CN109765315A - A kind of preparation method and detection method of polyoxin component C - Google Patents
A kind of preparation method and detection method of polyoxin component C Download PDFInfo
- Publication number
- CN109765315A CN109765315A CN201910089645.XA CN201910089645A CN109765315A CN 109765315 A CN109765315 A CN 109765315A CN 201910089645 A CN201910089645 A CN 201910089645A CN 109765315 A CN109765315 A CN 109765315A
- Authority
- CN
- China
- Prior art keywords
- polyoxin
- component
- detection method
- water
- phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to the preparation methods and detection method of a kind of polyoxin component C, pass through the component preparation of polyoxin C and efficient detection, the means such as the optimization especially by testing conditions further increase the content with specific composition and effectiveness, realize that the quality of polyoxin controls more representative and operability.
Description
Technical field
The present invention relates to the detection techniques of biological pesticide containing antibiotic, and in particular to a kind of preparation side of polyoxin component C
Method and detection method.
Background technique
Polyoxin is called Polyoxin, Japan be from cocoa streptocin Ah rope mutation (S.cacaoi var.asoensis) in be separated to a kind of nucleoside antibiotic.It and is golden chromogenic in China
Streptomycete (S.aurea chromogenes 4896) metabolite.
Polyoxin belongs to broad spectrum activity biological bactericide, has preferable absorbability conduction.Its mechanism of action is dry
The biosynthesis for disturbing somatic cells wall chitin makes somatic cells wall not can be carried out biosynthesis and lead to death, moreover it is possible to inhibit
Germ produces spore and scab expands.The ingredient is mainly used for preventing and treating alternaria leaf spot of apple, pear scab, grape grey mould, cucumber
The plurality of plant diseases such as downy mildew, tomato late blight, black fleck disease of ginseng.
Since the produced metabolite of streptomyces aureus is multi-component, component as many as totally 14 components from A to N, wherein
B, C and N is important component, and component C molecular formula C11H15N3O8, No. CAS is 21027-33-8, and the molecular structural formula of C is as follows
Figure:
Due to the produced metabolite of streptomyces aureus be it is multi-component, the present examination criteria in relation to polyoxin is most
Bactericidal effect of double dish bioassays identification polyoxin to instruction pathogenic bacteria of enterprise's use is counted, and gives birth to and surveys effect often because of each enterprise
Indicator bacteria difference, the different batches polyoxin each component content surveyed raw to polyoxin potency is different and surveys with life under batch real
Testing the factors such as error difference influences the measurement effect of polyoxin potency.
On the other hand, in controlling disease, the long-time service of a large amount of chemical pesticides will lead to the deterioration and agriculture of ecological environment
The generation of Product quality and safety problem, and polyoxin has efficient, safety, low-residual, to environment friend as biological bactericide
Well equal good characteristics, but since in production, each enterprise is further on the market using sod cultivation influence polyoxin in practice
It promotes and uses.The present invention is in " a kind of method of efficient liquid phase detection polyoxin B, grant number ZL20041003147.8 "
On the basis of further improvement, realize to the efficient detection of polyoxin C.
Summary of the invention
In order to solve the above-mentioned problems in the prior art, the present invention provides a kind of preparation side of polyoxin component C
Method and detection method realize that the quality control of polyoxin more represents by the component preparation of polyoxin C and efficient detection
Property and operability.
The present invention adopts the following technical scheme that a kind of preparation method of polyoxin component C dilutes raw medicine through distilled water
Afterwards by ion exchange resin, polyoxin component C solution then is obtained in cutting splitter, the solution is steamed by rotation
It sends out instrument and removes moisture content, after freeze-drying, obtain polyoxin component C.
In the preferred embodiments of the present invention, the raw medicine be by streptomyces aureus after everfermentation 144h,
Fermentation liquid is obtained, fermentation liquid is acidified, then passes through plate-frame filtering, ceramic membrane filter and nanofiltration respectively, is finally spray-dried institute
?.
The polyoxin component C that the present invention also protects the preparation method to be prepared.
The present invention also protects the detection method of the polyoxin component C, the highly effective liquid phase chromatography detection method of use, tool
The chromatographic condition of body:
Chromatographic column: to be embedded in the alkyl silane bonded silica gel of polar group as stationary phase；
Mobile phase: with volume basis, trifluoroacetic acid: (water+organic phase)=2~4 ‰ are 2.0~3.0 with phosphoric acid tune pH value；
Flow velocity: 0.5-1.5 mL/min；
Detection wavelength: 240-290nm；
Column temperature: 20~40 DEG C；
Sample volume: 5-20 μ L；
Detector: UV detector or diode array detector.
In a preferred embodiment of the present invention, the chromatographic column is selected from the octadecylsilane of insertion polar group
Bonded silica gel is the chromatographic column of stationary phase.
In a preferred embodiment of the present invention, in mobile phase, in the water+organic phase, with volume basis, water is
99.5%.Methanol is 0.5%；Trifluoroacetic acid: (water+organic phase)=3 ‰；It is 2.5 with phosphoric acid tune pH value.
In a preferred embodiment of the present invention, the flow velocity is 1.0 mL/min；The Detection wavelength is
260nm；The column temperature is 25 DEG C；The sample volume is 10 μ L.
In a preferred embodiment of the present invention, in mobile phase, with volume basis, trifluoroacetic acid: (water+organic phase)=
3‰。
Compared with prior art, the beneficial effects of the present invention are:
1, from 14 kinds of components such as polyoxin A-N, it can be determined by the test method to each component and obtain different component sample
Product, at the same to each sample isolation and purification after, carry out the correlative studys such as medicine efficacy relation and the pharmacokinetics of different component；
2, polyoxin fermenting and producing come out fermentation liquid is acidified, plate-frame filtering, ceramic membrane filter, nanofiltration and spray drying obtain
The raw medicine obtained is by the mixture of 14 kinds of components such as A-N, and being established by each component test method especially has several representativenesses
Component such as B, C and N detection method establish can measure the raw medicine mixture B, C and N component content and medicine efficacy relation；
3, it is established by the detection method to the produced secondary metabolite each component of streptomyces aureus fermentation, passes through testing conditions
The means such as optimization further increase the content with specific composition and effectiveness, further improve the drug effect of entire raw medicine and improve agriculture
The control efficiency of the specific pathogenic bacteria of crop.
Detailed description of the invention
It is described further with reference to the accompanying drawing:
Residence time of Fig. 1 polyoxin component C in HPLC；
The linear relationship of Fig. 2 polyoxin component C concentration and peak area；
The mass spectrogram of Fig. 3 polyoxin C.
Specific embodiment
In order to which the purpose of the present invention, technical solution and technical effect are more clearly understood, attached drawing and tool are now compareed
The present invention is described in more detail for body embodiment.
Means of testing of the present invention is as follows:
(1) reagent is as follows:
Trifluoroacetic acid: chromatographically pure；Water: secondary distilled water is newly steamed；Polyoxin C standard items: known quality score >=95.0%.
(2) instrument is as follows:
High performance liquid chromatograph: there is Variable wavelength UV detector；Chromatographic data processor；Chromatographic column: 4.6mm × 250mm
(id) stainless steel chromatographic column, Waters symetryshield RP18,5 μm of partial size；Microsyringe: 10 μ L.
(3) high performance liquid chromatography operating condition is as follows:
Mobile phase: trifluoroacetic acid: (VWater+VMethanol=99.5%+0.5%)=3 ‰ (v/v) are 2.5 with phosphoric acid tune pH value.
Flow velocity: 1.0mL/min；Detection wavelength: 260nm；Temperature: 25 DEG C；Sampling volume: 10 μ L；Retention time: more anti-mildews
Plain C about 5.38min.
(4) quantitative analysis of polyoxin
1. the configuration of the quasi- product solution of polyoxin mark C
Polyoxin C standard items about 50mg is weighed respectively into same 50ml volumetric flask, constant volume is diluted with water, then respectively with shifting
Liquid pipe easily takes 1mL into same 5mL volumetric flask, and constant volume is diluted with water.
2. the configuration of sample solution
The sample 100mg containing polyoxin is weighed into 10mL volumetric flask, with water dissolution and constant volume, with 0.45 μm of membrane filtration,
It is spare.
3. quantitative analysis
After instrument reaches balance and stability, it is molten that standard solution, two sample needle solution, standard items are carried out in the form of following sequence
Liquid, the final area for obtaining polyoxin B CN absorption peak.
It calculates:
The mass fraction ω of polyoxin C in polyoxin raw medicine (wettable powder, aqua etc.)1 (%) is calculated by formula (1):
In formula: ω1--- the mass fraction of polyoxin C in sample is indicated with %；
A2--- sample polyoxin C peak area；
m1--- the quality of standard specimen, g
ω --- polyoxin C mass fraction in standard specimen is indicated with %；
A1--- sample polyoxin C peak area；
m2--- the quality of sample, g；
N--- dilution gfactor, n=25.
Embodiment 1
Test apparatus and condition:
1525 high performance liquid chromatograph of Waters, 2487 UV detector of Waters；
Chromatographic column: being embedded in C18(250 × 4.6mm of polar group, and 5 μm)；
Mobile phase: trifluoroacetic acid: (VWater+VMethanol)=3 ‰ (v/v), wherein VWaterFor 99.5%, VMethanolIt is 0.5%；
Flow velocity: 1.0 mL/min；
Detection wavelength: 260nm；
Column temperature: 25 DEG C；
Sample volume: 10 μ L.
Test procedure:
It weighs the C of polyoxin containing 0.02g mark product with pure water dissolution, constant volume, to shake up in 50mL volumetric flask, crosses 0.45 μ
M filter membrane, available test-object product solution are used.
The C sample of polyoxin containing 0.02g is weighed, in 50mL volumetric flask, with pure water dissolution, constant volume, is shaken up, mistake
0.45 μm of filter membrane, available test agent.
It takes above-mentioned for test-object product and sample solution, injection high performance liquid chromatograph, according to above-mentioned condition progress efficient liquid phase
Chromatography records chromatogram, as shown in Figure 1.The result shows that polyoxin C main peak appearance time is 5.38min.
Embodiment 2
Test apparatus and condition:
1525 high performance liquid chromatograph of Waters, 2487 UV detector of Waters；
Chromatographic column: being embedded in C18(250 × 4.6mm of polar group, and 5 μm)；
Mobile phase: trifluoroacetic acid: (VWater+VMethanol)=3 ‰ (v/v), wherein VWaterFor 99.5%, VMethanolIt is 0.5%；
Flow velocity: 1.0 mL/min；
Detection wavelength: 260nm；
Column temperature: 25 DEG C；
Sample volume: 10 μ L.
Test procedure:
It weighs the C of polyoxin containing 0.02g mark product with pure water dissolution, constant volume, to shake up in 50mL volumetric flask, crosses 0.45 μ
M filter membrane, available test-object product solution are used.
The C sample of polyoxin containing 0.02g is weighed, in 50mL volumetric flask, with pure water dissolution, constant volume, is shaken up, mistake
0.45 μm of filter membrane, available test agent.
Mass Spectrometry Conditions
Instrument: Bruker 500MHz NMR with BBO probe；
Nucleus for channel:1H；
Frequency of observed channel:500MHz；
Temperature of sample : Ambient Temperature
Relaxation DI: 1.00s
Time domain size :65536
It is placed in 5mm nuclear magnetic tube with polyoxin standard items (sample) C of 10mg, the D of 0.50mL is then added2O is set in pipe,
It is uniformly mixed.Then whether test polyoxin C sample is consistent with the charge-mass ratio of C standard items, and concrete outcome is shown in Fig. 2, as a result table
Bright polyoxin C sample and the charge-mass ratio of C standard items are 317.95, that is, the sample prepared is identical as standard items charge-mass ratio, root
Category same substance is learnt according to map comparison.
Embodiment 3
Test apparatus and condition:
1525 high performance liquid chromatograph of Waters, 2487 UV detector of Waters；
Chromatographic column: being embedded in C18(250 × 4.6mm of polar group, and 5 μm)；
Mobile phase: trifluoroacetic acid: (VWater+VMethanol)=3 ‰ (v/v), wherein VWaterFor 99.5%, VMethanolIt is 0.5%；
Flow velocity: 1.0 mL/min；
Detection wavelength: 260nm；
Column temperature: 25 DEG C；
Sample volume: 10 μ L.
Test procedure:
It weighs the C of polyoxin containing 0.02g mark product with pure water dissolution, constant volume, to shake up in 50mL volumetric flask, crosses 0.45 μm of filter
Film, available test-object product solution are used.
The C sample of polyoxin containing 0.02g is weighed, in 50mL volumetric flask, with pure water dissolution, constant volume, is shaken up, mistake
0.45 μm of filter membrane, available test agent.
It takes above-mentioned for test-object product and sample solution, injection high performance liquid chromatograph, according to above-mentioned condition progress efficient liquid phase
Chromatography records chromatogram.
The linear dependence of analysis method is tested
It takes for test-object product solution, is analyzed according to detection method, sample volume is respectively 2,4,6,8,10 μ L, with more
Antimycin C mass is abscissa, and peak area is that ordinate makes standard curve, and obtaining linear equation is Y=5624.4X+12702, phase
Close coefficients R2It is 0.9997.
The accuracy test of analysis method
5 samples are weighed in the polyoxin C sample of known quality score, are separately added into a certain amount of polyoxin C mark
Product, are analyzed that the results are shown in Table 1 according to detection method, and measuring polyoxin C average recovery rate is
96.982%。
Table is tested in 1 component C sample accuracy of table
The precision test of analysis method
5 samples are accurately weighed from same sample, detection method according to the invention is analyzed, and polyoxin C is measured
Standard deviation be 0.0178%, relative standard deviation 1.16%.
2 component C sample accuracy of table tests table
As it can be seen that weighing 5 samples as known from Table 1, in C sample, a certain amount of polyoxin C mark product are separately added into, according to this hair
Bright detection method is analyzed, and measuring polyoxin C average recovery rate is 96.982%, as known from Table 2, measures polyoxin
The standard deviation of C is 0.0178%, relative standard deviation 1.16%, and the accuracy of explanation the method measurement and precision all compare
It is higher, it is reproducible many advantages, such as.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (8)
1. a kind of preparation method of polyoxin component C, which is characterized in that raw medicine is passed through ion exchange after distilled water dilutes
Then resin obtains polyoxin component C solution in cutting splitter, the solution removes moisture content by Rotary Evaporators,
After freeze-drying, polyoxin component C is obtained.
2. preparation method according to claim 1, which is characterized in that the raw medicine is by streptomyces aureus through everfermentation
After 144h, fermentation liquid is obtained, fermentation liquid is acidified, then passes through plate-frame filtering, ceramic membrane filter and nanofiltration respectively, finally sprays
The dry gained of mist.
3. the polyoxin component C that preparation method according to claim 1 or 2 is prepared.
4. the detection method of polyoxin component C according to claim 3, which is characterized in that the high-efficient liquid phase color of use
Detection method is composed, specific chromatographic condition:
Chromatographic column: to be embedded in the alkyl silane bonded silica gel of polar group as stationary phase；
Mobile phase: with volume basis, trifluoroacetic acid: (water+organic phase)=2~4 ‰ are 2.0~3.0 with phosphoric acid tune pH value；
Flow velocity: 0.5-1.5 mL/min；
Detection wavelength: 240-290nm；
Column temperature: 20~40 DEG C；
Sample volume: 5-20 μ L；
Detector: UV detector or diode array detector.
5. detection method according to claim 4, which is characterized in that the chromatographic column is selected from the ten of insertion polar group
Eight alkyl silane bonded silica gels are the chromatographic column of stationary phase.
6. detection method according to claim 4, which is characterized in that in mobile phase, in the water+organic phase, with body
Product is than meter, water 99.5%；Methanol is 0.5%；Trifluoroacetic acid: (water+organic phase)=3 ‰；It is 2.5 with phosphoric acid tune pH value.
7. detection method according to claim 4, which is characterized in that the flow velocity is 1.0 mL/min；The inspection
Survey wavelength is 260nm；The column temperature is 25 DEG C；The sample volume is 10 μ L.
8. detection method according to claim 4, which is characterized in that in mobile phase, with volume basis, trifluoroacetic acid: (water
+ organic phase)=3 ‰.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910089645.XA CN109765315A (en) | 2019-01-30 | 2019-01-30 | A kind of preparation method and detection method of polyoxin component C |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910089645.XA CN109765315A (en) | 2019-01-30 | 2019-01-30 | A kind of preparation method and detection method of polyoxin component C |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109765315A true CN109765315A (en) | 2019-05-17 |
Family
ID=66454463
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910089645.XA Pending CN109765315A (en) | 2019-01-30 | 2019-01-30 | A kind of preparation method and detection method of polyoxin component C |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109765315A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110483617A (en) * | 2019-09-17 | 2019-11-22 | 陕西绿盾环境工程研究院有限公司 | A kind of preparation method of high-content polyoxin original powder |
| CN111307965A (en) * | 2019-12-16 | 2020-06-19 | 陕西麦可罗生物科技有限公司 | Method for detecting polyoxin M by high performance liquid chromatography |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232525A (en) * | 2013-04-17 | 2013-08-07 | 南京工业大学 | Method for extracting polyoxin from streptomyces fermentation liquor |
| CN103613641A (en) * | 2013-11-11 | 2014-03-05 | 南京工业大学 | Method for separating and purifying polyoxins |
| CN103776929A (en) * | 2014-01-23 | 2014-05-07 | 陕西绿盾生物制品有限责任公司 | Method for detecting polyoxins B by high performance liquid chromatography method |
| CN109001328A (en) * | 2018-08-17 | 2018-12-14 | 山东省农业科学院农业质量标准与检测技术研究所 | A kind of LC-MS/MS detection method of polyoxin B |
-
2019
- 2019-01-30 CN CN201910089645.XA patent/CN109765315A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232525A (en) * | 2013-04-17 | 2013-08-07 | 南京工业大学 | Method for extracting polyoxin from streptomyces fermentation liquor |
| CN103613641A (en) * | 2013-11-11 | 2014-03-05 | 南京工业大学 | Method for separating and purifying polyoxins |
| CN103776929A (en) * | 2014-01-23 | 2014-05-07 | 陕西绿盾生物制品有限责任公司 | Method for detecting polyoxins B by high performance liquid chromatography method |
| CN109001328A (en) * | 2018-08-17 | 2018-12-14 | 山东省农业科学院农业质量标准与检测技术研究所 | A kind of LC-MS/MS detection method of polyoxin B |
Non-Patent Citations (3)
| Title |
|---|
| 中国农业科学院烟草研究所: "多抗霉素的生产工艺和应用", 《中国农业科学》 * |
| 许天委等: "多抗霉素在海滨雀稗草坪根系层土壤和水中的残留研究", 《热带农业工程》 * |
| 陶宁等: "稻田土壤及水中多抗霉素残留量的分析与检测方法研究", 《分析测试学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110483617A (en) * | 2019-09-17 | 2019-11-22 | 陕西绿盾环境工程研究院有限公司 | A kind of preparation method of high-content polyoxin original powder |
| CN111307965A (en) * | 2019-12-16 | 2020-06-19 | 陕西麦可罗生物科技有限公司 | Method for detecting polyoxin M by high performance liquid chromatography |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Macías et al. | Evidence for an allelopathic interaction between rye and wild oats | |
| Zhao et al. | Nitrate assay for plant tissues | |
| CN109765315A (en) | A kind of preparation method and detection method of polyoxin component C | |
| Latif et al. | Metabolomic approaches for the identification of flavonoids associated with weed suppression in selected Hardseeded annual pasture legumes | |
| Spielmeyer et al. | Dimethylsulfide sources from microalgae: Improvement and application of a derivatization-based method for the determination of dimethylsulfoniopropionate and other zwitterionic osmolytes in phytoplankton | |
| David et al. | Targeted metabolomics of plant hormones and redox metabolites in stomatal immunity | |
| CN109781886A (en) | A kind of preparation method and detection method of polyoxin N component | |
| CN112666280B (en) | Method for measuring main components of indigo naturalis before and after irradiation | |
| Hosaka et al. | Biochemical effects of sethoxydim in excised root tips of corn (Zea mays) | |
| CN111175389A (en) | Method for detecting zhongshengmycin F by high performance liquid chromatography | |
| CN106467739A (en) | The specificity fluorescent probe of catechol O-methyltransferase and its application | |
| CN109856280A (en) | Method that is a kind of while measuring tetrandrine and fangchinoline content in root of fangji medicinal material | |
| CN112666302B (en) | Method for identifying active flavone component group in barley seedling and rapidly detecting active flavone component group | |
| Yang et al. | Micro-nondestructive detection of the moisture and ion of rice seeds during germination under salt stress | |
| CN106092935A (en) | Method For Nitrate Content and application thereof in soil microorganism toxicity test | |
| CN111812237A (en) | Valproic acid drug concentration detection kit and application thereof | |
| RU2801102C1 (en) | Method for assessing completeness of seed treatment | |
| CN111307965A (en) | Method for detecting polyoxin M by high performance liquid chromatography | |
| Bayir et al. | In vitro human blood-brain barrier model for drug permeability testing | |
| CN103808545A (en) | Natural matrix standard sample for 13 pesticide residues in tea and preparation thereof | |
| CN114965772B (en) | Method for detecting content of S-elicitor in water body by utilizing HPLC-MS/MS method and application | |
| RU2748249C1 (en) | Method for determining concentration of antiviral drugs in biomaterials of laboratory animals | |
| CN108645927A (en) | A method of it measuring indigo plant using HPLC and spends plumbagin content in pellet | |
| CN113866299B (en) | Method for detecting neurotransmitters in dialysate | |
| Koca | The Cytogenetic Effects of Sheffer A, A Liquid Fertilizer and Growth Regulator in Root Tip Cells of Vicia Faba L. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190517 |