CN109134704A

CN109134704A - A kind of application of chitosan oligosaccharide thiourea derivatives, nano silver prepared therefrom and the nano silver

Info

Publication number: CN109134704A
Application number: CN201810939378.6A
Authority: CN
Inventors: 徐翠莲; 王彩霞; 樊素芳; 潘振良; 苏同福; 金秋; 姜松; 吴璐璐; 吴自豪; 陈亚鹏
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2018-08-17
Filing date: 2018-08-17
Publication date: 2019-01-04

Abstract

Chitosan oligosaccharide thiourea derivatives provided by the invention are the compounds such as following formula；Wherein 7≤n≤23, n are integer:

Description

A kind of chitosan oligosaccharide thiourea derivatives, nano silver prepared therefrom and the nano silver Using

Technical field

The invention belongs to chitosan oligosaccharide derivative applied technical fields, and in particular to a kind of chitosan oligosaccharide thiourea derivatives, by The application of its nano silver prepared and the nano silver.

Background technique

In plant three classes disease (fungi, bacterium and virus disease), disease sum is accounted for about as caused by disease fungus 70%-80%, every year because crop yield loss caused by fungal disease is very big.As wheat 200 multiple diseases in, fungal disease 90% is just accounted for, loses the 10-20% for accounting for total output caused by annual；The maize leaves as caused by ralstonia solanacearum are withered too early to be made Corn yield loses 20-30%, even serious loss is up to 50% or more total crop failure.Although constantly there is new bacteriostatic agent to develop Come, but easily drug resistance makes the development of new drug be still a highly important task.

Nano silver grain is because of the extensive use in the fields such as antibacterial, catalysis and surface-enhanced Raman and always by pass Note.Currently, since abuse of antibiotics causes threat caused by antibiotic antagonism increasingly serious, so that active to nano silver antibacterial Research interest recovery.Nano silver is to produce bacteriostasis of the silver under nm regime silver nanoparticle using forward position nanotechnology Qualitative leap is given birth to.The performance of nano silver and its partial size have direct relation.In general, partial size is smaller, bactericidal property is stronger.It receives Rice silver sterilization has the following characteristics that 1, broad-spectrum antibacterial, and nano-Ag particles are directly entered thallus and oxygen metabolism enzyme (- SH) combines, and makes The unique mechanism of action that thallus is choked to death can kill the microorganisms such as most of bacteriums, fungi, mould, the spore being in contact with it. 2, strong bactericidal, it is found by researches that nano silver can kill 650 various bacterias in several minutes.3, permeability is strong, nano-Ag particles Permeability with super strength can rapidly permeate into subcutaneous 2mm sterilization, draw to common bacteria, stubborn bacteria, drug-resistant bacteria and fungi The tissue infection of the higher depth risen has good bactericidal effect.4, reparative regeneration, nano silver can promote wound healing, promote by Damage the reparation and regeneration of cell.5, antibacterial persistently nano-Ag particles are produced using patented technology, have layer protecting film outside, in people It can gradually discharge in vivo, so fungistatic effect is lasting.6, it has no drug resistance, nano silver belongs to non-antibiotic fungicide, can kill each Kind pathogenic microorganisms, the nano-Ag particles unique antimicrobial mechanism of 10nm size can directly kill rapidly bacterium, its is made to lose breeding Ability can not produce the next generation of drug resistance, can effectively avoid leading to recurrent exerbation obstinate because of drug resistance.

These antibacterial features of nano silver undoubtedly open wide prospect to the extensive use of nano silver antibacterial, are newest The natural bacteriostatic agent of a generation.

The preparation method of nano silver can be mainly divided into two major classes by reaction mechanism point: physical method and chemical method.Object Reason method mainly makes silver-colored simple substance become nano-grade size with physical means such as mechanical lapping, radiation etc..Such as: Xu et al.- High-energy mechanical ball milling is carried out to silver powder in a low temperature of 196 DEG C, has obtained the silver particle powder that average grain diameter is about 20nm；Chang Et al. in NaCl solution use laser ablation plasma silver strip, make its rupture, at the same be stirred continuously prevent ablation formed nano silver Particle agglomeration can prepare the Argent grain of average grain diameter 26nm.Although various physical method principles are simple, due to instrument and equipment Requirement it is high, producing cost is expensive, is restricted its use.Chemical method is to be widely used in preparing the side of nano silver Method mainly has chemical reduction method, physics reduction method and biological reducing method.Chemical reduction method is current laboratory and industrial use Compare one of extensive method.Silver is restored from its compound or salt with reducing agent, the silver salt of use is mainly AgNO₃Or its complex, reducing agent mainly have sodium borohydride, hydrazine hydrate, formaldehyde, polyalcohol, ascorbic acid and more active than Ag Metal etc., be commonly incorporated into the control product such as dispersing agent or protective agent polyvinylpyrrolidone (PVP), sorbierite, polyvinyl alcohol Partial size and shape.In electronation agents useful for same in use may to environment or the harmful effect of organism, because This, environmental-friendly, green Nano silver grain preparation method urgently occurs.Zhang Shengmao etc. is environmental-friendly according to ionic liquid Characteristic, synthesizes silver nano-particle in ionic liquid at room temperature, and experiment intermediate ion liquid serves not only as solvent and as dressing agent Prevent the reunion of silver nano-particle.Chemical synthesis also can be used to prepare nano silver material, Zhou etc. utilizes ultraviolet radiation AgNO₃, Silver nanorod and dendroid nanocrystal are prepared under the protection of polyvinyl alcohol.Yin etc. utilizes microwave process for synthesizing, Trisodium citrate uses formaldehyde that can make the relatively narrow nano-silver powder of various particle diameter distributions as reducing agent in the state of existing.In general, Nanoparticle is easy to happen aggregation because of its hydrophobicity in forming process, in order to synthesize the metal nanoparticle of good dispersion, Need to be added stabilizer during the preparation process, such as: macromolecule polyalcohol, dendrimer, polyelectrolyte, surfactant. Due to there are a large amount of free amino and hydroxyls in chitosan oligosaccharide molecular structure, thus with pH controllable dissolubility polycation, The performance of chelating and easily film forming and hydrogel.Chitosan is often used as protective agent in the preparation process of metal nanoparticle, Because of its water solubility and property similar with hydrophilic polymer, can be adsorbed in the preparation process of nanoparticle metal from The surface of son forms complex, to play the role of stabilizer.

Chitosan is derivative of the chitin through deacetylated processing that it is obtained from the shell and cell wall of crustacean, It is from a wealth of sources, rich reserves, low-cost native biopolymer material.As a kind of biological polysaccharide, chitosan is same When there is the characteristics such as biodegradable, bio-compatible and nontoxic, antibacterial, anticoagulation, amino rich in molecular structure And hydroxyl.But since chitosan is not soluble in water, certain acid and a small number of organic solvents are dissolved only in, its use scope critical constraints is made. Degradation of chitosan is chitosan oligosaccharide, though water-soluble have improvement, the promotion of its bacteriostasis property has little effect.Therefore, to chitosan oligosaccharide It is modified very necessary with chemical modification.Current method of modifying has quaternization, acylation, carboxylated etc..Wherein in relation to one The report of serial chitooligosaccharidequaternary quaternary ammonium salt derivative antifungal activity is shown, compared with chitosan, all quaternized chitosan oligosaccharides spread out Biology has better bacteriostatic activity, and maximum suppression index is in vitro 86.7%.The antifungal mechanism of chitooligosaccharidequaternary quaternary ammonium salt More complicated, Tokura thinks the acid high score of the chitooligosaccharidequaternary quaternary ammonium salt with quaternary ammonium group cation and bacterial cell wall surface Son interaction, can form polyelectrolyte polymer, formed in cell peripheral and do not have infiltrative surface layer, prevent nutrition Material permeance cell wall and make bacterium not and can be carried out metabolism and cause its death.

In in the past few decades, chitosan oligosaccharide thiourea is because having good bacteriostasis property to have been a concern. Eweis, Elkholy and Elsabee are prepared for chitosan oligosaccharide thiourea derivatives (TUCS), biological activity test as a result, it has been found that these Compound, which has experiment fungi, significantly inhibits effect.The application is that bibliography method prepares oligosaccharides thiourea derivative, and benefit It uses it as reducing agent and protective agent and nitric acid silver reaction prepares stable, mono-dispersed nano silver solution at a lower temperature, and Study its inhibitory activity to common crop pathogens.

Summary of the invention

To solve the above-mentioned problems, the present invention provide a kind of chitosan oligosaccharide thiourea derivatives, nano silver prepared therefrom and The application of the nano silver synthesizes chitosan oligosaccharide thiourea derivatives using chitosan oligosaccharide as raw material, is then used as reducing agent and stabilizer, Prepare Nano silver solution.The optimum reaction condition for preparing nano silver is probed into, and its structure is characterized, finally to chitosan oligosaccharide Thiourea derivatives and the Nano silver solution of preparation carry out bacteriostatic activity test to several frequently seen plant pathogen.

The object of the present invention is achieved in the following manner:

A kind of chitosan oligosaccharide thiourea derivatives, the chitosan oligosaccharide thiourea derivatives are the compounds such as following formula；Wherein 7≤n≤ 23, n be integer:

R is

A method of above-mentioned chitosan oligosaccharide thiourea derivatives are prepared, chitosan oligosaccharide is first dissolved in ethanol solution and ammonium hydroxide In, sequentially add CS₂Solution, sodium chloroacetate and substrate, stirring while adding, chitosan oligosaccharide elder generation and CS₂It is thio that solution reaction generates two Carbamic acid chitosan oligosaccharide ammonium, aminodithioformic acid chitosan oligosaccharide ammonium is reacted with sodium chloroacetate generates carboxymethyl dithio carbamic acid Chitosan oligosaccharide sodium, carboxymethyl dithio carbamic acid chitosan oligosaccharide sodium generate chitosan oligosaccharide thiourea derivatives with substrate reactions again.

Such as the above-mentioned method for preparing chitosan oligosaccharide thiourea derivatives, comprising the following steps:

(1) it dissolves chitosan oligosaccharide: chitosan oligosaccharide being added in 95% ethanol solution and stirred evenly, obtain mixture I, by mixture I It is added in ammonium hydroxide, stirring while adding, after mixture I adds, solution is in dark-brown viscous liquid, continue to stir 0.5-1h, Obtain mixture II；Chitosan oligosaccharide unit and 95% ethanol solution molal volume ratio be (0.010-0.060) mol:(15-60) The molal volume ratio of mL, chitosan oligosaccharide unit and ammonium hydroxide is (0.010-0.060) mol:(10-40) mL；

(2) aminodithioformic acid chitosan oligosaccharide ammonium is synthesized: by CS₂Solution is added drop-wise in mixture II, stirs 2-3h, solution colour Deepen, obtains mixture III, chitosan oligosaccharide unit and CS₂Molar ratio be (0.010-0.060) mol:(0.03-0.10) mol；

(3) it synthesizes carboxymethyl dithio carbamic acid chitosan oligosaccharide sodium: sodium chloroacetate is added in mixture III, stir 0.5- 2h, obtains mixture IV, and the molar ratio of chitosan oligosaccharide unit and sodium chloroacetate is (0.010-0.060) mol:(0.03-0.15) mol；

(4) chitosan oligosaccharide thiourea derivatives are synthesized: aminated compounds is added in mixture IV, chitosan oligosaccharide unit and amine The molar ratio of object is closed as (0.010-0.060) mol:(0.03-0.12) mol, solution colour becomes dark brown, is stirred at room temperature 2-4h is reacted, reaction solution is added in 50-100mL dehydrated alcohol, has brown precipitate precipitation, decompression filters, with anhydrous second Alcohol washs filter cake repeatedly, obtains product；Product is dried in vacuo 10-12h at 50-60 DEG C, it is pure to obtain chitosan oligosaccharide thiourea derivatives Product.

The aminated compounds is hydrazine hydrate, ethylenediamine, ethanol amine, phenylhydrazine, 2-aminopyridine or methyl hydrazine.

The hydrazine hydrate aqueous solution that the hydrazine hydrate is 80%.

A method of nano silver being prepared using above-mentioned chitosan oligosaccharide thiourea derivatives, it is by chitosan oligosaccharide Thiourea Derivative solution and AgNO₃Solution reaction generates nano silver.

The following steps are included: chitosan oligosaccharide thiourea derivatives solution and AgNO₃Solution mixes, chitosan oligosaccharide sulphur in mixed solution Carbamide derivative and AgNO₃Unit molar ratio be (2-8): (3-9), at 20-60 DEG C react 30-100min obtain nano silver.

In the mixed solution, the concentration of chitosan oligosaccharide thiourea derivatives is 0.001-0.01mol/L, AgNO₃Concentration For 0.001-0.04

mol/L。

A kind of nano silver, it is to be prepared using the above method.

Such as application of the above-mentioned nano silver in terms of plant is antibacterial.

Compared with the existing technology, the present invention is using the chitosan oligosaccharide thiourea derivatives of synthesis as reducing agent and stabilizer system For nano silver, and phenylthiosemicarbazide chitosan oligosaccharide (A4) reduction is individually probed into and has prepared the molten influence factor of nano silver: certain item Under part, temperature is higher, and reaction is rapider；Phenylthiosemicarbazide chitosan oligosaccharide (A4) concentration is bigger under certain condition, generates nano silver (B4) Absorption peak is stronger；Under certain condition, the dosage of silver nitrate has optimal selection, not The more the better；

Using ultraviolet-visible absorption spectroscopy, Nano silver solution is prepared by tracking chitosan oligosaccharide-hydrazine hydrate thiourea derivative reduction Process, basic understandings have understood the forming process of nano silver；

The pattern of different nano silvers is observed by transmission electron microscope photo, 6 kinds of chitosan oligosaccharide thiourea derivatives are obtained to be received Rice Argent grain is the spheric granules less than 10nm or so, and Nano silver solution shows as transparent blood red.Demonstrate chitosan oligosaccharide Thiourea derivatives reduction prepares the feasibility of nano silver method；Bacteriostatic activity test is carried out to drug and nano silver to probe into, The power of the bacteriostatic activity of basic understanding chitosan oligosaccharide thiourea derivatives and Nano silver solution.Chitosan oligosaccharide derivative after modification And its Nano silver solution formed is higher than unmodified chitosan oligosaccharide activity.

Detailed description of the invention

Fig. 1 is chitosan oligosaccharide and ammonium sulfo-amino thiocarbamide chitosan oligosaccharide ultra-violet absorption spectrum, wherein 1 is chitosan oligosaccharide, 2 be that ammonium is thio Thiosemicarbazides chitosan oligosaccharide.

Fig. 2 is chitosan oligosaccharide, ammonium sulfo-amino thiocarbamide chitosan oligosaccharide and phenylthiosemicarbazide chitosan oligosaccharide (A4) ultra-violet absorption spectrum, In 1 be chitosan oligosaccharide, 2 be ammonium sulfo-amino thiocarbamide chitosan oligosaccharide, and 3 be phenylthiosemicarbazide chitosan oligosaccharide (A4).

Fig. 3 is chitosan oligosaccharide, ammonium sulfo-amino thiocarbamide chitosan oligosaccharide and pyridine -2- thiosemicarbazides chitosan oligosaccharide (A5) UV absorption light Spectrum, wherein 1 is chitosan oligosaccharide, 2 be ammonium sulfo-amino thiocarbamide chitosan oligosaccharide, and 3 be N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide (A5).

Fig. 4 is chitosan oligosaccharide, ammonium sulfo-amino thiocarbamide chitosan oligosaccharide and methylamino thiocarbamide chitosan oligosaccharide (A6) ultra-violet absorption spectrum, In 1 be chitosan oligosaccharide, 2 be ammonium sulfo-amino thiocarbamide chitosan oligosaccharide, and 3 be methylamino thiocarbamide chitosan oligosaccharide (A6).

Fig. 5 is chitosan oligosaccharide, phenylthiosemicarbazide chitosan oligosaccharide (A4) infrared absorption spectrum comparison diagram.

Fig. 6 is chitosan oligosaccharide, the comparison of pyridine -2- thiosemicarbazides chitosan oligosaccharide (A5) infrared absorption spectrum.

Fig. 7 is chitosan oligosaccharide, methylamino thiocarbamide chitosan oligosaccharide (A6) infrared absorption spectrum comparison diagram.

Fig. 8 is the UV-Vis absorption spectrum of thiosemicarbazides chitosan oligosaccharide (A1) reduction preparation of silver nanoparticle (B1), arrow in figure Head is followed successively by thiosemicarbazides chitosan oligosaccharide (A1) and AgNO from top to bottom₃Reaction starts 0min, 10min, 20min, 30min, 40min Generate the ultra-violet absorption spectrum of nano silver.

Fig. 9 is that thiosemicarbazides chitosan oligosaccharide (A1) reduction prepares nano silver (B1) solution with the color change of time, from a left side It is 0min, 10min, 20min, 30min, 40min since reaction to the right side.

Figure 10 is that the UV-Vis of various concentration phenylthiosemicarbazide chitosan oligosaccharide (A4) reduction preparation of silver nanoparticle (B4) absorbs Spectrum, wherein 1:0.001mol/L, 2:0.002mol/L, 3:0.003mol/L, 4:0.004mol/L, 5:0.005mol/L.

Figure 11 be various concentration phenylthiosemicarbazide chitosan oligosaccharide (A4) (1:0.001mol/L, 2:0.002mol/L, 3: 0.003mol/L, 4:0.004mol/L, 5:0.005mol/L) color for preparing nano silver (B4) compares.

Figure 12 is the uv-visible absorption spectra that N- (2- pyridine)-thiocarbamide (A5) prepares nano silver (B5) process, wherein 1.N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide (A5)；2.0min (reaction starts)；3.10min；4.20min；5.30min； 6.40min；7.48h.

Figure 13 is that N- (2- pyridine)-thiocarbamide (A5) prepares the color of nano silver (B5) and changes with time, from left to right according to Secondary is 0min, 10min, 20min, 30min, 40min.

Figure 14 is the uv-visible absorption spectra that methylamino thiocarbamide chitosan oligosaccharide (A6) prepares nano silver (B6) process, wherein 1. methylamino thiocarbamide chitosan oligosaccharide (A6)；2.0min (reaction starts)；3.10min；4.20min；5.30min；6.40min； 7.48h。

Figure 15 is that methylamino thiocarbamide chitosan oligosaccharide (A6) prepares the color of nano silver (B6) and changes with time, from left to right for 0min, 10min, 20min, 30min, 40min since reaction.

Figure 16 is the scanning electron microscope result that thiosemicarbazides chitosan oligosaccharide (A1) reduction prepares nano silver (B1).

Figure 17 is the electron-microscope scanning result that N- (2- aminoethyl)-thiocarbamide (A2) reduction prepares nano silver (B2).

Figure 18 is the electron-microscope scanning result that phenylthiosemicarbazide chitosan oligosaccharide (A4) does that reducing agent prepares nano silver (B4).

Figure 19 is the electron-microscope scanning result that N- (2- pyridine)-thiocarbamide (A5) does that reducing agent prepares nano silver (B5).

Figure 20 is the electron-microscope scanning result that methylamino thiocarbamide chitosan oligosaccharide (A6) does that reducing agent prepares nano silver (B6).

Specific embodiment

Because thiocarbamide structure has mild reproducibility, the present invention is with low molecular weight chitosan oligosaccharide (molecular weight 1100-3600) Primary raw material has synthesized the synthesis of chitosan oligosaccharide thiourea derivative, and is respectively that reducing agent exists with the chitosan oligosaccharide thiourea derivative of preparation It is prepared for stable Nano silver solution at room temperature, its structure is characterized.

Chemical equation of the invention is as follows:

A1 are as follows:A2 are as follows:A3 are as follows:A4 are as follows:A5 are as follows:A6 are as follows:

A kind of chitosan oligosaccharide thiourea derivatives, the chitosan oligosaccharide thiourea derivatives are the compounds such as following formula；Wherein 7≤ N≤23:

R is

(2) it synthesizes aminodithioformic acid chitosan oligosaccharide ammonium: solution is added drop-wise in mixture II, stir 2-3h, solution colour adds It is deep, obtain mixture III, chitosan oligosaccharide unit and molar ratio be (0.010-0.060) mol:(0.03-0.10) mol；

The hydrazine hydrate aqueous solution that the hydrazine hydrate is 80%.

In the mixed solution, the concentration of chitosan oligosaccharide thiourea derivatives is 0.001-0.01mol/L, AgNO₃Concentration For 0.001-0.04mol/L.

A kind of nano silver, it is to be prepared using the above method.

Embodiment: chitosan oligosaccharide derivative is synthesized under homogeneous phase condition:

Embodiment 1: the synthesis of thiosemicarbazides chitosan oligosaccharide A1 and Nano silver solution B1

Weigh chitosan oligosaccharide 8.0g (unit molal quantity 0.05mol) in a round bottom flask, after the ethyl alcohol dissolution of 20.0mL 95%, 15.0mL (0.04mol) ammonium hydroxide is slowly added in the solution under stirring, stirs 0.5h at room temperature；Then by CS2 (3.8mL, 0.06mol)) it is added drop-wise in above-mentioned solution, reaction 2h is stirred at room temperature；It is again that sodium chloroacetate (5.8g, 0.05mol) is slow Slowly it is added in the reaction solution, reacts 0.5h；Then measure again 80% hydrazine hydrate 6.0mL (0.12mol) be slowly added into it is above-mentioned In reaction solution, it is stirred to react 3h；Finally reaction solution is poured slowly into about 50mL anhydrous methanol, has steel gray flocculent deposit to analyse Out, it stands, removes supernatant, use dehydrated alcohol instead and wash repeatedly 7 times, filter, obtain product, be dried in vacuo (50 DEG C, 7h), obtain 5.97g (unit molal quantity 0.04mol) cinerous powder.

10mL (unit molal weight 235, unit molal quantity 0.034mmol) thiosemicarbazides chitosan oligosaccharide A1 solution and 1.5mL AgNO3 solution (10mg/mL, 0.088mmol) mixing, the concentration of thiosemicarbazides chitosan oligosaccharide A1 solution are 0.8mg/mL；It mixes molten In liquid, the unit molar concentration of thiosemicarbazides chitosan oligosaccharide A1 is 0.0029mol/L, and the concentration of AgNO3 is 0.0077mol/L, in 60min is reacted at 20 DEG C obtains nano silver B1.

Embodiment 2: the synthesis of thiosemicarbazides chitosan oligosaccharide A1 Nano silver solution B1

Weigh chitosan oligosaccharide 1.6g (unit molal quantity 0.01mol) in a round bottom flask, after the ethyl alcohol dissolution of 15.0mL 95%, 35.0mL (0.04mol) ammonium hydroxide is slowly added in the solution under stirring, stirs 0.8h at room temperature；Then by CS2 (1.9mL, 0.03mol)) it is added drop-wise in above-mentioned solution, reaction 2.5h is stirred at room temperature；Again by sodium chloroacetate (3.48g, 0.03mol) It is slowly added into the reaction solution, reacts 1h；Then measure again 80% hydrazine hydrate 1.5mL (0.03mol) be slowly added into it is above-mentioned In reaction solution, it is stirred to react 2h；Finally reaction solution is poured slowly into about 50mL anhydrous methanol, has steel gray flocculent deposit to analyse Out, it stands, removes supernatant, use dehydrated alcohol instead and wash repeatedly 7 times, filter, obtain product, be dried in vacuo (50 DEG C, 7h), obtain 1.19g (unit molal quantity 0.008mol) cinerous powder.

10mL (unit molal weight 235, unit molal quantity 0.034mmol) thiosemicarbazides chitosan oligosaccharide A1 solution and 1.0mL The mixing of AgNO3 (10mg/mL, 0.059mmol) solution, the concentration of thiosemicarbazides chitosan oligosaccharide A1 solution are 0.8mg/mL；It mixes molten In liquid, the unit molar concentration of thiosemicarbazides chitosan oligosaccharide A1 is 0.0029mol/L, and the concentration of AgNO3 is 0.0053mol/L, in 30min is reacted at 30 DEG C obtains nano silver B1.

The synthesis of embodiment 3:N- (2- aminoethyl)-thiocarbamide chitosan oligosaccharide A2 and Nano silver solution B2

Chitosan oligosaccharide (8g, unit molal quantity 0.05mol) is weighed to be slowly added in 95% ethyl alcohol (20mL) after stirring and dissolving, by solution It is slowly added in ammonium hydroxide (20mL, 0.52mol), is in dark-brown viscous liquid, stirs 0.5h at room temperature；Then by CS2 (3.8mL, It 0.06mol) is slowly dropped in mixed liquor and is stirred to react 2h；Weigh sodium chloroacetate (6g, 0.05mol) be slowly added into it is above-mentioned anti- It answers in liquid, continues to be stirred to react 0.5h；It measures ethylenediamine (6mL, 0.09mol) again to be added drop-wise in reaction solution, color becomes blackish green Color is stirred for reaction 3h at room temperature.Finally reaction solution is slowly added into about 50mL dehydrated alcohol, there have to be light grey cotton-shaped heavy Precipitation goes out, and stands, and removes supernatant, is washed repeatedly with dehydrated alcohol 8 times, filters, and is eluted 3 times with dehydrated alcohol, will obtain Product vacuum it is dry (55 DEG C, 8h), obtain khaki powder 9.96g (unit molal quantity 0.06mol).

The solution of 10mL (unit molal weight 262, unit molal quantity 0.032mmol) N- (2- aminoethyl)-thiocarbamide chitosan oligosaccharide A2 It is mixed with 1.5mL AgNO3 (10mg/mL, 0.088mmol) solution, the concentration of chitosan oligosaccharide derivative A2 solution is 0.85mg/mL； In mixed solution, N- (2- aminoethyl)-thiocarbamide chitosan oligosaccharide A2 unit molar concentration is 0.0028mol/L, the concentration of AgNO3 For 0.0077mol/L, 70min is reacted at 30 DEG C and obtains nano silver B2.

The synthesis of embodiment 4:N- (2- aminoethyl)-thiocarbamide chitosan oligosaccharide A2 and Nano silver solution B2

Chitosan oligosaccharide (9.6g, unit molal quantity 0.06mol) is weighed to be slowly added in 95% ethyl alcohol (60mL) after stirring and dissolving, it will be molten Liquid is slowly added in ammonium hydroxide (40mL, 0.52mol), is in dark-brown viscous liquid, is stirred 1h at room temperature；Then by CS2 (6.33mL, 0.10mol), which is slowly dropped in mixed liquor, is stirred to react 3h；Sodium chloroacetate (18g, 0.15mol) is weighed slowly to add Enter into above-mentioned reaction solution, continues to be stirred to react 2h；It measures ethylenediamine (6mL, 0.09mol) again to be added drop-wise in reaction solution, color Become blackish green, is stirred for reaction 4h at room temperature.Finally reaction solution is slowly added into about 100mL dehydrated alcohol, is had shallow Grey flocculent deposit is precipitated, and stands, and removes supernatant, is washed repeatedly with dehydrated alcohol 8 times, filters, and elutes 3 with dehydrated alcohol It is secondary, obtained product vacuum is dry (60 DEG C, 10h), obtain khaki powder 10.79g (unit molal quantity 0.065mol).

12mL (unit molal weight 262, unit molal quantity 0.046mmol) chitosan oligosaccharide derivative A2 solution and 1.0mL AgNO3 The mixing of (10mg/mL, 0.0059mmol) solution, the concentration of chitosan oligosaccharide derivative A2 solution are 1.0mg/mL；In mixed solution, shell The concentration of oligosaccharide derivative A2 is 0.0035moL/L, and the concentration of AgNO3 is 0.0045mol/L, and 70min is reacted at 25 DEG C and is obtained Nano silver B2.

The synthesis of embodiment 5:N- (2- ethoxy)-thiocarbamide chitosan oligosaccharide A3 and Nano silver solution B3

Chitosan oligosaccharide (8g, unit molal quantity 0.05mol) is weighed to be slowly added in 95% ethyl alcohol (25mL) after stirring and dissolving, by solution It is added in ammonium hydroxide (25mL, 0.65mol) under stirring, stirs 0.5h at room temperature；Then by CS2 (3.9mL, 0.065mol) Solution is slowly dropped in the mixed liquor and is stirred to react 2h；Sodium chloroacetate (6.2g, 0.053mol) is weighed again to be slowly added into It states in solution, continues to be stirred to react 0.5h；Then it measures ethanol amine (6mL, 0.10mol) to be slowly added into reaction solution, room temperature Under react 3h again；Finally reaction solution is slowly added into 55mL dehydrated alcohol, has brown flocculent deposit precipitation, stands, removal Supernatant is washed 6 times repeatedly with dehydrated alcohol, is filtered, and product vacuum is dry (60 DEG C, 9h), obtains light brown powder 4.22g (unit molal quantity 0.03mol).

8mL (unit molal weight 264, unit molal quantity 0.027mmol) chitosan oligosaccharide derivative A3 solution and 1.5mL AgNO3 The mixing of (10mg/mL, 0.088mmol) solution, the concentration of chitosan oligosaccharide derivative A3 solution are 0.9mg/mL；In mixed solution, shell The concentration of oligosaccharide derivative A3 is 0.0028moL/L, and the concentration of AgNO3 is 0.0093mol/L, and 80min is reacted at 40 DEG C and is obtained Nano silver B3.

Embodiment 6: the synthesis of phenylthiosemicarbazide chitosan oligosaccharide A4 and Nano silver solution B4

Chitosan oligosaccharide (8g, unit molal quantity 0.05mol) is weighed to be slowly added in 95% ethyl alcohol (30mL) after stirring and dissolving, by solution It is slowly added in ammonium hydroxide (30mL, 0.78mol), is in dark-brown viscous liquid, 0.5h is stirred at room temperature；Then by CS2 (4mL, It 0.066mol) is slowly dropped in above-mentioned mixed liquor and is stirred to react 2h, solution colour is deepened；Weigh sodium chloroacetate (6.5g, It 0.056mol) is added in reaction solution, continues to be stirred to react 0.5h；Phenylhydrazine (6mL, 0.061mol) is slowly dropped to again above-mentioned In reaction solution, 2h is reacted again at room temperature；Finally reaction solution is added in about 60mL dehydrated alcohol, there is brown flocculent deposit analysis Out, it stands, removes supernatant, washed repeatedly with dehydrated alcohol 8 times, filter, and eluted 2 times with dehydrated alcohol, the production that will be obtained Object is dried in vacuo (55 DEG C, 8h), obtains light brown powder 8.56g (unit molal quantity 0.053mol).

10mL (unit molal weight 297, unit molal quantity 0.032mmol) phenylthiosemicarbazide chitosan oligosaccharide A4 solution and 2.0mL The mixing of AgNO3 (10mg/mL, 0.12mmol) solution, the concentration of phenylthiosemicarbazide chitosan oligosaccharide A4 solution are 0.95mg/mL；Mixing In solution, the concentration of phenylthiosemicarbazide chitosan oligosaccharide A4 is 0.0027moL/L, and the concentration of AgNO3 is 0.0098mol/L, in 50 DEG C Lower reaction 90min obtains nano silver B4.

The synthesis of embodiment 7:N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide A5 and Nano silver solution B5

2-aminopyridine 4.7g (0.05mol) is dissolved in 95% ethyl alcohol (10mL), is added ammonium hydroxide (10mL, 0.26mol), solution In orange-yellow, 0.5h is stirred at room temperature；CS2 (4.4mL, 0.073mol) is slowly added dropwise in the solution, is stirred to react 2h, it is molten Liquid becomes orange red；Then sodium chloroacetate (7g, 0.06mol) is added in the reaction solution, the reaction was continued 0.5h, solution becomes orange Yellow；It weighs chitosan oligosaccharide (8g, unit molal quantity 0.05mol) to be dissolved in 95% ethyl alcohol (10mL), then be added dropwise to above-mentioned In reaction solution, and ammonium hydroxide (10mL, 0.26mol) is added, solution is in dark brown, reacts 4h again at room temperature；Finally reaction solution is delayed Slowly it pours into about 50mL dehydrated alcohol, has yellow flocculent deposit precipitation, stand, remove supernatant, wash 7 repeatedly with dehydrated alcohol It is secondary, it filters, and eluted 2 times with dehydrated alcohol, obtained product vacuum is dry (55 DEG C, 8h), obtain light yellow solid powder 7.78g (unit molal quantity 0.048mol).

N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide A5 solution of 10mL (unit molal weight 297, unit molal quantity 0.034mmol) It is the mixing of 1.0mL AgNO3 (10mg/mL, 0.059mmol) solution, N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide A5 solution with concentration Concentration be 1.0mg/mL；In mixed solution, the concentration of N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide A5 is 0.0031mol/L, The concentration of AgNO3 is 0.0053mol/L, and 80min is reacted at 40 DEG C and obtains nano silver B5.

Embodiment 8: the synthesis of methylamino thiocarbamide chitosan oligosaccharide A6 and Nano silver solution B6

It weighs after chitosan oligosaccharide (4g, unit molal quantity 0.025mol) is slowly added to 95% ethyl alcohol (10mL) stirring and dissolving, ammonia is added In water (10mL, 0.26mol), solution is in dark-brown, and 0.5h is stirred at room temperature；Then CS2 (2.1mL, 0.035mol) is slow Slowly it is added drop-wise in the mixed liquor, is stirred to react 2h, solution colour is deepened；Sodium chloroacetate (3.4g, 0.029mol) is weighed slowly to add Enter into above-mentioned reaction solution, continues to be stirred to react 0.5h；Then methyl hydrazine (3mL, 0.057mol) is taken to be added drop-wise in reaction solution again, React 3h again at room temperature；Finally reaction solution is poured slowly into about 40mL dehydrated alcohol, has light grey flocculent deposit to be precipitated, It stands, removes supernatant, washed repeatedly with dehydrated alcohol 8 times, filter, and eluted 3 times with dehydrated alcohol, obtained product is true Sky is dry (55 DEG C, 8h), obtains light gray solid powder 3.8g (unit molal quantity 0.024mol).

10mL (unit molal weight 249, unit molal quantity 0.040mmol) methylamino thiocarbamide chitosan oligosaccharide A6 solution and 1.0mL The mixing of AgNO3 (10mg/mL, 0.059mmol) solution, the concentration of methylamino thiocarbamide chitosan oligosaccharide (A6) solution are 1.2mg/mL；It is mixed It closes in solution, the concentration of methylamino thiocarbamide chitosan oligosaccharide A6 is 0.0036mol/L, and the concentration of AgNO3 is 0.0053mol/L, in 50 100min is reacted at DEG C obtains nano silver B6.

Chitosan oligosaccharide thiourea derivatives are made using method for the treatment of different things alike, and small molecule chitosan oligosaccharide is easy to reunite during the reaction Macromolecular chitosan oligosaccharide is formed, reaction is influenced and continues, reduce the yield of chitosan oligosaccharide derivative.We send out during the test It is existing: increase ammonia volume, form homogeneous phase solution after mixing chitosan oligosaccharide ethanol solution with ammonium hydroxide, in magnetic stirrer constantly into Row stirring, can reduce the appearance of agglomeration, improve the yield of chitosan oligosaccharide derivative.

1. the ultraviolet characterization of chitosan oligosaccharide derivative

The deacetylated chitosan oligosaccharide ammonium uv-visible absorption spectra of 1.1 aminodithioformic acids

As shown in Figure 1, the purple of the uv scan curve of the deacetylated chitosan oligosaccharide ammonium of aminodithioformic acid and chitosan oligosaccharide raw material Outer scanning curve is entirely different, this is because the ultraviolet absorption peak after addition reaction has occurred in chitosan oligosaccharide and ammonium hydroxide and carbon disulfide There is change compared with raw material chitosan oligosaccharide.This further demonstrates that the chemical reaction has occurred and that, there is ammonium sulfo-amino thiocarbamide shell Oligosaccharides generates.

As can be seen from Figure 1: chitosan oligosaccharide has a weak and wide absorption peak at 277nm, is being greater than 277nm wave-length coverage without obvious It absorbs.Ammonium sulfo-amino thiocarbamide chitosan oligosaccharide is the absorption peak of-CSS-, the absorption at 256nm, 288nm in the absorption of 205nm Peak is the thiocarbamide structure absorption peak for being influenced to occur red shift by chitosan oligosaccharide, illustrates with the presence of ammonium for thiosemicarbazides structure.Reaction obtains Target product.

The phenylthiosemicarbazide chitosan oligosaccharide ultra-violet absorption spectrum of 1.2 embodiments 6 preparation is as shown in Fig. 2, phenylthiosemicarbazide shell is few Sugared ultra-violet absorption spectrum has apparent difference, says compared with chitosan oligosaccharide, ammonium sulfo-amino thiocarbamide chitosan oligosaccharide ultra-violet absorption spectrum Bright three's structure is essentially different, and has reaction to give birth to.Phenylthiosemicarbazide chitosan oligosaccharide has stronger absorption at 276nm, is Phenyl ring B band, by phenyl ring conjugated double bond 270nm absorption, and-HN-NH- effect under red shift, illustrate there is phenylamino base junction Structure exists, and phenylhydrazine has added on ammonium sulfo-amino thiocarbamide, obtains target product.

1.3 N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide (A5) uv-visible absorption spectra such as Fig. 3 institute prepared by embodiment 7 Show, N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide (A5) has absorption peak at 254nm, 289nm, with ammonium sulfo-amino thiocarbamide 205nm, 256nm, 288nm absorption peak are compared, and absorption peak disappearance is because-CSS- structure is by broken ring at 205nm, and substituted reaction occurs. The absorption peak of pyridine corresponds to π → π * transition in 240~260nm with two: one；Another region in 270nm, accordingly In n → π * transition.The presence of pyridine makes ammonium sulfo-amino thiocarbamide chitosan oligosaccharide 256nm and two absorption peaks of 288nm that indigo plant occur respectively Shifting and red shift.

Methylamino thiocarbamide chitosan oligosaccharide (A6) ultra-violet absorption spectrum of 1.4 embodiments 8 preparation is as shown in figure 4, ammonium sulfo-amino Thiocarbamide chitosan oligosaccharide and methylamino thiocarbamide chitosan oligosaccharide have no significant difference, the former has obvious absorption peaks at 256nm, 288nm, are sulphur Urea structure absorption peak, the absorption peak position of the latter are 254nm, 286nm, are because the presence of methyl, which makes to absorb, has occurred blue shift. Methylamino thiocarbamide chitosan oligosaccharide destroys-CSS- key so the ultraviolet absorption peak at 205nm disappears.

2. the IR Characterization of chitosan oligosaccharide thiourea derivatives

Phenylthiosemicarbazide chitosan oligosaccharide (A4) infrared absorption spectrum of 2.1 embodiments 6 preparation is as shown in figure 5,3425.43cm^-1、 3329.91cm^-1、3133.99cm^-1Be become associate-OH stretching vibration absworption peak-NH stretching vibration absworption peak overlapping and Broadening multi-absorption peak, in 1083cm^-1What is located is then the angle vibration absorption peak of alcoholic extract hydroxyl group, in 852cm^-1There are β-types at place The characteristic absorption peak of glycosidic bond illustrates that chitosan is β-polysaccharide.Phenylthiosemicarbazide chitosan oligosaccharide is in 1450cm^-1And 1600cm^-1Near There is the skeletal vibration of phenyl ring, 700cm^-1Place is the mono-substituted characteristic peak of phenyl ring.Illustrate that, by series reaction, target produces Object is correct.

2.2 embodiments 7 preparation N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide (A5) infrared absorption spectrum as shown in fig. 6, 3170cm^-1、3324cm^-1、3418cm^-1Be become associate-OH stretching vibration absworption peak-NH stretching vibration absworption peak it is more Reabsorption peak, in 1073cm^-1That locate is then the angle vibration absorption peak of alcoholic extract hydroxyl group, 1616cm^-1For amino deformation vibration absorption Peak, 896cm^-1Place there are β-type glycosidic bond characteristic absorption peak, 1664,1241,1204,1154, be pyridine ring infrared signature Absorption peak.Reaction generates target product.

Methylamino thiocarbamide chitosan oligosaccharide (A6) infrared absorption spectrum of 2.3 embodiments 8 preparation is as shown in fig. 7,3133cm^-1It is to become The broadening absorption peak at the stretching vibration absworption peak overlapping of-OH stretching vibration absworption peak-NH of association, in 1082cm^-1Place is then It is the angle vibration absorption peak of alcoholic extract hydroxyl group, 1593cm^-1For amino deformation vibration the absworption peak, in 831cm^-1There are β-type glucosides at place The characteristic absorption peak of key illustrates that chitosan is β-polysaccharide, 1401cm^-1Strong absworption peak is the characteristic peak in curved (in the face) CH, compares shell Oligosaccharides absorbs stronger.Reaction generates target product.

3. the structural characterization that chitosan oligosaccharide and chitosan oligosaccharide derivative prepare nano silver

3.1 thiosemicarbazides chitosan oligosaccharides (A1) prepare the uv-visible absorption spectra of nano silver (B1) process

At 20 DEG C, the silver nitrate solution of 1.5ml 10mg/ml is added to 10mL 0.8mg/mL (unit molar concentration In thiosemicarbazides chitosan oligosaccharide (A1) solution 0.005moL/L), Fig. 8 is the ultraviolet absorption curve for scanning each time point, is reacted Color change in journey is as shown in Figure 9.

3.2 phenylthiosemicarbazide chitosan oligosaccharides (A4) prepare the uv-visible absorption spectra of nano silver (B4) process

At 20 DEG C, the silver nitrate solution of 1.5mL 10mg/mL is added to 10mL0.95mg/mL (unit molar concentration In phenylthiosemicarbazide chitosan oligosaccharide (homogeneous) solution 0.0059moL/L), Figure 10 various concentration phenylthiosemicarbazide chitosan oligosaccharide (A4)； 1:0.001mol/L；2:0.002mol/L；3:0.003mol/L；4:0.004mol/L；5:0.005mol/L) reduction preparation of silver receives The UV-Vis absorption spectrum of rice corpuscles (B4), Figure 11 are that various concentration phenylthiosemicarbazide chitosan oligosaccharide (A4) prepares nano silver (B4) Color compares.

3.3N- (2- pyridine)-N'- chitosan oligosaccharide thiocarbamide (A5) prepares the uv-visible absorption spectra of nano silver process

At 20 DEG C, N- (2- the pyridine)-N'- shell that the silver nitrate solution of 1.5mL 10mg/mL is added to 10mL 1mg/mL is few In sugared thiourea solution, Figure 12 is the ultraviolet absorption curve for scanning each time point, color change such as Figure 13 institute in reaction process Show.

3.4 methylamino thiocarbamide chitosan oligosaccharides (A6) prepare the uv-visible absorption spectra of nano silver process

At 20 DEG C, the methylamino thiocarbamide chitosan oligosaccharide that the silver nitrate solution of 1.5mL 10mg/mL is added to 10mL 1mg/mL is molten In liquid, Figure 14 is the ultraviolet absorption curve for scanning each time point, and the color change in reaction process is as shown in figure 15.

4. transmission electron microscope (TEM) result that chitosan oligosaccharide thiourea derivatives reduction prepares nano silver

Experiment characterizes the pattern of material using FEI TECNALG2 type transmission electron microscope (Dutch FEI Co.), adds Fast voltage is 200KV.It takes the Nano silver solution of 0.2ml to be added dropwise on the copper mesh grid for being covered with carbon film, is then placed in copper mesh red Dry 30min, uses the pattern of transmission electron microscope observing nano silver particles after the solvent is volatilized under outer lamp.

As shown in Figure 16, Figure 18, chitosan oligosaccharide-hydrazine hydrate thiourea derivative (A1) preparation nano silver (B1) partial size is in 10nm Nano silver (B1) nano particle diameter of left and right, phenylthiosemicarbazide chitosan oligosaccharide (A4) preparation is about 4-10nm, and distribution is relatively uniform, It can be measured ultraviolet absorption peak (see Fig. 8,10) in 410nm or so, chitosan oligosaccharide-ethylenediamine thiourea derivative (A2) prepares nano silver (B2) significant change occurs for solution colour when, and solution is blood red, but fails to measure ultraviolet absorption peak in 410nm or so, is but existed The spherical nanoparticle being evenly distributed can be seen under transmission electron microscope, it may be possible to which nanometer particle size is too small, due to reduction used Different distributions is presented in the difference of agent, obtained nano silver particles.

N- shown in Figure 17 (2- aminoethyl)-N'- chitosan oligosaccharide thiocarbamide (A2) reduction prepares nano silver (B2), N- shown in Figure 19 (2- pyridine)-N'- urea chitosan oligosaccharide thiocarbamide (A5) makees reducing agent and prepares methylamino thiocarbamide chitosan oligosaccharide shown in nano silver (B5) and Figure 20 (A6) do the electron-microscope scanning that reducing agent prepares nano silver (B6) has apparent nanoparticle under electron scanning Electronic Speculum as the result is shown In the presence of, have nano silver generation, nano particle diameter is about 4-10nm, be distributed it is relatively uniform.But N- (2- aminoethyl)-N'- shell is few Sugared thiocarbamide, methylamino thiocarbamide chitosan oligosaccharide reduction prepare on the ultraviolet-visible scanning optical spectrum of nano silver no absorption peak at 410nm (see Figure 12,14).

It is probed into 5. the nano silver antibacterial of chitosan oligosaccharide thiourea derivatives preparation is active

The nano silver B1 bacteriostatic activity of 5.1 thiosemicarbazides chitosan oligosaccharide A1 preparation is probed into

5.1.1 experimental principle

Using mycelial growth rate method is inhibited, the nano silver B1 of thiosemicarbazides chitosan oligosaccharide (A1) preparation is probed into wheat base rot disease Bacterium, tobacco black shank bacterium, Corn Green Wilt Disease, beading germ, tomato early blight bacterium, maize Curvularia leaf spot fungi, cereal reaping hook germ Inhibitory activity, the effect of its antifungal activity is compared as control using water, chitosan oligosaccharide, commercially available medicine triazolone.Calculation formula:

5.1.2 experimental procedure

Experiment the previous day sterilizing: culture dish wraps that (ten one piles), preparing a conical flask, (the inside adds 200mL pure with newspaper Water) test tube being sealed, wrapped with brown paper, the 2h that sterilizes is put in high-pressure sterilizing pot.Be put in after sterilizing in baking oven dry it is standby With.Culture medium is prepared: 1000mL water corresponds to 200g potato, 18g agar, 20g glucose.It prepares in proportion.Concrete operations, title Good potato is cut into slices, be put into the water of boiling boil to a certain extent (with glass bar potato can be pricked thoroughly can), water It filters in beaker, potato is thrown away.Continuation, which pours into filtrate in pot, boils, and glucose is first added, and stirs, is slow added into agar, and It is stirred continuously, (volume not enough initially requires to add appropriate hot water again) is poured into beaker after agar all dissolution.It is subsequently poured into cone It is sealed in shape bottle with brown paper, sterilizing (while sterilizing has tweezers, punch, transfer needle, pipette tips etc.) in high-pressure sterilizing pot. Compounding medicine.Ware: entering aseptic operating platform, and with alcohol cotton rub hand, culture dish number starts down culture medium after number, A conical flask is taken from pressure cooker, is taken quantitative drug from phial with liquid-transfering gun, is shaken up, culture solution is arrived first into 10mL's In test tube, then the culture solution in test tube poured into the culture dish of corresponding nomenclature of drug, obtains the culture medium of various concentration, it is of the same race Drug falls three wares, repetitive operation.Inoculation: being finished down and sterilize and cool down 0.5h after culture medium in the UV lamp, cooling to culture medium It after solidification, is then inoculated with, seals.Spawn incubation: constant temperature incubation a couple of days in Intelligent biochemistry incubator.Measurement: it is handed over cross Fork method measurement strain diameter record data are simultaneously taken pictures.Data preparation.

Note: all of above operation need to carry out sterile working in aseptic operating platform, and movement is rapid, in order to avoid microbiological contamination.

5.1.3 the bacteriostatic test result and analysis of the nano silver B1 of thiosemicarbazides chitosan oligosaccharide A1 preparation

5.1.3.1 inhibitory activity of the nano silver B1 of thiosemicarbazides chitosan oligosaccharide (A1) preparation to wheat stalk Phyllostachys pubescens

Inhibitory activity of the nano silver (B1) of 1 thiosemicarbazides chitosan oligosaccharide (A1) of table preparation to wheat stalk Phyllostachys pubescens

As shown in Table 1: nano silver is preferable to the fungistatic effect of wheat brown foot rot, low concentration (10 μ g/mL) nano silver just table Biocidal property is revealed, and bacteriostasis rate is positively correlated with nanometer silver concentration, increases with the increase of concentration, related coefficient reaches 0.9984, correlation is stronger.When nanometer silver concentration is 60 μ g/mL, bacteriostasis rate reaches 65.29%, but is still not so good as phase three Fungistatic effect (88.97%) when oxazolone (50 μ g/mL) is good.EC of the nano silver to wheat stalk Phyllostachys pubescens₅₀Value (μ g/mL) is 42.2364 fungistatic effect is preferable.

5.1.3.2 inhibitory activity of the nano silver (B1) of thiosemicarbazides chitosan oligosaccharide (A1) preparation to tobacco black shank bacterium

Inhibitory activity of the nano silver (B1) of 2 thiosemicarbazides chitosan oligosaccharide (A1) of table preparation to tobacco black shank bacterium

As shown in Table 2: nano silver is general to the fungistatic effect of the black shin of tobacco, and bacteriostasis rate is positively correlated with nanometer silver concentration, with dense The increase of degree and increase, related coefficient reaches 0.9984, and correlation is stronger.When nanometer silver concentration is 60 μ g/mL, bacteriostasis rate is only Have and reaches 46.55%, it is good lower than the fungistatic effect (80.31%) of triazolone (50 μ g/mL).Nano silver is to tobacco black shank bacterium EC₅₀Being worth (μ g/mL) is 60.7501.

5.1.3.3 inhibitory activity of the nano silver (B1) of thiosemicarbazides chitosan oligosaccharide (A1) preparation to Corn Green Wilt Disease

Inhibitory effect of the nano silver (B1) of 3 thiosemicarbazides chitosan oligosaccharide (A1) of table preparation to Corn Green Wilt Disease

As shown in Table 3: nano silver is preferable to the fungistatic effect that maize stalks are withered, and bacteriostasis rate is positively correlated with nanometer silver concentration, with dense The increase of degree and increase, related coefficient reaches 0.9847, and correlation is stronger.When nanometer silver concentration is 60 μ g/mL, bacteriostasis rate reaches To 56.01%, it is lower than triazolone (50 μ g/mL) fungistatic effect (89.89%).EC of the nano silver to Corn Green Wilt Disease₅₀It is worth (μ It g/mL) is 14.7149.

5.1.3.4 inhibitory activity of the nano silver (B1) of thiosemicarbazides chitosan oligosaccharide (A1) preparation to beading germ

Inhibitory activity of the nano silver (B1) of 4 thiosemicarbazides chitosan oligosaccharide (A1) of table preparation to beading germ

As shown in Table 4: nano silver is preferable to the fungistatic effect of a beading, and bacteriostasis rate is positively correlated with nanometer silver concentration, with concentration Increase and increases, related coefficient 0.9847.When nanometer silver concentration is 60 μ g/mL, bacteriostasis rate reaches 53.61%, is lower than three The bacteriostasis rate (85.28%) of oxazolone (50 μ g/mL).EC of the nano silver to beading germ₅₀Being worth (μ g/mL) is 51.1072.

5.1.3.5 inhibitory activity of the nano silver (B1) of thiosemicarbazides chitosan oligosaccharide (A1) preparation to tomato early blight bacterium

Inhibitory activity of the nano silver (B1) of 5 thiosemicarbazides chitosan oligosaccharide (A1) of table preparation to tomato early blight bacterium

As shown in Table 5: nano silver is fine to the fungistatic effect of tomato early epidemic, and bacteriostasis rate is positively correlated with nanometer silver concentration, with dense The increase of degree and increase, related coefficient 0.9720.When nanometer silver concentration is 60 μ g/mL, bacteriostasis rate reaches 67.31%, low Bacteriostasis rate (91.76%) when triazolone (50 μ g/mL).The EC that nano silver inhibits beading germ₅₀Value (μ g/mL) is 21.2889。

5.1.3.6 inhibitory activity of the nano silver (B1) of thiosemicarbazides chitosan oligosaccharide (A1) preparation to maize Curvularia leaf spot fungi

Inhibitory activity of the nano silver (B1) of 6 thiosemicarbazides chitosan oligosaccharide (A1) of table preparation to maize Curvularia leaf spot fungi

As shown in Table 6: nano silver is best to the fungistatic effect of maize curvularia, and the nano silver of low concentration (10 μ g/mL) just shows The more bacteriostasis rate (19.83%) of advantage out, and bacteriostasis rate is positively correlated with nanometer silver concentration, increases with the increase of concentration Greatly, related coefficient 0.9880.When nanometer silver concentration is 60 μ g/mL, bacteriostasis rate reaches 95.69%, fungistatic effect and triazole The advantages that fungistatic effect when ketone (50 μ g/mL) is identical, but nano silver has pollution small relative to triazolone, green, nano silver To the EC of maize curvularia₅₀Value is 54.3393 μ g/mL.

5.1.3.7 inhibitory activity of the nano silver (B1) of thiosemicarbazides chitosan oligosaccharide (A1) preparation to cereal reaping hook germ

Inhibitory activity of the nano silver (B1) of 7 thiosemicarbazides chitosan oligosaccharide (A1) of table preparation to cereal reaping hook germ

As shown in Table 7: nano silver is preferable to the fungistatic effect of cereal reaping hook, and the nano silver of low concentration (10 μ g/mL) is just shown Preferable bacteriostasis rate (5.43%), bacteriostasis rate is positively correlated with nanometer silver concentration, increases with the increase of concentration, related coefficient is 0.9450.When nanometer silver concentration is 60 μ g/mL, bacteriostasis rate 78.22% is higher than the bacteriostasis rate of triazolone (50 μ g/mL) (73.64%), EC of the nano silver to beading germ₅₀Value is 32.4018 μ g/mL.

By to chitosan oligosaccharide-hydrazine hydrate thiourea derivative A1 and the Nano silver solution pair prepared as reducing agent and stabilizer Six kinds of frequently seen plants fungi of curing the disease has carried out probing into for antibacterial activity, can Preliminary conclusion, shell is few compared with compareing chitosan oligosaccharide Sugared thiourea derivatives and its Nano silver solution of preparation enhance the inhibiting rate of fungi, chitosan oligosaccharide-hydrazine hydrate thiocarbamide of synthesis Derivative A1 has certain antibiotic property to different strains, also different to the antibiotic property of different strain, and with solution The concentration of middle drug itself increases and increases.And its antibacterial activity for restoring the Nano silver solution that silver nitrate obtains is than corresponding shell The antibacterial activity of oligosaccharides thiourea derivatives itself wants high, wherein having stronger antibiotic property to wheat pathogenic bacteria, such as to wheat Red mould bacteriostasis rate is up to 48.02%, to wheat brown foot rot bacteriostasis rate up to 55.6%.For plaque small for corn, nano silver resists Bacterium activity is higher by 15% or so than the antibacterial agent triazolone that market is sold, and as the concentration of Nano silver solution reduces, antibiotic property reduces.? Antibiotic property of the drug being combined to generally than heterogeneous synthesis is high, illustrates that degree of substitution plays an important role to the raising of antibiotic property.

The spy of 5.2 phenylthiosemicarbazide chitosan oligosaccharide A4 and the nano silver B4 bacteriostatic activity prepared as reducing agent and stabilizer Study carefully

5.2.1 experimental principle

Using mycelial growth rate method is inhibited, phenylthiosemicarbazide chitosan oligosaccharide A4 itself is probed into, as the nanometer of reducing agent preparation Silver-colored B4 is to cereal reaping hook germ, gibberella saubinetii, wheat stalk Phyllostachys pubescens, cotton-wilt fusarium, southern corn leaf blight, and tobacco is black The inhibitory activity of shin germ compares the effect of its antifungal activity using water, chitosan oligosaccharide, commercially available medicine triazolone as control.Meter Calculate formula:

5.2.2 experimental procedure

5.2.2.1 culture medium

PDA culture medium: potato 300g, glucose 30g, agar 27g, distilled water 1500mL.

Step: (1) potato is cut into the thin slice of thickness 1-2cm by, with the distillation boiling 10-15min of boiling；(2) is by Ma Ling Potato filters off, and leaves filtrate, and add to 1500mL；(3) continues to heat, and glucose is added；(4) is slowly added to agar powder；(5). High pressure sterilization 120min.

5.2.2.2 for trying fungi

Cereal reaping hook germ, gibberella saubinetii, wheat stalk Phyllostachys pubescens, cotton-wilt fusarium, southern corn leaf blight, the black shin of tobacco Germ, all strains are that this laboratory saves.

5.2.2.3 experimental method

Inhibit mycelial growth rate method: separately sampled product group and the PDA culture medium of control group solution 2mL and 18mL thawing mix, It uniformly pours into two sterile petri dish and band medicine culture medium flat plate is made, while being second of ten times of the dilution of each solution, to add The PDA culture medium for entering sterile water is growth control.Inoculation is sealed for trying fungi bacteria cake, sealed membrane, cultivates 4-5 in 28 DEG C of insulating boxs After it, it is repeated 3 times with crossing method measurement for trying bacterium colony diameter, every processing.Mycelial growth inhibition rate is calculated as follows: bacterium Silk growth inhibition ratio/%=(control colony growth diameter-processing colony growth diameter)/control colony growth diameter × 100%.

5.2.3 the bacteriostatic test of phenylthiosemicarbazide chitosan oligosaccharide A4 and the nano silver B4 prepared as reducing agent and stabilizer As a result with analysis

5.2.3.1 to the inhibitory activity of Fusarium graminearum kind

Bacteriostatic activity of the 8 phenylthiosemicarbazide chitosan oligosaccharide A4 of the table and its nano silver B4 of preparation to Fusarium graminearum

By table 8 it follows that when using gibberella saubinetii as strain, the Nano silver solution by the reduction preparation of phenylthiosemicarbazide chitosan oligosaccharide is dense Bacteriostasis rate (78.86%) highest when degree is 130 μ g/mL is higher than the bacteriostasis rate (65.04%) of triazolone (400ug/mL), nanometer Silver-colored solution bacteriostasis rate is positively correlated with its concentration.

5.2.3.2 to gibberella saubinetii bacteriostatic activity

9 phenylthiosemicarbazide chitosan oligosaccharide A4 of table and its nano silver B4 of preparation are to gibberella saubinetii bacteriostatic activity

By table 9 it follows that when using gibberella saubinetii as strain, the Nano silver solution of preparation is restored by phenylthiosemicarbazide chitosan oligosaccharide Bacteriostasis rate highest (87.30%) when concentration is 130 μ g/mL, it is antibacterial higher than the bacteriostasis rate (79.37%) of triazolone (400ug/mL) Rate is positively correlated with its concentration.

5.2.3.3 to the bacteriostatic activity of wheat brown foot rot bacterium

Bacteriostatic activity of the 10 phenylthiosemicarbazide chitosan oligosaccharide A4 of the table and its nano silver B4 of preparation to wheat brown foot rot bacterium

By table 10 it follows that when using wheat brown foot rot as strain, the nano silver by the reduction preparation of phenylthiosemicarbazide chitosan oligosaccharide is molten Bacteriostasis rate highest (78.40%) when liquid concentration is 130 μ g/mL, is higher than the bacteriostasis rate (69.60%) of triazolone (400ug/mL), Nano silver solution bacteriostasis rate is positively correlated with its concentration.

5.2.3.4 to the inhibitory activity of cotton wilt fusarium

Bacteriostatic activity of the 11 phenylthiosemicarbazide chitosan oligosaccharide A4 of the table and its nano silver B4 of preparation to cotton wilt fusarium

By table 11 it follows that when being withered with cotton for strain, by the Nano silver solution of phenylthiosemicarbazide chitosan oligosaccharide reduction preparation Concentration is the bacteriostasis rate (55.47%) of 130 μ g/mL, is lower than the bacteriostasis rate (69.53%) of triazolone (400ug/mL), nano silver Solution bacteriostasis rate is positively correlated with its concentration.

5.2.3.5 to the inhibitory activity of the small plaque of corn

12 phenylthiosemicarbazide chitosan oligosaccharide (A4) of table and its nano silver (B4) of preparation are to the bacteriostatic activity of the small plaque of corn

By table 12 it follows that when using corn stigma as strain, by the Nano silver solution of phenylthiosemicarbazide chitosan oligosaccharide reduction preparation Bacteriostasis rate (63.27%) highest when concentration is 130 μ g/mL is higher than the bacteriostasis rate (29.20%) of triazolone (400ug/mL), receives The silver-colored bacteriostasis rate of rice is positively correlated with its concentration.

5.2.3.6 to the inhibitory activity of the black shin bacterium of tobacco

Bacteriostatic activity of the 13 phenylthiosemicarbazide chitosan oligosaccharide A4 of the table and its nano silver B4 of preparation to the black shin bacterium of tobacco

By table 13 it follows that when using the black shin of tobacco as strain, by the Nano silver solution of phenylthiosemicarbazide chitosan oligosaccharide reduction preparation Bacteriostasis rate (35.66%) highest when concentration is 130 μ g/mL is lower than the bacteriostasis rate (66.67%) of triazolone (400ug/mL), receives The bacteriostasis rate of meter Yin is positively correlated with its concentration.

Find out that chitosan oligosaccharide and its simple derivatives have certain biocidal property in activity experiment, but few by phenylthiosemicarbazide shell The nano silver that sugar is reduced into has stronger broad-spectrum antibacterial: when strain is Fusarium graminearum, also by phenylthiosemicarbazide chitosan oligosaccharide Bacteriostasis rate (78.86%) highest of the Nano silver solution (130 μ g/mL) of original preparation, it is antibacterial higher than triazolone (400ug/mL) Rate (65.04%)；Nano silver solution (the 130 μ g/ of phenylthiosemicarbazide chitosan oligosaccharide reduction preparation when strain is gibberella saubinetii strain ML bacteriostasis rate (87.30%) highest) is higher than the bacteriostasis rate (79.37%) of triazolone (400ug/mL)；Strain is wheat stem foot When rotten bacterium, the bacteriostasis rate highest (73.2%) of the Nano silver solution (130 μ g/mL) of preparation is restored by phenylthiosemicarbazide chitosan oligosaccharide, Bacteriostasis rate (69.60%) higher than triazolone (400ug/mL)；When being withered with cotton for strain, also by phenylthiosemicarbazide chitosan oligosaccharide The bacteriostasis rate (55.47%) of the Nano silver solution (130 μ g/mL) of original preparation, is lower than the bacteriostasis rate of triazolone (400ug/mL) (69.53%)；When using corn stigma as strain, by the Nano silver solution (130 μ g/mL) of phenylthiosemicarbazide chitosan oligosaccharide reduction preparation Bacteriostasis rate (63.27%) highest is higher than the bacteriostasis rate (29.20%) of triazolone (400ug/mL)；When using the black shin of tobacco as strain, By Nano silver solution (130 μ g/mL) bacteriostasis rate (35.66%) highest of phenylthiosemicarbazide chitosan oligosaccharide reduction preparation, it is lower than triazole The bacteriostasis rate (66.67%) of ketone (400ug/mL).

What has been described above is only a preferred embodiment of the present invention, it is noted that for those skilled in the art, Without depart from that overall concept of the invention, several changes and improvements can also be made, these also should be considered as of the invention Protection scope.

Claims

1. a kind of chitosan oligosaccharide thiourea derivatives, it is characterised in that: the chitosan oligosaccharide thiourea derivatives are the chemical combination such as following formula Object；Wherein 7≤n≤23:

R is

2. a kind of method for preparing chitosan oligosaccharide thiourea derivatives as described in claim 1, it is characterised in that: first by chitosan oligosaccharide It is dissolved in ethanol solution and ammonium hydroxide, sequentially adds CS₂Solution, sodium chloroacetate and substrate, it is stirring while adding, chitosan oligosaccharide first and CS₂Solution reaction generates aminodithioformic acid chitosan oligosaccharide ammonium, and aminodithioformic acid chitosan oligosaccharide ammonium reacts life with sodium chloroacetate At carboxymethyl dithio carbamic acid chitosan oligosaccharide sodium, carboxymethyl dithio carbamic acid chitosan oligosaccharide sodium generates shell with substrate reactions again Oligosaccharides thiourea derivatives.

3. the method for preparing chitosan oligosaccharide thiourea derivatives as claimed in claim 2, it is characterised in that: the following steps are included:

4. the method according to claim 2 or 3 for preparing chitosan oligosaccharide thiourea derivatives, it is characterised in that: the amine Compound is hydrazine hydrate, ethylenediamine, ethanol amine, phenylhydrazine, 2-aminopyridine or methyl hydrazine.

5. the method according to claim 4 for preparing chitosan oligosaccharide thiourea derivatives, it is characterised in that: the hydrazine hydrate is 80% hydrazine hydrate aqueous solution.

6. a kind of method for preparing nano silver using chitosan oligosaccharide thiourea derivatives as described in claim 1, it is characterised in that: It is by chitosan oligosaccharide thiourea derivatives solution and AgNO₃Solution reaction generates nano silver.

7. the method according to claim 6 for preparing nano silver using chitosan oligosaccharide thiourea derivatives, it is characterised in that: packet Include following steps: chitosan oligosaccharide thiourea derivatives solution and AgNO₃Solution mixes, chitosan oligosaccharide thiourea derivatives in mixed solution With AgNO₃Unit molar ratio be (2-8): (3-9), at 20-60 DEG C react 30-100min obtain nano silver.

8. the method according to claim 7 for preparing nano silver using chitosan oligosaccharide thiourea derivatives, it is characterised in that: institute It states in mixed solution, the concentration of chitosan oligosaccharide thiourea derivatives is 0.001-0.01mol/L, AgNO₃Concentration be 0.001- 0.04mol/L。

9. a kind of nano silver, it is characterised in that: it is to be prepared using any one of claim 6-8 the method.

10. nano silver as claimed in claim 9 is in the application of the antibacterial aspect of plant.