CN109090115B

CN109090115B - Application of kaempferol (4-O-methyl) glucoside compound in insect-resistant medicament

Info

Publication number: CN109090115B
Application number: CN201810893964.1A
Authority: CN
Inventors: 徐玉泉; 张礼文; 王辰; 顿宝庆
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2021-03-05
Anticipated expiration: 2038-08-08
Also published as: CN109090115A

Abstract

The invention discloses kaempferol (4-O-methyl) glucosides as anti-insect agents, which exhibit insecticidal activity in biological activity tests, absent from the aglycone, while having significantly enhanced water solubility compared to the aglycone; compared with unmethylated kaempferol glucoside, the compound has obviously enhanced stability and wide application value.

Description

Application of kaempferol (4-O-methyl) glucoside compound in insect-resistant medicament

Technical Field

The invention belongs to the field of pesticides, and particularly relates to an application of a kaempferol- (4-O-methyl) glucoside compound in a pesticide.

Background art:

kaempferol (kaempferol) is a common secondary metabolite of natural flavonoids found widely in the roots, leaves and fruits of a variety of plants. Kaempferol has activities of resisting oxidation, resisting inflammation, inhibiting tumor growth, protecting liver cells, inhibiting platelet aggregation and adhesion, and the like, so that the kaempferol is widely concerned by people. However, kaempferol is only slightly soluble in water, has poor metabolic stability in mammals, is easily affected by various factors from organisms and the outside, causes self-degradation, and has reduced activity, thus greatly hindering the application thereof. Glucosylation and methylation are common modification reactions in organisms, and can change the water solubility, stability and other physicochemical properties of a compound, thereby influencing the bioavailability of the compound and obtaining a more valuable active compound.

The glycosylation products of kaempferol known at present are glucoside, rhamnoside, rutin glucoside, rutinoside and the like. According to research, the antioxidant activity of the glycoside kaempferol is obviously weakened compared with that of aglycone thereof. kaempferol-3-O-rutinoside can obviously reduce the activity of choline lipase in blood and brain tissues, reduce excitatory amino acid transmitter in hippocampus, and improve the content of acetylcholine, monoamines and inhibitory amino acid neurotransmitter in brain tissues, so that the kaempferol-3-O-rutinoside can be used for preparing the medicine for preventing and treating senile dementia. kaempferol-3-O-beta-D-glucoside has certain inhibitory effect on Escherichia coli, Pseudomonas aeruginosa, Streptococcus lactis, Staphylococcus aureus and Bacillus subtilis, and the Minimum Inhibitory Concentration (MIC) is 250, 125 and 125 μ g/mL respectively. Some kaempferol glycosides have HDAC inhibitory activity, such as kaempferol-3-O-alpha-L-arabinopyranoside (54.46%), kaempferol-3-O-beta-D-xylopyranoside (45.86%), kaempferol-3-O-beta-D-glucopyranoside (43.01%), kaempferol-3-O-beta-D-acetylglucoside (39.61%), etc., which is more active than its aglycones. However, few reports have been made on the anti-insect activity of kaempferol derivatives. Kaempferitrin is kaempferol 3, 7-rhamnoside (kaempferol-3, 7-dirhamoside), and has anti-Pieris brasiliensis (Pieris brassicae) activity.

Disclosure of Invention

The glucosyltransferase heterologous expression vector PRS425m-IfUGT and the oxygen methyltransferase heterologous expression vector PXW06F-IfOMT are transferred into an auxotrophic yeast receptor BJ5464 for heterologous expression, and the kaempferol is fed and the kaempferol derivative is detected by HPLC-HRMS. By increasing the fermentation scale, sufficient extract is obtained and then is separated and purified, so that three kaempferol (4-O-methyl) glucoside derivatives, namely kaempferol 7-O-beta-D- (4-O-methyl) glucoside (compound 1), kaempferol 3-O-beta-D- (4-O-methyl) glucoside (compound 2) and kaempferol 4' -O-beta-D- (4-O-methyl) glucoside (compound 3), are obtained, wherein the structures of the

compounds

2 and 3 are not reported. The solubility experiment results show that: the solubility of the compound 1 in various common solvents such as water, methanol, ethanol and the like is obviously higher than that of kaempferol, and is equivalent to that of the kaempferol glucoside compound 4. In addition, the stability test results show that: the stability of the compound 1 in escherichia coli, saccharomyces cerevisiae and mammalian cells is obviously stronger than that of a corresponding kaempferol glucoside compound 4, which indicates that the compound 1 has good solubility and metabolic stability.

The chemical structure is shown as formula (1):

therefore, the invention aims to provide the application of the kaempferol (4-O-methyl) glucoside compound, namely the compound 1, which has good water solubility and strong stability, as the pesticide. Thus, an insecticide containing the above compound is also provided. Preferably, the killing of insects is killing of helicoverpa armigera or diamondback moth.

Experiments show that the invention has beneficial technical effects. Specifically, the lethal and growth-inhibitory effects of kaempferol, kaempferol glucosides 4-6 and kaempferol (4-O-methyl) glucosides 1-3 on terminal-4-year-old cotton bollworms were evaluated by the blood cavity injection method. The results show that: the compounds 1-3 show insecticidal activity of different degrees at the dosage of 20ng/g, wherein the activity of the compound 1 is strongest, and the lethality reaches 85 percent (p)<0.05); compounds 1-3 were able to inhibit the growth of Helicoverpa armigera at 2ng/g and resulted in an antifeedant effect with Compound 1 being the most effective (p)<0.05). In addition, the growth inhibitory effect of compounds 1-3 on mammalian cells was evaluated by in vitro cytotoxic activity assay. The results show that: the compounds 1-3 did not show significant growth Inhibition (IC) on four human tumor cells (A549, HepG2, HeLa and MCF-7) and one Vero cell₅₀Not less than 25.0 mu M), which indicates that the compounds 1-3 have good safety to human body. Therefore, the compounds 1-3 are expected to be developed into green, environment-friendly, efficient and low-toxicity insecticidal pesticides and have wide application prospects.

Drawings

FIG. 1 chromatogram of kaempferol after biotransformation to yield 3 sugar methylated derivatives.

FIG. 2 Mass spectra of kaempferol after biotransformation yielding 1 glycosylated derivative and 3 sugar methylated derivatives.

FIG. 3 HPLC-MS chromatograms of Compound 4 (top) and Compound 1 (bottom) after 48h yeast culture.

FIG. 4 HPLC-MS chromatograms of Compound 4 (top) and Compound 1 (bottom) after 48h incubation in E.coli.

Figure 5 hydrolysis ratio of compound 4 and compound 1 after a549 culture for 6,12,24 h.

FIG. 6 hydrolysis ratio of Compound 4 and Compound 1 after 6,12,24h in MCF-7.

FIG. 7 hydrolysis ratio of Compound 4 and Compound 1 after 6,12,24h in Vero culture.

FIG. 8 is a graph of the lethal effect of kaempferol and its glucoside derivatives on Heliothis armigera at a dose of 20ng/(g body weight).

FIG. 9 the effect of kaempferol and its glucoside derivatives on weight gain of Heliothis armigera at a dose of 2ng/(g body weight).

FIG. 10 Effect of kaempferol and its glucoside derivatives on Heliothis armigera at a dose of 20ng/(g body weight).

FIG. 11 lethal effect of kaempferol and its glucoside derivatives on 1 st age plutella xylostella.

FIG. 12 lethal effect of kaempferol and its glucoside derivatives on 3-year-old diamond back moth.

FIG. 13 the weight effect of kaempferol and its glucosides on 3-year-old plutella xylostella.

Detailed Description

The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.

Example 1 use of glycosyltransferase recombinant vector PRS425m-IfUGT and O-methyltransferase recombinant vector PXW06F-IfOMT to achieve glycosylmethylation modification of Kaempferol

1.1 instruments and materials

Strains and vectors used in the experiments: isaria fumosorosea ACCC37775, microzyme S.cerevisiae BJ5464-NpgA, yeast expression vector PRS425m-IfUGT and PXW06F-IfOMT are all from the biotechnology institute of Chinese academy of agricultural sciences, and are stored by the inventor in a laboratory. The reagents used in the experiment are all domestic analytical pure products.Culture medium: the E.coli medium was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). SC-^-Leu deficient medium (1% glucose, 6.7% Difco)^TMYeast Nitrogen Base w/o Amino Acids, -Leu/-Trp DO Supplement). YPD medium (1% yeast extract, 2% Peptone, 2% glucose). YPD low-sugar medium (1% yeast extract, 2% Peptone, 1% glucose.) if solid medium is prepared, 2% agar powder is added.

The HPLC is Agilent 1290Infinity II, column RRHD Eclipse Plus C18,4.6X100 mm.

Other experimental procedures not specifically indicated for the conditions in the examples were carried out according to conventional methods, molecular cloning being carried out according to the conditions described in the Laboratory manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the conditions recommended by the manufacturer.

1.2 Experimental methods

Adopting a two-step fermentation technology, firstly inoculating a proper amount of yeast transformant thalli to corresponding 25-mL^-Leu/^-Culturing in Trp liquid-deficient culture medium at 30 deg.C for about 16h at 200 r.min-1, adding 25ml YPD low-sugar culture medium, and simultaneously adding 5mg kaempferol pure product for further culturing for 48 h; extracting the fermentation product with ethyl acetate at a ratio of ethyl acetate to fermentation broth of 1:1, namely extracting the fermentation product with 50ml of ethyl acetate; the ethyl acetate dry extract was recovered by rotary evaporator and the extract was redissolved in 1ml of methanol.

The fermentation product obtained above is subjected to high speed centrifugation and then detected by High Performance Liquid Chromatography (HPLC) and high resolution mass spectrometry (HRMS/MS). The HPLC detection conditions were as follows: performing gradient elution by using acetonitrile-water as a mobile phase, wherein the gradient elution condition comprises the following steps: 0-5 min, acetonitrile from 10% → 50%; 5-10 min, acetonitrile from 50% → 95%; 10-12 min, and the acetonitrile is 95%; 12-12.5 min, acetonitrile 95% → 10%; 12.5-13 min, and acetonitrile is 10%. The flow rate was 0.5 mL/min-1, and the detection wavelength was 300 nm. HRMS/MS detection conditions were as follows: negative ion mode, collision energy 25V.

1.3 Experimental results and analysis

HPLC analysis result shows: the feeding raw material kaempferol is almost completely converted to generate four derivatives. In the four peaks after conversion, the molecular weight of the compound at peak 1 was 162amu more than that of kaempferol, which is presumed to be a glucosylation product, and the molecular weight of the compound at peaks 2 to 4 was 176amu more than that of kaempferol, which is presumed to be a methyl glucosylation product, as determined by HRMS analysis. Through HRMS/MS analysis, the secondary mass spectrum base peaks of the compounds with the peaks 2-4 are all kaempferol [ M + H ] when the collision energy is 15eV]⁺Molecular weight of (1), no [ M + H ] was found after methylation of kaempferol]⁺The molecular weight of (a) indicates that the glycosyl group and the methyl group are simultaneously removed, so that the methyl modification is most likely on the hydroxyl group of the glycosyl group rather than on the hydroxyl group of kaempferol.

The chromatogram and mass spectrum of the fermentation product of the recombinant strain are shown in figures 1 and 2.

EXAMPLE 2 isolation and Structure characterization of Compounds 1-3

2.1 instruments and materials

The solvents chloroform, methanol and ethyl acetate used in the experiment are produced by Beijing chemical reagent factory and are all analytically pure. Chromatographically pure acetonitrile is produced by Fisher Chemical company. The column chromatography silica gel is produced by Qingdao ocean factory (200-300 mesh). The thin-layer silica gel chromatography plate is produced by a cigarette bench yellow affair silica gel development and test factory. BUCHI R-300 rotary evaporator, BUCHI I-300 touch control central control unit, BUCHI V-300 PTFE film vacuum pump, and BUCHI B-300 electric heating constant temperature water bath, all manufactured by BUCHI corporation of Switzerland. The low-temperature cooling liquid circulating pump is produced by Shanghai Xin laboratory instruments and technologies, Inc. The semi-preparative HPLC is Agilent 1260Infinity II, and the C18 column is Agilent ZORBAXRX-C18(5 μm,9.4 × 250 mm). The HRESI-MS instrument is an Agilent MS 6530Q-TOF type mass spectrometer, and is directly subjected to sample injection and measurement. The NMR spectrum is measured by Bruker AVIII 400M superconducting NMR spectrometer, and is calibrated by the peak of the residual solvent of deuterated dimethyl sulfoxide produced by Cambridge Company (CIL) in America.

2.2 Experimental methods

10L of fermentation liquor is extracted for three times by using equal volume of ethyl acetate, and the organic phase is decompressed and concentrated to obtain a total extract (1.9 g). The total extract is subjected to normal phase silica gel column chromatography,eluting with chloroform-methanol solutions with the volume ratios of 100:0, 98:2, 95:5 and 90:10 in sequence. Collecting 95:5 eluate, performing semi-preparative high performance liquid chromatography, and separating with acetonitrile-water solution at volume ratio of 23:77 as mobile phase to obtain compound 1(20mg, t)_R12.2min), compound 2(32mg, t)_R10.1min) and compound 3(50mg, t)_R＝14.2min)。

2.3 Experimental results and analysis

The structure of the compound 1-3 is identified as shown in the formula (1) by analyzing a high-resolution mass spectrum and one-dimensional and two-dimensional nuclear magnetic resonance spectrums. Wherein, the compound 1 is kaempferol 7-O-beta-D- (4-O-methyl) glucoside which is reported, the

compounds

2 and 3 are kaempferol 3-O-beta-D- (4-O-methyl) glucoside and kaempferol 4' -O-beta-D- (4-O-methyl) glucoside respectively, and the structure is not reported. The structural identification data for compounds 1-3 are as follows:

compound 1: high resolution mass spectrometry M/z 461.1009[ M-H [ ]]^-Calcd.461.1089 with molecular formula C₂₂H₂₂O₁₁；¹H NMR (400MHz) and¹³the C NMR (400MHz) data are shown in Table 1.

Compound 2: high resolution mass spectrometry M/z 461.1114[ M-H [ ]]^-Calcd.461.1089 with molecular formula C₂₂H₂₂O₁₁；¹H NMR (400MHz) and¹³the C NMR (400MHz) data are shown in Table 1.

Compound 3: high resolution mass spectrometry M/z 461.1106[ M-H [ ]]^-Calcd.461.1089 with molecular formula C₂₂H₂₂O₁₁；¹H NMR (400MHz) and¹³the C NMR (400MHz) data are shown in Table 1.

TABLE 1 NMR data for Compounds 1-3 (deuterated dimethyl sulfoxide as test solvent)

Example 3 solubility testing of Kaempferol and its glycoside derivatives

3.1 purpose of the experiment

The effect of sugar methylation on the solubility of kaempferol in different solvents was determined.

3.2 Experimental methods

Respectively weighing kaempferol and 1mg of compound, placing in 1ml of different solvents at 25 +/-2 ℃, and strongly shaking for 30 seconds every 5 minutes; dissolution was observed within 30 minutes, as solute particles or droplets were not visible, and was considered complete dissolution. The solvent is selected from distilled water, methanol, ethanol, etc.

3.3 Experimental results and analysis

The solubility of kaempferol and its glycosylated derivatives (compound 4), the sugar methylated derivatives (compound 1) in different solvents are shown in table 4.

TABLE 4 solubility of Kaempferol with Compound 1 in different solvents

Solvent(s)	Kaempferol	Compound	4	Compound 1
					Distilled water	-+	-+	-+
Methanol	++	+++	+++
				Ethanol	++	+++	+++
DMF	++	++	++
				1％DMSO	++	+++	+++
0.1％Tween 20	+	++	++
				0.1％Triton X100	+	++	++

Note: insoluble-; slightly soluble- +; partial dissolution +; mostly dissolved + +; completely dissolved + + ++

As shown in table 4, both glycosylation and sugar methylation significantly improved the solubility of kaempferol compared to before modification. However, according to HPLC liquid chromatogram, the water solubility of the product after sugar methylation is slightly lower than that of the glycosylated product.

Example 4 comparison of the stability of Compound 1 and Compound 4 in E.coli and Saccharomyces cerevisiae cells

4.1 purpose of the experiment

The hydrolytic stability of the sugar methylation modification product compound 1-3 and the glycoside compound 4-6 before methylation modification were compared under the same conditions under the action of yeast and the own enzyme system of Escherichia coli.

4.2 Experimental methods

To 50mL of yeast BJ5464 cultured in YPD, 2mg of the sugar methylation modification product compound 1 and the glycoside compound 4 before methylation modification were added, respectively, and the samples were dissolved in 100. mu.L of chromatographic methanol with the blank control being isometric chromatographic methanol. Respectively culturing at 30 deg.C and 200r min-1 for 48 hr, extracting with ethyl acetate at a ratio of ethyl acetate to fermentation liquid of 1:1, namely extracting with 50ml ethyl acetate; the ethyl acetate dry extract was recovered by rotary evaporator and the extract was redissolved in 1ml of methanol.

2mg of the sugar methylation modification product compound 1 and the glycoside compound 4 before methylation modification were added to 50mL of Escherichia coli DH5-alpha cultured in LB medium, and the samples were dissolved in 100. mu.L of chromatographic methanol with the blank control being the same volume of chromatographic methanol. Respectively culturing at 30 deg.C and 200r min-1 for 48 hr, extracting with ethyl acetate at a ratio of ethyl acetate to fermentation liquid of 1:1, namely extracting with 50ml ethyl acetate; the ethyl acetate dry extract was recovered by rotary evaporator and the extract was redissolved in 1ml of methanol.

And (3) centrifuging the obtained fermentation product at a high speed, and detecting by using a high performance liquid chromatography.

The HPLC detection conditions were as follows: the chromatographic column Kromasil 100-5-C18 adopts acetonitrile-H2O as a mobile phase to carry out gradient elution, and the gradient elution conditions are as follows: gradient elution conditions: 0-5 min, 10% of acetonitrile; 5-15 min, acetonitrile from 10% → 95%; 15-25 min, and the acetonitrile is 95%; 25-28 min, acetonitrile from 95% → 10%; 28-31 min, and acetonitrile is 10%. The flow rate was 0.8mL min-1, and the detection wavelength was 300 nm.

4.3 Experimental results and analysis

The chromatogram of the extraction products of compounds 1-3 and compounds 4-6 at the same incubation time was analyzed. The chromatogram of the extraction products of compound 1 and compound 4 after 48h yeast culture is shown in FIG. 3. The chromatogram of the extraction products of compound 1 and compound 4 after 48h of E.coli culture is shown in FIG. 4.

The chromatogram shows that the glycosylated derivatives are obviously hydrolyzed after 48 hours; however, the sugar methylation product is relatively stable in the culture process and has no obvious hydrolysis phenomenon, which shows that methylation on glucose can increase the stability of the glycosylation product and prevent the compound from being degraded by enzyme.

Example 5 comparison of the stability of Compound 1 and Compound 4 in mammalian cells

5.1 instruments and materials

Two human-derived tumor cell strains, namely human lung cancer A549 cells, human breast cancer MCF-7 cells and African green monkey kidney epidermis Vero cells, are purchased from Kunming animal research institute of Chinese academy of sciences; fetal bovine serum FBS, RPMI1640 medium, DMEM medium were purchased from Gibco, USA. The HPLC is Agilent 1290Infinity II and the column is RRHD Eclipse Plus C18,4.6X100 mm.

5.2 Experimental methods

Human lung cancer A549 cells, human breast cancer MCF-7 cells and African green monkey kidney epidermis Vero cells in the logarithmic growth phase are taken and added into a 96-well plate, and each well contains about 5000 cells. Then kaempferol glucoside compound 4 and kaempferol (4-O-methyl) glucoside compound 1 are respectively added, each well finally contains 100 mu L of culture solution, and the final concentration of each sample is 25 mu M. Each group was set with 4 parallel wells, and after incubation in a carbon dioxide incubator at 37 ℃ for 6,12 and 24 hours, respectively, the 96-well plate was removed and freeze-dried. Then adding 100 μ L of methanol into each well, performing ultrasonic extraction for 15min, repeating twice, mixing extractive solutions, centrifuging at 14000 r/min for 4min, and collecting supernatant.

And detecting the obtained supernatant by using a high performance liquid chromatography, and integrating peak areas of chromatographic peaks to calculate the hydrolysis ratio of the corresponding compound. Performing gradient elution by using acetonitrile-water as a mobile phase, wherein the gradient elution conditions are as follows: 0-5 min, acetonitrile from 10% → 50%; 5-10 min, acetonitrile from 50% → 95%; 10-12 min, and the acetonitrile is 95%; 12-12.5 min, acetonitrile 95% → 10%; 12.5-13 min, and acetonitrile is 10%. The flow rate was 0.5 mL/min-1, and the detection wavelength was 300 nm.

5.3 Experimental results and analysis:

the percent hydrolysis of compound 1 and compound 4 was compared in different cell lines under the same culture conditions. The results are shown in Table 3.

It can be seen from table 3 that the glycosylated derivatives all undergo significant hydrolysis after 24 h; however, the sugar methylation product is relatively stable in the culture process and does not have obvious hydrolysis phenomenon, which shows that methylation can increase the stability of the glucoside derivatives and prevent the compounds from being degraded by enzymes of mammalian cells.

TABLE 3 percent hydrolysis of Compound 1 and Compound 4 under mammalian cell culture conditions

See figure 5 for the hydrolysis ratio of compound 4 and compound 1 after a549 culture for 6,12,24 h.

The hydrolysis ratios of compound 4 and compound 1 after 6,12,24h in MCF-7 are shown in FIG. 6.

See figure 7 for the hydrolysis ratio of compound 4 and compound 1 after 6,12,24h in Vero culture.

Example 6 cytotoxic Activity of Kaempferol and its glycosides

6.1 instruments and materials

The microplate reader is a Genois microplate reader (Tecan GENios, Swizerland); four human-derived tumor cell lines, namely a human lung cancer A549 cell, a human hepatoma HepG2 cell, a human cervical cancer HeLa cell, a human breast cancer MCF-7 cell and an African green monkey kidney epidermis Vero cell, are purchased from Kunming animal institute of Chinese academy of sciences; fetal bovine serum FBS, RPMI1640 medium, DMEM medium purchased from Gibco, USA; 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide salt (MTT) was purchased from Amresco, USA.

6.2 Experimental methods

Human lung cancer A549 cells, human liver cancer HepG2 cells, human cervical cancer HeLa cells, human breast cancer MCF-7 cells and African green monkey kidney epidermis Vero cells in logarithmic growth phase are taken and added into a 96-well plate, and each well contains about 5000 cells. Adding kaempferol and compound 1-6 with different concentrations, using adriamycin as positive control, using cell hole without sample as control group, setting 4 parallel holes in each group, culturing at 37 deg.C for 72 hr in carbon dioxide incubator, adding 20 μ L MTT (5mg/mL) solution 4 hr before experiment termination, culturing for 4 hr, discardingAdding 150 mu L DMSO into the culture solution, dissolving the crystal, and detecting the OD value of each well at the wavelength of 570nm on an enzyme-linked detector. The growth inhibition rate was determined according to the following formula, and the median Inhibitory Concentration (IC) was determined by SPSS (17.0) software₅₀Value). The results are shown in Table 2, in which IC₅₀Expressed as Mean ± s.e.m.

6.2 Experimental results and analysis

As can be seen from the results in Table 2, compounds 1-3 showed no significant cytotoxicity against mammalian cells.

TABLE 2 tumor cell growth inhibitory Activity (IC) of Kaempferol and its glycoside derivatives₅₀,μM)

Example 7 Kaempferol and its glycoside Compounds ability to kill Helicoverpa armigera

7.1 purpose of the experiment

The influence of kaempferol and 6 glucoside compounds thereof on the growth of cotton bollworms is tested and compared by a blood cavity injection method, and the insecticidal activity of the compounds 1-6 is shown.

7.2 Experimental methods

The six compounds were diluted to a stock of 100. mu.g/mL with methanol before treatment with water to a solution of 10. mu.g/mL (20ng/(g body weight)) and 1. mu.g/mL (2ng/(g body weight)). The late-stage 4-year-old cotton bollworms were selected for injection treatment, 2. mu.L per bollworm, and 10% methanol was used as a control. Each treatment was injected with 10 beetles, 3 replicates. After 5d, the results were counted and sampled.

7.3 Experimental results and analysis

At the dosage of 20ng/(g body weight), kaempferol and glucosylated derivatives thereof (compounds 4-6) do not show significant insecticidal activity, and the cotton bollworm mortality rate is equivalent to that of a blank control; the compounds 1-3 after methylation modification show insecticidal effects with different degrees, and particularly the compound 1 has a remarkable lethal effect (p is less than 0.05).

At the dose of 2ng/(g body weight), kaempferol and the glucoside compounds (1-6) thereof do not show obvious lethal effect, but the compounds 1-6 cause the growth retardation of insects to different degrees, and the compounds 1-3 cause the insects to show antifeedant effect, particularly the compound 1 effect is the most obvious (p <0.05).

The lethal effect of kaempferol and its glucoside derivatives on Heliothis armigera at a dose of 20ng/(g body weight) is shown in FIG. 8.

The effect of kaempferol and its glucoside derivatives on weight gain of Heliothis armigera at a dose of 2ng/(g body weight) is shown in FIG. 9.

A picture of the effect of kaempferol and its glucoside derivatives on Heliothis armigera at a dose of 20ng/(g body weight) is shown in FIG. 10.

Example 8 Kaempferol and its glycosides test the insecticidal Capacity of diamondback moth

8.1 purpose of the experiment

The influence of kaempferol and 6 glucoside compounds thereof on the growth of the plutella xylostella is tested and compared by coating leaves with the compound and feeding the compound, and the insecticidal activity of the compounds 1-6 is shown.

8.2 Experimental methods

Kaempferol and 6 derivatives thereof were dissolved in DMSO to prepare a 10mM solution. Adding 10 μ l of the solution into 990 μ l of 1mL of 0.1% Triton X-100 aqueous solution to obtain a final concentration of 0.1mM, uniformly coating on cabbage leaves, air drying, placing into a culture dish filled with filter paper, and inoculating into larvae of diamondback moth of 1 or 3 instar. The mortality of the test insects was checked after 72 h. 15 insects were tested per treatment, 3 replicates.

8.3 Experimental results and analysis

Except for the compound 5, the insecticidal activity of other 5 glycosylated derivatives on the 1 st plutella xylostella is remarkably higher than that of a control group (kaempferol) (p <0.05).

For 3-year-old plutella xylostella, the mortality rate of the plutella xylostella treated by the compounds 1 to 6 is increased, but the difference is not obvious compared with a control; compound 1 and compound 2 resulted in a significant reduction in plutella xylostella body weight (p <0.05).

The lethal effect of kaempferol and its glucoside derivatives on 1 st diamond back moth is shown in FIG. 11.

The lethal effect of kaempferol and its glucoside derivatives on 3-year-old plutella xylostella is shown in FIG. 12.

The effect of kaempferol and its glucoside derivatives on the body weight of 3-year old plutella xylostella is shown in fig. 13.

Claims

1. The application of the kaempferol (4-O-methyl) glucoside compound as an anti-insect medicament is characterized in that the kaempferol (4-O-methyl) glucoside compound is as follows: kaempferol 7-O-beta-D- (4-O-methyl) glucoside; wherein the insect-resistant medicament is used for controlling cotton bollworms or diamondback moths.

2. An application of the insecticide containing kaempferol 7-O-beta-D- (4-O-methyl) glucoside in preventing and treating bollworm and plutella xylostella is disclosed.