Description:TITLE OF THE INVENTION
A bio-larvicidal composition using ethyl acetate extract of Cymodocea serrulata for mosquito control
TECHNICAL FIELD OF THE INVENTION
[001] The present invention relates to the field of bio-insecticides. More particularly, it discloses an eco-friendly mosquito larvicidal composition prepared from the ethyl acetate extract of the marine seagrass Cymodocea serrulata and its application in controlling mosquito larvae with minimal toxicity to non-target aquatic organisms.

BACKGROUND OF THE INVENTION
[002] Mosquitoes are responsible for transmission of malaria, dengue, chikungunya, yellow fever, and filariasis. Synthetic larvicides, though effective, suffer from problems of resistance development, bioaccumulation, environmental persistence, and adverse effects on non-target organisms and humans.

[003] Plant and marine-based bioactive compounds are being investigated as sustainable alternatives. Seagrasses are reservoirs of biologically active secondary metabolites. Cymodocea serrulata, a tropical seagrass, has shown medicinal potential. However, the larvicidal efficacy and biosafety profile of its ethyl acetate extract (CSEA) against mosquito vectors has not been previously established.

[004] There is therefore a need for a biodegradable, eco-friendly larvicide derived from marine resources that is effective against mosquito larvae and safe for non-target organisms.

OBJECT OF THE INVENTION
[005] One of the primary objects of the invention is to provide a bio-larvicidal composition comprising ethyl acetate extract of Cymodocea serrulata.

[006] Another object of the present invention is to develop a method for preparing the ethyl acetate extract of Cymodocea serrulata.

[007] Further object of the present invention is to use said extract as an eco-friendly larvicide against mosquito vectors including Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus.

[008] Yet another object of the present invention is to provide larvicidal formulations that are biodegradable, safe to non-target organisms, and suitable for field application.

SUMMARY OF THE INVENTION
[009] The following presents a simplified summary in order to provide a basic understanding of some aspects of the disclosed invention. This summary is not an extensive overview, and it is not intended to identify key/critical elements or to delineate the scope thereof. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[010] According to the basic aspects of the present invention, there is provided a bio-larvicidal composition comprising an ethyl acetate extract of Cymodocea serrulata, wherein the extract contains octadecadienoic acid as a major constituent, and wherein said composition is effective against mosquito larvae selected from Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus.

[011] In one aspect of the present invention, the extract is obtained by macerating dried powdered Cymodocea serrulata in ethyl acetate solvent for 5–15 days, filtering, and concentrating the solvent. The extract further comprises bioactive compounds selected from N-hexadecanoic acid, spinasterone, cholesta-22,24-dien-5-ol, hexatriacontane, and 2,7-octadiene-1,6-diol. The extract demonstrates LC50 values of about 10–11 mg/mL for Anopheles stephensi, 12–13 mg/mL for Aedes aegypti, and 14–15 mg/mL for Culex quinquefasciatus.

[012] In another aspect of the present invention, the extract causes histopathological changes in mosquito larvae including epithelial shrinkage, gut disruption, and muscle degeneration, and the extract exhibits negligible toxicity to non-target organisms including zebrafish embryos (Danio rerio) and brine shrimp (Artemia salina).

[013] In yet another aspect of the present invention, the composition further comprises a carrier medium selected from water, biodegradable polymers, oils, or emulsifiers to obtain a larvicidal formulation. The formulation is selected from aqueous sprays, slow-release pellets or briquettes, and oil-based emulsions.

[014] According to further aspects of the present invention, there is provided a method of controlling mosquito larvae comprising applying the composition of claim 1 to mosquito breeding sites at concentrations effective to achieve larval mortality. The composition can be applied to stagnant water bodies, drains, or breeding habitats at dosages of 10–50 mg/mL to achieve at least 80% larval mortality within 24 hours.

BRIEF DESCRIPTION OF THE DRAWINGS:

[015] Figure 1 depicts the graphical representation sowing Mosquito larvicidal activity of C. serrulata extracts against A. aegypti, C. quinquefasciatus and A. stephensi. The CS-EA showed highest mortality when compared to the other solvents extract of seagrass. CSH-hexane; CSDE‑diethyl ether; CSEA-ethyl acetate; CSC Chloroform; CSM-methanol; CSA-Acetone, according to the present invention.

[016] Figure 2 depicts light microscopic images of mosquitolarvae, control and CS-EA extract treated. Control (H- head; A-abdomen; T- thorax; RS-respiratory siphon; TG-tracheal gills; LH-lateral hairs and VB-ventral brush), according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION
[017] Referring now to Figure 1, the present invention will now be described in detail with reference to exemplary embodiments, which are provided for the purpose of understanding and enabling the invention. The description is not to be construed as limiting the scope of the invention. The present invention provides a bio-larvicidal composition comprising an ethyl acetate extract of the marine seagrass Cymodocea serrulata, which has been found to possess potent mosquito larvicidal activity. The extract contains a mixture of phytoconstituents, with octadecadienoic acid (linoleic acid) identified as the major compound, and demonstrates efficacy against mosquito larvae belonging to species Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. These species are major vectors of malaria, dengue, chikungunya, filariasis, and other vector-borne diseases, and the invention therefore provides an eco-friendly, plant-based alternative to conventional synthetic larvicides.

[018] In one embodiment of the present invention, the extract used in the invention is obtained from dried powdered plant material of Cymodocea serrulata. Freshly collected seagrass is shade-dried, pulverized into a fine powder, and subjected to maceration in ethyl acetate solvent. The ratio of plant material to solvent is maintained in the range of 1:2 to 1:5 by weight, and the maceration process is carried out for a period ranging from 5 to 15 days at ambient temperature. After maceration, the mixture is filtered through a sterile filter medium to remove debris and particulate matter, and the filtrate is concentrated under reduced pressure to obtain the ethyl acetate extract (CSEA). The concentrated extract is stored at temperatures between 0–10 °C to maintain its stability and bioactivity.

[019] In another embodiment, chemical characterization of the extract is performed using Gas Chromatography-Mass Spectrometry (GC–MS), Fourier Transform Infrared Spectroscopy (FTIR), and High performance Liquid Chromatography (HPLC). GC–MS analysis revealed that the extract contains multiple bioactive compounds, of which octadecadienoic acid is the major constituent, accounting for approximately 35–45% of the total peak area. Other significant compounds include N-hexadecanoic acid, spinasterone, cholesta-22,24-dien-5-ol, hexatriacontane, and 2,7-octadiene-1,6-diol, each contributing to the larvicidal efficacy. FTIR analysis confirmed the presence of functional groups such as amides, alkanes, alkyl halides, and phenolic compounds, which are associated with insecticidal activity. The phytochemical composition indicates the presence of secondary metabolites such as flavonoids, steroids, tannins, phenolics, alkaloids, and fatty oils, which collectively enhance the larvicidal potential of the extract.

[020] As shown in Figure 1, Larvicidal bioassays are conducted using fourth instar larvae of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The larvae were exposed to different concentrations of the CSEA extract in aqueous medium under controlled laboratory conditions. Mortality rates were calculated after 24 hours of exposure. The extract exhibited LC50 values of about 10–11 mg/mL for Anopheles stephensi, 12–13 mg/mL for Aedes aegypti, and 14–15 mg/mL for Culex quinquefasciatus. These values confirm that the extract is highly potent, with Anopheles stephensi being the most susceptible species. The larvicidal effects of the different crude extracts of Cymodocea serrulata namely hexane (CSH), chloroform (CSC), ethyl acetate (CSEA), diethyl ether (CSDE), methanol (CSM), and acetone (CSA) evaluated on Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi-larvae showed significant results. The values of the obtained lethal concentrations (LC50 and LC90) are shown in Table 1.

[021] Overall observations of larvicidal activity showed that C. serrulata ethyl acetate (CSEA) extract is found to be preponderant against An. Stephensi followed by Ae. aegypti and Cx. quinquefasciatus. After considering the CSEA, their LC50 and LC90 values (mg/mL) on A. stephensi is 10.663 and 117.013, respectively. Next, the CSEA extract on Ae. aegypti showed the following LC50 and LC90 values (mg/ml): 12.19 and 170.52. The minimum mortality rate was observed on Cx. quinquefasciatus with LC50 and LC90 values (mg/mL) of 14.32 and 326.43, respectively. Comparably from the different solvent extracts, CSEA extract showed the highest mortality rate than other extracts (Figure 1). Therefore, the active CSEA extract was selected for further studies. No malformations were observed from the control mosquito larvae and the bodies of CSEA-treated larvae showed shrinkage in the inner cuticles as shown in Figure 2.

[022] Histopathological studies of treated mosquito larvae revealed significant tissue alterations, including epithelial shrinkage, gut lumen disruption, midgut damage, and muscle degeneration. Such structural deformities result in impaired feeding, reduced motility, and eventual death of the larvae. No such abnormalities were observed in control larvae, thereby confirming the specific bioactivity of the CSEA extract.

[023] Furthermore, biosafety assessment of the extract is conducted on non-target aquatic organisms, specifically zebrafish embryos (Danio rerio) and brine shrimp (Artemia salina) nauplii. Zebrafish embryo toxicity studies demonstrated survival rates above 85% at concentrations up to 100 mg/mL, with no significant teratogenic malformations. Similarly, brine shrimp bioassays revealed high LC50 values (>400 mg/mL), confirming that the extract exhibits negligible toxicity to these organisms. This establishes the extract as a safe, eco-friendly larvicidal agent suitable for application in natural aquatic habitats.

[024] The bio-larvicidal composition of the present invention may be formulated with suitable carriers to facilitate large-scale application. Carriers may be selected from aqueous media, biodegradable polymers, oils, powders, or emulsifiers. Depending on the field requirement, the formulation may be provided as: aqueous sprays, wherein the extract is dissolved in water along with surfactants to ensure uniform dispersibility; slow-release pellets or briquettes, wherein the extract is adsorbed or encapsulated in biodegradable carriers such as starch, sawdust, or cellulose, allowing gradual release of the active compounds into stagnant water bodies; oil-based emulsions, wherein the extract is blended with oils such as neem oil or coconut oil to provide extended residual activity in mosquito breeding habitats.

[025] The invention further encompasses a method of controlling mosquito larvae by applying the bio-larvicidal composition to mosquito breeding sites. Application may be performed in stagnant water collections, drains, ponds, puddles, or other habitats favorable for mosquito breeding. The composition is preferably applied at a dosage of 10–50 mg/mL, which has been demonstrated to achieve at least 80% larval mortality within 24 hours of treatment. The method is safe for the environment and may be repeated at intervals of 7–14 days for sustained mosquito control. Thus, the present invention discloses a novel bio-larvicidal composition derived from the ethyl acetate extract of Cymodocea serrulata. The extract is rich in bioactive phytochemicals, shows high efficacy against vector mosquito larvae, causes specific histopathological alterations in larvae, and exhibits minimal toxicity to non-target organisms. The invention therefore provides a sustainable, eco-friendly, and cost-effective solution for mosquito vector management.

[026] While at least one exemplary embodiment has been presented in the foregoing summary and detailed description, it should be appreciated that a vast number of variations exist. It should also be appreciated that the exemplary embodiment or exemplary embodiments are only examples, and are not intended to limit the scope, applicability, or configuration in any way. Rather, the foregoing summary and detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment, it being understood that various changes may be made in the function and arrangement of elements described in an exemplary embodiment without departing from the scope as set forth in the appended claims and their legal equivalents.
, Claims:We claim:

1. A bio-larvicidal composition comprising an ethyl acetate extract of Cymodocea serrulata, wherein the extract contains octadecadienoic acid as a major constituent, and wherein said composition is effective against mosquito larvae selected from Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus.

2. The composition of claim 1, wherein the extract is obtained by macerating dried powdered Cymodocea serrulata in ethyl acetate solvent for 5–15 days, filtering, and concentrating the solvent.

3. The composition of claim 1, wherein the extract further comprises bioactive compounds selected from N-hexadecanoic acid, spinasterone, cholesta-22,24-dien-5-ol, hexatriacontane, and 2,7-octadiene-1,6-diol.

4. The composition of claim 1, wherein the extract demonstrates LC50 values of about 10–11 mg/mL for Anopheles stephensi, 12–13 mg/mL for Aedes aegypti, and 14–15 mg/mL for Culex quinquefasciatus.

5. The composition of claim 1, wherein the extract causes histopathological changes in mosquito larvae including epithelial shrinkage, gut disruption, and muscle degeneration.

6. The composition of claim 1, wherein the extract exhibits negligible toxicity to non-target organisms including zebrafish embryos (Danio rerio) and brine shrimp (Artemia salina).

7. The composition of claim 1, further comprising a carrier medium selected from water, biodegradable polymers, oils, or emulsifiers to obtain a larvicidal formulation.

8. The composition of claim 7, wherein the formulation is selected from aqueous sprays, slow-release pellets or briquettes, and oil-based emulsions.

9. A method of controlling mosquito larvae comprising applying the composition of claim 1 to mosquito breeding sites at concentrations effective to achieve larval mortality.

10. The method of claim 9, wherein the composition is applied to stagnant water bodies, drains, or breeding habitats at dosages of 10–50 mg/mL to achieve at least 80% larval mortality within 24 hours.