A biopesticide composition and method of preparation and use thereof

Abstract

The present invention relates to a biopesticide (18) composition comprising a mixture of a saponin (11) extract, an azadirachtin (12) extract, a citronella (13) extract, an ethoxylated castor oil surfactant (15), and a palmitic acid carrier (16) characterised in that the saponin (11) is extracted from Piper spp., the azadirachtin (12) is extracted from Azadirachta spp., and the citronella (13) is extracted from Backhousia spp. Accordingly, the invention also relates to a method (100) for preparing biopesticide composition to produce biopesticide (18) characterised in that the active ingredient (14) composition of saponin (11) and azadirachtin (12) is within the range of 1–30%, the inert ingredient (17) composition includes ethoxylated castor oil surfactant is within the range of 4–20% and palmitic acid carrier within the range of 40–80%, and the active ingredient (14) is 5–30% with the inert ingredient (17) of 70–95% to make the total biopesticide composition.

Description

[0001] A BIOPESTICIDE COMPOSITION AND METHOD OF PREPARATION AND USE THEREOF

[0002] FIELD OF INVENTION

[0003] The present invention generally relates to a biopesticide composition and the preparation method thereof by incorporating plant-based active ingredients comprising Backhousia spp., Azadirachta spp., and Piper spp. extracts and the preparation method thereof for use in agricultural applications.

[0004] BACKGROUND OF THE INVENTION

[0005] Crop loss in terms or quality and quantity reduction can occur in the field (preharvest) or in the storage (post-harvest) due to abiotic or biotic factors. Abiotic factors include climate (temperature, precipitation, humidity and others environmental factors), soil (such as soil composition, texture, structure, and fertility), water, topography (sloping terrain and altitude), and light. Meanwhile, biotic factors includes the types of plant and crops cultivated in the agricultural system, the microorganisms including bacteria and fungi presence in the soil, livestock, insects and pest which can be both positive and negative effect on agriculture as some insects act as crop pollination agent, while other can caused damage and reduced crop quality and quantity.

[0006] Malaysia's agriculture industry faces a number of insect-related challenges, which include the effect of pests on particular crops. For instance, different species of insect pests such as armyworms, caterpillars, beetles, aphids, whiteflies, mites, and thrips cause serious damage to different vegetable crops, which reduces yield and makes them unsuitable for human consumption. In another case, wireworms attack vegetables, especially at root stages, cutworms at the young seedlings, caterpillars, and beetles attack foliage and stems. While pests like aphids, whiteflies, and mites infested the young leaves and flowers of the plants. In order to control the pest in agricultural industry, insecticides are widely used in order to sustain crop yield and improve food quality. Unfortunately, excessive and inappropriate use of conventional broad spectrum insecticides have increased the number of insecticide resistance, secondary pest outbreaks, pesticide residues in food, public health effects, pollutions, and elimination of beneficial insects and non-targeted organisms. Synthetic pesticides are also often used to protect crops from pests and diseases. Nevertheless, their non-target toxicity, biodegradability, and residual consequences have become a major concern, which requires alternative adoption of environmentally friendly and reasonably priced pest control methods. The extensive use of insecticides in controlling this pest has resulted in the emergence of insecticide resistance insect and can caused major problem to the farmers. Therefore, it is crucial to identify potential bioinsecticide to control pest in agricultural land.

[0007] The developments of bioinsecticide has been studied wide around the globe as it is low-cost, safe for the environment, offer a targeted mode of action, and are sustainable. This means that organic pesticides typically breakdown in the environment, leaving no residual activity after a relatively short time. With proven efficacy against a wide range of pests and in numerous crop systems, essential oil-based insecticides have been used for well over a decade in the USA and, more recently, in the European Union (EU), Korea, and about a dozen other countries.

[0008] Nowadays, a great deal of insecticides is produced with active ingredients derived from rosemary, peppermint, cinnamon, clove, eucalyptus, and thyme oils, among others, either separately or in combination. Some products utilize a combination of oils, whereas others utilize a single essential oil or merely a single terpenoid component.

[0009] Numerous bioinsecticides have been investigated for their potential to manage golden apple snails. These comprise crude extracts from a variety of plants, such as Annona squamosa seed, Nerium indicum leaves, Stemona tuberose root, Cyperus rotundus corm, and Derris elliptica root. Bioinsecticides are more environmentally friendly than synthetic molluscicides. In addition, bioinsecticides are preferable compared to synthetic molluscicides, which may have adverse effects on the environment.

[0010] A prior Worldwide patent publication WO2022250524, with priority to Malaysian Patent MYPI2021002993A, discloses the invention of plant-based bioinsecticide composition. The invention comprises of 1% to 10% of saponin and azadirachtin extracted from Piper sarmentosum and Azadirachta indica, respectively. Addition to that, the invention discloses the application of 50% to 90% of carrier and 5% to 15% of surfactant of the invention are selected from palmitic acid and castor oil, respectively, similar to the current invention. However, the current invention did not disclose the citronella extracts extracted from Backhousia citriodora.

[0011] Another prior art European Patent EP3771335, discloses an invention that teaches a repellent composition containing margosa oil, lauric acid and a spreading agent. The compositions claimed by the invention include margosa extracts extracted from Azadirachta indica, saponin, citronella, palm oil, and castor oil. However, the invention did not disclosed the extraction of azarachtin from Azadirachta indica. Furthermore, saponin and citronella extracted from Piper sarmentosum and Backhousia citriodora are also not disclosed in the invention.

[0012] Meanwhile prior art Worldwide patent WO2021134127, discloses an invention of a composition and method of selection thereof. The invention includes a synergistic pesticidal complex compositions and methods for pesticidal active ingredients delivery. Some pesticidal compositions and methods disclosed in the invention is to enhanced fungicides, nematicides, and insecticides efficacies. The invention also disclosed a pesticidal composition comprising saponin, citronella, and azadirachtin from a wide range of plant sources, however the invention does not include Piper sarmentosum, Azadirachta indica, and Backhousia citriodora.

[0013] Another prior art from Worldwide patent WO2021041278A2, described a compositions and methods that relates to insecticides. The invention mentioned the insecticide comprises of azadirachtin and citronella extracts from Piper sarmentosum. However, the invention did not disclose incorporation of Azadirachta indica and the utilisation of saponins extract. In addition to that, the invention did not disclosed application of carrier from palmitic acid or palm oil and the use of ethoxylated castor oil as the surfactant.

[0014] Meanwhile prior art Australia patent AU2009350416, US patent US20220272987, and Worldwide patent WO2023052456 disclose the incorporation of saponins, azadirachtin, and citronella in the production of biopesticide with utilisation of ethoxylated castor oil as surfactant. However, none of the prior arts disclose the sources of the saponins, azadirachtin, and citronella extracts either from Piper sarmentosum, Azadirachta indica, and / or Backhousia citriodora. The prior arts also did not disclose the utilisation palmitic acid or palm oil as carrier in the biopesticide system.

[0015] At present, there is limited disclosure on the bioinsecticide formulation containing active ingredient extracted from Backhousia citriodora (lemon myrtle), Azadirachta indica (Neem), and Piper sarmentosum (wild betel). With this, the present invention shows the development of a new biopesticide and the composition preparation shows a necessity to combat emergence of pesticide resistance insects and to provide protection to the soil ecosystem.

[0016] SUMMARY OF THE PRESENT INVENTION

[0017] It is an objective of the present invention to provide a preparation method thereof for producing a biopesticide with the composition comprising plant-based bioactive extracts namely saponin, azadirachtin, and citronella, which is extracted from Piper sarmentosum (P. sarmentosum), Azadirachta indica (A. indica), and Backhousia citriodora (B. citriodora), respectively. The biopesticide also comprises of ethoxylated castor oil as surfactant and palmitic acid and / or palm oil as carrier for the extracts in the biopesticide system. Accordingly, these objectives may be achieved where the biopesticide composition is in the form of nanoemulsion with preferably having a size below 300 nm. The ratio between P. sarmentosum and A. indica is preferably between 2:8 to 5:5. Citronella extract is added as an additive with the percentage of 0.5% to 4.0%. The surfactant and carrier are from plant based whereby the surfactant is selected from natural triglycerides with a hydroxyl group, ethoxylated castor oil or castor beans, while the carrier is selected from palmitic acid derived from palm oil.

[0018] Accordingly, the amount of the saponin and azadirachtin is selected within the range of 1-30% with the amount of the surfactant and carrier is selected within the range of 4-20% and 40-80%respectively.

[0019] Accordingly, the embodiment of the present invention disclose a method of preparation of the production of biopesticide, characterised in that the active ingredient is composed of saponin and azadirachtin within a range of 1-30%, and the inert ingredient is composed of ethoxylated castor oil surfactant within the range of 4-20%, palmitic acid carrier within the range of 40-80%, and the total biopesticide composition includes 5-30%, preferably 25% of the active ingredients and 70-95%, preferably 75% of the inert ingredients.

[0020] The present invention shows advantage whereby the plant-based biopesticide composition is an eco-friendly and able to exhibit a synergistic effect against apple snail and whitefly.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The features of the invention will be more readily understood and appreciated from the following detailed description when read in conjunction with the accompanying drawing of the preferred embodiment of the present invention, in which:

[0023] Figure 1 is an illustration of a production method for biopesticide Figure 2 shows the effects of (a) F1 formulation, and (b) F2 formulation after 14 days at -20°C, 54°C and 2 months at 25°C

[0024] Figure 3 shows the bioassay effect against apple snail after (a) 24 hours, and (b) 96 hours

[0025] Figure 4 shows the bioassay effect against whitefly

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will be described in further details with the reference to the following examples in order to produce, to give technical solution and advantages of the present invention more apparent. It should be understood that certain preferred and optional embodiments described herein are for the purposes of illustration only and are not intended to limit the scope of the invention.

[0028] The embodiment of the present invention relates to a biopesticide formulation incorporated with active ingredients including saponin, azadirachtin and citronella extracted from Piper sarmentosum, Azadirachta indica and Backhousia citriodora respectively. The present invention shows that the combination of these three active ingredients exhibits positive effects against apple snail and whitefly, where the chemical substance or biological structures interact with the insect and mollusc results in a greater affect than the sum of the individual effect individually.

[0029] The embodiment of the present invention provides a biopesticide (16) which comprises the following components: saponin extract (11 ), azadirachtin extract (12), citronella extract (13), surfactant (14), and carrier (15).

[0030] The biopesticide provided by the embodiment of the present invention has the following composition after being improved: i) saponin extract from Piper sarmentosum (P. sarmentosum) ii) azadirachtin extract from Azadirachta indica (A.indica) iii) citronella extract as additive from Backhousia citriodora (B. citriodora) iv) surfactant from ethoxylated castor oil v) carrier from palmitic acid

[0031] According in one embodiment, the present invention discloses an application of plant-based extract comprising; a) Saponin: a naturally occurring compounds found in plants, which serves as an effective components in a biopesticide system. Saponin exhibits insecticidal, antifungal, and antimicrobial properties. Saponin mode of action is by disrupting cell membranes and vital processes in pests and pathogens. A part from that, saponin also acts as acting as a feeding deterrents and repellents which also protect crops from other herbivores. Their potential synergy with other pesticides enhances overall efficacy. Saponin can be integrated into integrated pest management (IPM) that offers a bio-based solution that is biodegradable and poses minimal harm to non-target organisms, contributing to a balanced and eco-friendly approach to pest control in agricultural systems. Furthermore, their environmentally friendly nature aligns with sustainable agriculture. b) Azadirachtin: a prominent component in neem tree extracts that can be as serves as a potent biopesticide with versatile functions. Azadirachtin can acts as a powerful insect growth regulator that can disrupt the insect molting process and inhibiting the development of larvae, nymphs, and pupae. Azadirachtin also exhibits antifeedant properties which can deter insect’s feeding activity thereby reducing damage to crops. Along with that, it possesses antifungal properties, contributing to the control of various plant diseases. As a biopesticide, azadirachtin able in targeting pests selectively while posing minimal harm to other beneficial organisms. It has a broad-spectrum effectiveness, low toxicity to humans and animals, and compatibility IPM strategies make azadirachtin a suitable substance for sustainable and eco-friendly pest control in agriculture. c) Citronella: can be derived from various plant species and is utilized in biopesticide system for its multifunctional in pest management. Citronella primary role in biopesticide is to act as a natural insect repellent. Citronella mode of action is by disrupting the sensory receptors of insects, deterring them from landing on treated surfaces and reducing feeding activity. Citronella is an effective biopesticide that can control the activity and infestation of flying insects. Additionally, citronella-based insect repellent provides low environmental impact and versatility contribute to its popularity as a biopesticide in I PM strategies for sustainable agriculture and public health. d) Ethoxylated castor oil: derived from castor beans and chemically modified, is used in biopesticides as a surfactant to improve pesticide effectiveness by enhancing their spreading, wetting, and penetration on plant surfaces. Ethoxylated castor oil can aids in an uniform distribution of active ingredients, maximizing pest contact. The emulsifying properties of ethoxylated castor oil ensure stability. It also able to reduce surface tension, promoting better and adherence to target organisms. e) Palmitic acid: Palmitic acid is a crucial carrier in biopesticides that can enhanced the delivery of active ingredients to pests. It improves solubility and stability of compounds, ensuring uniform dispersion in pesticide formulations. Palmitic acid also enhances biopesticide adhesion to plant surfaces, increasing coverage and persistence. Palmitic acid emulsifying properties provides a stable formulations, enhancing shelf life and ease of application. It promotes bioavailability and efficacy, aligning with environmentally friendly pest management practices in agriculture.

[0032] Ratio of the combined formulation of the active ingredients of P. sarmentosurrr.A. indica is preferably 2:8. B. citriodora extract acts as an additive with the range of preferably 0.5-4.0%. The composition of the biopesticide further comprises a plant-based carrier and a surfactant, whereby the carrier is palmitic acid derived from palm oil, and the surfactant is selected from ethoxylated castor oil, castor beans with carbon chain of C12-C18. The embodiment teaches the composition of the present invention is preferred to be in a nanoemulsion form with the size preferably below 300 nm with zeta potential of above -30 mV.

[0033] Another embodiment discloses that the present invention plant-based composition comprises a range of each substances, whereby the amount of saponins and azadirachtin are in the range of 1-30%. Accordingly, citronella as additive is within the range of 0.5-4%. In addition to that, the present invention comprises surfactant in the range of 4-20%, while the palmitic acid carrier is within the range of 40-80%. This composition is selected to ensure that the present invention can achieve high performance and functionality.

[0034] The embodiment of the present invention of a plant-based biopesticide composition showed synergistic effect towards apple snail and whitefly, indicating the present invention consist of a potent preparation composition. However, the present invention is harmless to human animal skin and safe for other mammals. Therefore, the present invention application does not cause environmental pollution, as it is biodegradable and resorbable by the plants.

[0035] In yet another embodiment, the final form of the present invention is in liquid form with the working concentration before application is 1 :2000. Addition to that, an alternative sources for saponin, azadirachtin, and citronella extracts can be from Helminthostachys zeylanica, Melia azadirachta, and Cymbopogon spp., respectively.

[0036] As shown in Figure 1 , the embodiment of the present invention disclose a preparation method (100) for the production of biopesticide (18), which comprises the following steps:

[0037] 101 , extraction of saponin (11 ), azadirachtin (12), and citronella (13) is done from P. sarmentosum, A.indica, and B. citriodora, whereby the amount of saponin and azadirachtin is in the range of 1-30%. 102, combination of saponin (11 ), azadirachtin (12), and citronella (13) to produce a mixture of active ingredient (14), whereby the ratio of the saponin to azadirachtin is betweeen 2:8 to 5:5 with 0.5-4% of citronella, preferably 4%.

[0038] 103, combination of surfactant (15), carrier (16), and water to produce inert ingredient (17), where the surfactant is ethoxylated castor oil and the carrier is palmitic acid and the ratio between surfactant:carrier:water is set at 14:60:26.

[0039] 104, mixing the active ingredient (14) and inert ingredient (17) to produce a biopesticide (18) where 25% of active ingredient is mixed with 75% of inert ingredient.

[0040] The embodiment of the present invention in Figure 2 discloses the effects of two formulation of the biopesticide namely, F1 formulation and F2 formulation after 14 days at -20°C, 54°C and 2 months at 25°C. The present invention discloses that F1 and F2 emulsion formulations had been produced as below:

[0041] Table 1 : Plant-based biopesticides compositions

[0042] Further characterisation of F1 and F2 emulsion formulations discloses that both formulations are in nanoemulsion form and the characteristics was evaluated based on particle size, zeta potential, polydispersity index (PDI), surface tension, viscosity, pH, and temperature stability as shown in Table 2.

[0043] Table 2: Characteristics of F1 and F2 biopesticide emulsion Therefore, as summary the biopesticide particles size is in the range of 180-230 nm, preferably less than 250 nm. Additon to that, have a zeta potential is in the range of -36.1 to -29.4 and PDI is between <0.2 to <0.4. Furthermore, the nanoemulsion have a surface tension between 33-34, have the viscosity of 77 to 910 mPa.s at pH 5, and have stability at temperature between -20-54°C. The stable formulation can be developed according to the results obtained to prove the the synergism efficacy of saponin from P. sarmentosum, azadirachtin from A. indica, and citronella from B. citriodora against apple snail and whitefly through emulsion formulations as joint toxicity could have a better potency in controlling pests.

[0044] Another embodiment of the present invention is shown in Figure 3 where the bioassay effect of the biopesticide is evaluated against apple snail after 24 and 96 hours. The combined formulation was diluted into three concentrations (half of the recommended concentration, recommended concentration, and double the recommended concentration) to evaluate the mortality bioassay of apple snail, where deionized water was used as control treatment. The mortality was evaluated at 24, 48, 72, and 96 hours. The snails are considered dead when they have no coordinated movement after being gently prodded.

[0045] Based on Figure 3, the formulation in the present invention results in a synergistic effect on the lethal concnetration (LC50) values when applied to the apple snail. In a time range between 24 to 72 hours, the LC50 required to control apple snail is between 0.008587 to 0.022129 ml / m2as disclosed in Table 3.

[0046] Table 3: The formulation LC50 values against apple snail at 24, 48 and 72 hours According to another embodiment of the present invention is shown in Figure 4 where The combination of three plants based extract in the present invention results in a synergistic effect on the LC50 values against whitefly. The evaluation is done in parts per million (mg / kg) of formulation at 24 to 72 hours showed that the LC50 required to control whitefly is between 296.067 ppm to 0.072 ppm as shown in Table 4.

[0047] Table 4: The formulation LC50 values against whitefly at 24, 48 and 72 hours

[0048] From the evaluation, the present invention also exhibit a time dependent factor whereby the longer exposure of pest to the present invention, the less formulation concentration to control the pests.

[0049] The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore indicated by the appended claims rather than by the foregoing description. All changes, which come within the meaning and range of equivalency of the claims, are to be embraced within their scope.

[0050] Description of the reference numerals used in the accompanying illustration according to the present invention:

[0051] Reference Numerals and Description

[0052] 11 Saponin

[0053] 12 Azadirachtin

[0054] 13 Citronella

[0055] 14 Active ingredient (saponimazadirachtin + 4% citronella)

[0056] 15 Ethoxylated castor oil surfactant 16 Palmitic acid carrier

[0057] 17 Inert ingredient (ethoxylated castor oil :palm itic acid)

[0058] 18 Biopesticide

[0059] 100 Production method of biopesticide 101 Extact (saponin, azadirachtin, and citronella)

[0060] 102 Combine (saponin, azadirachtin, and citronella)

[0061] 103 Combine (ethoxylated castor oil :palm itic acid)

[0062] 104 Mix (active and inert ingredient)

Claims

CLAIMS1 . A biopesticide (18) composition, comprising a mixture of: a saponin (11 ) extract; an azadirachtin (12) extract; a citronella (13) extract; an ethoxylated castor oil surfactant (15); and a palmitic acid carrier (16); characterised in that the saponin (11 ) is extracted from Piper spp., the azadirachtin (12) is extracted from Azadirachta spp., and the citronella (13) is extracted from Backhousia spp.

2. The biopesticide (18) composition according to claim 1 , wherein the saponin (11 ) is preferably extracted from Piper sarmentosum, the azadirachtin (12) is preferably extracted from Azadirachta indica and the citronella (13) is preferably extracted from Backhousia citriodora.

3. The biopesticide (18) composition according to claim 1 , wherein the composition is in a form of nanoemulsion.

4. The biopesticide (18) composition according to claim 2, wherein the nanoemulsion is with a particle size of less than 250 nm.

5. The biopesticide (18) composition according to claim 2, wherein the amount of palmitic acid carrier (16) is within the range of 40-80%.

6. The biopesticide (18) composition according to claim 1 , wherein the carrier is a vegetable-based palmitic acid (16) derived from palm oil with a carbon chain length of C12-C18.

7. The biopesticide (18) composition according to claim 1 , wherein the saponin extract (11 ) and the azadirachtin extract (12) is within a range of 1-30%.

8. The biopesticide (18) composition according to claim 1 , wherein the ethoxylated castor oil (15) surfactant is within a range of 4-20%.

9. The biopesticide (18) composition according to claim 1 , wherein the citronella (13) is added as an additive within the amount of 0.5-4.0%.

10. A method for using the biopesticide (18) composition of claim 1 for controlling apple snails (Pomacea canaliculata) or whiteflies (Bemisia tabaci), comprising: applying an amount of 0.08587-0.103767 ml / m2to a surface area having one or more apple snails for 24-72 hours; or applying an amount of 0.072-296.07 mg / kg to a surface area having one or more whiteflies for 24-72 hours.11 . A method (100) for preparing a biopesticide composition according to claim1 to produce biopesticide (18), comprising the steps of: extracting (101 ) a saponin (11 ) extract, an azadirachtin (12) extract, a citronella (13) extract from Piper spp., Azadirachta spp. and Backhousia spp.; combining (102) the saponin (11 ) extract, the azadirachtin (12) extract, the citronella (13) extract for producing an active ingredient (14); combining (103) an ethoxylated castor oil surfactant (15) with a palmitic acid carrier (16) with water for producing an inert ingredient (17); and mixing (104) the active ingredient (14) and inert ingredient (17) to produce a biopesticide (18), characterised in that the active ingredient (14) composition of saponin (11 ) and azadirachtin (12) is within a range of 1-30%, the inert ingredient (17) composition includes ethoxylated castor oil surfactant within the range of 4-20% and palmitic acid carrier within the range of 40-80%, wherein the active ingredient (14) is 5-30% with the inert ingredient (17) of 70-95% to make the total biopesticide composition.

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