JP3223795B2 - Tyrosinase inhibitor and hyaluronidase inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a tyrosinase inhibitor and a hyaluronidase inhibitor. More specifically, the present invention relates to a tyrosinase inhibitor and a hyaluronidase inhibitor comprising an extract from a plant as an active ingredient.

[0002]

2. Description of the Related Art There are still many unclear points about the mechanism of skin pigmentation such as spots and freckles, but it is generally caused by abnormalities in melanin metabolism due to exposure of skin to ultraviolet rays and abnormal secretion of hormones. It is thought that this is because production of melanin in the epidermis is enhanced and deposited on the skin.

[0003] Melanin, which is a major factor determining skin tone, is converted from tyrosine to dopa and from dopa to dopaquinone by tyrosinase in melanosomes in pigment cells called melanocytes, which are present in the basal layer of the epidermis, and then converted to melanin. It is said to be produced by oxidative polymerization via intermediate metabolites.

[0004] In addition, hyaluronidase is a hydrolase of hyaluronic acid, a biological membrane component present in various places such as lysosomes, testes and skin of various organs of higher animals, and its physiological significance is still unknown. Although there are many, it is considered that skin roughness and bulkiness occur due to enhanced activity of hyaluronidase which degrades hyaluronic acid, which is a moisturizing component.

Until now, various substances having a tyrosinase-inhibiting action or a hyaluronidase-inhibiting action have been used as agents for whitening and beautifying skin, but few of them are excellent in their action, and many of them are chemically active. It is synthesized by a method, and at present, it cannot avoid various side effects due to its continuous use due to its nature.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a tyrosinase inhibitor and a hyaluronidase inhibitor comprising as an active ingredient an extract from a plant having almost no concern about side effects. .

[0007]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
It is achieved by a tyrosinase inhibitor and a hyaluronidase inhibitor comprising an organic solvent or an aqueous extract of Chapeu de Couro as an active ingredient.

[0008]

DETAILED DESCRIPTION OF THE INVENTION Echinodor
us Macrophyllus Micaeli) has long been known as a plant effective in arthritis, gout, blood purification, nephritis, rheumatism and the like in South America. The plant is a member of the family Omodacae which grows naturally in ponds, swamps and streams, but cannot grow under very dirty water. In the present invention,
Preferably, the leaves reaching a size of about 30 to 70 cm are used for the extraction. Although found by the present applicant (JP-A-6-9416), it is not known at all to have a tyrosinase inhibitory action or a hyaluronidase inhibitory action.

Extraction of the leaves of Chapeu de Couro is carried out using an organic solvent and water (including hot water) as a solvent, if neat or after drying and grinding. Solvent extraction and water extraction are used in combination.
As the organic solvent, methanol, ethanol, n-butanol, acetone, ethyl acetate, chloroform, n-hexane and the like are used. In the case of a water-soluble organic solvent, an aqueous solution thereof is also used.

[0010] Of these extracted components, the tyrosinase inhibitory effect is remarkable in Sample 6 described later, and the hyaluronidase inhibitory effect is remarkable in Sample 7 described later.

The toxicity of each of these extracts is extremely low,
The acute toxicity of oral administration was examined in male Wistar rats, and no death occurred at 3000 mg / kg (po).

These extracts are provided in the form of a medicine or food. When used as a medicament, it is provided in the form of powders, granules, tablets, dragees, capsules, liquids, and the like, and when used as a food, it is provided in the form of gum, candy, jellies, tablet confections, beverages, etc. You. When used as a medicament, it is administered by oral administration, parenteral administration, inhalation, rectal administration, topical administration and the like. Parenteral administration includes subcutaneous injection, intravenous administration, intramuscular administration, nasal administration or infusion. The amounts used will generally be within the range of about 10-500 mg / kg body weight per dose, and will usually be administered 1-5 times daily. However, the exact dose is determined from the above range in consideration of the age, weight, symptoms, administration route and the like of the patient.

[0013]

According to the present invention, there are provided tyrosinase inhibitory agents and hyaluronidase inhibitory agents extracted from Chapeu de Couro.

[0014]

Next, the present invention will be described by way of examples.

Example 1 60 g of the dry powder of leaves of Chapeu de Couro was immersed in 1 L of acetone at 40 ° C. overnight, and the solvent was distilled off to obtain 1.30 g of a dark green acetone-eluting fraction (sample 1). obtain.

Example 2 1 L of 95% methanol was used as the residue after extraction with acetone in Example 1.
Then, 7.23 g of a brown powder (sample 2) is obtained by distilling off the solvent.

Example 3 The residue obtained after 95% methanol extraction in Example 2 was extracted with 1 L of hot water, and the aqueous solution was concentrated under reduced pressure to obtain 12.11 g of a brown powder (sample 3).

Example 4 The procedure of suspending 7.0 g of the sample 2 in 100 ml of water and distributing between solvents with 50 ml of n-butanol was repeated twice, and then the solvent was distilled off from the n-butanol extract. Then, 1.80 g of a brown powder (sample 4) is obtained. The aqueous solution is concentrated under reduced pressure to obtain 5.10 g of a light brown powder (sample 5).

Example 5 11.0 g of the sample 3 was dissolved in 175 ml of water, and 325 ml of methanol was added little by little with stirring. The resulting precipitate is centrifuged, and the obtained supernatant is concentrated under reduced pressure to obtain 8.84 g of a light brown powder (sample 6). When the precipitate is dried, 1.99 g of a brown powder (sample 7) is obtained.

The following tests were conducted on the above Samples 1 to 7 in order to confirm that these fractions had a tyrosinase inhibitory action and a hyaluronidase inhibitory action.

Tyrosinase inhibitory activity test: 200 μl of 0.2 M phosphate buffer (pH 6.8) was mixed with each sample dissolved in 180 μl of the same buffer, and 20 μl of a tyrosinase enzyme solution was added, followed by shaking at 35 ° C. for 10 minutes. I'm sorry. Thereafter, 20 μl of a tyrosine solution adjusted to a concentration of 0.3 mg / ml was added, and the change in absorbance at a wavelength of 475 nm was measured for 10 minutes with a spectrophotometer, and the amount of the reaction product was determined as the amount of change for 1 minute. Then, the IC ₅₀ value was calculated as the concentration at which each sample exhibited 50% tyrosinase inhibitory activity with respect to the blank control, and the results shown in the following Table 1 were obtained. Table 1 Sample IC ₅₀ (mg / ml) 1 4.6 2 0.8 3 0.3 4 0.22 5 0.71 6 0.15 7 1.20

Hyaluronidase inhibitory test: 1 mg of each sample suspended in 200 μl of 0.1 M acetate buffer (pH 3.5) and 0.7 mg
After mixing with 200 μl of a 5 M sodium chloride solution (prepared with the same buffer), 100 μl of hyaluronidase (2400 units / ml) derived from sheep testis was added thereto, and the mixture was shaken at 37 ° C. for 20 minutes. Thereafter, 500 μl of a potassium hyaluronate solution (concentration: 1.2 mg / ml) prepared with a 0.1 M acetate buffer was added, and an enzyme reaction was carried out at 37 ° C. for 40 minutes. After stopping the enzyme reaction by adding 180 μl of 0.4N sodium hydroxide aqueous solution, the reaction solution 500 μl
Was mixed with 100 μl of 0.8 M potassium tetraborate solution (pH 9.1) and heated at 100 ° C. for 2-3 minutes. After cooling with water, add 3 ml of a 1% dimethylbenzaldehyde solution as a coloring reagent, and add
The reaction was performed at 7 ° C. for 20 minutes. The amount of N-acetylglucosamine generated in the enzymatic reaction was quantified by measuring the absorbance of the reaction solution at a wavelength of 544 nm with a spectrophotometer, and the percentage of the hyaluronidase inhibitory activity of each sample with respect to the control group was determined. Calculated to obtain the results as shown in Table 2 below.

[0023]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 43/00 101 A61P 43/00 101 (56) References JP-A-6-9416 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) A61K 35/78 A61K 7/00 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (2)

(57) [Claims] 1. A tyrosinase inhibitor comprising, as an active ingredient, an organic solvent or an aqueous extract of Chapeu de Couro. 2. A hyaluronidase inhibitor comprising, as an active ingredient, an organic solvent or an aqueous extract of Chapeu de Couro.