JP2006062989A - Elastase activity inhibitor or maillard reaction suppressant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a Maillard reaction inhibitor effective as a wrinkle-formation suppressing agent, or to obtain an elastase activity inhibitor. <P>SOLUTION: The extracts of Phyllanthus emblica are effective for inhibiting elastase derived from dermal fibroblast and suppressing Maillard reaction. Several cutaneous aging symptoms such as wrinkles, skin roughening and skin somberness are found to be improved by compounding the extracts, with one or more of specific agents of humectants, cell activators, antiphlogistics, antioxidants, hyaluronidase inhibitors, antiplasmin agents, active oxygen suppressing agents, collagenase activity inhibitors, antihistaminic agents and elastase activity inhibitors. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

In the present invention, an extract extracted from a plant of a specific genus has an elastase inhibitory activity and a Maillard reaction inhibitory effect on fibroblasts of skin dermis, and a moisturizer, cell activator, anti-inflammatory agent, antioxidant By combining combinations of specific agents such as agents, hyaluronidase inhibitors, antiplasmin agents, active oxygen inhibitors, collagenase activity inhibitors, antihistamine agents, and elastase activity inhibitors, various skin conditions such as rough skin, wrinkles, sagging, dullness, etc. It relates to Item 2 elastase activity inhibitor or Maillard reaction inhibitor which improves aging symptoms.

Amla (Phyllanthus emblica) is a plant that belongs to the Euphorbiaceae family. The place of origin is India, Sri Lanka, and Malaysia, and it is a small tree that grows on sparse forests and mountain slopes and is widely distributed in regions such as southern China. The fruit is known for its high medicinal properties as one of the ancient medicine Ayurveda's Tri-Para (Tri-fruit Mix), and it is popular as the name of Amla. Its medicinal properties are known to be effective for chronic cough and lung disease, and to strengthen liver function.

The extract of Amla used in the present invention is recognized as an effect as a health food or an external preparation for skin (see Patent Document 1 for whitening and Patent Document 2 for anti-aging), but other effects Was not known.

The composition of the skin is roughly classified into three, and is formed from the outer layer from the epidermis, dermis, and subcutaneous tissue, each of which plays various roles such as maintaining the body's homeostasis and protecting from the outside world. The function of the skin dermis serves as a cushion that absorbs impacts from the outside world. If the cushion effect, that is, the thickness of the dermis and the flexibility are lost, wrinkles and sagging occur.

The dermis is mainly composed of collagen, elastin, and fibroblasts, and deterioration, decrease or degeneration of collagen, elastin causes aging symptoms such as disappearance of wrinkles, wrinkles and sagging . In healthy dermis, old collagen and elastin are absorbed and excreted, while new collagen and elastin are produced from fibroblasts, constantly maintaining a constant quality and quantity. However, with increasing age, the homeostasis of external and internal factors breaks down, and elastase derived from fibroblasts degrades elastin more than necessary, and the chemical bonds between the proteins and sugars that make up the dermal matrix. It is promoted by an abnormal crosslinking reaction (Maillard reaction).
Japanese Patent Laid-Open No. 12-615019 Japanese Patent Laid-Open No. 13-277716

The object of the present invention was to find an effective safe and effective substance as an elastase activity inhibitory effect and a Maillard reaction inhibitor, and to make various improvements in order to improve wrinkles and effectively utilize them.

In order to achieve the above-mentioned object, the present inventors have intensively studied and found that the effect is high on an extract of Amla (Phyllanthus emblica). In addition, certain drugs (humectants, cell activators, anti-inflammatory agents, antioxidants, hyaluronidase inhibitors, antiplasmin agents, active oxygen inhibitors, collagenase activity inhibitors, antihistamine agents, elastase activity inhibitors, etc.) are added. It was found that various skin aging symptoms such as wrinkles, rough skin, and dull skin were improved.

The form of the extract of Amla (Phyllanthus emblica) is not limited in any way, but preferably the whole Amla plant is shade-dried, ground, and then extracted with an extraction solvent (for example, alcohol such as ethanol, water or a mixture thereof). Use the filtered solution after immersion for 1 day or heating and cooling at 100 degrees for 1 hour.

Fibroblast-derived elastase inhibitory activity test Human-derived fibroblasts (NHDF) were planted in a 100 mm diameter petri dish, cultured until confluent, and then replaced with serum-free MEM medium containing 0.4 ng / mL IL-β. Incubate for 48 hours. After washing the cells twice with PBS (-), the cells are detached with cell scraper, dispersed in 1.5 mL of 100 mM Tris buffer (pH 8.3) containing 5 mM CaCl2, 0.1% TritonX, and sonicated and centrifuged at 10,000 rpm. The supernatant was used as an enzyme solution.
Enzyme reaction was carried out by placing 89 μL of enzyme solution in an Eppendorf tube and 10 μL of a test product dissolved in the above buffer (final concentration 0.01%), preincubating at 37 ° C., and then substrate (100 mM Suc-ala-ala-ala-pNa-N). -Methyl-2-pyrrolidone) 1 μL was added and reacted at 37 ° C. for 2 hours. After stopping the reaction with 50 μL of acetic acid, the reaction solution was centrifuged, and the resulting supernatant was transferred to a plate reader and the absorbance at 405 nm was measured.
The test products are shown below, and the test results are shown in Table 1.
Sample1: Amla (Phyllanthus emblica) whole plant (dried) (10 g) hot water (200 mL) extract
Sample2: Amla (Phyllanthus emblica) whole plant (dried) (10 g) 50% ethanol-water (200 mL) extract
Sample3: Amla (Phyllanthus emblica) whole plant (dried) (10 g) ethanol (200 mL) extract
Sample 4: Hot water (200 mL) extract of Phyllanthus urinaria (10 g)
Sample 5: Hot water (200 mL) extract of Menilan (Phyllanthus s niruri L.) (10 g)
Sample6: EDTA 10mM

Maillard reaction inhibitory test Maillard reaction inhibitory action was investigated about each extract by the NBT reduction method which measures the ketoamine which is an intermediate of the Maillard reaction. 100 μl of purified water and 500 μl of 0.1M phosphate buffer (pH 6.0) were added to 50 μl of a test product prepared with water, and 100 μl of 2M glucose aqueous solution was added. Further, 200 μl of 4 mg / ml BSA aqueous solution was added, stirred, and incubated at 60 ° C. for 30 hours. After 30 hours, add 2.7 ml of 0.2 mg / 0.9 ml [phosphate buffer (pH 10.3)] NBT solution to 300 μl of the reaction solution, incubate for 30 minutes at 37 ° C, and measure the absorbance (A) at Abs. did. Similarly, the absorbance (B) when water is used as an alternative to an aqueous BSA solution, the absorbance (C) when water is used as an alternative to a test product, water as an alternative to an aqueous BSA solution, and water as an alternative to a test product Absorbance (D) when used was measured, and Maillard reaction inhibition rate (%) was calculated from the following formula.
Maillard reaction inhibition rate (%) = [1- (AB) / (CD)] × 100
The test products are shown below, and the test results are shown in Table 2.
Sample1: Amla (Phyllanthus emblica) whole plant (dried) (10 g) 50% ethanol-water (200 mL) extract
Sample 2: Amla (Phyllanthus emblica) whole plant (dried) (10 g) ethanol (200 mL) extract
Sample 3: Hot water (200 mL) extract of whole plant (dried) (10 g) of Phyllanthus urinaria
Sample 4: hot water (200 mL) extract of Menilan (Phyllanthus s niruri L.) (10 g)

It is desirable that the whole extract of Amla (Phyllanthus emblica) be blended at 0.000001 to 20% by weight as a dry product.
Furthermore, certain drugs in the extract of Amla (humectant, cell activator, anti-inflammatory agent, antioxidant, hyaluronidase inhibitor, antiplasmin agent, active oxygen inhibitor, collagenase activity inhibitor, antihistamine agent, elastase activity inhibitor Etc.) was found to improve various skin aging symptoms such as wrinkles, rough skin, and firmness of the skin.
Listed in particular are the whole plant of morning glory, Asam wood, Adhatoda vasica wood, Yellow Merchant wood, Ixcate (Hippocratea celastroides) bark, Ibonashi ヅ rafu branch, Indian salsa root, bark of neem, Woodfordia fruticosa flowers and leaves, lumberwood, lagerstroemia speciosa leaves, CASCA-DOCE (CASCA-DOCE) (scientific name Pradosia lactescens or Lucumaglyciphloea Or Chrysophyllum buranhem), wig, kapool, stems and branches of the burdock, genus Kawaraketsume (Kemeko, Senna, Wangjiang South, mountain pea, Nanbansaikachi), whole spinach, cinnamon blossom, Cuachalalate (Cuachalalate) Juliania adstringens Schl) bark, guava leaf, Quercus pidankrate Quercus pedunculate fruit, cumin seeds, cubraco heartwood, coustigaya rhizome, costillas (Acalypha diversifolia Jacq.) Trunk and branch, bark of melanoleuca leucadendron, Gordolobo (Gnaphalium) sphacellatum), saffron pistil, Saraka indica bark, JEQUITIBA (Carinianabrasiliensis or Couratari legalis or Pyxidaria macrocarpa) bark, Shimoniro whole grass, Jatoba (Hymenaea couba) Bark of Eugenia jambolana, seeds of white-spotted morning glory, whole plant of Sphaeranthus indicus, bark of spruce, root of Smilax zeylanica, scepter wood, bark of barb tree, chaba Pepper root, Desmodium gangeticm Whole plant, Terminalia horrida fruit, Tragia involucrata whole plant, Nigella sativa seed, Herbushi whole plant, Hierba del sapo (Eryngium comosum) whole plant, Hibatsu root, Whole plant of Pirul (Schinus molle), whole plant of Phyllanthus nuriri, whole plant of Fusenkazura, PUCA (Cissus antiparaliticus), root of vetiver, Hedesalum austrocibilix (Hedysarum) austrosibiricus root, Herpestris monnies whole grass, VERONICA (Veronica officinalis) bark, Borrelia hispida root and stem, Pongamia glabra root, Mizuki bark , Mimusops elengi whole plant, Murraya koenigii shoot, Munronia Whole plant of mira, wood of melapie, bark of magnoliaceae genus genus, mountain cherry, lumbo, rumex cyprius leaf, wattle bark, aitamago, one root, fateless, fossil grass , Shellfish mother, intruder, Iwaben Kei root, Paeonia anomala root, chestnut butter, chestnut leaf, chicken blood wisteria, Saya, messenger, small gold crab, cinnamon bark, red spicy rice, Nishihagi, Ishirenko, Ishibuchi root bark , Stone persimmon skin, stone persimmon bark, sanctuary grass, pierced stone, persimmon, valley essence, Chinese white birch, iron wrapping, winter lion, persimmon leaves, Hishimi, Kashio grass, Ryo Wang, lotus leaf , Eggplant, cormorant sea, toothpick, coconut tree, samurai, sea urchin, seaweed of Ishigeaceae; pearl powder, conchiolin, conchyolin hydrolyzate, conchiolin hydrolyzate treated with succinic anhydride, shellfish Substances obtained by hydrolyzing mucus, sterols obtained from shellfish meat, acidic polysaccharides obtained from shellfish meat Collagen obtained from shellfish meat, glycogen obtained from shellfish meat, phospholipid obtained from shellfish meat, deoxyribonucleic acid obtained from shellfish meat, thiosalicylic acid and its salt, magnolol, honokiol, 1- (4 -Hydroxyphenthio) -2-propanol, 2- (4-hydroxyphenyl) -1,3-dithiane, 2- (4-acetoxyphenyl) -1,3-dithiane, 2,5-dihydroxybenzamide, 2,6 -Dihydroxybenzamide, 4-aminophenol, 4-hydroxythiophenol, 4-hydroxybenzamide, 5-hydroxyindole, 6-dihydroxy) -dihydrobenzofuran-3-yl- (3'4'-dihydroxy) -phenyl ketone, stilbengalo Ile glycoside, citracin C, ethylenediamine derivative, carahanaenone derivative, cashew nut shell oil, carda A peptide containing the sequence of nord, cardol, anacardic acid, sennoside, α-arbutin, Glu-Glu-Met-Gln-Arg-Arg, or the terminal of the carboxyl group of this peptide is converted to an amide or / and the amino group is acylated Peptides converted into isomers are mentioned.
Examples of ethylenediamine derivatives include N-phenyl-N- (2,3,3 trimethyl-1-cyclopentene-1-ethyl) -N ′, N′-dimethylethanediamine, N-2-pyridyl-N- (3 , 7-Dimethyl-6-octen-1-yl) -N ', N'-dimethylethanediamine, N-2-pyridyl-N- (2-pinen-10-yl) -N', N'-dimethylethane Mention may be made of diamines, N-2-pyridyl-N- (1,8-p-menthadien-7-yl) -N ', N'-dimethylethanediamine.
For example, N-p-hydroxyphenyl-3,3,6-trimethyl-5-cycloheptan-2-one-1-carboxamide, 2-hydroxy-Np-hydroxyphenyl-3,3 , 6-trimethyl-5-cycloheptene-1-carboxamide. Examples of stilbene galloyl glycosides include 3,5,4'-trihydroxystilbene 4'-0-β-D- (6 "galloyl) glucopyranoside, 3,5,4'-trihydroxystilbene 4'- Mention may be made of 0-β-D- (2 ″ galloyl) glucopyranoside.

Furthermore, it has also been found that the effect can be further obtained by blending one or more of polyhydric alcohols containing two or more hydroxyl groups, lower alcohols, surfactants, and low molecular betaines.

The polyhydric alcohol that can be used is not particularly limited, and examples thereof include glycerin, 1,3 butylene glycol, propylene glycol, isoprene glycol, sorbitol, trehalose, maltose and the like. These are blended in an amount of 0.1 to 80% by weight, preferably 1 to 30% by weight. Needless to say, the blending amount varies depending on the type of polyhydric alcohol, and a plurality of polyhydric alcohols can be used.

Absorbability improves by mix | blending a lower alcohol. Although there are various kinds of lower alcohols, ethanol is usually used. The blending amount is not particularly limited but is preferably 2 to 70% by weight.
It is also effective to add a surfactant to a combination of polyhydric alcohols containing an extract of Amla (Phyllanthus emblica), and it is also effective to add a lower alcohol.
There is no particular limitation on the surfactant, and the type and amount may be selected depending on the application and dosage form.
Surfactants are used for emulsification and solubilization of oils, etc., and are anionic (alkyl carboxylates, alkyl sulfonates, alkyl sulfate esters, alkyl phosphate esters), nonionic (ether type) Nonionic surfactant, ether ester type nonionic surfactant, ester type nonionic surfactant, block polymer type nonionic surfactant, nitrogen-containing type nonionic surfactant) and amphoteric (carboxylic acid type amphoteric surfactant) Agents (amino type, betaine type), sulfate ester type amphoteric surfactant, sulfonic acid type amphoteric surfactant, phosphate ester type amphoteric surfactant) and other surfactants (natural surfactants, protein hydrolysates) Derivatives, polymer surfactants, surfactants containing titanium and silicon, and fluorocarbon surfactants) can be used as appropriate.

Low molecular weight betaine is also one of the raw materials to be selected, and by blending this, the effect is further improved and the feeling of application is improved, and the effectiveness as an external preparation for skin is increased.
Low molecular weight betaines that can be utilized may include those that form zwitterions with internal salts such as quaternary ammonium bases, sulfonium bases, phosphonium bases and the like having a molecular weight of 200 or less. Examples include trimethylglycine, N, N, N-trimethylalanine, N, N, N-trimethylglutamic acid, triethylglycine, and the like, with trimethylglycine being preferred.
Those having a molecular weight of 200 or more have a surface-active ability, which is different from the effect of low molecular betaine. As described above, the use of a surfactant enhances the effect of the present invention, but the effect of low molecular betaine is Is different.

The external preparation for skin of the present invention can be prepared by blending with various forms of base materials known as normal external preparations for skin according to conventional methods.

It does not specifically limit as a form of an external preparation, For example, arbitrary dosage forms, such as a milky lotion, cream, aqueous solution, a pack, can be selected.

In the external preparation for skin of the present invention, in addition to the above-mentioned essential components, components that are blended in normal external preparations, such as oils, powders, purified water, polymer compounds, gelling agents, UV absorbers, UV scattering Agents, antioxidants, pigments, preservatives, fragrances, and cosmetic ingredients can be appropriately selected and used within a range not impairing the effects of the present invention.

EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not restrict | limited at all. Moreover, the extraction method about the extract of the used chemical | medical agent is not limited at all.
The formulations of the examples are shown in Tables 3 to 5. The preparation method was performed by a conventional method. Table 3 shows the lotion, Table 4 shows the serum, and Table 5 shows the cream. The amount is shown in parts by weight.

(Note 1) Amla extract 1 was prepared by adding 200 ml of purified water to 10 g of Amla whole plant (dried product) and boiling for 1 hour, followed by filtration.
(Note 2) Amla extract 2 was prepared by adding 100 ml of purified water and 100 ml of ethanol to 10 g of Amla whole plant (dried product) and allowing to stand for 10 days, followed by filtration.
(Note 3) As Amla extract 3, 200 ml of ethanol was added to 10 g of Amla whole plant (dried product) and allowed to stand for 10 days, and then the filtered extract was used.
(Note 4) The hydrolyzed conchiolin solution used was a hydrolyzed conchyolin that had a molecular weight of 1000 or less.
(Note 5) As the tablinus extract, a Matils extract [manufactured by Maruzen Pharmaceutical Co., Ltd.] was used.
(Note 6) Falco Rex Honoki E [Ichimaru Falcos Co., Ltd.] was used as the honoki extract.
(Note 7) As the guava extract, 300 ml of ethanol was added to 10 g of guava leaves and allowed to stand for 5 days, and then the filtered extract was used.
(Note 8) Nishi-Haruyanagi extract used Nishi-Haruyanagi extract [Nippon Seika Co., Ltd.].
(Note 9) Marine purge [manufactured by Ichimaru Falcos Co., Ltd.] was used as the seaweed extract.
(Note 10) As the acacia bark extract, 300 ml of ethanol was added to 10 g of bark and left for 5 days, and then the filtered extract was used.
(Note 11) The Murayako Engii extract used was the Murayako Engii extract [Yamakawa Trading Co., Ltd.].
(Note 12) As for the extract of the giant crape myrtle, the extract of the giant crape myrtle (produced by Yamakawa Trading Co., Ltd.) was used.

The skin beautification effect tests of Examples 1 to 12 and Comparative Examples 1 to 3 described in Tables 1 to 3 were performed. The test method consisted of 20 females aged 25 to 55 years old, and a suitable amount of a topical preparation was applied to the face after washing the face twice a day in the morning and night for 12 weeks. Table 6 shows the results of the skin beautifying effect by application.

From the results shown in Table 6, an extract of Amla (Phyllanthus emblica) having a fibroblast-derived elastase activity inhibitory effect and a Maillard reaction inhibitory effect and a specific drug (humectant, cell activator, anti-inflammatory agent, antioxidant, hyaluronidase inhibitor) 1 type or 2 or more types of agents, antiplasmin agents, active oxygen inhibitors, collagenase activity inhibitors, antihistamines, elastase activity inhibitors, etc., prevention, reduction and improvement of rough skin, skin firmness and wrinkles The effect became more effective. Furthermore, it turned out that the said effect increases synergistically.

Claims (5)

Elastase activity inhibitor characterized by containing an extract of Amla (Phyllanthus emblica) Maillard reaction inhibitor characterized by containing an extract of Amla (Phyllanthus emblica) Phyllanthus emblica extract and moisturizer, cell activator, anti-inflammatory agent, antioxidant, hyaluronidase inhibitor, anti-plasmin agent, active oxygen inhibitor, collagenase activity inhibitor, antihistamine agent, elastase activity inhibitor Or one or more skin external preparations Moisturizer, cell activator, anti-inflammatory agent, antioxidant, hyaluronidase inhibitor, antiplasmin agent, active oxygen inhibitor, collagenase activity inhibitor, antihistamine, elastase activity inhibitor Bark of Adhatoda vasica, bark of yellow ranch, Ixcate (Hippocratea celastroides) bark, Ibonashi tsumugi rakuji branch, Indian salsa root, bark of neem, Woodfordia fruticosa (Woodfordia fruticosa) Flowers and leaves, lumberwood, lagerstroemia speciosa leaves, CASCA-DOCE (Pradosia lactescens or Lucumaglyciphloea or Chrysophyllum buranhem), wigs, kapoor, stalks and branches, Kawarataketsugen (Kemeko, Senna, Noboru Minami, Yamashirazu, Namban Sai Kachi , Whole spinach, cinnamon blossom, Cuachalalate (Juliania adstringens Schl) bark, guava leaf, Quercus pedunculate fruit, cumin seed, cubraco heartwood, cous mussel rhizome, costillus ) (Acalypha diversifolia Jacq.) Trunk, branches, bark of Melaleuca leucadendron, Gordolobo (Gnaphalium sphacellatum) whole grass, saffron pistil, Saraka indica bark, Jekiba Bark of JEQUITIBA (Carinianabrasiliensis or Couratari legalis or Pyxidaria macrocarpa), whole plant of Simonil, bark of JATOBA (Hymenaea coubaril), bark of Jugeboran (Eugenia jambolana), seed of white butterfly, anthracnose indicus), Spruce bark, Sumilac Roots of Smilax zeylanica, scepter wood, bark of barb, pepper root, whole plant of Desmodium gangeticm, fruit of Terminalia horrida, whole of Tragia involucrata , Nigella sativa seeds, whole-bellied grass, Hierba del sapo (Eryngium comosum) whole plant, hi-hat root, Pirul (Schinus molle) whole plant, Phyllanthus nuriri whole plant , Whole plant of Fusenkazura, PUCA (Cissus antiparaliticus), root of vetiver, root of Hedysarum austrosibiricus, whole plant of Herpestris monnies, VERONICA ( Veronica officinalis) bark, Borrelia hispida root and stem, Pongamia glabra root, Mi Bark of bark, bark of earthworm, whole plant of Mimusops elengi (Mimusops elengi), shoot of Murraya koenigii, whole plant of Munronia pumila, bark of melody wood, bark of magnoliaceae, wood of Yamazakura, Rambo timber, Rumex cyprius leaves, Watle bark, Aitamago, Ichione root, Wang Shio, Fossil grass, Shellfish, Intruder, Iwabenkei root, Paeonia anomala root, Chestnut , Chestnut leaves, chicken blood wisteria, Saya, messenger, small gold cocoon, crust, spicy, Nishikawa willow, Ishirenko, sarcophagus bark, sarcophagus bark, sarcophagus bark, sanctuary, perforated stone, persimmon, valley essence , Chinese white camellia, iron wrapping, winter eggplant, cocoon leaf, Hishimi, Kashiwaso, Ryoo Wang, lotus leaf, coconut tree, 胖大海, 茘 枝, lion, Saito, 鵝 不 鵝 草, Seaweed of Ishigeaceae; pearl powder, conchiolin, conchiolin hydrolyzate, hydrolysis of conchiolin Substances obtained by treating succinic anhydride with products, substances obtained by hydrolyzing shellfish mucus, sterols obtained from shellfish meat, acidic polysaccharides obtained from shellfish meat, collagen obtained from shellfish meat, shellfish meat Glycogen obtained from phospholipid obtained from shellfish meat, deoxyribonucleic acid obtained from shellfish meat, thiosalicylic acid and its salt, magnolol, honokiol, 1- (4-hydroxyphenythio) -2-propanol, 2- (4-hydroxyphenyl) -1,3-dithiane, 2- (4-acetoxyphenyl) -1,3-dithiane, 2,5-dihydroxybenzamide, 2,6-dihydroxybenzamide, 4-aminophenol, 4 -Hydroxythiophenol, 4-hydroxybenzamide, 5-hydroxyindole, 6-dihydroxy) -dihydrobenzofuran-3-yl- (3'4 '-dihydroxy) -phenyl ketone, stilbenegaloyl glycoside, citracin C, ethylenediamine derivative, caranahanaone derivative, cashew nut shell oil, cardanol, cardol, anacardic acid, sennoside, α-arbutin, Glu-Glu-Met-Gln- The elastase activity inhibitor according to claim 1 or 2, which is a peptide comprising the Arg-Arg sequence, or a peptide in which the carboxyl group terminal of this peptide is converted to an amide form and / or the amino group terminal is converted to an acylated form. Maillard reaction inhibitor The elastase activity inhibitor or Maillard reaction inhibitor skin of claim 1, further comprising one or more of polyhydric alcohols, lower alcohols, surfactants, and low molecular betaines containing two or more hydroxyl groups. Topical preparation