CN107771866A - A kind of crowndaisy chrysanthemum bacteriostatic activity monomer and its application method - Google Patents
A kind of crowndaisy chrysanthemum bacteriostatic activity monomer and its application method Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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Abstract
The invention discloses a kind of crowndaisy chrysanthemum bacteriostatic activity monomer and its application method, belong to separation and the application method technical field of active material, the crowndaisy chrysanthemum bacteriostatic activity monomer is handled by pressing chromatographic isolation, the column chromatographies of sephadex LH 20, half to prepare liquid phase separation in the pretreatment of crowndaisy chrysanthemum cauline leaf, extraction, macroporous resin column chromatography, C18.A kind of crowndaisy chrysanthemum bacteriostatic activity monomer proposed by the invention, it can extract the monomer of efficient bacteriostatic activity, can suppress withered germ of water-melon well, have good wide application prospect.
Description
Technical field
The present invention relates to the separation of active material and application method technical field, more particularly to a kind of crowndaisy chrysanthemum is antibacterial
Activated monomer and its application method.
Background technology
Watermelon blight (Watermelon Fusariumwilt) also known as dead arm, stem wilt, wilt disease, it is referred to as " west
Melon cancer ", by Deuteromycotina fungi-watermelon pinch outs [Fusariumoxysporum f.sp.niveum
(E.F.Smith) Wollen] infect caused by, be a kind of destructive soil-borne disease for generally occurring both at home and abroad.Because watermelon is planted
Training area expands rapidly, causes continuous cropping watermelon area to increase, and the generation of watermelon blight is increasingly universal and serious, is produced to watermelon
Bring huge economic loss and the wasting of resources.Droop can cause the yield of watermelon to decline 30%~40%, the underproduction of grave illness plot
More than 80%, or even total crop failure.Therefore, effective preventing and treating of watermelon blight, directly influences the sound development of watermelon industry.
For a long time, people are mainly by preventing and treating west the methods of cultural control, breeding for disease resistance, biological control, chemical prevention
Cucurbit wilt.Chemical prevention has the characteristics of quick, effect is good.Chemical control work of the various regions to watermelon blight is all compared
Pay attention to, screen and have developed a collection of chemical agent with certain preventive effect, as octicin solution, KT emulsions, metalaxyl-mn-zn, evil are mould
Spirit, 40% melon are withered peaceful etc. to have certain prevention effect to watermelon blight.Progress and detection and analysis hand with science and technology
Section it is perfect, find it is considered that safety some chemical bactericides have carcinogenic or potential carcinogenic, mutagenesis, the possibility of teratogenesis
Property.In addition, long-term use of chemical bactericide, easily makes pathogen develop immunity to drugs it, prevention effect is reduced, pollutes environment.
The public is to heavy dose of harm brought using chemical bactericide to ecological environment and health also growing interest.Therefore, find
Wide spectrum, bactericide efficiently, less toxic, safe, studying and screen particularly from natural plants has fungistatic effect, and human body is good for
Health and ecological environment it is harmless bacteriostatic active ingredients tool be of great significance.
Crowndaisy chrysanthemum (Chrysanthemum Coronarium L.var.Spatiosum Bailer) is traditional composite family in China
Green vegetable, there is cultivation in north and south various regions.Modern pharmacological research shows, crowndaisy chrysanthemum have antibacterial, sterilization, antimicrobial, anticancer,
A variety of physiologically actives such as decompression, eliminating the phlegm.The plot of preceding stubble plantation crowndaisy chrysanthemum, then watermelon is planted, watermelon grows fine, and does not wither
Disease, and the yield of watermelon and quality all increase.The monomer with bacteriostatic activity is isolated from crowndaisy chrysanthemum, it can develop into one
Kind natural bactericidal agent application suppresses withered germ of water-melon, has good wide application prospect.
The content of the invention
The defects of in order to overcome prior art, the technical problems to be solved by the invention are to propose a kind of antibacterial work of crowndaisy chrysanthemum
Property monomer and its application method, it can extract the monomer of efficient bacteriostatic activity, can suppress withered germ of water-melon well,
With good wide application prospect.
To use following technical scheme up to this purpose, the present invention：
The invention provides a kind of crowndaisy chrysanthemum bacteriostatic activity monomer, comprise the following steps：
S1：Crowndaisy chrysanthemum cauline leaf pre-processes：Fresh crowndaisy chrysanthemum cauline leaf 20Kg is weighed, after naturally dry, in 45-55 DEG C of forced air drying
10-14h is dried in machine, the dried crowndaisy chrysanthemums of 1.7-2.3Kg is accurately weighed, is crushed in Chinese medicine material crushing machine, crosses 40 mesh sieves；
S2：Extraction：Add 48-52L 95% EtOH Sonicate ripple assisted extraction 2-4 times, each 1-3h, merging filtrate decompression
Concentration, obtains medicinal extract；
S3：Macroporous resin column chromatography：Medicinal extract is dissolved in 1.1-1.3L distilled water, filtering, in D101 macropores on supernatant
In resin column, successively with distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol elution gradient,
Each gradient elution 1.9-2.1L solvents, flow velocity 2.5-3.5mL/min, are separately recovered the eluent after each gradient elution, enter
Row is recovered under reduced pressure to obtain six cuts, and withered germ of water-melon is inoculated in the PDA culture medium containing different fractions, 28 is placed in and takes the photograph
The constant incubator culture 4d of family name's degree, filters out fraction A wherein corresponding to colony diameter minimum；
A diameter of 3.0-5.0cm of the D101 macroporous resin columns, macroreticular resin column length are 23-27cm；
S4：Chromatographic isolation is pressed in C18：S3 A cuts are taken through pressing chromatogram (MPLC) separation, methanol in C18:Water gradient is washed
It is de-, successively with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol elution gradient, each gradient elution
0.8-1.2L solvents, flow velocity 2.5-3.5mL/min, the eluent after each gradient elution detect through HPLC, and screening merges identical evaporates
Point, the B class cuts of different gradients are formed, withered germ of water-melon is inoculated in the PDA culture medium containing different B classes cuts, put
In 28 degrees Celsius of constant incubator culture 4d, one kind in colony diameter minimum B class cuts is screened, is defined as B1 cuts；
S5：Sephadex lh-20 column chromatography：B1 cuts in S4 are separated through SephadexLH-20 gel filtration chromatographies, according to
It is secondary to be eluted with 5% methanol, 15% methanol, 25% first and 35% methanol, each gradient elution 0.9-1.1L solvents, flow velocity 2.5-
The identical cut of 3.5mL/min, HPLC combining data detection, form the C class cuts of different gradients, by withered germ of water-melon be inoculated in containing
In the PDA culture medium of different C classes cuts, 23-33 degrees Celsius of constant incubator culture 3-5d is placed in, screening colony diameter is minimum
Cut, be defined as C1 cuts；
S6：Half prepares liquid phase separation：C1 cuts prepare liquid phase separation through half obtained by S5, successively with 18% methanol, 27% first
Alcohol, 36% first and the elution of 45% methanol, each gradient elution 0.9-1.1L solvents, flow velocity 2.5-3.5mL/min, HPLC detection are closed
And identical cut, the D class cuts of different gradients are formed, withered germ of water-melon is inoculated in the PDA containing different D classes cuts and cultivated
In base, 28 degrees Celsius of constant incubator culture 4d is placed in, the minimum cut of colony diameter is crowndaisy chrysanthemum bacteriostatic activity monomer.
In the present invention preferably technical scheme, in S1, crowndaisy chrysanthemum cauline leaf is dried into 12h, essence in 50 DEG C of blast driers
The dried crowndaisy chrysanthemums of 2.0Kg really are weighed, are crushed in Chinese medicine material crushing machine, cross 40 mesh sieves.
In the present invention preferably in technical scheme, in S2,50L 95% EtOH Sonicate ripple assisted extraction 3 times is added, every time
2h。
In the present invention preferably technical scheme, in S3, medicinal extract is dissolved in 1.2L distilled water；Each gradient elution 2L is molten
Agent, flow velocity 3.0mL/min.
In the present invention preferably technical scheme, in S3, a diameter of 4.0cm of the D101 macroporous resin columns, macropore tree
Fat column length is 25cm.
In the present invention preferably technical scheme, in S4, each gradient elution 1L solvents, flow velocity 3.0mL/min.
In the present invention preferably in technical scheme, in S5, flow velocity 3.0mL/min, culture medium is placed in 28 degrees Celsius of perseverance
Warm incubator culture 4d.
In the present invention preferably in technical scheme, in S6, each gradient elution 1L solvents, flow velocity 3.0mL/min, described half
It is 10 μm, internal diameter 10mm, length 250mm to prepare the packing material size that liquid phase uses.
A kind of application method of crowndaisy chrysanthemum bacteriostatic activity monomer, the compound for isolating and purifying to obtain is diluted to sterilized water
100mg·mL-1Stoste, poured in first 3-5 days of watermelon seedlings root, 1 time a day, afterwards per 3-4 days 1 time, then connected
It is continuous to pour 3-5 times, 8-12ml is poured every time.
In the present invention preferably technical scheme, stoste is poured in first 5 days of watermelon seedlings root, 1 time a day, afterwards
Every 3 days 1 time, then continuous pouring 5 times, 10ml is poured every time.
Beneficial effects of the present invention are：
Crowndaisy chrysanthemum bacteriostatic activity monomer provided by the invention isolate and purify and its application method, first by dried crowndaisy chrysanthemum powder
It is broken, 40 mesh sieves are crossed, then EtOH Sonicate ripple extraction is carried out, merging filtrate is simultaneously concentrated under reduced pressure, and then carries out macroporous resin column chromatography, first
Alcohol elutes, and takes the cut that fungistatic effect is best, then carries out pressing chromatographic isolation in C18, is eluted using methanol, detected by HPLC
Merge identical cut, then take the best cut of fungistatic effect, then eluted using glucose gel LH-20 column chromatographies, methanol,
The identical cut of HPLC combining data detections, takes the cut that fungistatic effect is best, finally liquid phase separation is prepared using half, equally using methanol
Elution, the identical cut of HPLC combining data detections, through experiment, the minimum cut of colony diameter is gained crowndaisy chrysanthemum bacteriostatic activity monomer, is led to
Multiple separating-purifying is crossed, extracts the monomer of efficient bacteriostatic activity, this crowndaisy chrysanthemum bacteriostatic activity monomer belongs to natural disinfection
Agent, also it is not easy to make pathogen develop immunity to drugs it even if long-term use, and environment is polluted, and it withers to watermelon
Germ has good inhibiting effect, protects the growth of watermelon.
Embodiment
Further illustrate technical scheme below and by embodiment.
The following example 1 to embodiment 3 is that the experiment of crowndaisy chrysanthemum bacteriostatic activity monomer is extracted from crowndaisy chrysanthemum cauline leaf：
Embodiment 1：
A kind of crowndaisy chrysanthemum bacteriostatic activity monomer is provided in embodiment 1, this method comprises the steps of：
S1：Crowndaisy chrysanthemum cauline leaf pre-processes：Fresh crowndaisy chrysanthemum cauline leaf 20Kg is weighed, after naturally dry, in 50 DEG C of blast driers
Middle dry 12h, the dried crowndaisy chrysanthemums of 2Kg are accurately weighed, are crushed in Chinese medicine material crushing machine, cross 40 mesh sieves；
S2：Extraction：Addition 50L 95% EtOH Sonicate ripple assisted extraction 3 times, each 2h, merging filtrate is concentrated under reduced pressure, and obtains
To medicinal extract；
S3：Macroporous resin column chromatography：Medicinal extract is dissolved in 1.2L distilled water, filtering, in D101 macroreticular resins on supernatant
In post, successively with distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol elution gradient, each
Gradient elution 2L solvents, flow velocity 3.0mL/min, are separately recovered the eluent after each gradient elution, are recovered under reduced pressure to obtain
Six cuts, withered germ of water-melon is inoculated in the PDA culture medium containing different fractions, is placed in 28 degrees Celsius incubated
Case culture 4d, filter out fraction A wherein corresponding to colony diameter minimum；
A diameter of 4.0cm of the D101 macroporous resin columns, macroreticular resin column length are 25cm；
S4：Chromatographic isolation is pressed in C18：S3 A cuts are taken through pressing chromatogram (MPLC) separation, methanol in C18:Water gradient is washed
It is de-, successively with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol elution gradient, each gradient elution 1L
Solvent, flow velocity 3.0mL/min, the eluent after each gradient elution detect through HPLC, and screening merges identical cut, forms different ladders
The B class cuts of degree, withered germ of water-melon is inoculated in the PDA culture medium containing different B classes cuts, is placed in 28 degrees Celsius of perseverance
Warm incubator culture 4d, one kind of colony diameter minimum B class cuts is screened, is defined as B1 cuts；
S5：Sephadex lh-20 column chromatography：B1 cuts in S4 are separated through SephadexLH-20 gel filtration chromatographies, according to
It is secondary to be eluted with 5% methanol, 15% methanol, 25% first and 35% methanol, each gradient elution 0.9-1.1L solvents, flow velocity 3.0mL/
The identical cut of min, HPLC combining data detection, the C class cuts of different gradients are formed, withered germ of water-melon is inoculated in containing different C
In the PDA culture medium of class cut, 28 degrees Celsius of constant incubator culture 4d is placed in, the minimum cut of screening colony diameter is fixed
Justice is C1 cuts；
S6：Half prepares liquid phase separation：C1 cuts prepare liquid phase separation through half obtained by S5, successively with 18% methanol, 27 | % first
Alcohol, 36% first and the elution of 45% methanol, each gradient elution 1L solvents, flow velocity 3.0mL/min, HPLC combining data detection is identical to be evaporated
Point, the D class cuts of different gradients are formed, withered germ of water-melon is inoculated in the PDA culture medium containing different D classes cuts, put
In 28 degrees Celsius of constant incubator culture 4d, the minimum cut of colony diameter is crowndaisy chrysanthemum bacteriostatic activity monomer, and described half prepares
The packing material size of liquid phase is 10 μm, internal diameter 10mm, length 250mm.
Embodiment 2
A kind of crowndaisy chrysanthemum bacteriostatic activity monomer that embodiment 2 provides, this method comprise the steps of：
S1：Crowndaisy chrysanthemum cauline leaf pre-processes：Fresh crowndaisy chrysanthemum cauline leaf 20Kg is weighed, after naturally dry, in 45 DEG C of blast driers
Middle dry 10h, the dried crowndaisy chrysanthemums of 1.7Kg are accurately weighed, are crushed in Chinese medicine material crushing machine, cross 40 mesh sieves；
S2：Extraction：Addition 48L 95% EtOH Sonicate ripple assisted extraction 2 times, each 1h, merging filtrate is concentrated under reduced pressure, and obtains
To medicinal extract；
S3：Macroporous resin column chromatography：Medicinal extract is dissolved in 1.1L distilled water, filtering, in D101 macroreticular resins on supernatant
In post, successively with distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol elution gradient, each
Gradient elution 1.9L solvents, flow velocity 2.5mL/min, are separately recovered the eluent after each gradient elution, be recovered under reduced pressure molten
Agent obtains six cuts, and withered germ of water-melon is inoculated in the PDA culture medium containing different fractions, is placed in 28 degrees Celsius of perseverance
Warm incubator culture 4d, filters out the fraction A wherein corresponding to colony diameter minimum；
A diameter of 3.0cm of the D101 macroporous resin columns, macroreticular resin column length are 23cm；
S4：Chromatographic isolation is pressed in C18：S3 A cuts are taken through pressing chromatogram (MPLC) separation, methanol in C18:Water gradient is washed
It is de-, successively with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol elution gradient, each gradient elution
0.8L solvents, flow velocity 2.5mL/min, the eluent after each gradient elution detect through HPLC, and screening merges identical cut, are formed not
With the B class cuts of gradient, withered germ of water-melon is inoculated in the PDA culture medium containing different B classes cuts, is placed in 28 degrees Celsius
Constant incubator culture 4d, screen colony diameter minimum B class cuts one kind, be defined as B1 cuts；
S5：Sephadex lh-20 column chromatography：B1 cuts in S4 are separated through SephadexLH-20 gel filtration chromatographies,
Eluted successively with 5% methanol, 15% methanol, 25% first and 35% methanol, each gradient elution 0.9-1.1L solvents, flow velocity
The identical cut of 2.5mL/min, HPLC combining data detection, form the C class cuts of different gradients, by withered germ of water-melon be inoculated in containing
In the PDA culture medium of different C classes cuts, 2328 degrees Celsius of constant incubator culture 3d is placed in, screening colony diameter minimum
Cut, it is defined as C1 cuts；
S6：Half prepares liquid phase separation：C1 cuts prepare liquid phase separation through half obtained by S5, successively with 18% methanol, 27 | % first
Alcohol, 36% first and the elution of 45% methanol, each gradient elution 0.9L solvents, flow velocity 2.5mL/min, HPLC combining data detection is identical to be evaporated
Point, the D class cuts of different gradients are formed, withered germ of water-melon is inoculated in the PDA culture medium containing different D classes cuts, put
In 28 degrees Celsius of constant incubator culture 4d, the minimum cut of colony diameter is crowndaisy chrysanthemum bacteriostatic activity monomer, and described half prepares
The packing material size of liquid phase is 10 μm, internal diameter 10mm, length 250mm.
Embodiment 3
A kind of crowndaisy chrysanthemum bacteriostatic activity monomer that embodiment 3 provides, this method comprise the steps of：
S1：Crowndaisy chrysanthemum cauline leaf pre-processes：Fresh crowndaisy chrysanthemum cauline leaf 20Kg is weighed, after naturally dry, in 55 DEG C of blast driers
Middle dry 14h, the dried crowndaisy chrysanthemums of 2.3Kg are accurately weighed, are crushed in Chinese medicine material crushing machine, cross 40 mesh sieves；
S2：Extraction：Addition 52L 95% EtOH Sonicate ripple assisted extraction 4 times, each 3h, merging filtrate is concentrated under reduced pressure, and obtains
To medicinal extract；
S3：Macroporous resin column chromatography：Medicinal extract is dissolved in 1.3L distilled water, filtering, in D101 macroreticular resins on supernatant
In post, successively with distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol elution gradient, each
Gradient elution 2.1L solvents, flow velocity 3.5mL/min, are separately recovered the eluent after each gradient elution, be recovered under reduced pressure
To six cuts, withered germ of water-melon is inoculated in the PDA culture medium containing different fractions, is placed in 28 degrees Celsius of constant temperature training
Case culture 4d is supported, filters out fraction A wherein corresponding to colony diameter minimum；
A diameter of 5.0cm of the D101 macroporous resin columns, macroreticular resin column length are 27cm；
S4：Chromatographic isolation is pressed in C18：S3 A cuts are taken through pressing chromatogram (MPLC) separation, methanol in C18:Water gradient is washed
It is de-, successively with 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol elution gradient, each gradient elution
1.2L solvents, flow velocity 3.5mL/min, the eluent after each gradient elution detect through HPLC, and screening merges identical cut, are formed not
With the B class cuts of gradient, withered germ of water-melon is inoculated in the PDA culture medium containing different B classes cuts, is placed in 28 degrees Celsius
Constant incubator culture 4d, screen colony diameter minimum B class cuts in one kind, be defined as B1 cuts；
S5：Sephadex lh-20 column chromatography：B1 cuts in S4 are separated through SephadexLH-20 gel filtration chromatographies, according to
It is secondary to be eluted with 5% methanol, 15% methanol, 25% first and 35% methanol, each gradient elution 0.9-1.1L solvents, flow velocity 3.5mL/
The identical cut of min, HPLC combining data detection, the C class cuts of different gradients are formed, withered germ of water-melon is inoculated in containing different C
In the PDA culture medium of class cut, 33 degrees Celsius of constant incubator culture 5d is placed in, the minimum cut of screening colony diameter is fixed
Justice is C1 cuts；
S6：Half prepares liquid phase separation：C1 cuts prepare liquid phase separation through half obtained by S5, successively with 18% methanol, 27 | % first
Alcohol, 36% first and the elution of 45% methanol, each gradient elution 1.1L solvents, flow velocity 3.5mL/min, HPLC combining data detection is identical to be evaporated
Point, the D class cuts of different gradients are formed, withered germ of water-melon is inoculated in the PDA culture medium containing different D classes cuts, put
In 28 degrees Celsius of constant incubator culture 4d, the minimum cut of colony diameter is crowndaisy chrysanthemum bacteriostatic activity monomer, and described half prepares
The packing material size of liquid phase is 10 μm, internal diameter 10mm, length 250mm.
The quality of embodiment 1, embodiment 2 and the gained compound of embodiment 3 is weighed in table 1 below
Table 1
Embodiment 4 is the inhibitory action test experiments to withered germ of water-melon by crowndaisy chrysanthemum bacteriostatic activity monomer：
Embodiment 4：
Test great Ye crowndaisy chrysanthemum of the crowndaisy chrysanthemum using Agricultural University Of Jiangxi's Vegetable Base plantation；PDA culture medium, Fusarium oxysporum west
Melon specialized form is withered germ of water-melon (Fusarium oxysporum), is carried by Agricultural University Of Jiangxi's bioengineering and science institute
For.The strain for being stored in slant medium is inoculated in PDA culture medium, 28 DEG C of constant incubator culture 4d is put, saves backup.
The crowndaisy chrysanthemum bacteriostatic activity monomer (i.e. chlorogenic acid) of the gained of embodiment 1 is diluted to doubling dilution under aseptic condition
250~1.95mgml-1 gradient solution, with 1：9 ratio is mixed into drug containing flat board with culture medium.Punched using diameter 8mm
Device, which is beaten, takes edge fungus block, and is transferred to the drug containing PDA culture medium center of each various concentrations respectively with loop-carrier, makes to carry disease germs
The one side of silk is fully contacted with culture medium, and each processing is repeated 3 times, and is put in 28 DEG C of constant incubators and cultivated.Seen after 2d
Examine, using the minimum concentration that contains of the culture dish of not long bacterium as minimal inhibitory concentration (MIC)；The culture dish of asepsis growth is continued to train
Observed after supporting 7d, using the minimum concentration that contains of the culture dish of not long bacterium as minimum bactericidal concentration (MFC).It is as a result as shown in table 2 below,
Its MIC and MFC to withered germ of water-melon is respectively 31.25 and 125mgml-1。
Virulence equation is determined with growth rate method, the crowndaisy chrysanthemum bacteriostatic activity monomer (i.e. chlorogenic acid) of the gained of embodiment 1 is pressed
100、50、25、12.5、6.25mg·ml-1Concentration dilution, aseptically with 1:9 ratio is mixed into drug containing with culture medium
Flat board.Beaten with diameter 8mm card punch and take edge fungus block, then be transferred to the drug containing of each various concentrations respectively with loop-carrier
PDA culture medium center, and the one side with mycelia is fully contacted with culture medium, each processing is repeated 3 times, and is placed on 28 DEG C of perseverances
3d is cultivated in warm incubator.Colony diameter is measured with crossing method, calculates bacteriostasis rate：
Correct colony diameter (mm)=colony diameter-card punch diameter
Bacteriostasis rate (%)=(control correction colony diameter-processing correction colony diameter)/control correction colony diameter × 100
It is computed, its EC to withered germ of water-melon50For 53.048mgml-1, it is as a result as shown in table 3 below.
MIC and MFC of the chlorogenic acid of table 2 to withered germ of water-melon
Table2 The MIC and MFC of chlorogenicacid on Fusarium oxysporum
Note："+" represents that bacterium colony has obvious growth；"-" represents that bacterium colony does not substantially grow.
Note："+"on behalf of the colonies have obvious growth,"-"on behalf of
the colonies did not grow significantly.
Table 3
Example 5 below to embodiment 7 and comparative example 1 is to act on the crowndaisy chrysanthemum bacteriostatic activity monomer of the gained of embodiment 1
In the experiment of the watermelon seedlings with wilt：
Embodiment 5
Embodiment 5 provides a kind of application method of crowndaisy chrysanthemum bacteriostatic activity monomer, and this method is as follows：
The crowndaisy chrysanthemum bacteriostatic activity monomer (i.e. chlorogenic acid) of the gained of embodiment 1 is diluted to 100mgmL with sterilized water-1Original
Liquid, poured in first 5 days of the watermelon seedlings root with withered germ of water-melon, 1 time a day, every 3 days 1 time afterwards, then connect
It is continuous to pour 5 times, 10ml is poured every time.
Embodiment 6
Embodiment 6 provides a kind of application method of crowndaisy chrysanthemum bacteriostatic activity monomer, and this method is as follows：
The crowndaisy chrysanthemum bacteriostatic activity monomer (i.e. chlorogenic acid) of the gained of embodiment 1 is diluted to 100mgmL with sterilized water-1Original
Liquid, poured in first 3 days of the watermelon seedlings root with withered germ of water-melon, 1 time a day, every 4 days 1 time afterwards, then connect
It is continuous to pour 3 times, 8ml is poured every time.
Embodiment 7
Embodiment 7 provides a kind of application method of crowndaisy chrysanthemum bacteriostatic activity monomer, and this method is as follows：
The crowndaisy chrysanthemum bacteriostatic activity monomer (i.e. chlorogenic acid) of the gained of embodiment 1 is diluted to 100mgmL with sterilized water-1Original
Liquid, poured in first 4 days of the watermelon seedlings root with withered germ of water-melon, 1 time a day, every 4 days 1 time afterwards, then connect
It is continuous to pour 4 times, 12ml is poured every time.
Comparative example 1
By distilled water, it is poured in first 5 days of the watermelon seedlings root with withered germ of water-melon, 1 time a day, afterwards every 3
It 1 time, then continuous pouring 5 times, 10ml is poured every time.
Observe growing state such as table 4 below in the watermelon seedlings 10 days after embodiment 5 is handled to embodiment 7 and comparative example 1
It is shown：
Table 4
| Embodiment 5 | Embodiment 6 | Embodiment 7 | Comparative example 1 | |
| First day | Normal growth | Normal growth | Normal growth | Branches and leaves are withered |
| 5th day | Normal growth | A little leaf is withered | Normal growth | It is withered |
| Tenth day | Normal growth | A little die back | A little leaf is withered | It is withered |
The present invention is described with reference to the preferred embodiments, and those skilled in the art know, is not departing from the present invention's
In the case of spirit and scope, various changes or equivalence replacement can be carried out to these features and embodiment.The present invention is not by this
The limitation of specific embodiment disclosed in place, other embodiments fallen into claims hereof belong to protection of the present invention
Scope.
Claims (10)
- A kind of 1. crowndaisy chrysanthemum bacteriostatic activity monomer, it is characterized in that comprising the following steps：S1：Crowndaisy chrysanthemum cauline leaf pre-processes：Fresh crowndaisy chrysanthemum cauline leaf 20Kg is weighed, after naturally dry, in 45-55 DEG C of blast drier 10-14h is dried, the dried crowndaisy chrysanthemums of 1.7-2.3Kg is accurately weighed, is crushed in Chinese medicine material crushing machine, crosses 40 mesh sieves；S2：Extraction：48-52L 95% EtOH Sonicate ripple assisted extraction 2-4 times, each 1-3h is added, merging filtrate decompression is dense Contracting, obtains medicinal extract；S3：Macroporous resin column chromatography：Medicinal extract is dissolved in 1.1-1.3L distilled water, filtering, in D101 macroreticular resins on supernatant In post, successively with distilled water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 90% methanol elution gradient, each Gradient elution 1.9-2.1L solvents, flow velocity 2.5-3.5mL/min, are separately recovered the eluent after each gradient elution, are subtracted Push back receipts and obtain six cuts, withered germ of water-melon is inoculated in the PDA culture medium containing different fractions, is placed in 28 degrees Celsius Constant incubator culture 4d, filter out fraction A wherein corresponding to colony diameter minimum；A diameter of 3.0-5.0cm of the D101 macroporous resin columns, macroreticular resin column length are 23-27cm；S4：Chromatographic isolation is pressed in C18：S3 A cuts are taken through pressing chromatogram (MPLC) separation, methanol in C18:Water gradient elution, according to Secondary 5% methanol, 15% methanol, 25% methanol, 35% methanol and 50% methanol elution gradient, each gradient elution 0.8-1.2L Solvent, flow velocity 2.5-3.5mL/min, the eluent after each gradient elution detect through HPLC, and screening merges identical cut, are formed not With the B class cuts of gradient, withered germ of water-melon is inoculated in the PDA culture medium containing different B classes cuts, is placed in 28 degrees Celsius Constant incubator culture 4d, screen colony diameter minimum B class cuts in one kind, be defined as B1 cuts；S5：Sephadex lh-20 column chromatography：B1 cuts in S4 are separated through SephadexLH-20 gel filtration chromatographies, used successively 5% methanol, 15% methanol, 25% first and the elution of 35% methanol, each gradient elution 0.9-1.1L solvents, flow velocity 2.5-3.5mL/ The identical cut of min, HPLC combining data detection, the C class cuts of different gradients are formed, withered germ of water-melon is inoculated in containing different C In the PDA culture medium of class cut, 23-33 degrees Celsius of constant incubator culture 3-5d is placed in, screening colony diameter is minimum to be evaporated Point, it is defined as C1 cuts；S6：Half prepares liquid phase separation：C1 cuts prepare liquid phase separation through half obtained by S5, successively with 18% methanol, 27 | % methanol, 36% first and the elution of 45% methanol, each gradient elution 0.9-1.1L solvents, flow velocity 2.5-3.5mL/min, HPLC combining data detection Identical cut, forms the D class cuts of different gradients, and withered germ of water-melon is inoculated in into the PDA culture medium containing different D classes cuts In, 28 degrees Celsius of constant incubator culture 4d is placed in, the minimum cut of colony diameter is crowndaisy chrysanthemum bacteriostatic activity monomer.
- 2. crowndaisy chrysanthemum bacteriostatic activity monomer according to claim 1, it is characterised in that：In S1, by crowndaisy chrysanthemum cauline leaf in 50 DEG C of drums 12h is dried in wind drying machine, the dried crowndaisy chrysanthemums of 2.0Kg is accurately weighed, is crushed in Chinese medicine material crushing machine, crosses 40 mesh sieves.
- 3. crowndaisy chrysanthemum bacteriostatic activity monomer according to claim 1, it is characterised in that：In S2,95% ethanol for adding 50L surpasses Sound wave assisted extraction 3 times, each 2h.
- 4. crowndaisy chrysanthemum bacteriostatic activity monomer according to claim 1, it is characterised in that：In S3, medicinal extract is dissolved in 1.2L distillations In water；Each gradient elution 2L solvents, flow velocity 3.0mL/min.
- 5. crowndaisy chrysanthemum bacteriostatic activity monomer according to claim 1, it is characterised in that：In S3, the D101 macroporous resin columns A diameter of 4.0cm, macroreticular resin column length is 25cm.
- 6. crowndaisy chrysanthemum bacteriostatic activity monomer according to claim 1, it is characterised in that：In S4, each gradient elution 1L solvents, Flow velocity 3.0mL/min.
- 7. crowndaisy chrysanthemum bacteriostatic activity monomer according to claim 1, it is characterised in that：In S5, flow velocity 3.0mL/min, it will train Foster base is placed in 28 degrees Celsius of constant incubator culture 4d.
- 8. crowndaisy chrysanthemum bacteriostatic activity monomer according to claim 1, it is characterised in that：In S6, each gradient elution 1L solvents, Flow velocity 3.0mL/min, the packing material size that the half preparation liquid phase uses is 10 μm, internal diameter 10mm, length 250mm.
- A kind of 9. application method of the crowndaisy chrysanthemum bacteriostatic activity monomer described in claim 1, it is characterised in that：It will isolate and purify to obtain Compound be diluted to 100mgmL with sterilized water-1Stoste, poured in first 3-5 days of watermelon seedlings root, daily 1 It is secondary, afterwards per 3-4 days 1 time, then continuous pouring 3-5 times, 8-12ml is poured every time.
- 10. the application method of crowndaisy chrysanthemum bacteriostatic activity monomer as claimed in claim 9, it is characterised in that：By stoste pour in First 5 days of watermelon seedlings root, 1 time a day, every 3 days 1 time afterwards, then continuous pouring 5 times, 10ml is poured every time.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113582757A (en) * | 2021-08-04 | 2021-11-02 | 安徽省司尔特肥业股份有限公司 | Special organic water-soluble fertilizer for melons and fruits and preparation method thereof |
| CN113875844A (en) * | 2021-09-29 | 2022-01-04 | 武夷山成隆天创茶业科技股份有限公司 | Application of spartina alterniflora extract in plant-derived functional tea product and functional tea product |
-
2017
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Non-Patent Citations (3)
| Title |
|---|
| CHUNPENG WAN, ET AL.: "Caffeoylquinic Acids from the Aerial Parts of Chrysanthemum coronarium L.", 《PLANT》 * |
| 李珊珊 等: "茼蒿提取物及其不同柱层析馏分对西瓜枯萎病菌的抑制作用", 《江西农业大学学报》 * |
| 林学政 等: "牛蒡叶内绿原酸抑制植物病原真菌的研究", 《植物保护》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113582757A (en) * | 2021-08-04 | 2021-11-02 | 安徽省司尔特肥业股份有限公司 | Special organic water-soluble fertilizer for melons and fruits and preparation method thereof |
| CN113875844A (en) * | 2021-09-29 | 2022-01-04 | 武夷山成隆天创茶业科技股份有限公司 | Application of spartina alterniflora extract in plant-derived functional tea product and functional tea product |
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