TW201028157A - Extracts and compounds for inhibiting tyrosinase activity - Google Patents

Abstract

The invention provides a Pyracantha koidzumii extract and new compounds isolated from the extract and their derivatives. This extract and these compounds have activity in inhibiting tyrosinase.

Description

201028157 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to Taiwanese spurt wood extract and novel compounds and derivatives thereof isolated from the extract. The extract and the compounds have activity to inhibit the activity of a cool acid enzyme. In particular, the present invention relates to an ethyl acetate extract of Taiwan spurs. [Prior Art] Melanin is a black pigment in hair and skin and is synthesized from amino acid tyrosine by melanosome. The melanosome is a organelle in melanocytes, a cell located at the junction of the dermal-epidermal junction. Melanin plays an important role in protecting the body from UV damage. Melanin is also very important in medicine and cosmetology. The biosynthetic pathway of melanin involves the catalyzed hydroxylation of tyrosine by tyrosinase to L-3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA Dopachrome 〇 lysinase (EC 1.14.18.1) is a copper-containing monooxygenase that is widely distributed in nature. Its basic metabolic function is to catalyze the oxidative degradation of amino acid tyrosine. This degradation is slightly different in animal, plant and microbial routes, but the rate-controlled first step of a tyrosinase catalysis is the same in all life species. In animals 'including humans, tyrosinase first converts tyrosine to 3,4-dihydroxyphenylalanine (dopa, d〇PA), which then becomes the corresponding brew (DOPAquinone), and then Conversion to 2-sulfo-2,3-dipyridin-5,6-stuff; (DOPAchrome), which is further converted from other enzymes to more oxidative substances' including melanin substances that cause skin pigmentation. 136342.doc 201028157 Over-formation of melanin after prolonged exposure to sunlight or erythema, sunburn, dark spots, freckles and pigmented cosmetic dermatitis due to melanin unit disease of the epidermis. There have been several reports on the use of tyrosine inhibitors (such as hydroquinone and its derivatives, ferulic acid, catechins, mercaptoamine, alpha acid, etc.) in cosmetic or pharmaceutical compositions. To regulate skin pigmentation. Taketsugu Tadokoro pointed out that glutamate is the most important enzyme for synthetic pigments, and its amount shows a response to UV (J Invest Dermato 124: 1326-1332, 2005). Medical experts have accepted the relationship between melanoma and inhibition of tyrosinase. Therefore, the development of an agent that modulates the activity of tyrosinase enzymes would be of great value in controlling the above-mentioned unwanted skin conditions (Hideya Ando et al., Journal of Investigative Dermatology (2007) 127, 751-761). The pharmaceutical, cosmetic and food industries and other similar industries have a need to develop agents that inhibit tyrosinase and prevent and treat symptoms caused by undesired tyrosinase activity. Non-toxic natural products used in cosmetics and pharmaceuticals are important considerations. Firetorn (Pyracantha) is a spiny evergreen shrub of Rosaceae and Apple subfamily. It is native to Southeast Europe to Southeast Asia and is closely related to the Cotoneaster, but has a mineral tooth margin and a majority of thorns. A. Falodun et al. pointed out that the active ingredient present in P. leaf extract may be a strategy for abortion and menstrual pain in traditional medicine (Pak J Pharm Sci. 2005 Oct; 18 (4): 31-5). Otsuka Η et al. reveal that the scallops (corpse yraea / ii / m creww / αία) have anti-inflammatory effects (Chem Pharm Bull (Tokyo). 1981 Nov; 29 (11): 3099-104) ° CN 1765912 reveals the mainland The extract of Pyracantha/oriwweflwa), 201028157 sterol, ethanol, propanol, butanol, acetone or a mixture thereof can remove oxygen free radicals. W〇013597U^ shows an oral bleaching composition containing a solvent extract of the genus Rosaceae and its application, wherein the water extract/ethanol extract of the Pyracantha fortune can effectively inhibit tyrosinase . The Japanese Patent Publication No. 05_05887A provides an extract of water, methanol, ethyl alcohol, propanol or a mixture thereof of the genus Rosaceae, which has a whitening effect. Furthermore, Jiang et al. found that the extract of Taiwanese firewood Aro/i/zMwn·) has lower toxicity and higher cell glutamate inhibitory activity. Other studies have indicated that Taiwan fire spurs can effectively inhibit tumors (Cancer Res. 1966; 26Β (11): 1302-453) and mainland firethorns have a blood-clotting effect (Zhong Yao Cai 2001; 24 (12): 874-6). The components of the genus Firefly, including carotenoids, flavonoids, glycosides, and sterol derivatives, have been isolated. In particular, Dai Y et al. found a tyrosinase-inhibiting biphenyl glycoside such as 3,3'-dihydroxy-5'-methoxy-(1,1'-phenyl)-4-oxime-0- (1,3'-dihydroxy-5'-methoxy-(l,l'-biphenyl)-4-0·beta-d-glucoside), 41-hydroxy-2,3·,5· -Trimethoxy-(1,Γ-biphenyl)-2'-Ο-β-d-glucophene (Uydroxydjy-trimethoxy-dr-biphenyG-Z'-O-beta-d-glucoside), 4'- Dimethoxy-(1, fluorene-biphenyl)-2-0-β-ί1-grass bitter (4'-]1)^(11*〇乂}/'-3'.,5'- (1111161;11〇\>^-(l'l'-biphenyD-SO-beta-d-glucoside),]〆'-:^*-]1〆-: 曱oxy-(1,1'- Biphenyl)-3-〇-0_(1-glucose (2,4'-£1111丫(11:〇乂7-3',5'-dimethoxy-(l,l,biphenyl)-3-0- Beta-d-glucoside) and 3,4·-dihydroxy-3',5|-dimethoxy-(1,1'-biphenyl)-4-〇-0-(1-glucoside (3, 4|-dihydroxy-3,,5,-dimethoxy-(l, l'-biphenyl)-4-0-beta-d- 136342.doc 201028157 glucoside) (J. Nat. Prod. 2006;69(7): 1022-1024). However, there is no Taiwanese firewood extract described in any of the above prior art documents showing excellent cheese Acidase inhibitory activity. There is still a need for a tyrosinase inhibitor which can effectively inhibit the activity of glutamate and can be used for a long time in human skin without side effects. SUMMARY OF THE INVENTION The present invention finds an acetic acid extract of Taiwanese spurt wood. The ethyl ester separated fraction and the two new compounds present in the fraction have a guanylase inhibitory activity. By inhibiting the tyrosinase' the isolated fraction and the compounds are effective for bleaching the skin and for preventing and treating undesired Symptoms caused by tyrosine activity. The present invention provides a Taiwanese spurt wood extract obtained by extracting Taiwan spurs from ethanol to obtain an ethanol extract and extracting the ethanol extract with ethyl acetate. The Taiwanese spurt plant (preferably its fruit) is crushed and extracted with ethyl acetate to obtain a crude extract. The crude extract is further extracted with ethyl acetate. Any known extraction technique can be used to prepare the extract of the present invention. The obtained ethyl acetate extract can be further separated by liquid chromatography using solvent extraction. Preferably, the liquid chromatography is high performance liquid chromatography (HpLC). Or reverse phase HPLC and the solvent is methanol or ethanol. According to the present invention, the ethyl acetate extract can be further prepared with a gradient of HpLC (18 column), borrowed from water to sterol, and Need to be washed with a gradient of methanol. According to one embodiment of the invention, 10% to 100% sterol (preferably from about 2% to about 5% by weight) is used for the gradient elution of methanol. More preferably, about 2%, 136, 342.doc 201028157 22% or 50% sterol is used. According to a specific embodiment of the present invention, Taiwanese spurs are extracted with ethanol to obtain an ethanol extract and the obtained ethanol extract is extracted with ethyl acetate. Next, the obtained ethyl acetate extract was separated by HPLC, a gradient of 100% water to 100% methanol to give 7 fractions, and the third fraction was collected and further separated. The third fraction was eluted with a gradient of HPLC, 100% water to 100% methanol to give 8 fractions, and the 5th fraction was collected and further separated. The 5th fraction was separated by HPLC, about 22% methanol to give the new compound, 3,4-dihydroxy-5-methoxybiphenyl)-2'-0-pD-glucopyranoside (3,4). -dihy droxy-5-methoxy biphenyl-2'-O-beta-D-glucopyranoside) (MHL-1). According to another embodiment of the present invention, Taiwanese spurs are extracted with ethanol to obtain an ethanol extract, and the obtained ethanol extract is extracted with ethyl acetate. Next, the obtained ethyl acetate extract was separated by HPLC, a gradient of 100% water to 100% methanol to obtain 7 fractions, and the 5th fraction was collected and further separated. The 5th fraction was eluted by HPLC, 100% water to 100% methanol to give 10 fractions, and the 4th fraction was collected and further separated. The 4th fraction was separated by HPLC, 100% water to 100% methanol to obtain 8 fractions, and the second fraction was collected and further separated. The second fraction was separated by HPLC, 100% water to 100% methanol to obtain 7 fractions, and the first fraction was collected and further separated. The first separated fraction was separated by HPLC, about 50% methanol to give a new compound, 3,6-di-based-2,4-dimethoxy-dibenzof (3,6-dihydroxy-2, 4-dimethoxy-dibenzofuran) (MHL-2) ° 136342.doc 201028157 According to the present invention, two new compounds were isolated and purified from Taiwan sapwood extract. The two new compounds are 3,4-dihydroxy-5-decyloxybiphenyl)-2'-0-0-indole-glucopyranoside (PCT 111^1) and 3,6-dihydroxy-2. 4-Dimethoxy-dibenzofuran (MHL-2). 3,4-dihydroxy-5-decyloxybiphenyl)-2'-〇-0-〇-glucopyranoside

3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (MHL-2)

According to the present invention, a compound isolated from Taiwan sapwood extract can be further modified to give a compound having the following formula:

among them

Xi&X3 is independently OH, Ο-lower alkyl, NH2, NH-lower alkyl, 136342.doc -8- 201028157 SH or S-lower grade; X is OH; and X4 is < glucosinolate , galactoside, μ mannose (four), „ 核 核 核 & 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 It is a pharmaceutically or cosmetically acceptable salt. f^Xn_^%Y/R4

among them

Ri is OH; and R2, R3 and β·4 are each independently OH, OC-lower alkyl, book 2, NH-lower alkyl, SH or S-lower alkyl; and pharmaceutically or cosmetically acceptable salts thereof . The present invention unexpectedly finds that the compound of the formula (1) having a hydroxyl group at the 3rd and 5th positions of the biphenyl group has excellent lunase inhibitory activity and the compound of the formula (II) has tyrosinase inhibitory activity. The term "lower alkyl" as used herein denotes a straight or branched saturated hydrocarbon chain of from 1 to 1 atom; preferably, the carbon number of the leuko group is selected from the group consisting of 1 to 8 carbon atoms; more preferably It is a C, .6 alkyl or CN4 alkyl group. Examples of the alkyl group include methyl (-CH3) 'ethyl (-CH2CH3), propyl (-(: Η2(:Η2(:Η3), isopropyl ((CH3)2CH), and butyl (-C4H9)) According to a specific embodiment of the compound of the formula (I) of the present invention, X!, 2 and 3 are preferably OH or OC!-6 alkyl and X4 is glucopyranoside. Preferably, ^ is 136342. .doc 201028157 OCH3; X2 is OH; X3 is OH and X4 is glucopyranoside. According to a specific embodiment of the compound of the formula (Π) of the present invention, Ri, R2, R3 and R4 are each independently OH or OCH3. R1 is OH; R2 is OCH3; R3 is OH and R4 is OCH3. As used herein, the phrase "pharmaceutically or cosmetically acceptable salts" includes those which are formed with or without a base or a base. Or cosmetically acceptable acid addition salts include those with mineral acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid) or carboxylic acids (such as citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid). Forming succinic acid, oxalic acid, citric acid, fumaric acid, malic acid, oxalic acid, decanesulfonic acid, ethanesulfonic acid, P-toluenesulfonic acid, benzenesulfonic acid and isethionate. medical Pharmaceutically or cosmetically acceptable base salts include ammonium salts, alkali metal salts (such as their sodium and potassium salts), alkaline earth metal salts (such as their calcium and magnesium salts), and basic base salts (including primary and secondary). And a tertiary amine. According to a specific embodiment of the compound of the formula (Π), R 2 and R 4 are each independently OH or OCu alkyl. Preferably, R 2 and R 4 are each independently OH or OCH 3 . According to the present invention, the compounds of the formulae (1) and (II) can be prepared by a synthetic procedure known in the prior art. The synthesis of the compound of the formula (1) is shown in the following Scheme 1: Scheme 1:

X1_, X2', X3, trifluoroacetyl protected X1, X2, Χ3 136342.doc -10- 201028157 About Reaction Scheme 1, will be dissolved in non-polar, aprotic solvents (such as benzene) and polar, protic solvents (such as ethanol) A mixture of compounds IB (wherein X4 is a glycosyl group) is added to a solution of a compound of formula IA wherein X, ', Χ2 and Χ3 are protecting groups such as trifluoroethenyl protecting groups. The resulting mixture is reacted at room temperature for 24 hours in the presence of a catalyst such as Pd(PPh3)4 to give compound 1C. Compound 1C was reacted with HOAc at room temperature for 6 hours to remove the protecting group to give a compound of formula (I).

The synthesis of the compound of formula (II) is shown in Scheme 2 below: Flowchart 2:

R4,

R4, r3·

Pd(OAc)2 Ν32〇〇3 DMA, 170°C 6h

R3'

R4 r3

Rf, 13⁄41 trifluoroacetyl protected R1t R2, R3, R4. For Reaction Scheme 2, a compound of formula IIA is dissolved in a non-polar, aprotic solvent (eg CH2C12) at room temperature in the presence of Cu(OAc)2. The reaction was carried out for 24 hours to give a compound of the formula IIC. a compound of the formula IIC wherein hydrazine, RV, RV and R4| are protecting groups, such as trifluoroethenyl protecting groups, in the presence of a catalyst such as Pd(0Ac)2 in a polar, aprotic solvent such as a dimercapto group Reaction in the amine, DMA) gives the compound IID. The compound IID was reacted with HOAc at 136342.doc -11 - 201028157 for 4 hours at room temperature to remove the protecting group to give a compound of the formula (π). Taiwanese spurt wood extract and compounds of formula (I), (Π) and pharmaceutically or cosmetically acceptable salts thereof are generally administered in the form of a pharmaceutical or cosmetic composition, wherein Taiwan sapwood extract or formula (I) The compound/compound of formula (II) is combined with a pharmaceutically or cosmetically acceptable adjuvant, diluent or carrier. The pharmaceutical composition of the present invention can be administered systemically, that is, orally administered in the form of a tablet, a capsule, a syrup, a powder or a granule, or a parenteral administration in the form of a solution or a suspension, or subcutaneous administration, or topical administration, or wearing. The skin is administered. Excessive melanin production or abnormal distribution can cause irregular over-coloration of the skin. In order to develop treatments or preventions that improve or avoid excessive pigmentation diseases such as dark spots and age spots, the goal is usually to destroy the activity of tyrosinase (Journal of Investigative Dermatology (2007), 127: 751-61). The Taiwanese firewood extract and compound have tyrosinase inhibitory activity, so they can be used for skin whitening agents and for preventing and treating symptoms caused by undesired tyrosinase activity, for example, age spots, melanoma , erythema, sunburn, dark spots, freckles and pigmented cosmetic dermatitis. [Examples] Example 1 Extraction of Taiwan fire thorn wood Pyrophanha koidzumii or its fruit was crushed and extracted with 40 ml of 95% ethyl yeast three times. The combined ethanol solution (120 ml total) was reduced. After concentration by pressure, 360 g of a crude extract of 95% ethanol was obtained. The crude extract was taken and suspended in water and extracted with ethyl acetate (EtOAc) to give ethyl acetate. Example 2 Separation of the ethyl acetate (EtOAc) extract and the compound of the invention 136342.doc •12- 201028157 Separation of the substance

The ethyl acetate extract of Example 1 was dissolved in water and separated on a Diaion HP-20 column, eluted with 100% water to 100% methanol gradient, and passed through a thin layer chromatography (TLC plate, CH2C12). / Methanol / Acetic acid = 7 : 1 : 0.1) Analysis, obtained as ΡΚ-ll~PK-1-7 seven parts. Further take ΡΚ-1-3 (1.99 g), ΡΚ-1_4 (1.15 g) and ΡΚ-1-5 (15.42 g) through; C18 chromatography column, with 100% water to 100% methanol gradient, They are divided into eight parts of PK-l-3-l~PK-l-3-8, and eight parts of ΡΚ-1-4-1~ΡΚ-1-4-® 8 and PK-1- 5-1~PK-1-5-10 total ten parts. These separated parts are further separated by different columns. Two new compounds were found, one in the PK 1-3-5 separation portion and the other in the PK-1-5-4 separation portion. PK1-3-5 was separated by high performance liquid chromatography (HPLC) under the condition of - reverse phase (RP), column Biosil 5 ODS-W 10 mmxI.D. 250 mm, mobile phase 22 % methanol, flow rate of 3 ml / min 'detector UV 280 nm separation and purification, 38 minutes to obtain a novel compound • 1^11^-1,3,4-dihydroxy-5-methoxybiphenyl) -2'-〇*^-〇-glucopyranoside; the identification data of this compound is as follows: Amorphous brown powder Md5-1〇.6° (c 0.5, MeOH) UV (MeOH) Xmax (logs): 265 ( 3.74) nm ESI-MS (negtive) m/z 393.1 [MH]- HRESIMS m/z 393.1202[MH]-(calculated for 394.1264) 1H-NMR (500 MHz, CD30D) δΗ 136342.doc •13- 201028157 6.67 ( 1H,d,J =1.8, H-2), 6.80 (1H,d,J=1.8 Hz, H-6), 7 22 (1H,m,H-3'), 7.23 (1H,dd,=8.5 , 1.4 Hz, H-4,), 7.02 (1H m, H-5'), 7·27 (1H, dd, J=8.5, 1.4 Hz, H-6'), 5.03 (13⁄4 d J=7.2, H-l"), 3·43 (1H, m, H-2"), 3.42 (1H, m, H-3",), 3 34 (1H, m, H-4"), 3.44 (1H, m, H-5"), 3.68 (1H, dd, J=5 4 12.0, H-6") 3.86 (1H, dd, J = 2.1,12.0, H-6 "), 3.86 (3H s 3OCH3)

13C-NMR (125 MHz, CD30D) 5C 130.6 (Cl), 111.4 (C-2), 146.0 (C-3), 134.6 (C-4), χ49 χ (C-5), 106.8 (C-6) , 133.0 (C-Γ), 155.4 (Cl), 116·4 (匕3,) 129.0 (C-4'), 123.4 (C-5'), 131.7 (C-6')} 101.8 (Cl') , ?5 Λ (C2,), 78.2 (C3,), 71.3 (C4'), 78.3 (C5,), 62.5 (C6·), 56 8 (3'-OCH3)

PK1-5-4 was firstly extracted by C18 chromatography column with 100% water to i00% methanol gradient and divided into PK-1-5-4-1~PK-1-5-4-8. And then take PK-1-5-4-2 through a C18 chromatography column, with 100% water to 1 〇〇〇 / 0 methanol gradient 'divided into PK-1-5-4-2-1~ PK-1-5-4-2-7 has seven parts. PK-1-5-4-1-1 was separated by high performance liquid chromatography (HPLC) under conditions 136342.doc •14· 201028157 for reverse phase (RP), column Biosil 5 ODS- W 10 mmxI.D. 250 mm, mobile phase 55% sterol, flow rate 3 ml / min, detector RI separation and purification, the retention time 12 minutes to obtain the novel compound MHL-2,3,6-dihydroxy- 2,4-Dimethoxy-dibenzofuran; the identification data of this compound are as follows: Amorphous granule powder UV (MeOH) Xmax (logs): 316 (3.78), 290 (3.91), 263 φ (3.91) Nm ESI-MS (positive) m/z 261.5 [M+H] + HREIMS m/z 260.0687 (calculated for 260.0679) 1H-NMR (500 MHz, CD30D) δΗ 7.21 (1H, s, Hl), 6.82 (1H, m, H-7), 7.08 (1H, m, H-8), 7.33 (1H, m, H-9), 3.91 (3H, s, 2-OCH3), 4.11 (3H, s, 4-OCH3)

13C-NMR (125 MHz, CD30D) 5C ® 98.3 (Cl), 147.4 (C-2), 140.0 (C-3), 134.7 (C-4), 144.6 (C-4a), 146.0 (C-5a) , 144.1 (C-6), 113.5 (C-7), 111.5 (C-8), 124.5 (C-9), 127.9 (C-9a), 117.4 (C-9b), 57.3 (2-OCH3), 61.3 (4-OCH3)

The separation of the ethyl acetate extract is summarized as follows: 136342.doc -15- 201028157

EtOAc layer (25.88 g) Dianion HP 20 gradient (H2OtoMeOH) 7 frs 1 ▼ r ψ PK-1-3 (1.99 g) Ci8 gradient (H20 to MeOH) 8 frs PK-l-4 (1.15 g)

Ci8 Gradient (H2OtoMeOH) 8 frs PK-1-5 (15.42 g) Ci8 Gradient (H2OtoMeOH) 10 firs ” The 6th ft

PK-1-4-1 to PK-1-4-8▼ ★ PK-1-3-5 PK-1-3-1 to Ci8 HPLC PK-1-3-4, 22% MeOH PK-1-5 -4 PK-1-5-1 to

Cjs gradient (H2〇to MeOH) PK-1-5-3 and PK-1-5-4 to PK-1-10 MHL-1 (11 mg) PK-1-,5-4-2 gradient (H2Oto MeOH ) 7 frs. PK-1-5-4-2-1

Ci8 HPLC 50% MeOH MHL-2 (6 mg) 136342.doc 16- 201028157 Example 3 Analysis of tyrosinase activity of human melanocytes Human melanocyte (HEMn) cells were cultured in Medium 254 mixed medium (included) Antibiotics and growth factors) (Cascade Biologies, Inc., Portland, OR, USA) were subcultured in a 5% C02 incubator, and passages from passage 3 to passage 12 were performed. After the HEMn cells were further cultured for 48 hours, the ethyl acetate extract of the present invention and the test compound (MHL-1: 3,4-dihydroxy-5-methoxybiphenyl)-2'-O-β- were added. D-0 is more than o-glucan^: and MHL-2.3,6-di-based-2,4-dimethoxy-dibenzofuran). After 24 hours of reaction, the cells were washed with pbs and lysis buffer (1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 pg/ml aprotinin and 10 pg/ml leupeptin) was added. To obtain cellular proteins, the cell proteins were stored at -80 ° C to be tested. Next, the cell protein was thawed, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was taken for protein quantification (BCA protein assay). Different concentrations of protein were added to a 96-well microplate followed by PBS buffer. Add L-DOPA to the microdisk at 37. (: react with protein for 1 hour. Detect the absorption of the solution at 475 nm. The activity of tyrosine (%) is: (OD475 of sample/〇D475 of control group) xl〇〇. Analysis of the above tyrosinase activity Arbutin was used as a positive control group and 1% DMSO as an experimental blank group. Figure 1 shows that the ethyl acetate extract of the present invention has more than 40% tyrosinase inhibitory activity. Figure 2 shows that the compound of the present invention MHL-2 has Similar to the tyrosinase inhibitory activity of arbutin, but MHL-1 has better activity than arbutin. MHL-1 and MHL-2 inhibit 136342.doc •17- 201028157 respectively, limiting 40% and 20% of tyrosine activity. The ethyl acetate extracts and compounds of the present invention are effective for inhibiting tyrosinase. Example 4 Analysis of tyrosinase oxidation activity by L-DOPA staining by SDS-PAGE gel Different concentrations of the compound of the present invention MHL-1 were added. In the HEMn cells, the supernatant as described in Example 1 was subjected to HEMn cell lysis, cell protein separation, and BCA protein analysis to determine the amount of protein. The resulting protein was mixed with sample buffer (60 mM Tris-HCl, pH 6.8). , 2% sodium dodecyl sulfat e (SDS), 10% glycerol, 0.01% bromophenol blue) 10% SDS-polyacrylamide gel electrophoresis (dd-H20; 10% Acrylamide mix; 1.5M Tris-base pH 8.8; 10% SDS; 10% Ammonium) Persulfate; TEMED) electrophoretic separation. The molecular weight of the lysinase is about 72kDa. After the electrophoresis, the gel is washed with 50 ml of 0.1 PBS (pH 6.8) for about 30 minutes, and then replaced with 0.1 M containing 5 mM L-DOPA. The PBS solution was placed at 37 ° C for 1 hour. After the reaction was completed, the amount of protein was determined by the analysis of Soft Zhao and the sputum by Quantity One 1-D analysis. As shown in Fig. 3, when the concentration of VIHL-1 was higher, The lower the activity of tyrosinase. [Simplified Schematic] Figure 1 shows the expression of tyrosinase inhibitory activity of EtOAc extract in HEMn cells. Figure 2 shows the compounds MHL-1 and MHL-2 of the present invention. The expression of tyrosinase inhibitory activity of HEMn cells. Figure 3 shows L-dopa staining of tyrosinase electrophoresis gel; lane 1: culture 136342.doc • 18 - 201028157 base, road 2: 2.5 arbutin, road 3-5: 100, 80 and 60 μΜ of MHL-

136342.doc -19-

Claims (1)

201028157 VII. Patent application scope: 1. A Taiwanese spurt wood (Pyracantha koidzumii) extract obtained by extracting Taiwan squid from ethanol to obtain ethanol extract and extracting ethanol extract with ethyl acetate. 2. The extract according to claim 1, which further comprises the step of partitioning the ethyl acetate extract by liquid phase precipitation of a solvent gradient. The extract according to claim 1, which further comprises a step of dividing the ethyl acetate extract by liquid chromatography with a C8 to C18 column, which is washed with a gradient of water to methanol. 4. The extract according to claim 3, wherein the separated fraction is further divided by liquid chromatography eluted with a methanol gradient. 5. The extract according to claim 4, wherein 10% to 100% of sterol is used for gradient elution of sterol. 6. The extract according to claim 5, wherein 2% to 5% methanol is used for gradient elution of methanol. • 7. According to the extract of claim 5, wherein about 鸠, coffee or ah • is extracted from the gradient of sterol. 8. A compound represented by the following formula (I), Wherein (1) 136342.doc 201028157 XI and X3 are independently OH, 0-lower alkyl, nh2, NH-lower alkyl, SH or S-lower alkyl; X2 is OH; and X4 is glucopyranoside, Galactoside, pyranomannosides, ribose ribose, alginate, glucopyranoside, furanoside, ribofuranoside, furano arabinoside or furanosylglucoside; and its medicinal or cosmetic properties Accept the salt. 9. The compound according to claim 8, wherein XI, χ2 and χ3 are preferably 〇11 or OC1-6 alkyl and χ4 is glucopyranoside. 1〇_ The compound according to claim 8, which is 3,4-dihydroxy-5-decyloxybiphenyl 2'-0-β-ϋ-ηpyranoside. 11_ a compound represented by the following formula (Π), R1 is ΟΗ; and R2, R3 and R4 are each independently OH, OC-lower alkyl, hydrazine 2, fluorene-lower alkyl, 8 η or S-lower alkyl; and pharmaceutically or cosmetically acceptable salts thereof. 12. A compound according to the claim, wherein R1, R2, R3 & R4 are each independently OH or 〇cH3. 13. The compound according to claim 11, wherein R2 and R4 are each independently 〇H or OCH3 〇 136342.doc 201028157 14. The compound according to claim ,, which is 3,6 dihydroxy-24 dimethoxy _ Benzofuran. A 15' on-line or cosmetic composition comprising the extract according to claim 1 or the compound of claim 4 or 7. 16. A method of preparing a Taiwanese spurt wood extract comprising the steps of: extracting an ethanol extract from Taiwan sapwood by ethanol and extracting the ethanol extract with ethyl acetate to obtain the Taiwan sapwood extract. 17. The method of claim 16, further comprising the step of fractionating the ethyl acetate extract by liquid chromatography on a solvent gradient. 18. The method of claim 16, which further comprises the step of dividing the ethyl acetate extract by liquid chromatography with a C8 to Cl8 column eluted from a gradient of water to methanol. V 19. The method of claim 18, wherein 10% to 100% methanol is used for the soaking of methanol. ^ 20. .21. The method of claim 18, wherein 20 〇 / 〇 to 5 〇 % methanol is used for the thyristing. The method of claim 18, wherein about 20 〇 / 〇, 22 〇 / 〇 or 5 % methanol is eluted with a gradient of alcohol. ? A 136342.doc