Repellent efficacy of water-balm formulation against Aedes aegypti based on zedoary, Curcuma zedoaria

Abstract

Essential oil from Curcuma zedoaria rhizomes is rich in bioactive sesquiterpenes with reported insect-repellent properties; however, its application as a topical mosquito repellent remains insufficiently explored. In this study, essential oil from C. zedoaria rhizomes was obtained by hydrodistillation and characterized using gas chromatography-mass spectrometry (GC-MS), followed by formulation into a hydroalcoholic water-balm containing 15% (w/w) essential oil. Repellent efficacy against adult female Aedes aegypti was evaluated using the Arm-in-Cage assay over a 180-min period, and cutaneous irritation was assessed in 12 adult volunteers using a standardized patch test. GC-MS analysis identified 37 compounds representing 99.98% of the total oil composition, predominantly sesquiterpenes, with AR-turmerone (23.66%), zingiberene (14.74%), and β-sesquiphellandrene (10.64%) as the major components. The formulation was well tolerated, with no skin irritation observed in any participant. Repellent activity was strong and sustained, providing 100% protection during the first 60 min post-application. Median repellency remained high at 98.50% at 120 min and 93.06% at 180 min, with an overall median repellency of 97.82%. The median complete protection time was 150 min (range: 90–180 min). These findings demonstrate that the C. zedoaria-based water-balm formulation provides effective protection against Ae. aegypti and supports the development of zedoary essential oil as a promising botanical alternative to synthetic mosquito repellents.

Graphical abstract

Highlights

• •Zedoary essential oil showed strong repellency against Aedes aegypti.
• •GC-MS revealed a sesquiterpene-rich chemotype dominated by AR-turmerone.
• •A zedoary-based water-balm roll-on was successfully developed.
• •The formulation provided > 93% repellency for up to 3 h.
• •No skin irritation was observed in human patch testing.

1. Introduction

Mosquito-borne diseases, including dengue, chikungunya, Zika, and yellow fever, continue to pose escalating public health threats, with notable global resurgences reported in recent years (Dahmana and Mediannikov, 2020; WHO, 2025). Among mosquito vectors, Aedes aegypti is of particular concern due to its strong anthropophilic behavior, adaptation to urban environments, and high efficiency in transmitting multiple arboviruses, contributing substantially to global disease burden (Kraemer et al., 2015). In the absence of universally effective vaccines and sustainable large-scale vector suppression strategies, personal protection measures continue to play a critical role in reducing human-vector contact and interrupting disease transmission. Topical mosquito repellents represent one of the most practical and widely adopted personal protection tools. These products function primarily by interfering with mosquito olfactory-mediated host-seeking behavior, thereby reducing landing and biting on human skin. Common synthetic repellents include N,N-diethyl-meta-toluamide (DEET), icaridin, and ethyl-butylacetylaminopropionate (IR3535). Although DEET remains the benchmark repellent due to its broad-spectrum and long-lasting efficacy, concerns related to unpleasant odor, skin discomfort, and rare but reported neurotoxic effects at high or prolonged exposure have been documented (Koren et al., 2003; Katz et al., 2008; Yadav et al., 2025). While icaridin and IR3535 exhibit improved safety profiles and are recommended by the World Health Organization, increasing awareness of environmental impact and consumer preference has driven growing interest in botanical and naturally derived alternatives (Islam et al., 2017; Feuser et al., 2020). Plant essential oils have attracted attention as eco-friendly mosquito repellents due to their complex mixtures of volatile bioactive compounds, rapid biodegradability, and generally low mammalian toxicity (Bakkali et al., 2008; Chellappandian et al., 2018; Ivanova et al., 2025). Many essential oil constituents, particularly terpenoids, have demonstrated repellent, insecticidal, or behavioral deterrent effects against medically important vectors by disrupting olfactory signaling pathways involved in host detection (Nerio et al., 2010; Yadav et al., 2025). Curcuma zedoaria Roscoe (Zingiberaceae), commonly known as zedoary or white turmeric, is a medicinal and culinary plant widely used in Asia. Its rhizome essential oil is rich in bioactive sesquiterpenes, including turmerones, zingiberene, humulene, bisabolene, and sesquiphellandrene, for which recent studies continue to report strong insecticidal, insect-repellent, anti-inflammatory, and antimicrobial activities (Hong et al., 2001; Lai et al., 2004; Lobo et al., 2009; Phukerd and Soonwera, 2013; Poudel et al., 2022; Panda et al., 2025). Previous studies have demonstrated that C. zedoaria essential oil possesses larvicidal, pupicidal, and adulticidal activities against several mosquito species, together with repellent activity against Ae. aegypti and Culex quinquefasciatus (Chaiyasit et al., 2006; Phukerd and Soonwera, 2013, 2014).

However, systematic evaluation of its repellency against adult Ae. aegypti, particularly in formulations designed for topical human application, remains limited. In addition, studies translating C. zedoaria essential oil into standardized topical delivery systems suitable for practical use are scarce. Therefore, this study aimed to characterize the chemical composition of C. zedoaria rhizome essential oil using gas chromatography-mass spectrometry (GC-MS), develop a hydroalcoholic zedoary-based water-balm formulation for topical application, and evaluate its skin compatibility and repellent efficacy against Ae. aegypti under controlled laboratory conditions. This work provides applied experimental evidence supporting the potential of C. zedoaria-based botanical formulations as plant-derived tools for personal protection against mosquito vectors of human disease.

2. Materials and methods

2.1. Plant material and chemical ingredients

Fresh rhizomes of zedoary used in this study were purchased from a reputable local herbal medicine supplier (Chao Krom Poe Dispensary Pharmacy, Bangkok, Thailand). The plant material was supplied as commercially labeled rhizomes intended for traditional medicinal and aromatic use. Mint essential oil (Mentha arvensis) was obtained from Chemipan Corporation Co., Ltd. (Bangkok, Thailand). All excipients and additional reagents used for formulation and chemical analysis were of cosmetic or analytical grade and were purchased from Chanjao Longevity Co., Ltd. (Bangkok, Thailand). All materials were stored under the manufacturers’ recommended conditions until use.

2.2. Extraction of zedoary essential oil

Fresh rhizomes of zedoary were subjected to hydrodistillation to obtain essential oil. The distillation apparatus consisted of an electromantle, a round-bottom flask, a Clevenger-type extraction column, and a water-cooled condenser. Approximately 500 g of freshly cut rhizomes were placed in the distillation flask containing 1600 ml of distilled water and heated to boiling. Hydrodistillation continued until no further oil was recovered. The resulting condensate was transferred to a separating funnel, where the essential oil layer was isolated, dried over anhydrous sodium sulfate (Na₂SO₄), and transferred into amber glass vials. The oil was stored at 4 °C in the dark until further analysis.

2.3. Gas chromatography-mass spectrometry (GC-MS) analysis

The zedoary essential oil was analyzed using an Agilent 6890N gas chromatograph coupled to a 5973N mass selective detector (MSD). Separation was achieved on an HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness), with helium as the carrier gas at a constant flow rate of 1.0 ml/min. The oven temperature program was initiated at 60 °C (held for 1 min), followed by an increase of 10 °C/min to 350 °C. The injector temperature was maintained at 250 °C and operated in splitless mode to enhance detection sensitivity for low-abundance constituents. Mass spectrometric detection was performed under electron ionization (EI), with the ion source and quadrupole temperatures set at 230 °C and 150 °C, respectively. These analytical conditions produced well-resolved chromatographic peaks and reproducible mass spectra suitable for qualitative characterization of the essential oil. Compound identification was carried out by comparison of mass spectra with authenticated reference standards, co-injection (spiking), and interpretation of fragmentation patterns. Spectral matching was performed using the National Institute of Standards and Technology (NIST) and Wiley GC-MS libraries. Chemical classification and structural taxonomy of identified compounds were further verified using the PubChem database and the ClassyFire chemical ontology (Djoumbou Feunang et al., 2016). Relative abundances of individual constituents were calculated by peak-area normalization of the total ion chromatogram, following standard essential-oil analytical procedures (Adams, 2007; Alvarez-Ospina et al., 2013).

2.4. Preparation of zedoary-based water-balm formulation

The composition of the mosquito-repellent water-balm formulation is presented in Table 1. The formulation consisted of essential oils, aromatic modifiers, and functional excipients selected to ensure repellency, physicochemical stability, and suitability for topical application in a hydroalcoholic system. Formulation was prepared in two phases. In the solvent phase, menthol, camphor, borneol, and vanillin were completely dissolved in a mixture of propylene glycol and ethanol under moderate stirring. In the oil-solubilizer phase zedoary and mint essential oils were blended with polyethylene glycol (PEG)-40 hydrogenated castor oil and polysorbate-20, and mixed at approximately 40 °C until a clear and homogeneous solution was obtained. The solvent phase was then gradually incorporated into the oil-solubilizer phase with continuous stirring at room temperature (approximately 800 rpm) until uniform. The preservative system (phenoxyethanol and ethylhexylglycerin) was subsequently added, followed by purified water with gentle mixing to adjust the formulation to 100% w/w. The final product was a clear, low-viscosity hydroalcoholic solution suitable for roll-on application. This delivery format was selected to align with the water-balm characteristics of the formulation. The roll-on applicator enables precise and localized application, reduces the risk of respiratory irritation associated with aerosol inhalation, and minimizes product loss resulting from rapid volatilization of the active components during application. The formulation was allowed to stabilize at room temperature for 24 h prior to further evaluation, including skin-patch irritation testing and repellency assessment using the Arm-in-Cage (AIC) method.

Table 1.

Formulation components of zedoary-based water-balm.

Ingredient	% w/w	Function
Zedoary essential oil	15.0	Active repellent
Mint essential oil	5.0	Aroma additive
Menthol	1.0	Cooling agent
Camphor	0.5	Cooling agent
Borneol	0.5	Aromatic agent
Vanillin	0.3	Fragrance fixative
PEG-40 hydrogenated castor oil	13.0	Solubilizer, emulsifier
Polysorbate-20	3.0	Co-solubilizer
Propylene glycol	5.0	Co-solvent, humectant
Phenoxyethanol & ethylhexylglycerin	1.0	Preservative
Ethanol (95%)	10.0	Solvent
Purified water	q.s. to 100	Vehicle

Abbreviation: q.s., quantum satis (sufficient quantity to make up to 100% of the formulation).

2.5. Human participants

Twelve healthy adult volunteers (aged 20–50 years; 6 males and 6 females) participated in the study after providing written informed consent. Participants were recruited randomly and took part in both the skin-patch irritation test and the repellency bioassay. Exclusion criteria included pregnancy or lactation, regular medication use, and known hypersensitivity to topical products. Prior to and during the study period, volunteers were instructed to refrain from using perfumes, fragranced cosmetics, insect repellents, and tobacco products for at least 12 h before testing.

2.6. Skin-patch irritation test

Skin irritation was assessed using a patch test procedure adapted from the International Contact Dermatitis Research Group (ICDRG) guidelines (Fregert, 1974; Bruze and Svedman, 2025) to evaluate the dermal tolerability of the water-balm formulation prior to repellency testing. For each volunteer, a 10 μl aliquot of the formulation was applied to a 0.6-cm-diameter filter paper disc and affixed to the inner upper arm using a waterproof occlusive dressing (3M Nexcare™). The patch remained in place for 48 h. Skin reactions were evaluated by a trained investigator at 30 min (immediate response) and 48 h (delayed response) after patch removal, under the supervision of a dermatologist at the Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University. Participants also conducted a self-photographic assessment at 96 h post-application. Clinical signs, including erythema, pruritus, edema, and vesiculation, were recorded and scored according to ICDRG criteria (Table 2). If any positive reaction was observed, volunteers were instructed to wash the test site immediately with clean water, report to the study supervisor, and discontinue further product use. Participants exhibiting adverse skin responses were excluded from subsequent repellency evaluation.

Table 2.

ICDRG criteria for assessing sensitization skin reactions.

Reaction description	ICDRG notation	Score
No reaction	–	0
Irritant reaction (faint erythema)	±	0.5
Erythema, with or without a few papules	+	1
Erythema, papules, and small vesicles	++	2
Intense erythema and coalescing vesicles (strong reaction)	+++	3

2.7. Mosquito rearing

The Bora Bora strain of Ae. aegypti was used in this study, is an established insecticide-susceptible reference colony. Periodic quality control using the WHO tube tests confirms that the colony remains fully susceptible to the WHO diagnostic insecticide doses (WHO, 2022). Mosquitoes were maintained at the Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, under controlled laboratory conditions (temperature of 25 ± 2 °C, relative humidity of 65 ± 10%, and a 12:12 h light-dark cycle). Larvae were reared in plastic trays containing 1000 ml of dechlorinated water and fed daily with powdered fish food. Pupae were collected and transferred to adult cages (20 × 20 × 30 cm) until emergence. Adult mosquitoes were provided with 5% sucrose solution ad libitum. For repellency testing, non-blood-fed female mosquitoes aged 5–7 days were selected and sugar-starved for 8–12 h prior to testing.

2.8. Repellency bioassay

Repellent efficacy of the zedoary-based water-balm formulation against Ae. aegypti was evaluated using the AIC method under controlled laboratory conditions, following WHO guidelines with minor modifications (WHO, 2009; Sutthanont et al., 2022). Twelve healthy adult volunteers participated in the bioassay. Approximately 250 unfed female mosquitoes (5–7 days-old) were released into a standard test cage (30 × 30 × 30 cm) and allowed to acclimate for 1–2 h before testing. Prior to exposure, volunteers washed both forearms thoroughly with unscented soap and water to remove potential confounding substances. A rectangular test area (3 × 10 cm; 30 cm²) was marked on the inner forearm between the wrist and elbow. A 100 μl aliquot of the water-balm formulation was evenly applied to the marked area and allowed to dry for 1–2 min. The contralateral untreated forearm served as the control. To confirm mosquito activity, the control arm was first exposed inside the cage for 3 min. If ≥ 10 mosquito landings were observed, testing proceeded. The treated arm was then exposed to the same cage for 3 min at 30-min intervals until repellency failure occurred. Complete protection time (CPT) was defined as the interval between product application and the observation of the second mosquito landing or probing on the treated arm, in accordance with Thai Food and Drug Administration (Thai FDA) criteria specifying a minimum CPT of 120 min for an effective topical repellent (TISI, 2010). After CPT determination, testing continued to assess percentage repellency over the full 180-min evaluation period. The treated and untreated arms were alternately exposed for 3 min at 30-min intervals, and the number of mosquito landings and probing attempts was recorded. All assays were conducted between 08:00 and 15:00 h, corresponding to the diurnal host-seeking activity period of Ae. aegypti, which typically exhibits daytime biting behavior (Jemberie et al., 2025), at a temperature of 25 ± 2 °C and relative humidity of 60 ± 10%. Because this study focused on absolute efficacy evaluation of a newly developed formulation, a synthetic reference repellent (e.g. DEET) was not included in the proof-of-concept design.

2.9. Data analysis

Repellent efficacy was expressed as the percentage reduction in mosquito landings on the treated arm relative to the untreated control. Percentage repellency (%R) was calculated as:

$$\%\mathrm{R}=\frac{(\mathrm{C}-\mathrm{T})}{\mathrm{C}}\times 100$$

where C represents the number of mosquito landings on the control arm, and T represents the number of landings or probing attempts on the treated arm. Volunteers were exposed for 3 min at 30-min intervals over a 3-h test period. As this study evaluated a single formulation with a limited sample size, data for percentage repellency and CPT were summarized as median and range (minimum-maximum), in accordance with WHO and Thai FDA reporting recommendations. No statistical comparisons were performed.

3. Results

3.1. Physical properties and extraction yield of zedoary essential oil

The essential oil obtained from zedoary rhizomes was a light yellow, low-viscosity liquid with a characteristic cineolic and aromatic odor. The oil exhibited a lower density than water, allowing clear phase separation during hydrodistillation. The extraction yield of zedoary rhizome essential oil was 3.94% (w/w), calculated on a fresh-weight basis.

3.2. GC-MS chemical composition of zedoary essential oil

GC-MS analysis of zedoary rhizome essential oil resulted in the identification of 37 volatile compounds, representing 99.98% of the total oil composition. The oil was predominantly composed of sesquiterpenes, comprising 43.40% sesquiterpene hydrocarbons and 31.56% oxygenated sesquiterpenes. Monoterpenes were present at lower proportions, including 13.05% oxygenated monoterpenes and 7.56% monoterpene hydrocarbons, while aromatic hydrocarbons (2.43%) and miscellaneous compounds (1.98%) represented minor fractions. The major compounds identified were AR-turmerone (23.66%), zingiberene (14.74%), β-sesquiphellandrene (10.64%), and 1,8-cineole (10.14%), collectively representing the dominant chemotype of the oil. Other notable constituents included α-humulene (7.42%), α-terpinolene (5.85%), α-turmerone (4.85%), β-bisabolene (2.90%), trans-caryophyllene (2.46%), α-curcumene (3.99%), and zingiberenol (1.43%). A complete list of all identified compounds, including retention times, relative peak areas, and chemical classifications, is provided in Table 3.

Table 3.

Chemical components of C. zedoaria essential oil as determined by GC-MS.

RT (min)	Compound	Area (%)	Classification
4.30	α-Pinene	0.57	Monoterpene hydrocarbon
4.51	Camphene	0.24	Monoterpene hydrocarbon
4.89	β-Pinene	0.29	Monoterpene hydrocarbon
5.27	L-Phellandrene	0.26	Monoterpene hydrocarbon
5.45	α-Terpinolene	0.35	Monoterpene hydrocarbon
5.57	p-Cymene	0.29	Aromatic hydrocarbon
5.67	1,8-Cineole	10.14	Oxygenated monoterpene
6.49	α-Terpinolene	5.85	Monoterpene hydrocarbon
6.60	p-Isopropenyltoluene	0.22	Aromatic hydrocarbon
6.64	2-Isopropenyltoluene	0.30	Aromatic hydrocarbon
7.33	Camphor	0.47	Oxygenated monoterpene
7.52	Isoborneol	0.48	Oxygenated monoterpene
7.65	Borneol	0.36	Oxygenated monoterpene
7.82	4-Terpineol	0.39	Oxygenated monoterpene
7.99	β-Fenchyl alcohol	1.21	Oxygenated monoterpene
11.64	Azulene derivative	0.31	Sesquiterpene hydrocarbon
12.01	trans-Caryophyllene	2.46	Sesquiterpene hydrocarbon
12.49	3,6-dimethoxy-2-methylnaphthalene	0.29	Aromatic hydrocarbon
12.72	α-Humulene	7.42	Sesquiterpene hydrocarbon
13.23	α-Curcumene	3.99	Sesquiterpene hydrocarbon
13.55	Zingiberene	14.74	Sesquiterpene hydrocarbon
13.72	trans-α-Bergamotene	0.32	Sesquiterpene hydrocarbon
13.80	β-Bisabolene	2.90	Sesquiterpene hydrocarbon
14.17	β-Sesquiphellandrene	10.64	Sesquiterpene hydrocarbon
14.33	trans-γ-Bisabolene	0.29	Sesquiterpene hydrocarbon
15.01	Nerolidol	0.31	Oxygenated sesquiterpene
15.42	tert-Amylbenzene	0.30	Aromatic hydrocarbon
15.95	4-Methyl-β-methylenebenzene	1.03	Aromatic hydrocarbon
16.18	Zingiberenol	1.43	Oxygenated sesquiterpene
16.49	β-Guaiene	0.37	Sesquiterpene hydrocarbon
16.58	Azulene derivative	0.95	Sesquiterpene hydrocarbon
16.69	2,3-Dihydrobenzofuran	0.60	Miscellaneous
16.97	2,3,5-Trimethylfuran	0.44	Miscellaneous
17.55	AR-Turmerone	23.66	Oxygenated sesquiterpene
17.96	Cedr-8-ene	0.28	Sesquiterpene hydrocarbon
18.26	α-Turmerone	4.85	Oxygenated sesquiterpene
19.33	Bisabolon	1.02	Oxygenated sesquiterpene

Abbreviation: RT, retention time.

3.3. Physical characteristics of the zedoary-based water-balm

The zedoary-based water-balm formulation was a clear, low-viscosity hydroalcoholic solution with a pale-yellow appearance and a pleasant aromatic camphoraceous cineolic-mint odor. The formulation exhibited a uniform and homogeneous consistency with no visible phase separation and demonstrated good spreadability upon application. A mild cooling sensation, attributable to menthol, camphor, and borneol, was perceived immediately after application, followed by a smooth, non-sticky finish. The formulation maintained stable clarity, odor, and appearance after 24 h under ambient conditions, indicating good physical compatibility among the essential oils, solubilizers, and solvent system. The formulation exhibited viscosity, homogeneity, and sensory properties appropriate for roll-on topical application (Fig. 1).

Fig. 1.

The final zedoary-based water-balm formulation prepared for topical application. The formulation is a clear, low-viscosity hydroalcoholic solution packaged in a roll-on container, as used for skin irritation and AIC repellency evaluations.

3.4. Skin-patch irritation

All twelve volunteers completed the skin-patch irritation test without adverse events. No participants exhibited erythema, pruritus, edema, vesiculation, or other signs of cutaneous irritation at any observation time point (30 min, 48 h, and 96 h). ICDRG scores were uniformly 0 for all subjects, indicating the absence of measurable skin reactions. These results demonstrate that the water-balm formulation was well tolerated and appropriate for subsequent repellency evaluation.

3.5. Repellent efficacy against Aedes aegypti

The zedoary-based water-balm formulation demonstrated strong repellent activity against Ae. aegypti under AIC conditions. As shown in Fig. 2, complete protection (100% repellency) was observed in all volunteers during the first 60-min post-application. Repellency declined gradually thereafter but remained high throughout the 180-min evaluation period. At 120-min post-application, the median repellency was 98.50% (range: 93.33–100%), and at 180 min it remained robust at 93.06% (range: 86.54–100%). Despite minor inter-individual variation, the median response and interquartile range remained consistently high, indicating uniform protection across volunteers. Across all participants, the CPT, defined as the interval until observation of the second mosquito landing or probing on the treated arm, ranged from 90 to 180 min, with a median CPT of 150 min. The overall median repellency across the 0–180 min period was 97.82% (range: 86.54–100%). Detailed data are provided in Supplementary Table S1.

Fig. 2.

Percent repellency over time following application of the zedoary-based water balm formulation. The dark red line indicates the median repellency, the shaded band represents the interquartile range (IQR), and grey dotted lines show individual volunteer responses. Median complete protection time (CPT) was 150 min (range: 90–180 min).

4. Discussion

Substances of natural origin, particularly plant-derived materials, have long been used to repel or control medically important arthropods, predating the widespread adoption of synthetic chemical repellents (Tavares et al., 2018). Among these botanical resources, C. zedoaria rhizome essential oil has been reported to exhibit diverse bioactivities, including larvicidal and pupicidal effects against multiple mosquito species (Phukerd and Soonwera, 2013) as well as adulticidal activity against female Ae. aegypti (Chaiyasit et al., 2006). Despite these documented insecticidal properties, evidence supporting the use of C. zedoaria essential oil as a topical repellent against adult mosquitoes, particularly in formulated products suitable for personal protection, has remained limited. In the present study, we demonstrate that zedoary essential oil, when incorporated into a zedoary-based water-balm formulation, provides strong and sustained repellency against adult Ae. aegypti under controlled laboratory conditions. This finding extends previous reports by showing that the bioactive constituents of C. zedoaria can be effectively translated into a topical delivery system capable of reducing mosquito landing and probing. The observed repellency highlights the potential of zedoary essential oil not only as an insecticidal agent but also as a practical botanical repellent for personal protection against arbovirus vectors.

GC-MS analysis revealed a sesquiterpene-rich chemotype dominated by AR-turmerone, zingiberene, β-sesquiphellandrene, α-humulene, and α-turmerone, compounds widely reported to possess insect-repellent, insecticidal, anti-inflammatory, and antimicrobial activities (Hong et al., 2001; Antonious and Kochhar, 2003; Rao et al., 2022; Damodharan et al., 2024). The predominance of AR-turmerone is particularly noteworthy. Recent computational and molecular docking studies have identified AR-turmerone as a promising natural mosquito repellent candidate, demonstrating DEET-like binding to mosquito odorant-binding protein 1 (OBP1), with stronger predicted binding affinity and lower skin permeability than DEET, suggesting high repellent potential combined with reduced dermal toxicity (Rao et al., 2022). The chemical profile observed in this study is consistent with previous reports from Thailand, India, Indonesia, Nepal, and the USA, which describe C. zedoaria rhizome oil as exhibiting turmerone- and zingiberene-rich chemotypes influenced by genetic background, geographic origin, and extraction conditions (Champakaew et al., 2007; Widyowati and Agil, 2018; Dosoky et al., 2019; Poudel et al., 2022; Liu et al., 2023). In addition, the presence of monoterpenes such as 1,8-cineole (eucalyptol) is relevant, as this compound has documented repellent, fumigant, and insect-deterrent activities across several plant species and may further contribute to the overall protective effect of the formulation (Andreazza et al., 2023; Mattar et al., 2024; Barrozo et al., 2025). Collectively, the dominance of bioactive sesquiterpenes, together with supportive monoterpenes, likely underpins the strong repellency observed for the zedoary-based water-balm formulation. While various repellent products, including creams, sprays, and roll-ons, are commercially available for personal protection against mosquitoes, this study specifically focused on the development of a zedoary-based water-balm roll-on formulation. The formulation formed a clear and physically stable hydroalcoholic system through effective solubilization of essential oils without phase separation and exhibited favorable sensory characteristics suitable for topical use. Importantly, the product demonstrated excellent cutaneous compatibility, as all volunteers completed the skin-patch test without signs of irritation or sensitization. This observation is consistent with previous reports describing generally low dermal toxicity of plant-derived terpenoids when applied at cosmetic-grade concentrations (Bleasel et al., 2002; Herman and Herman, 2015; Kopečná et al., 2019; Kłos and Chlubek, 2022).

Repellent efficacy was evaluated using the AIC bioassay following the 180-min evaluation framework recommended by the U.S. Environmental Protection Agency and adapted from WHO guidelines. This approach ensures adequate mosquito biting pressure, methodological reproducibility, volunteer safety and ethical acceptability, and comparability with other laboratory-based repellent studies (US EPA, 1999; Trongtokit et al., 2005; WHO, 2009). Under these conditions, the zedoary-based water-balm formulation provided complete protection during the first 60 min post-application and maintained high repellency (> 93%) throughout the full 180-min test period. The median CPT of 150 min exceeded the Thai FDA requirement of ≥ 120 min for an effective topical repellent (TISI, 2010). These results are comparable to those reported for other botanical repellents, including citronella, lemongrass, clove, makaen, and patchouli essential oils, which typically provide 60–180 min of protection depending on formulation type and active concentration (Trongtokit et al., 2005; Maia and Moore, 2011; Diaz, 2016). In addition to the intrinsic repellency of C. zedoaria essential oil, several co-ingredients may have contributed to the observed protective performance. Mint oil (M. arvensis) and menthol-rich monoterpenes have been reported to exert repellent or deterrent effects against mosquitoes and other insects (Kamatou et al., 2013; Manh and Tuyet, 2020; Alonso Leite dos Santos et al., 2024). Camphor and borneol are volatile monoterpenoids known to possess mild insect-repellent activity and enhance sensory acceptability through cooling effects (Watt et al., 2008; Chen et al., 2013; Zielińska-Błajet and Feder-Kubis, 2020). Vanillin, used as a fragrance fixative, has been shown to reduce the evaporation rate of volatile terpenes and extend the duration of protection in essential oil-based repellents (Arya et al., 2021). Although the individual contributions of these auxiliary components were not independently evaluated in the present study, their physicochemical and biological properties suggest potential synergistic effects that may enhance repellent persistence. All remaining formulation components were included to ensure solubilization, physical stability, microbial safety, and suitability for topical application and are not expected to contribute directly to repellent activity.

Several limitations should be acknowledged. This study evaluated a single formulation using a cohort of 12 volunteers under controlled laboratory conditions; therefore, repellency performance under field conditions may differ due to environmental factors such as wind, perspiration, and temperature. In addition, the specific repellent contributions of auxiliary ingredients were not quantified. Furthermore, while the formulation demonstrated strong repellency under controlled conditions, the absence of a standard reference repellent (e.g. DEET) limits direct comparison with conventional synthetic products. Future studies should assess formulation stability, concentration-dependent effects, field efficacy, and repellency against additional vector and nuisance species, including Culex spp., Anopheles spp., biting midges, ticks, and fleas. In addition, consumer acceptability studies addressing sensory perception, particularly odor preference, ease of use, and overall user satisfaction, as well as market-oriented evaluations of product positioning and adoption potential, would support translational development. Overall, the findings indicate that the zedoary-based water-balm roll-on is a safe, well-tolerated, and effective botanical repellent capable of providing sustained protection against Ae. aegypti. Its favorable sensory attributes, strong repellency, and natural origin highlight its potential as a viable alternative to conventional synthetic repellents within integrated vector control and personal protection strategies.

5. Conclusion

This study presents the first development and laboratory evaluation of zedoary-based water-balm roll-on formulated for personal protection against Ae. aegypti. The formulation was physically stable, well-tolerated by all volunteers, and exhibited strong repellent efficacy under controlled conditions. Chemical characterization confirmed a sesquiterpene-rich chemotype dominated by AR-turmerones and zingiberene, compounds that likely underpin the observed repellency. The water-balm provided complete protection during the first 60 min post-application and maintained high repellency (> 93%) throughout the 180-min AIC evaluation, with a median CPT of 150 min. These findings highlight the potential of C. zedoaria essential oil as a safe and effective botanical ingredient for topical mosquito repellents. Further investigations involving larger cohorts, additional vector species, formulation stability, field-based efficacy, and comparative trials with synthetic standards, as well as user acceptability, are warranted to support product optimization and facilitate translation to real-world vector-borne disease prevention.

Ethical approval

Studies involving human participants and animals were reviewed and approved by the Faculty of Tropical Medicine, Mahidol University, Thailand. Human volunteer testing was approved under Certificate of Ethical Approval No. MUTM 2024-091-01, and all participants provided written informed consent prior to their inclusion in the study. Animal use was approved by the Institutional Animal Care and Use Committee (FTM-ACUC) under Certificate No. FTM-ACUC 020/2024.

CRediT authorship contribution statement

Nataya Sutthanont: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Atchara Phumee: Data curation, Formal analysis, Project administration, Resources, Validation, Writing - review & editing. Nutticha Silakom: Data curation, Formal analysis, Visualization, Writing - review & editing. Jitrawadee Intirach: Data curation, Methodology, Validation, Writing - review & editing.

Statement on the use of AI-assisted technologies

During the preparation of this work, the authors used Grammarly (https://www.grammarly.com/) and QuillBot (https://quillbot.com/) in order to correct grammatical errors and improve readability. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Funding

This research project has been supported by Mahidol University (Fundamental Fund: fiscal year 2025 by National Science Research and Innovation Fund (NSRF)).

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

The data supporting the conclusions of this article are included within the article and its supplementary files.