm ^-1 indicates the C = O stretch of the ketone group. The broad band at 3200 cm ^-1 may be due to the OH stretch. The IR-data revealed that the fluorophore had an amino group, a carbonyl group, and a hydroxy group.

(B) Mass spectrum analysis of compound (1) Compound (1) was analyzed by JEOL HX 110/110 ta.
F by ndem massspectrometer
The AB mass spectrum (M + 1) was measured. Sample is 5
% Acetic acid, glycerol, thioglycerol and p-nitrobenzyl alcohol as matrix 1:
Analysis was performed using 1: 1 (volume ratio). Ionization was performed with a 10 kV beam of xenon atoms. FAB CAD
MS / MS analysis (collisionary act)
Ivated dissociation tande
The spectrum generated by the M mass spectroscopy is a JEOL scan with a constant B / E.
DA 5000 data system-gene
Obtained using the rate linked. From Figure 6,
(M + 1) is 314.1140, showing that this compound has a molecular weight of 313. The fragment pattern of this compound is shown in FIG.

(C) NMR spectrum analysis of compound (1) The ¹ H-NMR spectrum of compound (1) was measured. The model is VARIAN VXR-500S spectro
meter, using sodium 3- (trimethylsilyl) propionate as an external standard, in D ₂ O at 30 ° C.
At 500 MHz (FIGS. 8 and 9). A doublet was observed at 7.68 and 7.33 ppm and a triplet at 6.78 ppm in the spectrum. And, each double line is COSY (correlation sp
As evidenced by the experiment,
Coupling with 6.78ppm. This indicates that this configuration shows the presence of three adjacent aromatic protons (Figure 10). NOESY (nuclear o
verhauser effect spectros
copy) and the singlet at 2.63 ppm and 7.6
Since a cross peak was observed with the doublet at 8 ppm, the presence of an acetyl group was confirmed at the 1-position of the benzene ring (Fig. 11). 5.0 in the NOESY experiment
The anomeric proton at 4 ppm was found to be close to the proton at 7.33 ppm, indicating the presence of glucose at the 3-position of the benzene ring. The coupling constant of the anomeric proton is J = 7.5.
Since it indicates Hz, it can be said that glucose has a β-conformation. To further confirm the presence of acetyl groups, ¹³ C-NMR was run on a VARIAN GEM.
30 using IN-300 spectrometer
It was confirmed under the conditions of 0 MHz, 30 ° C. and D ₂ O (FIG. 12). The results show its response at 207.18 ppm, indicating the presence of a carbonyl group. From the above results, it was confirmed that the amino group could exist only at the 2-position of the benzene ring.

From the above results, it is understood that the compound (1) is 2-amino-3-hydroxy-acetophenone-O-β-D-glucoside [the above formula (1)].

Example 2 (1) Synthesis of tetra-O-acetyl-α-D-glucopyranosyl bromide: 60 ml of acetic anhydride was placed in a three-necked flask, and 70% perchloric acid of 0.1% was added while cooling with ice water. Add 36 ml dropwise. Then, anhydrous glucose 15 at 30-40 ° C
After adding g and cooling to 20 ° C., 4.5 g of red phosphorus, 8.7 ml of bromine and 5.4 ml of water are added. This was left at room temperature for 2 hours and then extracted with chloroform to give colorless crystals 32.4.
g (yield 95%) was obtained. Recrystallize this with ether,
Colorless needle crystals were obtained. ^It was identified as the title compound by ¹ H-NMR and IR (FIGS. 13 and 14).

(2) 3-acetyl-2-nitrophenoki
Ci-tetra-O-acetyl-β-D-glucopyranoside
Synthesis of 3-hydroxy-2-nitroacetophenone 2
7 g, silver (I) oxide 37.5 g, calcium sulfate 150
g, 100 ml of chloroform, 50 ml of pyridine
Put it in a Rasco and shade the flask, 0.375 g of iodine
Add. To this, tetra-O-acetyl-α-D-glu
Copyranosyl bromide 62 g dissolved in 200 ml chloroform
The liquid was added dropwise with stirring and the reaction was carried out for 24 hours.
The reaction solution is filtered and the solvent is distilled off, followed by silica gel (waco
-Gel C-200), 56.2 g of crystals (yield 7
4%). This is recrystallized with ethanol and colorless needles
I got crystals. ¹Identified as the title compound by 1 H-NMR and IR
(FIGS. 15 and 16).

(3) Synthesis of 3-acetyl-2-aminophenoxy-tetra-O-acetyl-β-D-glucopyranoside: 23 g of 3-acetyl-2-nitrophenoxy-tetra-O-acetyl-β-D-glucopyranoside After dissolving in 150 ml of chloroform and 50 ml of ethanol,
500 mg of platinum dioxide was added, and the reaction was carried out under hydrogen pressure.
After the reaction was completed, the reaction mixture was filtered, the solvent was distilled off, and the residue was purified by silica gel (Wakogel C-200) to give 18.0 g of crystals.
(Yield 83%) was obtained. This is recrystallized with ethanol,
Colorless needle crystals were obtained. ^It was identified as the title compound by ¹ H-NMR and IR (FIGS. 17 and 18).

(4) 2-amino-3-hydroxy-acetate
Tophenone-O-β-D-glucoside (the compound of the present invention
Synthesis of (1)): 3-acetyl-2-aminophenoxy
-Tetra-O-acetyl-β-D-glucopyranoside 1
0 g, sodium hydroxide 3.5 g water-methanol 20
It was dissolved in 0 ml, heated for 1 hour and purified to give the title compound 6.1.
g (yield 94%) was obtained. this ¹H-NMR and IR
It was identified by (FIGS. 19 and 20).

Example 3 0.08 mol of 2-amino-3-hydroxy-acetophenone (manufactured by Tokyo Kasei) and uridine-5'-diphospho-α
-D-glucoside disodium salt (manufactured by SIGMA)
0.08 mol was dissolved in 160 μl of 0.02M tris-maleic acid buffer solution (pH 6.5) containing 1 mM EDTA and 2 mM β-mercaptoethanol, and this was used as a reaction solution. An enzyme solution extracted from wheat germ (YAMAHA, T., & CARDINI, C.I.
E. Arch. Biochem. Biophys. ，
86, 127 (1960)) 40 μl, and added at 37 ° C.
And reacted for 1 hour. After this, 1-butanol: acetic acid:
Thin layer chromatography (TLC) was developed using water = 4: 1: 1 as a solvent, and this was irradiated with a UV-lamp,
The compound of the present invention that emits fluorescence was confirmed. Furthermore, Example 1
Similarly to the above, the compound of the present invention was confirmed by HLPC.

Example 4 (1) The photooxidation suppressing effect of the compound of the present invention based on the ultraviolet scattering action was examined. 50μ dissolved in 1.5ml of purified water
The sample solution designated as M was irradiated with ultraviolet rays ( ² mW / cm ² , 25 ° C.) with a UV-lamp having a peak at 365 nm to obtain a reaction solution. 20 μl of 2-methyl-p-methoxyphenyl-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA) was adjusted to 30 μM with a buffer (0.3 M Tris-HCl pH 7.8, 0. 6 mM ED
TA) was dissolved in 500 μl of purified water and 2280 μl of purified water, and 200 μl of the reaction solution was sampled and added thereto, and the luminescence intensity was measured with a luminescence reader (Aloka). The case where distilled water was added instead of the reaction solution was used as a blank, and the difference in luminescence intensity was determined. In addition, it was confirmed that this difference in intensity is proportional to the existing concentration of superoxide. As a sample, the compound (1) of the present invention and its fluorophore 2-amino-3-hydroxy-acetophenone (AHA) were used. As a result, when the sample solution is AHA, the amount of superoxide generated increases as the ultraviolet irradiation time increases, whereas in the case of the compound of the present invention, superoxide is generated even when ultraviolet irradiation is performed. It turned out not to do (Fig. 21).

(2) Next, the photooxidation reaction of tryptophan residues in proteins was examined. 365 nm in a solution containing 1 mg / ml bovine serum albumin (manufactured by Sigma) and 50 μM sample
UV irradiation with a UV-lamp having a peak at 2 mW / cm
² , 25 ° C) to obtain a reaction solution. 100 μl of this reaction solution
Were collected over time and buffered (10 mM Tris-HCl
2900 μl of pH 7.6, 0.1 M KCl) was added to prepare a test solution. The progress of the photooxidation reaction of tryptophan residues was determined by measuring the decrease in the fluorescence intensity at 350 nm using the properties of tryptophan (excitation at 290 nm and fluorescence emission at 350 nm). As the sample, the compound (1) of the present invention and AHA were used as described above.

As a result, when AHA is used as the sample, the amount of tryptophan present decreases as the ultraviolet irradiation time increases, whereas in the case of the sample of the present invention, the amount of tryptophan existing does not change even when the sample is irradiated with ultraviolet light. There was no change (Figure 22).

From the above results, AHA, which is a compound in which glucose is desorbed from the compound of the present invention, is excited and sensitized by the absorbed ultraviolet ray and promotes the production of superoxide, so that the tryptophan residue becomes a photooxidation product. Have an effect. In contrast, the compound of the present invention (1) (AHA-
It was found that Glc) has a function of absorbing ultraviolet rays and scattering as harmless fluorescence, and thus suppresses the oxidation reaction of tryptophan residues by light.

[0030]

INDUSTRIAL APPLICABILITY The compound (1) of the present invention has a U of around 260 nm.
Since it strongly absorbs V-C and UV-A around 370 nm and scatters the energy of these harmful ultraviolet rays as harmless fluorescence, it is useful as an ultraviolet absorber and an ultraviolet scatterer, and is stable to light. Therefore, it can be used as a blending component for external skin preparations such as cosmetics and pharmaceuticals.

[Brief description of drawings]

FIG. 1 is a diagram showing a gel filtration pattern of a centrifugal supernatant in Example 1.

FIG. 2 is a diagram showing a reversed phase high performance liquid chromatograph pattern in Example 1.

FIG. 3 is a diagram showing an ultraviolet absorption spectrum of the compound of the present invention obtained in Example 1.

FIG. 4 shows excitation light for the compound of the present invention obtained in Example 1,
It is a figure which shows and the fluorescence spectrum generated as a result.

5 is a diagram showing an infrared absorption spectrum of a β-glucosidase degradation product (fluorophore) of the compound of the present invention obtained in Example 1. FIG.

6 is a diagram showing a FAB-mass spectrum of the compound of the present invention obtained in Example 1. FIG.

FIG. 7 is a view showing a fragment pattern in a FAB-mass spectrum of the compound of the present invention obtained in Example 1.

FIG. 8 is a chart showing ¹ H-NMR spectrum of the compound of the present invention obtained in Example 1.

FIG. 9 is a partially enlarged view of the ¹ H-NMR spectrum shown in FIG.

10 is a view showing a COZY spectrum of the compound of the present invention obtained in Example 1. FIG.

11 is a diagram showing a NOESY spectrum of the compound of the present invention obtained in Example 1. FIG.

¹³ C-NMR of [12] the compound of the present invention obtained in Example 1
It is a figure which shows a spectrum.

FIG. 13 ¹ H-NMR of the compound obtained in Example 2 (1)
It is a figure which shows a spectrum.

FIG. 14 is a diagram showing an infrared absorption spectrum of the compound obtained in Example 2 (1).

FIG. 15 ¹ H-NMR of the compound obtained in Example 2 (2)
It is a figure which shows a spectrum.

FIG. 16 is a view showing an infrared absorption spectrum of the compound obtained in Example 2 (2).

FIG. 17 ¹ H-NMR of the compound obtained in Example 2 (3)
It is a figure which shows a spectrum.

FIG. 18 is a view showing an infrared absorption spectrum of the compound obtained in Example 2 (3).

FIG. 19 shows ¹ H-of the compound of the present invention obtained in Example 2 (4).
It is a figure which shows a NMR spectrum.

FIG. 20 is a diagram showing an infrared absorption spectrum of the compound of the present invention obtained in Example 2 (4).

FIG. 21 is a graph showing a change with time in emission intensity when the compound of the present invention and AHA are irradiated with ultraviolet rays.

22 is a diagram showing the time-dependent degree of progress of the photooxidation reaction in Example 4. FIG.

─────────────────────────────────────────────────── ─── Continuation of front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) C07H 15/203 C09K 3/00 104 A61K 7/42 REGISTRY (STN) CA (STN) CAOLD (STN) JISST file (JOIS)

Claims (3)

(57) [Claims] 1. 2-Amino-3-hydroxy-acetophenone-O-β-D-glucoside. 2. An ultraviolet absorber containing 2-amino-3-hydroxy-acetophenone-O-β-D-glucoside as an active ingredient. 3. An ultraviolet scatterer containing 2-amino-3-hydroxy-acetophenone-O-β-D-glucoside as an active ingredient.