Biopesticide composition as well as preparation method and application thereof

Abstract

The invention discloses a biopesticide composition as well as a preparation method and application thereof, and the biopesticide composition comprises the following components in parts by weight: 10-30 parts of xyveratrane diterpenes; and 10 to 50 parts of a flavonoid compound. According to the present invention, the resveratrol diterpenes extracted from the pieris formosa or the vaccinium bracteatum thunb and the flavonoid compounds are adopted as the active components, are mixed according to the specific ratio, and are compounded with the specific auxiliary components so as to provide a certain synergistic effect, such that the pesticide composition has significant insecticidal and antibacterial effects, can be directly sprayed on crops, and further has characteristics of environmental protection and environmental protection. And plant diseases caused by pests and pathogenic bacteria can be effectively prevented and treated.

Description

Technical Field

[0001] The present invention relates to the field of biological pesticides, and in particular to a biological pesticide composition, a preparation method and application thereof. Background Art

[0002] In traditional crop pest management, chemical pesticides are widely used due to their rapid and efficient control effects. However, long-term reliance on chemical pesticides has led to many problems, including increased pest resistance, environmental pollution, disruption of ecological balance, and potential threats to human health. With the increasing awareness of food safety and environmental protection, the development of new and environmentally friendly biopesticides has become an important topic in the field of agricultural science and technology.

[0003] Biopesticides are derived from nature, including living organisms or their metabolites, such as microorganisms and plant derivatives. Compared with traditional chemical pesticides, biopesticides have low toxicity, less residue, are less likely to cause bioaccumulation, and have little impact on the ecological environment. Biopesticides are highly selective and only effective against specific pests and diseases, and safe for other organisms. They are considered an effective alternative to chemical pesticides because of their environmental friendliness, low toxicity, and low residue.

[0004] However, the current application of biopesticides is still subject to great restrictions. First, because the control of biopesticides is a biological process, they usually cannot take effect immediately like chemical pesticides. The effect is relatively slow, so farmers may not easily accept it. Second, biopesticides are not easy to preserve, and the efficacy of the drugs may be unstable. The shelf life is limited, and the active ingredients in the pesticides are very easy to decompose, which increases the difficulty and uncertainty of their use. Third, the extraction efficiency of the active ingredients of biopesticides is low and the cost is high. Summary of the invention

[0005] The purpose of the present invention is to provide a biological pesticide composition and a preparation method and application thereof. The composition has the dual effects of insecticide and antibacterial, and can be used as a green prevention and control of crop diseases and insect pests.

[0006] In order to solve the problems existing in the prior art, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a biopesticide composition comprising the following components in parts by weight:

[0008] 10-30 parts of veratrane-type diterpenes; 10-50 parts of flavonoid compounds.

[0009] Preferably, the following components are included in parts by weight:

[0010] 20 parts of veratranolane-type diterpenes; 40 parts of flavonoids.

[0011] Furthermore, the veratrol-type diterpenes and flavonoid compounds are extracts of the beautiful lilacinus or the orchid tree.

[0012] Furthermore, auxiliary ingredients are included, including: synergist, surfactant, dispersant, stabilizer, preservative.

[0013] Further, the synergist is peppermint oil, eucalyptus oil, saponin or its derivatives, preferably peppermint oil;

[0014] The surfactant is selected from nonionic, cationic or anionic surfactants, preferably polyvinyl pyrrolidone;

[0015] The dispersant is selected from polyvinyl alcohol or cellulose derivatives; preferably xanthan gum;

[0016] The stabilizer is sodium hydroxymethyl cellulose. Sodium carboxymethyl cellulose as a stabilizer can increase the viscosity of the preparation and prevent the components from settling.

[0017] The preservative is a paraben.

[0018] Furthermore, the auxiliary ingredients include, by weight: 2-20 parts of synergist, 0.5-2 parts of surfactant, 0.5-2 parts of dispersant, 0.1-0.5 parts of stabilizer, 0.05-0.2 parts of preservative. Preferably, 2 parts of synergist, 0.8 parts of surfactant, 1.5 parts of dispersant, 0.3 parts of stabilizer, 0.1 parts of preservative.

[0019] Furthermore, a pH adjuster is used to adjust the pH value of the composition to 5-9, preferably 7.

[0020] Furthermore, the pH regulator is 1% hydrochloric acid or 1% sodium hydroxide solution.

[0021] In a second aspect, the present invention provides a method for preparing the biopesticide composition described in the first aspect, comprising the following steps:

[0022] S1. Extracting active ingredients, comprising the following steps:

[0023] ① Take fresh / dried leaves of the beautiful dwarf dwarf dwarf dwarf or southern candle, crush them to 40-80 mesh; use 60% (v / v) ethanol solution as the extraction solvent, the mass ratio of the raw material to the ethanol solution is 1:3-1:10, and extract in an ultrasonic extractor, the power is 300-500W, the temperature is 35-45°C, and the time is 30-70 minutes; repeat the extraction process 3 times, combine the extracts, and concentrate them to 1 / 5 of the original volume by a rotary evaporator;

[0024] ②Purification of crude extract:

[0025] The concentrated extract is purified by medium pressure liquid chromatography to collect components with biological activity;

[0026] ③ Using high performance liquid chromatography to monitor and accurately collect fractions with specific absorption wavelengths, the biologically active components were further accurately separated to obtain veratrol-type diterpenes and flavonoid compounds respectively;

[0027] S2. Preparation of active ingredient mixture:

[0028] Mixing the extracted veratrol-type diterpenes and flavonoid compounds according to the weight proportions to obtain an active ingredient mixture;

[0029] S3. Mix the active ingredient mixture, synergist, surfactant, dispersant, stabilizer and preservative in the above-mentioned proportions by weight under sterile conditions and adjust the pH value to 5-9.

[0030] In a third aspect, the present invention provides the use of the biopesticide composition described in the first aspect in drugs for preventing and controlling plant diseases.

[0031] Furthermore, the plant diseases include rice blast, wheat fusarium wilt, cotton wilt, aphids and whiteflies of vegetable crops.

[0032] The advantages and beneficial effects of the present invention are as follows: the present invention uses the veratrol type diterpenes and flavonoid compounds extracted from the beautiful dwarf ...

[0033] The present invention adopts a method combining ultrasound-assisted extraction and ethanol reflux extraction, and finely optimizes the extraction conditions, including solvent concentration, extraction temperature, extraction time and solid-liquid ratio, to ensure efficient and high-purity acquisition of active ingredients. The extraction efficiency and purity of active ingredients in Ericaceae plants are significantly improved, and production costs are reduced.

[0034] The present invention takes the extracted active ingredients as the core, supplemented with appropriate amounts of synergists, surfactants, dispersants and stabilizers. The above ingredients have a synergistic effect. Combining the ratio of active ingredients with synergists, surfactants, dispersants and stabilizers, the efficacy and bioavailability are significantly improved, while the toxicity to non-target organisms and the environment is reduced.

[0035] The aseptic preparation process of the preparation includes mixing, dissolving, dispersing, homogenization, and key pH adjustment and stability adjustment steps, which ensure the quality and safety of the preparation. The present invention adopts a sodium hydroxide solution with a mass volume concentration of 1% as a pH adjuster, which can not only effectively adjust the pH value of the preparation, but also has the insecticidal and bacteriostatic effect of the synergistic component. The present invention adopts high shear mixing technology and homogenization treatment to ensure the uniformity and stability of the preparation; the pesticide composition of the present invention is efficient, environmentally friendly, safe to non-target organisms, and easily biodegradable. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a graph showing the antibacterial effect results of each test group; wherein: a is Bacillus subtilis; b is Staphylococcus aureus; c is Escherichia coli, 1 is a blank control (sterile water), 2 is the biological pesticide composition prepared in Example 1, 3 is commercially available (penicillin), and 4 is the biological pesticide composition prepared in Comparative Example 3. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments in the present application belong to the scope of protection of this application.

[0038] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0039] Example 1

[0040] This embodiment provides a method for preparing a biopesticide composition, comprising the following steps:

[0041] S1. Extracting active ingredients, comprising the following steps:

[0042] ① Take 1000 grams of naturally dried Pieris formosa leaves and crush them to 60 mesh using a grinder. Use a 60% (v / v) ethanol solution as the extraction solvent. The mass ratio of the crushed Pieris formosa leaves to the ethanol solution is 1:5. Extract in an ultrasonic extraction tank with a power of 400W, a temperature of 40°C, and a time of 1 hour. Keep the extraction tank sealed to reduce solvent volatilization. Repeat the extraction process 3 times, repeat the ultrasonic extraction of the residue twice, each time for 30 minutes, and combine all the extracts. After the extraction, filter the extract with filter paper, collect the filtrate, and concentrate it to 1 / 5 of the original volume at 40°C using a rotary evaporator to increase the concentration of active ingredients.

[0043] ②Purification of crude extract:

[0044] The concentrated extract is separated by silica gel column chromatography, and a normal hexane-ethyl acetate gradient elution system is used in the gradient elution process of medium pressure liquid chromatography (MPLC). First, a mixture of normal hexane and ethyl acetate is set as the mobile phase, and the initial ratio is a mass ratio of normal hexane and ethyl acetate of 70:30. After the elution begins, the proportion of ethyl acetate is gradually increased according to a preset gradient program, for example, 5% is increased every 10 minutes until the mobile phase becomes 100% ethyl acetate. In this process, the flow rate is maintained at 1-5 ml / min to ensure the uniformity of elution. The fractions are collected automatically or manually, and 50 ml of fractions are collected every 5 minutes. Thin layer chromatography (TLC) is used to monitor the active ingredients in the fractions. According to the TCL results, the fractions containing similar components are merged, and the merged fractions are subjected to secondary purification to collect components with biological activity.

[0045] ③Use high performance liquid chromatography (HPLC) to monitor and accurately collect fractions with specific absorption wavelengths, and then use freeze-drying technology to obtain veratrol-type diterpenes and flavonoid compound quercetin:

[0046] A semi-preparative high performance liquid chromatography (HPLC) system was used, equipped with an ODS-C18 column (5 micron particle size, 250 mm length, 10 mm inner diameter), and separated at a flow rate of 2 ml / min. The mobile phase of methanol and water was used, and the initial ratio was set to a mass ratio of methanol to water of 60:40. The methanol ratio was increased to 90% in a linear gradient within 40 minutes, maintained for 5 minutes, and then restored to the initial ratio within 5 minutes to achieve the regeneration of the chromatographic column. The components extracted from the euphorbia cerifera were developed with 0.5% vanillin sulfuric acid. According to the characteristics of the initial color development of purple-red and then gradually turning blue, and the color turning green after standing for 12 hours, it was directional judged that it was a diterpenoid component; the absorbance at 280 nanometers was monitored by an ultraviolet detector, and the retention time of quercetin was 6.35-6.92min compared with the known literature chromatographic conditions and peak time. The fraction of the quercetin target compound was collected according to the known retention time. Subsequently, the fractions were concentrated to a small volume using a rotary evaporator at 40°C, and two different solid powdered compounds were obtained by freeze drying. The purity of the two components was tested by HPLC-UV to ensure that the purity was above 95%.

[0047] The two compounds isolated from the extract were used 1 H-NMR, 13 The structure was determined by spectral methods such as C-NMR and ESI-MS combined with references. The structure of the obtained limonene-type diterpene compound 1 is shown in Formula 1;

[0048] Formula 1:

[0049]

[0050] The structure of the obtained flavonoid compound 2 is shown in Formula 2:

[0051] Formula 2:

[0052]

[0053] Morinane-type diterpene compound 1: white amorphous powder. [α] 20 D -7.20(c0.1, MeOH), ESI-MS m / z:448[M+Na] + ; 1 H-NMR (600MHz, methanol-d4)δ H :5.45(1H,s), 1.36(3H,s), 1.34(3H,s), 1.19(3H,s), 0.96(3H,s); 13 C-NMR (150MHz, methanol-d4)δ C : 175.3(COCH2CH3), 85.3(C-5), 83.8(CH-3), 83.1(CH-14), 79.0(C-16), 78.9(C-10 ), 73.6(CH-6), 60.9(CH2-15), 56.9(CH-9), 54.9(CH-13), 52.5(C-4), 51.7(C-8), 5 1.3 (CH-1), 43.3 (CH2-7), 35.7 (CH2-2), 28.9 (COCH2CH3), 27.7 (CH3-20), 28.0 (CH2-12), 23.8 (CH3-17), 23.4 (CH3-19), 22.6 (CH2-11), 19.1 (CH3-18), 9.5 (COCH2CH3). The above data are consistent with those reported by WANG et al. [1] The comparison was basically consistent, so compound 1 was identified as pierisformosin B (C 23 H 38 O7).

[0054] Flavonoid compound 2: bright yellow needle-shaped crystals, mp: 310-315℃. Spray 1% AlCl3 ethanol solution on TLC thin layer plate, bright yellow fluorescence, positive reaction of HCl-Mg powder, positive reaction of FeCl3, negative reaction of Molish, UVλ max (nm):257,367(MeOH); ESI-MSm / z:300[M +](100),273(14),245(6),228(10),153(8),137(20),109(7); IR(KBr)cm -1 :3438, 2914, 1706, 1687, 1566, 1556, 1503, 1326, 1284; 1 H-NMR (400MHz, CH3COCH3-d6)δ H :7.68(1H,dd,J=8.8Hz,2.0Hz,H-6'),6.94(1H,d,J=8.8Hz,H-5'),6.32(1H ,d,J=2.0Hz,H-8),6.23(1H,d,J=2.0Hz,H-6),7.84(1H,d,J=8.8Hz,H-2'); 13 C-NMR (100 MHz, CH3COCH3-d)δ C :146.6(C-2), 135(C-3), 178.9(C-4), 153.5(C-5), 96.7(C-6), 164.0(C-7), 96.04(C-8), 157.6(C-9), 102.0(C-10), 124(C-1'), 116(C-2'), 147.6(C-3'), 144.1(C-4'), 116.2(C-5'), 121.4(C-6'). Data and literature [2-3] The reported comparisons were basically consistent, so it was identified as the flavonoid compound quercetin (CHCl3-CH3OH).

[0055] In this example, a total of 3.056 g of veratrol-type diterpene solid powder and 5.107 g of quercetin solid powder were obtained.

[0056] Further analysis by HPLC confirmed that the purity of veratrol-type diterpenes was 98.0% and that of quercetin was 97.3%. These results demonstrated the high efficiency and selectivity of our purification process, ensuring that the obtained active ingredients have high purity and stability, providing high-quality raw materials for further application research and the development of biopesticide formulations.

[0057] Calculate the extraction yield:

[0058] Extraction yield of terpenoids from oleander = weight of pure compound / weight of starting material used in the extraction process

[0059] =3.056 / 1000=0.3056%.

[0060] Extraction yield of quercetin = weight of pure compound / weight of starting material used in the extraction process

[0061] =5.107 / 1000=0.5107%.

[0062] S2. Preparation of active ingredient mixture:

[0063] 20 parts by weight of purified veratrol-type diterpenes and 40 parts by weight of quercetin were mixed to obtain an active ingredient mixture.

[0064] S3. Mix the auxiliary ingredients with the active ingredient mixture and adjust the pH value:

[0065] 2 parts by weight of synergist peppermint oil, 0.8 parts by weight of nonionic surfactant polyvinylpyrrolidone (PVP), 1.5 parts by weight of dispersant xanthan gum, 0.3 parts by weight of stabilizer sodium hydroxymethylcellulose, and 0.1 parts by weight of preservative methyl paraben are added to the active ingredient mixture in sequence, and stirred with a magnetic stirrer until completely dissolved to obtain a mixed solution. A pH meter is used to monitor the pH value of the mixed solution, and a mass volume concentration of 1% (g / mL) sodium hydroxide solution is used to adjust the pH value of the mixed solution to 7.0. Under aseptic conditions, the mixed solution is transferred to a high shear mixer and emulsified at a speed of 15000-20000 rpm for 20 minutes until a uniform suspension is formed. Homogenization is carried out by a homogenizer to further refine the emulsion droplet diameter and improve the uniformity and stability of the preparation. The preservative can be propyl paraben or methyl paraben, and the preservative in this embodiment is methyl paraben.

[0066] Example 2

[0067] This embodiment provides a method for preparing a biopesticide composition. The difference between this embodiment and embodiment 1 is only the amount of quercetin used. The weight portion of quercetin in this embodiment is 50 parts, and the rest is the same as embodiment 1.

[0068] In this example, a total of 2.933 g of veratrol-type diterpene solid powder and 4.952 g of quercetin solid powder were obtained.

[0069] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 98.0% and the purity of the flavonoids was 98.1%.

[0070] Calculate the extraction yield:

[0071] The extraction yield of diterpenes of the oleander tree = 2.933 / 1000 = 0.2933%.

[0072] The extraction yield of quercetin = 4.992 / 1000 = 0.4952%.

[0073] Example 3

[0074] This embodiment provides a method for preparing a biopesticide composition. The only difference between this embodiment and embodiment 1 is the amount of quercetin used. The weight portion of quercetin in this embodiment is 10 parts, and the rest is the same as embodiment 1.

[0075] In this example, a total of 2.951 g of veratrol-type diterpene solid powder and 4.947 g of quercetin solid powder were obtained.

[0076] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 98.1% and the purity of quercetin was 97.1%.

[0077] Calculate the extraction yield:

[0078] The extraction yield of diterpenes of the oleander tree = 2.951 / 1000 = 0.2951%.

[0079] The extraction yield of quercetin = 4.987 / 1000 = 0.4947%.

[0080] Example 4

[0081] This embodiment provides a method for preparing a biopesticide composition. The only difference between this embodiment and Example 1 is that this embodiment extracts naturally shade-dried Lyonia ovalifolia leaves, and the rest is the same as Example 1.

[0082] The purified active ingredients were compared using liquid chromatograms and compared with known literature and databases, and the active ingredients were identified as veratrol-type diterpenes such as quercetin and flavonoid compound quercetin.

[0083] In this example, a total of 3.005 g of veratrol-type diterpene solid powder and 4.991 g of quercetin solid powder were obtained.

[0084] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 98.2% and the purity of quercetin was 97.1%.

[0085] Calculate the extraction yield:

[0086] The extraction yield of diterpenes of oleander = 3.005 / 1000 = 0.3005%.

[0087] The extraction yield of quercetin = 4.991 / 1000 = 0.4991%.

[0088] Example 5

[0089] This embodiment provides a method for preparing a biopesticide composition. The only difference between this embodiment and embodiment 1 is the preparation method. Step ① in step S1 of this embodiment uses a 60% (v / v) ethanol solution as an extraction solvent, and the mass ratio of the crushed Aglaonema serrata leaves to the ethanol solution is 1:10. The rest is the same as embodiment 1.

[0090] Finally, a total of 2.894 g of veratrol-type diterpene solid powder and 4.963 g of quercetin solid powder were obtained in this example.

[0091] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 98.1% and the purity of quercetin was 97.6%.

[0092] Calculation formula:

[0093] The extraction yield of diterpenes of the oleander tree = 2.894 / 1000 = 0.2894%.

[0094] The extraction yield of quercetin = 4.963 / 1000 = 0.4963%.

[0095] Example 6

[0096] This embodiment provides a method for preparing a biopesticide composition. The difference between this embodiment and embodiment 1 is only in the amount of auxiliary components. In this embodiment, the auxiliary components include, by weight:

[0097] The synergist is peppermint oil 20 parts, the nonionic surfactant polyvinylpyrrolidone (PVP) 0.5 parts, the dispersant xanthan gum 0.5 parts, the stabilizer 0.1 parts, the preservative 0.05 parts. The rest is the same as in Example 1.

[0098] Finally, a total of 2.901 g of veratrol-type diterpene solid powder and 4.947 g of quercetin solid powder were obtained in this example.

[0099] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 98.1% and the purity of quercetin was 97.3%.

[0100] Calculation formula:

[0101] The extraction yield of diterpenes of the oleander tree = 2.901 / 1000 = 0.2901%.

[0102] The extraction yield of quercetin = 4.947 / 1000 = 0.4947%.

[0103] Example 7

[0104] This embodiment provides a method for preparing a biopesticide composition. The difference between this embodiment and embodiment 1 is only in the amount of auxiliary components. In this embodiment, the auxiliary components include, by weight:

[0105] The synergist is peppermint oil 5 parts, the nonionic surfactant polyvinylpyrrolidone (PVP) 2 parts, the dispersant xanthan gum 2 parts, the stabilizer 0.5 parts, the preservative 0.2 parts. The rest is the same as in Example 1.

[0106] Finally, a total of 2.917 g of veratrol-type diterpene solid powder and 4.935 g of quercetin solid powder were obtained in this example.

[0107] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 97.9% and the purity of quercetin was 97.4%.

[0108] Calculation formula:

[0109] The extraction yield of diterpenes of the oleander tree = 2.917 / 1000 = 0.2917%.

[0110] The extraction yield of quercetin = 4.935 / 1000 = 0.4935%.

[0111] Example 8

[0112] This embodiment provides a method for preparing a biopesticide composition. The only difference between this embodiment and Example 1 is that a hydrochloric acid solution with a mass volume concentration of 1% is used to adjust the pH value of the composition to 5.0, and the rest is the same as Example 1.

[0113] Finally, a total of 2.978 g of veratrol-type diterpene solid powder and 5.021 g of quercetin solid powder were obtained in this example.

[0114] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 98.1% and the purity of quercetin was 97.4%.

[0115] Calculation formula:

[0116] The extraction yield of diterpenes of the genus Asteraceae = 2.978 / 1000 = 0.2978%.

[0117] The extraction yield of quercetin = 5.021 / 1000 = 0.5021%.

[0118] Example 9

[0119] This embodiment provides a method for preparing a biopesticide composition. The only difference between this embodiment and Example 1 is that a hydrochloric acid solution with a mass volume concentration of 1% is used to adjust the pH value of the composition to 6.0, and the rest is the same as Example 1.

[0120] Finally, a total of 2.952 g of veratrol-type diterpene solid powder and 4.931 g of quercetin solid powder were obtained in this example.

[0121] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 97.3% and the purity of quercetin was 97.2%.

[0122] Calculation formula:

[0123] The extraction yield of diterpenes of the genus Asteraceae = 2.952 / 1000 = 0.2952%.

[0124] The extraction yield of quercetin = 4.931 / 1000 = 0.4931%.

[0125] Example 10

[0126] This embodiment provides a method for preparing a biopesticide composition. The only difference between this embodiment and Example 1 is that a sodium hydroxide solution with a mass volume concentration of 1% is used to adjust the pH value of the composition to 8.0, and the rest is the same as Example 1.

[0127] Finally, a total of 2.954 g of veratrol-type diterpene solid powder and 4.935 g of quercetin solid powder were obtained in this example.

[0128] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 98.1% and the purity of quercetin was 97.2%.

[0129] Calculation formula:

[0130] The extraction yield of diterpenes of the oleander tree = 2.954 / 1000 = 0.2954%.

[0131] The extraction yield of quercetin = 4.935 / 1000 = 0.4935%.

[0132] Embodiment 11

[0133] This embodiment provides a method for preparing a biopesticide composition. The only difference between this embodiment and Example 1 is that a sodium hydroxide solution with a mass volume concentration of 1% is used to adjust the pH value of the preparation to 9.0. The rest is the same as Example 1.

[0134] Finally, a total of 2.971 g of veratrol-type diterpene solid powder and 4.928 g of quercetin solid powder were obtained in this example.

[0135] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 98.1% and the purity of quercetin was 97.2%.

[0136] Calculation formula:

[0137] The extraction yield of diterpenes of the oleander tree = 2.971 / 1000 = 0.2971%.

[0138] The extraction yield of quercetin = 4.928 / 1000 = 0.4928%.

[0139] Example 12

[0140] This embodiment provides a method for preparing a biopesticide composition. The only difference between this embodiment and Example 1 is that eucalyptus oil is used as a synergist, and the rest is the same as Example 1.

[0141] Finally, a total of 2.939 g of solid powder of veratrol-type diterpenes and 4.974 g of solid powder of flavonoid compounds were obtained in this example.

[0142] Further analysis by HPLC confirmed that the purity of the veratranoid diterpenes was 98.2% and the purity of the flavonoids was 97.1%.

[0143] Calculation formula:

[0144] The extraction yield of diterpenes of the oleander tree = 2.939 / 1000 = 0.2939%.

[0145] The extraction yield of quercetin = 4.974 / 1000 = 0.4974%.

[0146] Example 13

[0147] This embodiment provides a method for preparing a biopesticide composition. The difference between this embodiment and embodiment 1 is only that saponin is used as a synergist, and the rest is the same as embodiment 1.

[0148] Finally, a total of 2.914 g of veratrol-type diterpene solid powder and 4.956 g of quercetin solid powder were obtained in this example.

[0149] Further analysis by HPLC confirmed that the purity of the veratranoid-type diterpenes was 97.9% and the purity of quercetin was 97.4%.

[0150] Calculation formula:

[0151] The extraction yield of diterpenes of the genus Asteraceae = 2.914 / 1000 = 0.2914%.

[0152] The extraction yield of quercetin = 4.956 / 1000 = 0.4956%.

[0153] Comparative Example 1

[0154] This comparative example provides a method for preparing a biopesticide composition. The only difference between this comparative example and Example 1 is that the amount of veratrol-type diterpene used in step S2 of this comparative example is 5 parts by weight, and the rest is the same as Example 1.

[0155] Comparative Example 2

[0156] This comparative example provides a method for preparing a biopesticide composition. The difference between this comparative example and Example 1 is that the amount of veratrol-type diterpene used in step S2 of this comparative example is 60 parts by weight, and the rest is the same as Example 1.

[0157] Comparative Example 3

[0158] This comparative example provides a method for preparing a biopesticide composition. The only difference between this comparative example and Example 1 is that in step S3 of this comparative example, no auxiliary ingredients are added, but the pH value of the active ingredient mixture is directly adjusted to 7 under sterile conditions. The rest is the same as Example 1.

[0159] The biopesticide compositions prepared in Examples 1-13 and Comparative Examples 1-3 were tested for stability, and the test method was as follows:

[0160] The biopesticide compositions prepared in Examples 1-13 and Comparative Examples 1-3 were stored at room temperature for 3 months, the initial amount of the active ingredient was defined as 100%, the pH value and the active ingredient content were tested regularly, and the measured content / initial content*100% was calculated to obtain the relative percentage content, which was used to evaluate the stability of the formulation. The results are shown in Table 1:

[0161] Table 1

[0162]

[0163]

[0164] According to Table 1, the relative content of the effective active ingredient in the preparation is constantly decreasing during storage. When stored to the 90th day, the content of the effective ingredient in Examples 1-13 accounts for more than 90% of the initial amount, the content of the effective ingredient in Comparative Examples 1-2 accounts for less than 65% of the initial amount, and the content of the effective ingredient in Comparative Example 3 accounts for less than 20% of the initial amount, indicating that the biological pesticide composition prepared in Examples 1-13 has strong stability. Compared with other examples, the content of the effective ingredient in Examples 8-11 decays faster, which may be related to the change in pH value. The stability of the preparation is best when the pH value is 7.0, and the active ingredient content decreases by no more than 5% within 3 months.

[0165] The stability of Comparative Examples 1 and 2 is much lower than that of all the embodiments, which may be because the stability of the drug in the suspension mainly depends on the effect of the stabilizer. Under the conditions of the same content of surfactant, dispersant and stabilizer, too little or too much Veratrane-type diterpenes will affect the stability. The presence of stabilizers helps to maintain the physical stability of the suspension and prevent drug particles from aggregating or settling. When there are too many stabilizers and less extracts in the suspension, although the stability of the suspension can be enhanced, it will also cause the suspension to be too viscous, and too much stabilizer may also change the physical and chemical properties of the suspension. If there are too many extracts and insufficient stabilizers, it will lead to problems such as unstable drug crystal forms and accelerated degradation. Therefore, maintaining an appropriate ratio of extracts to stabilizers is crucial to ensuring the stability and effectiveness of the suspension. The stability of Comparative Example 3 is the worst among all the embodiments and comparative examples, which may be because in the absence of surfactants, dispersants, stabilizers, and preservatives, the extracted natural products Veratrane-type diterpenes and flavonoids are susceptible to chemical instability, thermal instability, microbial contamination, light sensitivity, and temperature sensitivity. Veratran-type diterpenes usually have multiple chiral centers, ring structures, and active functional groups, such as hydroxyl, carbonyl, and olefin bonds, which make them prone to chemical changes. Chemical reactions such as hydrolysis, oxidation, and rearrangement may occur, resulting in changes in their structure and properties, which may affect the biological activity of veratran-type diterpenes; flavonoids may undergo oxidation and decomposition, and the slowly precipitated white precipitate may be due to the reduced solubility of flavonoids or reactions with other components. Sodium hydroxymethylcellulose (CMC) is a highly polymerized linear compound, and its performance is not only related to the degree of substitution, but also to the uniformity of the distribution of carboxyl groups in cellulose molecules and the uniformity of the substitution of hydroxymethyl groups with C2, C3, and C6 in each unit of each molecule. Due to this characteristic of CMC, when there is shear force in the solution, the long axis of its linear molecules tends to turn to the flow direction, and as the shear rate increases, this tendency becomes stronger until it is finally completely oriented. This property is called pseudoplasticity, which helps stabilize the components in the solution. Therefore, by adding CMC to the extract, its stability can be enhanced.

[0166] Application Examples

[0167] The biopesticide composition prepared by the present invention can be used to prepare drugs for preventing and controlling plant diseases, including rice blast, wheat head blight, cotton wilt, aphids and whiteflies of vegetable crops, and the use method thereof is as follows:

[0168] 1. For the prevention and control of rice blast, the following methods are adopted:

[0169] The biopesticide composition of the present application is diluted with water at a mass ratio of 1:500. The application period is selected at the early stage of the disease or before the rainy season, and 50 liters of the diluted solution is applied per mu. Depending on the disease condition, the application is once every 7 to 10 days for 3 to 4 consecutive times, and the sprayer is used to evenly spray the front and back sides of the rice leaves to ensure that the solution contacts.

[0170] 2. For wheat fusarium rust, the application method is as follows:

[0171] The biopesticide composition of the present application is mixed with water at a mass ratio of 1:800. The pesticide is applied during the wheat heading to flowering period, and 60 liters of diluted liquid is used per mu. Depending on the disease situation, the pesticide is applied once every 10 to 14 days for 2 to 3 times in a row, and the spray is evenly sprayed using a mist machine to cover the entire wheat plant.

[0172] 3. The prevention and control methods of cotton wilt are as follows:

[0173] The biopesticide composition of the present application is diluted with water at a mass ratio of 1:1000. Apply the pesticide during the vigorous growth period of cotton or the early stage of the disease, and apply 40 liters of the diluted solution per mu. The frequency of application is once every 15 days, adjusted according to the condition, for 2 to 4 consecutive times, using a low-pressure sprayer to focus on spraying the leaves and stems.

[0174] 4. The methods for controlling aphids in vegetable crops are:

[0175] The biopesticide composition of the present invention is diluted with water at a mass ratio of 1:300. Apply the pesticide at the early stage of aphid discovery, and apply 30 liters of the diluted solution per mu. Depending on the number of aphids, apply the pesticide once every 5 to 7 days for 2 to 3 times in a row, and use a handheld sprayer to carefully spray on the vegetable leaves, especially the back of the leaves.

[0176] 5. The whitefly control methods for vegetable crops are as follows:

[0177] The biopesticide composition of the present invention is diluted with water at a mass ratio of 1:400. The pesticide is applied at the early stage of whitefly occurrence or when signs of pests are observed, and 40 liters of the diluted solution is applied per mu. The frequency of application is once every 7 days, adjusted according to the pest density, and repeated 2 to 3 times, using a high-pressure sprayer to ensure that the solution evenly covers the leaves and stems of the crops.

[0178] Experimental example

[0179] 1. Field application test:

[0180] The field test was conducted in Mingba Village, Mingxin Temple Town, Zizhong County, Neijiang City in summer. The pesticide compositions prepared in Examples 1-13 and Comparative Examples 1-3 and commercially available chemical pesticides (avermectin) were respectively used for field tests in a tomato-aphid system. The pesticide composition preparations prepared in Examples 1-13 and Comparative Examples 1-3 were diluted with water at a mass ratio of 1:300. The pesticide was applied at the early stage of aphid discovery, and 30 liters of the diluted solution was applied per mu. Depending on the number of aphids, the pesticide was applied once every 7 days for 3 consecutive times, using a handheld sprayer to carefully spray on the vegetable leaves, especially the back of the leaves. The commercially available chemical pesticide (avermectin) is a 1.8% emulsifiable concentrate, which is diluted and sprayed at a volume concentration of 1:5000 as recommended in the instructions. Insect population reduction rate = (number of live insects before application - number of live insects after application) / number of live insects before application × 100%;

[0181] Control effect = (insect population reduction rate in the pesticide-treated area - insect population reduction rate in the blank control area) / (1 - insect population reduction rate in the blank control area) × 100%.

[0182] The number of aphids before and after application was recorded, and the control effect is shown in Table 2.

[0183] Table 2

[0184]

[0185] It can be seen from Table 2 that the pesticide composition provided by the present invention has a good effect of killing aphids, and its composition and preparation method have an important influence on its efficacy.

[0186] The results of the field test showed that the number of aphids in Examples 1-13 decreased significantly after three consecutive applications, all reaching more than 75%. The average decrease rate of aphids in Comparative Example 1 after three consecutive applications was 67.26%; the average decrease rate of aphids in Comparative Example 2 after three consecutive applications was 64.85%; the average decrease rate of aphids in Comparative Example 3 after three consecutive applications was 66.47%;

[0187] The average reduction rate of aphid number after three consecutive applications of 5000 times diluted 1.8% avermectin emulsifiable concentrate was 74.25%. The control effect of Examples 1-13 was significantly better than that of Comparative Examples 1-3 and 5000 times diluted 1.8% avermectin emulsifiable concentrate available on the market.

[0188] 2. Antibacterial test

[0189] Test groups:

[0190] Test group 1: the biopesticide composition prepared in Example 1;

[0191] Test group 2: the biological pesticide composition prepared in Comparative Example 3;

[0192] Test group 3: commercially available (penicillin);

[0193] Test group 4: blank control (sterile water).

[0194] Slant activation method: Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were inoculated in half on nutrient agar medium and cultured at 30-35°C for 18 hours to activate the bacteria. A 0.9% volume concentration of sterile sodium chloride solution was added to the above bacterial culture in a sterile test tube to make a bacterial concentration of 10 8 cfu / mL bacterial suspension was placed at 4°C in a dark environment for later use. Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were all commercially available strains.

[0195] Determination of antibacterial effect: The antibacterial effect is judged by the diameter of the inhibition zone. The diameter of the inhibition zone of the pesticide composition is determined by the filter paper agar plate diffusion method. The filter paper is made into a circular paper with a diameter of 6 mm. After sterilization and cooling, it is placed in the pesticide composition and soaked for 12 hours for use. Sterile water is used as a blank control and penicillin solution is used as an antibacterial control. In the clean bench, 0.2 mL is drawn from the test bacterial suspension and evenly applied on the surface of the NA solid culture medium to prepare a bacterial plate; the filter paper soaked with the pesticide composition, sterile water and penicillin solution is evenly placed on the surface of the bacterial plate, and the prepared plate is placed in a 30°C constant temperature incubator for 18 hours. The filter paper is taken out and the diameter of the inhibition zone is measured by the cross-cross method. The test is repeated 3 times to take the average value. The results are shown in Tables 3 and Figure 1 .

[0196] Table 3

[0197]

[0198] Note: The diameter of the inhibition zone is the mean ± SD. Different lowercase letters in each column indicate significant differences (P < 0.05).

[0199] As shown in Table 3, the commercially available penicillin in test group 3 has the strongest inhibitory effect on Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The pesticide compositions in test group 1 (Example 1) and test group 2 (Comparative Example 3) have inhibitory effects on Bacillus subtilis and Staphylococcus aureus, but have no inhibitory effect on Escherichia coli. The effect of Example 1 is significantly stronger than that of Comparative Example 3, which may be related to the rapid loss of the active ingredient content of the pesticide composition in the comparative example. The inhibitory effect of Example 1 on Bacillus subtilis and Staphylococcus aureus is not significantly different from that of the commercially available pesticide penicillin.

[0200] According to the inhibition zone judgment standard, among the three bacterial species, the pesticide composition of test group 1 (Example 1) has the strongest antibacterial effect on Staphylococcus aureus, has a better inhibitory effect on Bacillus subtilis, and has no significant inhibitory effect on Escherichia coli. This indicates that the pesticide composition has a targeted inhibitory effect on Gram-positive bacteria, but has no inhibitory effect on Gram-negative bacteria.

[0201] The minimum inhibitory concentration (MIC) of the biological pesticide composition was determined by the microbroth dilution method: the pesticide composition prepared in Example 1 was set to 7 concentration gradients, namely 0.0002, 0.0004, 0.008, 0.016, 0.032, 0.125, and 0.5 μg / mL. 10 μL of bacterial suspension was added to the Mueller-Hinton liquid culture medium containing 1 mL of the pesticide composition and then placed in a 30°C incubator for 18 h. The bacterial suspension test tube and the control tube were evenly spread on the corresponding solid culture plates for culture and counting, and the minimum inhibitory concentration was determined by observing the number of colonies on the nutrient plate.

[0202] The solid nutrient plates used for Escherichia coli are VRBA and VRBA-MUG medium. First, take VRBA and VRBA-MUG medium, weigh and dissolve according to the label instructions, heat and boil VRBA and VRBA-MUG medium for sterilization, boil 2-3 times, and then place in a 45℃±0.5℃ water bath for standby. Select 2 to 3 appropriate sample homogenates with continuous dilutions. This time, the sample homogenates with the original solution, -1, and -2 gradients are used for the experiment. Each dilution is inoculated into 2 sterile plates, 1mL per plate. At the same time, take 1mL of the dilution and add it to the sterile plate as a blank control. At the same time, use known MUG-positive strains (such as Escherichia coli 25922) and Enterobacter aerogenes (such as ATCC 13048) as positive and negative controls. Pour 10mL to 15mL of crystal violet neutral red bile agar (VRBA) cooled to 45℃±0.5℃ into each plate. Carefully rotate the plate to mix the culture medium and sample solution thoroughly. After the agar solidifies, add 3mL~4mL VRBA-MUG to cover the surface of the plate. After solidification, turn the plate over and incubate at 36℃±1℃ for 18h~24h. Select a plate with a colony count between 10CFU and 100CFU, and count the colonies on the plate that emit light blue fluorescence under 360nm~366nm wavelength ultraviolet light in a dark room.

[0203] Staphylococcus aureus was cultured on Baird-Parker plates. The nutrient plates for Bacillus subtilis were prepared in the laboratory, and the solid culture medium consisted of: 1L distilled water, 20g glucose, 15g peptone, 5g sodium chloride, 0.5g beef extract, and 20g agar.

[0204] The results are shown in Table 4.

[0205] Table 4

[0206] Bacteria name Bacteria Type MIC / (μg / mL) Escherichia coli G- - Staphylococcus aureus G+ O.004 Bacillus subtilis G+ 0.016

[0207] As shown in Table 4, the pesticide composition of the present invention has a significant antibacterial effect. The MICs of Staphylococcus aureus and Bacillus subtilis are 0.004 and 0.016 μg / mL, respectively. Among the three bacterial species, the pesticide composition has the strongest antibacterial effect on Staphylococcus aureus.

[0208] References:

[0209] [1]WANG LQ,DING BY,QIN GW,et al.,1998.Grayanoids from Pieris formosa[J].Phytochemistry,49(7):2045–2048.

[0210] [2] Kong Lingyi, Min Zhida. Study on chemical components of Euphorbia pulegata root[J]. Acta Pharmaceutica Sinica, 1996, 31(7): 524.

[0211] [3] Yu Dequan, Yang Junshan. Handbook of Analytical Chemistry, Volume 7: Nuclear Magnetic Resonance Spectroscopy [M]. Beijing: Chemical Industry Press, 1999.820.

[0212] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A biopesticide composition, characterized in that: According to weight parts, it includes the following components: 10-30 parts of veratrane-type diterpenes; 10-50 parts of flavonoid compounds.

2. A biopesticide composition according to claim 1, characterized in that: According to weight parts, it includes the following components: 20 parts of veratranolane-type diterpenes; 40 parts of flavonoids.

3. A biopesticide composition according to claim 1 or 2, characterized in that: The veratrolane-type diterpenes and flavonoid compounds are all extracts of the beautiful dwarf ...

4. A biopesticide composition according to claim 1, characterized in that: The invention also comprises auxiliary ingredients, which include: synergist, surfactant, dispersant, stabilizer and preservative.

5. A biopesticide composition according to claim 4, characterized in that: The synergist is peppermint oil, eucalyptus oil, saponin or its derivatives, preferably peppermint oil; The surfactant is selected from nonionic, cationic or anionic surfactants, preferably polyvinyl pyrrolidone; The dispersant is selected from polyvinyl alcohol or cellulose derivatives; preferably xanthan gum; The stabilizer is sodium hydroxymethyl cellulose; The preservative is a paraben.

6. A biopesticide composition according to claim 5, characterized in that: In terms of weight, the auxiliary ingredients include: 2-20 parts of synergist, 0.5-2 parts of surfactant, 0.5-2 parts of dispersant, 0.1-0.5 parts of stabilizer, 0.05-0.2 parts of preservative, preferably: 2 parts of synergist, 0.8 parts of surfactant, 1.5 parts of dispersant, 0.3 parts of stabilizer, and 0.1 parts of preservative.

7. A biopesticide composition according to any one of claims 1 to 6, characterized in that: The pH value of the composition is adjusted to 5-9 with a pH adjuster, preferably 7.

8. A biopesticide composition according to claim 7, characterized in that: The pH regulator is 1% hydrochloric acid or 1% sodium hydroxide solution.

9. The method for preparing a biopesticide composition according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. Extracting active ingredients, comprising the following steps: ① Take fresh / dried leaves of Psoralea corylifolia or Psoralea corylifolia and crush them into 40-80 mesh; use 60% (v / v) ethanol solution as the extraction solvent, the mass ratio of raw material to ethanol solution is 1:3-1:10, and extract in an ultrasonic extractor with a power of 300-500W, a temperature of 35-45°C, and a time of 30-70 minutes; repeat the extraction process 3 times, combine the extracts, and concentrate to 1 / 5 of the original volume by rotary evaporator; ② Purification of crude extract: purifying the concentrated extract by medium pressure liquid chromatography to collect the biologically active components; ③ Using high performance liquid chromatography to monitor and accurately collect fractions with specific absorption wavelengths, the biologically active components were further accurately separated to obtain veratrol-type diterpenes and flavonoid compounds respectively; S2. Preparation of active ingredient mixture: Mixing the extracted veratrol-type diterpenes and flavonoid compounds according to the weight proportions to obtain an active ingredient mixture; S3. Mix the active ingredient mixture, synergist, surfactant, dispersant, stabilizer and preservative in the above-mentioned proportions by weight under sterile conditions and adjust the pH value to 5-9.

10. Use of the biopesticide composition according to any one of claims 1 to 8 in medicines for preventing and controlling plant diseases.

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