Trichophyllum multiforme extract and application thereof in preparation of pest repellent

Abstract

The invention discloses a lass multiforme extract and application of the lass multiforme extract in preparation of a pest repellent, and belongs to the field of pest repellent. The preparation method comprises the following steps: carrying out reflux extraction on the hairy moss for several times by using ethanol, and merging extract liquids; the preparation method comprises the following steps: extracting a leaf moss extract, carrying out rotary evaporation on the extract to obtain an extract paste, dissolving the extract paste, carrying out liquid separation extraction by using petroleum ether, ethyl acetate, n-butyl alcohol and / or water as an extraction solvent, carrying out extraction to obtain a leaf moss multiform extract, and carrying out evaporation to obtain a leaf moss multiform extract, namely the leaf moss multiform extract which has insect antifeedant activity. According to the invention, the antifeedant active components of the multiform leaf moss insects are screened, the research of the insect antifeedant experiment mainly depends on the scientific method for the insect antifeedant experiment when the insect pest resistance of the multiform leaf moss plants is explored, and the research shows that the multiform leaf moss different-phase extracting solution has the antifeedant activity on the insects.

Description

Technical Field

[0001] This invention belongs to the field of insect repellents, specifically relating to an extract of Polymorpha foliata and its application in the preparation of insect repellents. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Chemical pesticides still dominate the field of pest control, and their potential harm to humans and livestock is becoming increasingly apparent. Therefore, the development of natural insecticides is more urgent. Bryophytes are rich and diverse, but there are no reports of natural enemies of this plant group. Therefore, it is an important exploration path to find substances with insect-repelling activity and develop green and environmentally friendly pesticide products.

[0004] Bryophytes are an ancient group in the plant kingdom. Their secondary metabolites possess unique structures and diverse biological activities. However, in the field of pest control, especially in their development as pest repellents, they remain a treasure trove yet to be fully explored. Polymorphous leafy bryophytes (… Porella oblongifolia It is widely distributed in China and has a relatively large biomass, but whether it contains highly effective pest-repelling active ingredients and how to apply it to pest control are currently unknown, and there are no publicly available reports.

[0005] Therefore, based on the urgent need for the development of biopesticides, exploring the application potential of *Lysimachia polymorpha* in pest repellency not only helps to broaden the plant library of plant-derived pesticides, but also provides the possibility for developing new green pesticides with novel mechanisms of action. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this paper provides an extract of *Polymorpha foliata* and its application in the preparation of insect repellents.

[0007] The technical solution adopted in this invention is as follows: In a first aspect of the invention, a polymorphic leafy moss extract is provided, which is prepared by the following method: The polymorphic leaf moss was extracted several times by heating and reflux with an ethanol solution, and the extracts were combined. The extracts were then rotary evaporated to obtain an extract paste. The extract paste was dissolved and extracted by separation. The extraction solvents were petroleum ether, ethyl acetate, n-butanol and / or water. The resulting extract was then evaporated to obtain the polymorphic leaf moss extract, which is the polymorphic leaf moss extract. This polymorphic leaf moss extract has insect antifeedant activity.

[0008] In one or more embodiments of the present invention, the volume fraction of the ethanol solution is 80-85%, preferably 85%.

[0009] In one or more embodiments of the present invention, the liquid-liquid extraction method is as follows: the dissolved extract solution is first extracted with petroleum ether to obtain an aqueous phase I and a petroleum ether phase; then, ethyl acetate is added to aqueous phase I for extraction to obtain an aqueous phase II and an ethyl acetate phase; finally, n-butanol is added to aqueous phase II for extraction to obtain an aqueous phase III and a n-butanol phase. The obtained petroleum ether phase, ethyl acetate phase, n-butanol phase, and aqueous phase III are evaporated separately to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract, and aqueous extract, which are the extracts of *Polygonum multiforme*. The petroleum ether extract and n-butanol extract have a particularly significant effect in inhibiting insect feeding behavior.

[0010] Preferably, the volume ratio of the extract solution to petroleum ether is (1~4):4.

[0011] In one or more embodiments of the present invention, the extraction solvent is petroleum ether or n-butanol.

[0012] In a second aspect of the invention, the use of the polymorphic leafy moss extract in the preparation of pest repellents is provided.

[0013] In one or more embodiments of the present invention, the pest repellent has insect antifeeding activity.

[0014] In one or more embodiments of the present invention, the pests repelled by the pest repellent are diamondback moth larvae and / or beet armyworm larvae.

[0015] In a third aspect of the invention, an insect repellent is provided, which is prepared by dissolving the extract of the Polymorpha foliata in methanol to a concentration of 1-1000 μg / mL.

[0016] In one or more embodiments of the present invention, the concentration is 100~500 μg / mL.

[0017] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: This invention screens for an insect-repellent active ingredient in a polymorphic leaf moss. The exploration of the plant's ability to resist insect pests primarily relies on the scientific method of insect repellent experiments. Studies show that different phase extracts of this polymorphic leaf moss exhibit insect-repellent activity. In the leaf-dish method experiment, the high-concentration petroleum ether phase extract showed a particularly significant effect in inhibiting insect feeding behavior, indicating that the high-concentration petroleum ether phase extract of polymorphic leaf moss has strong insect-inhibiting activity. This provides laboratory data for further development of this plant-derived pesticide. Attached Figure Description

[0018] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 : Leaf disc area after insect feeding in petroleum ether phase at 24h, 48h, and 72h.

[0020] Figure 2 Bar chart of feeding area of ​​petroleum ether leaf butterfly. Different letters indicate significant differences between means. P≤0.05 (test). The values ​​are the mean (n=3).

[0021] Figure 3 : Leaf disc area after insect feeding in ethyl acetate phase at 24h, 48h, and 72h.

[0022] Figure 4 : Bar chart of feeding area of ​​Ethyl acetate leaf butterfly. Different letters indicate significant differences between means, P≤0.05 (test), and the values ​​are the mean (n=3).

[0023] Figure 5 Leaf disc area after insect feeding in the n-butanol phase at 24h, 48h, and 72h.

[0024] Figure 6 Bar chart of n-butanol feeding area of ​​the Butterfly phyllodes. Different letters indicate significant differences between means, P≤0.05 (test), and the values ​​are the mean (n=3).

[0025] Figure 7 Leaf disc area after feeding by aquatic insects at 24h, 48h, and 72h.

[0026] Figure 8 Bar chart of feeding area of ​​water-phase leaf butterflies. Different letters indicate significant differences between means, P≤0.05 (test). The values ​​are the average (n=3).

[0027] Figure 9 : Leaf disc area after insect feeding in petroleum ether phase at 24h, 48h, and 72h.

[0028] Figure 10 Bar chart of feeding area of ​​petroleum ether leaf butterfly. Different letters indicate significant differences between means. P≤0.05 (test). The values ​​are the mean (n=3).

[0029] Figure 11 : Leaf disc area after insect feeding in ethyl acetate phase at 24h, 48h, and 72h.

[0030] Figure 12: Bar chart of feeding area of ​​Ethyl acetate leaf butterfly. Different letters indicate significant differences between means, P≤0.05 (test), and the values ​​are the mean (n=3).

[0031] Figure 13 Leaf disc area after insect feeding in the n-butanol phase at 24h, 48h, and 72h.

[0032] Figure 14 Bar chart of n-butanol feeding area of ​​Phyllostachys nigra. Different letters indicate significant differences between means, P≤0.05 (test). Values ​​are averages (n=3).

[0033] Figure 15 Leaf disc area after feeding by aquatic insects at 24h, 48h, and 72h.

[0034] Figure 16 Bar chart of feeding area of ​​water-phase leaf butterflies. Different letters indicate significant differences between means. P≤0.05 (test). Values ​​are averages (n=3). Detailed Implementation

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: 1. Experimental Materials 1.1 Test materials The polymorphic variegated bryophyte was collected in July 2024 by Professor Zhou Jinchuan of Linyi University in Guanshan National Nature Reserve, Jiangxi Province, China, and identified as *Variegata*, a plant belonging to the family Variegataceae in the order Variegataales. Currently, the polymorphic variegated bryophyte is preserved in the research group's collection.

[0039] 1.2 Test Insects Diamondback moth larvae are raised indoors. The rearing method is as follows: First, select some larvae with good growth and development from the farm's cabbage field, inoculate them onto fresh cabbage leaves that have not been treated with pesticides, and place them in a suitable environment with a temperature of 20 to 26 degrees Celsius and a relative humidity of 50% to 70%. After the larvae pupate, carefully transfer the pupae to a petri dish lined with moist filter paper with tweezers, and then place the petri dish in a rearing box. After the pupae emerge as adults, place clean, moist filter paper with 10% honey in the rearing box to supplement the adults' nutrition. Also, place fresh, tender cabbage leaves of suitable size for the adults to lay eggs. Repeat this rearing operation to cultivate three generations of diamondback moths. Select 3rd instar larvae from each generation for activity testing experiments.

[0040] The rearing method for the beet armyworm is as follows: Purchase beet armyworm egg masses from an egg culture company, place them in a culture dish with good humidity control, and wait for the egg masses to hatch into larvae. Once the larvae hatch, transfer them to a special rearing box. Under indoor conditions, young leaves of Shanghai bok choy are used as the main food source for the larvae. Carefully feed the larvae until they grow into adults. To ensure that the adults have sufficient nutrition, prepare a 10% concentration of honey water to encourage the adults to lay eggs. Afterward, carefully collect these precious eggs, gently pick up the hatched larvae with a brush, and place them in a rearing box that is 20 cm long, 10 cm wide, and 5 cm high. Continue to feed these larvae with fresh young leaves at room temperature. Carefully select larvae that are in the same physiological state and in the 3rd instar for subsequent experiments.

[0041] 2 Experimental Methods 2.1 Extraction of polymorphic leafy mosses: First, material preparation is fundamental to the experiment. Fresh, healthy *Polygonum multiflorum* were selected, ensuring they were free from contamination and pests. Anhydrous ethanol, distilled water, and filter paper were also prepared. The choice of extraction method is crucial; this experiment employed the traditional solvent extraction method. The specific procedure was as follows: 5799 g of fresh *Polygonum multiflorum* was taken, washed with distilled water to remove surface impurities, and drained. The treated *Polygonum multiflorum* was placed in a 10-liter round-bottom flask, and 85% ethanol solution was added until it just covered the plant material. The mixture was soaked at room temperature for 12 hours, then refluxed for 2.5 hours. After cooling, it was filtered, and the filtrate was collected. The residue was then refluxed twice more using the same method. The filtrates were combined and rotary evaporated at 45°C under reduced pressure until the solvent was completely evaporated, yielding a thick, paste-like crude extract of *Polygonum multiflorum*. This experiment successfully extracted the crude extract of *Polygonum multiflorum* and preliminarily assessed its insect antifeedant activity. This provides basic data for further research on the application of the crude extract of *Polygonum multiflorum*. The obtained thick paste-like crude extract of Polymorpha polyphylla was completely dissolved in 1000 mL of purified water to obtain an aqueous solution of Polymorpha polyphylla, which was used as the sample for subsequent liquid-liquid extraction.

[0042] 2.2 Polar solvent extraction of polymorphic leafy mosses: 200 ml of petroleum ether and 50 ml of purified water containing the extract of Polymorpha polyphylla were poured into a 500 ml separatory funnel. The stopper was tightened and the funnel was inverted five times to thoroughly mix the petroleum ether and water. The funnel was placed on an iron stand until the boundary between the petroleum ether and water was clear and transparent. At this point, the aqueous phase was slowly released from the bottom of the separatory funnel and the petroleum ether phase was poured out from the top. This process was repeated three times. Then, 200 ml of ethyl acetate and the previously separated aqueous phase were placed back into the 500 ml separatory funnel and the same steps were repeated three times to obtain the ethyl acetate phase. Then, 200 ml of n-butanol was used to extract the aqueous phase after the ethyl acetate extraction. The same procedure as the petroleum ether extraction was performed, and the extraction was repeated three times. Finally, the n-butanol phase and the remaining aqueous phase were obtained.

[0043] The remaining aqueous solution of the polymorphic leafy moss was subjected to liquid-liquid extraction using this method.

[0044] Following the extraction process, we obtained petroleum ether, ethyl acetate, n-butanol, and aqueous layers. These layers were then concentrated one by one by distillation using a rotary evaporator to obtain an extract. The results are as follows: Table 1 Drug Quality

[0045] The four-phase extract obtained by rotary evaporation was dissolved in methanol, transferred to a vial, dried, and then the sample powder of the polymorphic leafy moss four-phase was obtained, sealed and stored for later use.

[0046] The four-phase extract obtained by rotary evaporation was dissolved in methanol, transferred to a vial, dried, and then the sample powder of the polymorphic leafy moss four-phase was obtained, sealed and stored for later use.

[0047] 2.3 Experimental Methods for Insect Antifeeding Activity Test Diamondback moth larvae and beet armyworm larvae showed the most significant feeding preference for rapeseed leaves, with comparative analysis revealing that their feeding rate ranked first. The leaf disc method is frequently used by researchers to study insect antifeeding activities. The specific procedure is as follows: First, 210 third-instar larvae were selected and starved for 4 hours. Then, 672 leaf discs were punched from fresh rapeseed leaves using a 1.5 cm diameter punch. Finally, 42 holes were punched in a 9 cm diameter petri dish. Experimental group procedure: Take 10 mg of sample and add 2 ml of petroleum ether. Rinse with 18 ml of ultrapure water until all the sample is poured into an Erlenmeyer flask to prepare a 500 μg / ml petroleum ether solution. Take 6 ml of this solution and add 9 ml of water to prepare a 200 μg / ml petroleum ether solution. Finally, take 3 ml of the 500 μg / ml petroleum ether solution and add 12 ml of water to prepare a 100 μg / ml petroleum ether solution. Similarly, take ethyl acetate, n-butanol, and water, and prepare ethyl acetate, n-butanol, and aqueous solutions of different concentrations using the same method. Control group procedure: Add 18 ml of water to 2 ml of acetone.

[0048] The drug was added to a round petri dish containing leaflets. Using a small brush, all 24 leaflets (8 leaflets per experiment, with three parallel experiments) were thoroughly soaked in the drug, and then allowed to dry. The leaflets were then placed in plastic petri dishes lined with absorbent filter paper, with 8 leaflets in each dish and a third-instar diamondback moth larva placed on each leaflet. The area of ​​these leaflets was precisely measured at 24, 48, and 72 hours to obtain the final result of the non-selective feeding rejection rate.

[0049] 3. Results of the insect refusal-to-feed experiment: The following are the data results for the petroleum ether phase, ethyl acetate phase, n-butanol phase, and aqueous phase, respectively showing leaf disc illustrations 24 hours, 48 ​​hours, and 72 hours after insect feeding, as well as a bar chart of the average feeding area of ​​insects and the insect refusal rate.

[0050] In the significance test, if P(F≤f) is greater than 0.05, it means that the difference is not significant. When 0.01 is less than P(F≤f) and P(F≤f) is less than 0.05, the difference is relatively significant. When P(F≤f) is less than 0.01, the difference is extremely significant. The relationship between the F value and the F-critical value (Fcrit) must also be considered. If the actual F value is greater than the F-critical value, there is a significant difference between the two groups of data. If the actual F value is less than the F-critical value, there is no significant difference between the two groups of data. Such difference can be marked by symbols such as a, b, c, d, etc.

[0051] The algorithm for calculating the refusal-to-feed ratio is as follows: subtract the corresponding area of ​​the treatment group from the feeding area of ​​the leaves in the blank control group, divide the difference by the feeding area of ​​the blank group, and multiply by 100%. In this case, the total feeding area data of eight leaves is substituted into the above formula for accurate calculation.

[0052] 3.1 Results of petroleum ether phase analysis of diamondback moth Conclusion: Medium to high concentrations of petroleum ether extract from *Polygonum multiforme* can be observed from the bar chart (...). Figure 2 It is clearly visible that there is a significant insect-resistant and anti-feeding effect. Compared with the blank control group, the diamondback moth significantly reduced the feeding area on the leaf discs and showed a clear tendency to anorexia. Figure 1 And Table 2.

[0053] Table 2. Refusal rate of petroleum ether phase

[0054] 3.2 Results of Ethyl Acetate Phase Analysis of Diamondback Moth Conclusion: Figure 3 , Figure 4 As shown in Table 3, when diamondback moths are active in an ethyl acetate environment, medium and high concentrations of ethyl acetate have a significant inhibitory effect on their activity, while low concentrations of ethyl acetate can also produce an inhibitory effect, but this effect is relatively weak.

[0055] Table 3. Ethyl acetate phase rejection rate

[0056] 3.3 Results of n-Butanol Phase in Diamondback Moth Conclusion: Figure 5 , Figure 6 As shown in Table 2, lower and medium concentrations of n-butanol extract of Polymorpha spp. had a slight inhibitory effect on the feeding behavior of Diamondback moth, while higher concentrations significantly hindered its feeding activity.

[0057] Table 4. Refusal rate in the n-butanol phase

[0058] 3.4 Results of aqueous phase analysis of diamondback moth Conclusion: Figure 7 , Figure 8 As shown in Table 5, aqueous extracts of Polymorpha polymorpha at different concentrations inhibited the feeding behavior of Diamondback moth. The low-concentration solution showed the most significant and prominent insect-resistant and feeding-rejecting effect, while the high-concentration solution was less effective in this regard.

[0059] Table 5. Aquatic phase feeding rejection rate

[0060] 3.5 Results of petroleum ether phase analysis of Spodoptera litura Conclusion: Observe the bar chart ( Figure 10 It is clearly visible that medium and high concentrations of petroleum ether extract of *Spodoptera exaltata* exhibit significant insect-resistant and antifeedant effects. Compared with the blank control group, the feeding area of ​​leaf discs containing these concentrations of extract by *Spodoptera exaltata* was significantly reduced. Figure 9 As shown in Table 6.

[0061] Table 6. Refusal rate of petroleum ether phase

[0062] 3.6 Results of Ethyl Acetate Phase Analysis of Spodoptera litura Conclusion: Figure 11 , Figure 12 As shown in Table 7, the activity of Spodoptera litura is inhibited to varying degrees in the presence of ethyl acetate. Specifically, the inhibition of Spodoptera litura activity is particularly significant when ethyl acetate is present at medium to high concentrations, while low concentrations of ethyl acetate also have an inhibitory effect, but the effect is relatively weaker.

[0063] Table 7. Ethyl acetate phase rejection rate

[0064] 3.7 Results of n-Butanol Phase Analysis of Spodoptera litura Conclusion: Figure 13 , Figure 14 As shown in Table 8, the low concentration of the n-butanol phase extract of Polymorpha spp. did not significantly inhibit the feeding behavior of Spodoptera litura, but the increased concentration of the extract significantly hindered the feeding behavior of Spodoptera litura.

[0065] Table 8. Refusal rate in the n-butanol phase

[0066] 3.8 Results of aqueous phase analysis of Spodoptera litura Conclusion: Figure 15 , Figure 16 As shown in Table 9, aqueous extracts of Polymorpha polymorpha at different concentrations inhibited the feeding behavior of Diamondback moth. The low-concentration solution showed the most significant and prominent insect-resistant and feeding-rejecting effect, while the high-concentration solution was less effective in this regard.

[0067] Table 9. Aquatic phase feeding rejection rate

[0068] 4. Conclusion and Analysis This invention uses insect feeding refusal experiments to study the natural active substances in *Polygonum multiflorum*. After careful processing, *Polygonum multiflorum* extract yielded various extracts. The feeding behavior of third-instar diamondback moth larvae was significantly inhibited by the petroleum ether and n-butanol extracts of the *Polygonum multiflorum* ethanol extract. As the extract concentration gradually increased, the feeding refusal rate significantly increased, fully verifying the excellent insect feeding refusal activity of the petroleum ether and n-butanol phase extracts. The ethyl acetate and aqueous phases showed large variances and random fluctuations in experimental results, making them difficult to use as reliable scientific evidence. However, the bar chart visually showed that these two phases also had a significant inhibitory and avoidance effect on the feeding behavior of diamondback moths. The discovery that the *Polygonum multiflorum* ethanol extract does indeed contain insect feeding refusal active substances lays a preliminary experimental foundation for further development of this plant as a green and environmentally friendly insecticide.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An extract of a polymorphic leafy moss, characterized in that, It is obtained through the following preparation method: The polymorphic leaf moss was extracted several times by heating and reflux with an ethanol solution, and the extracts were combined. The extracts were then rotary evaporated to obtain an extract paste. The extract paste was dissolved and extracted by separation. The extraction solvents were petroleum ether, ethyl acetate, n-butanol and / or water. The resulting extract was then evaporated to obtain the polymorphic leaf moss extract, which is the polymorphic leaf moss extract. This polymorphic leaf moss extract has insect antifeedant activity.

2. The extract of *Polygonum multiforme* as described in claim 1, characterized in that, The separation extraction method is as follows: the dissolved extract solution is first extracted with petroleum ether to obtain aqueous phase one and petroleum ether phase; then ethyl acetate is added to aqueous phase one for extraction to obtain aqueous phase two and ethyl acetate phase; finally, n-butanol is added to aqueous phase two for extraction to obtain aqueous phase three and n-butanol phase. The obtained petroleum ether phase, ethyl acetate phase, n-butanol phase and aqueous phase three are evaporated respectively to obtain petroleum ether phase extract, ethyl acetate phase extract, n-butanol phase extract and aqueous phase extract, which are the extracts of Polymorpha foliata.

3. The extract of *Polygonum multiforme* as described in claim 1, characterized in that, The volume ratio of the extract solution to petroleum ether is (1~4):

4.

4. The extract of *Polygonum multiforme* as described in claim 1, characterized in that, The extraction solvent is petroleum ether or n-butanol.

5. The use of the extract of Polymorpha foliata according to any one of claims 1 to 4 in the preparation of pest repellents.

6. The application as described in claim 5, characterized in that, The pest repellent has insect antifeeding activity.

7. The application as described in claim 6, characterized in that, The pests repelled by the pest repellent are diamondback moth larvae and / or beet armyworm larvae.

8. An insect repellent, characterized in that, The pest repellent is prepared by dissolving the extract of Polymorpha foliata as described in any one of claims 1 to 4 in methanol to a concentration of 1 to 1000 μg / mL.

9. The pest repellent as described in claim 8, characterized in that, The concentration is 100~500μg / mL.

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