TW201204408A - Tyrosinase polypeptide inhibitor - Google Patents

Abstract

The present invention relates to a tyrosinase polypeptide inhibitor. The inhibitor is composed of a polypeptide. One or more amino acids of the polypeptide can be further modified through acetylation, amidation, hydroformylation, hydroxylation, lipid modification, methylation, or phosphorylation. The tyrosinase inhibitor of the present invention can be used as a medicinal composition and comprises the above mentioned polypeptide and further comprises a medically acceptable carrier. The medicinal composition of the present invention suppresses the generation of melanin by suppressing the activity of tyrosinase and is applicable to cosmetics, such as whitening agent.

Description

201204408 IV. Designated representative map: (1) The representative representative of the case is: (1). (2) A brief description of the symbol of the representative figure: No. 5. If there is a chemical formula in this case, please disclose the chemical formula which best shows the characteristics of the invention. 6. Description of the invention: [Technical field of the invention] The present invention relates to a tyrosine The enzyme inhibitors are specifically related to a polypeptide-containing glutaminase inhibitor. [Prior Art] 1. Source and function of melanin During embryonic development, melanocytes differentiated from neuralcrest are distributed in the skin (the basal layer of the epidermis), the eye-retinal pigment epithelium, and the uvea ( Uveal tract), hair follicle stroma, stria vascularis, mucosa and central nervous system - leptomeninges. In melanocytes, a membrane-bound cell called melanomas synthesizes melanin. When melanin is synthesized, it moves to adjacent keratinocytes by dendrites of melanocytes ( Keratinocytes), in turn, present the color of the skin, and the difference in skin color is mainly due to the different production and distribution of melanin. The main function of melanin is to absorb ultraviolet light (UV) in the sun. 201204408 Protects the skin from ultraviolet light and removes reactive oxygen species (ROS) to reduce the damage of free radicals to normal cells. 2. The biosynthesis of melanin tyrosine is an important substrate in the synthesis of melanin. In the reaction, tyrosine is first hydroxylated: by catalyzing tyrosinase (tyr〇sinase), a hydroxyl group is added to the ortho carbon of the hydroxyl group to form dopa (L-3). , 4-dihydr〇xyPhenylalanine, L-dopa), followed by conversion of dopa to dopaquinone. After that, Dopa will take two paths. The first pathway will form a brownish-black eumelanin via catalysis of a tyrosinase-related protein. In another pathway, Dopa wakes up through the action of glutathione or cystehe to produce pheomelanin with yellow and red. (Figure 1). Catalytic mechanism of tyrosinase Luteinase (EC 1.14.18.1) plays an important role in the biosynthesis of melanin. It is a multifunctional, copper-containing transmembrane protein found in melanocytes. It has a molecular weight of about 6 kD in mammals and is usually expressed in melanocytes and belongs to the third type of copper-containing protein ( Type 3 copper protein), the peak is a very important cofactor in the catalytic reaction. In the catalytic shawl, the structure of the living tilt has three different types, each playing a different role. · Oxy-form, deoxyxy (De〇xy_f〇rm), and stationary state (Met-f0rm). . Deoxy-hydrazide _ is not active, the copper ion of its activation site is positively valence and forest oxygen ion. In the catalytic reaction, the deoxygenation state needs to be oxidized, and the two electrons are lost to form an oxidized amine. This hybrid has a positive digal ion and a negative monovalent oxygen 201204408 ion, and the oxidized tyrosinase accepts two receptors, tyrosine and dopa, when combined with tyrosine, The static tyrosinase-dopa complex (Met-E-Dopa) is formed, and after the release of dopaquinone, the tyrosinase is again deoxygenated to continue to circulate; on the other hand, when combined with dopa, In addition to the formation of dopaquinone, tyrosinase also changes from an oxidized state to a stationary state. This form of tyrosinase can form dopaquinone if it reacts with dopa. If it combines with tyrosine, it will form a non-catalytic effect. The complex does not continue the melanin production pathway. It can be seen that the oxidative tyrosinase plays an important role in the catalytic process (Fig. 2). 4. The effect of acid gull tyrosinase is the cause of enzymatic browning of fruits and vegetables during storage or processing. Browning can produce special color and flavor for certain products, such as black tea. , raisins' but for most fresh fruits and vegetables, 'tyrosinase is an enzyme that changes the color and odor of vegetables and fruits, softens tissue, reduces its digestibility, inhibits proteolysis and sugar decomposition, and tyrosinase is in the catalytic process. The non-nutritional and toxic compounds produced in the diet may further deprive the nutritional value of 'and even affect the safety of the food. In 2003, Dr. Asanuma (Reference Neurotox Res 5, pl65_76, 2003) pointed out that the gibberase may be associated with Parkinson's disease and other neurodegenerative diseases. 5. Application of tyrosinase inhibitors In the agricultural and food industries: Browning of fruits and vegetables affects the appearance, smell and taste of food. This is a concern of growers and food industry. The rate of enzymatic browning is determined according to the concentration of activated tyrosinase and phenolic compounds in the tissue, available oxygen, pH and temperature, etc. 201204408. Therefore, it is necessary to use different methods to stop the enzyme browning caused by the glutaminase. Cool aminase inhibitors have also been used in the food industry for many years. Traditionally used browning reaction inhibitors include ascorbic acid, citric acid and sulfite, although sulfites. The inhibitory effect is excellent 'but there are serious side effects, so it is necessary to find a natural and harmless glutaminase inhibitor. In the cosmetics industry: • Tyrosinase inhibitors are becoming more and more important in the cosmetic industry because of the whitening function. Natural inhibitors that have been previously discovered include Arbutin, decanoic acid addic acid, linoleic acid, hydroquinones, etc., but only a few are used as skin whitening agents, mainly Because these natural tyrosinase inhibitors can cause side effects. At present, the Department of Health has approved the age of Linmei's cosmetics, such as Magnesium Asewbyl Phosphate and Vitamin C. Sodium Ascorbyl.

Phosphate), vitamin c glycoside (Asc〇rbyl Gluc〇side), face acid (Jiao Chong (8) eight coffee), arbutin (such as 丨11), Ellagic Acid, Tranexamic acid and Chamomile The extract (mi>mileET) is a tyrosine and arbutin which is a tyrosinase inhibitor and can be used to inhibit the function of the glutamate. It is also a popular ingredient in whitening skin care products. Arbutin is extracted from the bearberry leaves of the bilberry family. Traces of arbutin can also be found in some fruits, such as pears and hills. The structure of arbutin is similar to that of the Department of Health. It is similar to the benzene age of the drug. After being absorbed by the skin, it will be hydrolyzed into benzophenone (tetra) sugar. Therefore, its action is transferred to the same benzene-(four). Amino acid _ activity, lighten the formed melanin 'but benzene - while inhibiting melanin formation and destroying melanocytes, the use of the early US 201204408 white and spot removal effect is very good, but it will cause permanent damage to the skin, such as Other spots or corrosive skin, because the structure of arbutin has more glucose molecules than hydroquinone, so it is less irritating and can be found in skin care products, but because it still has to be reduced to benzodiazepines, Some people are skeptical that there is still a chance to produce side effects similar to those of benzodiazepines. However, the Department of Health has so far announced that arbutin has been approved as a whitening ingredient and that the concentration of arbutin in skin care products must be below 7%. Avoid high concentrations of arbutin to reduce skin immunity. Facial acid is a component extracted from the surface bacteria. During the formation of melanin, copper ions play a catalytic role, while tannic acid can seize copper ions, inhibit the formation of melanin, normalize the metabolism of pigment-producing cells, and inhibit the formation of dark spots or freckles. Whitening effect. Dr. Noh was in 2〇〇7 years (reference Bi〇p〇lymers 88, p300-7, 2007) and pointed out that adding an amino group to the carboxylic acid end of the tannic acid-tripeptide will help improve its acid resistance. (4) Sexuality, depositing the reliance on the money and the effect of the face. However, the Japanese Ministry of Health and Welfare recently suspected that face acid is a carcinogenic hazard as a food additive and has been discontinued in foods. Cosmetics require clinical testing to assess whether it is banned. Since the Japanese government announced the food additives for the forbidden acid, the products that contain the touch are all-in-one, and there are many sayings. Until now, there is no clear data and research to prove that it is harmful. The Taiwan Department of Health is still facing The acid announcement is approved as a whitening ingredient and the concentration of tannic acid in the skin care product must be below 2%. , ': At present, the Department of Health has announced that there are eight kinds of ingredients approved for whitening cosmetics, but some of them have *all doubts. 'We will be sure to be correct when it comes to whitening, and the idea that it will not work immediately, for healthy and fair skin. How to do effective and safe whitening is an important issue in the field of whitening cosmetics. The problem encountered in the field of cosmetic whitening, U.S. Patent Application No. 201204408 20090099093 A1 provides a gibberase inhibitor which consists of a polymorphism comprising 6 to 8 amino acids. Compared with the compounds used in the prior art as tyrosinase inhibitors, the polypeptide used in this case is safer, mild in effect and easy to be absorbed and metabolized by the human body. However, the cost of polypeptide synthesis is tyrosinase inhibition by the manufacturer. One of the major considerations for the commercialization of the drug, the longer the length of the peptide synthesis, the higher the cost, and the way the peptide is transported is also the focus of consideration, 2 to 8 years.

Dr. Agarwal S (Reference Int J Pharm 359, 7-14, 2008) proposes to deliver the appropriate pharmaceutical composition to improve the water solubility, penetration, etc. of the peptide prodrug. The peptide precursor is decomposed by the protease during the process, thereby maintaining the concentration of the drug. Therefore, the development of a tyrosinase inhibitor that is both effective, safe, and has a shorter polypeptide length will be an urgent problem to be solved. SUMMARY OF THE INVENTION In view of the above-mentioned background of the invention, R&D is a kind of natural glutaminase which is effective, safe and has a shorter polypeptide length. The present invention is based on the structural characteristics of _acidase. - fine mysterin Wei Wei noodles, through the test of the steroidal secret amine test, safety test silk to the black and ampere content analysis of the cryptophore experiment, confirmed that the prolinease inhibiting shed of the present invention has (four) Ship fruit. And the secret ship _ 卩 surface is more efficient, safer and shorter than the peptide sequence proposed by the prior art. Amino acid sequence name definition reference table cited in the present invention: English Chinese abbreviation Glycine Glycine Gly (G) Alanine Alanine Ala (A) 201204408

Valine Vale Val (V) Leucine Leucine Leu (L) Isoleucine Isoleucine He (I) Serine Serine Ser (S) Threonine Threonine Thr (T) Cystine Cystine Cys (C) Methionine 曱 methionine Met (M) Proline lysine Pro (P) Lysine lysine Lys (K) Arginine arginine Arg (R) Phenylalanine phenylalanine Phe (F) Tyrosine tyrosine Tyr (Y) Tryptophan color Amino acid Trp (W) Histidine Histidine His (H) Aspartate Aspartate Asp (D) Glutamate Glu (E) Asparagine Aspartic acid Asn (N) Glutamine Glycine Gin (Q) Classification of amino acids referred to in the context of the present invention: 1. Aliphatic includes: alanine (A), isoleic acid (Isoleucine, I), leucine (Leucine, L) , Valine (V), Proline (P) ° 2. Aromatic Aromatic includes: Phenylalanine (F), Tryptophan (W), Road Amino acid (Tyrosine, Y). 3. Acidic acid includes: Aspartic acid (D), glutamic acid 201204408 (Glutamic acid, E) 〇 4 · Basic amino acids (Basic) including: arginine (Arginine , R), histidine (H), lysine (Lysine, K). 5. Hydroxylic acids include: serine (Srine), and threonine (Treonine, T). 6. Sulfur-containing acid (SulfUrcontaining) includes: cysteine (Cysteine, C), methionine (Methionine, Μ) 〇 Lu 7. Amidic acid (Amidic) includes: aspartic acid (Asparagine, Ν), glutamic acid (Glutamine, Q) 〇 The present invention relates to a tyrosinase inhibitor having a polypeptide sequence of R1 η 1 -Xaa-R2n2-Yaa-R3n3 wherein Xaa or Yaa is tyramine Acid, cysteine, glycine, glutamic acid or arginine, the substituent R1, R2 or R3 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, a group consisting of a basic amino acid, a hydroxyl amino acid, a sulfur-containing amino acid, and a guanamine amino acid, wherein n1, n2 or n3 is the number of substituents, the numbers being independently equal to 0 or 1. The present invention relates to a tyrosinase inhibitor, the polypeptide sequence comprising: (1) Rlnl_Tyr-R2n2-Cys-R3n3 or Rlnl_Cys-R2n2-Tyr-R3n3 (b) Rlnl-Cys-R2n2-Gly-R3n3 or Rlnl- Gly-R2n2-Cys-R3n3 (III) Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Glu-R3n3 (4) Rlnl-Arg-R2n2-Tyr-R3n3 or Rlnl-Tyr-R2n2-Arg- R3n3 The present invention relates to a tyrosinase inhibitor whose polypeptide sequence comprises: 201204408

Tyr-Cys-Cys, Glu-Cys-Val, Cys-Arg, Cys-Asp-Tyr, Arg-Tyr-Cys-Arg, Asp-Cys-Gly, Arg-Cys-Tyr-Arg, Cys-Gly-Ser, Asn-Cys-Tyr, Phe-Tyr-Cys, Arg-Cys-Tyr-Val, Val-Cys-Gly, Cys-Gly-Tyr, Phe-Tyr-Cys, Tyr-Phe-Cys, Cys-Val, Arg- Phe-Tyr-Cys, Gly-Cys-Tyr, Phe-Tyr-Cys-Arg, Cys-Tyr-Gly, Arg-Cys-Tyr, Val-Ser-His-Tyr, Gly-Cys-Tyr, Tyr-Phe- Arg, Tyr-Asp. Detailed description of the polypeptide sequence of the tyrosinase inhibitor of the present invention: (1) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3 wherein the substituent R1, R2 or R3 is selected from glycine a group consisting of a fatty amino acid, an aromatic amino acid, an acidic amino acid, an acid-based amino acid, a transbasic acid, a sulfur-containing amino acid, and a guanamine amino acid; and nl thereof , n2 or n3 is the number of substituents', the number is independently equal to 〇 or 卜. As can be seen from the table below, the polypeptide sequence design is represented by the type of each amino acid functional group, and the short polypeptides are arranged in different sequences. . Table 1 The design of the polypeptide sequence and examples thereof

Rlnl -Tyr-R2n2-Cys-R3n3 sequence 2. 3. 4. nl=〇, n2=0, η3=0 nl=l, η2=0, η3=0 η 1=0, η2=1, η3=0 η 1=0, η2=0, η3=1

YC

Rl-Y-C

Y-R2-C Y-C-R3

GYC

Rl-C-Y

GCY

CSY

CYN 201204408 5. nl=l, n2=l, n3=0 R1-Y-R2-C AYGC R1-C-R2-Y RCGY b. nl=l, n2=0, n3=l R1-YC-R3 - ----- NYCR R1-CY-R3 RCYN 7· nl=0, n2=l, n3=l Y-R2-C-R3 ------ YDCD C-R2-Y-R3 CFYF 8· nl =l, n2=l, n3=l R1-Y-R2-C-R3 GYGCG R1-C-R2-Y-R3 gcgya (b) Rlnl-Cys-R2n2-Gly-R3n3 or Rlnl-Gly-R2n2-Cys -R3n3 wherein the substituent R1, R2 or R3 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, basic amino acids, hydroxyl amino acids, thioamino acids And a group consisting of a guanamine amino acid and wherein n1, n2 or n3 are the number of substituents, the numbers being independently equal to 〇 or 】. As can be seen from the table below, the polypeptide sequence design is represented by the type of each amino acid functional group, and the short polypeptides of different sequences are arranged. Table 2: Design and examples of polypeptide sequences

No. nl=〇, n2=〇, , n3=〇------ nl=l, n2=〇, n3=〇Rlnl-Cys-R2n2-Gly-R3n3 sequence such as Rlnl.Gly-R2n2-Cys-R3n3 The sequence is, for example, 1 · CG ere Rl-CG DCG Rl-Gc DGC 3· 4. η1=〇Ρ n2=l, n3=〇C-R2-G CFG G-R2-C ] GFC nl=〇, n2=〇, N3=l ---- CG-R3 CGK GC-R3 GCK 5, nl=l, n2=l, n3=〇R1-C-R2-G ACYG R1-G-R2-C AGYC 201204408

6. nl=l, n2=0, n3=l R1-CG-R3 NCGR R1-GC-R3 RGCY 7. nl=0, n2=l, n3=l C-R2-G-R3 CDGY G-R2- C-R3 GYCR 8. nl=l, n2=l, n3=l R1-C-R2-G-R3 RCYGR R1-G-R2-C-R3 RGYCW (III) Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-GliKR3n3 wherein the substituent R1, R2 or R3 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, basic amino acids, hydroxyl amino acids, a group consisting of a sulfaminic acid and a guanamine amino acid; and wherein n1, n2 or n3 are the number of substituents, the numbers being independently equal to 〇 or i. As can be seen from the table below, the polypeptide sequence design is represented by the type of each amino acid functional group, and the short polypeptides which are arranged in different sequences are cross-linked. Table III Design of polypeptide sequences and examples thereof

Rlnl-Glu-R2n2-Cys-R3n3, for example, Rlnl-Cys-R2n2-Glu-R3n3, for example, sequence sequence sequence 1. nl=0, EC CE n2=0, n3=0 2. nl=l, Rl-EC DEC Rl -CE DCE n2=0, n3=0 3. nl=0, E-R2-C EVC C-R2-E CVE n2=l, n3=0 4. nl=0, EC-R3 ECR CE-R3 CER n2 =0, n3=l 5. nl=l, R1-E-R2-C VESC R1-C-R2-E VCSE n2=l, n3=0 12 201204408

6. nl=l, n2=0, n3=l r1-EC-R3 — HECY R1-CE-R3 HCEY L nl=〇, n2=l, n3=l t-R2-C_R3 EFCR C-R2-E- R3 CFER 8. nl=l, n2=l, n3=l ^1-E-R2-C-R3 -----... REYCW R1-C-R2-E-R3 RCYEW (4) Rlnl-Arg -R2n2-Tyr-R3n3 or Rlnl-Tyr-R2n2-Arg-R3n3 wherein the substituent R1, R2 or R3 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, alkaline The group consisting of amino acids, hydroxyl amino acids, thioamino acids and guanamine amino acids and wherein nl, n2 or n3 are the number of substituents, the numbers are each independently equal to 〇 or i. As can be seen from the table below, the polypeptide sequence design is represented by the type of each amino acid functional group, and the short polypeptides of different sequences are arranged.

Table IV Design of the polypeptide sequence and its example number----- R1 nl -Arg-R2n2-Tyr-R3n3 sequence such as R1 nl -Tyr-R2n2-Arg-R3n3 sequence, for example 1. - 2. 2. - 3 · —— 4. 〜 5. 5. nl=〇, η2=〇, η3=〇RY YR η1=ΐ, η2=〇, η3=〇—Rl-RY CRY Rl-YR CYR ~VVR YRK η1=〇, η2= 1, η3=〇------ R-R2-Y RVY Y-R2-R — η1=〇, η2=〇, η3=1 ---- RY-R3 RYK YR-R3 , nl=l, Η2=ΐ, _η3=〇_ R1-R-R2-Y VRSY R1-Y-R2-R n vysr 13 201204408

6. nl=l, n2=0, n3=l R1-RY-R3 WRYC R1-YR-R3 WYRC 7. nl=0, n2=l, n3=l R-R2-Y-R3 RFYW Y-R2- R-R3 YFRW 8. nl=l, n2=l, n3=l R1-R-R2-Y-R3 VRCYE R1-Y-R2-R-R3 VYCRE In a preferred embodiment, the tyramine of the present invention Acidase inhibitors (1) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3, (2) Rlnl-Cys-R2n2-Gly-R3n3 or Rlnl-Gly-R2n2-Cys-R3n3, (iii) Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Glu-R3n3 or (iv) Rlnl-Arg-R2n2-Tyr-R3n3 or Rlnl-Tyr-R2n2-Arg-R3n3, where nl=0 When n2=0 and n3=l, the substituent R3 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, basic amino acids, hydroxyl amino acids, sulfur-containing A group consisting of amino acids and guanamine amino acids. In a preferred embodiment, the tyrosinase inhibitor of the invention (I) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3, (b) Rlnl-Cys-R2n2-Gly -R3n3 or Rlnl-Gly-R2n2-Cys-R3n3, (S)Rlnl-Glu-R2n2-Cys-R3n3 4Rlnl-Cys-R2n2-Glu-R3n3 or (iv) Rlnl-Arg-R2n2-Tyr_R3n3 or Rlnl-Tyr- R2n2-Arg-R3n3 wherein nl=0, n2=l and n3=0, the substituent R2 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, and amines A group consisting of a base acid, a hydroxyl amino acid, a sulfur-containing amino acid, and a guanamine amino acid. 14 201204408 In a preferred embodiment, the tyrosinase inhibitor of the invention (I) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3, (b) Rlnl_Cys-R2n2-Gly -R3n3 or Rlnl, Gly-R2n2-Cys-R3n3, (3) Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl, Cys-R2n2-Glu-R3n3 or (iv) Rlnl-Arg-R2n2-Tyr-R3n3 or Rlnl -Tyr-R2n2-Arg-R3n3, wherein nl = Bu n2 = 0 and n3 = 0, the substituent R1 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, alkalis A group consisting of an amino acid, a hydroxyl amino acid, a sulfur-containing amino acid, and a 醯φ amine amino acid. In a preferred embodiment, the tyrosinase inhibitor of the invention (I) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3, (b) Rlnl-Cys-R2n2_Gly-R3n3 Or Rlnl-Gly-R2n2-Cys-R3n3, (iii) Rlnl-Glu-R2n2_Cys-R3n3 or Rlnl-Cys-R2n2-Glu-R3n3 or (iv) Rlnl-Arg-R2n2-Tyr-R3n3 or Rlnl-Tyr-R2n2 -Arg-R3n3 wherein, when nl==l, n2=0 and n3=l, the substituent R1 or R3 is selected from the group consisting of glycine, fatty Φ amino acid, aromatic amino acid, acid amino acid, A group consisting of a basic amino acid, a hydroxyl amino acid, a sulfur-containing amino acid, and a guanamine amino acid. In a preferred embodiment, the tyrosinase inhibitor of the invention (I) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3, (b) Rlnl-Cys-R2n2-Gly -R3n3 or Rlnl-Gly-R2n2-Cys-R3n3, (3) Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Glu-R3n3 or (iv) Rlnl-Arg-R2n2-Tyr-R3n3 or Rlnl -Tyr-R2n2_Arg-R3n3, wherein nl=l, n2=l and n3=0, the substituent R1 or R2 is selected from the group consisting of glycine, fatty 15 201204408 amino acid, aromatic amino acid, acid amine group A group consisting of an acid, a basic amino acid, a hydroxyl amino acid, a sulfur-containing amino acid, and a guanamine amino acid. In a preferred embodiment, the tyrosinase inhibitor of the invention (I) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr_R3n3, (b) Rlnl-Cys-R2n2-Gly-R3n3 Or Rlnl-Gly-R2n2-Cys-R3n3, (iii) Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Glu-R3n3 or (iv) Rlnl-Arg-R2n2-Tyr-R3n3 or Rlnl-Tyr -R2n2-Arg-R3n3 'wherein nl=0, n2=l and n3=l, the substituent R2 or R3 is selected from the group consisting of glycine, fatty acid, aromatic amino acid, acid amino acid A group consisting of an amino acid, a hydroxyl amino acid, a sulfhydryl acid, and a guanamine amino acid. In a preferred embodiment, the tyrosinase inhibitor of the invention (I) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys_R2n2-Tyr-R3n3, (b) Rlnl-Cys-R2n2-Gly-R3n3 Or Rlnl-Gly-R2n2-Cys-R3n3, (iii) Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Glu-R3n3 or (iv) Rlnl-Arg-R2n2-Tyr-R3n3 or Rlnl-Tyr -R2n2-Arg-R3n3, wherein when nl=l, n2=l and n3=l, the substituent R1, R2 or R3 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acid amines A group consisting of a base acid, a basic amino acid, a hydroxyl amino acid, a sulfur-containing amino acid, and a guanamine amino acid. In a preferred embodiment, the tyrosinase inhibitor of the invention wherein the polypeptide sequence comprises: Tyr-Cys-Cys 'Glu-Cys-Val ' Cys-Arg ' Cys-Asp-Tyr ' Arg-Tyr- Cys-Arg 'Asp-Cys-Gly, Arg-Cys-Tyr-Arg, Cys-Gly-Ser, Asn-Cys-Tyr, Phe-Tyr-Cys, 16 201204408

Arg-Cys-Tyr-Val ^ Val-Cys-Gly ^ Cys-Gly-Tyr > Phe-Tyr-Cys ^ Tyr-Phe-Cys > Cys-Val > Arg-Phe-Tyr-Cys ^ Gly-Cys -Tyr > Phe-Tyr-Cys-Arg ^ Cys-Tyr-Gly >

Arg-Cys-Tyr, Val-Ser-His-Tyr, Gly-Cys-Tyr, Tyr-Phe-Arg, Tyr* In a preferred embodiment, the tyrosinase inhibitor of the invention, wherein the polypeptide sequence The one or more amino acids may be further subjected to Acetylated, Amidoxime (Blood (4), Formylated, Hydmxylated, Lipid M〇dified, Sulfhydryl) (Methylated) or Phosphorylated modification, in 2 years and 9 years, Man Abu said that the polypeptide sequence also found in the same can be used as an inhibitor of tyrosinase, while the patent was published at the same time (W02009003034) (A1)) It is also mentioned that the modified polypeptide sequence is also considered in the patent range, and it is considered that the modified polypeptide sequence does not change the active mechanism of its action. The present invention further relates to a method for inhibiting tyrosinase activity. a pharmaceutical composition comprising the above-mentioned tyrosinase inhibitor, and further comprising: a pharmaceutical acceptable, wherein the _ can be an excipient, a diluent, a build-up, a filler, a binder, a disintegrant, Lubricant, grease or non-grease base, • surfactant, suspension Agent, gelling agent, adjuvant, preservative, antioxidant, stabilizer, coloring agent or perfume. The hair composition of the present invention is inhibited by the activity of lysin, thereby inhibiting the production of melanin and reducing the melanin content in the cell. It can be applied to cosmetics or agricultural products, and can be used as a whitening agent when it is applied to cosmetics. The present invention further relates to a method for reducing melanin precipitation in the skin, the mechanism mainly by inhibiting the activity of the road amine, the method comprising the above The thief enzyme inhibiting ship is transported to a mammal by oral, percutaneous absorption, injection or inhalation. The mammal includes humans and the like. 0 17 201204408 [Embodiment] The following examples are non-limiting and only serve as the present invention. Representative examples of various aspects. Example 1: In vitro tyrosinase activity test I. Drug I. Mushroom tyrosinase (1〇〇u/ml), arbutin (Agutin), lysine ( L-tyrosine, 0.5 mM), Kojic acid and 2 to 5 short peptides of different sequences (0.5 mM). The short polypeptide sequence is Rlnl-Xaa-R2n2-Yaa-R3n3, Xaa or Yaa is lysine Cysteine Each of the five amino acids of amino acid, glycine acid, facial acid and arginine, the amino acid of the substituent R1, R2 or R3 is divided into the following eight types: glycine, fatty Amino acid, aromatic amino acid, acid amino acid, basic amino acid, hydroxyl amino acid, sulfur-containing amino acid and guanamine amino acid, each type, nl, n2 or n3 is substituted The number of bases, which are individually independently equal to 〇 or 1, and the above amino acids are cross-aligned into short polypeptides of 2 to 5 different sequences. II. Experimental Procedure The OD value of dopachrome was measured at 475 nm using a Varian cary-50 Bio UV-Visible spectrophotometer. Calculate the remaining active percentage of tyrosinase by the following formula:

Tyrosinase activity (%) = (D - C) / (B - A) x 100% A : absorbance at 475 nm before control group reaction; B: absorbance at 475 nm after control group reaction; 201204408 C. Control reaction The absorbance at 475 nm before; D. The absorbance at 475 nm after the reaction in the control group. ΙΠ·Experimental results The arbutin and citric acid (previous experimental results show that citrate can inhibit the activity of myrmase at a concentration of 0.4 mM, the test time is 3G minutes) as a positive control group. Table 5 lists the effects of short peptides of different cores inhibiting tyrosinase activity at 5 minutes. Comparing the short polypeptide designed by the present invention with the commercially available whitening ingredient arbutin and citric acid, the activity of arbutin inhibitory lumenase is 322, and the short polypeptide designed by the invention is far superior to arbutin, and the facial acid inhibition The aminase activity is 〇.10, which has the same superior effect as the short polypeptide of the present invention, but it is considered to be safe for use in humans. The short polypeptide of the present invention is safer than citric acid. Table 5, 5 minutes, the effect of short peptides of different sequences on inhibition of tyrosinase activity (1) Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3: sequence Tyrosinase activity (%) sequence Tyrosinase activity YCC 0 ECY 0.074653674 CDY 0 DYC 0.087482961 YCN 0 VYCR 0.091795925 CVY 0 CGY 0.096860964 YGC 0 CYN 0.097461159 RYC 0 DYCR 0.101490686 RFYC 0 KA 0.10591521 YNC 0 VCY 0.109361087 RCYV 0 NYCR 0.121654937 RCYC 0 YCS 0.140878234 NCY 0 CYS 0.148467869 VYC 0 CNY 0.152249737 YSC 0 CCY 0.170952487 RCYD 0 YCV 0.201675903 RYCR 0 RCYW 0.240532622 SYCR 0 YC 0.282109105 CSY 0 RCYL 0.306704013 YCG 0 RCYI 0.315981809 201204408 YFC 0 RCY 0.356030441 RCYN 0 CYR 0.383016975 FYC 0 GCY 0.388680245 GYC 0 RCYG 0.422501613 RCYR 0 RCYA 0.422863366 CYC 0 WCY 0.433679474 GYCR 0 RCGY 0.439602446 CYG 0 YCE 0.440729397 YEC 0 CYD 0.445290753 VRCY 0 CY 0.516055046 SCY 0 YCD 0.67074304 YRC 0 CFY 0.872799404 YDC 0 CRY 1.102088681 RCYF 0.019169001 RCYCR 1.146788991 FYCR 0.020633759 YVC 4.196121435 YCR 0.036315672 Arb 32.242669 RCYS 0.0382263 control 100 YCF 0.047937278

(II) Rlnl-Cys-R2n2-Gly-R3n3 or Rlnl-Gly-R2n2-Cys-R3n3 : Sequence Tyrosinase activity (0/〇) Sequence Tyrosinase activity (%) CGS 0 VCG 0.16501671 CGR 0 CGN 0.184239081 DCG 0 GCG 0.194596694 CGD 0 CGV 0.20573982 CGG 0 ECG 0.332101409 CGC 0 NCG 0.440803923 SCG 0 CG 0.496941896 CCG 0.00758032 Arb 32.242669 RCG 0.028104759 control 100 KA 0.1059152

(iii) Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Glu-R3n3 : sequence Tyrosinase activity (3⁄4) sequence Tyrosinase activity (%) ECV 0 REC 0.244581269 ECR 0 CEC 0.268719348 ECN 0 ECG 0.332101409 ECC 0 DEC 0.350414781 ECS 0.046361766 NEC 0.39675514 GEC 0.05283631 ECD 0.39964605 20 201204408 KA 0.10591521 Arb 32.242669 VEC 0.207898268 control 100 (IV) Rlnl-Arg-R2n2-Tyr-R3n3 or Rlnl-Tyr-R2n2-Arg-R3n3 : Sequence Tyrosinase Activity (%) Sequence Tyrosinase Activity (%) RSY 0 WRFY 9.830538813 RFYW 0 RYY 15.8876895 KA 0.10591521 Arb 32.242669 RWY 1.04109589 YR 44.9087323 RFY 1.259403349 YFR 47.37618551 RIY 2.4216621 RGY 51.92132116 RLY 2.525159817 RY 54.22843594 RTY 2.608200147 RPYR 82.384621 RMY 2.708666667 RFYE 83.06501065 RAY 3.633065449 control 100 RVY 4.551878234 (V) Val -Ser-His-Tyr sequence Tyrosinase activity (%) KA 0.105915208 VSHY 1.366590214 Arb 32.24266895 HY 47.81793167 YH 48.56735741 VY 68.98319048 VH 69.42109648 control 100 Further test 5 and 30 minutes, no Short polypeptide sequences inhibition effect of enzyme activity of tyrosine (list only a partial core Rlnl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3 a). The results listed in Table 6 below show that the 5 and 3 〇 minutes have different inhibitory effects, respectively. The short polypeptide of the present invention has an inhibitory effect at 5 minutes, and continues to be effective until 3 〇 minutes, while the time is economically considered, the present invention is at 5 There is enough suppression in minutes. Comparing the short peptide and arbutin designed by the present invention with citric acid, the activity of arbutin inhibiting tyrosinase 21 201204408 is restored to 49 M at 32.24 ' to 30 minutes in 5 minutes, and it can be inferred that arbutin has been metabolized in 3 minutes. (4) 妓 has slowly recovered, the secret of the object _• sex is 0.1 in 5 minutes, 0.24 in 30 minutes, the effect is very significant, but in terms of effect, the short peptide designed by the invention is far superior to arbutin for safety. As a matter of sexual consideration, the short polypeptide of the present invention is safer than citric acid, and therefore, the present invention is superior to the commercially available whitening ingredient arbutin and citric acid. Table 6. Rabbits at 5 and 30 minutes, the inhibitory effect of tyrosinase activity on short peptides of different sequences. Tyrosim ise activity sequence Tyrosinase activity · 5 min 30 min 5 min 30 min YCC 0 0 ECY 0.074653674 0.130554158 CDY 0 0 DYC 0.087482961 0.302584335 YCN 0 0.140488897 VYCR 0.091795925 0.504389473 CVY 0 0.05923513 CGY 0.096860964 0 YGC 0 3.530247154 CYN 0.097461159 0.243517324 RYC 0 0.312252027 DYCR 0.101490686 0.273056365 RFYC 0 0.150098847 KA 0.10591521 0.24267145 YNC 0 0.861511469 VCY 0.109361087 0 RCYV 0 0 NYCR 0.121654937 0.208785075 RCYC 0 0.400263592 YCS 0.140878234 0.335180982 NCY 0 0 CYS 0.148467869 0.429436199 VYC 0 0.034048589 CNY 0.152249737 0 YSC 0 0 CCY 0.170952487 0.356391264 RCYD 0 0.269577529 YCV 0.201675903 0.546470525 RYCR 0 0 RCYW 0.240532622 0.415660095 SYCR 0 0.206994126 YC 0.282109105 0.337364536 CSY 0 0.125723179 RCYL 0.306704013 0.432102741 YCG 0 0.065545331 RCYI 0.315981809 0.753837345 YFC 0 0.038542855 RCY 0.356030441 1.008505766 RCYN 0 0.21 4637204 CYR 0.383016975 0.421953156 FYC 0 0.007075857 GCY 0.388680245 0.163539516 GYC 0 0.251992166 RCYG 0.422501613 0.044462887 RCYR 0 0 RCYA 0.422863366 0.513454042 CYC 0 0.013077983 WCY 0.433679474 0.975242241 GYCR 0 0.167987598 RCGY 0.439602446 0.82807942 CYG 0 0.248010189 YCE 0.440729397 0.506883249 YEC 0 11.08214699 CYD 0.445290753 0.370091693 VRCY 0 1.192089591 CY 0.516055046 1.731438788 SCY 0 0.056270458 YCD 0.67074304 0.729086611

22 201204408 YRC 0 0.412643849 CFY 0.872799404 1.034434046 YDC 0 0.817724888 CRY 1.102088681 0.262336753 RCYF 0.019169001 0.418457391 RCYCR 1.146788991 1.402089019 FYCR 0.020633759 0.165538253 YVC 4.196121435 40.71233186 YCR 0.036315672 0.779613411 Arb 32.242669 49.1479689 RCYS YCF 0.0382263 0.81396443 control 100 100 0.047937278 14.91477258 Example 2: Short peptides of different sequences Conduct safety test results I·Drug source 1. Polypeptide sequence is YD, YFR, GCY and YC • II. Experimental step-by-step use of MTT assay to determine whether the inhibitor contains cytotoxicity, the chlorophyll in the mitochondria of living cells Desiccate dehydrogenase can reduce yellow MTT to purple formazan, then add DMSO to dissolve formazan, and measure the absorbance of the solution under OD595nm by spectrophotometer and estimate the test substance to the cell. The impact of growth. (1) First prepare a culture medium containing MTT. Add 80 #1 MTT (original concentration of 5 mg/ml) per 500 /zl of culture medium. (2) All the cell culture fluids are aspirated, and 100% of the culture medium containing MTT is added to each % well plate (2 of the winter hole plate is added with 5 〇〇" 1 culture medium containing MTT), at 37 丫The 5% c〇2 incubator was allowed to stand for one hour. (3) Remove the medium from the cell culture plate, add an appropriate volume of DMS(R) (296#1 in 96-well plate) and let stand for 10 5 minutes. The absorbance at a wavelength of 550 nm was measured by a continuous wavelength microplate analysis system (Molecular Devices > SPECTRAMAX M2), and then the average absorbance of the untreated control group was 100%, and the percentage of absorbance of the experimental group was calculated. 23 201204408 III·Experimental results Figure 2 shows the safety test results for short peptide inhibitors of different sequences. Effect of different short peptides inhibiting tyrosinase activity at 5 minutes In the eMMTTassay experiment, when 5 mM arbutin or 1 mM of facial acid was added, the cell activity had dropped to the lowest threshold of 95%. In the results of the MTTassay experiment, the addition of GCY1〇〇 "Μ still does not affect cell viability. While other inhibitors YC, YD, and YFR have the same situation, the above experiments confirmed that the four short polypeptides were not toxic and did not affect cell viability. Example 3: In vivo melanin content analysis In this patent, there are some cis-transverse compounds whose inhibitory effects are not affected by the reverse direction. However, some of the forward and reverse compounds have inhibitory effects. The difference is about 50〇/〇. On the other hand, some compounds have a coloration after being dissolved in the solution. Therefore, in order to eliminate the coloration, the absorbance may be too high, and it is confirmed that the inhibitor designed in this study is also in the human body. Can have the same good whitening effect, so we selected some compounds YD, yfr, GCY, YC for melanin content analysis. I. Drug source 1. The peptide sequence is YD, YFR, GCY and YC II. Experimental procedure

Melanocytes were cultured in 24-well plates, and different inhibitors were cultured for seven days at concentrations of 1, 5, 10, 25, 50 and 1 〇〇 βΜ, respectively. Seven days later, trypsin/EDTA (0.25°/c>/0.10/〇 in phosphate buffered saline) was added for brief culture to separate the cells. After that, all the cell culture fluids were sucked up, washed twice with HBSS, and the cells were shaken by ultrasonic waves, and then 0.3 ml 1N 24 201204408 was added.

NaOH (70 ° C), and placed in an oven at 50 ° C for 10 minutes. Finally, the eluted melanin was mixed, and 0.2 ml to 96-well plate was taken, and the absorbance of OD 405 nm melanin was measured by a spectrophotometer. ΙΠ.Experimental results

Table 7. Analysis of melanin content of short peptides with different sequences YD Relative melanin content SD Control 100.00 11.24 1 80.57 1.65 5 78.20 2.98 10 77.94 2.98 25 73.02 10.87 50 80.37 7.97 100 75.72 1.08 kojic acid (0.5mM) 81.87 3.01 kojic acid ( LmM) 67.63 2.87 Arbutin (5 mM) 74.52 4.19 YFR Relative melanin content SD Control 100.00 10.93 1 91.65 6.06 5 86.78 2.53 10 86.12 0.81 25 84.15 3.29 50 84.48 1.70 100 81.02 1.75 kojic acid (1 mM) 67.63 2.87 kojic acid (0.5 mM 81.87 3.01 Arbutin (5 mM) 74.52 4.19 GCY Relative melanin content SD Control 100.00 7.84 1 107.05 18.26 5 90.40 2.83 10 85.93 5.66 25 92.75 3.28 50 90.47 0.71 Ϊ00 82.41 2.19 kojic acid (lmM) 67.63 2.87 25 201204408 kojic acid (0.5mM 81.87 3.01 Arbutin (5 mM) 74.52 4.19 YC Relative melanin content SD Control __ 100 5.75 1 96.33 5.13 5 __ 95.00 2.53 10 95.39 1.60 25 100.97 2.19 50 92.26 1.86 100 ' 91.21 4.28 kojic acid (ImM) 67.63 2.87 kojic acid (0.5 mM) 81.87 3.01 Arbutin (5 mM) 74.52 4.19 From the results of the experiment When, as it can be found Arbutin added 5 mM, so that the amount can be reduced to 74.52% containing melanin, whereas kojic acid was added 1 mM, can be reduced to the content of meianin 67.63 ° /. Therefore, the inhibitory effect of noodle acid is also better than that of arbutin in human tyrosinase.

From the experimental results of YD, we can see that when adding milk 1〇〇_, only 0.02 times the dose of arbutin can achieve the inhibition effect of phase $, so that the age of the age is reduced to about 75%, and when When YD1 //M is 'the same inhibition effect as adding 5 mM bismuth citrate', the content of melanin is reduced to 80%. When 1 加入 is added, only 0.2 times the dose of noodle acid can be used to achieve the same inhibitory effect as the addition of sputum, so that her blood content is reduced to 80%. Therefore, it is known from the above data that melon human acidase has a inhibitory effect, and its inhibitory power is even better than thieves and arbutin. Therefore, the reverse push & vi plus mushroom tyrosinase transfer vegetarian, _ try (four) fruit, the reason why the % anger compound is misjudged as the inhibition effect is not good, mainly due to the compound in the solution i color situation 26 201204408 ring In addition, the above data also confirmed that the inhibitory effect of the cis-transverse compound on tyrosinase is not much different. According to the experimental yarn of GCY, it can be known from the data that when GCY 1〇〇 is added, only a small dose can be used to achieve the same inhibitory effect as the addition of bismuth citrate, which reduces the content of melanin. Up to 80%. On the other hand, the experimental results of Yc are compared with the experimental data of GCY. 'We can find that in human tyrosinase, the inhibitory effect of GCY is also excellent in YC. This situation is related to the in vitro mushroom tyrosi enzyme. The results of the activity test are consistent. Therefore, based on the above experimental results, we conclude that compounds with inhibitory effects on tyrosinase may have the same good inhibition in human tyrosinase. From the results of the MTT assay, 'imM's citrate or 5 mM arbutin, the results of the in vitro mushroom tyrosinase activity test test 'learned to add YD 100' Μ can reach seven similar to limmic acid lmM or arbutin 5 mM It has an inhibitory effect, but the addition of TO 1〇〇 “〇〇 does not affect its fine cell activity” is confirmed by the above two experiments. These compounds are not only good compared to the tannic acid and arbutin commonly used in the cosmetics industry. It inhibits the effect and is not cytotoxic. [Simple description of the diagram] Figure 1 shows the biosynthetic pathway of melanin. Figure 2 shows the catalytic process of tyrosinase. Figure 3 shows the safety test results for short peptide inhibitors of different sequences. [Main component symbol description] None 27

Claims (1)

201204408 VII. Scope of application: 1. A glutaminase inhibitor whose polypeptide sequence comprises: Rlnl -Xaa-R2n2-Yaa-R3n3 Its ten Xaa or Yaa is tyrosine, cysteine, glycine, A glutamic acid or a arginine, the substituent R1, R2 or R3 is selected from the group consisting of glycine, a fatty amino acid, an aromatic amino acid, an acidic amino acid, a basic amino acid, a hydroxyl amino acid, A group consisting of a thioaminic acid and a guanamine amino acid and wherein nl, n2 or n3 are the number of substituents, each number being independently equal to 0 or 1. 2. According to the tyrosinase inhibitor of claim 1, the polypeptide sequence comprises: Lunl-Tyr-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Tyr-R3n3* wherein the substituents R1, R2 or R3 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, organic amines, amino-based amino acids, sulfur-containing amino acids, and amine amino acids. a group, and wherein nl, n2 or n3 are the number of substituents, the numbers being independently equal to 0 or 1. Xin 3. According to the tyrosinase inhibitor of claim 1, the polypeptide sequence comprises: Rlnl-Cys-R2n2-Gly-R3n3 or Rlnl-Gly-R2n2-Cys-R3n3 > wherein the substituents R1, R2 Or R3 is selected from the group consisting of glycine, fatty amino acids, aromatic amino acids, acidic amino acids, basic amino acids, hydroxyl amino acids, thioamino acids, and guanamine amino acids. Group ' and where nl, n2 or n3 are the number of substituents, each number being independently equal to 〇 or 1. 28 201204408 4. The root shot please follow the 丨 丨 _ amine___, shirt: Rlnl-Glu-R2n2-Cys-R3n3 or Rlnl-Cys-R2n2-Glu-R3n3 > which replace fine, R2 fine Wei from (four) _ a group consisting of blue (10) grade, aromatic amine grade, acid amino acid, tester amino acid, amide amino acid, sulphur-containing amino acid and tyrosine amino acid, and wherein η Π 2 or moor As the number of substituents, the numbers are each independently equal to 〇 or 1. 5. According to the scope of the patent, the first part of the patent, the polypeptide sequence includes: R1 η 1 -Arg-R2n2-Tyr-R3n3 or Rlnl-Tyr-R2n2-Arg-R3n3 > wherein the substituents IU, R2 Or a group consisting of R3 health free thief, arginous amine grade, aromatic amino acid, acid amino acid, test amino acid, trans-amino acid, thiol acid and tyrosine amino acid Group, and wherein Μ, η2 or n3 are the number of substituents, the numbers being independently equal to 0 or 1. 6. The tyrosinase inhibitor's polypeptide sequence is selected from Tyr_Cys_Cys, Glu Cys-Val, Cys-Arg, Cys-Asp-Tyr, Arg-Tyr-Cys-Arg, Asp-Cys-Gly, Arg Cys -Tyr-Arg ' Cys-Gly-Ser ' Asn-Cys-Tyr ' Phe-Tyr-Cys ' Arg-Cys-Tyr-Val , Val-Cys-Gly , Cys-Gly-Tyr , Phe-Tyr-Cys , Tyr -Phe-Cys ' Cys-Val ' Arg-Phe-Tyr-Cys ' Gly-Cys-Tyr ' Phe-Tyr-Cys-Arg, Cys-Tyr-Gly, Arg_Cys-Tyr, Val-Ser-His-Tyr, Gly a group consisting of -Cys-Tyr, Tyr-Phe-Arg and Tyr-Asp. 29 201204408 7. 8. 9. 10. 11. 12. 13. 14. A tyrosine peak inhibitor whose polypeptide sequence is selected from the group consisting of “rg_Cys_Tyr, patent scopes 2, 3, 4, 5, 6 or Seven of the statin inhibitors, the § sequence of one or more amino acids are undergoing acetylation, guanidation, formazanization, thiolation, thiolation or phosphorylation A pharmaceutical composition for inhibiting the activity of aglyase, which comprises a glutaminase inhibitor of the scope of the patent, in the second, third, third, fifth, sixth or seventh aspect of the patent. The pharmaceutical composition further comprises a medically acceptable carrier. The pharmaceutical composition according to the scope of the patent application, wherein the scaly agent, the diluent, the thickener, the filler, the binder, the disintegrating agent , lubricant, grease or non-greasy base, surfactant, suspending agent, gelling agent, adjuvant, preservative, antioxidant, stabilizer, colorant or fragrance. Pharmaceutical combination according to claim 9 Applied to cosmetics, agricultural products or whitening agents. A type of black that reduces the skin of mammals. And a method for transferring a glutaminase inhibitor according to claim 1, 2, 3, 4, 5, 6 or 7 to a mammal, according to the method of claim 13 The tyrosinase inhibitor is delivered by means of alpha, percutaneous absorption, injection or inhalation. The method of claim 13, wherein the tyrosinase inhibitor further comprises a pharmaceutically acceptable carrier. The method of claim 13, wherein the mammal is a human. 31