CN101352180A - Macleaya cordata alkaloids for inhibiting plant pathogenic bacteria and preparation method thereof - Google Patents
Macleaya cordata alkaloids for inhibiting plant pathogenic bacteria and preparation method thereof Download PDFInfo
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Abstract
The invention relates to macleaya cordata alkaloid used for inhibiting plant pathogens and the preparation method thereof. The alkaloid of the invention is TA total alkaloids which are extracted from macleaya cordata whole grass and four main monomer alkaloids: sanguinarine, chelerythrine, protopine, alpha-allocryptopine. The alkaloid clears that macleaya cordata alkaloid has obvious inhibitory activity on plant pathogens and epiphyte for the first time, wherein, the inhibitory activity of sanguinarine and chelerythrine is strongest. Macleaya cordata TA total alkaloids and monomer alkaloids can be used for controlling plant diseases.
Description
Technical field
The present invention relates to the alkaloidal preparation of macleaya cordata biology total alkali and principal monomer and, belong to technical field of pesticide the inhibitory action of plant pathogenic fungi and bacterium.
Background technology
Macleaya cordata (Macleaya cordata (Willd.) R.Br.) belongs to Papaveraceae (Papaveraceae) Macleaya in classification, be perennial tall and big draft, mainly grow in the shrubbery or thick grass of the hills of height above sea level 120-180 rice or low mountain forest, be distributed in the provinces such as Guizhou, Guangxi, Guangdong, Fujian, Jiangxi, Hunan, Hubei, Anhui, Zhejiang, Jiangsu, Henan, Shaanxi, SOUTH OF GANSU of China, be distributed in the Japan middle part on a small quantity.Macleaya cordata is important medicinal plant, has desinsection, multiple biologically active such as antibiotic, antitumor.Total alkaloid content accounts for 1% of herb dry weight in the macleaya cordata, is isoquinoline alkaloid.Alkaloid is considered to the main bioactive ingredients of macleaya cordata.
Have not yet to see Macleaya cordata alkaloids to plant pathogenetic bacterias such as Agrobacterium tumefaciens, angular leaf spot of cucumber bacterium, tomato bacterial spot germs, and the inhibitory action of plant pathogenic fungis such as tomato early blight bacterium, botrytis cinerea, cucumber fusarium axysporum, tomato wilt bacterium, cotton-wilt fusarium, gibberella saubinetii, Rhizoctonia solani Kuhn, ring rot of apple bacterium report.
The present invention focuses on macleaya cordata total alkali and the alkaloidal preparation of principal monomer thereof, and alkaloid is to the inhibition activity of above-mentioned plant pathogenetic bacteria and fungi, for the research and development of novel plant source bactericide provide foundation.
In order to protect the environment and the mankind, and the sustainable development of keeping agricultural, be badly in need of that constantly development is efficient, the environmentally compatible type agricultural chemicals of low toxicity, low-residual.The purpose of this invention is to provide Macleaya cordata alkaloids and suppressing examples of applications on plant pathogenic fungi and the pathogenetic bacteria as high-efficient antibacterial agent.
Summary of the invention
The present invention relates to the alkaloidal preparation of macleaya cordata biology total alkali and principal monomer and, provide following technical method the inhibitory action of plant pathogenic fungi and bacterium.
1, the preparation of macleaya cordata biology total alkali
Adopt acid to put forward the heavy method of alkali and get macleaya cordata total alkaloid, yield is 0.7% (g/g dry weight).
2, the separation of macleaya cordata main alkaloid and structure are identified
Adopt multiple column chromatography and in conjunction with the active method of separating of following the trail of, from the macleaya cordata total alkali, separate and identify 4 isoquinoline alkaloids such as sanguinarine, Chelerythrine, Biflorine and α-allocryptopine.
3, Macleaya cordata alkaloids is to the inhibitory action of bacterium
Adopt the porous plate growth rate method, measure sanguinarine and Chelerythrine Agrobacterium tumefaciens, angular leaf spot of cucumber bacterium and tomato bacterial spot germ are had obvious suppression effect (table 3).
4, Macleaya cordata alkaloids is to the inhibitory action of rice blast fungus spore germination
Macleaya cordata principal monomer alkaloid: spore germination has the obvious suppression effect to rice blast fungus for sanguinarine, Chelerythrine, Biflorine and α-allocryptopine, 503nhibiting concentration at 10 μ g/mL to (table 4) between the 40 μ g/mL.
5, Macleaya cordata alkaloids is to the inhibitory action of conk
Adopt the liquid nutrient medium growth rate method, mensuration sanguinarine, Chelerythrine, Biflorine and α-allocryptopine all have obvious suppression effect (table 5) to the growth of botrytis cinerea, ring rot of apple bacterium, gibberella saubinetii, tomato wilt bacterium, cotton-wilt fusarium, Rhizoctonia solani Kuhn.
Advantage of the present invention: the present invention is clear and definite first, and Macleaya cordata alkaloids has the anti-plant pathogenetic bacteria of wide spectrum and the activity of fungi, and main antibacterial activity alkaloid is: sanguinarine, Chelerythrine, Biflorine and α-allocryptopine.Purpose is to utilize plant resources, obtains the antibacterial activity composition, is the research and development of botanical pesticide, for the physiological ecological function of inquiring into the Secondary Metabolism of Plant composition provides foundation.
In order to understand the present invention better, further specify essentiality content of the present invention below in conjunction with embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment
Embodiment 1: the preparation of macleaya cordata total alkali
Gather the macleaya cordata complete stool autumn, pulverize behind the airing, get the 3kg powder, with extracting 3 times (each 3 days) under 2 times of volumes, the 1% sulfuric acid room temperature, filtrate gets sediment with sodium hydroxide adjust pH to 9.Use 1L 95% ethanol in 80 ℃ of following refluxing extraction 3 times (each 5 hours) sediment, to pH 2, alkaloid becomes to salt out extract with 50% sulfuric acid acidation, filters, and promptly gets macleaya cordata total alkaloid 21g, and calculating total alkaloid content is 0.7% of plant dry weight.
Embodiment 2: the macleaya cordata total alkali is to the inhibition activity of plant pathogenetic bacteria
Be prepared into total alkaloids with the method described in the embodiment 1, adopt punching medicament diffusion process, measure the inhibition activity of macleaya cordata total alkaloid plant pathogenetic bacteria.
(1) the plant pathogenetic bacteria bacterial strain of determination of activity: comprise for the examination bacterium: Agrobacterium tumefaciens (Agrobacterium tumefaciens ATCC11158), angular leaf spot of cucumber bacterium (Pseudomonas lachrymans ATCC 11921) and tomato bacterial spot germ (Xanthomonasvesicatoria ATCC 11633).Above-mentioned strains tested long preservation is under-20 ℃, before determination of activity, need be to carrying out (28 ℃ of activation culture on the LB flat board for the examination bacterium, secretly) 48h, picking list bacterium colony shakes (28 ℃ of trainings in the LB liquid nutrient medium then, secretly, 150rpm) 24h, successive transfer culture 8-12h then, bacterial concentration reaches 10
9Cfu/mL promptly can be used for determination of activity.
(2) medium: adopt the LB agar medium (yeast extract 5g/L, peptone 10g/L, sodium chloride 5g/L, agar 20g/L, pH7.0) and LB liquid nutrient medium (sodium chloride 5g/L, pH 7.0 for yeast extract 5g/L, peptone 10g/L).
(3) antibacterial activity is measured: adopt punching medicament diffusion process to measure.Concrete determination step is: contain 3% water agar 10mL in the culture dish of 90mm diameter, add 15mL after the cooled and solidified and contain 150 μ L bacterium liquid (10
9Cfu/mL) LB medium (45 ℃), after treating that the upper strata is solidified, with aseptic card punch punching (diameter 5mm), adding concentration in every hole is the macleaya cordata biology total alkali solution 40 μ L of 5mg/mL (preparing with DMSO) or streptomycin sulphate solution 40 μ L (positive control) or the DMSO solution 40 μ L (negative control) of 0.2mg/mL (preparing with DMSO), after 28 ℃ of following overnight incubation, adopt the right-angled intersection method to measure the diameter in antibacterial zone.Each is handled and repeats 3 times.
(4) experimental result: the results are shown in Table 1, macleaya cordata total alkaloid is to having better inhibited activity for the examination bacterium.
The antibacterial activity of table 1 macleaya cordata total alkaloid
Annotate: the amount of macleaya cordata total alkali is 0.2mg in every hole, and the amount of streptomycin sulphate is 8.0 μ g.
Embodiment 3: macleaya cordata total alkaloid is to the inhibition activity of plant pathogenic fungi
Prepare total alkaloids with the method described in the embodiment 1, adopt the malicious dull and stereotyped mycelial growth rate method of being with, measure the inhibition activity of macleaya cordata total alkaloid plant pathogenic fungi.
(1) potato dextrose agar (PDA): potato dextrose agar (the Potato dextrose agar of preparation 1L, PDA) step is: the potato 200g that will clean peeling, be cut into small pieces, add deionized water 1000mL, boil 30min, filter, get filtrate, add agar 20g and glucose 20g, after heating is melted agar, use filtered through gauze while hot, add deionized water then and be settled to 1000mL.121 ℃ of moist heat sterilization 20min.This medium is used for activation culture, successive transfer culture and the antibacterial activity of fungi and measures.
(2) for the plant pathogenic fungi of trying: comprise for the examination fungi: tomato early blight bacterium (Alternaria solani), cucumber fusarium axysporum (Fusariumoxysporum f.sp.cucumerinum), tomato wilt bacterium (Fusarium oxysporum f.sp.lycopersici), cotton-wilt fusarium (Fusarium oxysporum f.sp vasinfectum), Rhizoctonia solani Kuhn (Rhizoctonia solani).The above-mentioned plant pathogenic fungi bacterial strain long preservation that supplies examination before determination of activity, need be carried out activation culture (25 ℃, dark) 5-10 days to supplying to try fungi on the PDA flat board under 4 ℃, successive transfer culture is 1-2 time then, promptly can be used for determination of activity.
(3) experimental design and operation: adopt the malicious dull and stereotyped mycelial growth rate method of being with, measure the inhibitory action of biology total alkali to the hypha,hyphae growth.Biology total alkali is made into the mother liquor of series concentration: 200mg/mL, 100mg/mL, 25mg/mL, 10mg/mL, 5mg/mL (at different confession examination fungies, select different solvents, i.e. tomato wilt bacterium, Rhizoctonia solani Kuhn, rhizoctonia cerealis determination of activity adopts acetone to make solvent; Cucumber fusarium axysporum and cotton-wilt fusarium use DMSO as solvent; Tomato early blight bacterium is used ethanol as solvent), get a certain amount of (that is: 0.5mL biology total alkali mother liquor is in 50mL PDA medium) then in fungi culture medium (50-60 ℃), fully mix, the concentration of biology total alkali this moment in culture fluid is respectively: 2000 μ g/mL, 1000 μ g/mL, 250 μ g/mL, 100 μ g/mL, 50 μ g/mL.The medium that mixes is poured in the sterile petri dish of 90mm diameter, treat culture medium solidifying after, the fresh mycelia of picking (diameter is the bacterium cake of 5mm) 25 ℃, was secretly cultivated 5-10 days in the center of PDA flat board (have one of mycelia to face down and medium is fitted).Negative control (being the solvent contrast), blank (water) and positive control (carbendazim) are established in experiment, and each is handled and repeats 3 times.
(4) result observes and the data processing: observed result when treating the blank bacterium colony near the culture dish edge, take out culture dish kind of calliper colony diameter, and diameter is surveyed in each bacterium colony right-angled intersection twice.Calculate inhibiting rate (comparing with solvent when calculating inhibiting rate) with following formula:
The average expansion diameter (mm)-5 (bacterium cake diameter mm) of bacterium colony expansion diameter (mm)=measurement bacterium colony
Result of the test adopts Microsoft Excel software to carry out data preparation, in analysis, for test agent concentration (μ g/mL) take the logarithm (X), inhibiting rate is converted into biostatistics probit value (Y), try to achieve and suppress active regression equation (virulence regression equation) (Y=aX+b), thus can be in the hope of 503nhibiting concentration (IC
50).
(5) experimental result: see Table 2.The macleaya cordata biology total alkali has obvious inhibiting activity to tomato early blight bacterium, cucumber fusarium axysporum and cotton-wilt fusarium.
The antifungal activity of table 2 macleaya cordata biology total alkali
Annotate: carbendazim in the medium (positive control) concentration is 10 μ g/mL, is 60-100% to the inhibiting rate for the examination conk.
Embodiment 4: the alkaloidal separation of macleaya cordata principal monomer
Macleaya cordata total alkaloid 20g, with filtering behind the acetic acid ethyl dissolution, insoluble matter elder generation water fully dissolves, and the sodium hydroxide solution titration aqueous solution with 50% constantly shakes, and sees that solution is got final product by salmon pink browning look and appearance precipitation, filters, and removes moisture.(chloroform: methyl alcohol: acetone=100: 3: 3 v/v), obtains each 1 of yellow color component and red component to adopt silica gel column chromatography then.Red component through gel (Sephadex LH-20) column chromatography (chloroform: methyl alcohol=1: 1, v/v), obtain purer compound, through reverse phase silica gel (RP-18) column chromatography, gradient elution (water: methyl alcohol=90: 10; 80: 20; 70: 30; 50: 50; 30: 70; 10: 90, v/v; Each gradient elution is 2 column volumes), obtain a red powder shape material, i.e. compound 1 (sanguinarine) 1800mg.Above-mentioned yellow color component is through gel (Sephadex LH-20) column chromatography (chloroform: methyl alcohol=1: 1, v/v), obtain purer compound, again through preparation of lamina chromatography (chloroform: ethyl acetate: methyl alcohol=50: 5: 1, v/v), obtain a yellow needle shape material, be compound 2 (Chelerythrine) 900mg.
The ethyl acetate filtrate of above-mentioned total alkali, concentrate back water suspendible, use chloroform extraction then, extract concentrates back purification on normal-phase silica gel (300-400 order) column chromatography (chloroform: methyl alcohol: ammoniacal liquor=60: 4: 1.5, v/v) wash-out, obtain two main components, use the chloroform-methanol repeated crystallization respectively, obtain compound 3 (Biflorine) 150mg and compound 4 (α-allocryptopine) 120mg.
Embodiment 5: the structure of compound 1 (sanguinarine) is identified
Be prepared into compound 1 with embodiment 4 described methods, be in red crystallization (water).M.p.242-243 ℃; Bismuth potassium iodide shows Chinese red, points out this compound may be alkaloid.
EI-MS：m/z?332[M]
+。
1H-NMR(CD
3OD，500MHz)δ(ppm)：7.58(1H，s，H-1)，8.24(1H，s，H-4)，9.95(1H，s，H-6)，8.66(1H，d，J＝9.0Hz，H-9)，7.97(1H，d，J＝8.5Hz，H-10)，8.56(1H，d，J＝9.0Hz，H-11)，8.24(1H，d，J＝9.0Hz，H-12)，4.95(3H，s，H-13)，6.27(2H，s，H-14)，6.52(2H，s，H-15)。
13C-NMR(CD
3OD，125MHz)δ(ppm)：105.5(C-1)，148.5(C-2)，148.5(C-3)，131.0(C-4)，119.9(C-4a)，131.0(C-4b)，149.5(C-6)，131.9(C-6a)，146.0(C-7)，147.3(C-8)，118.5(C-9)，119.7(C-10)，126.8(C-10a)，125.3(C-10b)，117.1(C-11)，104.0(C-12)，109.2(C-12a)，52.1(C-13)，102.7(C-14)，104.8(C-15)。
Compound 1 physicochemical constant and spectral data and document (Yang Xiuwei chief editor.Alkaloid.Beijing: Chemical Industry Press, 2005, pp.211-212; Marek R et al.
1H and
13C NMR study of quaternary benzo[c] phenathridine alkaloids.Magnetic Resonance inChemistry, 1999,37:781-787.) sanguinarine (Sanguinarine) of report compares, basically identical, so definite compound 1 is a sanguinarine, molecular formula is C
20H
14NO
4, its structural formula is as follows:
Embodiment 6: the structure of compound 2 (Chelerythrine) is identified
Be prepared into compound 2 with embodiment 4 described methods, be yellow needle (EtOH).M.p.207 ℃ (containing a part ethanol); Bismuth potassium iodide shows Chinese red, points out this compound may be alkaloid, and its hydrochloride is yellow needle, m.p.202-204 ℃.
EI-MS：m/z?348[M]
+。
1H-NMR(CD
3OD，500MHz)δ(ppm)：4.17(3H，s，H-15)，4.31(3H，s，H-16)，5.03(3H，s，H-13)，6.30(2H，s，H-14)，7.62(1H，s，H-1)，8.27(1H，d，H-4)，8.23(1H，d，J＝4.25Hz，H-9)，8.29(1H，d，J＝8.6Hz，H-12)，8.74(2H，d，J＝9.25Hz，H-10，H-11)，10.00(1H，s，H-6)。
13C-NMR(DMSO-d
6，125MHz)δ(ppm)：105.7(C-1)，149.6(C-2)，149.4(C-3)，103.7(C-4)，120.5(C-4a)，131.3(C-4b)，150.7(C-6)，118.5(C-6a)，146.2(C-7)，150.4(C-8)，125.8(C-9)，119.6(C-10)，126.1(C-10a)，124.3(C-10b)，118.2(C-11)，128.9(C-12)，133.0(C-12a)，51.5(C-13)，102.9(C-14)，61.4(C-15)，56.2(C-16)。
Compound 2 physicochemical constants and spectral data and document (Yang Xiuwei chief editor.Alkaloid.Beijing: Chemical Industry Press, 2005, R et al. p.203:Marek
1H and
13C NMR study of quaternary benzo[c] phenathridine alkaloids.Magnetic Resonance inChemistry, 1999,37:781-787) Bao Dao Chelerythrine (Chelerythrine) is compared, basically identical, so determine that compound 2 is a Chelerythrine, molecular formula is C
21H
18NO
4, its structural formula is as follows:
Embodiment 7: the structure of compound 3 (Biflorine) is identified
Be prepared into compound 3 with embodiment 4 described methods, the prismatic crystallization (EtOH-CHCl of yellow monocline
3).M.p.208 ℃; Bismuth potassium iodide shows salmon pink, points out this compound may be alkaloid.
EI-MS：m/z?353[M]
+。
1H-NMR (DMSO-d
6, 300MHz) δ (ppm): 6.95 (1H, s, H-1), 6.67 (1H, s, H-4), 6.69 (2H, s, H-11, H-12), 6.00 and 5.96 (each 1H, s, H-16), 3.32 (2H, s, H-13), 3.29 (2H, s, H-8), 2.51 (4H, br, H-5 and H-6), 1.82 (3H, s, H-15).
13C-NMR(DMSO-d
6，75MHz)δ(ppm)：107.36(C-1)，145.84(C-2)，147.34(C-3)，110.44(C-4)，30.52(C-5)，57.49(C-6)，50.78(C-8)，118.34(C-8a)，145.39(C-9)，145.28(C-10)，106.31(C-11)，125.07(C-12)，129.65(C-12a)，46.12(C-13)，194.75(C-14)，136.10(C-14a)，41.17(C-15)，101.16(C-16)，100.78(C-17)。
Compound 3 physicochemical constants and spectral data and document (Yang Xiuwei chief editor.Alkaloid.Beijing: Chemical Industry Press, 2005, p.201.) Biflorine (Protopine) of report compares, basically identical, so definite compound 3 is a Biflorine, molecular formula is C
20H
19NO
5, its structural formula is as follows:
Embodiment 8: the structure of compound 4 (α-allocryptopine) is identified
Be prepared into compound 4 with embodiment 4 described methods, colourless prism-shaped crystallization (MeOH-CHCl
3), m.p.159.6-161.4 ℃; Prism-shaped crystallization (EtOAc), m.p.158-160 ℃.Bismuth potassium iodide shows salmon pink, points out this compound may be alkaloid.
EI-MS：m/z?369[M]
+。
1H-NMR(CDCl
3，500MHz)δ(ppm)：6.95(1H，s，H-1)，6.91(1H，d，J＝8.5Hz，H-12)，6.79(1H，d，J＝8.5Hz，H-11)，6.63(1H，s，H-4)，5.94(2H，s，H-16)，3.85(3H，s，H-17)，3.78(3H，s，H-18)，1.88(3H，s，H-15)。
13C-NMR(CDCl
3，125MHz)δ(ppm)：110.4(C-1)，146.0(C-2)，148.0(C-3)，109.2(C-4)，135.8(C-4a)，32.2(C-5)，57.5(C-6)，50.2(C-8)，128.4(C-8a)，151.5(C-9)，147.3(C-10)，110.6(C-11)，127.6(C-12)，129.4(C-12a)，46.1(C-13)，132.7(C-14a)，41.2(C-15)，101.2(C-16)，60.7(C-17)，55.6(C-18)。
Compound 3 physicochemical constants and spectral data and document (Yang Xiuwei chief editor.Alkaloid.Beijing: Chemical Industry Press, 2005, p.193-194.) (so definite compound 4 is α-allocryptopine, molecular formula is C for α-allocryptopine) compare, basically identical for the α-allocryptopine of report
21H
23NO
5, its structural formula is as follows:
Embodiment 9: the antibacterial activity of Macleaya cordata alkaloids
With embodiment 3 described methods, to 4 alkaloid monomers that are separated to from the macleaya cordata biology total alkali: sanguinarine (1), Chelerythrine (2), Biflorine (3), α-allocryptopine (4) carry out antibacterial activity and measure.
(1) the plant pathogenetic bacteria bacterial strain of determination of activity: see embodiment 2.
(2) medium: see embodiment 2.
(3) antibacterial activity is measured
The mensuration of minimum inhibitory concentration (MIC): adopt porous plate-mtt assay to measure the MIC value of compound, the results are shown in Table 3 to bacterium.Concrete steps are as follows.It is 10 that every hole adds concentration
6Cfu/mL for examination bacterium liquid 90 μ L, add then variable concentrations (20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 160 μ g/mL, 320 μ g/mL, 640 μ g/mL, 1280 μ g/mL) compound soup 10 μ L (40% ethanolic solution, v/v).And positive control be variable concentrations (12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 400 μ g/mL) streptomycin sulphate mother liquor 10 μ L (40% ethanolic solution, v/v).Negative control (being that final concentration is the solvent control of 4.0% ethanol), blank (water) and positive control (streptomycin sulphate) are established in experiment, and each is handled and repeats 3 times.The concentration of compound in bacterium liquid is 1/10th of mother liquid concentration like this.
With porous plate after 24h is cultivated in 28 ℃ of vibrations (secretly), the MTT solution 10 μ L (pH 7.2,0.2mol/LPBS solution) that add 5.0mg/mL in every hole, after continuing to cultivate 4h, centrifugal 20min under the 3000g, abandoning supernatant, add 100% dimethyl sulfoxide (DMSO) (DMSO), 100 μ L, vibration 30min is with cessation reaction and help its dissolving.Naked eyes can be observed the MIC value of judging compound.For further checking MIC value, from each hole of porous plate, draw 10 μ L culture fluids and be applied on the LB flat board, observation statistics flat-plate bacterial colony number gets final product after cultivating 24h under 28 ℃.
503nhibiting concentration (IC
50) mensuration: adopt porous plate-mtt assay to measure the IC of compound to bacterium
50, the results are shown in Table 3.Concrete steps are: it is 10 that every hole adds concentration
6The confession examination bacterium liquid 90 μ L of cfu/mL, soup 10 μ L (40% ethanolic solutions that add variable concentrations (20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 160 μ g/mL, 320 μ g/mL, 640 μ g/mL, 1280 μ g/mL) then, v/v), establish positive control, blank and solvent control simultaneously, behind 28 ℃ of dark cultivation 24h, the MTT solution 10 μ L that add 5mg/mL in every hole, continue to cultivate 4h, centrifugal 20min under the 3000g, abandon supernatant, add 100% dimethyl sulfoxide (DMSO), 100 μ L again, continue vibration 30min, with cessation reaction and help its dissolving.With bacteria suspension centrifugal 20min under 3000g of colour developing, supernatant forwards in another porous plate, measures its light absorption value (OD at the 590nm place with microplate reader
590nm), be calculated as follows medicine inhibiting rate (%):
According to inhibiting rate, look into the probit value table, can get inhibiting rate probit value (Y), (μ g/mL) is converted into concentration logarithm (X) with liquor strength, can obtain linear equation Y=aX+b, can obtain 503nhibiting concentration (IC according to linear equation
50).Data among the present invention are 3 times and repeat mean value.
(4) experimental result: the results are shown in Table 3, sanguinarine and Chelerythrine have tangible antibacterial activity.
The antibacterial activity of table 3 porous plate-MTT colorimetric method for determining Macleaya cordata alkaloids
Annotate: NA shows that at Cmax be 128 μ g/mL, does not detect antibacterial activity;-show the active regression equation of the inhibition of failing to obtain compound.
Embodiment 10: Macleaya cordata alkaloids is to the inhibitory action of rice blast fungus spore germination
With embodiment 3 described methods, to 4 alkaloid monomers that from the macleaya cordata biology total alkali, are separated to: sanguinarine (1), Chelerythrine (2), Biflorine (3), α-allocryptopine (4), carry out the inhibition determination of activity of rice blast fungus spore germination.
(1) medium: employing oat tomato medium (Oat-tomato agar medium, OTA).The step of oat tomato medium of preparation 1L is: with the tomato chopping of maturation, squeeze the juice with juice extractor, juice is with four layers of filtered through gauze, and it is stand-by to get the juice 150mL that has filtered.Simultaneously, take by weighing oats flakes 30g, add the 1000mL deionized water, boil 30min, note stirring while boiling.The two-layer filtered through gauze of well-done oats flakes.Collect filtrate, add off-the-shelf tomato juice 150mL and 20g agar then in filtrate, heating and melting is used filtered through gauze while hot, adds deionized water then and is settled to 1000mL.121 ℃ of moist heat sterilization 20min after the packing.This medium is used for the cultivation of rice blast fungus.
(2) for the plant pathogenic fungi of trying: rice blast fungus (Magnaporthe oryzae).
(3) rice blast fungus activation culture: the transfer needle a small amount of mycelia of picking from the test tube of preserving bacterial classification with sterilization, be inoculated on the oat tomato juice culture medium flat plate, seal with sealing film, cultivate down for 25 ℃.
(4) rice blast fungus successive transfer culture: the tomato juice of falling oat culture medium flat plate, the every ware pact of falling medium 40mL.Drip sterile water 1mL with liquid-transfering gun in rice blast fungus bacterium colony central authorities, clean the bacterium colony surface gently with the transfer needle after the sterilization then, note firmly wanting light, in order to avoid the galling media surface.
After treating that sterile water becomes muddiness, draw 100 μ L with liquid-transfering gun again and be added drop-wise to good oat tomato juice culture medium flat plate surface, be coated with rod with glass then and it be coated with evenly diffusing.After treating the media surface drying, build and be inverted 25 ℃ of cultivations down.
(5) the product spore is induced in mechanical stimulus: after cultivating 48h under the rice blast fungus room temperature of inoculation activation, drip the about 3mL of sterile water on its surface, clean mycelia gently with the cotton swab after the sterilization then, mycelia machinery is wiped broken, clean continuously twice, treat to get final product after the grey bacterium colony becomes black, the water on surface is outwelled, and back-off dries moisture on the blotting paper after the sterilization then, with the gauze after three layers of sterilization the ware mouth is sealed, placed drying to have to cultivate 48h under the condition of light to obtain the rice blast fungus spore.
(6) preparation compound mother liquor: take by weighing each 2mg of compound, add dissolve with ethanol, be made into the mother liquor of 2000 μ g/mL, be diluted to the mother liquid concentration gradient that contains 20% ethanol then: 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 20 μ g/mL, 15 μ g/mL, 10 μ g/mL and 5 μ g/mL.
Positive control adopts carbendazim (Aldrich), takes by weighing carbendazim 1.0mg in the Eppendorf pipe, and to wherein adding straight alcohol solution 1.0mL, vibration is dissolved, and obtains the carbendazim mother liquor of 1000 μ g/mL.Then the carbendazim mother liquor being mixed with series concentration is 30 μ g/mL, 20 μ g/mL, 16 μ g/mL, 10 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, and solvent is the solution of 20% ethanol.
(7) preparation spore suspension: produce dropping sterile water 3.0mL on the good flat board of spore in mechanical induction, clean the bacterium colony surface gently with aseptic cotton carrier then, wash spore, the spore washing lotion is installed in the centrifuge tube of 1.0mL, centrifugal 3 times (centrifugal force 8000g, each centrifugal 10min).Prepare spore suspension then, utilize blood cell counting plate to measure spore concentration, spore concentration is formulated as 2 * 10
6Individual/mL.
(8) carry out determination of activity: prepare clean culture dish, in each culture dish, put four layers of blotting paper then, sterilize.After the sterilization, in every ware, add sterile water 5.0mL, two aseptic toothpicks of parallel placement on blotting paper.Other is ready to concave slide, and the alcohol immersion 24h with 75% dries then.On each pothole, drip the rice blast fungus spore suspension (2 * 10 that 25 μ L prepare
6Individual/mL) compound solution with 25 μ L series concentration gradients (should fully shake on oscillator before dripping, to guarantee spore suspension and the abundant mixing of liquid medicine), obtaining alkaloidal detection final concentration like this is 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 10 μ g/mL, 7.5 μ g/mL, 5 μ g/mL and 2.5 μ g/mL.And the detection final concentration of positive control is 15 μ g/mL, 10 μ g/mL, 8 μ g/mL, 5 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL.Establish blank and solvent control (10% ethanol) simultaneously, each handles 3 repetitions.Then concave slide is placed on the toothpick in the culture dish, observes behind the cultivation 7h under the room temperature.Negative control (being the solvent contrast), blank (water) and positive control (carbendazim) are established in experiment, and each is handled and repeats 3 times.
(9) calculating of observed and recorded experimental result and spore germination rate
Spore germination situation on doubly following each concave slide of observation of microscope 10 * 10 (eyepiece * object lens), select 3 different visuals field, 100 above spores in each visual field, be total to several 300 spores, write down the spore count of sprouting with counter, repeat to be incorporated into one with 3 and calculate spore germination rate (%).
(10) calculating of data processing and 503nhibiting concentration
Result of the test adopts Microsoft Excel software to carry out data preparation, in analysis, to take the logarithm (X) for test agent concentration (μ g/mL), inhibiting rate is converted into biostatistics probit value (Y), try to achieve and suppress active regression equation (Y=aX+b), thus can be in the hope of 503nhibiting concentration (IC
50).Blank and solvent control are established in experiment, and (10% ethanol, v/v), each handles two repetitions.
(11) experimental result
Compound suppresses activity to the rice blast fungus spore germination and the results are shown in Table 4, is separated to from the macleaya cordata total alkali in 4 monosomic alkali, and the inhibition activity of sanguinarine is the strongest, secondly is Chelerythrine.
Table 4 compound suppresses active to the rice blast fungus spore germination
Embodiment 11: Macleaya cordata alkaloids is to the inhibition activity of hypha,hyphae growth
(1) potato dextrose agar (PDA) and potato glucose liquid nutrient medium (PDB): potato dextrose agar (the Potato dextrose agar of preparation 1L, PDA) step is: the potato 200g that will clean peeling, be cut into small pieces, add deionized water 1000mL, boil 30min, filter, get filtrate, add agar 20g and glucose 20g, after heating is melted agar, use filtered through gauze while hot, add deionized water then and be settled to 1000mL.121 ℃ of moist heat sterilization 20min.This medium is used for activation culture and the successive transfer culture of fungi.
PDB culture medium preparation step is same as the PDA medium except that not adding the agar.This medium is used for the inhibition determination of activity of Macleaya cordata alkaloids to the hypha,hyphae growth.
(2) for the plant pathogenic fungi of trying: botrytis cinerea (Botrytis cinerea), ring rot of apple bacterium (Botryosphaeriaberengeriana), gibberella saubinetii (Fusarium graminearum), tomato wilt bacterium (Fusarium oxysporum f.sp.lycopersici), cotton-wilt fusarium (Fusarium oxysporum f.sp vasinfectum), Rhizoctonia solani Kuhn (Rhizoctonia solani).
The above-mentioned plant pathogenic fungi bacterial strain long preservation that supplies examination before determination of activity, need be carried out activation culture (25 ℃, dark) 5-10 days to supplying to try fungi on the PDA flat board under 4 ℃, successive transfer culture is 1-2 time then, promptly can be used for determination of activity.
(3) experimental design and operation: adopt mycelial growth rate method in the liquid nutrient medium, measure the inhibitory action of compound to the hypha,hyphae growth.Compound is made into (comprising the positive control carbendazim) mother liquor (solvent is a methyl alcohol) of series concentration: 2.0mg/mL, 1.5mg/mL, 1.0mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL and 0.0625mg/mL, get a certain amount of (that is: 300 μ L compound mother liquors are in the 30mL fungus culture medium) then in fungus culture medium, the concentration of compound this moment in culture fluid is respectively: 20 μ g/mL, 15 μ g/mL, 10 μ g/mL, 5.0 μ g/mL, 2.5 μ g/mL, 1.25g/mL, 0.625 μ g/mL.Simultaneously, the fresh mycelia of picking (diameter is the bacterium cake of 5mm) (triangular flask of 1 bacterium cake/30mL medium/100mL volume) in the PDB culture fluid, the shaking table cultivation (25 ℃, dark, 150rpm) 5-6 days, adopt filtration method to collect mycelia, under 60 ℃, dry to constant weight.Negative control (being the solvent contrast), blank (water) and positive control (carbendazim) are established in experiment, and each is handled and repeats 3 times.Being calculated as follows of inhibiting rate:
(4) data are handled: result of the test adopts Microsoft Excel software to carry out data preparation, in analysis, for test agent concentration (μ g/mL) take the logarithm (X), inhibiting rate is converted into biostatistics probit value (Y), try to achieve and suppress active regression equation (Y=aX+b), thus can be in the hope of 503nhibiting concentration (IC
50).
(5) experimental result
Adopt mycelial growth rate inhibition method in the liquid nutrient medium to measure in the macleaya cordata inhibitory action (the results are shown in Table 5) of 4 kinds of main alkaloid to 6 plant species disease funguses, sanguinarine and Chelerythrine are better than the inhibition activity of Biflorine and α-allocryptopine to the inhibition activity for the examination fungi.
Table 5 Macleaya cordata alkaloids is to the inhibition activity of plant pathogenic fungi
Claims (7)
1, the application of Macleaya cordata alkaloids in preparation inhibition phytopathogen medicine.
2, a kind of by the described Macleaya cordata alkaloids of claim 1, it is characterized in that the macleaya cordata herb pulverizes after drying, adopt acid to put forward the heavy method of alkali and obtain total alkaloids.
3, a kind of by the described Macleaya cordata alkaloids of claim 1, it is characterized in that total alkaloid contents is 0.7% of a plant dry weight.
4, a kind of from by the method that is separated to sanguinarine the described macleaya cordata total alkaloid of claim 1, this compound has plant pathogenetic bacteria and disease fungus and suppresses active.
5, a kind of from by the method that is separated to Chelerythrine the described macleaya cordata total alkaloid of claim 1, this compound has plant pathogenetic bacteria and disease fungus and suppresses active.
6, a kind of from by the method that is separated to Biflorine the described macleaya cordata total alkaloid of claim 1, this compound has plant pathogenetic bacteria and disease fungus and suppresses active.
7, a kind of from by the method that is separated to α-allocryptopine the described macleaya cordata total alkaloid of claim 1, this compound has plant pathogenetic bacteria and disease fungus and suppresses active.
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