Plant extract for killing pests and preparation process thereof

Abstract

The invention discloses a plant extract for killing pests and a preparation process of the plant extract, and relates to the technical field of biopesticides, and the technical key points are as follows: the plant extract is selected from one or more of adhatoda vasica Nees, azadirachta indica, nigra sativa, parthenium hysterium and oriental arborvitae Thuja orientia, and the extract contains phytosterol, saponin, diterpene, flavone and alkaloid. Contact and residual toxicity tests are carried out on the cigarette beetles cultured in a laboratory, the experimental result shows that the neem and adhatoda vasica extracts have remarkable toxicity and repelling effects on the cigarette beetles, the neem has the highest toxicity, and the LC50 value is the lowest. The method comprises the steps of plant collection, drying, grinding, solvent extraction, rotary evaporation and the like so as to obtain high-concentration active ingredients. The plant extract of the invention can be incorporated into the manufacture of novel biological insecticides to reduce the harm of cigarette beetles.

Description

Technical Field

[0001] The invention relates to the technical field of biological pesticides, and in particular to a plant extract for killing pests and a preparation process thereof. Background Art

[0002] The tobacco beetle (Lasioderma serricorne) is a major global stored-grain pest that poses a serious threat to food and other plant-derived products, with the most severe economic losses in the tobacco industry. Traditional pest control methods rely on chemical insecticides, such as phosphine, but long-term use has led to insecticide resistance, destruction of natural enemy biological control, and environmental and human health problems. Therefore, researchers have begun to seek more environmentally friendly alternatives, among which plant-derived insecticides have become the focus of research due to their biodegradability, low toxicity to mammals, and low harm to the environment. These botanical insecticides usually contain active ingredients such as alkaloids, flavonoids, saponins, diterpenes, phytosterols, and phenolic compounds, which can damage the insect's nervous and hormonal systems, or interfere with mitochondrial function, thereby effectively controlling pests.

[0003] Although plant extracts show potential as an environmentally friendly alternative to insecticides, existing technologies still face some challenges. First, the efficacy of plant extracts is affected by multiple factors such as application dose, technology, and insect development stage, and further research is needed to determine the optimal formulation and application method. Second, the comprehensive component analysis of plant extracts has not yet been fully clarified, which is crucial for understanding their insecticidal mechanism and improving their efficacy. In addition, the large-scale production and application technology of plant extracts need to be further optimized to ensure their effectiveness and feasibility in actual pest management. Finally, although plant extracts have less impact on the environment and human health, the potential risks of their long-term use still need to be evaluated. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a plant extract for killing insects and a preparation process thereof, so as to solve the problems of the prior art such as the enhancement of insect resistance and the destruction of biological control caused by the long-term use of phosphides to control tobacco beetles.

[0005] In order to achieve the above-mentioned object, the technical scheme of the present invention is as follows: a plant extract for killing insects, wherein the extract is selected from one or more of Adhatoda vasica Nees, Azadirachta indica, Nigellasativa, Parthenium hysterophorus and Thuja orientalis, and the extract contains phytosterols, saponins, diterpenes, flavonoids and alkaloids.

[0006] Furthermore, the extract is used to control the tobacco beetle Lasioderma serricorne.

[0007] Furthermore, the concentration of the extract is 250 mg / L to 1500 mg / L.

[0008] Another object of the present invention is to provide a method for preparing a plant extract for killing insects, the method comprising the following steps:

[0009] a. Collect leaves of neem, black cumin, guayule and oriental arborvitae;

[0010] b. The collected leaves were air-dried at room temperature for 15 days;

[0011] c. Grind the dried leaves into a fine powder;

[0012] d. The powder was mixed with a solvent and extracted at room temperature using a 50% concentration by soaking at 12 hour intervals;

[0013] e. Use a rotary evaporator to remove the solvent at low temperature and pressure to obtain the plant extract.

[0014] Furthermore, the concentration of the extract is adjusted to a desired concentration by a rotary evaporator.

[0015] Another object of the present invention is to provide a method for killing pests using the above plant extract, the method comprising applying the extract to the pest-infected area at a concentration of 750 mg / L.

[0016] Another object of the present invention is to provide a method for evaluating the toxicity and repellent effect of plant extracts on tobacco beetles, the method comprising the following steps:

[0017] S1: plant extracts were applied to tobacco beetles at different concentrations;

[0018] S2: Check the mortality of tobacco beetles after 24, 48, 72, and 96 h;

[0019] S3: Determine LC50 and LC90 using Probit analysis.

[0020] Compared with the prior art, the beneficial effects of the present scheme are as follows: the plant extracts used in the present invention are derived from naturally grown plants, such as Daffodil, Azadirachta indica, Black cumin, Argyle and Oriental Platycladus orientalis. The active ingredients such as phytosterols, saponins, diterpenes, flavonoids and alkaloids contained in these plants have significant toxicity and repellent effects on the tobacco beetle Lasioderma serricorne. This method of utilizing natural resources not only reduces dependence on chemical synthetic pesticides and reduces environmental pollution, but also reduces adverse effects on non-target organisms, thereby protecting the ecological balance. The concentration range of the extract in the present invention is 250 mg / L to 1500 mg / L, which provides a flexible dosage adjustment space for practical applications and can be optimized according to the specific situation of the pests and environmental conditions to achieve the best control effect while reducing the excessive use of chemical substances.

[0021] The present invention can efficiently extract active ingredients from plants and prepare the required plant extracts by collecting plant leaves, drying, grinding, solvent extraction and rotary evaporation. This method is not only simple to operate, but also low in cost and suitable for large-scale production and application. The present invention also provides a specific method for killing pests using plant extracts, that is, applying them to pest-infected areas at a concentration of 750 mg / L, which provides a clear operating guide for actual pest management. At the same time, the present invention determines LC50 and LC90 by mortality inspection and Probit analysis at different time points, providing accurate data support for scientific evaluation of the effect of the extract.

[0022] The invention not only improves the efficiency of pest control, but also focuses on cost-effectiveness and ease of operation, providing a new and economical solution for agricultural pest management. This method based on natural plant extracts is not only environmentally friendly, but also an economically viable alternative for farmers, helping to reduce the use of chemical pesticides, reduce the cost of agricultural production, and improve the safety of agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a complete flow chart from plant extraction to biological activity evaluation in an embodiment of the present invention;

[0024] Figure 2 is the direct toxic effect of different plant extracts in the examples of the present invention on adult tobacco beetles;

[0025] in, Figure 2 a-2d are the mortality rates of adult tobacco beetles after exposure to different plant extracts for 24, 48, 72, and 96 hours, respectively;

[0026] Figure 3 is the residual toxicity effect of different plant extracts on adult tobacco beetles in the examples of the present invention;

[0027] in, Figure 3 a-3d are the mortality rates of different plant extracts on adult tobacco beetles after 24, 48, 72, and 96 hours of residual toxicity test;

[0028] Figure 4 is the repellent effect of different plant extracts on tobacco beetles in the embodiments of the present invention;

[0029] in, Figure 4 a-4f are the repellent effects of different plant extracts on adult tobacco beetles after 24, 48, 72, and 96 hours of exposure. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0031] Lasioderma serricorne collected from Pak Hill (private company) warehouse were cultured in jars containing diet (wheat feed / yeast, 10:1, w / w) in an incubator under dark conditions (29 ± 1°C, 70-80% rh). Insects used in all experiments were of similar age and size, regardless of sex.

[0032] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0033] Example 1: A plant extract for killing pests and its preparation process, as shown in the attached Figure 1 As shown, the following steps are included:

[0034] Plant and Sample Preparation: Leaves of five different plant species used in the present invention were collected in and around Haripur University, Pakistan during the growth stage as shown in Table 1 below. Identification of plant species was done by botanists of the Department of Biology, Haripur University and plant specimens were deposited in the herbarium (voucher number F.No.UH / Hort / 2514). The plant leaves were air dried at room temperature for 15 days. The dried samples were ground into fine powder. One kg of each sample was separated by soaking (1*2L) at 12 hours interval using 50% concentration at room temperature. The filtered samples were freed from solvents using a rotary evaporator (Buchi, R-210) at low temperature and pressure in the Food Science Laboratory, Haripur University. The yields obtained were 46 g of Parthenium hysterophorus, 63 g of Thuja orientalis, 51 g of Azadirachtaindica, 48 g of Adhatoda vasica and 55 g of Nigella sativa, respectively, and then stored at a temperature below 4°C.

[0035] Table 1 List of plant species tested for effectiveness against tobacco beetles at six different concentrations

[0036]

[0037] Initial Screening: Extracts from five different plant species were screened for efficacy against adult tobacco beetles based on yield (500-4000 mg / L). Based on the preliminary data, six different concentrations (250 mg / L, 500 mg / L, 750 mg / L, 1000 mg / L, 1250 mg / L, and 1500 mg / L) were selected and evaluated against tobacco beetles in the final bioassay.

[0038] Example 2: Qualitative analysis of the plant extract described in Example 1

[0039] The present invention, when performing qualitative analysis of plant species extracts, first needs to prepare the plant extract. The whole or coarsely ground plant material is contacted with a solvent in a covered container and stirred for a predetermined time until a homogenous mixture is formed. Next, the Wegener test is performed to detect the presence of alkaloids. The herbal extract sample is mixed with dilute hydrochloric acid (1.5%) and filtered, and then a small amount of potassium iodide is added to the filtrate. If alkaloids are present in the sample, a reddish-brown precipitate will appear, which is a clear sign of the presence of alkaloids.

[0040] The present invention uses the iron chloride test to detect phenolic compounds. A few drops of iron chloride solution are added to the extract. The blue-black color in the solution indicates the presence of phenolic compounds. Next, the Salkovsky test is performed to detect phytosterols. Chloroform is added to the plant extract solution. After filtering, 3-4 drops of concentrated H 2 SO4, after standing for a period of time, the solution turns golden yellow, indicating the presence of phytosterols. Finally, the diterpene test is performed, copper acetate solution (2-3 drops) is added to the plant extract, and the appearance of bright green indicates the presence of diterpenes.

[0041] After completing the above tests, the next step is to test for saponins and flavonoids. For the saponin test, 2 ml of the plant extract is diluted with distilled water and vortexed for 5 minutes. If white foam appears on the surface for more than 10 minutes, the presence of saponins is confirmed. For the detection of flavonoids, an alkaline reagent test is used. A few drops of lead acetate solution and dilute acid are added to the plant water extract. The appearance of a bright yellow color indicates the presence of flavonoids in the extract.

[0042] The results of phytochemical screening of plant extracts showed that all tested plant species aqueous extracts contained phytochemicals. Among the plant species, kummer had the highest content of alkaloids, flavonoids, saponins, diterpenes, phytosterols, and phenolic compounds compared to other plant species. Guayule also showed higher alkaloid and saponin contents. Saponins and flavonoids were higher in Dalbergia orientalis compared to Thuja orientalis and Azadirachta indica. The percentage contents of all phytochemicals were lower in Azadirachta indica.

[0043] Example 3: Residual toxicity test of plant extracts to tobacco beetles

[0044] This example uses a 6x6 factorial design to evaluate the residual toxicity of five plant extracts to adult tobacco beetles. The experiment was conducted by the Potter spray method, involving six different concentrations of plant extracts (from 250 mg / L to 1500 mg / L). Filter paper was placed at the bottom of each culture dish, and 10 adult tobacco beetles were placed in the center. The treatment of each concentration was repeated three times to ensure the reliability of the results. Under controlled conditions, six different concentrations from 25 mg / L to 1500 mg / L were incubated, and the mortality of the beetles was observed at 24-hour intervals until 96 hours. Distilled water was used as a control group in the experiment to exclude the influence of non-plant extract factors. The Abbott formula was used to correct the mortality data, and Probit analysis was used to determine the LC50 and LC90 values, i.e., the median lethal concentrations, which represent the concentrations of plant extracts that cause 50% and 90% of tobacco beetles to die. The Abbott formula is shown below:

[0045] Mortality (%) = Number of dead insects × 100 / Number of introduced insects

[0046] The experimental results showed that as the concentration of plant extracts increased, the LC50 and LC90 values ​​showed a decreasing trend. After 24 hours, Parthenium hysterophorus showed the highest toxicity, with an LC50 of 10.02 mg / L and an LC90 of 108.61 mg / L, while Azadirachta indica had the lowest efficacy, with an LC50 of 14.12 mg / L and an LC90 of 188.12 mg / L, as shown in Table 2 below. At a concentration of 3.0%, Parthenium hysterophorus had the highest average mortality rate, reaching 77.00%, while Azadirachta indica had the lowest average mortality rate, only 40.00%, as shown in Table 2. Figure 2 After 48 hours, the efficacy of black cumin was still the highest, with LC50 of 2.60 mg / L and LC90 of 28.77 mg / L. Figure 2 b. After 72 hours, the toxicity of black cumin was the highest, with LC50 of 1.33 mg / L and LC90 of 12.08 mg / L. Figure 2 c, while neem remained the least toxic plant species.

[0047] Table 2 Residual toxicity of plant extracts to tobacco beetles after different periods of time

[0048]

[0049]

[0050] Among all the plant species tested, Artemisia annua showed the greatest toxicity to tobacco beetles, with an LC50 of 0.95 mg / L and an LC90 of 8.70 mg / L, followed by black cumin. In contrast, neem had the lowest toxicity, with an LC50 of 3.40 mg / L and an LC90 of 40.50 mg / L. At a concentration of 3.0%, Artemisia annua had the highest average mortality rate, reaching 90.00%, while neem had the lowest average mortality rate, at 50.83%, as shown in Figure 2. Figure 2 The above results indicate that guayule and black cumin may be effective botanical insecticides for controlling tobacco beetles, while the effect of neem is relatively weak.

[0051] Example 4: Direct (contact) toxicity test of plant extracts on tobacco beetles

[0052] This example uses a 7x7 factorial design to evaluate the direct (contact) toxicity of plant extracts to adult tobacco beetles. In this experiment, a total of 210 adult tobacco beetles were used, 30 adult tobacco beetles were used for each treatment, and each treatment was repeated three times, with each repeat containing 10 beetles. The purpose of the experiment was to determine the lethal effects of different plant extracts on tobacco beetles, especially the mortality rate after different time points. Tobacco beetle adults are considered dead when they do not respond to slight touch, and this criterion is used to assess mortality at intervals of 24 hours to 96 hours. The Abbott formula is used to correct the mortality data, and logarithmic probability analysis is used to determine the LC50 and LC90 values, which represent the concentrations of plant extracts that cause 50% and 90% of tobacco beetle deaths, respectively.

[0053] The experimental results showed that the lethal effect of the plant extracts increased with increasing concentration. After 24 hours of exposure, Parthenium hysterophorus showed the lowest LC50 = 6.80 mg / L and LC90 = 95.78 mg / L, as shown in Table 3 below. At a concentration of 3.0%, the mortality rate of the black cumin extract against tobacco beetles was 52.50%, while neem (Azadirachta indica) showed the lowest mortality rate of 35.00%, as shown in the attached Figure 3 a. After 48 hours, Artemisia annua showed the lowest LC50 = 3.40 mg / L and LC90 = 25.60 mg / L, followed by black cumin. The average mortality rate of Artemisia annua was the highest, reaching 69.00%, as shown in the attached Figure 3 b. After 72 hours, the LC50 of guayule = 1.15 mg / L and LC90 = 8.30 mg / L were significantly lower than those of other plant species extracts, while neem was the least toxic plant species, showing LC50 = 1.85 mg / L and LC90 = 31.29 mg / L. At the maximum concentration of 3.0%, the average mortality of guayule to tobacco beetles was 87.00%, as shown in the attached Figure 3 As shown in c.

[0054] After 96 hours of exposure, black cumin showed the greatest toxicity with LC50 = 0.46 mg / L and LC90 = 6.62 mg / L, followed by guayule with LC50 = 0.92 mg / L and LC90 = 6.58 mg / L. Neem remained the least effective plant species, showing the lowest toxicity with LC50 = 1.82 mg / L and LC90 = 12.86 mg / L. At 3.0% concentration, black cumin and guayule had an average mortality rate of 90.00% and 89.00% against tobacco beetles, respectively. Figure 3These results suggest that black cumin and guayule may be effective botanical insecticides for controlling tobacco beetles, whereas neem is less effective.

[0055] Table 3 Direct toxicity of plant extracts to tobacco beetles at different times

[0056]

[0057]

[0058] Example 5: Test on the repellent effect of plant extracts on tobacco beetles

[0059] The present invention examines the repellent effect of six different concentrations of five plant species on tobacco beetles. A 16 cm diameter culture dish was used, and the bottom of the culture dish was cut in half, affixed with Whatman filter paper No.1, and then evenly placed. Half of the filter paper was treated with plant species extracts (250 mg / L, 500 mg / L, 750 mg / L, 1000 mg / L, 1250 mg / L and 1500 mg / L), and the other half was treated with distilled water. The filter paper was dried for 20 minutes and then carefully placed in the culture dish, evenly placed edge to edge.

[0060] Twenty newly emerged adult beetles, mostly of the same size and age, were arranged in 10 pairs and placed in the center of the treatment area in a Petri dish. To prevent the insects from escaping, the Petri dish was covered with gauze and maintained in an incubator at 27±°C with a relative humidity of 65±5%. Thirty beetles were used for each treatment, and each treatment contained three replicates with 10 beetles in each replicate. The distribution of the insects was observed every hour, after 1 hour, 12 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours. After this exposure period, the percent repellency (PR%) was calculated using the following formula:

[0061] PR(%)=(Nc-Nt) / (Nc+Nt)×100

[0062] Nc = number of beetles in the untreated area

[0063] Nt = number of beetles in the treatment area

[0064] Where Nc represents the number of insects in the control segment, and Nt represents the number of insects in the treatment segment. Different grades are assigned according to the average repellency percentage, including Class 0 = PR 0-0.1%; Class I (0.2-10%); Class II (20.1-40%); Class III (40.1-60%); Class IV (60.1-80%); and Class V (80.1-100%).

[0065] In the one-hour plant extract exposure test on tobacco beetles, black cumin showed the greatest repellency at concentrations of 250mg / L, 500mg / L, 750mg / L and 1000mg / L, which is classified as Class III. Guayule recorded the repellency of the test insects at higher concentrations of 1250mg / L and 1500mg / L, as shown in Table 4 below. Although there was no statistically significant difference in the repellency of black cumin and guayule at different concentrations, the repellency of both was significantly higher than that of neem, oriental arborvitae and gentian (classified as Class II). The average percentage repellency of black cumin extract was the highest at 34.9%, while that of neem was the lowest at only 24.1%, as shown in the attached Figure 4 As shown in a.

[0066] After 12 and 24 hours, tobacco beetles showed the greatest repellency to all concentrations of black cumin, with percentages ranging from 31.44% to 63.54% (classified as Class IV), while neem caused the least repellency, with percentages ranging from 20.50% to 43.92% (classified as Class III). The average percentage repellency of black cumin was significantly greater than that of the other plant extracts, at 47.8% and 53.30%. Neem had significantly lower repellency to tobacco beetles during this time period, at 31.6% and 41.5%, respectively, as shown in the attached Figure 4 b-4c. After 72 hours, the average percentage repellency of tobacco beetles to black cumin extract was significantly greater than that to neem and daffodil extracts, while there was no significant difference in the repellency of black cumin, oriental arborvitae, and guayule extracts. The repellency caused by neem and daffodil was significantly lower than that caused by the extracts of the other three plant species, such as Figure 3 As shown in d.

[0067] After 72 and 96 hours, tobacco beetles were significantly more repellent to the six concentrations of black cumin: 54.50%, 63.23%, 71.12%, 79.10%, 85.50% and 93.20% and 62.10%, 78.72%, 84.52%, 92.10%, 100.00% and 100.00% (Class V). Although the repellency of neem increased with increasing exposure time, its repellency remained significantly lower at all six concentrations (35.225%, 40.62%, 46.32%, 52.50%, 58.22% and 65.22% and 42.22%, 51.23%, 58.64%, 62.70%, 71.33% and 78.33%) (Class IV). Likewise, the mean percentage repellency of the test insects to black cumin was significantly higher (74.4% and 86.2%), while the mean percentage repellency to neem was significantly lower (49.7% and 60.7%), as shown in the attached Figure 4e-4f. The average percentage repellency of black cumin was significantly higher than that of neem and daffodil extracts, but not significantly different from that of oriental arborvitae and guayule extracts. Overall, black cumin and guayule extracts had the strongest repellency against tobacco beetles, while neem had the weakest effect.

[0068] Table 4 Repellency of plant extracts to tobacco beetles after exposure for different time periods

[0069]

[0070]

[0071] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A plant extract for killing pests, characterized in that: The extract is selected from one or more of Adhatodavasica Nees, Azadirachta indica, Nigella sativa, Parthenium hysterophorus and Thuja orientalis, and contains phytosterols, saponins, diterpenes, flavonoids and alkaloids.

2. The plant extract according to claim 1, characterized in that: The extract is used to control the tobacco beetle Lasiodermaserricorne.

3. The plant extract according to claim 1, characterized in that: The concentration of the extract is 250 mg / L to 1500 mg / L.

4. A method for preparing a plant extract for killing pests, characterized in that: The method comprises the following steps: a. Collect leaves of neem, black cumin, guayule and oriental arborvitae; b. The collected leaves were air-dried at room temperature for 15 days; c. Grind the dried leaves into a fine powder; d. The powder was mixed with a solvent and extracted at room temperature using a 50% concentration by soaking at 12 hour intervals; e. Use a rotary evaporator to remove the solvent at low temperature and pressure to obtain the plant extract.

5. The method according to claim 4, characterized in that: The concentration of the extract was adjusted to the desired concentration by rotary evaporation.

6. A method for killing pests using the plant extract according to any one of claims 1 to 3, characterized in that: The method comprises applying the extract to the pest infested area at a concentration of 750 mg / L.

7. A method for evaluating the toxicity and repellent effect of plant extracts on tobacco beetles, characterized in that: The method comprises the following steps: S1: plant extracts were applied to tobacco beetles at different concentrations; S2: Check the mortality of tobacco beetles after 24, 48, 72, and 96 h; S3: Determine LC50 and LC90 using Probit analysis.

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