Papain-based formulation for tick control.

Description

/ 8 Papain-based formulation for tick control. Field of invention

[001] The present invention consists of formulating a product from the enzyme papain, an enzyme from the papaya tree (Carica papaya), and its use in tick control.

[002] The present technology has the merit of using papain for a purpose not yet described, in a simple and low-cost process. The present invention will be used in the livestock sector as an alternative to the use of toxic pesticides in controlling tick infestations in various types of herds and any other types of animal breeding, without the risk of toxicity and contamination of humans, other animals and the environment.

[003] Furthermore, the present technology adds value to an enzyme that is already used in the pharmaceutical sector, providing added value to papain. The use of papain with a formulation suitable for use as an acaricide allows it to be used as a sustainable and complementary alternative to chemical acaricides. Fundamentals of the invention

[004] Ticks are hematophagous ectoparasites of the phylum Arthropoda, which feed on various animals, such as birds, mammals, reptiles and amphibians (FERNANDES, É.KK; BITTENCOURT, VR Entomopathogenic fungi against South American tick species. Experimental and Applied Acarology, v.46, p. 7193, 2008).

[005] Among the predominant Ixodidae tick species in South America, some are vectors of zoonoses, such as the species Rhipicephalus microplus (cattle tick), Rhipicephalus sanguineus sl (brown dog tick) and Dermacentor nitens (horse ear tick) Petition 870250066458, dated 07 / 31 / 2025, page 4 / 12 / 8 (ANDREOTTI, R. Ticks of importance in One Health and animal production in Brazil. Campo Grande, Mato Grosso do Sul: Embrapa Gado de Corte, 2021). The species Amblyomma sculptum acts as a vector of the bacterium Rickettsia rickettsii (Rickettsiales: Rickettsiaceae), the causative agent of Brazilian spotted fever (GERARDI, M. et al. Comparative Susceptibility of Different Populations of Amblyomma sculptum to Rickettsia rickettsii. Frontiers in Physiology, v.10, 2019). Beyond the medical and veterinary fields, the cattle tick causes economic losses exceeding 3 billion dollars worldwide, as the effects of its parasitism include anemia, reduced milk production, and damage to hides (GRISI, L. et al. Reassessment of the potential economic impact of cattle parasites in Brazil. Brazilian Journal of Veterinary Parasitology, v. 23, n. 2, p. 150-156, 2014).

[006] This tick is also a vector of the hemoparasites Anaplasma marginale (causative agent of anaplasmosis) and Babesia spp. (causative agent of babesiosis), which together are responsible for the condition of bovine parasitic tristeza, which can lead to the animal's death quickly (FERREIRA, GCM et al. Prevalence of bovine Babesia spp., Anaplasma marginale, and their co-infections in Latin America: Systematic review-meta-analysis. Ticks and Tick-borne diseases, v. 13, n. 4, 101967, 2022).

[007] Tick management is predominantly done with chemical acaricides. However, there are already reports of populations of different tick species resistant to a wide variety of chemical compounds (ANDREOTTI, R. Current situation of resistance of the cattle tick Rhipicephalus (Boophilus) microplus to acaricides in Brazil. Campo Grande, Mato Grosso do Sul: Embrapa Gado de Corte, 2010; RODRIGUEZ-VIVAS, RI et al. First report of amitraz and cypermethrin resistance in Rhipicephalus sanguineus sensu lato infesting dogs in Mexico. Medical and Veterinary Entomology, v. 31, p. 72-77, 2017.) An alternative to the exclusive use of synthetic pesticides is the biological control of pests. Petition 870250066458, dated 07 / 31 / 2025, page 5 / 12 / 8

[008] Entomopathogenic fungi are the most promising biological control agents for tick management (FERNANDES, É.KK; BITTENCOURT, VR Entomopathogenic fungi against South American tick species. Experimental and Applied Acarology, v.46, p. 71-93, 2008). The infection mechanism occurs through the active penetration of the arthropod cuticle, which occurs through the combination of mechanical pressure from the hyphae added to the enzymatic action of enzymes secreted by the fungus. The main ones are lipases, proteases, and chitinases, with the fungus's ability to produce these enzymes being directly linked to the virulence of the isolate (SCHRANK, A., VAINSLEIN, MH Metarhizium anisopliae enzymes and toxins. Toxicon, v. 56, p. 1267-1274, 2010). Proteases, mainly Pr1 (subtilisin), Pr2 (trypsin), and Pr4 (cysteine ​​protease), are the main proteases linked to fungal virulence, since the arthropod cuticle can be composed of up to 70% protein (CHARNLEY, AK).Fungal pathogens of insects: Cuticle degrading enzymes and toxins. Advances in Botanical Research, v. 40, p. 241-321, 2003; FERREIRA, J.M.; SOARES, F.E.F. Entomopathogenic fungi hydrolytic enzymes: a new approach to biocontrol? Journal of Natural Pesticides Research, v. 3, 100020, 2023).

[009] Even in the absence of hyphae, the extracellular enzymes produced by the fungus can damage the cuticle of arthropods (FERREIRA, JM; SOARES, FEF. Entomopathogenic fungi hydrolytic enzymes: a new approach to biocontrol? Journal of Natural Pesticides Research, v. 3, 100020, 2023). In addition to the enzymes produced by entomopathogenic fungi, there are others that may have acaricidal activity. Papaya latex (Carica papaya), for example, has demonstrated acaricidal activity against the cattle tick (Rhipicephalus microplus) (Filgueiras, MDG et al. Activity of papaya (Carica papaya) latex on Rhipicephalus microplus (Acari: Ixodidae) under laboratory conditions. International Journal of Acarology, v. 47, n. 7, p. 638-642. 2021).

[010] This latex contains many cysteine ​​proteases, mainly papain (EC 3.4.22.2). This enzyme is already used by the pharmaceutical industry with Petition 870250066458, dated 07 / 31 / 2025, page 6 / 12 / 8 frequency due to its debriding and healing action (LEITE, AP et al. Use and effectiveness of papain in the wound healing process: a systematic review. Revista Gaúcha de Enfermagem, v. 33, n. 3, p. 198-207, 2012). It is also used in the food industry as a meat tenderizer, as well as for the manufacture of dairy hydrolysates and bioactive peptides, among others. In addition to these known uses, new research indicates the potential of latex in controlling pests, such as arthropods and even mollusks (JAISWA, P. et al. Carica papaya Linn: a potential source for various health problems. Journal of Pharmacy Research, v. 3, p. 998-1003, 2010; Filgueiras, MDG et al. Activity of papaya (Carica papaya) latex on Rhipicephalus microplus (Acari: Ixodidae) under laboratory conditions. International Journal of Acarology, v. 47, n. 7, p. 638-642, 2021).

[011] Enzymes are proteins that accelerate biochemical reactions by acting as catalysts, lowering the activation energy required for the reaction to occur. This is crucial because activation energy is the barrier that must be overcome for the transformation of the substrate into product. The higher the activation energy, the slower the reaction. Enzymes act by reducing this barrier, thus increasing the reaction rate. They do this by providing a favorable environment for the substrate to reach the transition state, which is highly energetic. The site where this interaction occurs is called the active site, which is highly specific to the substrate, thus ensuring the enzyme's specificity. Although they bind to the substrate during the reaction, enzymes are not consumed or altered, and can catalyze multiple molecules (Nelson, DL; Cox, MM Lehninger Principles of Biochemistry. In: Enzymes. Artmed. Porto Alegre, 2014).

[012] Enzymes are classified according to the type of reaction they catalyze, receiving a name and an EC (Enzyme Commission number), assigned by the International Union of Biochemistry and Molecular Biology (IUBMB). This number usually consists of four digits, with the first two providing more general information: the first indicates the Petition 870250066458, dated 07 / 31 / 2025, page 7 / 12 / 8 class and the second indicates the subclass (Nelson, DL; Cox, MM Lehninger Principles of Biochemistry. In: Enzymes. Artmed. Porto Alegre, 2014).

[013] In the case of papain, it belongs to the class of hydrolases (EC 3), that is, it catalyzes the hydrolysis reaction of covalent bonds in a molecule. In this specific case, it acts on the cleavage of peptide bonds, being called a peptidase (EC3.4). Papain contains a cysteine ​​residue in its active site, which plays a crucial role in catalyzing the cleavage reaction within the protein molecule; therefore, it is classified as a cysteine ​​endopeptidase (EC 3.4.22) (National Center for Biotechnology Information. PubChem Enzyme Summary for Enzyme 3.4.22.2, Papain (EC 3.4.22.2). Available at: https: / / pubchem.ncbi.nlm.nih.gov / protein / EC:3.4.22.2. Accessed November 2024).

[014] It is estimated that the protease market will reach approximately US$2 billion in 2024 alone, with an annual growth rate for the industrialized enzyme market exceeding 6% per year (MEGHWANSHI, GK et al. Enzymes for pharmaceutical and therapeutic applications. Biotechnology and Applied Biochemistry, v. 67, p. 586-601, 2020). This demonstrates that the enzyme market still has significant room for growth.

[015] Papain plays a vital role in protecting papaya trees against caterpillars, especially lepidopteran larvae, providing a defense mechanism against insects (Konno. K. et al. Papain protects papaya trees from herbivorous insects: role of cysteine ​​proteases in latex. Plant Journal, v. 37, n. 3, p. 370-378, 2004). Since papain has insecticidal action, it also presents acaricidal potential to be explored.

[016] A search of national and international patent databases did not find any documents related to the present patent application. Thus, the present invention consists of the use of product formulations from the enzyme papain, from papaya latex (Carica papaya) and its use in tick control. It is worth noting that at low concentrations, Petition 870250066458, dated 07 / 31 / 2025, page 8 / 12 / 8 no risk of toxicity to humans, other animals and contamination of the environment. Brief description of the drawings

[017] The figures detail the investigation process that led to this patent application and are related to the claims.

[018] Figure 1: Mortality of engorged female Rhipicephalus microplus treated with papain and with Metarhizium anisopliae ss conidia. Females were treated by immersion and incubated at 27°C. Mortality was assessed daily. Different letters indicate a statistically significant difference (p<0.05) between treatments on the same day.

[019] Figure 2: Mortality of engorged female Rhipicephalus microplus treated with papain and with a chemical acaricide (Colosso®). Females were treated by immersion and incubated at 27°C. Mortality was assessed daily. Different letters indicate a statistically significant difference (p<0.05) between treatments on the same day.

[020] Figure 3: Engorged female Rhipicephalus microplus treated with papain. Figure 3A shows damage to the tick's cuticle. Figure 3B shows rupture of the cuticle and leakage of contents inside the tick. Description of the invention

[021] The invention proposes using papain in the preparation of a pesticide formulation. Various types of preparations and additives can be used for this purpose.

[022] The invention seeks to use these formulations in order to control ticks in a wide variety of animal breeding. Therefore, a description of the steps adopted in the methodology follows below.

[023] Several extraction methods can be applied to extract papain. Then, different purification and concentration methodologies. Petition 870250066458, dated 07 / 31 / 2025, page 9 / 12 / 8 of the enzymes can be applied to generate a suitable, enzyme-rich formulation for tick control. Examples of embodiments of the invention

[024] The use of the invention is exemplified below. The example is described solely for illustrative purposes.

[025] The acaricidal effect of the industrialized enzyme (crude powder extract) on engorged females of R. microplus was evaluated. The assays were performed in triplicate, according to the methodology of Muniz, ER et al. Efficacy of Metarhizium anisopliae conidia in oil-in-water emulsion against the tick Rhipicephalus microplus under heat and dry conditions. BioControl v. 65, p. 339-351, 2020.

[026] The females were weighed and sorted by weight to homogenize the control treatment groups, each with 20 ticks. Three groups were prepared: Control (Tween 80 ® 0.01%), papain 1%, papain 1% associated with M. anisopliae ss (1*107 conidia mL-1), papain 1% associated. The females were incubated in a humid chamber at 27±1°C in the dark. Mortality was assessed daily.

[027] The results show (Figure 1) a significant difference between the control group and the groups treated with papain (p<0.05). The papain treatment demonstrated a mortality rate of 62.5% immediately after 24h. The papain treatment associated with the fungus M. anisopliae obtained a mortality rate of 65% at the same time, reaching 77.5% after 5 days of treatment, while the treatment with only conidia showed only 21.9% mortality at the same time.

[028] These results demonstrate the potential of formulations containing papain as an alternative to tick control, given the high mortality over a significant period of time. It was also shown that the enzyme can be used in conjunction with entomopathogenic fungi that are already used in integrated pest management. Petition 870250066458, dated 07 / 31 / 2025, page 10 / 12 / 8

[029] Another trial was conducted with papain and a chemical acaricide on engorged female R. microplus. The objective was to compare mortality between the most commonly used chemical acaricide currently compared to papain and to evaluate a possible synergism between the action of both. Figure 2 shows that there was a significant difference (p<0.05) between the treatments. Using the dose recommended by the manufacturer, mortality with the chemical treatment was 75% after 24h, but the combination with papain resulted in 97.5% mortality in the same period. All results were subjected to analysis of variance (ANOVA) and compared with the control using the SNK test. This result demonstrates that papain can be used in conjunction with chemical acaricides, increasing treatment efficiency and eliminating individuals resistant to the synthetic compound.

[030] Figure 3 shows the damage caused to the cuticle by the action of the enzyme. After 24 hours it is possible to see the rupture of the cuticle and leakage of the contents from inside the female. Five days after treatment it was already possible to see the presence of the fungus M. anisopliae on the outside of the cadaver. Petition 870250066458, dated 07 / 31 / 2025, page 11 / 12

Claims

CLAIMS 1. Pesticide composition for tick control, characterized by comprising papain as the active ingredient, optionally combined with an entomopathogenic fungus selected from the Metarhizium anisopliae group, and a suitable application vehicle.

2. The composition according to claim 1, characterized in that the entomopathogenic fungus is present at a concentration of 1 10Γ conidia / mL.

3. The composition according to claim 1, characterized in that papain is present at a concentration of 1% (w / v).

4. The composition according to claim 1, characterized in that it is applied in liquid form, emulsified in oil or in an aqueous dispersion.

5. The composition according to claim 1, characterized by the fact that papain causes rupture of the tick cuticle, enhancing the action of the entomopathogenic fungus.

6. The composition according to claim 1, characterized by the fact that it is applied to selected livestock from the bovine, ovine and caprine groups.

7. The composition according to claim 1, characterized by the fact that papain is associated with a chemical acaricide for synergistic action and elimination of resistant populations.

8. Tick control process, characterized by comprising the application of the pesticide composition claimed in claim 1 on engorged females of Rhipicephalus microplus, resulting in mortality greater than 62.5% after 24 hours and 77.5% after 5 days. Petition 870250012324, dated 02 / 14 / 2025, page 8 / 112

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