CN107771866B - Preparation method of crowndaisy chrysanthemum bacteriostatic active monomer - Google Patents
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Abstract
The invention discloses a crowndaisy chrysanthemum bacteriostatic activity monomer and a use method thereof, belonging to the technical field of separation and use methods of active substances. The Crowndaisy chrysanthemum bacteriostatic activity monomer provided by the invention can be used for extracting a high-efficiency bacteriostatic activity monomer, can well inhibit watermelon wilt germs, and has a good and wide application prospect.
Description
Technical Field
The invention relates to the technical field of separation and use methods of active substances, in particular to a preparation method of antibacterial activity of crowndaisy chrysanthemum.
Background
Watermelon Fusarium wilt, also known as a leaf blight, wilting disease, and wilting disease, is called "Watermelon cancer" and is a devastating soil-borne disease commonly occurring at home and abroad due to infection with Fusarium oxysporum f.sp.niveum (e.f. smith) wolfen, a fungi of the deuteromycotina. The area of continuous cropping watermelon is increased due to the rapid expansion of the cultivation area of watermelon, the occurrence of watermelon wilt is becoming more common and more serious, and huge economic loss and resource waste are brought to watermelon production. The blight can cause the watermelon yield to be reduced by 30-40%, and the yield of the seriously ill plot is reduced by more than 80%, even the watermelon is out of production. Therefore, the effective prevention and treatment of watermelon fusarium wilt directly influences the healthy development of watermelon industry.
For a long time, people mainly prevent and treat the watermelon fusarium wilt by methods such as agricultural prevention and treatment, disease-resistant breeding, biological prevention and treatment, chemical prevention and treatment and the like. The chemical prevention and treatment has the characteristics of quick response and good effect. The prevention and treatment work of the watermelon fusarium wilt is emphasized in various places, and a batch of chemical agents with certain prevention and treatment effects, such as the bactericidin, KT emulsion, metalaxyl manganese zinc, hymexazol, 40% melon withering and the like, are screened and developed to have certain prevention and treatment effects on the watermelon fusarium wilt. With the progress of scientific technology and the improvement of detection and analysis means, some chemical bactericides considered safe in the past are found to have the possibility of carcinogenicity or potential carcinogenesis, mutagenicity and teratogenicity. In addition, long-term use of chemical bactericides can easily cause pathogenic bacteria to generate drug resistance to the bactericide, reduce the control effect and pollute the environment. There is also a growing public concern over the hazards to the ecological environment and human health associated with the use of chemical biocides in large doses. Therefore, broad-spectrum, efficient, low-toxicity and safe bactericides are searched, and particularly bacteriostatic active ingredients which have bacteriostatic effects and are harmless to human health and ecological environment are researched and screened from natural plants, so that the bactericides have very important significance.
Chrysanthemum Coronarium L.var.Spatiospm Bailer is a traditional green-leaf vegetable of Compositae in China and is cultivated in all parts of the south and the north. Modern pharmacological research shows that the crowndaisy chrysanthemum has various physiological activities of antibiosis, disinfection, microorganism resistance, cancer resistance, blood pressure reduction, phlegm elimination and the like. The field where the chrysanthemum coronarium is planted in the previous crop and then the watermelon is planted, the watermelon grows well, the blight does not occur, and the yield and the quality of the watermelon are improved. Monomers with bacteriostatic activity are separated from the crowndaisy chrysanthemum, and the monomer can be developed into a natural bactericide for inhibiting watermelon wilt germs and has good and wide application prospect.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a preparation method of a crowndaisy chrysanthemum bacteriostatic activity monomer, which can extract a high-efficiency bacteriostatic activity monomer, can well inhibit watermelon wilt germs and has a good and wide application prospect.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a crowndaisy chrysanthemum bacteriostatic active monomer, which comprises the following steps:
s1: preprocessing stems and leaves of crowndaisy chrysanthemum: weighing 20Kg of fresh stem and leaf of crowndaisy chrysanthemum, drying naturally, drying in a blast drier at 45-55 ℃ for 10-14h, accurately weighing 1.7-2.3Kg of dried crowndaisy chrysanthemum, crushing in a Chinese medicinal material crusher, and sieving with a 40-mesh sieve;
s2: leaching: adding 48-52L 95% ethanol, ultrasonic extracting for 2-4 times (each for 1-3 hr), mixing filtrates, and concentrating under reduced pressure to obtain extract;
s3: macroporous resin column chromatography: dissolving the extract in 1.1-1.3L of distilled water, filtering, putting the supernatant on a D101 macroporous resin column, performing gradient elution by using distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol in sequence, eluting 1.9-2.1L of solvent by each gradient at the flow rate of 2.5-3.5mL/min, respectively recovering the eluent after each gradient elution, performing reduced pressure recovery to obtain six fractions, inoculating the fusarium oxysporum onto PDA culture media containing different fractions, culturing for 4D in a constant-temperature incubator at 28 ℃, and screening out a fraction A with the smallest colony diameter;
the diameter of the D101 macroporous resin column is 3.0-5.0cm, and the length of the macroporous resin column is 23-27 cm;
s4: c18 medium pressure chromatographic separation: separating fraction A of S3 by C18 medium pressure chromatography (MPLC), eluting with water in a gradient manner, sequentially eluting with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol in a gradient manner, wherein each gradient elutes 0.8-1.2L of solvent at the flow rate of 2.5-3.5mL/min, detecting eluates after each gradient elution by HPLC, screening and combining the same fractions to form different gradient type B fractions, inoculating the fusarium oxysporum f.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.in PDA culture medium containing different type B fractions, culturing for 4d in a constant temperature incubator at 28 ℃, screening one of the type B fractions with the smallest colony diameter to define as the type B1 fraction;
s5: sephadex LH-20 column chromatography: separating the fraction B1 in S4 by SephadexLH-20 gel column chromatography, eluting with 5% methanol, 15% methanol, 25% methanol and 35% methanol sequentially, eluting 0.9-1.1L solvent in each gradient, and the flow rate is 2.5-3.5mL/min, detecting by HPLC, combining the same fractions to form C fractions with different gradients, inoculating the Fusarium oxysporum F.sp.sp.citrulli in PDA culture medium containing different C fractions, culturing in a constant temperature incubator at 23-33 deg.C for 3-5d, and screening the fraction with the smallest colony diameter to obtain C1 fraction;
s6: semi-preparative liquid phase separation: separating the C1 fraction obtained in S5 by a semi-preparative liquid phase, eluting by 18 percent methanol, 27 percent methanol, 36 percent methanol and 45 percent methanol in sequence, eluting 0.9 to 1.1L of solvent by each gradient, combining the same fractions by HPLC detection at the flow rate of 2.5 to 3.5mL/min, forming D fractions with different gradients, inoculating the fusarium oxysporum f.sp.sp.citrulli into PDA culture media containing different D fractions, culturing in a constant-temperature incubator at 28 ℃ for 4 days, and separating the fraction with the smallest colony diameter into the crowndaisy chrysanthemum bacteriostatic active monomer.
In a preferred technical scheme of the invention, in S1, the stem and leaf of the crowndaisy chrysanthemum are dried for 12 hours in a blowing drier at 50 ℃, 2.0Kg of dried crowndaisy chrysanthemum is accurately weighed, crushed in a Chinese medicinal material crusher and sieved by a 40-mesh sieve.
In a preferred technical scheme of the invention, 50L of 95% ethanol is added into S2 for ultrasonic-assisted extraction for 3 times, and each time lasts for 2 hours.
In a preferred technical scheme of the invention, in S3, the extract is dissolved in 1.2L of distilled water; 2L of solvent were eluted per gradient at a flow rate of 3.0 mL/min.
In a preferred technical scheme of the invention, in the S3, the diameter of the D101 macroporous resin column is 4.0cm, and the length of the macroporous resin column is 25 cm.
In the preferred embodiment of the present invention, in S4, 1L of solvent is eluted per gradient at a flow rate of 3.0 mL/min.
In a preferred technical scheme of the invention, in S5, the flow rate is 3.0mL/min, and the culture medium is placed in a constant temperature incubator at 28 ℃ for culture for 4 d.
In a preferred embodiment of the present invention, in S6, 1L of solvent is eluted at each gradient, the flow rate is 3.0mL/min, and the semi-preparative liquid phase uses a filler with a particle size of 10 μm, an inner diameter of 10mm, and a length of 250 mm.
A method for using antibacterial monomer of caulis et folium Chrysanthemi Segeti comprises diluting the compound obtained by separation and purification with sterile water to 100 mg/mL-1The stock solution is irrigated for 1 time per day in the first 3-5 days of the watermelon seedling root, and then irrigated for 3-5 times per 3-4 days, and 8-12ml of the stock solution is irrigated for each time.
In the preferred technical scheme of the invention, the stock solution is irrigated for 1 time every day in the first 5 days of the watermelon seedling roots, then irrigated for 1 time every 3 days and irrigated for 5 times continuously, wherein 10ml of the stock solution is irrigated for each time.
The invention has the beneficial effects that:
the invention provides a separation and purification of garland chrysanthemum bacteriostatic activity monomer and a using method thereof, firstly crushing dried garland chrysanthemum, sieving with a 40-mesh sieve, then carrying out ethanol ultrasonic extraction, merging filtrate and carrying out reduced pressure concentration, then carrying out macroporous resin column chromatography, methanol elution, taking the fraction with the best bacteriostatic effect, then carrying out C18 medium pressure chromatographic separation, adopting methanol elution, merging the same fractions through HPLC detection, then taking the fraction with the best bacteriostatic effect, then adopting glucose gel LH-20 column chromatography, methanol elution, HPLC detection, merging the same fractions, taking the fraction with the best bacteriostatic effect, finally adopting semi-preparative liquid phase separation, similarly adopting methanol elution, HPLC detection, merging the same fractions, and through experiments, the fraction with the smallest colony diameter is the obtained garland chrysanthemum bacteriostatic activity monomer, and extracting the high-efficiency bacteriostatic activity monomer through multiple times of separation and purification, the garland chrysanthemum bacteriostatic active monomer belongs to natural bactericide, is not easy to cause pathogenic bacteria to generate drug resistance to the garland chrysanthemum bacteriostatic active monomer even if the garland chrysanthemum bacteriostatic active monomer is used for a long time, causes pollution to the environment, has good inhibition effect on fusarium oxysporum f.sp.sp.citrullus, and protects the growth of watermelon.
Detailed Description
The technical solution of the present invention is further explained below by the specific embodiments.
The following examples 1 to 3 are experiments for extracting monomers with bacteriostatic activity from chrysanthemum coronarium from stems and leaves of chrysanthemum coronarium:
example 1:
the embodiment 1 provides a preparation method of a crowndaisy chrysanthemum bacteriostatic activity monomer, which comprises the following steps:
s1: preprocessing stems and leaves of crowndaisy chrysanthemum: weighing 20Kg of fresh stem and leaf of crowndaisy chrysanthemum, naturally drying, drying in a 50 ℃ blast drier for 12h, accurately weighing 2Kg of dried crowndaisy chrysanthemum, crushing in a Chinese medicinal material crusher, and sieving with a 40-mesh sieve;
s2: leaching: adding 50L 95% ethanol, ultrasonic extracting for 3 times, each for 2 hr, mixing filtrates, and concentrating under reduced pressure to obtain extract;
s3: macroporous resin column chromatography: dissolving the extract in 1.2L of distilled water, filtering, putting the supernatant on a D101 macroporous resin column, carrying out gradient elution by using distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol in sequence, eluting 2L of solvent by each gradient, carrying out flow rate of 3.0mL/min, respectively recovering the eluent after each gradient elution, carrying out reduced pressure recovery to obtain six fractions, inoculating the fusarium oxysporum f.sp.sp.sp.citrulli in PDA culture media containing different fractions, culturing in a constant-temperature incubator at 28 ℃ for 4D, and screening out a fraction A corresponding to the smallest colony diameter;
the diameter of the D101 macroporous resin column is 4.0cm, and the length of the macroporous resin column is 25 cm;
s4: c18 medium pressure chromatographic separation: separating fraction A of S3 by C18 medium pressure chromatography (MPLC), eluting with water in gradient, eluting with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol in sequence in gradient with 1L solvent per gradient and flow rate of 3.0mL/min, detecting eluates after each gradient elution by HPLC, screening and combining the same fractions to form B fractions with different gradients, inoculating Fusarium oxysporum onto PDA culture medium containing different B fractions, culturing at 28 deg.C in constant temperature incubator for 4d, and screening one of the B fractions with minimum colony diameter to obtain B1 fraction;
s5: sephadex LH-20 column chromatography: separating the fraction B1 in S4 by SephadexLH-20 gel column chromatography, eluting with 5% methanol, 15% methanol, 25% methanol and 35% methanol sequentially, eluting 0.9-1.1L solvent in each gradient at the flow rate of 3.0mL/min, combining the same fractions by HPLC detection to form C fractions with different gradients, inoculating the fusarium oxysporum f.sp.sp.cubense in PDA culture media containing different C fractions, culturing in a constant-temperature incubator at 28 ℃ for 4d, and screening the fraction with the smallest colony diameter to define the fraction C1;
s6: semi-preparative liquid phase separation: separating the C1 fraction obtained in the S5 by using a semi-prepared liquid phase, eluting by using 18% methanol, 27% methanol, 36% methanol and 45% methanol in sequence, eluting 1L of solvent in each gradient at the flow rate of 3.0mL/min, combining the same fractions by HPLC (high performance liquid chromatography) detection to form D fractions with different gradients, inoculating watermelon wilt germs into PDA (personal digital assistant) culture media containing different D fractions, culturing for 4D in a constant-temperature incubator at the temperature of 28 ℃, wherein the fraction with the smallest colony diameter is a crowndaisy chrysanthemum bacteriostatic active monomer, and the filler particle size of the semi-prepared liquid phase is 10 mu m, the inner diameter is 10mm, and the length is 250 mm.
Example 2
The preparation method of the crowndaisy chrysanthemum bacteriostatic activity monomer provided by the embodiment 2 comprises the following steps:
s1: preprocessing stems and leaves of crowndaisy chrysanthemum: weighing 20Kg of fresh stem and leaf of crowndaisy chrysanthemum, naturally drying, drying in a blast drier at 45 ℃ for 10h, accurately weighing 1.7Kg of dried crowndaisy chrysanthemum, crushing in a Chinese medicinal material crusher, and sieving with a 40-mesh sieve;
s2: leaching: adding 48L 95% ethanol, ultrasonic extracting for 2 times (each for 1 hr), mixing filtrates, and concentrating under reduced pressure to obtain extract;
s3: macroporous resin column chromatography: dissolving the extract in 1.1L of distilled water, filtering, putting the supernatant into a D101 macroporous resin column, carrying out gradient elution by using distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol in sequence, eluting 1.9L of solvent by each gradient, carrying out flow rate of 2.5mL/min, respectively recovering the eluent after each gradient elution, carrying out reduced pressure recovery on the solvent to obtain six fractions, inoculating the fusarium oxysporum f.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp;
the diameter of the D101 macroporous resin column is 3.0cm, and the length of the macroporous resin column is 23 cm;
s4: c18 medium pressure chromatographic separation: separating fraction A of S3 by C18 medium pressure chromatography (MPLC), eluting with water in gradient, eluting with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol in sequence in gradient with flow rate of 2.5mL/min for 0.8L solvent, detecting eluates after each gradient elution by HPLC, screening and combining the same fractions to form B fractions with different gradients, inoculating Fusarium oxysporum onto PDA culture medium containing different B fractions, culturing in constant temperature incubator at 28 deg.C for 4d, and screening one of the B fractions with minimum colony diameter to obtain B1 fraction;
s5: sephadex LH-20 column chromatography: separating the fraction B1 in S4 by SephadexLH-20 gel column chromatography, eluting with 5% methanol, 15% methanol, 25% methanol and 35% methanol in sequence, eluting 0.9-1.1L solvent in each gradient at the flow rate of 2.5mL/min, combining the same fractions by HPLC detection to form C fractions with different gradients, inoculating the watermelon fusarium wilt pathogen into PDA culture media containing different C fractions, culturing in a constant-temperature incubator at 2328 ℃ for 3d, and screening the fraction with the smallest colony diameter to define the fraction C1;
s6: semi-preparative liquid phase separation: separating the C1 fraction obtained in the S5 by using a semi-prepared liquid phase, eluting by using 18% methanol, 27% methanol, 36% methanol and 45% methanol sequentially, eluting 0.9L of solvent in each gradient at the flow rate of 2.5mL/min, combining the same fractions by HPLC (high performance liquid chromatography) detection to form D fractions with different gradients, inoculating the watermelon wilt germs into PDA (personal digital assistants) culture media containing different D fractions, culturing for 4 days in a constant-temperature incubator at 28 ℃, wherein the fraction with the smallest colony diameter is a crowndaisy chrysanthemum bacteriostatic active monomer, and the filler particle size of the semi-prepared liquid phase is 10 mu m, the inner diameter is 10mm, and the length is 250 mm.
Example 3
The preparation method of the crowndaisy chrysanthemum bacteriostatic activity monomer provided by the embodiment 3 comprises the following steps:
s1: preprocessing stems and leaves of crowndaisy chrysanthemum: weighing 20Kg of fresh stem and leaf of crowndaisy chrysanthemum, naturally drying, drying in a blast drier at 55 ℃ for 14h, accurately weighing 2.3Kg of dried crowndaisy chrysanthemum, crushing in a Chinese medicinal material crusher, and sieving with a 40-mesh sieve;
s2: leaching: adding 52L 95% ethanol, ultrasonic extracting for 4 times, each for 3 hr, mixing filtrates, and concentrating under reduced pressure to obtain extract;
s3: macroporous resin column chromatography: dissolving the extract in 1.3L of distilled water, filtering, putting the supernatant into a D101 macroporous resin column, carrying out gradient elution by using distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol in sequence, eluting 2.1L of solvent by each gradient, carrying out flow rate of 3.5mL/min, respectively recovering the eluent after each gradient elution, carrying out reduced pressure recovery to obtain six fractions, inoculating the fusarium oxysporum f.sp.sp.sp.citrulli in a PDA culture medium containing different fractions, culturing in a constant-temperature incubator at 28 ℃ for 4D, and screening out a fraction A corresponding to the smallest colony diameter;
the diameter of the D101 macroporous resin column is 5.0cm, and the length of the macroporous resin column is 27 cm;
s4: c18 medium pressure chromatographic separation: separating fraction A of S3 by C18 medium pressure chromatography (MPLC), eluting with water in gradient, eluting with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol in sequence in gradient with flow rate of 3.5mL/min for 1.2L of solvent, detecting eluates after each gradient elution by HPLC, screening and combining the same fractions to form B fractions with different gradients, inoculating Fusarium oxysporum onto PDA culture medium containing different B fractions, culturing in constant temperature incubator at 28 deg.C for 4d, and screening one of the B fractions with minimum colony diameter to obtain B1 fraction;
s5: sephadex LH-20 column chromatography: separating the fraction B1 in S4 by SephadexLH-20 gel column chromatography, eluting with 5% methanol, 15% methanol, 25% methanol and 35% methanol sequentially, eluting 0.9-1.1L solvent in each gradient at the flow rate of 3.5mL/min, combining the same fractions by HPLC detection to form C fractions with different gradients, inoculating the fusarium oxysporum f.sp.sp.cubense in PDA culture media containing different C fractions, culturing in a constant-temperature incubator at 33 ℃ for 5d, and screening the fraction with the smallest colony diameter to define the fraction C1;
s6: semi-preparative liquid phase separation: separating the C1 fraction obtained in S5 by a semi-preparative liquid phase, eluting with 18% methanol, 27% methanol, 36% methanol and 45% methanol in sequence, eluting 1.1L of solvent in each gradient at the flow rate of 3.5mL/min, combining the same fractions by HPLC detection to form D fractions with different gradients, inoculating watermelon wilt pathogen in PDA culture media containing different D fractions, culturing in a constant-temperature incubator at 28 ℃ for 4D, wherein the fraction with the smallest colony diameter is a crowndaisy chrysanthemum bacteriostatic active monomer, and the semi-preparative liquid phase has a filler particle size of 10 mu m, an inner diameter of 10mm and a length of 250 mm.
The compounds obtained in example 1, example 2 and example 3 were weighed out in the following Table 1
TABLE 1
Example 4 is a test experiment for the inhibition effect of the chrysanthemum coronarium bacteriostatic active monomer on watermelon fusarium oxysporum:
example 4:
the test crowndaisy chrysanthemum adopts big-leaf crowndaisy chrysanthemum planted in a vegetable base of Jiangxi agricultural university; PDA culture medium, Fusarium oxysporum watermelon specialization type namely watermelon Fusarium oxysporum (Fusarium oxysporum), is provided by biological engineering and scientific college of Jiangxi agricultural university. Inoculating the strain preserved in the slant culture medium into a PDA culture medium, culturing in a constant temperature incubator at 28 deg.C for 4d, and preserving for use.
Diluting the chrysanthemum coronarium bacteriostatic active monomer (chlorogenic acid) obtained in example 1 into a gradient solution of 250-1.95 mg/ml & lt-1 & gt by a two-fold dilution method under an aseptic condition, wherein the gradient solution is prepared by mixing 1:9 is mixed with the culture medium to form a drug-containing plate. Punching edge fungus blocks with a puncher with the diameter of 8mm, respectively transferring the edge fungus blocks to the centers of PDA culture media containing medicines with different concentrations by using a seed inoculating rod, fully contacting one surface with hyphae with the culture media, repeating the treatment for 3 times, and culturing in a constant-temperature incubator at 28 ℃. Observing after 2d, and taking the lowest drug-containing concentration of the culture dish with no bacteria as the Minimum Inhibitory Concentration (MIC); the culture dishes grown aseptically were further cultured for 7 days, and then observed, with the lowest drug-containing concentration of the culture dishes with no bacteria being grown being the minimum bactericidal concentration (MFC). The results are shown in Table 2 below, which have MICs for Fusarium oxysporum and MFC of 31.25 and 125 mg-ml, respectively-1。
Determining toxicity equation by growth rate method, mixing the monomer (chlorogenic acid) obtained in example 1 at 100, 50, 25, 12.5, 6.25 mg/ml-1Diluted in concentration and mixed with culture medium in the ratio of 1:9 under aseptic condition to form drug-containing plate. Punching edge fungus blocks with a puncher with the diameter of 8mm, respectively transferring the edge fungus blocks to the centers of PDA culture media containing medicines with different concentrations by using a seed inoculating rod, fully contacting one surface with hyphae with the culture media, repeating the treatment for 3 times, and culturing in a constant-temperature incubator at 28 ℃ for 3 days. Measuring the diameter of bacterial colony by cross methodCalculating the antibacterial rate:
corrected colony diameter (mm) — colony diameter-punch diameter
The inhibition ratio (%) (control corrected colony diameter-treatment corrected colony diameter)/control corrected colony diameter × 100 was calculated, and EC thereof against fusarium oxysporum f.sp.citrulli50Is 53.048 mg/ml-1The results are shown in Table 3 below.
TABLE 2 MIC and MFC for chlorogenic acid to Fusarium oxysporum f.sp.citrulli
Table2The MIC and MFC of chlorogenicacid on Fusarium oxysporum
Note: "+" indicates that colonies had significantly grown; "-" indicates that the colonies did not grow significantly.
Note："+"on behalf of the colonies have obvious growth,"-"on behalf ofthe colonies did not grow significantly.
TABLE 3
The following examples 5 to 7 and comparative example 1 are experiments in which the chrysanthemum coronarium bacteriostatic active monomer obtained in example 1 was applied to watermelon seedlings with fusarium oxysporum:
example 5
Embodiment 5 provides a method for using a crowndaisy chrysanthemum bacteriostatic activity monomer, which comprises the following steps:
the chrysanthemum coronarium bacteriostatic active monomer (chlorogenic acid) obtained in the example 1 is diluted to 100 mg/mL by sterile water-1The stock solution is irrigated for 5 times every day and 1 time every 3 days after the stock solution is irrigated to the roots of the watermelon seedlings with the fusarium oxysporum, and the stock solution is irrigated for 5 times continuously, wherein 10ml of the stock solution is irrigated for each time.
Example 6
Embodiment 6 provides a method for using a crowndaisy chrysanthemum bacteriostatic activity monomer, which comprises the following steps:
the chrysanthemum coronarium bacteriostatic active monomer (chlorogenic acid) obtained in the example 1 is diluted to 100 mg/mL by sterile water-1The stock solution is irrigated for 1 time every day and 1 time every 4 days after the stock solution is irrigated for the first 3 days of the roots of the watermelon seedlings with the fusarium oxysporum, and then is irrigated for 3 times continuously, and 8ml is irrigated for each time.
Example 7
Embodiment 7 provides a method for using a crowndaisy chrysanthemum bacteriostatic activity monomer, which comprises the following steps:
the chrysanthemum coronarium bacteriostatic active monomer (chlorogenic acid) obtained in the example 1 is diluted to 100 mg/mL by sterile water-1The stock solution is irrigated for 1 time every day and 1 time every 4 days later on at the root of the watermelon seedling with the fusarium oxysporum, and is irrigated for 4 times continuously, and 12ml is irrigated for each time.
Comparative example 1
The distilled water is irrigated for 5 times every day and 1 time every 3 days after the distilled water is irrigated to the roots of the watermelon seedlings with the fusarium oxysporum, and the water is irrigated for 5 times continuously, wherein 10ml of the distilled water is irrigated for each time.
The 10-day in-growth of watermelon seedlings after the treatments of examples 5 to 7 and comparative example 1 was observed as shown in the following table 4:
TABLE 4
| Example 5 | Example 6 | Example 7 | Comparative example 1 | |
| Day one | Normal growth of the plant | Normal growth of the plant | Normal growth of the plant | Withering of branches and leaves |
| The fifth day | Normal growth of the plant | A few leaves withered | Normal growth of the plant | Withered and dead |
| The tenth day | Normal growth of the plant | A few branches and leaves withered | A few leaves withered | Withered and dead |
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. The present invention is not to be limited by the specific embodiments disclosed herein, and other embodiments that fall within the scope of the claims of the present application are intended to be within the scope of the present invention.
Claims (8)
1. A preparation method of a crowndaisy chrysanthemum bacteriostatic activity monomer is characterized by comprising the following steps:
s1: preprocessing stems and leaves of crowndaisy chrysanthemum: weighing 20Kg of fresh stem and leaf of crowndaisy chrysanthemum, drying naturally, drying in a blast drier at 45-55 ℃ for 10-14h, accurately weighing 1.7-2.3Kg of dried crowndaisy chrysanthemum, crushing in a Chinese medicinal material crusher, and sieving with a 40-mesh sieve;
s2: leaching: adding 48-52L 95% ethanol, ultrasonic extracting for 2-4 times (each for 1-3 hr), mixing filtrates, and concentrating under reduced pressure to obtain extract;
s3: macroporous resin column chromatography: dissolving the extract in 1.1-1.3L of distilled water, filtering, putting the supernatant on a D101 macroporous resin column, performing gradient elution by using distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol in sequence, eluting 1.9-2.1L of solvent by each gradient at the flow rate of 2.5-3.5mL/min, respectively recovering the eluent after each gradient elution, performing reduced pressure recovery to obtain six fractions, inoculating the fusarium oxysporum onto PDA culture media containing different fractions, culturing for 4D in a constant-temperature incubator at 28 ℃, and screening out a fraction A with the smallest colony diameter;
the diameter of the D101 macroporous resin column is 3.0-5.0cm, and the length of the macroporous resin column is 23-27 cm;
s4: c18 medium pressure chromatographic separation: separating fraction A of S3 by C18 medium pressure chromatography (MPLC), eluting with water in a gradient manner, sequentially eluting with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol in a gradient manner, wherein each gradient elutes 0.8-1.2L of solvent at the flow rate of 2.5-3.5mL/min, detecting eluates after each gradient elution by HPLC, screening and combining the same fractions to form different gradient type B fractions, inoculating the fusarium oxysporum f.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.in PDA culture medium containing different type B fractions, culturing for 4d in a constant temperature incubator at 28 ℃, screening one of the type B fractions with the smallest colony diameter to define as the type B1 fraction;
s5: sephadex LH-20 column chromatography: separating the fraction B1 in S4 by SephadexLH-20 gel column chromatography, eluting with 5% methanol, 15% methanol, 25% methanol and 35% methanol sequentially, eluting 0.9-1.1L solvent in each gradient, and the flow rate is 2.5-3.5mL/min, detecting by HPLC, combining the same fractions to form C fractions with different gradients, inoculating the Fusarium oxysporum F.sp.sp.citrulli in PDA culture medium containing different C fractions, culturing in a constant temperature incubator at 23-33 deg.C for 3-5d, and screening the fraction with the smallest colony diameter to obtain C1 fraction;
s6: semi-preparative liquid phase separation: separating the C1 fraction obtained in S5 by a semi-preparative liquid phase, eluting with 18% methanol, 27% methanol, 36% methanol and 45% methanol in sequence, eluting 0.9-1.1L of solvent in each gradient at a flow rate of 2.5-3.5mL/min, combining the same fractions by HPLC detection to form D fractions with different gradients, inoculating watermelon wilt germs into PDA culture media containing different D fractions, culturing in a constant-temperature incubator at 28 ℃ for 4D, wherein the fraction with the smallest colony diameter is a crowndaisy chrysanthemum bacteriostatic active monomer which is chlorogenic acid.
2. The preparation method of the crowndaisy chrysanthemum bacteriostatic-active monomer according to claim 1, which is characterized in that: s1, drying stems and leaves of caulis et folium Chrysanthemi Segeti in a forced air drier at 50 deg.C for 12h, accurately weighing 2.0Kg of dried caulis et folium Chrysanthemi Segeti, pulverizing in a Chinese medicinal material pulverizer, and sieving with 40 mesh sieve.
3. The preparation method of the crowndaisy chrysanthemum bacteriostatic-active monomer according to claim 1, which is characterized in that: in S2, 50L of 95% ethanol was added for ultrasonic assisted extraction for 3 times, each time for 2 h.
4. The preparation method of the crowndaisy chrysanthemum bacteriostatic-active monomer according to claim 1, which is characterized in that: in S3, dissolving the extract in 1.2L of distilled water; 2L of solvent were eluted per gradient at a flow rate of 3.0 mL/min.
5. The preparation method of the crowndaisy chrysanthemum bacteriostatic-active monomer according to claim 1, which is characterized in that: in S3, the diameter of the D101 macroporous resin column is 4.0cm, and the length of the macroporous resin column is 25 cm.
6. The preparation method of the crowndaisy chrysanthemum bacteriostatic-active monomer according to claim 1, which is characterized in that: in S4, 1L of solvent was eluted per gradient at a flow rate of 3.0 mL/min.
7. The preparation method of the crowndaisy chrysanthemum bacteriostatic-active monomer according to claim 1, which is characterized in that: in S5, the culture medium was cultured in a constant temperature incubator at 28 ℃ for 4d at a flow rate of 3.0 mL/min.
8. The preparation method of the crowndaisy chrysanthemum bacteriostatic-active monomer according to claim 1, which is characterized in that: in S6, 1L of solvent is eluted in each gradient at a flow rate of 3.0mL/min, and the semi-preparative liquid phase uses a filler with a particle size of 10 μm, an inner diameter of 10mm and a length of 250 mm.
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