CN109608392A - Stilbene class similar compound and its preparation method and application - Google Patents

Stilbene class similar compound and its preparation method and application Download PDF

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CN109608392A
CN109608392A CN201811534599.1A CN201811534599A CN109608392A CN 109608392 A CN109608392 A CN 109608392A CN 201811534599 A CN201811534599 A CN 201811534599A CN 109608392 A CN109608392 A CN 109608392A
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similar compound
stilbene class
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CN109608392B (en
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周涛
陈凯
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Zhejiang Gongshang University
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Abstract

The invention discloses stilbene class similar compound, general structure is following any:The present invention further simultaneously discloses the preparation method of above-mentioned stilbene class similar compound: using kojic acid as raw material, reacting to obtain quaternary phosphonium salt with triphenylphosphine after benzyl protection, chlorination；Quaternary phosphonium salt occurs wittig with benzaldehyde and reacts；Reaction gains are deprotected through Boron tribromide or reaction gains are reacted with the fatty amine of different carbon chain lengths and are deprotected again through Boron tribromide, obtain stilbene class similar compound.Stilbene class similar compound can serve as tyrosinase inhibitor, antibacterial agent, antioxidant etc..

Description

Stilbene class similar compound and its preparation method and application
Technical field
The present invention relates to a kind of agent for preservation of shrimp and its preparation method with stilbene class similar structures, the stilbene class similar compounds With tyrosinase inhibitory activity, antibacterial and the multiple biological activities such as anti-oxidant.
Background technique
Shrimps are a kind of high-moisture protein foods, are after death highly prone to the influence of the factors such as polyphenol oxidase, bacterium and send out Raw blacking and putrid and deteriorated.Polyphenol oxidase (PPO) such as tyrosinase in shrimp body is after death activated in shrimp, in aerobic conditions Under, single phenol substance contained in shrimps is oxidized to bisphenol by tyrosinase catalysis, and then is catalyzed and generates coloured quinone Then substance occurs a series of biochemical reactions and generates melanin, to cause the blacking of shrimp body.After shrimp body is dead, newly Protein, amino acid and other nitrogen substances in fresh shrimp, are easily decomposed into a variety of nitrogen-containing basics under the action of bacterium Product, and niff is generated, such as hydrogen sulfide, bad smell.Xi Washi spoilage organisms is advantage corruption common in aquatic products Bacterium can quickly breed after shrimp body is dead, have the rotten ability of extremely strong cause.In addition, protein, how unsaturated rouge in shrimps Oxidation deterioration easily occurs for the substances such as fat acid, while generating a large amount of free radicals, highly unstable, the chemical reaction with height Property, it is easy to it reacts with other molecules, and then generates more free radicals, further promote the rotten of shrimp.
Current existing agent for preservation of shrimp is numerous, multi-pass cross inhibit tyrosinase activity, antibacterial or antioxidation reach To preservation, such as 4- hexyl resorcin, arbutin tyrosinase inhibitor, the bacteriostatic agents such as ε polylysine, chitosan, The antioxidants such as resveratrol, ascorbic acid.But most antistaling agents only have single creature activity, and its fresh-keeping application is by certainly The limitation of body defect, such as price it is higher, be more toxic.Therefore, it is obtained by research and safe and efficient, cheap has anti-junket The antistaling agent of the multiple biological activities such as propylhomoserin enzyme, antibacterial is the work with huge meaning and value.
There is stronger tyrosinase to inhibit to live for kojic acid, entitled 5- hydroxyl -2- (methylol) -4 hydrogen-pyrans -4- ketone of chemistry The multiple biological activities such as property, antioxidant activity, are not only applicable in skin-lightening cosmetic, used also as food additives.Therefore, It is to obtain an important way of high activity food preservative, bacteriostatic agent and antioxidant by the structural modification to kojic acid molecule Diameter.
The structural formula with the compound compared with strong biological activity obtained by kojic acid molecular modification existing at present are as follows:
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of stilbene class similar compound and preparation method thereof, such compounds With good tyrosinase inhibitory activity, antibacterial activity and antioxidant activity；It can be used for the fresh-keeping of food in the food industry, It can be used as antibacterial agent in field of medicaments, be alternatively arranged as whitening agent for cosmetics industry.
In order to solve the above technical problem, the present invention provides a kind of stilbene class similar compounds (to have antityrosinase, antioxygen Change the stilbene class similar compound with multiple biological activities such as antibacterials), general structure is following general formula 1,2:
R in compound1Respectively 4-OH, 3,4-di-OH, 3,5-di-OH, R2For CnH2n+1(n=1-12).
As the improvement of stilbene class similar compound of the invention, structural formula be it is following any one:
The present invention also and meanwhile provide the preparation method of above compound, successively the following steps are included:
1), using kojic acid as raw material, react after benzyl protection, chlorination with triphenylphosphine quaternary phosphonium salt (that is, compound 6)；
2), quaternary phosphonium salt is reacted with benzaldehyde generation Wittig；
3), step 2) gains (that is, compound 7) are deprotected through Boron tribromide, and it is similar to obtain the stilbene class containing free hydroxyl group Compound 1；
Alternatively, step 2) gains (that is, compound 7) are reacted with the fatty amine of different carbon chain lengths, then through Boron tribromide The stilbene class similar compound 2 for being deprotected containing free hydroxyl group.
The present invention also simultaneously provide the purposes of above-mentioned stilbene class similar compound: as tyrosinase inhibitor, antibacterial agent, Antioxidant, the fresh-keeping or cosmetics for food.
The usage and dosage of stilbene class similar compound of the invention are identical as kojic acid.
Stilbene class similar compound of the invention is better than kojic acid to the preservation of shrimps.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is inhibiting effect comparison diagram of the compound 1c and 2h to diphenolase；
Fig. 2 is influence diagram of the compound 1c and 2h to bacterium S1 growth curve；
Fig. 3 is that compound 2h and citric acid cooperate with bacteriostasis figure；Concentration of the compound 2h in I-III group is respectively 10,20,40 μ g/mL, the concentration of citric acid are 50 μ g/mL.
Fig. 4 is Penaeus Vannmei L during 4 DEG C of storages*(A), a*(B), b*(C) variation diagram；
Fig. 5 is the variation diagram of Penaeus Vannmei Volatile Base Nitrogen (TVB-N) during 4 DEG C of storages；
Fig. 6 is the variation diagram of Penaeus Vannmei total number of bacteria during 4 DEG C of storages.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:
With kojic acid (3) be Material synthesis compound 1 (a-c) and the route of 2 (a-i) is as shown in Scheme1.
The preparation method of embodiment 1-1, compound 1c successively carry out following steps:
The synthesis of compound 4 is to be raw material with kojic acid (3) and synthesize (Design and by literature procedure synthesis of hydroxypyridinone-L-phenylalanine conjugates as potential tyrosinase inhibitors.Journal of Agricultural and Food Chemistry 2013,61(27), 6597-6603)。
Compound 4:5- benzyloxy -2- methylol-pyrans -4- ketone, yield 82.7%.1H NMR(400MHz,DMSO-d6,δ Ppm) δ: 4.30 (d, J=6.0Hz, 2H, CH2),4.96(s,2H,CH2), 5.67 (t, J=6.0Hz, 1H, OH), 6.34 (s, 1H, ), Py-H 7.34-7.44 (m, 5H, Ar-H), 8.17 (s, 1H, Py-H) .ESI-HRMS:m/z, calculated value C13H13O4([M+H]+) 233.0808 measured value 233.0830.
A, the synthesis of compound 5
It weighs 30g (129.72mmol) compound 4 (5- benzyloxy -2- methylol-pyrans -4- ketone) and is dissolved in 100mL chlorination In sulfoxide, in room temperature reaction 2h, after reaction, filters, obtain pale yellow precipitate, room temperature is dry after being washed with acetone (about 100mL) It is dry to constant weight, obtain white solid -- compound 5.Yield is 72.5%.
Compound 5:5- benzyloxy -2- chloromethyl-pyrans -4- ketone, yield 72.5%.1H NMR(400MHz,DMSO)δ: 4.68(s,2H,CH2),4.96(s,2H,CH2),6.58(s,1H,Py-H),7.38(m,5H,Ar-H),8.30(s,1H,Py-H) .ESI-HRMS:m/z, calculated value C13H12ClO3([M+H]+) 251.0469, measured value 251.0480.
B, the synthesis of compound 6
20g (79.9mmol) compound 5 is weighed respectively and 20g (79.9mmol) triphenylphosphine is added to equipped with 150mL first In the round-bottomed flask of benzene, the monitoring of heating reflux reaction 12h, TLC contact plate.A large amount of white precipitates are generated in reaction process, after suction filtration It is washed with paraxylene (about 100mL), drying at room temperature to constant weight, obtains white compound crude product (that is, 6 crude product of compound) 28.64g is not purified and is directly used in the next step.
C, the synthesis of compound 7c
It weighs 6 crude product of 3.078g (6mmol) compound to be added into 10mLDMF, uncle 0.808g is added under nitrogen protection Butanol potassium, is stirred at room temperature 30min.1.097g 3,5- dimethoxy benzaldehyde, in 70 DEG C of reaction 7h is added.It passes through after reaction Silica gel column chromatography obtains compound 7c, yield 78.6%.
The specific steps of compound 7c crude product silica gel column chromatography purifying are as follows: with petrol ether/ethyl acetate (volume ratio of 4:1) As eluent；Collect Rf=0.45 solution, boils off solvent, obtains sterling 7c.
The structural formula of 7c are as follows:
5- benzyloxy -2- (2 (3,5- dimethoxy-phenylf)-vinyl)-pyrans -4- ketone (7c): yield 78.6%.1H NMR(500MHz,CDCl3,δppm)δ:3.84(s,6H,2OCH3),5.10(s,2H,CH2),6.40(s,1H,Py-H),6.48 (s, 1H, Ar-H), 6.63 (d, J=16.5Hz, 1H, CH=CH), 6.64 (d, J=1.0Hz, 2H, Ar-H), 7.26 (d, J= 16.5Hz, 1H, CH=CH), 7.38 (m, 5H, Ar-H), 7.55 (s, 1H, Py-H) .ESI-HRMS:m/z, calculated value C22H21O5 ([M+H]+) 365.1384, measured value 365.1377.
D, the synthesis of compound 1c
It weighs 0.364g (1mmol) compound 7c to be dissolved in the three-neck flask equipped with 10mL methylene chloride, connection nitrogen dress It sets, makes reaction system full of nitrogen.Under the conditions of 0 DEG C, 10mL is slowly added dropwise using constant pressure funnel and contains 1mmol/mL tri- The dichloromethane solution (time for adding is about 10~12 minutes) of boron bromide, is then gradually warmed up (heating-up time is about 2 hours) To room temperature, react overnight (12h).Appropriate methanol (about 10mL) is slowly added dropwise after reaction is completely dissolved precipitating, then revolves Solvent (that is, removing methylene chloride, methanol) is evaporated off, with methanol ether (methanol: ether=1:2 volume ratio) recrystallization three It is secondary, obtain compound 1c, yield 92.6%.
5- hydroxyl -2- [2- (3,5- dihydroxy-phenyl)-vinyl]-pyrans -4- ketone (1c) yield 92.6%.1H NMR (400MHz,DMSO-d6, δ ppm) and δ: 6.50 (m, 2H, Ar-H), 6.54 (s, 1H, Py-H), 6.69 (d, J=2.8Hz, 1H, Ar- H) 6.93 (d, J=16.4Hz, 1H, CH=CH), 7.56 (d, J=16.4Hz, 1H, CH=CH), 8.10 (s, 1H, Py-H)13C NMR(100MHz,CD3OD,δppm)102.43,104.88,105.36,113.37,122.99,133.56,136.16, 139.91,146.35,155.75,157.67,160.83,174.55.ESI-HRMS:m/z calculated value C13H11O4([M+H]+) 247.0601 measured value 247.0611.
Embodiment 1-2,
3,5- dimethoxy benzaldehyde in embodiment 1-1 step C is changed to 4-methoxybenzaldehyde, 3,4- diformazan respectively Oxygroup benzaldehyde, remaining is equal to embodiment 1-1；To obtain compound 1a, 1b accordingly.
The structural formula of compound 1a are as follows:
The structural formula of compound 1b are as follows:
The preparation method of embodiment 2, compound 2h successively carries out following steps:
The synthesis of compound 4 is to be raw material with kojic acid (3) and synthesize (Design and by literature procedure synthesis of hydroxypyridinone-L-phenylalanine conjugates as potential tyrosinase inhibitors.Journal of Agricultural and Food Chemistry 2013,61(27), 6597-6603)。
Compound 4:5- benzyloxy -2- methylol-pyrans -4- ketone: yield 82.7%.1H NMR(400MHz,DMSO-d6,δ Ppm) δ: 4.30 (d, J=6.0Hz, 2H, CH2),4.96(s,2H,CH2), 5.67 (t, J=6.0Hz, 1H, OH), 6.34 (s, 1H, ), Py-H 7.34-7.44 (m, 5H, Ar-H), 8.17 (s, 1H, Py-H) .ESI-HRMS:m/z, calculated value C13H13O4([M+H]+) 233.0808 measured value 233.0830.
A, the synthesis of compound 5
It weighs 30g (129.72mmol) compound 4 (5- benzyloxy -2- methylol-pyrans -4- ketone) and is dissolved in 100mL chlorination In sulfoxide, in room temperature reaction 2h, a large amount of pale yellow precipitates are filtered to obtain after reaction, after being washed with a large amount of acetone (100mL) White solid -- compound 5, yield 72.5%.
Compound 5:5- benzyloxy -2- chloromethyl-pyrans -4- ketone (4): yield 72.5%.1H NMR(400MHz,DMSO) δ:4.68(s,2H,CH2),4.96(s,2H,CH2),6.58(s,1H,Py-H),7.38(m,5H,Ar-H),8.30(s,1H,Py- H) .ESI-HRMS:m/z, calculated value C13H12ClO3([M+H]+) 251.0469, measured value 251.0480.
B, the synthesis of compound 6
20g (79.9mmol) compound 5 is weighed respectively and 20g (79.9mmol) triphenylphosphine is added to equipped with 150mL first In the round-bottomed flask of benzene, the monitoring of heating reflux reaction 12h, TLC contact plate.A large amount of white precipitates are generated in reaction process, after suction filtration It is washed with paraxylene (about 100mL), drying at room temperature obtains white compound crude product (that is, 6 crude product of compound) 28.64g, does not purify It is directly used in the next step.
C, the synthesis of compound 7c
It weighs 6 crude product of 3.078g (about 6mmol) compound to be added into 10mL DMF, 0.808g is added under nitrogen protection 30min is stirred at room temperature in potassium tert-butoxide.1.097g 3,5- dimethoxy benzaldehyde, in 70 DEG C of reaction 7h is added.After reaction Compound 7c, yield 78.6% are obtained through silica gel column chromatography.
The specific steps that compound 7c crude product silica gel column chromatography is purified are as follows: with the petrol ether/ethyl acetate (volume of 4:1 Than) it is used as eluent；Collect Rf=0.45 solution boils off solvent and obtains sterling 7c.
The structural formula of 7c are as follows:
5- benzyloxy -2- [2 (3,5- dimethoxy-phenylf)-vinyl]-pyrans -4- ketone (7c): yield 78.6%.1H NMR(500MHz,CDCl3,δppm)δ:3.84(s,6H,2OCH3),5.10(s,2H,CH2),6.40(s,1H,Py-H),6.48 (s, 1H, Ar-H), 6.63 (d, J=16.5Hz, 1H, CH=CH), 6.64 (d, J1=1.0Hz, 2H, Ar-H), 7.26 (d, J= 16.5Hz, 1H, CH=CH), 7.38 (m, 5H, Ar-H), 7.55 (s, 1H, Py-H) .ESI-HRMS:m/z, calculated value C22H21O5 ([M+H]+) 365.1384, measured value 365.1377.
D, the synthesis of compound 8h
It weighs 1.82g (5mmol) compound 7c and (ethyl alcohol: water=1:1) is added into 20mL ethanol water, add 1g Sodium hydroxide and 0.56g (5.5mmol) n-hexylamine, are condensed back 8h, and TLC monitors reaction process.After reaction, big portion is boiled off Divide solvent (that is, most of ethyl alcohol, water being boiled off, until about the 30% of original volume), resulting reaction solution is extracted with dichloromethane 3 It is secondary, merge organic layer, through saturated common salt water washing 2-3 times, boils off solvent after crossing anhydrous sodium sulfate, obtain chemical combination through silica gel column chromatography Object 8h, yield 40.8%.
The specific steps of compound 8h crude product silica gel column chromatography purifying are as follows: with methylene chloride/methanol (volume ratio of 50:1) As eluent；Collect Rf=0.42 solution boils off solvent and obtains sterling 8h.
The structural formula of 8h are as follows:
5- benzyloxy -2- [2 (3,5- dimethoxy-phenylf)-vinyl] -1- hexyl-pyridine -4- ketone (8h): yield 40.8%.1H NMR(500MHz,CDCl3, δ ppm) and δ: 0.87 (t, J=5.5Hz, 3H, CH3),1.23(m,6H,3CH2),1.62 (m,2H,CH2), 3.78 (t, J=6.5Hz, 2H, CH2),3.85(s,6H,2OCH3),5.24(s,2H,CH2), 6.49 (t, J= 1.5Hz, H, Ar-H), 6.62 (d, J=2.0Hz, 2H, Ar-H), 6.70 (s, 1H, Py-H), 6.82 (d, J=16.0Hz, 1H, CH =CH), 6.92 (s, 1H, Py-H), 7.02 (d, J=16.0Hz, 1H, CH=CH), 7.38 (m, 6H, Ar-H) .ESI-HRMS:m/ Z, calculated value C28H34NO4([M+H]+) 448.2482, measured value 448.2495.
E, the synthesis of compound 2h
It weighs 0.447g (1mmol) compound 8h to be dissolved in the three-neck flask equipped with 10mL methylene chloride, connection nitrogen dress It sets, makes reaction system full of nitrogen.Under the conditions of 0 DEG C, 10mL is slowly added dropwise using constant pressure funnel and contains 1mmol/mL tri- The dichloromethane solution (time for adding is about 10~12 minutes) of boron bromide, is then gradually warmed up (heating-up time is about 2 hours) To room temperature, react overnight (12h).Appropriate methanol (10mL) is slowly added dropwise after reaction is completely dissolved precipitating, then rotates Solvent (that is, removing methylene chloride, methanol) is removed, three times with methanol ether (methanol: ether=1:2 volume ratio) recrystallization, Obtain compound 2h, yield 95.6%.
5- hydroxyl -2- [2 (3,5- dihydroxy-phenyl)-vinyl] -1- hexyl-pyridine -4- ketone (2h): yield 95.6% .1H NMR(500MHz,CD3OD, δ ppm) δ: 0.91 (t, J=7.5Hz, 3H, CH3),1.36(m,6H,3CH2),1.85(m,2H, CH2), 4.47 (t, J=7.5Hz, 2H, CH2), 6.34 (s, 1H, Ar-H), 6.62 (d, J=2.0Hz, 2H, Ar-H), 7.20 (d, J =16.0Hz, 1H, CH=CH), 7.32 (d, J=16.0Hz, 1H, CH=CH), 7.43 (s, 1H, Py-H), 8.12 (s, 1H, Py- H).13C NMR(125MHz,CD3OD,δppm)12.87,22.10,25.47,29.93,30.89,56.26,104.23, 105.90,110.35,116.64,130.77,136.71,140.60,144.82,146.39,159.69,160.00.ESI- HRMS:m/z, calculated value C19H24NO4([M+H]+) 330.1700, measured value 330.1713.
Embodiment 2-2,
3,5- dimethoxy benzaldehyde in embodiment 2-1 step C is changed to 4-methoxybenzaldehyde or 3,4- bis- respectively N-hexylamine in step D is changed to n-butylamine or n-octyl amine by methoxybenzaldehyde respectively, remaining is equal to embodiment 2-1；To It is corresponding to obtain compound 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2i.
The structural formula of compound 2a are as follows:
The structural formula of compound 2b are as follows:
The structural formula of compound 2c are as follows:
The structural formula of compound 2d are as follows:
The structural formula of compound 2e are as follows:
The structural formula of compound 2f are as follows:
The structural formula of compound 2g are as follows:
The structural formula of compound 2i are as follows:
Experiment 1, the measurement of compound 1 and 2 pair Mushroom Tyrosinase single phenol enzyme inhibition activity
Tyrosinase single phenol enzyme activity determination bibliography (Design and synthesis of hydroxypyridinone-L-phenylalanine conjugates as potential tyrosinase inhibitors.Journal of Agricultural and Food Chemistry 2013,61(27),6597-6603) Method and slightly improve: tyrosinase single phenol enzyme activity determination is with 2mMLTyrosine is substrate, and total live body system is 300 μ L is separately added into (30 DEG C of substrate solution of 100 μ L 2mM in the phosphate buffer (PBS (pH 6.86)) that 180 μ L are prepared in advance Heat preservation) and a series of 10 various concentrations of μ L compound 1 or 2 solution (0.0313-1.0mM is previously dissolved in DMSO), finally it is fast 10 μ L Mushroom Tyrosinase solution (enzyme activity 500u/mL) are added in speed, and 30 DEG C of guarantors in constant incubator are transferred quickly to after mixing Warm 10min measures absorbance value at 475nm.Solution needed for testing follows ready-to-use principle, tests parallel 3 times and makes even Mean value.The composition of the reaction system of difference group is specifically such as table 1.
1 reaction solution system of table
Compound calculates the active inhibiting rate of tyrosinase monophenolase as follows:
Inhibiting rate (%)=[1- (OD3-OD4)/(OD1-OD2)] × 100%
Wherein OD1、OD2、OD3And OD4The absorbance value of respectively first to the 4th group of solution.
Compound increases, single phenol enzyme activity the active inhibiting effect of tyrosinase monophenolase with the increase of compound concentration Property inhibiting rate be 50% when compound concentration (IC50) it is shown in Table 2.The single phenol enzyme inhibition activity of compound 1c and 2h are most strong, respectively It is 8.03 and 4.60 times of kojic acid.
The tyrosinase inhibitory activity of 2 compound 1 and 2 of table
It chooses the preferable compound 1c and 2h of tyrosinase single phenol enzyme inhibition activity and measures it to tyrosinase diphenolase Inhibitory activity (experiment 2) inhibits type and inhibition constant (experiment 3).
Test 2, measurement of the compound 1c and 2h to Mushroom Tyrosinase diphenol enzyme inhibition activity
Diphenol enzyme activity determination bibliography (Design and synthesis of hydroxypyridinone-L- phenylalanine conjugates as potential tyrosinase inhibitors.Journal of Agricultural and Food Chemistry 2013,61 (27), 6597-6603) method: tyrosinase diphenol enzyme activity Power measurement is with 0.5mMLDOPA is substrate, and total live body system is 300 μ L, in the phosphate buffer that 180 μ L are prepared in advance The change of substrate solution (the 30 DEG C of heat preservations) and a series of 10 various concentrations of μ L of 100 μ L 2mM is separately added into (PBS (pH 6.86)) It closes object 1c or 2h solution (0.0313-1.0mM is previously dissolved in DMSO), is eventually adding 10 μ L Mushroom Tyrosinase solution (enzyme activity It for 500u/mL), mixes rapidly, the reaction system change of absorbance value at any time at 475nm is detected under 30 DEG C of constant temperatures Change, determining instrument is the full-automatic microplate reader of Infinite M200.It is plotted against time with absorbance value, the straight line portion of curve obtained The slope divided is enzyme activity, is mapped with the relative surplus vigor of enzyme to inhibitor concentration, and the relative surplus vigor of enzyme is 50% When corresponding inhibitor concentration be inhibitor rate IC half-suppressed50.Acquired results are as shown in Figure 1.
The relative activity of enzyme reduces to some extent with the increase of compound 1c or 2h concentration, illustrates that 1c or 2h are inhibited The vigor of tyrosinase diphenolase, and concentration is bigger, rejection ability is stronger.Suppression of the compound 1c to tyrosinase o-diphenolase activity Concentration (the IC of compound when rate processed is 50%50) be 7.15 μm of ol/L, 2h concentration (IC50) it is 15.86 μm of ol/L；Through than Relatively know that 1c is better than 2h to the rejection ability of diphenolase.Concentration (the IC of existing pyridone ketone derivatives 9a, 9b, 9c50) respectively For 4.00,8.97,26.20 μm of ol/L.
Experiment 3,1c and 2h inhibit the measurement of type and inhibition constant to tyrosinase diphenolase
Measuring method and 2 measuring methods of experiment are essentially identical, and the concentration of fixed tyrosinase changes additionLDOPA Amount measures influence of the various concentration 1c or 2h to enzyme activity.It is dense with substrate using the bis- counting backward technique mappings of Lineweaver-Burk The inverse 1/ [S] of degree is X-axis, using 1/ υ of inverse of reaction speed as Y-axis, to judge the inhibition type of inhibitor.With the oblique of straight line Rate and the intercept in Y-axis map again to inhibitor concentration, find out inhibitor to the inhibition constant K of resolvase (E)IWith it is right The inhibition constant K of enzyme-substrate complex (ES)IS。
With the increase of 1c and 2h concentration, not only make the maximum reaction velocity (V of enzymatic reactionmax) become smaller, also result in Michaelis Constant (Km) become larger, it is known that 1c and 2h is mixed type to the inhibition type of tyrosinase diphenolase.Compound 1c and 2h is to junket ammonia Sour enzyme diphenolase inhibits type and inhibition constant to be shown in Table 3.
The tyrosinase diphenolase of table 3 compound 1c and 2h inhibit type and inhibition constant
Test the Determination of Antibacterial Activity of 4, compound 1c and 2h
It is raw by measurement antibacterial circle diameter size, minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and bacterium Long curve experiments research compound 1c and 2h is for being isolated from the dominant spoilage organisms genus Shewanella S1 of Penaeus Vannmei The antibacterial activity of (Shewanella amazonensis).And compound 2h and citric acid are studied for the Synergistic antimicrobial of bacterium S1 Effect.
The preparation of bacteria suspension: the freezing for being stored in -80 DEG C is taken out for examination strain and is thawed in room temperature.Use liquid-transfering gun (pipette tips sterilizing) is drawn 200 μ L bacterium solutions and is added separately in sterilized 50mL pancreas peptone soybean broth, 30 DEG C, 180r/min Constant-temperature table culture 18-24h.A small amount of bacteria suspension is dipped in solid agar medium table after sterilization and cooling with aseptic inoculation ring Face scribing line, has single colonie generation after guaranteeing culture.Inoculated solid medium is placed in 30 DEG C of constant incubators Cultivate 16-18h.With the good single colonie secondary inoculation of aseptic inoculation ring picking thalli morphology into soybean broth, again in 30 DEG C, 180r/min constant-temperature table culture 18-24h.Activated bacterium solution is subjected to 10 times of gradient method dilutions, until reaching most suitable It is spare to be placed in 4 DEG C of refrigerators for growth concentration.
1, minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) measurement
After minimal inhibitory concentration (MIC) refers in vitro culture bacterium 18-24h, it is able to suppress the minimum drug of bacterial growth Concentration, for measuring bacterium for the sensitivity of drug.The specific method is as follows: pipetting 900 μ L using liquid-transfering gun and has sterilized greatly A series of compound solution (the compound 1c of 80 various concentrations of μ L is then added into teat glass (13 × 100mm) in beans meat soup And the final concentration of 0-1mg/mL of 2h, the final concentration of 0-4mg/mL of kojic acid), being subsequently added into 20 μ L bacteria suspensions, (bacterial concentration is 105CFU/mL), so that mixed solution final volume is 1mL, rock mixing and be placed in 30 DEG C of constant incubators and cultivate for 24 hours.Culture After detect by an unaided eye, it is minimal inhibitory concentration (MIC) that sample solution final concentration representated by muddy test tube does not occur, often Group test is parallel three times.
Do not occur to move respectively in muddy test tube (sample solution final concentration >=MIC) from minimal inhibitory concentration (MIC) test Take 100 μ L solution coatings on solid medium, constant temperature incubation observes on culture medium whether have bacterium colony for 24 hours afterwards under the conditions of 30 DEG C Growth.If there is bacterium colony is grown, illustrate still there is bacteria living in corresponding MIC test tube, if sterile length of being born on culture medium, Then compound solution final concentration representated by this test tube is minimum bactericidal concentration (MBC), and every group of test is in parallel three times.
Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) test result are shown in Table 4.
Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 4 compound of table to S1 bacterial strain
2, inhibition zone test (filter paper agar diffusion method)
This test is using filter paper agar diffusion method test compound 1c and 2h for the inhibiting effect of bacterium S1.Specific side Method is as follows:
(1) filter paper is broken into the circular filter paper piece that diameter is 6mm with punch, is dried for standby after sterilizing.
(2) certain initial concentration solution is prepared according to test requirements document, carries out gradient dilution, wherein compound 1c and 2h concentration For 10,20,40mg/mL, kojic acid 10,20,40,80mg/mL.It is molten that filter paper after sterilizing is soaked in various concentration compound In liquid, drying is placed in spare under aseptic condition after immersion 2h.To be soaked in the filter paper of sterile water as blank control.
(3) drawing 1mL concentration is 105CFU/mL bacteria suspension sterilizes to 20mL and is cooled to 50 DEG C of solid agar medium In, it is poured into immediately into plate after mixing, to its cooled and solidified under aseptic condition.
(4) it with the susceptibility filter paper of tweezers clamping various concentration, is put down gently on corresponding solid agar medium, gently Light press filter paper is bonded it completely with culture medium.Test uses 4 methods, i.e., places 4 small filter paper dicks in each plate, Respectively test-compound 1c and 2h, kojic acid (positive control) and sterile water (negative control).Then each group plate is placed in It is cultivated in 30 DEG C of constant incubators for 24 hours, using vernier caliper measurement antibacterial circle diameter, each processing is parallel three times.Test result It is shown in Table 5.
Inhibition zone test result of 5 compound of table to S1 bacterial strain
By table 4, table 5 it is found that 1c and 2h is obviously stronger than that kojic acid to the inhibiting effect of genus Shewanella bacterium S1.
3, to the influence of growth curve of bacteria
It takes a certain amount of test bacterium solution in the sterile soy broth of 50mL, is separately added into the compound of 1mL 5mg/mL 1c and 2h, makes its final concentration of 100 μ g/mL after mixing, 30 DEG C, 180r/min shaking table culture measured bacteria suspension every 2 hours OD600Value, with incubation time (hour) for abscissa, OD600Value is ordinate, the growth curve of bacterium S1 is drawn, not add The bacteria suspension of compound is as blank control.
From Figure 2 it can be seen that the absorbance value of 1c processing group is significantly less than blank control group each culture period (removing 0h), Illustrate that compound 1c has stronger inhibiting effect for bacterium S1.In addition, the bacterium of 2h processing group in entire culture period not There is apparent OD value variation, also do not occur logarithmic phase, illustrates that concentration is that the compound 2h of 100 μ g/mL has been completely inhibited The growth of bacterium S1.
4, bacteriostatic test is cooperateed with
270 μ L are pipetted with liquid-transfering gun and have sterilized soybean broth into the 96 clean orifice plates that sterilize, and it is molten that 12 μ L citric acids are added Liquid (50 μ g/mL of final concentration) and 12 μ L various concentration compound 2h solution (final concentration of 10,20,40 μ g/mL), are eventually adding 6 μ L bacteria suspension (105CFU/mL), level rock mixing after in 30 DEG C of constant incubator cultures for 24 hours.After culture, from each examination It tests in group to pipette 100 μ L reaction solutions and be diluted to suitable concentration with 10 times of methods and carry out plate coating and count, measurement is added different dense When spending 2h, bacteriostasis is cooperateed with citric acid.Every group of test is in parallel three times.Blank control group replaces compound with sterile water 2h and citric acid solution.
As seen from Figure 3, citric acid cooperates with bacteriostasis with significant with when 2h compatible use.Concentration is 40 μ g/mL's The other bacterial concentration of group that inhibitor 2h and citric acid use simultaneously is 0Log10(CFU/mL), illustrate the other bacterial growth of the group Breeding has been totally constrained.
Experiment 5, the measurement of antioxidant activity
By measurement compound 1c and 2h to the clearance rate of DPPH free radical, hydroxyl radical free radical and ultra-oxygen anion free radical To evaluate its antioxidant activity.
1, the measurement of DPPH free radical scavenging activity
Bibliography method (Mei-Jia Shi, Xiaoyi Wei, Jie Xu, Bing-Jie Chen, De-Yin Zhao, Shuai Cui,Tao Zhou.Carboxymethylated Degraded Polysaccharides from Enteromorpha prolifera:Preparation and in Vitro Antioxidant Activity.Food Chemistry 2017,215,76-83.) it is measured.
2, the measurement of hydroxyl radical free radical clearance rate
Bibliography method (Mei-Jia Shi, Xiaoyi Wei, Jie Xu, Bing-Jie Chen, De-Yin Zhao, Shuai Cui,Tao Zhou.Carboxymethylated Degraded Polysaccharides from Enteromorpha prolifera:Preparation and in Vitro Antioxidant Activity.Food Chemistry 2017,215,76-83.) it is measured.
3, the measurement of ultra-oxygen anion free radical clearance rate
Bibliography method (Bing-Jie Chen, Mei-Jia Shi, Shuai Cui, Shu-Xian Hao, Robert C Hider,Tao Zhou.Improved antioxidant and anti-tyrosinase activity of polysaccharide from Sargassum fusiforme by degradation.International Journal Of Biological Macromolecules.2016,92,715-722.) it is measured.
Compound 1c and 2h shows good Scavenging activity to three kinds of free radicals, they are to DPPH and hydroxyl radical free radical Scavenging capacity much stronger than 9b and 9c (table 6).
Free radical scavenging activity (the IC of table 6. compound 1c and 2h50(mg/mL))
Experiment 6, the fresh-keeping experiment of Penaeus Vannmei
Make by taking compound 2h as an example, and with kojic acid and commercially available " the fresh treasured of shrimp " (principle active component 4- hexyl resorcin) For positive control.The sample pretreatment of Penaeus Vannmei: by fresh and alive prawn (the long 10.5 ± 0.5cm of average body, weight 10.0 ± It 0.5g) is immersed in 30min in ice water to clean, then be grouped sample at random spare.
The fresh-keeping experiment of Penaeus Vannmei: kojic acid solution (1mg/mL) is prepared respectively, 4- hexyl resorcin solution (1mg/ ML), compound 2h solution (1mg/mL), 1mg/mL compound 2h+0.1% ascorbic acid solution, 1mg/mL compound 2h+ 0.1% citric acid solution, using ultrapure water as blank control.Each group shrimp sample is soaked in respectively in 6 kinds of different solutions, It pulls out to drain to be filled in sterilized antistaling bag after 10min and be stored in 4 DEG C of refrigerators.It measures within (2,4,6,8,10,12 days) in storage phase The indices of different group shrimp samples.
1, color change measures
The color at each test group difference storage phase shrimp sample carapace is measured using color difference analysis instrument, parallel 3 times.It uses CIE Lab system indicates the color of sample, L*The brightness of representative sample, range 0-100, numerical value illustrate sample closer to 0 Color is darker, brighter closer to 100；a*The red value of green of representative sample indicates color sample from-a*(green) arrives+a*(red) Variation；b*The champac value of representative sample indicates color sample from-b*(blue) arrives+b*The variation of (yellow).
By Fig. 4 A it is found that the L of blank group and each processing group*All decline as time increases.The L of blank control group*Under Drop quickly, its L at the 4th day*Value has dropped to 37.66.The L of treated with kojic acid group*Value starts to be decreased obviously on day 4, the 6th day and Respectively reach 37.06 and 33.63 within 12nd day.In storage phase, it is 4-HR < 2h < 2h+ that each processing group, which inhibits the ability of shrimp body blacking, 0.1%AA < 2h+0.1%CA, L*Value respectively 36.05,37.03,37.83,38.04, the above results explanation at the 12nd day Compound 2h has the stronger ability for inhibiting the blacking of shrimp body compared to 4-HR；The addition of ascorbic acid and citric acid then further mentions The high anti-shrimp body blacking ability of formula, substantially improves the visual experience of shrimp body.
By Fig. 4 B it is found that all groups of a*Value all rises with the increase of storage time, illustrates shrimp body carapace color Gradually turn red, wherein the variation of blank control group is most obvious, its a at the 4th day*Value has risen to 0.90, hence it is evident that is greater than everywhere Reason group (P < 0.05).At the 12nd day, a of treated with kojic acid group*Value is 3.23, less than the 4.26 of blank control group, but is far longer than 2.62,2.41,2.13 and the 2.05 of other 4 processing groups.Storage phase 0-12 days, 2h+0.1%AA and 2h+0.1%CA processing group a*The ascendant trend of value is significantly less than 4-HR, 2h, a of the processing group of 4-HR, 2h at the 12nd day*Value is 2.62 and 2.41, and 2h+ 0.1%AA and 2h+0.1%CA processing group is only 2.13 and 2.05.
As shown in Figure 4 C, b*The variation tendency and a of value*It is worth similar, shrimp body all xanthochromias with the extension of time, and 2h+ 0.1%AA and 2h+0.1%CA processing group has strongest inhibition shrimp body xanthochromia ability.At the 12nd day, 2h+0.1%AA and 2h+ The b of 0.1%CA processing group*Value is respectively 3.86 and 3.78, is lower than blank control group, treated with kojic acid group, 4-HR and 2h processing group 9.70,6.36,5.18 and 4.26.
2, the measurement of total number of bacteria
GB 4789.2- is referred to the measuring method of the total number of bacteria in the shrimp sample of each test group difference storage time 2016 " Food Microbiology detects total plate count measurement ".Method particularly includes: weigh 5g shrimp in sterile homogenizing bag, then plus Enter 45mL sterile saline, obtains the even liquid of 1:10 sample after patting 1-2min with sterile homogenizer.Using 10 times of dilution methods, use The even liquid of sample is diluted to 10 by sterile saline-2、10-3、10-4、10-5、10-6Deng the even liquid of sample.It is a suitable dilute to choose 2-3 The even liquid of sample for releasing concentration draws 100 μ L respectively and adds on the counting solid culture medium of sterilized solidification and be coated, often A dilution makees two plates, in 37 DEG C of 48 ± 2h of culture.Plate of the clump count within the scope of 30CFU~300CFU is chosen to carry out It counts, result is the average value of two plates.
The variation of total number of bacteria (Total bacterial count, TBC) of the Penaeus Vannmei sample in storage phase is such as Shown in Fig. 5, the initial bacterial count of Penaeus Vannmei sample is about 3.24lg (CFU/g), and the TBC of each processing group all with when Between increase and increase.In storage phase (0-12 days), secondly it is treated with kojic acid group that the TBC increase of blank control group is most fast.2h The TBC of processing group is significantly less than 4-HR processing group (P < 0.05) in storage phase 8-12 days, illustrates that compound 2h has compared with 4-HR Stronger bacteriostasis.In 0-12 days, the TBC of 2h and 2h+0.1%AA processing group is without significant difference (P > 0.05).Entirely storing up Hide interim, the TBC of 2h+0.1%CA processing group is both less than other processing groups.General total number of bacteria reaches 6lg (CFU/g) and is recognized To reach corrupt limit, blank control group, treated with kojic acid group, 4-HR, 2h and 2h+0.1%AA processing group are respectively at storage phase 6,8,10 and 12 days are more than corrupt limit, and TBC at 2h+0.1%CA processing group the 12nd day is only 5.82, still not up to corruption Limit shows strong bacteriostasis.
3, the measurement of Volatile Base Nitrogen (TVB-N)
Bibliography method (Chen B J, Zhou Y J, Wei X Y, et al.Edible antimicrobial coating incorporating a polymeric iron chelator and its application in the preservation of Surimi product[J].Food&Bioprocess Technology,2016,9(6):1031- 1039.) be measured.
Penaeus Vannmei sample in TVB-N value situation of change in storage phase as shown in fig. 6, each group TVB-N value all with The increase of time dramatically increases (P < 0.05).Blank control group and the TVB-N value of kojic acid control group were respectively at the 2nd, 4 day Shi Mingxian Increase, the 8th day when respectively reaches 47.22 and 37.36mg/100g, alreadys exceed national standard GB2733-2015 " food safety country Standard is fresh, freezes animality aquatic products " specified in the TVB-N values of seawater fishes and shrimps must not exceed the requirement of 30mg/100g.4-HR, The TVB-N value of 2h, 2h+0.1%AA and 2h+0.1%CA processing group started to occur in storage phase the 8th day significantly to increase (P < 0.05), the 12nd day when 4-HR, 2h, 2h+0.1%AA processing group TVB-N value be more than 30mg/100g, respectively reach 40.09, 37.42,34.16, and the TVB-N value (27.33mg/100g) of 2h+0.1%CA processing group is still below 30mg/100g.
In summary multiple index of fish freshness, the compound of the present invention 2h prevent refrigeration (4 DEG C) South America white right with stronger The effect of shrimp corruption blacking, and be better than widely used shrimp antistaling agent 4- hexyl resorcin, and by the shelf of Penaeus Vannmei Phase extends to 10 days.And 2h compatible use 0.1%CA can further extend the shelf life of shrimp to 12 days.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure All deformations for directly exporting or associating, are considered as protection scope of the present invention.

Claims (7)

1. stilbene class similar compound, it is characterized in that general structure is following any:
R in compound1It is following any: 4-OH, 3,4-di-OH, 3,5-di-OH；
R2For CnH2n+1(n=1-12).
2. stilbene class similar compound according to claim 1, it is characterized in that structural formula is following any:
3. the preparation method of stilbene class similar compound as claimed in claim 1 or 2, it is characterized in that:
1), using kojic acid as raw material, quaternary phosphonium salt is reacted to obtain with triphenylphosphine after benzyl protection, chlorination；
2), quaternary phosphonium salt is reacted with benzaldehyde generation wittig；
3), step 2) gains are deprotected through Boron tribromide, obtain the stilbene class similar compound 1 containing free hydroxyl group；
Alternatively, step 2) gains are reacted with the fatty amine of different carbon chain lengths, then it is deprotected containing free through Boron tribromide The stilbene class similar compound 2 of hydroxyl.
4. the purposes of stilbene class similar compound as claimed in claim 1 or 2, it is characterized in that: being used as tyrosinase inhibitor.
5. the purposes of stilbene class similar compound as claimed in claim 1 or 2, it is characterized in that: being used as antibacterial agent.
6. the purposes of stilbene class similar compound as claimed in claim 1 or 2, it is characterized in that: being used as antioxidant.
7. the purposes of stilbene class similar compound as claimed in claim 1 or 2, it is characterized in that: for the fresh-keeping of food or makeup Product.
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