Larvicidal Efficacy Against Dengue Vector Aedes aegypti and Antibacterial Activity of Leaf Ethyl Acetate Extract of Clerodendrum indicum (L.) Kuntze

Larvicidal Efficacy Against Dengue Vector Aedes aegypti and Antibacterial Activity of Leaf Ethyl Acetate Extract of Clerodendrum indicum (L.) 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Kuntze Gobinda Dhibar, Souvik Bag, Dibyendu Saha, Biswajit Ghosh, Rupayan Chatterjee, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8162261/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Clerodendrum indicum , a traditionally used medicinal plant in South and Southeast Asia, was evaluated for the bioactivity of its ethyl acetate leaf extract (EAECI). The extract showed strong larvicidal and ovicidal effects against Aedes aegypti , producing dose-dependent larval mortality with LC₅₀ values of 187.427 µg/mL at 12 hours and 106.019 µg/mL at 24 hours. Complete ovicidal activity occurred at 350 µg/mL. EAECI exposure induced oxidative stress in larvae, evidenced by elevated levels of CAT, SOD, and MDA. The extract also exhibited notable antibacterial activity against Bacillus megaterium and B. cereus , with MICs between 100 and 500 µg/mL, and SEM imaging confirmed membrane deformation in treated bacteria. Phytochemical and FT-IR analyses verified the presence of hydroxyl, alkene, ester, and carboxylic acid groups. GC-MS profiling identified major components, particularly tetracosanoic acid methyl ester (36.17%) and 3,3-dimethylcyclohexene. Overall, the findings demonstrate that EAECI contains potent bioactive compounds with significant insecticidal and antimicrobial potential, supporting its use as an eco-friendly, plant-based agent for integrated vector and microbial disease control. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Drug discovery Biological sciences/Microbiology Biological sciences/Plant sciences Clerodendrum indicum GCMS Aedesaegypti oxidative stress enzymes larvicide antibacterial activity 1. Introduction Clerodendrum indicum (L.) Kuntze, belonging to the family Verbenaceae, is an annual shrub that can reach heights up to 2 to 3 meters. The lanceolate leaves can grow to be 23 cm long. The plant has big flower clusters with tubular white or yellow blooms. Its fruits are spherical and pulpy, and they change colour from green to blue-black or reddish-black when they are ripe (Manandhar and Manandhar, 2002 ). This plant is native to Sri Lanka and the Andaman Islands, and is widely distributed across tropical Asia. The plant holds a prominent place in traditional medicinal systems, particularly in Northeast India, where it is frequently used for its therapeutic properties (Somwong and Suttisri, 2018 ; Wang et al., 2018 ). Traditionally, C. indicum has been used for treating a broad spectrum of ailments, including respiratory disorders (asthma, bronchitis, colds), gastrointestinal issues (gastric tumors, intestinal worms), neurological conditions (epilepsy, hysteria, febrile convulsions), urogenital disorders (hematuria, painful urination, impotence), and musculoskeletal problems (arthritis, rheumatism) (Khare, 2007 ; DeFilipps and Krupnick, 2018 ). Phytochemical investigations have revealed that the plant contains a variety of bioactive compounds, including flavonoids, phenolics, terpenoids, saponins, and alkaloids, which contribute to its pharmacological effects such as antioxidant (Kar et al., 2014 ; Arokiyaraj et al., 2012 ), anti-inflammatory (Wang et al., 2018 ; Wahba et al., 2011 ; Saha et al., 2025 ), and antimicrobial activities (Pal et al., 2012 ). Despite its well-documented traditional uses and preliminary pharmacological evidence, the vector control potential of C. indicum remains underexplored. Only a few studies have been reported the bioactivities of its extracts such as the analgesic effect of crude ethanolic leaf extracts (Raihan et al., 2012 ; Das et al., 2025a ; Das et al., 2025b ; Mandal et al., 2023 ) and the in vitro antibacterial activity of root and stem extracts (Rahman et al., 2000 ). Mosquitoes (Diptera: Culicidae) are vectors of some of the deadliest human diseases, including malaria, dengue hemorrhagic fever, filariasis, Japanese encephalitis and yellow fever, making them one of the leading causes of global mortality (Mittal, 2003 ). According to the World Health Organization ( 2012 ), mosquito-borne diseases are responsible for approximately one million deaths annually. In this context, vector control emerges as the most effective strategy for reducing mortality. The primary methods of mosquito control include the use of larvicidal chemicals and the application of repellents or insecticide-treated bed nets to deter adult mosquitoes. Arbovirus transmission to humans primarily occurs through the bites of infected female Aedes mosquitoes, which are known for their daytime feeding behaviour. The efficiency of Ae. aegypti as a dengue virus vector is partly due to the extended time infected females take to obtain a blood meal compared to uninfected ones (Hardy, 2020 ; Almadiy, 2020). Given the absence of vaccines or targeted therapies for many mosquito-borne diseases, pesticide use remains a cornerstone of control efforts. However, the indiscriminate use of chemical pesticides raises serious concerns about environmental harm, risks to non-target organisms, and human health (Yeguerman et al., 2022 ). Additionally, the overuse of conventional insecticides has led to the development of resistance in mosquito populations and negative environmental impacts. In recent years, researchers have increasingly focused on developing eco-friendly and cost-effective alternatives, with particular emphasis on plant-derived mosquitocidal compounds (Chatterjee et al., 2023a ; Adhikary et al., 2024 ; Bag et al., 2025a ). An ideal pesticide should be efficient, precise, durable, environmentally sustainable, minimally harmful to mammals, and economically viable (Bag and Chatterjee, 2025 ). Among these alternatives, Bacillus thuringiensis , a soil bacterium, has gained prominence for its entomopathogenic activity against mosquito larvae (Roy et al., 2021 ; Bag et al., 2022a ; Bag et al., 2022b ; Bag et al., 2025b ; Saha et al., 2024 ). Furthermore, botanicals have attracted significant attention as natural insecticides against arthropod pests, including mosquitoes. Numerous studies have shown that plant extracts can act as larvicides, adulticides, pupicides, ovicides, repellents, and growth inhibitors (Fouda et al., 2022 ; Panneerselvam&Murugan, 2013 ; Chatterjee et al., 2023a ; Chatterjee et al., 2023b ).However, systematic evaluations focusing on its efficacy as a mosquito larvicide or ovicide, particularly against key disease vectors like Ae. aegypti , are notably lacking. Moreover, its antimicrobial spectrum has not been comprehensively studied using organic solvent extracts, which are known to improve the recovery of non-polar bioactive compounds. Given the global demand for eco-friendly, plant-based biocontrol agents, C. indicum represents a promising candidate for further investigation. Assessing its larvicidal, ovicidal, and antimicrobial potential could not only validate traditional knowledge but also contribute to the development of novel botanical formulations for integrated vector management and antimicrobial therapy. 2. Material methods 2.1. Plant material collection and preparation of extracts Fresh green leaves of C. indicum , were collected from Joypur forest, West Bengal, India, and were taxonomically authenticated by the Botanical Survey of India (BSI), Kolkata. The voucher specimen (SR/REN/BWN/2018/01) is preserved at the Department of Zoology, The University of Burdwan. The freshly collected leaves were thoroughly washed with distilled water and air-dried for 10 days at room temperature in a well-ventilated space. Once dried, the leaves were pulverized using an electronic grinder (Philips Mixture Grinder HL 1605, Kolkata, India), then stored in an airtight container for future use (Dutta and Ray, 2015 ; Dhibar et al., 2025). A powdered sample (20 g) of C. indicum leaves were properly packed within the soxhlet apparatus and add 500 mL ethyl acetate solvent (SA3F7005, MERCK) for the preparation of EAECI. The extract was coded as EAECI and kept in the refrigerator at 4°C for future use (Chipiti et al., 2015; Barman et al., 2020 ). 2.2. Mosquito rearing Mosquito larvae of Ae. aegypti were collected from the outskirts of Burdwan, West Bengal, India. The larvae were cultured in plastic bowls under laboratory conditions, maintained at 29 ± 3°C and 75–85% relative humidity (RH). The rearing process was designed to mimic natural conditions and support optimal larval development. Adult mosquitoes were reared in wooden cages measuring 30×30×30 inches. They were provided with cotton pads soaked in a 10% glucose solution, while adult females were periodically blood-fed to sustain their reproductive cycle. Petri dishes lined with damp cotton were placed at the bottom of each cage to facilitate egg-laying. The development of mosquito larvae was managed according to the standard protocol established by the World Health Organization (WHO, 2014). All experimental trials were conducted using third instar larvae, as recommended by WHO guidelines. 2.3. Larvicidal bioassay Laboratory bioassay experiments were conducted on third instar larvae of laboratory-reared mosquitoes under aseptic conditions, following WHO guidelines (WHO, 2005) with minor modifications. Twenty-five early instar larvae were placed in 50 mL disposable plastic cups and treated with plant extracts prepared in a suitable solvent and diluted with 0.01% DMSO as an emulsifier. EAECI extracts were tested at various concentrations (100, 200, 300, 400, 500and 600 µg/mL). For comparison, an equal number of larvae were maintained as controls, treated with 0.01% DMSO. The larvae were not fed during the experiment, and all other growth conditions were kept constant. Larval mortality was observed and recorded at 12 and 24 hours after treatment. Nonresponsive larvae were considered dead. Mortality data were converted into percentage mortality using the following formula: $$\:\text{L}\text{a}\text{r}\text{v}\text{a}\text{l}\:\text{m}\text{o}\text{r}\text{t}\text{a}\text{l}\text{i}\text{t}\text{y}\:\left(\text{\%}\right)=\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{d}\text{e}\text{a}\text{d}\:\text{l}\text{a}\text{r}\text{v}\text{a}\text{e}\:}{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{t}\text{e}\text{s}\text{t}\:\text{l}\text{a}\text{r}\text{v}\text{a}\text{e}}\times\:100$$ The mortality was calculated, determined the lethal concentrations (LC 50 and LC 90 ) and their 95% upper and lower confidence limit. 2.4. Ovicidal activity Ovicidal activity was assessed following Su and Mulla's (1998) method, with minor modifications. Mosquito eggs or egg rafts were collected from a breeding cage (30 × 30 × 30 inches) containing an established mosquito colony. The eggs were then exposed to ten concentrations of EAECI (100, 150, 200, 250, 300, 350, 400, 450, 500 and 1000 µg/mL) in 100 mL plastic containers for a duration of 120 hours. After treatment, individual eggs or egg rafts from each concentration were transferred to glasses of distilled water for hatching evaluation. The eggs were counted under a microscope before transferring. Each experiment was conducted six times on different days, with six replicates, using the appropriate solvent as a control. Following the 120 hours treatment period, hatch rates were calculated using the following formula: $$\:\text{E}\text{g}\text{g}\:\text{m}\text{o}\text{r}\text{t}\text{a}\text{l}\text{i}\text{t}\text{y}\:\:\left(\text{\%}\right)=\frac{\text{N}\text{o}\:\text{o}\text{f}\:\text{h}\text{a}\text{t}\text{c}\text{h}\text{e}\text{d}\:\text{e}\text{g}\text{g}\:}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{N}\text{o}\:\text{o}\text{f}\:\text{e}\text{g}\text{g}\text{s}}\times\:100$$ 2.5. Oxidative stress biomarkers Mosquito larvae from each treatment group were homogenized individually in 200 µL of phosphate buffer (0.1M, pH 7.6) at 4°C. The homogenates were centrifuged using a HERMLE Labortechnik centrifuge at 10,000 rpm for 10 minutes at 4°C. The resulting supernatant was collected and stored at − 20°C for use as an enzyme extract. The supernatant was immediately utilized as the enzyme source for biochemical assays. Each biochemical experiment was conducted in triplicate, with 25 larvae used for each replicate. Protein content was quantified using the Bradford method (1976), with bovine serum albumin (BSA) as the standard reference. Catalase (CAT) activity was determined by measuring the absorbance of residual H₂O₂ (Beers & Sizer, 1952 ), while superoxide dismutase (SOD) activity was assessed based on the inhibition of nitrobluetetrazolium (NBT) photoreduction (Beauchamp and Fridovich, 1971 ). Malondialdehyde (MDA) levels, indicative of lipid peroxidation, were measured by determining thiobarbituric acid-reactive substances (TBARS) (Ohkawa et al., 1979 ). SOD and CAT activities were expressed as U/mg protein, and MDA levels were reported as nMol TBARS/min/mg protein. All parameters were measured at room temperature (29°C) using a UV-visible spectrophotometer (Cecil Aquarius CE 7400). Detailed information regarding analytical instrument models, detection limits, and operational parameters for the detected biomarkers is provided in the supplementary materials. 2.6. Assays to determine the antibacterial activity 2.6.1. Minimum inhibitory concentration (MIC) The minimum inhibitory concentrations (MICs) of the EAECI were determined using the agar dilution method (Wiegand et al. 2008 ). Briefly, stock solutions of 1000 µg/mL were prepared for each extract, which were then serially diluted to produce concentrations of 0,100, 200, 300, 400, 500 µg/mL. These diluted extracts were poured into sterile petri dishes and allowed to solidify. A 2 µL drop of the prepared test and control organisms was then inoculated onto the surface of each plate using a template. After incubation at 35°C for 24 hours, the plates were examined, and the MIC was defined as the lowest concentration of the extract that prevented visible growth of the organism. For positive control tetracycline (30 µg) was used. 2.6.2. Scanning Electron Microscopy (SEM) study According to Hayat ( 1981 ), The most susceptible bacterial strain to the EAECI was selected for scanning electron microscopy (SEM) analysis. Bacterial cells were collected from the inhibition zone after 24 hours of treatment and fixed in 2.5% glutaraldehyde solution for 45 min at room temperature. The fixed cells were dehydrated through a graded ethanol series (50% for 7–5 min, 70% for 10 min, 90% for 5 min, and absolute ethanol for 5 min), followed by transfer to a 1:1 isoamyl alcohol:absolute ethanol mixture for 5 min and subsequently to pure isoamyl alcohol for 5 min. The samples were then air-dried, mounted onto aluminum stubs, sputter-coated with gold, and examined under a Hitachi S-530 SEM to observe morphological alterations induced by the EAECI. 2.7. Functional group analysis by FTIR (Fourier transform infrared) We used FT-IR spectroscopy to find the functional groups, various bonds, and chemical makeup of the substance in the EAECI. FT-IR spectroscopy was performed using the positive response band compound in an FT-IR spectrophotometer (JASCO FT-IR Model-4700) at room temperature (25 ± 5°C), with a scanning range of 400 to 4500 cm − 1 , to identify the functional groups of the EAECI. The peaks have been analysed using KnowItAll software (serial no. 107733-00001F44). The functional groups were identified by comparing the characteristic peak frequencies with those reported in the reference literature (Stuart, 2021 ; Socrates, 2004 ). 2.9. GC-MS analysis The EAECI was analyzed using GC-MS (Model: Clarus 680 GC) and the software used in the system is TurboMass Version 6.4.2 to identify and characterize bioactive components. The capillary column used is ‘Elite- 5MS’ having dimensions length 60 m, ID 0.25 mm and film thickness 0.25 µm and the stationary phase is 5% diphenyl 95% dimethyl polysiloxane. At a flow rate of 1 mL/min, helium gas (99.99%) was utilised as the mobile phase. The run time was around 40 min. An 8 min solvent delay was maintained. Data analysis library (NIST-2014) was used to identify the peak. 2.10. Statistical analysis Analysis of the obtained data was performed by using Graph pad-prism 9 software. All data presented are mean ± SEM values of triplicates (n = 3) and obtained from separate experiments. Probit analysis was used to calculate the 12, 24 and 48 hours LC 50 and LC 90 values (Finney, 1971 ). Student’s t-test were used to compare means of control and the oxidative stress biomarkers, namely CAT (Catalase), SOD (Superoxide dismutase), and MDA (Malondialdehyde). 3. Results 3.1. Larvicidal activity The EAECI demonstrated dose-dependent larvicidal activity against Ae. aegypti larvae after 24 hours of treatment. The data were analyzed, and statistical values including LC 50 , LC 90 , lower confidence limit (LCL), upper confidence limit (UCL), were recorded. The larvicidal efficacy varied across different extracts of C. indicum , with distinct LC 50 and LC 90 values. For the 12-hour treatment, the EAECI showed an LC 50 of 187.427 (125.078- 280.856) µg/mL and an LC 90 of 857.438 (572.204 -1284.857) µg/mL. In the 24-hour treatment, the LC 50 was 106.019 (66.604-168.759) µg/mL and the LC 90 was 539.890 (339.173-859.388) µg/mL. These results indicate that Ae. aegypti larvae were susceptible to the EAECI treatment (Fig. 1). 3.2. Ovicidal activity The egg hatchability of dengue vector mosquitoes was tested across different concentrations of the extract. The hatchability percentage was found to be directly proportional to the number of eggs and inversely proportional to the concentration of the toxicant. The ovicidal activity of EAECI was observed against Ae. aegypti , where 100% egg mortality was recorded at a concentration of 350 µg/mL. In the control experiment, the eggs exhibited 100% hatchability. 3.3. Oxidative stress enzymes Exposure of Ae. aegypti mosquito larvae to lethal concentrations of EAECI resulted in significant changes in total protein concentration and oxidative stress enzyme levels when compared to the control group. The total protein concentration in the control group was measured at 137.27 µg/mL, while in the EAECI-treated group, it increased to 220.02 µg/mL. Additionally, the levels of CAT, SOD, and MDA were elevated upon exposure to lethal concentrations of EAECI, with measurements of 57.04 µg/mL protein, 91.51 µg/mL protein, and 1.65 nMol TBARS/min/mg protein, respectively. In comparison, the control group showed values of 16.42 µg/mL protein, 14.36 µg/mL protein, and 0.92 nMol TBARS/min/mg protein. Statistical analysis revealed significant changes in oxidative stress parameters, with t-test results showing CAT (P < 0.0001), SOD (P < 0.0001), and MDA (P = 0.6311) levels significantly different from the control group. 3.4. Antibacterial activity The antimicrobial activity of EAECI was assessed against B. megaterium (MTCC 2412) and B. cereus (MTCC 1272). EAECI demonstrated moderate inhibitory effects, with both bacterial strains showing a minimum inhibitory concentration (MIC) of 200 µg/mL (Fig. 2). However, their susceptibility varied slightly: B. megaterium exhibited a zone of inhibition (ZOI) of 10 mm, whereas B. cereus was more sensitive, showing the largest ZOI among tested strains at 12 mm. Scanning electron microscopy (SEM) further confirmed the bactericidal action of EAECI displayed significant structural damage: B. megaterium exposed to EAECI (Fig. 3a) exhibited clear membrane disintegration and cell surface roughening, while B. cereus cells (Fig. 3b) showed pronounced shrinkage, irregular surfaces, and partial lysis. These ultrastructural alterations indicate that EAECI compromises bacterial cell envelope integrity, ultimately leading to cell death. The combined MIC, ZOI, and SEM findings validate the bactericidal mechanism of EAECI against these pathogenic Bacillus species. 3.5. FTIR analysis FTIR (Fourier-transform infrared) spectroscopy was employed to analyze the EAECI, aiming to identify the functional groups potentially responsible for their bioactivity. In the EAECI spectrum, the characteristic absorption peaks were observed at 3432.67 cm⁻¹, corresponding to O–H stretching. Peaks at 2975.62 cm⁻¹ indicated C–H stretching, the peak at 1642.09 cm⁻¹ indicated C = C stretching, while the peak at 1046.19 cm⁻¹ suggested the presence of an ester group (Fig. 4). These FTIR spectra confirm the presence of various functional groups, including hydroxyl, carboxylic acid, alkane, alkene, ester, aromatic, and ether groups, as outlined by (Socrates, 2004 ; Stuart, 2005 ). 3.6. GC-MS analysis The chemical composition of the EAECI was analyzed using GC-MS, revealing the presence of main two compounds 3,3 dimethylcyclohexene (Fig. 5) and tetracosanoic acid, methyl ester (Fig. 6) (Table 1 ). Table 1 GC-MS Spectral Analysis of EAECI No. RT (min) Name of the Compound Molecular Formula Molecular Weight Peak Area (%) 1 26.586 3,3-dimethylcyclohexene C 8 H 14 110.1094 1.74 2 36.98 Tetracosanoic acid, methyl ester C 25 H 50 O 2 382.6633 36.173 4. Discussion Recently, plant-derived bio-pesticides have gained significant attention as safer alternatives to traditional synthetic insecticides, particularly in the control of disease-carrying vectors. These plant-based chemicals are favoured for their safety profile, as they are non-toxic to humans and animals, lack phototoxic effects, and do not leave harmful residues in the environment (Schmutterer, 1990 ; Chatterjee et al., 2023b ). As the development of resistance to conventional synthetic insecticides becomes an increasing challenge in vector mosquito management, there is a pressing need for innovative solutions or novel insecticides (Chandre et al., 1998 ). The emergence of insecticide-resistant mosquito populations calls for the exploration of alternative control methods, such as the use of plant-derived bio-pesticides, which could play a key role in integrated vector management strategies. Bowers et al. ( 1995 ), emphasized that screening locally available medicinal plants for their mosquito control potential could stimulate local economies, reduce dependence on costly imported chemicals, and support community-driven public health initiatives. Plants contain a wide range of bioactive compounds with diverse biological activities, which are believed to be responsible for their pesticidal properties. These compounds, such as toxins and secondary metabolites, act as effective mosquito control agents, contributing to the larvicidal and adulticidal effects observed in many plant extracts (Niraimathi et al., 2010 ). This is particularly important for managing disease vectors, where reducing the population of mosquitoes is crucial in preventing the transmission of diseases like dengue, malaria, and Zika virus. Our study supports these findings, as it demonstrated that an increase in the concentration of EAECI significantly contributed to the mortality of Ae. aegypti larvae. While the medicinal properties of C. indicum have been well-documented (Shrivastava and Patel, 2007 ), the mosquito larvicidal effects of its leaves have not been extensively explored. Our results provide valuable new insights into the potential of C. indicum as an effective natural bio-pesticide. Similar studies, such as the work by Mathew et al. ( 2009 ), who investigated the methanol extract of C. ternatea seeds, found notable larvicidal activity against Ae. aegypti larvae with an LC 50 of 154.5 ppm. Likewise, Ansari et al. ( 2005 ), demonstrated that Pinus longifolia oil exhibited larvicidal efficacy against Ae. aegypti mosquitoes, with an LC 50 value of 82.1 ppm. These findings align with our results, highlighting the potential of plant extracts for mosquito control. In addition, numerous other recent studies have corroborated our findings regarding the larvicidal activity of various plant extracts against Ae. aegypti larvae (Onah et al., 2022 ; Ajaegbu et al., 2022 ; Bakar et al., 2018 ). Mechanistic insights into its larvicidal effect were provided by biochemical assays, which revealed significant elevations in catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels in treated larvae, suggesting that EAECI induces oxidative stress that disrupts normal metabolic functions and damages cellular components, a mode of action consistent with earlier observations that phytochemicals trigger redox imbalance leading to larval death (Govindarajan and Benelli, 2016 ). This dual activity direct larvicidal toxicity along with oxidative stress induction indicates that EAECI may exert synergistic pressure on mosquito larvae, thereby reducing the likelihood of resistance development that is common with conventional synthetic insecticides (Chandre et al., 1998 ). In parallel to its mosquitocidal efficacy, EAECI also displayed moderate antibacterial activity against two Gram-positive pathogens, B. megaterium and B. cereus , with a minimum inhibitory concentration (MIC) of (200 µg/mL) for both strains. The bactericidal nature of EAECI was further confirmed through scanning electron microscopy, where untreated control cells maintained smooth, intact rod-shaped morphologies, while treated cells showed dramatic ultrastructural alterations, including membrane disintegration, surface shrinkage, and partial lysis, thereby suggesting that EAECI compromises bacterial envelope integrity and leads to cell death. These observations align with earlier studies demonstrating that plant-derived secondary metabolites such as alkaloids, tannins, and saponins disrupt bacterial cell walls and interfere with critical physiological processes (Hassan et al., 2004 ). The moderate antimicrobial activity observed in our study is particularly noteworthy as bacterial pathogens like B. cereus are associated with food borne diseases, and plant-derived agents with membrane-disruptive properties offer promising natural alternatives to chemical antibiotics, especially in the context of increasing antimicrobial resistance. The phytochemical basis of EAECI’s bio efficacy was further elucidated by FT-IR and GC-MS analyses. FT-IR spectra revealed functional groups including hydroxyl, ester, alkene and carboxylic acids, which are well known for contributing to biological activity by enabling hydrogen bonding, disrupting microbial proteins and enhancing lipophilicity to facilitate cell penetration (Sofowara, 1993; Stuart, 2005 ). GC-MS analysis identified two major compounds, 3,3-dimethylcyclohexene and tetracosanoic acid, methyl ester, the latter constituting 36.17% of the total peak area. Fatty acid esters like tetracosanoic acid derivatives are widely recognized for their antimicrobial and surface-active properties, and their high abundance in EAECI likely contributes substantially to the bactericidal and larvicidal activities observed in the present study (Rahman et al., 2000 ; Raihan et al., 2012 ). Taken together, these results demonstrate that EAECI exerts its bioactivity through a combination of biochemical, structural and molecular mechanisms, including oxidative stress induction, membrane disruption, and the action of fatty acid derivatives and other phytochemicals. Importantly, the multifunctionality of EAECI highlights its potential for integration into eco-friendly vector and microbial control strategies, offering a sustainable alternative to chemical pesticides and antibiotics, which often face challenges of resistance, environmental persistence and non-target toxicity (Schmutterer, 1990 ; Bowers et al., 1995 ). Moreover, the dual larvicidal and antibacterial activities of EAECI underscore its relevance in tropical and subtropical regions where mosquito-borne diseases and bacterial infections frequently coexist as public health burdens, thereby providing a holistic approach to disease management. By validating the ethnomedicinal applications of C. indicum with modern scientific evidence, the present study not only supports its traditional therapeutic use (Shrivastava and Patel, 2007 ) but also contributes to the growing framework of plant-based integrated pest and pathogen management. Ultimately, the findings suggest that C. indicum EAECI, with its demonstrated efficacy against both insect vectors and bacterial pathogens, may serve as a promising natural bioagent capable of addressing urgent challenges in public health, including vector-borne disease transmission and antimicrobial resistance, while minimizing ecological risks and fostering sustainable disease control practices. 5. Conclusion The present investigation highlights the ethyl acetate leaf extract of C. indicum (EAECI) as a potent multifunctional biocontrol agent with promising applications in integrated vector and microbial disease management. The extract exhibited strong larvicidal activity against Ae. aegypti , with complete ovicidal efficacy at relatively low concentrations, and induced significant oxidative stress in larvae, suggesting a dual mechanism of direct toxicity and redox imbalance. Alongside its mosquitocidal potential, EAECI demonstrated moderate antibacterial activity against B. megaterium and B. cereus , with a minimum inhibitory concentration (MIC) of 200 µg/mL, while scanning electron microscopy confirmed its bactericidal nature through observable ultrastructural damage to bacterial cell membranes. Phytochemical characterization by FTIR revealed the presence of hydroxyl, ester, alkene, and carboxylic acid groups, while GC-MS identified 3,3-dimethylcyclohexene and tetracosanoic acid, methyl ester the latter representing a major component likely responsible for its bioactivity. These findings collectively validate the traditional medicinal uses of C. indicum and provide mechanistic evidence supporting its efficacy as an eco-friendly alternative to synthetic insecticides and antibiotics, which are increasingly challenged by resistance and environmental concerns. By combining larvicidal, ovicidal, oxidative stress-inducing, and antimicrobial properties within a single botanical extract, C. indicum demonstrates multifunctionality that is particularly valuable in tropical regions burdened with vector-borne diseases and bacterial infections. Future research should focus on formulation development, field trials, and safety evaluations to facilitate its translation into practical, sustainable biocontrol solutions. Declarations Funding Declaration First author, Gobinda Dhibar, acknowledges financial support from the Council of Scientific and Industrial Research (CSIR), Government of India, in the form of Junior Research Fellowship (JRF) and Senior Research Fellowship (SRF), Grant No. 09/025(0283)/2019-EMR-I Dated: 30.11.2019. No other external funding was received for this study. Declaration of Competing Interest The authors declare no conflict of interest. Author Contribution G.D.: Investigation, Methodology, Antimicrobial Analysis, Writing – Original Draft.G.D., S.B., and D.S.: Scanning Electron Microscopy Analysis.S.B. and D.S.: Larvicidal Analysis, Writing – Review & Editing.B.G., R.C., and R.D.: Investigation, Writing – Review & Editing.S.R. and S.C.: Conceptualization, Investigation, Formal Analysis, Supervision, Writing – Review & Editing.All authors reviewed and approved the final manuscript. Acknowledgement The authors gratefully acknowledge Dr. Karthigeyan Kaliyamurthy, Scientist-E, BSI, Howrah, India, for authenticating the plant species. The facilities sponsored by UGC-MRP, DST-PURSE, DST-FIST, and UGC-DRS in the Department of Zoology, The University of Burdwan, were utilized for this research. The authors also express their gratitude to IIT Guwahati and Biotech Park for conducting the GC–MS analysis, and to USIC, The University of Burdwan, for providing FE–SEM facilities. ChatGPT was used by the author to enhance linguistic clarity and improve readability of the manuscript. Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. All the FTIR and GC-MS data are represented with Figure 4,5,6 and 7 in supplementary file attached. References Adhikary, K. et al. Larvicidal activity of β-Citral: An in-vitro and in-silico study to understand its potential against mosquito. 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A., Sulaiman, S., Omar, B. & Ali, R. M. Screening of five plant extracts for larvicidal efficacy against larvae of Aedes aegypti (L.) and Aedes albopictus (Skuse). ASM Sci. ; 11 (2). (2018). Barman, M., Roy, S. & Ray, S. Colchicine like metaphase and cell cycle delay inducing effects of leaf aqueous extract of Clerodendrum inerme (L.) Gaertn. in Allium cepa root apical meristem cells. Cytologia 85 (3), 197–201 (2020). Beauchamp, C. & Fridovich, I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44 , 276–287. 10.1016/0003-2697(71)90370-8 (1971). Beers, R. F. & Sizer, I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide. J. Biol. Chem. 195 , 133–140 (1952). Bowers, W. S., Sener, B., Evans, P. H., Bingol, F. & Erdogan, I. Activity of Turkish medicinal plants against mosquitoes Aedes aegypti and Anopheles gambiae . Int. J. Trop. Insect Sci. 16 (3–4), 339–342 (1995). Chandre, F. et al. Pyrethroid resistance in Culex quinquefasciatus from West Africa. Med. Vet. Entomol. 12 (4), 359–366 (1998). Chatterjee, S. et al. Mitigating the public health issues caused by the filarial vector, Culex quinquefasciatus (Diptera: Culicidae) through phytocontrol and larval source marker management. Appl. Biochem. Biotechnol. 1–32. (2023b). Chatterjee, S. et al. Neem-based products as potential eco-friendly mosquito control agents over conventional eco-toxic chemical pesticides - a review. Acta Trop. 240 , 106858 (2023a). Das, R. et al. Assessment of antibacterial activity of leaf extracts of Crinum asiaticum L. against five gram-negative fish pathogenic bacterial strains. Microbe 7 , 100319 (2025a). Das, R. et al. Bioprospecting nonpolar solvent extracts from Scadoxus multiflorus (Martyn) Raf. for antibacterial activity against fish pathogenic bacteria. Microbe 100543. (2025b). DeFilipps, R. & Krupnick, G. The medicinal plants of Myanmar. PhytoKeys 102 , 1–341 (2018). Dhibar, G. & Ray, S. Antimitotic and cytotoxic effects of Clerodendrum indicum leaf aqueous extract in Allium cepa root tip cells. CYTOLOGIA 90 (3), 173–178 (2025). Dutta, S. & Ray, S. Evaluation of in vitro free radical scavenging activity of leaf extract fractions of Manilkara hexandra (Roxb.) Dubard in relation to total phenolic contents. Int. J. Pharm. Pharm. Sci. 7 (10), 296–301 (2015). Finney, D. J. Probit analysis: A statistical treatment of the sigmoid (Cambridge Univ, 1971). Fouda, A. et al. Enhanced antimicrobial, cytotoxicity, larvicidal, and repellence activities of brown algae, Cystoseira crinita-mediated green synthesis of magnesium oxide nanoparticles. Front. Bioeng. Biotechnol. 10 , 849921 (2022). Govindarajan, M. & Benelli, G. Artemisia absinthium-borne compounds as novel larvicides: effectiveness against six mosquito vectors and acute toxicity on non-target aquatic organisms. Parasitol. Res. 115 (12), 4649–4661 (2016). Hardy, J. L. Susceptibility and resistance of vector mosquitoes. In: The Arboviruses. CRC; 87–126. (2020). Hassan, M. M., Oyewale, A. O., Amupitan, J. O., Abduallahi, M. S. & Okonkwo, E. M. Preliminary phytochemical and antibacterial investigation of crude extracts of the root bark of Detarium microcarpum . J. Chem. Soc. Nigeria . 29 (1), 26–29 (2004). Hayat, M. A. Principles and techniques of electron microscopy. London: Edward Arnold; p.522. (1981). Kar, P., Goyal, A. K., Das, A. P. & Sen, A. Antioxidant and pharmaceutical potential of Clerodendrum L.: An overview. Int. J. Green. Pharm. 8 , 4 (2014). Khare, C. P. Indian medicinal plants: An illustrated dictionary. New York: Springer Press; p.900. (2007). Manandhar, N. & Manandhar, S. Useful plants of Nepal. In: Plants and People of Nepal. London: Timber; 153–160. (2002). Mandal, A. et al. Soil bacterial diversity in the tropical dry deciduous forest of Ajodhya hills, Purulia, West Bengal. Acta Ecol. Sin . 43 (5), 899–906 (2023). Mathew, N. et al. Larvicidal activity of Saraca indica , Nyctanthes arbor-tristis, and Clitoria ternatea extracts against three mosquito vector species. Parasitol. Res. 104 , 1017–1025 (2009). Mittal, P. K. Prospects of using herbal products in the control of mosquito vectors. ICMR Bull. 33 , 1–10 (2003). Niraimathi, S., Balaji, N., Venkataramanan, N. & Govindarajan, M. Larvicidal activity of alkaloid from Sida acuta against Anopheles subpictus and Culex tritaeniorhynchus . Int. J. Curr. Res. 11 , 34–38 (2010). Ohkawa, H., Ohishi, N. & Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95 , 351–358. 10.1016/0003-2697(79)90738-3 (1979). Onah, G. T. et al. Larvicidal and synergistic potentials of some plant extracts against Aedes aegypti . J. Entomol. Zool. Stud. ; 10 (1). (2022). Pal, A., Al Mahmud, Z., Akter, N., Islam, S. & Bachar, S. C. Evaluation of antinociceptive, antidiarrheal and antimicrobial activities of leaf extracts of Clerodendrum indicum . Pharmacogn J. 4 , 41–46 (2012). Panneerselvam, C. & Murugan, K. Adulticidal, repellent, and ovicidal properties of indigenous plant extracts against the malarial vector, Anopheles stephensi (Diptera: Culicidae). Parasitol. Res. 112 , 679–692 (2013). Rahman, A. A., Azam, A. T. M. Z. & Gafur, M. A. Brine shrimp lethality bioassay with extracts and two flavonoids from Clerodendrum indicum Linn. Pak J. Pharmacol. 17 , 1–6 (2000). Raihan, S. Z. et al. Phytochemical investigation and in vitro antinociceptive activity of Clerodendrum indicum leaves. Pak J. Biol. Sci. 15 (3), 152–155 (2012). Roy, M., Chatterjee, S. & Dangar, T. K. Characterization and mosquitocidal potency of a Bacillus thuringiensis strain of rice field soil of Burdwan, West Bengal, India. Microb. Pathog . 158 , 105093 (2021). Saha, B., Bag, S., Mandal, A. & Chatterjee, S. Diversity of aerobic bacterial groups in Chandur forest areas of West Bengal, India. Asian J. Microbiol. Biotechnol. Environ. Sci. ; 26 (3). (2024). Saha, S. et al. Are biopesticides really safe? Impacts on gut microbiota and intestinal health in freshwater fish. J. Contam. Hydrol. ;104727. (2025). Schmutterer, H. Properties and potential of natural pesticides from the neem tree, Azadirachta indica . Annu. Rev. Entomol. 35 (1), 271–297 (1990). Shrivastava, N. & Patel, T. Clerodendrum and healthcare: an overview. Med. Aromat. Plant. Sci. Biotechnol. 1 (1), 142–150 (2007). Socrates, G. Infrared and Raman characteristic group frequencies: tables and charts (Wiley, 2004). Somwong, P. & Suttisri, R. Cytotoxic activity of the chemical constituents of Clerodendrum indicum and Clerodendrum villosum roots. J. Integr. Med. 16 , 57–61 (2018). Stuart, B. Infrared spectroscopy (Wiley Online Library, 2005). Stuart, B. Infrared spectroscopy. In: Analytical techniques in forensic science. Wiley; 145–160. (2021). Su, T. & Mulla, M. S. Ovicidal activity of neem products (azadirachtin) against Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae). J. Am. Mosq. Control Assoc. 14 (2), 204–209 (1998). Trease, G. E. & Evans, W. C. Pharmacognosy 13th edn (Brailliar Tiridel Can, 1989). Wahba, H. M. et al. Chemical and biological investigation of some Clerodendrum species cultivated in Egypt. Pharm. Biol. 49 , 66–72 (2011). Wang, J. H., Luan, F., He, X. D., Wang, Y. & Li, M. X. Traditional uses and pharmacological properties of Clerodendrum phytochemicals. J. Tradit Complement. Med. 8 , 24–38 (2018). Wiegand, I., Hilpert, K. & Hancock, R. E. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3 (2), 163–175. 10.1038/nprot.2007.521 (2008). World Health Organization. Guidelines for laboratory and field testing of mosquito larvicides (WHO, 2005). WHO/CDS/WHOPES/GCDPP/2005.13. World Health Organization. Handbook for integrated vector management (WHO, 2012). Yeguerman, C. A. et al. Essential oils loaded on polymeric nanoparticles: bioefficacy against economic and medical insect pests and risk evaluation on terrestrial and aquatic non-target organisms. Environ. Sci. Pollut Res. 29 (47), 71412–71426 (2022). Additional Declarations No competing interests reported. Supplementary Files supplimentary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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12:48:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":877348,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8162261/v1/31edf2c7-9e0d-4029-82cb-9dc76f40cdf6.pdf"},{"id":98900782,"identity":"488be5dd-e0f6-42b5-92d4-1aedcbc56dda","added_by":"auto","created_at":"2025-12-23 19:16:14","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4305491,"visible":true,"origin":"","legend":"","description":"","filename":"supplimentary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8162261/v1/89676c20a4308e3458cb7a5d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Larvicidal Efficacy Against Dengue Vector Aedes aegypti and Antibacterial Activity of Leaf Ethyl Acetate Extract of Clerodendrum indicum (L.) Kuntze","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eClerodendrum indicum\u003c/em\u003e (L.) Kuntze, belonging to the family Verbenaceae, is an annual shrub that can reach heights up to 2 to 3 meters. The lanceolate leaves can grow to be 23 cm long. The plant has big flower clusters with tubular white or yellow blooms. Its fruits are spherical and pulpy, and they change colour from green to blue-black or reddish-black when they are ripe (Manandhar and Manandhar, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). This plant is native to Sri Lanka and the Andaman Islands, and is widely distributed across tropical Asia. The plant holds a prominent place in traditional medicinal systems, particularly in Northeast India, where it is frequently used for its therapeutic properties (Somwong and Suttisri, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Traditionally, \u003cem\u003eC. indicum\u003c/em\u003e has been used for treating a broad spectrum of ailments, including respiratory disorders (asthma, bronchitis, colds), gastrointestinal issues (gastric tumors, intestinal worms), neurological conditions (epilepsy, hysteria, febrile convulsions), urogenital disorders (hematuria, painful urination, impotence), and musculoskeletal problems (arthritis, rheumatism) (Khare, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; DeFilipps and Krupnick, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Phytochemical investigations have revealed that the plant contains a variety of bioactive compounds, including flavonoids, phenolics, terpenoids, saponins, and alkaloids, which contribute to its pharmacological effects such as antioxidant (Kar et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Arokiyaraj et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), anti-inflammatory (Wang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wahba et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Saha et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and antimicrobial activities (Pal et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Despite its well-documented traditional uses and preliminary pharmacological evidence, the vector control potential of \u003cem\u003eC. indicum\u003c/em\u003e remains underexplored. Only a few studies have been reported the bioactivities of its extracts such as the analgesic effect of crude ethanolic leaf extracts (Raihan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Das et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e; Das et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e; Mandal et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and the \u003cem\u003ein vitro\u003c/em\u003e antibacterial activity of root and stem extracts (Rahman et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Mosquitoes (Diptera: Culicidae) are vectors of some of the deadliest human diseases, including malaria, dengue hemorrhagic fever, filariasis, Japanese encephalitis and yellow fever, making them one of the leading causes of global mortality (Mittal, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). According to the World Health Organization (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), mosquito-borne diseases are responsible for approximately one million deaths annually. In this context, vector control emerges as the most effective strategy for reducing mortality. The primary methods of mosquito control include the use of larvicidal chemicals and the application of repellents or insecticide-treated bed nets to deter adult mosquitoes. Arbovirus transmission to humans primarily occurs through the bites of infected female \u003cem\u003eAedes\u003c/em\u003e mosquitoes, which are known for their daytime feeding behaviour. The efficiency of \u003cem\u003eAe. aegypti\u003c/em\u003e as a dengue virus vector is partly due to the extended time infected females take to obtain a blood meal compared to uninfected ones (Hardy, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Almadiy, 2020). Given the absence of vaccines or targeted therapies for many mosquito-borne diseases, pesticide use remains a cornerstone of control efforts. However, the indiscriminate use of chemical pesticides raises serious concerns about environmental harm, risks to non-target organisms, and human health (Yeguerman et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, the overuse of conventional insecticides has led to the development of resistance in mosquito populations and negative environmental impacts. In recent years, researchers have increasingly focused on developing eco-friendly and cost-effective alternatives, with particular emphasis on plant-derived mosquitocidal compounds (Chatterjee et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Adhikary et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Bag et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). An ideal pesticide should be efficient, precise, durable, environmentally sustainable, minimally harmful to mammals, and economically viable (Bag and Chatterjee, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Among these alternatives, \u003cem\u003eBacillus thuringiensis\u003c/em\u003e, a soil bacterium, has gained prominence for its entomopathogenic activity against mosquito larvae (Roy et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bag et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Bag et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Bag et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e; Saha et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, botanicals have attracted significant attention as natural insecticides against arthropod pests, including mosquitoes. Numerous studies have shown that plant extracts can act as larvicides, adulticides, pupicides, ovicides, repellents, and growth inhibitors (Fouda et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Panneerselvam\u0026amp;Murugan, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chatterjee et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Chatterjee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).However, systematic evaluations focusing on its efficacy as a mosquito larvicide or ovicide, particularly against key disease vectors like \u003cem\u003eAe. aegypti\u003c/em\u003e, are notably lacking. Moreover, its antimicrobial spectrum has not been comprehensively studied using organic solvent extracts, which are known to improve the recovery of non-polar bioactive compounds. Given the global demand for eco-friendly, plant-based biocontrol agents, \u003cem\u003eC. indicum\u003c/em\u003e represents a promising candidate for further investigation. Assessing its larvicidal, ovicidal, and antimicrobial potential could not only validate traditional knowledge but also contribute to the development of novel botanical formulations for integrated vector management and antimicrobial therapy.\u003c/p\u003e"},{"header":"2. Material methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant material collection and preparation of extracts\u003c/h2\u003e \u003cp\u003eFresh green leaves of \u003cem\u003eC. indicum\u003c/em\u003e, were collected from Joypur forest, West Bengal, India, and were taxonomically authenticated by the Botanical Survey of India (BSI), Kolkata. The voucher specimen (SR/REN/BWN/2018/01) is preserved at the Department of Zoology, The University of Burdwan. The freshly collected leaves were thoroughly washed with distilled water and air-dried for 10 days at room temperature in a well-ventilated space. Once dried, the leaves were pulverized using an electronic grinder (Philips Mixture Grinder HL 1605, Kolkata, India), then stored in an airtight container for future use (Dutta and Ray, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Dhibar et al., 2025). A powdered sample (20 g) of \u003cem\u003eC. indicum\u003c/em\u003e leaves were properly packed within the soxhlet apparatus and add 500 mL ethyl acetate solvent (SA3F7005, MERCK) for the preparation of EAECI. The extract was coded as EAECI and kept in the refrigerator at 4\u0026deg;C for future use (Chipiti et al., 2015; Barman et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Mosquito rearing\u003c/h2\u003e \u003cp\u003eMosquito larvae of \u003cem\u003eAe. aegypti\u003c/em\u003e were collected from the outskirts of Burdwan, West Bengal, India. The larvae were cultured in plastic bowls under laboratory conditions, maintained at 29\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C and 75\u0026ndash;85% relative humidity (RH). The rearing process was designed to mimic natural conditions and support optimal larval development. Adult mosquitoes were reared in wooden cages measuring 30\u0026times;30\u0026times;30 inches. They were provided with cotton pads soaked in a 10% glucose solution, while adult females were periodically blood-fed to sustain their reproductive cycle. Petri dishes lined with damp cotton were placed at the bottom of each cage to facilitate egg-laying. The development of mosquito larvae was managed according to the standard protocol established by the World Health Organization (WHO, 2014). All experimental trials were conducted using third instar larvae, as recommended by WHO guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Larvicidal bioassay\u003c/h2\u003e \u003cp\u003e Laboratory bioassay experiments were conducted on third instar larvae of laboratory-reared mosquitoes under aseptic conditions, following WHO guidelines (WHO, 2005) with minor modifications. Twenty-five early instar larvae were placed in 50 mL disposable plastic cups and treated with plant extracts prepared in a suitable solvent and diluted with 0.01% DMSO as an emulsifier. EAECI extracts were tested at various concentrations (100, 200, 300, 400, 500and 600 \u0026micro;g/mL). For comparison, an equal number of larvae were maintained as controls, treated with 0.01% DMSO. The larvae were not fed during the experiment, and all other growth conditions were kept constant. Larval mortality was observed and recorded at 12 and 24 hours after treatment. Nonresponsive larvae were considered dead. Mortality data were converted into percentage mortality using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{L}\\text{a}\\text{r}\\text{v}\\text{a}\\text{l}\\:\\text{m}\\text{o}\\text{r}\\text{t}\\text{a}\\text{l}\\text{i}\\text{t}\\text{y}\\:\\left(\\text{\\%}\\right)=\\frac{\\text{N}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}\\:\\text{o}\\text{f}\\:\\text{d}\\text{e}\\text{a}\\text{d}\\:\\text{l}\\text{a}\\text{r}\\text{v}\\text{a}\\text{e}\\:}{\\text{N}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}\\:\\text{o}\\text{f}\\:\\text{t}\\text{e}\\text{s}\\text{t}\\:\\text{l}\\text{a}\\text{r}\\text{v}\\text{a}\\text{e}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe mortality was calculated, determined the lethal concentrations (LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e) and their 95% upper and lower confidence limit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Ovicidal activity\u003c/h2\u003e \u003cp\u003eOvicidal activity was assessed following Su and Mulla's (1998) method, with minor modifications. Mosquito eggs or egg rafts were collected from a breeding cage (30 \u0026times; 30 \u0026times; 30 inches) containing an established mosquito colony. The eggs were then exposed to ten concentrations of EAECI (100, 150, 200, 250, 300, 350, 400, 450, 500 and 1000 \u0026micro;g/mL) in 100 mL plastic containers for a duration of 120 hours. After treatment, individual eggs or egg rafts from each concentration were transferred to glasses of distilled water for hatching evaluation. The eggs were counted under a microscope before transferring. Each experiment was conducted six times on different days, with six replicates, using the appropriate solvent as a control. Following the 120 hours treatment period, hatch rates were calculated using the following formula:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{E}\\text{g}\\text{g}\\:\\text{m}\\text{o}\\text{r}\\text{t}\\text{a}\\text{l}\\text{i}\\text{t}\\text{y}\\:\\:\\left(\\text{\\%}\\right)=\\frac{\\text{N}\\text{o}\\:\\text{o}\\text{f}\\:\\text{h}\\text{a}\\text{t}\\text{c}\\text{h}\\text{e}\\text{d}\\:\\text{e}\\text{g}\\text{g}\\:}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{N}\\text{o}\\:\\text{o}\\text{f}\\:\\text{e}\\text{g}\\text{g}\\text{s}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Oxidative stress biomarkers\u003c/h2\u003e \u003cp\u003eMosquito larvae from each treatment group were homogenized individually in 200 \u0026micro;L of phosphate buffer (0.1M, pH 7.6) at 4\u0026deg;C. The homogenates were centrifuged using a HERMLE Labortechnik centrifuge at 10,000 rpm for 10 minutes at 4\u0026deg;C. The resulting supernatant was collected and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for use as an enzyme extract. The supernatant was immediately utilized as the enzyme source for biochemical assays. Each biochemical experiment was conducted in triplicate, with 25 larvae used for each replicate. Protein content was quantified using the Bradford method (1976), with bovine serum albumin (BSA) as the standard reference. Catalase (CAT) activity was determined by measuring the absorbance of residual H₂O₂ (Beers \u0026amp; Sizer, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1952\u003c/span\u003e), while superoxide dismutase (SOD) activity was assessed based on the inhibition of nitrobluetetrazolium (NBT) photoreduction (Beauchamp and Fridovich, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Malondialdehyde (MDA) levels, indicative of lipid peroxidation, were measured by determining thiobarbituric acid-reactive substances (TBARS) (Ohkawa et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). SOD and CAT activities were expressed as U/mg protein, and MDA levels were reported as nMol TBARS/min/mg protein. All parameters were measured at room temperature (29\u0026deg;C) using a UV-visible spectrophotometer (Cecil Aquarius CE 7400). Detailed information regarding analytical instrument models, detection limits, and operational parameters for the detected biomarkers is provided in the supplementary materials.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Assays to determine the antibacterial activity\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Minimum inhibitory concentration (MIC)\u003c/h2\u003e \u003cp\u003eThe minimum inhibitory concentrations (MICs) of the EAECI were determined using the agar dilution method (Wiegand et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Briefly, stock solutions of 1000 \u0026micro;g/mL were prepared for each extract, which were then serially diluted to produce concentrations of 0,100, 200, 300, 400, 500 \u0026micro;g/mL. These diluted extracts were poured into sterile petri dishes and allowed to solidify. A 2 \u0026micro;L drop of the prepared test and control organisms was then inoculated onto the surface of each plate using a template. After incubation at 35\u0026deg;C for 24 hours, the plates were examined, and the MIC was defined as the lowest concentration of the extract that prevented visible growth of the organism. For positive control tetracycline (30 \u0026micro;g) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Scanning Electron Microscopy (SEM) study\u003c/h2\u003e \u003cp\u003eAccording to Hayat (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1981\u003c/span\u003e), The most susceptible bacterial strain to the EAECI was selected for scanning electron microscopy (SEM) analysis. Bacterial cells were collected from the inhibition zone after 24 hours of treatment and fixed in 2.5% glutaraldehyde solution for 45 min at room temperature. The fixed cells were dehydrated through a graded ethanol series (50% for 7\u0026ndash;5 min, 70% for 10 min, 90% for 5 min, and absolute ethanol for 5 min), followed by transfer to a 1:1 isoamyl alcohol:absolute ethanol mixture for 5 min and subsequently to pure isoamyl alcohol for 5 min. The samples were then air-dried, mounted onto aluminum stubs, sputter-coated with gold, and examined under a Hitachi S-530 SEM to observe morphological alterations induced by the EAECI.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Functional group analysis by FTIR (Fourier transform infrared)\u003c/h2\u003e \u003cp\u003eWe used FT-IR spectroscopy to find the functional groups, various bonds, and chemical makeup of the substance in the EAECI. FT-IR spectroscopy was performed using the positive response band compound in an FT-IR spectrophotometer (JASCO FT-IR Model-4700) at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C), with a scanning range of 400 to 4500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, to identify the functional groups of the EAECI. The peaks have been analysed using KnowItAll software (serial no. 107733-00001F44). The functional groups were identified by comparing the characteristic peak frequencies with those reported in the reference literature (Stuart, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Socrates, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9. GC-MS analysis\u003c/h2\u003e \u003cp\u003eThe EAECI was analyzed using GC-MS (Model: Clarus 680 GC) and the software used in the system is TurboMass Version 6.4.2 to identify and characterize bioactive components. The capillary column used is \u0026lsquo;Elite- 5MS\u0026rsquo; having dimensions length 60 m, ID 0.25 mm and film thickness 0.25 \u0026micro;m and the stationary phase is 5% diphenyl 95% dimethyl polysiloxane. At a flow rate of 1 mL/min, helium gas (99.99%) was utilised as the mobile phase. The run time was around 40 min. An 8 min solvent delay was maintained. Data analysis library (NIST-2014) was used to identify the peak.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eAnalysis of the obtained data was performed by using Graph pad-prism 9 software. All data presented are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM values of triplicates (n\u0026thinsp;=\u0026thinsp;3) and obtained from separate experiments. Probit analysis was used to calculate the 12, 24 and 48 hours LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e values (Finney, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Student\u0026rsquo;s t-test were used to compare means of control and the oxidative stress biomarkers, namely CAT (Catalase), SOD (Superoxide dismutase), and MDA (Malondialdehyde).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Larvicidal activity\u003c/h2\u003e \u003cp\u003eThe EAECI demonstrated dose-dependent larvicidal activity against \u003cem\u003eAe. aegypti\u003c/em\u003e larvae after 24 hours of treatment. The data were analyzed, and statistical values including LC\u003csub\u003e50\u003c/sub\u003e, LC\u003csub\u003e90\u003c/sub\u003e, lower confidence limit (LCL), upper confidence limit (UCL), were recorded. The larvicidal efficacy varied across different extracts of \u003cem\u003eC. indicum\u003c/em\u003e, with distinct LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e values. For the 12-hour treatment, the EAECI showed an LC\u003csub\u003e50\u003c/sub\u003e of 187.427 (125.078- 280.856) \u0026micro;g/mL and an LC\u003csub\u003e90\u003c/sub\u003e of 857.438 (572.204 -1284.857) \u0026micro;g/mL. In the 24-hour treatment, the LC\u003csub\u003e50\u003c/sub\u003e was 106.019 (66.604-168.759) \u0026micro;g/mL and the LC\u003csub\u003e90\u003c/sub\u003e was 539.890 (339.173-859.388) \u0026micro;g/mL. These results indicate that \u003cem\u003eAe. aegypti\u003c/em\u003e larvae were susceptible to the EAECI treatment (Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Ovicidal activity\u003c/h2\u003e \u003cp\u003eThe egg hatchability of dengue vector mosquitoes was tested across different concentrations of the extract. The hatchability percentage was found to be directly proportional to the number of eggs and inversely proportional to the concentration of the toxicant. The ovicidal activity of EAECI was observed against \u003cem\u003eAe. aegypti\u003c/em\u003e, where 100% egg mortality was recorded at a concentration of 350 \u0026micro;g/mL. In the control experiment, the eggs exhibited 100% hatchability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Oxidative stress enzymes\u003c/h2\u003e \u003cp\u003eExposure of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquito larvae to lethal concentrations of EAECI resulted in significant changes in total protein concentration and oxidative stress enzyme levels when compared to the control group. The total protein concentration in the control group was measured at 137.27 \u0026micro;g/mL, while in the EAECI-treated group, it increased to 220.02 \u0026micro;g/mL. Additionally, the levels of CAT, SOD, and MDA were elevated upon exposure to lethal concentrations of EAECI, with measurements of 57.04 \u0026micro;g/mL protein, 91.51 \u0026micro;g/mL protein, and 1.65 nMol TBARS/min/mg protein, respectively. In comparison, the control group showed values of 16.42 \u0026micro;g/mL protein, 14.36 \u0026micro;g/mL protein, and 0.92 nMol TBARS/min/mg protein. Statistical analysis revealed significant changes in oxidative stress parameters, with t-test results showing CAT (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), SOD (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and MDA (P\u0026thinsp;=\u0026thinsp;0.6311) levels significantly different from the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Antibacterial activity\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of EAECI was assessed against \u003cem\u003eB. megaterium\u003c/em\u003e (MTCC 2412) and \u003cem\u003eB. cereus\u003c/em\u003e (MTCC 1272). EAECI demonstrated moderate inhibitory effects, with both bacterial strains showing a minimum inhibitory concentration (MIC) of 200 \u0026micro;g/mL (Fig.\u0026nbsp;2). However, their susceptibility varied slightly: \u003cem\u003eB. megaterium\u003c/em\u003e exhibited a zone of inhibition (ZOI) of 10 mm, whereas \u003cem\u003eB. cereus\u003c/em\u003e was more sensitive, showing the largest ZOI among tested strains at 12 mm. Scanning electron microscopy (SEM) further confirmed the bactericidal action of EAECI displayed significant structural damage: \u003cem\u003eB. megaterium\u003c/em\u003e exposed to EAECI (Fig.\u0026nbsp;3a) exhibited clear membrane disintegration and cell surface roughening, while \u003cem\u003eB. cereus\u003c/em\u003e cells (Fig.\u0026nbsp;3b) showed pronounced shrinkage, irregular surfaces, and partial lysis. These ultrastructural alterations indicate that EAECI compromises bacterial cell envelope integrity, ultimately leading to cell death. The combined MIC, ZOI, and SEM findings validate the bactericidal mechanism of EAECI against these pathogenic \u003cem\u003eBacillus\u003c/em\u003e species.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. FTIR analysis\u003c/h2\u003e \u003cp\u003eFTIR (Fourier-transform infrared) spectroscopy was employed to analyze the EAECI, aiming to identify the functional groups potentially responsible for their bioactivity. In the EAECI spectrum, the characteristic absorption peaks were observed at 3432.67 cm⁻\u0026sup1;, corresponding to O\u0026ndash;H stretching. Peaks at 2975.62 cm⁻\u0026sup1; indicated C\u0026ndash;H stretching, the peak at 1642.09 cm⁻\u0026sup1; indicated C\u0026thinsp;=\u0026thinsp;C stretching, while the peak at 1046.19 cm⁻\u0026sup1; suggested the presence of an ester group (Fig.\u0026nbsp;4). These FTIR spectra confirm the presence of various functional groups, including hydroxyl, carboxylic acid, alkane, alkene, ester, aromatic, and ether groups, as outlined by (Socrates, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Stuart, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6. GC-MS analysis\u003c/h2\u003e \u003cp\u003eThe chemical composition of the EAECI was analyzed using GC-MS, revealing the presence of main two compounds 3,3 dimethylcyclohexene (Fig.\u0026nbsp;5) and tetracosanoic acid, methyl ester (Fig.\u0026nbsp;6) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGC-MS Spectral Analysis of EAECI\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRT (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eName of the Compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecular Formula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMolecular Weight\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePeak Area (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26.586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3,3-dimethylcyclohexene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e110.1094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTetracosanoic acid, methyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e50\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e382.6633\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36.173\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRecently, plant-derived bio-pesticides have gained significant attention as safer alternatives to traditional synthetic insecticides, particularly in the control of disease-carrying vectors. These plant-based chemicals are favoured for their safety profile, as they are non-toxic to humans and animals, lack phototoxic effects, and do not leave harmful residues in the environment (Schmutterer, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Chatterjee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). As the development of resistance to conventional synthetic insecticides becomes an increasing challenge in vector mosquito management, there is a pressing need for innovative solutions or novel insecticides (Chandre et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The emergence of insecticide-resistant mosquito populations calls for the exploration of alternative control methods, such as the use of plant-derived bio-pesticides, which could play a key role in integrated vector management strategies. Bowers et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), emphasized that screening locally available medicinal plants for their mosquito control potential could stimulate local economies, reduce dependence on costly imported chemicals, and support community-driven public health initiatives. Plants contain a wide range of bioactive compounds with diverse biological activities, which are believed to be responsible for their pesticidal properties. These compounds, such as toxins and secondary metabolites, act as effective mosquito control agents, contributing to the larvicidal and adulticidal effects observed in many plant extracts (Niraimathi et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This is particularly important for managing disease vectors, where reducing the population of mosquitoes is crucial in preventing the transmission of diseases like dengue, malaria, and Zika virus. Our study supports these findings, as it demonstrated that an increase in the concentration of EAECI significantly contributed to the mortality of \u003cem\u003eAe. aegypti\u003c/em\u003e larvae. While the medicinal properties of \u003cem\u003eC. indicum\u003c/em\u003e have been well-documented (Shrivastava and Patel, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), the mosquito larvicidal effects of its leaves have not been extensively explored. Our results provide valuable new insights into the potential of \u003cem\u003eC. indicum\u003c/em\u003e as an effective natural bio-pesticide. Similar studies, such as the work by Mathew et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), who investigated the methanol extract of \u003cem\u003eC. ternatea\u003c/em\u003e seeds, found notable larvicidal activity against \u003cem\u003eAe. aegypti\u003c/em\u003e larvae with an LC\u003csub\u003e50\u003c/sub\u003e of 154.5 ppm. Likewise, Ansari et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), demonstrated that \u003cem\u003ePinus longifolia\u003c/em\u003e oil exhibited larvicidal efficacy against \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes, with an LC\u003csub\u003e50\u003c/sub\u003e value of 82.1 ppm. These findings align with our results, highlighting the potential of plant extracts for mosquito control. In addition, numerous other recent studies have corroborated our findings regarding the larvicidal activity of various plant extracts against \u003cem\u003eAe. aegypti\u003c/em\u003e larvae (Onah et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ajaegbu et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bakar et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Mechanistic insights into its larvicidal effect were provided by biochemical assays, which revealed significant elevations in catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels in treated larvae, suggesting that EAECI induces oxidative stress that disrupts normal metabolic functions and damages cellular components, a mode of action consistent with earlier observations that phytochemicals trigger redox imbalance leading to larval death (Govindarajan and Benelli, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This dual activity direct larvicidal toxicity along with oxidative stress induction indicates that EAECI may exert synergistic pressure on mosquito larvae, thereby reducing the likelihood of resistance development that is common with conventional synthetic insecticides (Chandre et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn parallel to its mosquitocidal efficacy, EAECI also displayed moderate antibacterial activity against two Gram-positive pathogens, \u003cem\u003eB. megaterium\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e, with a minimum inhibitory concentration (MIC) of (200 \u0026micro;g/mL) for both strains. The bactericidal nature of EAECI was further confirmed through scanning electron microscopy, where untreated control cells maintained smooth, intact rod-shaped morphologies, while treated cells showed dramatic ultrastructural alterations, including membrane disintegration, surface shrinkage, and partial lysis, thereby suggesting that EAECI compromises bacterial envelope integrity and leads to cell death. These observations align with earlier studies demonstrating that plant-derived secondary metabolites such as alkaloids, tannins, and saponins disrupt bacterial cell walls and interfere with critical physiological processes (Hassan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The moderate antimicrobial activity observed in our study is particularly noteworthy as bacterial pathogens like \u003cem\u003eB. cereus\u003c/em\u003e are associated with food borne diseases, and plant-derived agents with membrane-disruptive properties offer promising natural alternatives to chemical antibiotics, especially in the context of increasing antimicrobial resistance. The phytochemical basis of EAECI\u0026rsquo;s bio efficacy was further elucidated by FT-IR and GC-MS analyses. FT-IR spectra revealed functional groups including hydroxyl, ester, alkene and carboxylic acids, which are well known for contributing to biological activity by enabling hydrogen bonding, disrupting microbial proteins and enhancing lipophilicity to facilitate cell penetration (Sofowara, 1993; Stuart, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). GC-MS analysis identified two major compounds, 3,3-dimethylcyclohexene and tetracosanoic acid, methyl ester, the latter constituting 36.17% of the total peak area. Fatty acid esters like tetracosanoic acid derivatives are widely recognized for their antimicrobial and surface-active properties, and their high abundance in EAECI likely contributes substantially to the bactericidal and larvicidal activities observed in the present study (Rahman et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Raihan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Taken together, these results demonstrate that EAECI exerts its bioactivity through a combination of biochemical, structural and molecular mechanisms, including oxidative stress induction, membrane disruption, and the action of fatty acid derivatives and other phytochemicals. Importantly, the multifunctionality of EAECI highlights its potential for integration into eco-friendly vector and microbial control strategies, offering a sustainable alternative to chemical pesticides and antibiotics, which often face challenges of resistance, environmental persistence and non-target toxicity (Schmutterer, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Bowers et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Moreover, the dual larvicidal and antibacterial activities of EAECI underscore its relevance in tropical and subtropical regions where mosquito-borne diseases and bacterial infections frequently coexist as public health burdens, thereby providing a holistic approach to disease management. By validating the ethnomedicinal applications of \u003cem\u003eC. indicum\u003c/em\u003e with modern scientific evidence, the present study not only supports its traditional therapeutic use (Shrivastava and Patel, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) but also contributes to the growing framework of plant-based integrated pest and pathogen management. Ultimately, the findings suggest that \u003cem\u003eC. indicum\u003c/em\u003e EAECI, with its demonstrated efficacy against both insect vectors and bacterial pathogens, may serve as a promising natural bioagent capable of addressing urgent challenges in public health, including vector-borne disease transmission and antimicrobial resistance, while minimizing ecological risks and fostering sustainable disease control practices.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present investigation highlights the ethyl acetate leaf extract of \u003cem\u003eC. indicum\u003c/em\u003e (EAECI) as a potent multifunctional biocontrol agent with promising applications in integrated vector and microbial disease management. The extract exhibited strong larvicidal activity against \u003cem\u003eAe. aegypti\u003c/em\u003e, with complete ovicidal efficacy at relatively low concentrations, and induced significant oxidative stress in larvae, suggesting a dual mechanism of direct toxicity and redox imbalance. Alongside its mosquitocidal potential, EAECI demonstrated moderate antibacterial activity against \u003cem\u003eB. megaterium\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e, with a minimum inhibitory concentration (MIC) of 200 \u0026micro;g/mL, while scanning electron microscopy confirmed its bactericidal nature through observable ultrastructural damage to bacterial cell membranes. Phytochemical characterization by FTIR revealed the presence of hydroxyl, ester, alkene, and carboxylic acid groups, while GC-MS identified 3,3-dimethylcyclohexene and tetracosanoic acid, methyl ester the latter representing a major component likely responsible for its bioactivity. These findings collectively validate the traditional medicinal uses of \u003cem\u003eC. indicum\u003c/em\u003e and provide mechanistic evidence supporting its efficacy as an eco-friendly alternative to synthetic insecticides and antibiotics, which are increasingly challenged by resistance and environmental concerns. By combining larvicidal, ovicidal, oxidative stress-inducing, and antimicrobial properties within a single botanical extract, \u003cem\u003eC. indicum\u003c/em\u003e demonstrates multifunctionality that is particularly valuable in tropical regions burdened with vector-borne diseases and bacterial infections. Future research should focus on formulation development, field trials, and safety evaluations to facilitate its translation into practical, sustainable biocontrol solutions.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003cb\u003eFunding Declaration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst author, Gobinda Dhibar, acknowledges financial support from the Council of Scientific and Industrial Research (CSIR), Government of India, in the form of Junior\u003c/p\u003e \u003cp\u003eResearch Fellowship (JRF) and Senior Research Fellowship (SRF), Grant No. 09/025(0283)/2019-EMR-I Dated: 30.11.2019. No\u003c/p\u003e \u003cp\u003eother external funding was received for this study.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eG.D.: Investigation, Methodology, Antimicrobial Analysis, Writing \u0026ndash; Original Draft.G.D., S.B., and D.S.: Scanning Electron Microscopy Analysis.S.B. and D.S.: Larvicidal Analysis, Writing \u0026ndash; Review \u0026amp; Editing.B.G., R.C., and R.D.: Investigation, Writing \u0026ndash; Review \u0026amp; Editing.S.R. and S.C.: Conceptualization, Investigation, Formal Analysis, Supervision, Writing \u0026ndash; Review \u0026amp; Editing.All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge Dr. Karthigeyan Kaliyamurthy, Scientist-E, BSI, Howrah, India, for authenticating the plant species. The facilities sponsored by UGC-MRP, DST-PURSE, DST-FIST, and UGC-DRS in the Department of Zoology, The University of Burdwan, were utilized for this research. The authors also express their gratitude to IIT Guwahati and Biotech Park for conducting the GC\u0026ndash;MS analysis, and to USIC, The University of Burdwan, for providing FE\u0026ndash;SEM facilities. ChatGPT was used by the author to enhance linguistic clarity and improve readability of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information. All the FTIR and GC-MS data are represented with Figure 4,5,6 and 7 in supplementary file attached.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdhikary, K. et al. 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Essential oils loaded on polymeric nanoparticles: bioefficacy against economic and medical insect pests and risk evaluation on terrestrial and aquatic non-target organisms. \u003cem\u003eEnviron. Sci. Pollut Res.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (47), 71412\u0026ndash;71426 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Clerodendrum indicum, GCMS, Aedesaegypti, oxidative stress enzymes, larvicide, antibacterial activity","lastPublishedDoi":"10.21203/rs.3.rs-8162261/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8162261/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eClerodendrum indicum\u003c/em\u003e, a traditionally used medicinal plant in South and Southeast Asia, was evaluated for the bioactivity of its ethyl acetate leaf extract (EAECI). The extract showed strong larvicidal and ovicidal effects against \u003cem\u003eAedes aegypti\u003c/em\u003e, producing dose-dependent larval mortality with LC₅₀ values of 187.427 \u0026micro;g/mL at 12 hours and 106.019 \u0026micro;g/mL at 24 hours. Complete ovicidal activity occurred at 350 \u0026micro;g/mL. EAECI exposure induced oxidative stress in larvae, evidenced by elevated levels of CAT, SOD, and MDA. The extract also exhibited notable antibacterial activity against \u003cem\u003eBacillus megaterium\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e, with MICs between 100 and 500 \u0026micro;g/mL, and SEM imaging confirmed membrane deformation in treated bacteria. Phytochemical and FT-IR analyses verified the presence of hydroxyl, alkene, ester, and carboxylic acid groups. GC-MS profiling identified major components, particularly tetracosanoic acid methyl ester (36.17%) and 3,3-dimethylcyclohexene. Overall, the findings demonstrate that EAECI contains potent bioactive compounds with significant insecticidal and antimicrobial potential, supporting its use as an eco-friendly, plant-based agent for integrated vector and microbial disease control.\u003c/p\u003e","manuscriptTitle":"Larvicidal Efficacy Against Dengue Vector Aedes aegypti and Antibacterial Activity of Leaf Ethyl Acetate Extract of Clerodendrum indicum (L.) 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