CN101144808A - Trichodermisin determination method and its uses - Google Patents
Trichodermisin determination method and its uses Download PDFInfo
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- CN101144808A CN101144808A CNA2007101563920A CN200710156392A CN101144808A CN 101144808 A CN101144808 A CN 101144808A CN A2007101563920 A CNA2007101563920 A CN A2007101563920A CN 200710156392 A CN200710156392 A CN 200710156392A CN 101144808 A CN101144808 A CN 101144808A
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Abstract
The present invention discloses a sample determining method for the wood mildew ester element. The present invention uses a Waters C18 analysis column, a duality high pressure gradient pump, and a Waters 2484 variable wavelength detector to conduct efficiency liquid phase analysis on the yew mildew secondary metabolism production wood mildew ester element. The test condition uses the acetonitrile and the water with the volume ratio of 3 to 2 as the flowing phase, the flowing speed is 1.0mL/min, the sample intake is 20 micro liters, the test wavelength is 193nm, and the test temperature is the room temperature. The present invention also discloses a wood mildew ester element consistence acquiring method in the sample using the above determining method. Defining the peak area obtained by the determining method as y, and according to the formula y=32797x+150488 to calculate the consistence of the wood mildew ester element in the flowing phase, then the content of the wood mildew ester element in the sample is obtained. The method of the present invention can be used to solve the problems of the improvement of the producing technical parameters of the wood mildew ester element and the control of the product quality.
Description
Technical field
The present invention relates to chemical analysis field, specially refer to a kind of trichodermisin HPLC analytical method, the sample after being applicable to mould (Trichoderma taxi) fermentation liquor of southerm yew endogenetic fungus yew and extracting purifying carries out the check and analysis of trichodermisin.
Background technology
Banded sclerotial blight that is caused by Rhizoctonia solani Kuhn (Rhizoctonia solani) and damping-off, the gray mold that is caused by Botrytis cinerea bacterium (Botrytiscinerea) are the important diseases in the agricultural production, produce at present to go up mainly and control its harm, mainly rely on chemical agents such as Shi Jiale, Sukeling as the gray mold chemical prevention by cultural control and chemical prevention.Because resistance to the action of a drug problem etc. is sought the novel bioactive material from natural animal-plant, microorganism, and the research and development of carrying out novel biopesticide are important research field of modern pesticide industry.Separate endogenetic fungal bacterial strain ZJUF0986 (the China Committee for Culture Collection of Microorganisms common micro-organisms center that obtains from southerm yew, preserve numbering: CGMCC1672) belong to a kind of trichoderma novel species: yew is mould, and the novel compound trichodermisin (trichodermisin) of its generation has very strong inhibiting effect (application number 200610050963.5) to important plant pathogenic fungi Rhizoctonia solani Kuhn and Botrytis cinerea bacterium.Also there is not detection method both at home and abroad at trichodermisin.
High performance liquid chromatography (High Performance Liquid Chromatography, HPLC) be a branch of red, orange, green, blue, yellow (ROGBY), be in phase late 1960s, on the basis of classical liquid chromatography and gas chromatography, the novel separate analytical technique that grows up.Since the appearance, because of it has the separation efficiency height, analysis speed is fast, detection sensitivity good, can analyze higher boiling but the characteristics of the thermally labile physiological activator that can not gasify are widely used in biological chemistry, food analysis, medicine and clinical analysis.In recent years, along with the continuous development of chromatographic technique, the exploitation of various workstation softwares, and with the coupling of instruments such as mass spectrum, widened the range of application of HPLC greatly.
How to use high performance liquid chromatography and be used to detect the trichodermisin sample, thereby accurately learn the content of trichodermisin in the sample, become people urgently to wish the problem that solves.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of assay method of trichodermisin, utilizes this method can accurately learn the content of trichodermisin in the sample; Therefore can be used for solving the improvement of processing parameter of trichodermisin and the problem of production quality control.
In order to solve the problems of the technologies described above, the invention provides a kind of method of rp-hplc trichodermisin: use Waters C18 analytical column (sunfire C185 μ m 4.6 * 250mm), the binary geopressure gradient pump of Waters (1525binary HPLC pump) and Waters 2487 variable-wavelenght detectors (Waters 2487 Dual Absorbance Detector), the mould secondary metabolite trichodermisin of yew is carried out high-efficient liquid phase analysis, testing conditions: moving phase is acetonitrile: water=3: 2 (V: V), flow velocity 1.0mL/min, sample size 20 μ L, the detection wavelength is 193nm, detected temperatures is a room temperature.
Improvement as the assay method of trichodermisin of the present invention: this trichodermisin be present in the mould fermentation liquor of endogenetic fungus yew trichodermisin or for extracting the trichodermisin behind the purifying.
The present invention also provides the method for utilizing the said determination method to obtain the concentration of trichodermisin in the sample: will be defined as y according to the peak area of assay method gained, again according to formula: y=32797x+150488, calculate the concentration x of trichodermisin in moving phase, and then obtain the content of trichodermisin in the sample.
The inventor has carried out following invention work in the invention process:
After the trichodermisin reference substance is dissolved in acetonitrile, do blank, do uv absorption scanning, determine the maximum absorption wavelength of sample with acetonitrile, with this maximum absorption wavelength as HPLC analyzing and testing wavelength.Uv absorption scanning shows, trichodermisin has maximum absorption band at the 205nm place, consider the difference between the instrument, we are testing the actual maximum absorption wavelength of trichodermisin under Waters 2487 Dual λ Absorbance Detector with efficient liquid phase chromatographic analysis near the 205nm wavelength, determine that by test of many times the maximum absorption wavelength of trichodermisin under this detecting device is 193nm, so select the detection wavelength of 193nm for use as sample analysis.Use acetonitrile: water (1: 9, V: V) to acetonitrile: water (9: 1, V: V) do the continuous gradient wash-out, determine proportion of mobile phase according to going out the peak situation.By selection to proportion of mobile phase, take into account degree of separation and analysis time, by the optimization experiment condition, determine at last with acetonitrile: water=3: 2 (V: V) as analysis condition.Under this testing conditions, trichodermisin retention time 11.38min, peak width is 0.6min, chromatographic peak peak shape symmetry is better.The chromatogram of the reference substance of gained is seen accompanying drawing 2 under optimal detection condition.
The present invention is applicable to the analysis of the trichodermisin sample that the mould liquid fermentation of yew produces, and comprises the analysis of fermentation broth sample, crude extract and refining sample.The present invention is removing impurities effectively, and the symmetry of peak shape is better, and method is easy, highly sensitive, favorable reproducibility, result are accurate.
The present invention provides effective analytical approach for the improvement and the production quality control of the technological parameter of trichodermisin, to research, the industrialization of trichodermisin and apply and have positive prograding.
Foregoing is specifically realized by following steps:
1, the preparation of trichodermisin reference substance:
One, another piece patent of applying on the same day according to the applicant " method of fermenting and producing trichodermisin and used fermentation medium " can contain the wooden mould fermentation liquor of trichodermisin.
Particular content is as follows:
A, making fermentation medium:
With the water-soluble formation mixed liquor of glucose, starch, peptone, magnesium sulphate, sodium nitrate, diammonium hydrogen phosphate and potassium chloride, glucose, starch, peptone, magnesium sulphate, sodium nitrate, diammonium hydrogen phosphate and the potassium chloride concentration in mixed liquor is respectively 16.0g/L, 25.8g/L, 2.0g/L, 1.0g/L, 0.5g/L, 2.0g/L and 0.5g/L.
Be the consisting of of fermentation medium of gained: glucose 16.0g/L, starch 25.8g/L, peptone 2.0g/L, magnesium sulphate 1.0g/L, sodium nitrate 0.5g/L, diammonium hydrogen phosphate 2.0g/L and potassium chloride 0.5g/L, all the other are water.
The method of B, a kind of fermenting and producing trichodermisin, carry out following steps successively:
1), earlier the yew trichoderma strain ZJUF0986 that is numbered CGMCC1672 with low temperature (promptly-20 ℃) preservation is seeded on the PDA nutrient culture media,, after the activation processing, cuts colony edge bacterium cake with card punch in 28 ℃～30 ℃ activation culture 5 days;
2), be (the bottled 200mL liquid seed culture medium of the triangle of 1000mL) in the 1000mL triangular flask of dress liquid seed culture medium in 10 of the bacterium cakes of 5mm are linked into above-mentioned diameter, 28 ℃～30 ℃, 180r/min lucifuge were cultivated 72 hours, seed liquor.Liquid seed culture medium is formed: glucose 20.0 g/L, starch 15.0 g/L, peptone 2.0 g/L, urea 0.5g/L, magnesium sulphate 1.0g/L, sodium nitrate 0.5g/L, diammonium hydrogen phosphate 2.0g/L, potassium chloride 0.5g/L, lime carbonate 0.4g/L, all the other are water.
3), select for use 10 liters of mechanical ventilation fermentation tanks (FJG-10, Jiangsu Ge Rui Bioisystech Co., Ltd), to select above-mentioned fermentation medium for use as fermentation tank.
With above-mentioned steps 2) seed liquor of gained insert with the ratio of fermentation medium 10% (volume ratio) in the fermentation tank of dress fermentation medium, carry out following fermentation step (tank pressure is controlled between 0.03MPa～0.05MPa in whole fermentation process, and incubation time is 240 hours altogether) successively:
Initial stage: temperature is controlled at 28 ℃～30 ℃, and bubbling air stirs, and the ventilation ratio: 1: 0.5 VN/min, speed of agitator are 200r/min, and pH regulates naturally; Incubation time is 120 hours,
Mid-term: temperature is reduced to 25 ℃, improve the ventilation ratio, make ventilation than being: 1: 1 VN/min, when concentration of reduced sugar drops to about 10g/L, add mass concentration when dissolved oxygen DO (DO) beginning is slowly risen is 20% starch solution, disposable additional amount be the starch in the starch solution be in the fermentation medium starch weight 30%, the pH value of controlling in the fermentation tank with the mode of NaOH manual shift is 5.0～6.0, and incubation time is 48 hours;
Later stage: stop to add carbon source, reduce the ventilation ratio, make ventilation than being 1: 0.5VN/min; When reducing sugar drops to 1g/L when following, microscopy finds that mycelia begins self-dissolving, the fermentation liquor of gained is put jar after forming arthrospore; Incubation time is 72 hours.
Two, the another piece of writing patent " method of purification of trichodermisin " of applying on the same day according to the applicant can get pure product trichodermisin, is required trichodermisin reference substance.
Particular content is as follows:
A kind of method of purification of trichodermisin, carry out following steps successively:
1), selects above-mentioned fermentation liquor for use;
2), in above-mentioned fermentation liquor, add aluminium potassium sulfate and stir static 12～24 hours protein precipitations in back, the aluminium potassium sulfate consumption is 0.5% of a fermentation liquor weight; It is centrifugal to remove precipitation to get supernatant then, and centrifugal condition is: 5000～10000rmp/min, centrifugal 5～10min under the room temperature.
3), take the supernatant after the above-mentioned centrifugal treating of 1000mL, be extractant with sherwood oil (60～90); At ambient temperature the supernatant after the centrifugal treating is divided with sherwood oil (60～90) to extract for three times, when extracting: the consumption of sherwood oil (60～90) is 300mL at every turn, and duration of oscillation is 10min, and be 2 hours rest time; Separate static back, gets water A and organic phase A.
4), in organic phase A, add anhydrous sodium sulfate and carry out drying, the weight of anhydrous sodium sulfate is 7% of organic phase A; Again with behind the filter paper filtering, under negative pressure 0.08MPa, 50 ℃ condition, rotate evaporation and concentration, to reclaim the organic solvent sherwood oil.Get the faint yellow crude extract of 1.25g.
5), at first with faint yellow crude extract under 10～50 ℃ temperature with sherwood oil (60～90) dissolving, the volume ratio of faint yellow crude extract and sherwood oil (60～90) is 1: 100; Be extractant then at ambient temperature with the deionized water, the total consumption of deionized water is 315 times of faint yellow crude extract volume, is divided into 3 times and extracts, and in each extraction: duration of oscillation is 10min, and be 2 hours rest time.Separate static back, gets aqueous phase B and organic phase B;
6) anhydrous sodium sulfate drying that, in organic phase B, adds organic phase B10% weight ratio; Then with behind the filter paper filtering, under negative pressure 0.08MPa, 50 ℃ condition, rotate evaporation and concentration, reclaim the organic solvent sherwood oil, bring up again the colourless thing of bringing up again of 1.08g.
The colourless main composition trichodermisin content of bringing up again thing reaches about 98%.
7), for obtaining pure product, the colourless thing of bringing up again is crossed silicagel column repurity.Concrete operations are as follows: 200～300 purpose chromatographic silica gels (Qingdao Haiyang chemical industry group) are placed 120 ℃ of activation 1h, take by weighing silica gel after the about 200 g activation and the chloroform mixing of 500mL, and with ultrasound wave catch up with except that pack into diameter 4cm, length of wet method behind the steam bubble be the glass column of 50cm.Colourless the bringing up again of 1.08g admixed 200～300 purpose chromatographic silica gels (not being the oil stain shape is advisable) after thing dissolves with 5mL～10mL chloroform, treat that solvent volatilizes the back and goes up sample.Behind the balance 2h, emit chloroform to the silicagel column upper surface, by polarity order from small to large, select the chloroform/methanol (1/0～1.0) of different gradients to carry out wash-out as eluant, eluent, 50mL is that unit carries out eluent and collects, receive 40 parts of eluents.Be mixed with methanol solution after respectively the constituent parts eluent being concentrated, adopt efficient thin-layer chromatography chromatogram (TLC) to follow the trail of spot, select optimization developping agent system, with ultraviolet light (254nm and 365nm) irradiation or iodine steam tracing, simultaneously mould with ultimate corruption, Rhizoctonia solani Kuhn, Botrytis cinerea are indicator bacteria, carry out the activity tracking with the filter paper method.
Pure product trichodermisin is that chloroform/methanol is 1/0.2 component, concentrating the back detects with high performance liquid chromatography (HPLC), retention time about 11.4min be exactly to extract purifying after the trichodermisin that obtained, obtain pure product trichodermisin 0.836g behind the silicagel column purifying altogether, purity can reach more than 99.5%.
2, the preparation of trichodermisin reference substance solution:
(acetonitrile: water=3: 2) dissolving back constant volume is 10mL, is mixed with the storing solution that concentration is 1000 μ g/mL with moving phase to take by weighing the homemade trichodermisin reference substance of 10mg.
3, the preparation of trichodermisin test sample:
1), (acetonitrile: water=3: 2) being mixed with final concentration respectively is 1,2,5,10,20,50,100,200, the 500 and 1000 μ g/mL test sample of totally 10 gradients with moving phase with above-mentioned storing solution.If precipitation then needs centrifugal or be the organic membrane filter of 0.22 μ m with the aperture.
2), for guaranteeing the safe and efficient of test column, fermentation broth sample will carry out necessary pre-service before entering test column, guarantee that albumen, polysaccharide and granule foreign do not enter chromatographic column, the method for processing is that test sample adds isopyknic trifluoroacetic acid aqueous solution, centrifugal removal precipitation behind the mixing.
4, chromatographic condition
1), Waters C18 analytical column (sunfire C185 μ m 4.6 * 250mm)
2), the binary geopressure gradient pump of Waters (1525 binary HPLC pump)
3), Waters 2487 detecting devices (Waters 2487 Dual λ Absorbance Detector)
4), 77251 manual injectors (KlT72251 Manualinjector 1500 series)
5), moving phase is acetonitrile: water=3: 2 (V: V)
6), flow velocity 0.8 mL/min～1.2 mL/min, for example be 1.0 mL/min
7), sample size 20 μ L
8), detecting wavelength is 193nm
9), temperature: room temperature
5, the used instrument and equipment of the present invention
1), the Waters liquid chromatograph, binary geopressure gradient pump (1525 binary HPLC pump), Waters2487 detecting device (Waters 2487 Dual Absorbance Detector), 77251 manual injectors (KIT72251 Manual injector 1500 series) of joining Waters
2), chromatographic work station: Breeze
3), high speed tabletop centrifuge: Sigma 1-15K
4), electronic balance: METTLERAB204-E
5), rotary evaporator: RE-52AA
6), PH analyzer: METTLER TOLEDO 320
6, agents useful for same of the present invention
1), acetonitrile: HPLC U.S. Weston Scientific
2), water: tri-distilled water (self-control)
3), trichodermisin reference substance (self-control)
4) it is pure that, other agents useful for same is homemade analysis.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is a trichodermisin liquid-phase chromatographic analysis working stamndard curve map;
Fig. 2 is that trichodermisin reference substance solution high performance liquid chromatography detects collection of illustrative plates;
Fig. 3 is the high-efficient liquid phase chromatogram spectrum of the refining sample determination of trichodermisin;
Fig. 4 is the high-efficient liquid phase chromatogram spectrum that trichodermisin is measured in the mould fermentation liquor of yew.
Embodiment
The foundation of embodiment 1, trichodermisin working stamndard curve
With 1,2,5,10,20,50,100,200,500 and 1000 μ g/mL are the test sample solution of the trichodermisin of totally 10 concentration, at selected Waters C18 analytical column (sunfire C185 μ m 4.6 * 250mm), moving phase is acetonitrile: water=3: 2, flow velocity 1.0mL/min, sample size 20 μ L, the detection wavelength is 193nm, detect under the room temperature condition, each concentration repeats sample introduction 3 times, the sample peak is carried out integration, calculate the average peak area at each concentration sample peak, with the sample concentration is horizontal ordinate, the average peak area at sample peak is that ordinate is drawn typical curve, as shown in Figure 1.
Sample peak integration to each concentration sample obtains its peak area, each concentration is calculated its average peak area and standard deviation, by standard deviation as can be seen, in the concentration range of being tested, all the other each concentration peak area relative standard deviation RSD≤0.5% (as shown in table 1) except that 1 μ g/mL, all in claimed range, still select data in the 2 μ g/mL-1000 μ g/mL concentration ranges for use, with the sample concentration is horizontal ordinate, and the average peak area at sample peak is that ordinate is drawn typical curve.Simulating regression equation according to trichodermisin concentration and average peak area is: y=32797x+150488, its regression coefficient R=0.9995, its concentration and absorption peak area are good linear relationship, illustrate that this regression equation model is meaningful, and testing result is accurately credible.
The HPLC of each concentration trichodermisin reference substance solution of table 1 analyzes data
| Concentration (μ g/mL) | Retention time (min) | Peak area (μ Vsec) | Average peak area (μ Vsec) | RSD/％ |
| 1 | 7.647 | 35390 | 35376.33 | 0.55 |
| 1 | 7.614 | 35176 | ||
| 1 | 7.492 | 35563 | ||
| 2 | 7.561 | 70962 | 70927.33 | 0.09 |
| 2 | 7.563 | 70970 | ||
| 2 | 7.567 | 70850 | ||
| 5 | 7.561 | 174088 | 174748.7 | 0.37 |
| 5 | 7.568 | 174788 | ||
| 5 | 7.558 | 175370 | ||
| 10 | 7.548 | 362181 | 361793.7 | 0.10 |
| 10 | 7.562 | 361484 | ||
| 10 | 7.568 | 361716 | ||
| 20 | 7.568 | 704268 | 704716.3 | 0.08 |
| 20 | 7.574 | 705352 | ||
| 20 | 7.567 | 704539 | ||
| 50 | 7.577 | 1710771 | 1714686 | 0.22 |
| 50 | 7.576 | 1715027 | ||
| 50 | 7.573 | 1718261 |
| 100 | 7.572 | 3453265 | 3437987 | 0.45 |
| 100 | 7.575 | 3438329 | ||
| 100 | 7.574 | 3422366 | ||
| 200 | 7.568 | 7292164 | 7298261 | 0.13 |
| 200 | 7.575 | 7309187 | ||
| 200 | 7.586 | 7293433 | ||
| 500 | 7.542 | 17067795 | 17030686 | 0.19 |
| 500 | 7.611 | 17020053 | ||
| 500 | 7.604 | 17004211 | ||
| 1000 | 7.598 | 32559400 | 32596026 | 0.10 |
| 1000 | 7.609 | 32622224 | ||
| 1000 | 7.598 | 32606453 |
Embodiment 2, reversed-phase high-performance liquid chromatography detect trichodermisin method reappearance, precision and recovery test (moving phase of the following stated all is equal to embodiment 1):
1), to take by weighing 10mg trichodermisin sample be 10mL with moving phase dissolving back constant volume, making its concentration is 1000 μ g/mL, afterwards again with 10 times of constant volumes of moving phase dilution, even concentration is 100 μ g/mL.With the aperture is the organic membrane filter survey fully of 0.22 μ m, and condition determination is the same.
Repeat sample introduction 6 times, absorption peak area (μ Vsec) mean value is 3374132, relative standard deviation RSD=0.10%, as seen in Figure 3, detect removing impurities effectively with the method, the symmetry of peak shape is also fine, highly sensitive, favorable reproducibility, and the content that records trichodermisin in this sample is 98.32%.
2), the accurate absorption with the moving phase concentration of ordinary dissolution be to be diluted to 1000 μ L with moving phase again behind 1000 μ g/mL trichodermisin samples, 80 μ L, 100 μ L, the 120 μ L, three gradients totally 9 samples.Assay method is the same, calculates the recovery of trichodermisin, the results are shown in Table 2.
Table 2, trichodermisin sample determination recovery test result
| Sample number into spectrum | Addition/μ g | Yield/μ g | The recovery/% | Average recovery rate/% | RSD/％ |
| 1 | 78.66 | 78.74 | 100.10 | 100.11 | 0.41 |
| 2 | 78.66 | 78.79 | 100.17 | ||
| 3 | 78.66 | 78.96 | 100.20 | ||
| 4 | 98.32 | 98.93 | 100.29 | ||
| 5 | 98.32 | 98.11 | 100.20 | ||
| 6 | 98.32 | 98.41 | 100.09 | ||
| 7 | 117.98 | 117.86 | 99.90 | ||
| 8 | 117.98 | 118.23 | 99.96 | ||
| 9 | 117.98 | 118.64 | 99.56 |
The mensuration of embodiment 3, the mould fermentation liquor of yew:
Draw the fermentation broth sample of 1mL, the acetonitrile that adds the HPLC level is settled to 2mL, and mixing is placed on Sigma1-15K high speed tabletop centrifuge centrifugal 10min under 4 ℃, the condition of 10000rpm/min, and it is standby to draw supernatant.Condition determination is the same, and Fig. 4 is the high-efficient liquid phase chromatogram spectrum that trichodermisin is measured in the mould fermentation liquor of yew, removing impurities effectively, and the symmetry of peak shape is better, and the concentration that records trichodermisin in the mould fermentation broth sample of this yew is 294.06 μ g/mL.
Computing method are specific as follows: the peak area y of gained is 4972630.91, according to formula y=32797x+150488, extrapolates X=147.03; Again owing to be that fermentation broth sample has been diluted 1 times, so concentration=2X=294.06 μ g/mL.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1. the assay method of a trichodermisin sample, it is characterized in that using binary geopressure gradient pump and Waters 2487 variable-wavelenght detectors of Waters C18 analytical column, Waters, the mould secondary metabolite trichodermisin of yew is carried out high-efficient liquid phase analysis, testing conditions: as moving phase, flow velocity 1.0mL/min, sample size 20 μ L, detection wavelength are that 193nm, detected temperatures are room temperature with the acetonitrile of volume ratio=3: 2 and water.
2. the assay method of trichodermisin according to claim 1 is characterized in that: described trichodermisin be present in the mould fermentation liquor of endogenetic fungus yew trichodermisin or for extracting the trichodermisin behind the purifying.
3. utilize claim 1 or 2 described assay methods to obtain the method for the concentration of trichodermisin in the sample, it is characterized in that: will be defined as y according to the peak area of assay method gained, again according to formula: y=32797x+150488, calculate the concentration x of trichodermisin in moving phase, and then obtain the content of trichodermisin in the sample.
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| CN110361469A (en) * | 2019-07-17 | 2019-10-22 | 长春中医药大学 | A kind of Chinese medicine mildew detection method |
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| CN110361469A (en) * | 2019-07-17 | 2019-10-22 | 长春中医药大学 | A kind of Chinese medicine mildew detection method |
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