CN102232954A - Use of thiobabituric acid for inhibiting generation of melanin and biological pesticide - Google Patents

Abstract

The invention discloses the use of thiobabituric acid for inhibiting generation of melanin and a biological pesticide. In the invention, the activity of tyrosinase is measured by spectrophotometry, the inhibiting factor Km of the tyrosinase is measured by using L-DOPA as a substrate and by a Lineweaver-Burk method, the activity of the tyrosinase is measured by reaction of typically 1 milliliter of reaction substrate system and 10 milliliters of enzyme solution, and in the research, the activity of the tyrosinase is measured by using a PerkinElmerLambdaBio ultraviolet and visible spectrophotometer to detect an absorption value, namely the value of v is indicated by the change per mine of absorption value at a 492-nanometer position. In the use of the thiobabituric acid as the tyrosinase for inhibiting generation of melanin, a fluorescence emission spectrum is detected by using a cuvette with a 1-centimeter optical path for a JascoFP750 fluorescence spectrometer.

Description

Thiobarbituricacid is used to suppress melanin and generates and biological insecticides

Technical field

(Thiobarbituric acid is TBA) as the application of tyrosinase inhibitor at pharmaceuticals, cosmetics and food, agriculture field to the present invention relates to thiobarbituricacid.

Background technology

(EC 1. 14. 18. 1 for tryrosinase, Tyrosinase) be a kind of copper bearing metalloenzyme, being distributed widely in microorganism, animals and plants and the human body. tryrosinase is the important enzyme that has multiple catalysis during skin pigment synthesizes, this enzyme has oxygenase and oxidasic dual-use function concurrently, playing a crucial role in the melanin building-up process, is the rate-limiting enzyme of melanin building-up process.Tryrosinase belong to the 3rd class cuprein family ( 1. Yoon J, Fujii S, Solomon EI. Proc Natl Acad Sci U S A. 2009 Apr 21; 106 (16): 6585-90; 2. Li Y, Wang Y, Jiang H, Deng J. Proc Natl Acad Sci U S A. 2009 Oct 6; 106 (40): 17002-6.), link to each other with 3 histidine respectively at 2 copper ions of active site, and these 2 copper ions are directly related with different catalytic reactions, as single phenolic hydroxyl group turn to diphenol (cresolase activity) and diphenol be oxidized to diquinone (catechol oxidase active) ( 3. Decker, H. and Tuczek, F. (2000) Trends Biochem. Sci. 25,392-397.).The activity of tryrosinase and melanin synthetic quantity are closely related, the intravital activity of people increase back diseases such as melanin over-deposit such as causing freckle, chloasma generation ( 4. Olivares C, Solano F. Pigment Cell Melanoma Res. 2009 Dec; 22 (6): 750-60; 5. Jimbow K, Park JS, Kato F, Hirosaki K, Toyofuku K, Hua C, Yamashita T. Pigment Cell Res. 2000 Aug; 13 (4): 222-9.).Tryrosinase in the food (fruit and vegerable, shrimp Eriocheir sinensis etc.) can cause the brown stain in food processing and the storage, cause food spoilage ( 6. Kim YJ, Uyama H. Cell Mol Life Sci. 2005 Aug; 62 (15): 1707-23; 7. Rescigno A, Sollai F, Pisu B, Rinaldi A, Sanjust E. J Enzyme Inhib Med Chem. 2002 Aug; 17 (4): 207-18.).Tryrosinase also ubiquity in insect bodies, the insecticide wound healing ( 8. Kanost MR, Jiang H, Yu XQ. Immunol Rev. 2004 Apr; 198:97-105; 9. Lai SC, Chen CC, Hou RF. J Med Entomol. 2002 Mar; 39 (2): 266-74.) and epidermis formation ( 10. Guerrero, A. and Rosell, G. (2005) Curr. Med. Chem. 12,461-469.) in play an important role.

Since the potential application foreground of tryrosinase on medicine, cosmetics and agricultural, the focus of its activity regulation research becoming research.Up to now, develop polytype tyrosinase inhibitor, can be used as the candidate of exploitation brightening agent and biological insecticides.

At present, existingly much derive from the tyrosinase inhibitor of chemosynthesis and natural product as medicine, cosmetics or food additive and biological insecticides.The tyrosinase inhibitor of widespread usage has hydroquinone, kojic acid and derivant thereof, Azelaic Acid, arbutin, flavone compound, Vc and derivant thereof, green tea extract, Radix Glycyrrhizae extract etc.But existing tyrosinase inhibitor is in the defective of many-sided various degrees such as activity, stability or safety, and therefore active height is sought in continuation, safety tyrosinase inhibitor good, stable in properties still has crucial meaning.

Thiobarbituricacid (TBA) is a kind of antioxidant, as blood plasma with organize the index frequent application of inner lipid peroxidization and oxidative pressure, be called the TBARS identification and analysis ( 11. Y.J. Garcia et al./Journal of Neuroscience Methods 144 (2005) 127-135; 12. Gutteridge JM, Halliwell B. Trends Biochem Sci (1990) 15:129-34; 13. Janero DR/ Free Radic Biol Med (1990) 9:515-40.).It can analyze malonaldehyde, and as the variation of determining the oxidation of food medium oil alicyclic diradical ( 14. Guilllen-Sans R/ Crit Rev Food Sci Nutr. (1998) 38:315-30).TBA also be used to screen the HIV (human immunodeficiency virus) integrase inhibitor ( 15. S. Rajamake et al./Bioorg. Med. Chem. Lett. 19 (2009) 3615-3618).. therefore, we propose the inhibitory action of TBA to tryrosinase, and propose a kind of dynamics research and computer forecast TBA research approach that effect research combines in the tyrosinase catalysis reaction of suppressing.

Summary of the invention

The objective of the invention is to overcome the deficiency of above-mentioned existence, a kind of defective that overcomes existing tyrosinase inhibitor in stability, safety is provided, a kind of new good stability, the tyrosinase inhibitor that cytotoxicity is very small are provided, thereby suppress the application that the melanin in the melanocyte generates.

The objective of the invention is to finish by following technical solution, the used tryrosinase (M.W. 128 kDa) of the present invention is available from Sigma-Aldrich company.

The used thiobarbituricacid of the present invention (2-Thiobarbituric acid, TBA), molecular weight: 144.15, little yellow or little orange red powdery crystallization are dissolved in boiling water, the alkali liquor, storage at normal temperature, its chemical structural formula such as formula 1:

1. tyrosinase activity analysis

The mensuration of tryrosinase vigor adopts spectrophotography.We as substrate, adopt the Lineweaver-Burk method to record tryrosinase K with L-DOPA _m=0.51 ± 0.03 mM (V _Max=0.3 ± 0.01).Reaction is adopted typical 1ml reaction substrate system to add the 10ml enzymatic solution and is measured tyrosinase activity.In this research, detect absorption value and calculate enzymatic activity, promptly be used in 492nm place per minute light absorption value and change and indicate the v value by Perkin Elmer Lambda Bio ultraviolet-uisible spectrophotometer.

2. the tryrosinase noncompetitive suppresses dynamic analysis

Noncompetitive suppresses the Lineweaver-Burk equation such as the formula 2 of dynamic analysis: formula 3:

With [I] is abscissa, 1/V _MaxFor vertical coordinate carries out secondary mapping, as obtain straight line, illustrate in single position or similar position suppresses.

3. endogenous and external source fluoremetry

Fluorescence emission spectrum is detected with 1cm optical path cuvette by Jasco FP750 fluorescence spectrophotometer.Excitation wavelength detects tryptophan fluorescence at 280nm, detect wave-length coverage 300 to 410nm.Add final concentration in the enzymatic solution and hatch the external source fluorescence intensity situation of change that detects tryrosinase liquid behind the 30min for 40M ANS.Excitation wavelength is 390nm during the external source fluoremetry, detect wave-length coverage 400 to 520nm.50 mM buffer solution of sodium phosphate (pH 7.0) are all adopted in all kinetic reactions and detection.

4. the detection of thiobarbituricacid (TBA) and tryrosinase binding constant and binding site quantity

According to previous report ( 16. M.X. Xie, XY. Xu, Y.D. Wang, Biochim. Biophys. Acta. 1724 (2005) 215-224.), when the micromolecule equivalence was attached on the macromole, the equilibrium equation of free molecular flow and binding molecule was as follows, can be used for calculations incorporated constant (K) and binding site quantity (n), in the formula 4, and F ₀Relative intensity of fluorescence when reaction reaches balance when being respectively no fluorescence quencher and fluorescence quencher is arranged with F.[Q] is the concentration of fluorescence quencher (TBA).Mapping obtains intercept and slope based on equation [3], can be used to the value of calculating K and n.

5. the homology model of tryrosinase

According to report in the past carry out Mushroom Tyrosinase 3D structure prediction ( 17. Guo L, L ü ZR, Park D, Oh SH, Shi L, Park SJ, Bhak J, Park YD, Ren ZL, Zou F. J Biomol Struct Dyn. 2008 Dec; 26 (3): 395-402; 18. L ü ZR, Shi L, Wang J, Park D, Bhak J, Yang JM, Park YD, Zhou HW, Zou F. Appl Biochem Biotechnol. 2010 Apr; 160 (7): 1896-908.), adopt based on the mimic software MODELLER9v1 of homology generate comprise 556 amino acid whose tryrosinase threedimensional models ( 19. John, B. and Sali, A. (2003) Nucleic Acids Res. 31,3982-3992.).We are from the known tryrosinase homologous structure of Protein Data Bank Protein Data Bank (PDB) (http://www.pdb.org) retrieval, find 4 clauses and subclauses (PDB entry:1wxc, 1xom, 2oic, 2oid) stay in place form (average 26% sequence unanimity) for being suitable for has homeologous with tryrosinase.Adopt the ALIGN2D calibration tryrosinase of MODELLER program package and the sequence of template.Based on this sequence calibration, the 3D structure formation of tryrosinase has very high confidence level.Thereupon, we have adopted as discrete optimization protein energy (DOPE) score calculation of stability metric standard the structure energy of tryrosinase structural model.

6. the butt joint of computer simulation tryrosinase and TBA

In the computer simulation application tool of being useful on protein-ligand butt joint, AutoDock4 and DOCK6 are because its automatic butt performance applications is the most extensive.This program utilizes the default target protein 3D grid of a cover to finish the connection of part, AutoDock adopt at random search technique ( 20. Huey, R., Morris, G.M., Olson, A.J., and Goodsell, D.S. (2007) J. Comput. Chem. 28,1145-1152.) and DOCK adopt system search technique ( 21. Moustakas, D.T., Lang, P.T., Pegg, S., Pettersen, E., Kuntz, I.D., Brooijmans, N., and Rizzo, R.C. (2006) J. Comput. Aided Mol. Des. 20,601-619.).Therefore, we adopt two kinds of slightly different approach assessment tryrosinases and the combination of TBA.Primary TBA structure stem from PubChem data base (Compound ID:2723628) (http://www.pubchem.org) ( 22. Xie, X.Q. and Chen, J.Z. J. (2008) Chem. Inf. Model 48,465-475.).Need carry out before the butt joint step: 1) the 2D structure is converted to 3D structure, 2) the estimation electric charge, 3) the interpolation hydrogen atom, 4) the sunk area location.We use Transformation Program and OpenBabel (http://openbabel.sourceforge.org) in the J-Chem software kit (http://www.chemaxon.com/) in the above step.

7. HaCaT cell culture and MTT analyze

HaCaT cell line be the unlimited horn cell commonly used that goes down to posterity of a kind of energy ( 23. Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. J Cell Biol. 1988 Mar; 106 (3): 761-71.).(Gibco, Rockville cultivate in cell culture medium MD) this cell containing 10% hyclone FBS and 1% antibiotic-mould resistant.With the cytotoxicity of TBA in the MTT test kit analysis HaCaT cell of Sigma-Aldrich company purchase, operating process is carried out according to supplier's explanation.

The present invention will be aspect architectural feature, and TBA belongs to sulfur alcohol compound, and our result is similar to the remarkable inhibition phenomenon of tryrosinase with the sulfur alcohol compound of reporting before.We find that TBA is all highly stable in the aqueous solution of different temperatures, and very small to the skin keratin cytotoxicity.TBA suppresses to have represented to be different from the active another kind of pattern of copper ion chelating agen restraint of tyrosinase.Compare with the copper ion chelating agen restraint of tyrosinase activity of reporting before, TBA brings out the mechanism difference of inhibitory reaction, mainly contains two reasons: one, TBA is directly connected on the copper ion of avtive spot, therefore, does not have the competition with L-DOPA.Two, two of avtive spot copper ions are shared the tryrosinase residue that is derived from organism, but are present in the different connecting portions at avtive spot place as the connection residue of inhibitor.TBA can cause the complete deactivation of tryrosinase, does not follow the variation of tertiary structure.

TBA does not cause that the adjustment phenomenon reflection TBA of tryrosinase tertiary structure just is connected on the specific residue relevant with the L-DOPA catalytic action, and this position is present in the tyrosinase activity position, so this inhibitory action is not followed significant structural change.Because suppressing type is the noncompetitive suppressor mode, in avtive spot inside, the TBA binding site is different from the binding site of L-DOPA.TBA is not direct and the L-DOPA competition, but has influenced a series of catalytic process, and the connection site of hint TBA binding site and L-DOPA is very approaching.Based on above result, we have further carried out the aminoacid that the computer simulation prediction is connected with TBA, with the TBA inhibitory action mechanism that proves that we predict.

Computer simulation shows that TBA can directly form aglucon with the tryrosinase residue and combine complex.In the bonded first step of TBA, these amino acid residues are extremely important.Binding site is in active site, and the noncompetitive inhibitory action under TBA and L-DOPA substrate exist does not cause the significant change of tryrosinase conformation.

In sum, the result of the present invention's research is summarized as follows: 1) the TBA aglucon combines with tryrosinase, and the noncompetitive that causes tryrosinase suppresses, and it is obvious to suppress effect; 2) even in the presence of the TBA of high concentration, the tertiary structure that tryrosinase does not take place yet changes; 3) by the combining of computer simulation tryrosinase and TBA, dope the aminoacid of binding site, and these residues are positioned at active site; 4) TBA has represented the active newtype of restraint of tyrosinase to the inhibitory action of tryrosinase, and the new inhibitor and the biological insecticides for the treatment of cutaneous pigmentation for exploitation provide the research basis.

Description of drawings

Fig. 1 is a formula 1:

Fig. 2 is a formula 2:

Fig. 3 is a formula 3:

Fig. 4 formula 4:

Fig. 5 is TBA influences figure to the inhibition of tryrosinase;

Fig. 6 is v and [E figure];

Fig. 7 is the Lineweaver-Burk double reciprocal plot;

Fig. 8 be under the different temperatures during long-term storage TBA inhibition of tryrosinase is influenced figure;

The fluorescence intensity variation diagram of tryrosinase under the different TBA concentration of Fig. 9;

Figure 10 is F ₀/ (F ₀-F) vs. [Q] ^-1Double reciprocal plot;

The variation diagram of tryrosinase external source fluorescence among Figure 11 variable concentrations TBA

The bonded computer mould graphoid of Figure 12 tryrosinase and TBA;

The TBA toxicity test figure of Figure 13 people HaCaT keratinocyte.

The specific embodiment

Embodiment 1

TBA is to the influence of tyrosinase activity: suppress kinetics

The activity of tryrosinase is significantly suppressed (Fig. 5) in dose-dependent mode by TBA.When TBA concentration is higher than 40 mM, the activity of tryrosinase is suppressed fully.The IC that records ₅₀Value is 8.0 ± 1.0 mM.In order to analyze the reversibility that TBA suppresses, with residual enzyme vigor and [E] mapping (Fig. 6).The diagram result obtains 4 straight lines by initial point, shows that TBA is a reversible process to the inhibitory action of tryrosinase.For assessment suppresses type, with the two counting backward technique mappings of Lineweaver-Burk (Fig. 7).

The result shows, surperficial V _MaxChange, and K _mBe worth constantly, illustrate that TBA is that noncompetitive suppresses to the inhibition type of tryrosinase.Use Eq. [2] calculating K _iValue is 14.0 ± 8.5 mM.The experimental data match meets the expectation equation.

Then, be the stability of assessment TBA aqueous solution, we select for use-20,4 and 25 ℃ of three temperature conditions to preserve the TBA aqueous solutions respectively, and detect after the condition of different temperatures long preservation TBA to the inhibition situation of change (Fig. 8) of tryrosinase.The result shows that TBA is equal significantly restraint of tyrosinase activity after long preservation under these three temperature, proves that TBA is a kind of stable antioxidant.

Embodiment 2

TBA is to the influence of tryrosinase tertiary structure: spectrofluorimetry

The tertiary structure of tryrosinase in the presence of TBA changes by endogenous fluorescence and the bonded external source fluoroscopic examination of ANS.We find that TBA has caused the endogenous Quenching of fluorescence of tryrosinase, increase the fluorescence intensity peak value with TBA concentration and reduce, but displacement (Fig. 9) does not appear in peak value place wavelength.The fluorescence intensity peak value is to TBA concentration mapping display line sexual relationship (Figure 10).From above data, according to Eq.[3]., we calculate binding constant K=16.37 ± 1.3 x 10 ³M ^-1, become bond number n=2.12 ± 0.9.These results show that in the system that does not contain substrate, TBA has very strong adhesion to tryrosinase, and possible binding site has 2.

Next step, we have detected TBA and have had down the hydrophobic variation of tryrosinase (Figure 11).In the presence of TBA, even its concentration is when reaching 80mM, and the external source fluorescence intensity of tryrosinase does not have significant change, proves that the hydrophobic surface of tryrosinase under the TBA effect does not expose.Generally speaking, ANS can combine with hydrophobic amino acid residues as a kind of fluorescent dye, therefore can be used for detecting the destruction situation of enzyme tertiary structure in the presence of inhibitor.Even our result shows that the bonded external source fluorescence of ANS is no change still in (enzymatic activity is obviously suppressed under this concentration as can be seen from Figure 5) under the higher TBA inhibitor concentration, illustrate that TBA does not cause the significant change of tryrosinase tertiary structure.

Embodiment 3

The 3D structure of computer forecast tryrosinase and with the TBA docking simulation

Because the crystal structure of tryrosinase is not also illustrated, we select sequence identity respectively from the PDB Protein Data Bank be 25%, 29%, 26% and 25% 1wxc, 1xom, and 2oic and 2oid are as the 3D structure of stay in place form simulation tryrosinase.In the predict of tryrosinase, we are enlarged into 496 wxc with binding site ³, shown in yellow area among Figure 12.

Tryrosinase is simulated extremely successful (Autodock4 gained binding energy-4.75 kcal/mol, Dock6 binding energy-23.07 kcal/mol) with combining of TBA.Adopt Autodock4 and Dock6, we search for the residue that combines with TBA on tryrosinase, and these residues are very close on the position.We find that combining very important residue with TBA on the tryrosinase is PHE170 with Autodock4, THR175, VAL177, GLY251, PHE261 and ASP536, and be GLU250 and ASP536 (red frame part among Figure 13) with Dock6, the site of all finding in two programs is ASP536.

Locating in conjunction with residue and to calmodulin binding domain CaM of TBA on the tryrosinase identified in the computer docking simulation, for TBA produces evidence by the Mechanism Study that the noncompetitive suppressor mode acts on tryrosinase.And to record the result consistent for endogenous fluorescence among DOCK6 analog result and Fig. 5: binding site is 1 or 2 closely similar sites.

Embodiment 4

Adopt MTT to detect the toxicity of TBA to HaCaT cell line

After adopting variable concentrations TBA to cultivate the HaCaT cell, carry out the MTT test.We use TBA and act on the HaCaT cell, detect the toxicity of TBA to Skin Cell.The result shows that TBA does not have toxic effect to the HaCaT keratinocyte.This result is indicating that TBA can be applied to alleviate the synthetic imbalance of pigment (as hyperpigmentation) as a kind of effective brightening agent as the inhibitor of tryrosinase.

Shown in Figure 5: as to get the average and match of three secondary data.Tryrosinase joins in the TBA test system that contains respective concentration with after the TBA of variable concentrations mixes 2 hours.The final concentration of L-DOPA and enzyme is respectively 2 mM and 4 g/ml.

Shown in Figure 6: the representative of v value is in the variation of 492nm wavelength place per minute light absorption value.1 to No. 4 TBA concentration is respectively 0,5,10 and 15 mM.The final concentration of L-DOPA is 2 mM in the system.

Shown in Figure 7: TBA concentration is 0 10 (+) in the system, 20 and 27 mM.The tryrosinase final concentration is 4 g/ml.

X axle shown in Figure 8 is respectively 25 (a), the resting period of 4 (b) and-20 ℃ of (c) systems.The final concentration of L-DOPA and tryrosinase is respectively 2 mM and 4 g/ml in the system.

Endogenous change in fluorescence shown in Figure 9; Figure 10 is F ₀/ (F ₀-F) vs. [Q] ^-1Double reciprocal plot.

(A) endogenous change in fluorescence.Before the test, tryrosinase and TBA were hatched 3 hours jointly.Label 0 expression space state.Label 1 to 6 expression TBA concentration is respectively 0.02,0.03,0.04,0.05,0.08 and 0.1 mM.(B) F ₀/ (F ₀-F) vs. [Q] ^-1Double reciprocal plot.F ₀, blank fluorescence intensity peak value.F, sample fluorescence intensity peak value.Q, quencher TBA.Data come from (A).The final concentration of tryrosinase is 4 g/ml in the system.

Shown in Figure 11: test adds ANS (final concentration 40 M) in the protyrosinase and hatched 30 minutes in the darkroom, is used for the labelling hydrophobic surface.The final concentration of tryrosinase is 4 g/ml in the system.

Shown in Figure 12: the prediction of Mushroom Tyrosinase 3D structure is based on previous report.Yellow area indication active site, the TBA binding site of red block indication supposition.Light blue part is represented the combination of TBA under the AutoDock4 program, and pink colour is represented the combination of TBA under the Dock6 program.

Claims (3)

1. thiobarbituricacid suppresses the application that melanin generates as tryrosinase, and the mensuration of tryrosinase vigor adopts spectrophotography, and we as substrate, adopt the Lineweaver-Burk method to record tryrosinase K with L-DOPA _m=0.51 ± 0.03 mM, reaction is adopted typical 1ml reaction substrate system to add 10 l enzymatic solution and is measured tyrosinase activity, in this research, detect absorption value and calculate enzymatic activity, promptly be used in 492nm place per minute light absorption value and change and indicate the v value by Perkin Elmer Lambda Bio ultraviolet-uisible spectrophotometer.

2. suppress the application that melanin generates according to the described thiobarbituricacid of claim 1 as tryrosinase, it is characterized in that described fluorescence emission spectrum is detected with 1cm optical path cuvette by Jasco FP750 fluorescence spectrophotometer.

3. thiobarbituricacid is used for biological insecticides, uses TBA and acts on the HaCaT cell, detects the toxicity of TBA to Skin Cell.

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