CN101863933B - Chitosan oligosaccharide Schiff base phosphonate as well as preparation method and application thereof - Google Patents
Chitosan oligosaccharide Schiff base phosphonate as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to chitosan oligosaccharide Schiff base phosphonate, which has the structural formula shown in the specification, and in the formula, n is 2-20. The preparation method comprises the following steps of: carrying out chemical modification on chitosan oligosaccharide with salicylaldehyde and substituted salicylaldehyde by adopting a microwave radiation method to obtain a chitosan oligosaccharide Schiff base (S-COS), and then carrying out addition reaction on the S-COS and diethyl phosphite to obtain the chitosan oligosaccharide Schiff base phosphonate. The chitosan oligosaccharide Schiff base phosphonate has better activity for resisting tobacco mosaic virus.
Description
Technical field
The invention belongs to the Biological resources pesticide developing and utilize technical field, be specifically related to a kind of oligochitosan Schiff base phosphonate and preparation method thereof and application.
Background technology
(Tobacco Mosaic Virus TMV) is one of the main disease of tobacco to tobacco mosaic virus disease, and the tobacco virus kind of having reported at present has 16 kinds, and general sickness rate is 10%～20%, and indivedual serious fields pieces can reach more than 50%.After mosaic disease was infected in the cigarette strain, it is bad that quality of tobacco becomes, and grade descends; Visual appearance and inherent composition influence to tobacco leaf are bigger, have reduced the utilizability of tobacco leaf, thus serious harm yield of tobacco and quality; Often cause enormous economic loss, become one of restraining factors of sound tobacco production.In the period of 1989～1991, the annual onset area of China's main cigarette district mosaic disease reaches 18.7 ten thousand hm
2, production loss 88650kg, the output value is lost 2.3 hundred million yuan.Comparatively serious mosaic disease disease had taken place in Henan tobacco region in 1996, and wherein the hazard rating of tafelberg Yan Qu is the heaviest.The nearly ten thousand mu of tobacco leaves in not cooperation high-quality of cigarette one phenanthrene cigarette production base receive the mosaic disease bad attack in tafelberg in 1996, and part county base tobacco leaf almost has no harvest.Though both at home and abroad the existing institute of the anti-TMV biotechnological formulation of natural or synthetic of some report still do not have the efficacious therapy medicament so far, therefore, seek a kind of economy, effective tobacco mosaic disease prophylactico-therapeutic measures becomes the urgent task in the tobacco production.
Contemporary society; The attention that Along with people's environmental pollution, food safety and mankind itself are healthy; Modernized agricultural-ecological agriculture of carrying forward vigorously to protect, to improve agroecological environment has become the key subjects that China's agricultural faces, and the natural environmental-protective type degradable product of therefore seeking new biologically active has become the problem that people pay close attention to.
Oligochitosan (chitooligosaccharides; COS) also claim chitin oligo saccharide; Be to be raw material with the chitosan; The lower molecular weight that forms through biotechnology degraded, oligomeric β-(1,4)-2-deoxidation-2-GS that the polymerization degree is 2-20 have good water solubility, function is strong, biological activity is high characteristics.Oligochitosan is the non-specific signal of plant identification pathogenic fungi invasion, and many plants are shown that all the intensive immune induction is active, can excite the disease-resistant gene of plant to express, thereby reach disease-resistant purpose.Oligochitosan is used existing research in agricultural, its inducing anti-disease activity also has been successfully applied to the control of plurality of plant diseases, like the disease that also is difficult to prevent and treat in soil-borne disease and these productions of the viroses of plant the good effect of preventing and treating is arranged promptly.
On the other hand, Schiff's base and schiff base metal complex also are one type and have extensive bioactive compound, about the existing many reports of its research in sterilization, antiviral activity.α-An Jilinsuan is the 3rd amino acid of behind aminocarboxylic acid, thionamic acid, finding in vivo as the phosphoramidate analog that contains of natural amino acid, is the important component part that organophosphorus chemistry grows up in recent decades.People have carried out extensive studies to its compound method and biological activity; Find that it has weeding activity, plant growth regulating activity, anti-phytoviral activity, anti-tumor activity, hiv protease suppresses activity and alanine racemase suppresses multiple biological activitys such as activity, is one of important directions of novel pesticide research and development in the world today.
Summary of the invention
The object of the present invention is to provide a kind of prevent and treat tobacco mosaic virus(TMV), degradable oligochitosan Schiff base phosphonate and preparation method thereof.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is following:
A kind of oligochitosan Schiff base phosphonate, this compound has following general structural formula:
N=2-20 in the formula;
R is
The preparation method of oligochitosan Schiff base phosphonate of the present invention is following:
(1) oligochitosan is soaked swelling in the absolute ethanol solvent after, be benchmark with the acetyl monose that takes off of oligochitosan, add the substituted salicylic aldehydes of 1～2 times of molar weight, adding NaOH regulator solution pH value is 7.5～9, microwave heating back flow reaction 1.5～2.5h.After reaction finished, suction filtration was dry, and with ethanol Soxhlet extraction product 24～28h, to remove unnecessary salicylic aldehyde and substituted salicylic aldehydes in the product, product is subsequent use after being lower than 50 ℃ of following vacuum-dryings.
(2) the oligochitosan Schiff's base and the diethyl phosphite microwave heating back flow reaction 1.5～2.5h that step (1) are obtained; The mol ratio of oligochitosan Schiff's base and diethyl phosphite is 1: 20～1: 30, after reaction finishes, and suction filtration vacuum-drying; Use the ethanol Soxhlet extraction product; To remove diethyl phosphite unnecessary in the product, product is being lower than 50 ℃ of following vacuum-dryings, gets finished product through 120 ℃ of sterilising treatment 20min.
Described oligochitosan Schiff's base 5-chloro-salicylic aldehyde oligochitosan Schiff's base, 3,5-dichloro-salicylaldehyde oligochitosan Schiff's base, 5-nitrosalicylaldehyde oligochitosan Schiff's base, 5-bromosalicylaldehyde oligochitosan Schiff's base.
Described microwave heating power is 400W.
The application of oligochitosan Schiff base phosphonate in the treatment tobacco mosaic virus disease.
Oligochitosan Schiff base phosphonate of the present invention has activity of resisting tobacco mosaic virus preferably: 4 kinds of oligochitosan Schiff base phosphonates (S-COS-P) medicament all has the inhibition effect to withered spot; 5-nitrosalicylaldehyde oligochitosan Schiff base phosphonate and 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate pharmacy effect all are superior to 800 times of 5% 2 (octyl amine ethyl) glycinate AS, and wherein 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate pharmacy effect more is superior to 300 times of Ningnanmycin aquas; (3) oligochitosan Schiff base phosphonate (S-COS-P) medicament has remarkable inducing anti-disease effect to tobacco TMV, and the drug effect base table reveals 200 μ gmL-1＞100 μ gmL-1＞50 μ gmL-1 (wherein 3,5-dichloro-salicylaldehyde oligochitosan Schiff base phosphonate medicament except); (4) generally speaking, the 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate pharmacy effect of 200 μ gmL-1 concentration is best, and the withered spot inhibiting rate to TMV in the prophylactic effect reaches 90.79%.
The present invention adopts microwave heating method to synthesize above-mentioned oligochitosan Schiff base phosphonate, have short, temperature condition of reaction times be prone to control, easy and simple to handle, pollute characteristics such as few, be a green synthesis method that is worthy to be popularized.
Description of drawings
The uv scan curve of Fig. 1 oligochitosan (COS) and oligochitosan Schiff's base (S-COS);
Among the figure: a. oligochitosan, b. salicylic aldehyde oligochitosan Schiff's base, bromosalicylaldehyde oligochitosan Schiff's base c.5-, chloro-salicylic aldehyde's oligochitosan Schiff's base d.5-, e.3,5-dichloro-salicylaldehyde oligochitosan Schiff's base, f.5-nitrosalicylaldehyde oligochitosan Schiff's base.
Fig. 2 is the ir spectra of oligochitosan (COS) and oligochitosan Schiff's base (S-COS).
Among the figure: a. oligochitosan, b. salicylic aldehyde oligochitosan Schiff's base, bromosalicylaldehyde oligochitosan Schiff's base c.5-, chloro-salicylic aldehyde's oligochitosan Schiff's base d.5-, e.3,5-dichloro-salicylaldehyde oligochitosan Schiff's base, f.5-nitrosalicylaldehyde oligochitosan Schiff's base.
Fig. 3 is the thermogravimetric change curve of oligochitosan (COS) and oligochitosan Schiff's base (S-COS);
Among the figure: a. oligochitosan, b. salicylic aldehyde oligochitosan Schiff's base, chloro-salicylic aldehyde's oligochitosan Schiff's base c.5-, d.3,5-dichloro-salicylaldehyde oligochitosan Schiff's base, e.5-nitrosalicylaldehyde oligochitosan Schiff's base, f.5-bromosalicylaldehyde oligochitosan Schiff's base.
Fig. 4 is the uv scan curve of oligochitosan (COS), oligochitosan Schiff's base (S-COS) and oligochitosan Schiff base phosphonate (S-COS-P).
Fig. 5 is the withered spot contrast that sprays 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament and clear water contrast;
Among the figure: a left side: spray 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament; Right: as to spray clear water.
Fig. 6 sprays the regulating effect of 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament to chlorophyll a, b content in the tobacco leaf that infects TMV.
Fig. 7 sprays 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament to the active regulating effect of SOD in the tobacco leaf that infects TMV.
Fig. 8 sprays 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament to the active regulating effect of CAT in the tobacco leaf that infects TMV.
Fig. 9 sprays 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament to the active regulating effect of POD in the tobacco leaf that infects TMV.
Figure 10 sprays 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament to the active regulating effect of PPO in the tobacco leaf that infects TMV.
Figure 11 sprays 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament to the active regulating effect of PAL in the tobacco leaf that infects TMV.
Embodiment
Through embodiment the present invention is elaborated below, but and do not limit the present invention in any way.
1. the preparation of oligochitosan Schiff base phosphonate (S-COS-P)
1.1 key instrument and reagent
XH-100A type microwave synthesizes/abstraction instrument (Beijing XiangHu Science and Technology Development Co., Ltd.); Heat collecting type thermostatically heating magnetic stirring apparatus (Zhengzhou Greatwall Scientific Industrial & Trading Co., Ltd., DF-101S); SHB-3 type recirculated water is used vacuum pump (Zhengzhou Tu Fu instrument plant) more; RE-52AA rotatory evaporator (Shanghai Yarong Biochemical Instrument Plant); The twin-beam ultraviolet-visible pectrophotometer (Beijing Puxi General Instrument Co., Ltd, TU-1901); Electronic balance (Minqiao Precision Scientific Instruments Co., Ltd., Shanghai, SL-202); Ultrasonic cleaner (BRANSON, 83200S-T); Nicolet IS10 Fourier infrared spectrograph, KBr powder pressing method (Thermo company); Flash EA 1112 elemental analysers (Thermo company); Thermo iCAP 6500 spectrographs (Thermo company).
Oligochitosan, molecular weight＜3000, deacetylation＞90%, Shandong aokang bio tech ltd; 5-nitrosalicylaldehyde, 5-bromosalicylaldehyde, 5-chloro-salicylic aldehyde and 3,5-dichloro-salicylaldehyde, technical grade, sea, Shanghai Qu Huagong ltd (using preceding recrystallization); Diethyl phosphite, analytical pure, the brilliant pure reagent in Shanghai ltd; Sodium hydroxide, analytical pure, Tianjin moral grace chemical reagent ltd; Absolute ethyl alcohol, analytical pure, reagent Manufacturing Co., Ltd (with before doing absolute anhydrous processing) is learned in permanent Xinghua, Tianjin.
1.2 preparation method
The preparation of embodiment 1 5-chloro-salicylic aldehyde oligochitosan Schiff base phosphonate
(1) preparation of salicylic aldehyde oligochitosan Schiff's base
The 7.0186g oligochitosan soaked swelling in 20mL absolute ethanol solvent after, add 8.0538g salicylic aldehyde and 0.2445gNaOH (regulator solution pH value is 7.5～9), microwave heating back flow reaction 1.5h.After reaction finished, suction filtration was dry, and with ethanol Soxhlet extraction product 24h, to remove unnecessary salicylic aldehyde and substituted salicylic aldehydes in the product, product is being lower than 50 ℃ of following vacuum-dryings.Output 4.2815g.
(2) preparation of 5-chloro-salicylic aldehyde oligochitosan Schiff base phosphonate (S-COS-P)
Salicylic aldehyde oligochitosan Schiff's base (S-COS) and 2.2766g diethyl phosphite microwave heating back flow reaction 2.5h with 1.8339g above-mentioned steps (1) preparation gained.After reaction finished, the ethanol Soxhlet extraction product was used in suction filtration vacuum-drying, and to remove diethyl phosphite unnecessary in the product, product is being lower than 50 ℃ of following vacuum-dryings, preserved subsequent use in the bottle of behind 120 ℃ of sterilising treatment 20min, packing into.Output 1.5360g.
The preparation of embodiment 2 5-chloro-salicylic aldehyde oligochitosan Schiff base phosphonates
(1) preparation of 5-chloro-salicylic aldehyde oligochitosan Schiff's base
The 6.5852g oligochitosan soaked swelling in 50mL absolute ethanol solvent after, add 4.6473g 5-chloro-salicylic aldehyde and 0.9910gNaOH (regulator solution pH value is 7.5～9), microwave heating back flow reaction 2h.After reaction finished, suction filtration was dry, and with ethanol Soxhlet extraction product 26h, to remove unnecessary salicylic aldehyde and substituted salicylic aldehydes in the product, product is being lower than 50 ℃ of following vacuum-dryings.Output 7.1272g.
(2) the preparation 3.2071g of 5-chloro-salicylic aldehyde oligochitosan Schiff base phosphonate (S-COS-P)
5-chloro-salicylic aldehyde oligochitosan Schiff's base (S-COS) and 3.2071g diethyl phosphite microwave heating back flow reaction 1.8h with 2.0484g above-mentioned steps (1) preparation gained.After reaction finished, the ethanol Soxhlet extraction product was used in suction filtration vacuum-drying, and to remove diethyl phosphite unnecessary in the product, product is being lower than 50 ℃ of following vacuum-dryings, preserved subsequent use in the bottle of behind 120 ℃ of sterilising treatment 20min, packing into.Output 1.2972g.
Embodiment 33,5-dichloro-salicylaldehyde oligochitosan Schiff base phosphonate
The preparation of (1) 3,5-dichloro-salicylaldehyde oligochitosan Schiff's base
The 3.6080g oligochitosan soaked swelling in 40mL absolute ethanol solvent after, add 3.0732g 3,5-dichloro-salicylaldehyde and 0.2038gNaOH (regulator solution pH value is 7.5～9), microwave heating back flow reaction 2.5h.After reaction finished, suction filtration was dry, and with ethanol Soxhlet extraction product 27h, to remove unnecessary salicylic aldehyde and substituted salicylic aldehydes in the product, product is being lower than 50 ℃ of following vacuum-dryings.Output 3.756g.
The preparation of (2) 3,5-dichloro-salicylaldehyde oligochitosan Schiff base phosphonates (S-COS-P)
With 3 of 1.4653g above-mentioned steps (1) preparation gained, 5-dichloride base salicylic aldehyde oligochitosan Schiff's base (S-COS) and 2.7600g diethyl phosphite microwave heating back flow reaction 2.3h.After reaction finished, the ethanol Soxhlet extraction product was used in suction filtration vacuum-drying, and to remove diethyl phosphite unnecessary in the product, product is being lower than 50 ℃ of following vacuum-dryings, preserved subsequent use in the bottle of behind 120 ℃ of sterilising treatment 20min, packing into.Output 0.6913g.。
Embodiment 4 5-nitrosalicylaldehyde oligochitosan Schiff base phosphonate oligochitosan Schiff base phosphonates
(1) preparation of 5-nitrosalicylaldehyde oligochitosan Schiff's base
The 3.0802g oligochitosan soaked swelling in 40mL absolute ethanol solvent after, add 2.3382g 5-nitrosalicylaldehyde and 0.2gNaOH (regulator solution pH value is 7.5～9), microwave heating back flow reaction 1.8h.After reaction finished, suction filtration was dry, and with ethanol Soxhlet extraction product 25h, to remove unnecessary salicylic aldehyde and substituted salicylic aldehydes in the product, product is being lower than 50 ℃ of following vacuum-dryings.Output 2.9461g.
(2) preparation of 5-nitrosalicylaldehyde oligochitosan Schiff base phosphonate (S-COS-P)
5-nitrosalicylaldehyde oligochitosan Schiff's base (S-COS) and 2.9527g diethyl phosphite microwave heating back flow reaction 2.0h with 1.3352g above-mentioned steps (1) preparation gained.After reaction finished, the ethanol Soxhlet extraction product was used in suction filtration vacuum-drying, and to remove diethyl phosphite unnecessary in the product, product is being lower than 50 ℃ of following vacuum-dryings, preserved subsequent use in the bottle of behind 120 ℃ of sterilising treatment 20min, packing into.Output 1.085g.
(1) preparation of 5-bromosalicylaldehyde oligochitosan Schiff's base
The 6.4517g oligochitosan soaked swelling in 40mL absolute ethanol solvent after, add 7.1139g 5-bromosalicylaldehyde salicylic aldehyde and 0.2000gNaOH (regulator solution pH value is 7.5～9), microwave heating back flow reaction 2.3h.After reaction finished, suction filtration was dry, and with ethanol Soxhlet extraction product 28h, to remove unnecessary salicylic aldehyde and substituted salicylic aldehydes in the product, product is being lower than 50 ℃ of following vacuum-dryings.Output 5.8034g.
(2) preparation of 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate (S-COS-P)
5-bromosalicylaldehyde oligochitosan Schiff's base (S-COS) and 2.1152g diethyl phosphite microwave heating back flow reaction 1.5h with 1.9083g above-mentioned steps (1) preparation gained.After reaction finished, the ethanol Soxhlet extraction product was used in suction filtration vacuum-drying, and to remove diethyl phosphite unnecessary in the product, product is being lower than 50 ℃ of following vacuum-dryings, preserved subsequent use in the bottle of behind 120 ℃ of sterilising treatment 20min, packing into.Output 1.6138g.
2. oligochitosan Schiff base phosphonate (S-COS-P) medicament is to the inducing anti-disease property of tobacco TMV
2.1 supply the examination material
Choosing respectively has the tobacco bred of different resistances to try material as confession to TMV.Different varieties is following:
Nonsystematic infects the type kind: coral west cigarette (Xanthi-NN), Nicotiana glutinosa (N.glutinosa)
Systemic infection type kind: K326, No. five, Henan cigarette
Wherein coral west cigarette (Xanthi-NN) is provided by doctor Jiang Shijun for plant protection institute of Agricultural University Of He'nan, and Nicotiana glutinosa (N.glutinosa) is bought in Tobacco Institute, Chinese Academy of Agricultural Science.
2.2 TMV poison source
Supply prelibation source TMV virus effluent south doctor Jiang Shijun of plant protection institute of agriculture university to be so kind as to give, breeding obtains on common cigarette.Venom preparation: take by weighing the sick tobacco leaf of about 0.5g and in mortar, grind, add the 0.01molL of pH=7.0
-1Phosphate buffer solution be mixed with 25mL solution after with two-layer filtered through gauze, under wavelength 200～300nm, carry out UV scanning to confirm its purity and concentration.It is subsequent use to place 5 ℃ of refrigerators to preserve.
2.3 oligochitosan Schiff base phosphonate (S-COS-P) infects in the cigarette strain retarding effect to TMV at nonsystematic
Adopt full leaf method.In 25 ℃ of room temperatures, select for use and transplant back 3 months consistent potted plant Nicotiana glutinosas of growing way, spray clear water or the processing of spray medicine earlier, inoculate TMV virus by conventional juice frictional inoculation method behind the 24h.With spray inoculate behind the clear water be treated to contrast (CK), each processing repetition 3 times.In about the 10d of inoculation back, observe incidence, calculate withered spot inhibiting rate with (1) formula.
2.4 oligochitosan Schiff base phosphonate (S-COS-P) in the strain of systemic infection cigarette to the active regulating and controlling effect of its defensive ferment
.2.4.1 oligochitosan Schiff base phosphonate (S-COS-P) is to the active influence of cigarette strain defensive ferment
Select that TMV is suppressed the best medicament of effect is 5-bromosalicylaldehyde oligochitosan for use, the effect analysis of on No. five, Henan cigarette, carrying out.Select the growing way homogeneous for use; No. five, the potted plant Henan cigarette of 7～8 true leaves, spray plant 1d with 5-bromosalicylaldehyde oligochitosan after, adopt conventional juice rubbing method inoculation TMV; And behind 2d, carry out coating once more, test is provided with: (1) is inoculated after spraying 5-bromosalicylaldehyde oligochitosan; (2) inoculate after spraying 300 times of Ningnanmycin aquas; (3) inoculation contrast (virus inoculation behind the sprinkling clear water);
(4) normal healthy controls (spraying not virus inoculation of clear water).The the 1st, 4,7,10 and 13 day processing leaf sample of gathering respectively in the inoculation back, in the masking foil of packing into, marked is preserved subsequent use in-80 ℃ of refrigerators.
2.4.2 the measuring method of physiological and biochemical index
Soluble proteins adopts Xylene Brilliant Cyanine G G-250 dye binding method; Chlorophyll content adopts spectrophotometry; Hemocuprein (SOD) is slightly revised with reference to the method in the desolate wave book; Px (POD) adopts guaiacol method; Katalase (CAT) is with reference to the method in the literary compositions such as Wang Xiaoqing; Polyphenoloxidase (PPO) is slightly revised with reference to colourimetry; Phenylalanine ammonia lyase (PAL) adopts spectrophotometer method.
2.5 key instrument is used in test
High speed freezing centrifuge, UV-9200 ultraviolet-visible pectrophotometer, Ultralow Temperature Freezer, refrigerator, electric-heated thermostatic water bath, magnetic stirring apparatus, micro sample adding appliance etc.
2.6 statistical analysis technique
Data processing softwares such as utilization Excel and SPSS carry out statistical study.
3. result and analysis
3.1 the structural characterization of oligochitosan Schiff's base (S-COS)
3.1.1 uv-absorbing analysis
Uv absorption spectrum data (table 1) by oligochitosan (COS) and oligochitosan Schiff's base (S-COS) can be found out: COS has 2 strong and narrow absorption peaks at 212nm and 205nm place, is not having obvious absorption greater than the 212nm wavelength region.S-COS is the absorption of phenol imine group at the absorption peak at 297～327nm place, belongs to the charge transfer transition from the phenyl ring to the imido grpup, is characterized in that transition probability is little, a little less than the absorption intensity; The absorption peak at 241～269nm place is the distinctive B band of an aromatic compounds absorption peak, and this is because overlapping the causing of π → π * transition and phenyl ring vibration; 215～220nm place is the K band absorption peak of phenyl ring, is characterized in that absorption intensity is big, is to be produced by π in the conjugated double bond → π * transition.
The uv absorption spectrum data of table 1 oligochitosan (COS) and oligochitosan Schiff's base (S-COS)
As can beappreciated from fig. 1; The uv scan curve of oligochitosan Schiff's base (S-COS) is different fully with the UV scanning curve of oligochitosan (COS) raw material, and this is because the ultraviolet absorption peak after COS and salicylic aldehyde and the substituted salicylic aldehydes generation addition reaction has been compared change with the raw material oligochitosan.This has confirmed that further chemical reaction has taken place for oligochitosan and salicylic aldehyde and substituted salicylic aldehydes.
3.1.2 IR spectroscopy
Table 2 is infrared spectrograms of oligochitosan (COS) and oligochitosan Schiff's base (S-COS).In the infrared spectrum of COS, 3416cm
-1Be the O-H stretching vibration, 3237cm
-1Be that N-H goes up stretching vibration, 1721cm
-1Be the C=O stretching vibration, 1401cm
-1Be C-C stretching vibration on the phenyl ring, 1082cm
-1It is the C-O stretching vibration; In the ir spectra of S-COS, 785,774cm
-1Be the C-Cl stretching vibration, 538cm
-1Be the C-Br stretching vibration, 1298cm
-1Be-NO
2Symmetrical stretching vibration.O-H compares with COS with the stretching vibration on the N-H red shift has taken place, at 1635～1640cm
-1Near the stretching vibration peak of C=N has appearred, this is the characteristic absorbance of Schiff's base phenol imines, and at 1452～1520cm
-1With 1510～1617cm
-1Near the skeletal vibration of phenyl ring has appearred, at 1270cm
-1Near phenolic hydroxyl group appearred and flexural vibration absorb, these new peaks all are to be produced by the Schiff's base of substituted salicylic aldehydes with COS reaction generation.Particularly the appearance of C-X and-NO2 absorption peak has also confirmed the upper of substituted salicylic aldehydes.
Ir data (the v/cm of table 2 oligochitosan (COS) and oligochitosan Schiff's base (S-COS)
-1)
Fig. 2 is the ir spectra of oligochitosan (COS) and oligochitosan Schiff's base (S-COS).Find out that from collection of illustrative plates the ir spectra curve of S-COS compared considerable change with raw material COS, explain that chemical reaction has taken place for raw material oligochitosan and salicylic aldehyde and substituted salicylic aldehydes.In addition, S-COS is at 2820～2720cm
-1The place do not have to absorb honeybee, does not promptly have-c h bond in the CHO group
[125], explain through operations such as washing, backflows and excessive aldehyde removed fully that the target compound structure is accurate.
3.1.3 ultimate analysis
Ultimate analysis test result (table 3) shows that the content of principal element changes along with the substituent variation of salicylic aldehyde in the oligochitosan Schiff's base (S-COS), and wherein the C element accounts for the largest percentage, and then is N or H element.Thus, confirmed that further reaction has taken place for substituted salicylic aldehydes and oligochitosan.Consider the influence of the water absorbability and the intramolecularly combination water of oligochitosan, actual element content can be a little less than calculated value.
The ultimate analysis data of table 3 oligochitosan Schiff's base (S-COS)
3.1.4 thermogravimetric analysis
Select oligochitosan (COS) and oligochitosan Schiff's base (S-COS) to do thermogravimetric analysis.As shown in Figure 3, the weightlessness before 100 ℃ are due to the dehydration, COS 150～250 ℃ weightless comparatively slowly, and S-COS sharply descends 200～280 ℃ of weight, forms sharp contrast with COS.S-COS the weightless time occurs early than COS.
3.2 the structural characterization of oligochitosan Schiff base phosphonate (S-COS-P)
3.2.1 uv-absorbing analysis
After oligochitosan Schiff's base (S-COS) and the addition of diethyl phosphite phosphonic acid ester (table 4); The absorption peak of phenol imido grpup roughly shows violet shift situation, and this is because two keys of C=N are opened, and has destroyed the order of molecule; Thereby the transition energy level is increased, and violet shift has taken place in absorption spectrum.
The absorption peak position (UV-Vis/nm) of C=N in the table 4 oligochitosan Schiff base phosphonate (S-COS-P)
Can find out by Fig. 4; Oligochitosan Schiff's base (S-COS) through with the diethyl phosphite addition reaction after; Uv absorption spectrum curve and raw material oligochitosan and oligochitosan Schiff's base have obvious difference; This can explain that the oligochitosan Schiff's base with diethyl phosphite reaction has taken place, and reactor product awaits next step structural validation.
3.2.2 IR spectroscopy
As shown in table 5, in the infrared spectrum of oligochitosan Schiff base phosphonate (S-COS-P), 3394～3415cm
-1Be the O-H stretching vibration, 2934～3231cm
-1Be that N-H goes up stretching vibration, 1451～1693cm
-1Near be the skeletal vibration of phenyl ring, 1399～1400cm
-1Be C-C stretching vibration on the phenyl ring, 1274～1321cm
-1Be the P=O stretching vibration, 1064～1075cm
-1It is C-P-O stretching vibration absorption peak.Wherein P=O stretching vibration and C-P-O stretching vibration absorption peak are the positions of the ft-ir characteristic absorption peak of oligochitosan Schiff base phosphonate (S-COS-P), indicate that addition reaction has taken place for diethyl phosphite and oligochitosan Schiff's base.
Ir data (the v/cm of table 5 oligochitosan Schiff base phosphonate (S-COS-P)
-1)
3.2.3 ultimate analysis and inductively coupled plasma (ICP) spectrometry
(table 6) result that ultimate analysis and inductively coupled plasma (ICP) spectrum test is analyzed shows that C element content proportion in compound is maximum, and is about about 33%, H, N, P element than weight average about 5%.Consider the influence of the water absorbability and the intramolecularly combination water of oligochitosan, actual element content can be a little less than calculated value.
The ICP spectrum test of table 6 oligochitosan Schiff base phosphonate (S-COS-P) constituent content is analyzed (%)
3.3 oligochitosan Schiff base phosphonate (S-COS-P) is to the inducing anti-disease Journal of Sex Research of tobacco TMV
3.3.1 oligochitosan Schiff base phosphonate (S-COS-P) medicament on Nicotiana glutinosa to the retarding effect of TMV
Table 7 oligochitosan Schiff base phosphonate (S-COS-P) medicament on Nicotiana glutinosa to the retarding effect of TMV
Annotate: mark identical lowercase person in the table behind the same column data and represent multiple range test, at P through DuncanShi
0.05Difference is not remarkable on the level.
Table 7 is the result show, (1) is compared with the clear water contrast, and 15 kinds of oligochitosan Schiff base phosphonates (S-COS-P) medicament all has the inhibition effect to withered spot; (2) oligochitosan Schiff base phosphonate (S-COS-P) medicament has remarkable inducing anti-disease effect to tobacco TMV, and the drug effect base table reveals 200 μ gmL
-1＞100 μ gmL
-1＞50 μ gmL
-1(3) concentration is 200 μ gmL
-1Oligochitosan Schiff base phosphonate pharmacy effect all be superior to 800 times of 5% 2 (octyl amine ethyl) glycinate AS, also be superior to 300 times of Ningnanmycin aquas; (4) generally speaking, 200 μ gmL
-1The 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate pharmacy effect best (Fig. 5) of concentration.
3.3.2 oligochitosan Schiff base phosphonate medicament chlorophyll and active research of defensive ferment in No. five, Henan cigarette
3.3.2.1 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament is to the chlorophyllous influence of tobacco
Fig. 6 result shows that chlorophyll a, the b content of (1) inoculation control treatment descend gradually behind inoculation 7d, wherein the chlorophyll a content range of decrease is bigger, reaches 22.85%; (2) spray chlorophyll a content that 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate handles and after inoculation, reached peak 0.3678mgmg on the 7th day
-1(albumen) sprays 300 times of Ningnanmycin aquas than the same period and handles growth by 8.02%; The chlorophyll b that sprays the processing of 5-bromosalicylaldehyde oligochitosan reached peak 0.7752mgmg at the 10th day
-1(albumen) sprays 300 times of Ningnanmycin aquas than the same period and handles growth by 32.63%, inoculates control treatment than the same period and increases by 13.89%; (3) spray 5-bromosalicylaldehyde oligochitosan and handle the reduction amplitude that can significantly reduce chlorophyll content in the inoculation later stage, the range of decrease of its chlorophyll a, b is respectively 16.35% and 10.15%, and the range of decrease of inoculation contrast reaches 22.85% and 22.56% respectively; (4) because tobacco is in the growth phase in seedling stage, illumination is sufficient
[127,128], chlorophyll content increases at preseed stage to some extent.
3.3.2.2 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament is to the influence of tobacco leaf SOD enzyme
Fig. 7 result shows; Spraying 5-bromosalicylaldehyde oligochitosan handles with the SOD activity change that sprays 300 times of Ningnanmycin aquas processing similar; All risings to some extent in the 4th day after inoculation; Reduced at the 7th day, wherein spraying the range of decrease that 5-bromosalicylaldehyde oligochitosan handles at the 7th day is 5.40%, and spray that 300 times of Ningnanmycin aquas handle decrease by 34.44%.Compare with contrast, the SOD activity that sprays the processing of 5-bromosalicylaldehyde oligochitosan is significantly increased, and performance in the 7th day is the most obvious after inoculation: compare with spraying 300 times of Ningnanmycin aquas processing the same period, increase 33.93mgmg
-1(albumen); Compare with inoculating control treatment the same period, increase 29.35mgmg
-1(albumen); Compare with the normal healthy controls processing same period, increase 15.68mgmg
-1(albumen).Thus it is clear that, spray 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate chemicals treatment and can after tobacco leaf is infected by TMV, significantly improve tobacco leaf SOD activity.
3.3.2.3 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament is to the influence of tobacco leaf CAT enzyme
Can know by Fig. 8; Spraying 5-bromosalicylaldehyde oligochitosan handles with the CAT activity change that sprays 300 times of Ningnanmycin aquas processing similar; Be rising-reduction-rising-reduction, present bimodal state, their CAT activity all reached peak value in the 4th day and the 10th day after inoculation; And the 4th day be the whole inoculation peak in period, be respectively 1122.21mgmg
-1(albumen) and 1363.77mgmg
-1(albumen); Inoculation control treatment CAT activity reduces along with the increase of inoculation time, removes the 10th beyond the highest heavens, and all the other time enzymic activitys are linear decrease trend; The CAT activity change that normal healthy controls is handled obviously presents the unimodal curve rule, peak after inoculation the 4th day, and the CAT activity reaches 1251.53mgmg
-1(albumen).It is thus clear that, spray 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate chemicals treatment and can after tobacco leaf is infected by TMV, significantly improve tobacco leaf CAT activity, particularly evident in the lifting in inoculation later stage.
3.3.2.4 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament is to the influence of tobacco leaf POD enzyme
Fig. 9 result shows that the POD activity change amplitude that normal healthy controls is handled is little, and the enzymic activity of testing period is roughly at 73mgmg
-1Fluctuate near (albumen), state generally tends towards stability; The POD activity of inoculation control treatment sharply dropped to 22.92mgmg at the 4th day
-1(albumen) decreases by 65.91%, and sharply rises at the 7th day, and increasing degree reaches 260.40%, and is bigger in the rangeability in whole inoculation period; Spray 5-bromosalicylaldehyde oligochitosan and handle, all appear and reduce back rising trend earlier, wherein spray the processing of 5-bromosalicylaldehyde oligochitosan and after inoculation, reached peak 72.43mgmg on the 13rd day with to spray the POD activity that 300 times of Ningnanmycin aquas handle similar
-1(albumen) sprays 300 times of Ningnanmycin aquas than the same period and handles growth by 20.42%, inoculates control treatment than the same period and increases by 21.49%.It is thus clear that spraying 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate chemicals treatment can receive TMV to infect back tobacco leaf POD activity obvious raising of inoculation later stage.
3.3.2.5 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament is to the influence of tobacco leaf PPO enzyme
Can know by Figure 10, spray 5-bromosalicylaldehyde oligochitosan and handle, being first the rising and afterwards reducing that present the unimodal curve rule, their peak is all at the 7th day with to spray the PPO activity that 300 times of Ningnanmycin aquas handle similar.Wherein spray the processing of 5-bromosalicylaldehyde oligochitosan and reach 108.89mgmg
-1(albumen) sprays 300 times of Ningnanmycin aquas than the same period and handles growth by 7.18%, inoculates control treatment than the same period and increases by 23.19%, increases by 10.96% than the normal healthy controls processing same period.Remove the 1st beyond the highest heavens, spray 5-bromosalicylaldehyde oligochitosan and handle at the trial that the PPO activity of phase all is higher than the inoculation contrast, wherein the 10th day raising is the most obvious, reaches 36.91mgmg
-1(albumen) increases by 84.30 percentage points on year-on-year basis.It is thus clear that, compare with inoculation contrast, spray 5-bromosalicylaldehyde oligochitosan and handle and can significantly improve that to receive TMV to infect back tobacco leaf PPO active.
3.3.2.6 5-bromosalicylaldehyde oligochitosan Schiff base phosphonate medicament is to the influence of tobacco leaf PAL enzyme
Shown in figure 11, spray 5-bromosalicylaldehyde oligochitosan and handle, spray 300 times of Ningnanmycin aquas to handle the PAL activity of handling with normal healthy controls similar, be rising-reduction-rising-reduction, present the bimodal curve rule.Their peak all at the 4th day the 1st peak value place, is respectively 1678.58mgmg
-1(albumen), 1517.40mgmg
-1(albumen) and 1785.02mgmg
-1(albumen).The PAL activity of inoculation control treatment shows the Changing Pattern that raises and afterwards reduce earlier, and its peak was 1842.31mgmg at the 4th day
-1(albumen), the 5-bromosalicylaldehyde oligochitosan that sprays that is higher than the same period is handled, but sharply descends in inoculation its enzymic activity of later stage, and the 5-bromosalicylaldehyde oligochitosan that sprays that all is lower than the same period is handled.It is thus clear that, compare with inoculation contrast, spray 5-bromosalicylaldehyde oligochitosan and handle and can significantly improve that to receive TMV to infect back tobacco leaf PAL active in the inoculation later stage.
Claims (5)
1. oligochitosan Schiff base phosphonate, it is characterized in that: this compound has following general structural formula:
N=2-20 in the formula;
R is
2. the preparation method of the described oligochitosan Schiff base phosphonate of claim 1 is characterized in that:
(1) oligochitosan is soaked swelling in the absolute ethanol solvent after, be benchmark with the acetyl monose that takes off of oligochitosan, add the salicylic aldehyde of 1～2 times of molar weight, adding NaOH regulator solution pH value is 7.5～9, microwave heating back flow reaction 1.5～2.5h.After reaction finished, suction filtration was dry, and with ethanol Soxhlet extraction product 24～28h, to remove unnecessary salicylic aldehyde in the product, product is subsequent use after being lower than 50 ℃ of following vacuum-dryings;
(2) the oligochitosan Schiff's base and the diethyl phosphite microwave heating back flow reaction 1.5～2.5h that step (1) are obtained; The mol ratio of oligochitosan Schiff's base and diethyl phosphite is 1: 20～1: 30, after reaction finishes, and suction filtration vacuum-drying; Use the ethanol Soxhlet extraction product; To remove diethyl phosphite unnecessary in the product, product is being lower than 50 ℃ of following vacuum-dryings, gets finished product through 120 ℃ of sterilising treatment 20min.
3. the preparation method of oligochitosan Schiff base phosphonate according to claim 2; It is characterized in that: described oligochitosan Schiff's base is selected from 5-chloro-salicylic aldehyde oligochitosan Schiff's base, 3,5-dichloro-salicylaldehyde oligochitosan Schiff's base, 5-nitrosalicylaldehyde oligochitosan Schiff's base, 5-bromosalicylaldehyde oligochitosan Schiff's base.
4. the preparation method of oligochitosan Schiff base phosphonate according to claim 2 is characterized in that: described microwave heating power is 400W.
5. the application of the described oligochitosan Schiff base phosphonate of claim 1 in the treatment tobacco mosaic virus disease.
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| CN102516413B (en) * | 2011-12-09 | 2014-02-12 | 中国科学院海洋研究所 | Furyl alpha-aminophosphonate chitosan derivative and its preparation method |
| CN102942590B (en) * | 2012-11-01 | 2015-12-16 | 武汉三聚生物技术有限公司 | α-(diamantane) aminophosphonic acid and its production and use |
| CN103182516A (en) * | 2013-03-27 | 2013-07-03 | 河南农业大学 | Chitosan oligosaccharide derivative and nano-silver preparation method |
| CN103819274B (en) * | 2014-03-05 | 2015-07-01 | 福建永宸生物科技开发有限公司 | Preparation method for phosphite- chitosan oligosaccharide composite biological medical fertilizer |
| CN106496359A (en) * | 2016-11-11 | 2017-03-15 | 中国科学院海洋研究所 | A kind of shitosan toloxyl p diethylaminobenzoic acid ester derivant and preparation method thereof |
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