Evaluation of Asparagus racemosus Root Extract and Eucalyptus Oil Formulations as Plant-Based Repellents Against Mosquitoes and Cockroaches

Vector-borne diseases and domestic pest infestations continue to impose a substantial global burden, particularly in densely populated and tropical regions. Mosquitoes act as vectors of malaria, dengue, chikungunya, yellow fever, and filariasis, while cockroaches are recognized as mechanical carriers of pathogens and potent indoor allergens (WHO, 2020; Pai et al., 2022). Their control remains a public health priority, as both groups significantly contribute to morbidity, mortality, and reduced quality of life.

Conventional control strategies rely heavily on synthetic insecticides and repellents such as N,N-diethyl-meta-toluamide (DEET), pyrethroids, and organophosphates. While effective, their long-term and widespread use has raised serious concerns. Reported drawbacks include the emergence of insecticide resistance, contamination of soil and aquatic ecosystems, and adverse health impacts ranging from skin irritation to neurotoxicity and endocrine disruption (Hemingway et al., 2016; Norris & Coats, 2017). These limitations necessitate the exploration of safer, sustainable alternatives.

Plant-based repellents and insecticidal agents are increasingly recognized as promising candidates due to their safety, rapid biodegradability, and environmental compatibility (Isman, 2020). Medicinal and aromatic plants contain diverse phytochemicals—such as alkaloids, terpenoids, flavonoids, and phenolics—that exhibit repellent, insecticidal, and larvicidal properties with minimal side effects. Importantly, the chemical complexity and multi-target modes of action of botanicals reduce the likelihood of resistance development compared to single-molecule synthetic agents (Regnault-Roger et al., 2012).

Several plants have been documented as effective repellents against mosquitoes and cockroaches. Examples include Azadirachta indica (azadirachtin), Cymbopogon citratus (citronellal), Ocimum sanctum (eugenol), Mentha piperita (menthol), and Eucalyptus globulus (1,8-cineole), which interfere with insect behavior, feeding, and development (Maia & Moore, 2011; Nerio et al., 2010).

Asparagus racemosus (Shatavari), a member of the family Asparagaceae, is traditionally valued in Ayurvedic medicine for its adaptogenic, antioxidant, and immunomodulatory properties (Alok et al., 2013). Interestingly, recent entomological studies have reported that A. racemosus root extracts exhibit ovicidal, larvicidal, and adulticidal activity against disease-vector mosquitoes (Aedes aegypti, Anopheles stephensi, Culex quinquefasciatus) (Govindarajan & Sivakumar, 2014). However, its potential as a repellent remains underexplored.

The present study was therefore designed to evaluate the repellent efficacy of A. racemosus root extract, in combination with E. globulus essential oil, against mosquitoes and cockroaches. Two formulations were developed: a topical cream targeting mosquito repellency and a surface spray aimed at cockroach control. The formulations were subjected to physicochemical evaluation, stability testing, and laboratory bioassays to provide scientific validation for the use of A. racemosus as a plant-based repellent alternative to synthetic agents.

MATERIAL AND METHODS

Plant Material and Extraction

Roots of Asparagus racemosus Willd. (Asparagaceae) were collected from local areas of Hyderabad, Telangana, India, during the flowering season (August–September 2024). The plant material was authenticated by a taxonomist at [Institution/University name], and a voucher specimen (No. XXXX) was deposited in the departmental herbarium for reference.

The roots were shade-dried at room temperature, coarsely powdered, and subjected to Soxhlet extraction using 70% ethanol for 48 h. The ethanolic extract of A. racemosus roots (EARR) was concentrated under reduced pressure using a rotary evaporator at 40 °C, yielding a semisolid residue. The extract was stored in airtight containers at 4 °C until further analysis.

Phytochemical Screening and Quantitative Analysis

Preliminary Phytochemical Screening

The crude extract was subjected to preliminary phytochemical tests for alkaloids, saponins, tannins, flavonoids, glycosides, and phenolics using standard qualitative methods described by Harborne (1998) and Trease and Evans (2002).

Determination of Total Phenolic Content (TPC)

The TPC of EARR was determined using the Folin–Ciocalteu reagent colorimetric method (Singleton, Orthofer, and Lamuela-Raventós, 1999). Briefly, 0.5 mL of extract solution (1 mg/mL) was mixed with 2.5 mL of Folin–Ciocalteu reagent (diluted 1:10) and incubated for 5 min. Thereafter, 2 mL of sodium carbonate (7.5% w/v) was added, and the mixture was incubated for 30 min in the dark at room temperature. Absorbance was recorded at 765 nm using a UV–Vis spectrophotometer. Gallic acid was used as the standard, and results were expressed as milligrams of gallic acid equivalents per gram of extract (mg GAE/g).

Determination of Total Flavonoid Content (TFC)

The TFC of EARR was estimated using the aluminium chloride colorimetric method (Chang, Yang, Wen, and Chern, 2002). A 0.5 mL aliquot of extract solution (1 mg/mL) was mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminium chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. After incubation at room temperature for 30 min, absorbance was measured at 415 nm. Quercetin was used as the standard, and results were expressed as milligrams of quercetin equivalents per gram of extract (mg QE/g).

Mosquito Rearing

Mosquito larvae were collected from stagnant ponds in Hyderabad and identified morphologically. They were reared in glass containers containing dechlorinated water under laboratory-controlled conditions (27 ± 2 °C, 75–85% relative humidity, 14:10 h light/dark photoperiod). Larvae were fed daily with a mixture of dog biscuits and yeast in a 3:1 ratio until pupation. Pupae were transferred to emergence cages, and adults were maintained under the same conditions. Female mosquitoes were blood-fed on rodents to stimulate oviposition and colony establishment.

Formulation of Topical Creams

Two oil-in-water (O/W) emulsion cream formulations were prepared using the fusion method.

• Test formulation: contained 10% w/w EARR and 5% w/w Eucalyptus globulus oil.
• Control formulation: contained 5% w/w Eucalyptus globulus oil only.

Other excipients included stearic acid, cetostearyl alcohol, glycerin, potassium hydroxide, methylparaben, and distilled water. The oil phase (stearic acid, cetostearyl alcohol, eucalyptus oil) and aqueous phase (glycerin, potassium hydroxide solution, methylparaben in distilled water) were separately heated to 70 °C. The aqueous phase was slowly added to the oil phase with continuous stirring to form an emulsion. The resultant cream was cooled to room temperature, transferred into amber glass jars, and stored until evaluation.

Table: 1 Composition of herbal cream for mosquito repellent action

Mosquito Repellent Efficacy

The repellent activity of the formulations was evaluated using the Arm-in-Cage (AIC) method in accordance with World Health Organization (WHO) guidelines (WHO, 2009). In this assay, healthy volunteers introduced their treated forearm into a cage containing 20 unfed female mosquitoes maintained under controlled laboratory conditions (27 ± 2 °C, 75–85% RH, and a 14:10 h light/dark cycle). The number of mosquito landings and bites on the treated arm was recorded at fixed time intervals and compared with those on an untreated control arm.

Figure 1:Arm-In-Cage Method

The percentage protection was calculated using the formula:

Protection (%) = U (U−T) ?× 100

where U = number of landings/bites on the untreated control and T = number on the treated arm.

The cream formulation containing EARR (10%) and eucalyptus oil (5%) exhibited a higher repellency rate, offering longer protection duration and significantly fewer mosquito landings compared to the control formulation with only eucalyptus oil.

Cockroach Repellent Formulation and Testing

Adult cockroaches were collected from local residential kitchens and bathrooms in Hyderabad, where infestation levels were observed to be moderate to high. Specimens were captured using non-toxic sticky traps and carefully transferred to clean laboratory containers. Prior to experimentation, the cockroaches were acclimatized for 24 hours under laboratory conditions to minimize stress and standardize behavioral responses.

For the repellent study, a herbal spray formulation was prepared containing 10% ethanolic extract of Asparagus racemosus roots (EARR) and 5% eucalyptus oil. These active components were dispersed in 60% ethanol with the addition of 2% Tween 80 as a surfactant, and the final volume was adjusted to 100 mL with distilled water. A control spray formulation, without EARR, was prepared in the same manner.

All formulations were homogenized thoroughly, filtered through muslin cloth, and stored in amber-colored PET spray bottles to prevent phytochemical degradation due to light exposure.

Table 2: Composition of herbal cockroach repellent spray

The prepared cockroach repellent sprays were subjected to physicochemical evaluation. Visual inspection was carried out to assess clarity, homogeneity, and phase separation, while the pH was measured using a digital pH meter to ensure suitability for both skin contact and household surface applications. Both formulations exhibited good physical stability during storage, with no signs of sedimentation, odor alteration, or phase separation. The incorporation of Tween 80 as a surfactant enhanced emulsion stability and facilitated uniform dispersion of eucalyptus oil within the ethanolic extract. These findings confirmed that the EARR-based spray maintained its physical integrity and was appropriate for domestic use.

Repellent efficacy was evaluated using the two-choice chamber method. A rectangular arena was divided into two equal zones, with one surface treated with the test formulation and the other left untreated as control. A group of 10–15 healthy adult cockroaches was released at the center of the arena, and their movement patterns were monitored over a period of 15–60 minutes. The number of cockroaches present in each zone was recorded, and the percentage repellency was calculated using the formula:

Repellency  (%) = Nc ?(Nc?−Nt?) ?× 100

where Nc represents the number of cockroaches in the control zone and Nt represents the number in the treated zone.

The formulation containing EARR (10%) and eucalyptus oil (5%) demonstrated strong repellency (>80%), which was significantly higher compared to the eucalyptus-only spray, confirming its superior efficacy for household pest management.

Molecular Docking and Statistical Analysis

Molecular docking studies were carried out using CB-Dock2 to predict the binding interactions of selected phytoconstituents from Asparagus racemosus with insect acetylcholine receptors (AChRs), which are critical for neural signaling and survival. Inhibition or disruption of AChRs interferes with normal neurotransmission, thereby contributing to insecticidal and repellent effects. AutoDock Vina was employed to calculate binding affinities, where more negative Vina scores indicated stronger ligand–receptor interactions.

All experimental data from the in vivo repellent assays—including the Arm-in-Cage test for mosquitoes and the Two-choice chamber test for cockroaches—were subjected to statistical analysis. Results were expressed as Mean ± Standard Deviation (SD) from three independent replicates (n = 3). A two-way Analysis of Variance (ANOVA) was performed to determine the individual and interactive effects of time and formulation on repellent efficacy. Statistical significance was accepted at p < 0.05. Data analysis and graphical representations were carried out using GraphPad Prism version 9.0 (GraphPad Software, San Diego, USA).

RESULTS

The percentage yield of the Asparagus racemosus ethanolic extract was found to be 18% w/w. Preliminary phytochemical screening confirmed the presence of diverse bioactive compounds including alkaloids, flavonoids, saponin glycosides, tannins, steroids, triterpenoids, amino acids, carbohydrates, fats and fixed oils, proteins, and starch, which may contribute to the pharmacological and repellent properties of the plant.

Quantitative estimation showed that the total phenolic content of the extract was 5.419 mg gallic acid equivalents (GAE)/g of dried root, as determined from the gallic acid calibration curve (y = 0.0026x + 0.5491, R² = 0.9697). The total flavonoid content was 3.515 mg quercetin equivalents (QE)/g, calculated from the quercetin standard curve (y = 0.002x – 0.0043, R² = 0.9914). GC-MS analysis revealed several phytoconstituents, including methyl sulfidtiole, xanthone, oxirane, n-hexadecanoic acid, flavone, 1,8-nonadiene, epoxyhexanol, pentadecane-2,4-dione, and stigmasterol.

Herbal cream formulations prepared with A. racemosus root extract and eucalyptus oil, and a control cream with eucalyptus oil alone, were assessed for physicochemical parameters. Both formulations displayed smooth and homogeneous textures without evidence of phase separation. The pH values were 6.2 ± 0.1 for the test cream and 6.1 ± 0.2 for the control, which fall within the acceptable range for topical application. Both formulations exhibited satisfactory spreadability and washability, although the test cream showed slightly reduced spreadability. The viscosity values were 22,500 ± 300 cP (test cream) and 21,800 ± 350 cP (control), consistent with desirable topical cream properties. Stability testing under accelerated conditions (40 ± 2 °C, 75 ± 5% RH) for four weeks revealed no significant changes in pH, color, odor, or consistency, confirming the stability of both creams.

The mosquito repellent efficacy, determined using the Arm-in-Cage method, demonstrated superior activity of the A. racemosus + eucalyptus cream compared to the eucalyptus-only cream. At 30 minutes post-application, the test cream achieved 81.32% repellency versus 71.43% for the control. Repellency decreased progressively over time, but the test cream maintained 50.00% repellency at 120 minutes, compared to 31.33% for the control. At 180 minutes, repellency values declined to 33.11% (test cream) and 11.92% (control), indicating that while efficacy reduced over time, the A. racemosus formulation provided longer-lasting protection.

Table 3: Repellent Efficacy of Herbal and Eucalyptus Creams in Arm-in-Cage Mosquito  Test

(Mean ± SD, n = 20 mosquitoes per trial)

Figure 2: Comparison of Percentage Mosquito Repellency between Herbal Cream (Asparagus racemosus + Eucalyptus) and Eucalyptus Alone Cream Over Time

Similarly, evaluation of the cockroach repellent sprays showed that both the herbal and control formulations produced stable emulsions with acceptable clarity and sprayability. The herbal spray containing A. racemosus extract and eucalyptus oil demonstrated a maximum repellency of 77.89% at 30 minutes, significantly higher than the control. At the peak repellency point, the number of cockroach entries in the treated area was only 4.2 ± 0.7. Although repellency declined with time, it remained statistically significant across the 180-minute study. At 90 minutes, repellency was 53.93%, and it persisted at 27.04% after 180 minutes, underscoring its potential for prolonged household application. Physicochemical evaluation of both sprays, including pH, viscosity, spray delivery rate, and mist pattern, confirmed their suitability for indoor use.

Table 4: Repellent Efficacy of Herbal and Eucalyptus spray in  Two choice chamber for cockroach  repellent Test

(Mean± SD, n = 20 cockroaches per trial)

Figure 3: EARR + Eucalyptus sprayed paper in left side chamber and control treatment in right side at 30 minutes.

Figure 4: Eucalyptus sprayed paper in right side chamber and  control treatment in the left  side at 30 minutes.

Molecular docking studies were performed to investigate the potential interactions of key phytoconstituents from Asparagus racemosus root extract (EARR) with insect acetylcholinesterase (AChE), a critical enzyme in the nervous system. Compounds such as stigmasterol, asparanin B, shatavarin I, flavone, and n-hexadecanoic acid were docked using CB-Dock2 and analyzed with AutoDock Vina. The docking results revealed that stigmasterol and asparanin B exhibited the strongest binding affinities, with Vina scores of –11.6 and –11.5, respectively, indicating high potential for AChE inhibition. Other compounds, including shatavarin I and flavone, also demonstrated significant binding interactions, suggesting that multiple constituents may contribute synergistically to neuroinhibition. The binding poses showed stable interactions within the active site of AChE, including hydrogen bonding and hydrophobic interactions, which likely disrupt normal neurotransmission in insects. These findings support the hypothesis that the repellent activity observed in vivo against mosquitoes and cockroaches may be mediated, at least in part, through the neurotoxic effects of EARR phytoconstituents.

Figure 5: Molecular analysis of the binding affinities and receptor interaction of the selected active compounds toward the hub target Acetyl Choline Esterase (1EEA).