Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of a mosquito larvicidal control, and in specific, relates to a bio-degradable mosquito larvicidal composition that utilizes enzymes (Papain-like cysteine proteases (PLCP)) and ricin protein derived from plants to enhance larvicidal activity, thereby presenting a viable alternative to chemical insecticides.
Background of the invention:
[0002] Mosquitoes that breed in habitats made of containers can spread viral diseases. The viral diseases that cause multiple illnesses, including dengue fever, malaria, yellow fever and Japanese encephalitis, are spread by mosquitoes. With an estimated 2.5 billion people worldwide and 50–100 million new cases each year, dengue fever is prevalent in more than 100 countries in Africa, America, and the eastern Mediterranean.

[0003] Aedes aegypti, the primary vector, is a Cosmo tropical species that thrives in water containers near and inside homes. It is grown in a variety of household containers, including plant or flower pots, barrels, tanks and jars for storing water. The development of new sedatives and mosquito control tactics is essential in order to solve the issues. Due to their eco-safety, target specificity, lack of resistance development, reduced number of applications, increased acceptability and suitability for rural regions, mosquito repellents are an excellent choice for repelling mosquitoes.

[0004] Mosquitoes are known vectors for a range of debilitating diseases such as malaria, dengue fever, Zika virus, and chikungunya. Traditional chemical insecticides, while effective, pose significant risks to the environment and human health due to their toxicity and persistence. Consequently, there is an increasing demand for alternative solutions that minimize ecological impact while providing effective pest control.

[0005] Natural properties of papain-like cysteine protease enzymes extracted from the latex of Carica papaya, a plant known for its defense mechanisms against pests. These enzymes are proteolytic in nature, meaning they hydrolyze peptide bonds and disrupt the structural integrity of proteins. Experimental evidence has shown that these enzymes exhibit larvicidal activity by degrading the peritrophic membrane in mosquito larvae, which is essential for their survival.

[0006] Carica papaya latex with Ricin protein from castor seeds to enhance the larvicidal effect. Ricin, a potent protein, works synergistically with the papain-like enzymes to improve efficacy. The composition not only demonstrates high effectiveness in killing mosquito larvae but is also biodegradable and environmentally friendly, offering a cost-effective alternative to conventional chemical insecticides. This combination addresses the pressing need for safer pest control methods, making a significant advancement in the fields of environmental management, biopesticides, and public health.

[0007] In existing technology, an insecticide composition is known. The composition comprises at least one essential oil with at least one acid function, at least one proteolytic enzyme, a surfactant, and amphoteric. The composition is characterized with one or more alcoholates of fresh leaves, dried and crushed. The crushed leaves, berries, and bulbs of plant species selected from the group comprises Allium sativum, Julgans regia, Azadirachta indica, Sambucus nigra, Quillaja sapaonaria, and mixtures. Preparing the composition is characterized in that essential oils are mixed with constant stirring for 6 hours at a room temperature, then the surfactant, the proteolytic enzymes, and optionally the alcoholates are added gradually and separately with constant stirring for at least 1 hour. The composition destroys flying and crawling insects with their eggs, larvae, and nymphs. However, the composition might cause environmental issues. Moreover, the composition is prepared by a complex process.

[0008] Therefore, there is a need for a composition that utilizes enzymes (Papain-like cysteine proteases (PLCP)) and ricin protein derived from plants to enhance larvicidal activity, thereby presenting a viable alternative to chemical insecticides. There is also a need for a composition that is easily adapted for various application methods, including direct application in water bodies or incorporation into other pest control systems. There is also a need for a composition that is cost-effective, involving readily available raw materials and simple processing steps, thereby making it accessible for large-scale production. Further, there is also a need for a composition that combines raw carica papaya latex with ricin protein from castor seeds with enhanced larvicidal activity, thereby offering a synergistic effect that improves efficacy.
Objectives of the invention:
[0009] The primary objective of the present invention is to provide a composition that utilizes enzymes (Papain-like cysteine proteases (PLCP)) and ricin protein derived from plants to enhance larvicidal activity, thereby presenting a viable alternative to chemical insecticides.

[0010] Another objective of the present invention is to provide a composition that is effective in killing mosquito larvae even in small amounts of "fresh" milky latex, whether diluted with deionized water or dried and reconstituted.

[0011] The other objective of the present invention is to provide a composition that dries the collected latex at a temperature for grinding into a fine powder, and mixes the powder with a distilled water, thereby ensuring a consistent and practical composition method.

[0012] The other objective of the present invention is to provide a composition that is easily adapted for various application methods, including direct application in water bodies or incorporation into other pest control systems.

[0013] The other objective of the present invention is to provide a composition that is cost-effective, involving readily available raw materials and simple processing steps, thereby making it accessible for large-scale production.

[0014] The other objective of the present invention is to provide a composition that causes viability of mosquito larvae in test tubes with dried latex indicating that the larvae are killed by the active enzymatic contents rather than by asphyxiation caused by latex viscosity.

[0015] Yet another objective of the present invention is to prepare a composition that combines raw carica papaya latex with ricin protein from castor seeds with enhanced larvicidal activity, thereby offering a synergistic effect that improves efficacy.

[0016] Further objective of the present invention is to provide a composition that achieves optimal larvicidal effects at a latex-to-ricin ratio of 1:3 and 1:2, thereby balancing effectiveness with minimal degradation of ricin, thereby ensuring a potent and stable larvicidal agent.
Summary of the invention:
[0017] The present disclosure proposes a bio-degradable mosquito larvicidal composition and method of preparing the same. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0018] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a bio-degradable mosquito larvicidal composition that utilizes enzymes (Papain-like cysteine proteases (PLCP)) and ricin protein derived from plants to enhance larvicidal activity, thereby presenting a viable alternative to chemical insecticides.

[0019] According to one aspect, the invention provides the mosquito larvicidal composition. In one embodiment, the mosquito larvicidal composition comprises 30 to 35 weight percentage of Carica papaya latex, and 65 to 70 weight percentage of ricin protein from castor seeds. The mosquito larvicidal composition is bio-degradable with cysteine protease of Carica papaya having a larvicidal preventive effect that destroys peritrophic membrane of the mosquito larvae in water bodies to which it is applied, thereby enhancing the larvicidal activity.

[0020] In one embodiment, the Carica papaya latex comprises Papain-like cysteine proteases (PLCP), which is effective in breaking down the peritrophic membrane in the mosquito larvae. In another embodiment, the Carica papaya latex is a form of fresh milky latex that is diluted with deionized water to effectively kill mosquito larvae. In one embodiment, the ricin protein is extracted using a standard ammonium sulfate precipitation method.

[0021] In one embodiment, the mosquito larvicidal composition is prepared by a method. At one step, the Carica papaya latex dries at a temperature of at least 450 °C for a time period of at least 24 hr. At another step, the dried Carica papaya latex grinds to obtain a fine powder.

[0022] At another step, the at least 6.84 gm of the fine powder mixes with at least 200 mL of distilled water to form a latex extract. Further, at another step, the latex extract mixes with the ricin protein to create the mosquito larvicidal composition. The fine powder of Carica papaya latex is mixed with the ricin protein in an aqueous medium.

[0023] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0024] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0025] FIG. 1 illustrates a schematic view of a mosquito larvicidal composition, in accordance to an exemplary embodiment of the invention.

[0026] FIG. 2A illustrates a schematic view of a Carica papaya allow a user to extract papaya latex, in accordance to an exemplary embodiment of the invention.

[0027] FIG. 2B illustrates a schematic view of a fine powder of the Carica papaya latex, in accordance to an exemplary embodiment of the invention.

[0028] FIG. 2C illustrates a schematic view of mosquito larvae dipped in a test tube with a distilled water, in accordance to an exemplary embodiment of the invention.

[0029] FIG. 2D illustrates a schematic view of the mosquito larvicidal composition that effectively kill the mosquito larvae, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0030] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0031] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a bio-degradable mosquito larvicidal composition 100 that utilizes enzymes (Papain-like cysteine proteases (PLCP)) and ricin protein 104 derived from plants to enhance larvicidal activity, thereby presenting a viable alternative to chemical insecticides.

[0032] According to one exemplary embodiment of the invention, FIG. 1 refers to a schematic view of the mosquito larvicidal composition 100. The mosquito larvicidal composition 100 is effective in killing mosquito larvae even in small amounts of "fresh" milky latex, whether diluted with deionized water or dried and reconstituted. The mosquito larvicidal composition 100 is easily adapted for various application methods, including direct application in water bodies or incorporation into other pest control systems. The mosquito larvicidal composition 100 achieves optimal larvicidal effects at a latex-to-ricin ratio of 1:3 and 1:2, thereby balancing effectiveness with minimal degradation of ricin, thereby ensuring a potent and stable larvicidal agent.

[0033] In one example embodiment herein, the fresh latex was obtained from developing green fruits directly picked from nearby trees. Three or four vertical incisions were made in the fruits with a sharp stainless-steel instrument to a depth of 2 to 3 mm. The latex that surfaces after incision lasts only 1 to 2 minutes and then rapidly coagulates and can be collected into a glass container by a user. After extraction, the latex was immediately used for the purification of papain in its native state or stored at -8 °C protected under a nitrogen atmosphere.

[0034] In one embodiment herein, the ricin protein 104 is extracted from castor seeds using a standard ammonium sulfate precipitation method. Additionally, the ammonium sulfate precipitation method is used to extract and purify ricin protein 104 from the castor seeds (Ricinus communis) extracts. At one step, grind the castor beans and extract the proteins with a buffer (example: phosphate buffer). At another step, add ammonium sulfate to the crude extract to a saturation point of 40 to 50 percent. At another step, centrifuge and collect the supernatant containing ricin and other soluble proteins.

[0035] At another step, add more ammonium sulfate to the supernatant to increase the saturation point of 60-80 percent. This precipitates ricin and other proteins. At another step, centrifuge and collect the pellet containing ricin. Further, at another step, dissolve the pellet in the buffer and dialyze the same buffer to remove excess ammonium sulfate.

[0036] In another embodiment herein, the extracted ricin might be composed of impurities and further purification might be achieved using techniques including gel filtration chromatography, ion exchange chromatography, and affinity chromatography. The user handles the ricin with extreme caution due to its high toxicity.

[0037] In one embodiment herein, the mosquito larvicidal composition 100 comprises 30 to 35 weight percentages of Carica papaya latex 102, and 65 to 70 weight percentages of ricin protein 104 from castor seeds. The mosquito larvicidal composition 100 is bio-degradable with cysteine protease of Carica papaya having a larvicidal preventive effect that destroys peritrophic membrane of the mosquito larvae in water bodies to which it is applied, thereby enhancing the larvicidal activity.

[0038] The biodegradable nature of the mosquito larvicidal composition 100 prevent reinfestation, thereby making sustainable and long-term solution for mosquito control in various aquatic environments. The reinfestation of mosquitoes in water bodies utilizing the mosquito larvicidal composition 100 derived from the Carica papaya latex 102 and the ricin protein 104. The reinfestation disrupts the structural integrity of the mosquito larvae's midgut, leading to their death.

[0039] In one embodiment herein, the Carica papaya latex 102 includes Papain-like cysteine proteases (PLCP), which is effective in breaking down the peritrophic membrane in the mosquito larvae. The Carica papaya latex 102 is a form of fresh milky latex that is diluted with deionized water to effectively destroy the mosquito larvae.

[0040] According to another exemplary embodiment of the invention, FIG. 2A refers to a schematic view 200 of the Carica papaya that allow the user to extract papaya latex. In one embodiment herein, the fresh latex was collected from developing green fruits that were directly picked from nearby trees. To obtain the latex, three or four vertical incisions were made on the fruits using a sharp stainless-steel instrument, about 2-3 mm deep. The latex emerged from the incisions and remained fluid for only 1-2 minutes before rapidly coagulating. It was then collected into a container by the user. For example, the extracted latex was immediately used for native-state papain purification to preserve its properties.

[0041] In one example, the physical properties of the Carica papaya latex 102 are a thick, milky liquid with a viscosity similar to that of honey. The Carica papaya latex 102 is white or slightly yellowish in color. The pH value of Carica papaya latex 102 is slightly acidic to neutral (pH 6.5-7.5). The Carica papaya latex 102 comprises a mixture of proteolytic enzymes, including papain, chymopapain, and caricain, which break down proteins into smaller peptides and amino acids. In one example, the Carica papaya latex 102 exhibits antimicrobial activity against various microorganisms, including bacteria, fungi, and viruses. The Carica papaya latex 102 having anti-inflammatory properties, which may contribute to its traditional use in wound healing and pain relief.

[0042] According to another exemplary embodiment of the invention, FIG. 2B refers to a schematic view 202 of a fine powder of the Carica papaya latex 102. In one embodiment herein, the Carica papaya latex 102 is dried at a temperature of 450 °C for a time period of at least 24 hr. The dried Carica papaya latex 102 grinds into a fine powder (as depicted in FIG. 2B) using a mortar and pestle.

[0043] For example, the grinding parameters include time, speed, and powder characteristics. The fine powder is configured to improve solubility, enhanced larvicidal activity, easier application, and enhance shelf life. The fine powder is used in various applications, including mosquito control services, public health programs, agricultural applications, and household usages.

[0044] In one embodiment herein, the method for preparing the mosquito larvicidal composition 100. At one step, the Carica papaya latex 102 dries at least temperature of 450 °C for at least the time period of at least 24 hr. At another step, the dried Carica papaya latex 102 grinds into the fine powder using the grinding machines. At another step, the at least 6.84 gm of fine powder mixes with 200 mL of distilled water to form the latex extract. Further, at another step, the latex extract combines with ricin protein 104 in order to create the mosquito larvicidal composition 100.

[0045] According to another exemplary embodiment of the invention, FIG. 2C refers to a schematic view 204 of the mosquito larvae dipped in a test tube with distilled water. In one embodiment herein, the test container is a transparent test tube filled with the distilled water, either dechlorinated water, and is equipped with several small mosquito larvae. The mosquito larvae are visible, dark specks during erratically within the water column. The test tube is held upright in a test tube rack. In one embodiment herein, the mosquito larvae are intended to be effective and provide a clear visual reference for controlling mosquito populations by eliminating their larval stage.

[0046] In one embodiment herein, the experimental is demonstrated the larvicidal activity of the enzymes against mosquito larvae. The mode of action involves the enzymes breaking down the peritrophic membrane in the mosquito’s midgut, which includes chitin and proteins, thereby leading to the larvae’s death.

[0047] To evaluate the efficacy of latex in killing mosquito larvae, both undried and dried latex preparations were used in separate experiments. In the undried latex experiment, adding different volumes of latex to test tubes containing mosquito larvae resulted in 100 percent mortality within 24 hours. However, it was unclear whether the larvae died due to the latex's viscosity or its active components. To address this, a second experiment used dried latex preparation, which ruled out the possibility of asphyxiation caused by latex viscosity. The results conclusively showed that the mosquito larvae were killed by the bioactive compounds present in the latex, the papain-like cysteine protease from Carica papaya. The Carica papaya latex 102 is a promising, biodegradable, and cost-effective alternative to chemical insecticides for controlling mosquito populations and combating vector-borne diseases.

[0048] In one example, the mosquito larvicidal composition 100 for killing mosquito larvae, both undried and dried latex preparations were used in separate test tubes. Next, the test tubes are incubated along with the mosquito larvae and latex preparations for at a time period of 24 hr. Next, the observes the viability of the mosquito larvae in each test after the incubation period, thereby determining the mosquito larvae are killed. Next, the evaluated mosquito larvae in the undried latex samples are non-viable, thereby confirming the larvicidal activity by the latex contents and not due to viscosity.

[0049] Next, the mosquito larvae in the test tubes are confirmed with dried latex preparation are non-viable, thereby ruling out asphyxiation caused by latex viscosity and coagulation as a cause of larval mortality. Finally, the mosquito larvicidal composition 100 suggests effective killing of mosquitoes, thereby making it promising biodegradable, and effective alternative to chemical insecticides for controlling mosquito populations and combating vector-borne diseases.

[0050] According to another exemplary embodiment of the invention, FIG. 2D refers to a schematic view 206 of the mosquito larvicidal composition 100 that effectively kills the mosquito larvae. In one embodiment herein, the experiment demonstrates the larvicidal activity of the mosquito larvicidal composition 100. The cloudy liquid in the petri dish is a diluted form of the latex extract or a composition containing both Carica papaya latex 102 and ricin protein 104. The presence of dead mosquito larvae within this liquid provides visual evidence of the mosquito larvicidal composition 100 efficacies in killing the mosquito larvae.

[0051] The mosquito larvicidal composition 100 is suggestive of the active larvicidal agent, which is likely derived from papaya latex. The presence of dead mosquito larvae indicates the successful application of the mosquito larvicidal composition 100 in controlling mosquito populations. The quantifying number of dead larvae could provide additional data to support the efficacy claims using a microscope image to show the morphological changes in the dead larvae due to the action of the larvicidal agent could provide deeper insights into the mode of action.

[0052] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, the mosquito larvicidal composition 100 is disclosed. The proposed mosquito larvicidal composition 100 is effective in killing mosquito larvae even in small amounts of "fresh" milky latex, whether diluted with deionized water or dried and reconstituted. The proposed mosquito larvicidal composition 100 dries the collected latex at a temperature for grinding into a fine powder, and mixes the powder with a distilled water, thereby ensuring a consistent and practical composition method.

[0053] The proposed mosquito larvicidal composition 100 is easily adapted for various application methods, including direct application in water bodies or incorporation into other pest control systems. The proposed mosquito larvicidal composition 100 is cost-effective, involving readily available raw materials and simple processing steps, thereby making it accessible for large-scale production. The proposed mosquito larvicidal composition 100 is non-viability of mosquito larvae in test tubes with dried latex, which indicates that the larvae are killed by the active enzymatic contents rather than by asphyxiation caused by latex viscosity.

[0054] The proposed mosquito larvicidal composition 100 combines raw Carica papaya latex 102 with ricin protein 104 from castor seeds with enhanced larvicidal activity, thereby offering a synergistic effect that improves efficacy. The proposed mosquito larvicidal composition 100 achieves optimal larvicidal effects at a latex-to-ricin ratio of 1:3 and 1:2, thereby balancing effectiveness with minimal degradation of ricin, thereby ensuring a potent and stable larvicidal agent.

[0055] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
, Claims:CLAIMS:
I/We Claim:
1. A mosquito larvicidal composition (100), comprising:
30 to 35 weight percentage of Carica papaya latex (102); and
65 to 70 weight percentage of ricin protein (104) from castor seeds,
wherein the mosquito larvicidal composition (100) is bio-degradable with cysteine protease of Carica papaya having a larvicidal preventive effect that destroys peritrophic membrane of the mosquito larvae in water bodies to which it is applied, thereby enhancing the larvicidal activity.
2. The mosquito larvicidal composition (100) as claimed in claim 1, wherein the Carica papaya latex (102) comprises Papain-like cysteine proteases (PLCP), which is effective in breaking down the peritrophic membrane in the mosquito larvae.
3. The mosquito larvicidal composition (100) as claimed in claim 1, wherein the Carica papaya latex (102) is a form of fresh milky latex that is diluted with deionized water to effectively kill mosquito larvae.
4. The mosquito larvicidal composition (100) as claimed in claim 1, wherein the ricin protein (104) is extracted using a standard ammonium sulfate precipitation method.
5. The mosquito larvicidal composition (100) as claimed in claim 1, wherein the mosquito larvicidal composition (100) is prepared by a method, wherein the method comprises:
drying the Carica papaya latex (102) at a temperature of at least 450 °C for a time period of at least 24 hr;
grinding the dried Carica papaya latex (102) to obtain a fine powder;
mixing at least 6.84 gm of the fine powder with at least 200 mL of distilled water to form a latex extract; and
mixing the latex extract with the ricin protein (104) to create the mosquito larvicidal composition (100).
6. The mosquito larvicidal composition (100) as claimed in claim 4, wherein the fine powder of Carica papaya latex (102) is mixed with the ricin protein (104) in an aqueous medium.