The fabrication and assessment of mosquito repellent cream for outdoor protection

Mosquito-borne infections like dengue, malaria, chikungunya, etc. are a nuisance and can cause profound discomfort to people. Due to the objectional side effects and toxicity associated with synthetic pyrethroids, N,N-diethyl-3-methylbenzamide (DEET), N,N-diethyl phenylacetamide (DEPA), and N,N-di ethyl benzamide (DEBA) based mosquito repellent products, we developed an essential oil (EO) based mosquito repellent cream (EO-MRC) using clove, citronella and lemongrass oil. Subsequently, a formulation characterization, bio-efficacy, and safety study of EO-MRC were carried out. Expression of Anti-OBP2A and TRPV1 proteins on mosquito head parts were studied by western blotting. In-silico screening was also conducted for the specific proteins. An FT-IR study confirmed the chemical compatibility of the EOs and excipients used in EO-MRC. The thermal behaviour of the best EOs and their mixture was characterized by thermogravimetric analysis (TGA). GC–MS examination revealed various chemical components present in EOs. Efficacy of EO-MRC was correlated with 12% N,N-diethyl benzamide (DEBA) based marketed cream (DBMC). Complete protection time (CPT) of EO-MRC was determined as 228 min. Cytotoxicity study on L-132 cell line confirmed the non-toxic nature of EO-MRC upon inhalation. Acute dermal irritation study, acute dermal dose toxicity study, and acute eye irritation study revealed the non-toxic nature of EO-MRC. Non-target toxicity study on Danio rerio confirmed EO-MRC as safer for aquatic non-target animals. A decrease in the concentration of acetylcholinesterase (AChE) was observed in transfluthrin (TNSF) exposed Wistar rats. While EO-MRC did not alter the AChE concentrations in the exposed animals. Results from western blotting confirmed that Anti-OBP2A and TRPV1 proteins were inhibited in TNSF exposed mosquitoes. Mosquitoes exposed to EO-MRC showed a similar expression pattern for Anti-OBP2A and TRPV1 as the control group. In silico study revealed eight identified compounds of the EOs play significant roles in the overall repellency property of the developed product. The study emphasizes the mosquito repellent activity of EO-MRC, which could be an effective, eco-friendly, and safer alternative to the existing synthetic repellents for personal protection against mosquitoes during field conditions.

Rosemary oil showed peak at 982/cm for C=C bending 1465/cm for C-H bending 1750/cm for C=O stretching 2923/cm for C-H stretching respectively.
Polyethylene glycol showed peak at 1308/cm for C-O stretching; 1475/cm for C-H bending; 1714/cm for C=O stretching 2844/cm and 2927/cm for C-H stretching respectively. Tween 20 showed peak at 1117/cm for C-O stretching; 1722/cm for C=O stretching; 2876/cm and 2916/cm for C-H stretching respectively.
Glycerol showed peak at 1110/cm for C-O stretching, which might be due to the presence of secondary alcohol, and a characteristic peak at 1420/cm for O-H bending might be correspondents to alcoholic group respectively. Table 1. Screening of essential oils and their blends (best four oils) for mosquito repellency under K&D module. CNL citronella, CLV clove, LVDR lavender, LMG lemon grass.

Essential oils and their blends
Percentage of mosquito repellency with respect to applied doses of essential oils (mg/cm 2 ) ED 50 ( www.nature.com/scientificreports/ Propyl paraben showed peak at 960/cm for C=C bending; 1269/cm for C-O stretching; 1436/cm for O-H bending; 1690/cm for C=O stretching; 1937/cm for C-H bending 2964/cm for C-H stretching and peak at 3305/ cm for O-H stretching respectively.
Physical mixture exhibited peak at 1085/cm for C-O stretching, which might be due to the presence of primary alcohol; 1167/cm for C-O stretching corresponds to ester group; 1603/cm for C=C stretching due to the presence of conjugated alkene; 1722/cm for C=O stretching; 1921/cm for C-H bending for aromatic compound; 2852/cm and 2947 for C-H stretching; 3600/cm for O-H stretching might be due to the presence of alcoholic group respectively.
FT-IR study confirmed the compatibility of the formulation ingredients. The peaks of the physical mixture were intact and correlated to the individual components and no interaction was observed.
Thermogravimetric analysis (TGA). Thermal stability of citronella oil, clove oil, lemon grass oil and their mixture were investigated by TGA and results are presented in Supplementary Fig. S2. Citronella oil showed 98.09% mass loss below 300 ℃ which might be caused by decomposing the organic content of the sample. The weight of clove oil remained constant up to 60 ℃ and started decomposition/mass loss from 60 to 171.7 ℃. At second weight loss region, from 171.7 to 599.6 ℃, clove oil left a residual mass of about 0.87% respectively. Lemon grass oil showed decomposition ranging from 60 to 228 ℃. The blend of citronella, clove and lemon grass oil showed first weight loss region from 30 to 260 ℃, respectively, where all EO components got decomposed. A residual mass of about − 16.18% was remained at 599.6 °C under second weight loss region (228-600 °C). The TGA profile for EO-MRC showed considerable variations in the weight loss behaviour. Within a temperature range from 50 to 260 °C, removal of loaded EO molecules with water might occurred from repellent cream bases. The second weight loss region was evidenced from 270 to 600 °C which might be due to the decomposition of excipients used to formulate EO-MRC.

Formulation optimization. Optimization was done by setting the criteria for desirability functions in
Design-Expert software. A three-dimensional response surface plot was generated by the software (Fig. 1) which are very useful to study the interaction effects of the factors on the responses. Response surface plots describe the effect of various factors on the response at a time 36 . Here, Fig. 1 indicates the effect of different concentration of EOs on complete protection time (CPT) against mosquitoes. The optimized formula was based on the set criteria of maximum protection time. Therefore, seventeen cream formulations with the predicted levels of formulation factors were prepared. The non-linear computer-generated quadratic model was: where, 'Y' was the complete protection time (CPT), the response variable, associated with each factor level combination; and citronella oil (A), lemongrass oil (B) and clove oil (C) were the coded levels (at 2 levels; low and as high level) of dependent variables.
The variables, along with their levels, were selected based on the results from preliminary experimentation are shown in Table 2. The experimental design matrix for the amounts of A, B and C, used to prepare each of the seventeen formulations generated by the software and the responses were observed. ANOVA was performed using the same software to obtain the most effective formulation (Table 3). Nine formulation solutions were generated, following 17-run, 3-factor, 3-level Box-Behnken design (BBD) ( Table 4). The best EO-based mosquito repellent cream formulation was found to have the following compositions: 8.13% citronella oil, 4% clove oil, and 4% lemon grass oil, and excipients q.s. to 100% (w/w), and the optimized formula for 50 g EO-MRC are shown in Table 5.
Physicochemical parameters. EO-MRC and placebo formulation were prepared to obtain a uniform and stable oil in water emulsion with aesthetic appearance. EO-MRC showed a good uniformity and spread www.nature.com/scientificreports/  www.nature.com/scientificreports/ ability with acceptable odour and colour. pH of EO-MRC and placebo formulation was found to be 7.3 ± 0.08 and 6.93 ± 0.13 respectively. Values of density (1.02 ± 0.01 g/mL) and viscosity (28,878.33 ± 594.99) indicated sufficient stability of EO-MRC (data are shown in Supplementary Table S1). Further, studies regarding the effect of long-term storage are required to establish the shelf life of the formulations.
Cytotoxicity by MTT assay. A decreasing pattern of human lung epithelial cell (L-132) mortality was observed with increasing concentrations of the EO-MRC, while there was a noticeable decrease in cell viability after exposure to the different EO-MRC concentrations. Figure 4 represents the estimated cell viability, by MTT assay, to control (untreated) and 62 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, and 1000 µg/mL, of EO-MRC formulation in L-132 cultures. 500 µg/mL and 1000 µg/mL concentration were evidenced with significant decrease (p < 0.001) in cell viability, however, 250 µg/mL also inhibited cell viability (p < 0.05) and EO-MRC concentration of 62 µg/mL and 125 µg/mL did not show any significant differences in cell viability as compared to un-treated L-132 cell lines. ANOVA followed by Dunnett's multiple comparison tests, where, NS = p > 0.05; *p ˂ 0.05; ***p ˂ 0.001 respectively.
Acute dermal irritation study. In Table 4. Nine solutions for formulation was generated by Design-Expert, following 17-run, 3-factor, 3-level Box-Behnken design. CPT against mosquitoes were determined and percentage error was calculated. www.nature.com/scientificreports/ Repeated dose dermal toxicity study. No treatment-related clinical signs were observed during the study period. Neither any erythema or oedema was present, nor the treated area of skin was abraded. Aberrations in the locomotor activity were also absent. The daily food and water consumption were normal. All rats had normal body weight, and appeared active and healthy. Histopathological observations of skin evidenced with normal architecture of epidermis (ED), dermis (D), sebaceous gland (SG) and hair follicles (HF) shown in Fig. 5a control skin; Fig. 5b EO-MRC treated skin. Repeated dermal treatment of EO-MRC did not cause any adverse effects on the body weight and feed consumption of the animals as compared to control group. All animals were found to be healthy throughout the study period.

Number CNL (%) LMG (%) CLV (%) Predicted CPT (min) Experimental CPT (min) % error
Acute eye irritation. From acute eye irritation study on rabbits, our results categorize EO-MRC as nonirritant ( Fig. 6). However, capsaicin (negative control group), as a known irritant specially used in defence utilization, was categorized in Category 2B (as per the UN-GHS system), as the irritation responses was observed within 48 h.
Acute toxicity test on Danio rerio (D. rerio). The most frequent pathological modifications were epithelial lifting, erythrocyte infiltration, and basic hyperplasia. Under all observed morphological changes, deltamethrin (DLM) showed more severe effects on gill structure. Minimal toxicity to the gill tissues were observed in EO-MRC exposed group, and was evidenced with erythrocyte infiltration, and basis hyperplasia. Results are shown in Fig. 5c control gill; Fig. 5d EO-MRC exposed gill; Fig. 5e deltamethrin exposed gill.  www.nature.com/scientificreports/ AChE activity assay. AChE enzymes are located at the synaptic cleft that hydrolyse the neurotransmitter acetylcholine (ACh) to acetate and choline and terminates synaptic transmission. Changes in AChE activity might results from exposure to certain chemicals or insecticides, which acts as cholinesterase inhibitors. From the values shown in Fig. 7a, it was clear that the activity level of AChE decreased (83 ± 15 units) significantly (p < 0.01) in transfluthrin (TNSF) exposed rats as compared to control (177 ± 28 units) respectively. In EO-MRC exposed rats, no significant decrease (p > 0.05) in AChE was recorded and the value was found to be 132 ± 13 units respectively. ANOVA followed by Dunnett's multiple comparison tests. Where, NS = p > 0.05; **p < 0.01.

Protein expression in mosquito head part. Protein estimation. Protein concentrations obtained for
EO-MRV, EO-MRC, TNSF and control mosquitoes were 7.14 mg/mL, 3.03 mg/mL, 9.146 mg/mL and 3.275 mg/ mL, respectively. Based on these concentrations, the amount of each treatment sample containing 50 μg protein was calculated and loaded into the respective SDS PAGE gels. Hence, based on the aforementioned concentrations, 6.68 μL, 16.4 μL, 5.46 μL and 15.26 μL of EO-MRV, EO-MRC, TNSF and control samples were loaded for SDS PAGE respectively.
Western blotting. Relative expression levels of TRPV1 and Anti-OBP2A, as quantified by ImageJ, are shown in Fig. 7b. As a control, the housekeeping gene, β-actin, was experimented on prior to blotting of the other antibodies and showed profound expression in the mosquito head part. Expression of anti-OBP2A was shown in 22 kDa, showed its higher expression (p < 0.01) in EO-MRV exposed mosquito head part (13.02 ± 2.05), but in case of TNSF exposed group, expression level was inhibited (3.37 ± 0.93), (p > 0.05). EO-MRC exposed mosquitoes showed similar expression levels (6.6 ± 1.5) as control mosquito (6.62 ± 1.43), no any significant differences were observed (p > 0.05). There were no any significant changes (p > 0.05) recorded in EO-MRC exposed group of mosquitoes for TRPV1 (2.9 ± 0.28) as compared to the control. TRPV1 in the mosquito head part showed bands at an expected molecular weight of 100 kDa. In terms of band intensity, WB showed that TRPV1 expression was lower, however it was not significant (p > 0.05) in TNSF exposed mosquitoes (2.07 ± 0.58) as compared to the control samples (3.23 ± 0.88). But, in case of EO-MRV exposed mosquitoes, a higher expression (p > 0.05) of TRPV1 was recorded (4.95 ± 1.44). ANOVA followed by Dunnett's multiple comparison tests. Where, NS = p > 0.05; **p < 0.01.

Molecular docking.
From molecular docking studies, we found eight compounds, namely, betula oil, cinnamaldehyde, citronellal, citronellol, estragole, eugenol, methyl eugenol and o-cymene, that showed better tendency to bind with the active site of all the three selected target proteins (data available in Supplementary Table S3

Discussion
Synthetic repellents are always problematic and have negative public perceptions due to their side effects and toxicity to users as well as harmful effects to ecology and non-target organisms [38][39][40] . EOs are rich in terpenoids and effective against mosquitoes due to their insecticidal and repellent property. They are economic, safe, easily available, less toxic, and decrease the chances of resistance to insects due to their complex chemistry. In our previous research, we reported the attraction and repellency assay of fourteen EOs against Musca domestica using 'Y'-tube olfactometer. The blend of clove, citronella and lemongrass oil exhibited maximum flight orientation response. In this study also, the blend of citronella, clove and lemon grass oil showed best results against mosquitoes under K&D study. The efficacy of EO depends on the different phytoconstituents present in each oil. Under characterization, FT-IR study confirmed the EOs and other auxiliary formulation ingredients are compatible to each other and peaks of the physical mixture are intact and correlated to the individual chemicals. TGA thermogram showed the weight loss rates of EOs as a function of temperature elevation from 30 to 600 ℃. Onset weight loss of clove oil was reported as 80-90 ℃ 41 previously. In our study, EO-MRC showed first weight loss region from 50 to 260 °C, which might be due to the removal of loaded EO molecules with water contents Table 7. Identified compounds of essential oils (EOs) and the selected blend with retention indices (RI) and relative peak area percentage. RI Exp. : Experimentally determined linear retention indices using homologous series of C 7 -C 30 Pinene  935  937  -----------05.3  ---Camphene  955  952  ------ Methyl eugenol 1399 1402  44 . To measure the fraction of subjects living or survived or protected after treatment, Kaplan-Meier estimate is one of the best options to be utilized 45 . In this study, mosquito bioassay was carried out to determine CPT as per standard test guidelines, recommended by WHO 46 . Efficacy of EOs have been previously reported by Azeem et al. 47 . They revealed more than 45 min of protection time under arm in cage bioassay 47 . Our study results confirm that, after processing the best blend of EOs into formulations, the developed product gave complete protection by up to 228 min, respectively.  www.nature.com/scientificreports/ Lipophilic molecules present in EOs might have a direct entry into the lung cells of the user as there is only one cell membrane to pass through. For which, the effect of breathing EO fragrances from EO-MRC can be considerable. Chances of toxicity to the lungs might be there due to excessive use of EO, but the actual concentration has not been worked out and very few studies are available 48 . The Flavour and Extract Manufacturers Association (FEMA) granted generally recognized as safe (GRAS) status to most of the EOs and they are approved by the US Food and Drug Administration (US FDA) for utilization in food, cosmetics and pharmaceutical products. After doing evaluation by the expert panel of the FEMA, this was reviewed in 1996. Basically, most flavour ingredients at less than 100 ppm can be used for exposure, and predictions regarding their safety can be assessed 49 . Thus, from our study results on L-132, EO-MRC might be considered appropriate for effective mosquito repellent without producing any harmful effect to the lung cells.
Determination of acute dermal irritation study is useful where formulation to be exposed in skin. It provides information on health hazards likely to arise from a short-term exposure by the dermal route 50 . Acute dermal irritation study of EO-MRC on rabbit and repeated dose dermal toxicity study on Wistar rats confirms that EO-MRC is safe and do not produce any harmful effects to the skin. Under acute eye irritation study, EO-MRC proved its non-irritative nature to any ocular tissues. A test substance is categorized as United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) eye irritant category 2A when it shows a positive response of conjunctival redness ≥ 2; conjunctival edema ≥ 2 corneal opacity ≥ 1; iritis ≥ 1; estimated as the mean scores after grading in different time intervals as 24 h, 48 h and 72 h following instillation of the test substance, and which entirely reverses within an observation time of 21 days. If the above-mentioned effects are entirely reversible within an observation time of 7 days, than the test substance would be categorized under category 2B 51 .
Synthetic pyrethroids and organophosphates are becoming a threat to fish and other aquatic animals 52 . Hedayati et al., reported hematological and gill histopathology data on iridescent shark (Pangasius hypophthalmus) exposed to organophosphate and pyrethroid insecticides 53 . In our study, non-target toxicity on D. rerio confirms non-toxic nature of EO-MRC. Here also, DLM was used as negative control. The Fish gill histopathology of EO-MRC was found to be same tissue architecture as the un-treated (control) gills. Our finding on D. rerio gill has similar results as previously reported on fish gill histopathology.
A decreased AChE was determined in rats brain exposed to TNSF, which might be associated in increase in lipid peroxidation 54 , and possibility of disruption of metabolic and nervous activities 55 . However, EO-MRC exposed rats did not show any significant difference with the control rats. Increased activity of AChE causes a decreased amount of ACh, which causes a reduction in blood flow and vasodilatation 56 . In brain hypoperfusion by ligating common carotid arteries, reduction in ACh in the hippocampal area is responsible for memory Figure 5. Histopathology of skin tissue under repeated dermal toxicity study for EO-MRC on rats (a) control skin; (b) EO-MRC treated skin. No remarkable changes had been observed for EO-MRC exposed skin tissues under histopathology study. Histopathology of D. rerio gill tissue (c) control gill; (d) EO-MRC exposed gill; (e) deltamethrin exposed gill. EO-MRC exposed gill shows same tissue architecture as control group. DLM exposed (negative control) group shows abnormality in tissue architecture. White arrow: basis hyperplasia; EI: erythrocyte infiltration; EL: epithelial lifting. www.nature.com/scientificreports/ and learning impairment 57 . In various research studies, the loss of perivascular cholinergic terminals has been reported in Alzheimer's disease patients 58,59 . Moreover, AChE inhibitor medication is known to affect cholinergic function in subjects treated with hypobaric hypoxia (↓AChE activity, ↑ACh levels and upregulation of choline acetyltransferase, enzyme that has a role in ACh formation) and eventually memory function 60 . Odorant binding proteins (OBPs) are the first relay in semi-chemical reception in insects as they are the liaison between the air medium that broadcasts chemical signals and odorant receptors which are located in olfactory structures (mainly the antenna and maxillary pulps) of insect's peripheral sensory system 61 . No significant expression of OBP-2A in EO-MRC exposed rats head part have been seen, however, mosquitoes exposed to EO-MRV, containing the same EOs showed overexpression of OBP-2A. It might be due to the exposure pattern of EO to the target groups. It confirms that, EOs have the capability to overexpress OBP of mosquitoes. In case of synthetic TNSF exposed mosquito head part, OBP-2A was inhibited. In case of TRPV1, EO-MRV exposed mosquito head part showed overexpression of this protein, where EO-MRC exposed group did not show any changes and TNSF exposed group showed inhibition of TRPV1 respectively. This might be an indication of TRPV1 being expressed more in inflamed tissues, thereby associating its significance in the antennal inflammatory cascade of mosquitoes. Same results were observed in case of anti-OBP2A antibody also. This is the first report that demonstrates the expression of TRPV1 and anti-OBP2A antibody in mosquito head part. Insect use at least three families of olfactory receptors, viz., Odorant Receptors (ORs), Ionotropic Receptors (IRs) and Gustatory Receptors (GRs) 12,62 . DEET masks the olfactory receptor neurons (ORNs) to attractants and decreases the sensitivity to lactic acid, a human sweat component by decreasing the response of lactic acid excited neurons and increasing the inhibition of lactic acid inhibited neurons 12 .
EO-MRC showed very promising results against mosquitoes. WB analysis supported the significant expression of the target proteins when treated with developed formulation containing the EOs as active ingredients. Hence, in the in-silico study, we tried to find out the exact molecules present in the EOs responsible for the repellent activity. From the in silico docking study, eight compounds were identified which showed better affinity to bind with the target proteins of the mosquitoes and rat species to form stable protein-ligand complex. The in Figure 6. Acute eye irritation study on rabbit eye after 1 h, 24 h, and 48 h of control (untreated), EO-MRC, capsaicin (negative control) exposed groups. Capsaicin was used here as a known irritant, specially used in capsigranade under defence utilization, belonging to the Category 2B, as the reversibility of animal responses. EO-MRC exposed group was evidence with non-irritant to ocular tissues. www.nature.com/scientificreports/ Figure 7. Change in AChE activity may result from exposure to certain chemicals or insecticides, which acts as cholinesterase inhibitors. Most of the EO components have been reported as AChE inhibitors. (a) AChE activity assay on wistar rats after TNSF and EO-MRC exposure; significant decrease (p < 0.01) in AChE was observed in TNSF exposed group. EO-MRC exposed group did not show any significant differences (p > 0.05) in AChE concentrations as compared with the control group; (b) relative expression levels of TRPV1 and Anti-OBP2A, in mosquito head part after exposure to TNSF and EO-MRC as quantified by ImageJ. Significant (p < 0.01) over expression of anti-OBP2A was observed in EO-MRV exposed mosquito head part (13.02 ± 2.05) but, in case of TNSF exposed group, expression level was inhibited (p > 0.05). EO-MRC exposed mosquitoes showed similar expression levels as the control mosquito (p > 0.05). Similar expression pattern has been recorded for TRPV1 also (p > 0.05). TRPV1 expression was lower in TNSF exposed mosquitoes as compared to the control (p > 0.05). But, in case of EO-MRV exposed mosquitoes, a higher expression of TRPV1 was observed, however, there was no any significant change (p > 0.05) has been recorded in EO-MRC exposed group of mosquitoes as compared to the control. ANOVA followed by Dunnett's multiple comparison tests. Where, NS = p > 0.05; **p < 0.01. Original blots/gels are presented in Supplementary Figs. S9-Figs. S11. www.nature.com/scientificreports/ silico study revealed that the eight identified compounds of the EOs play significant role in the overall repellency property of the developed product.  where, C is the number of mosquitoes landing, or biting at the control area; T is the number of mosquitoes landing or biting at the treated area. www.nature.com/scientificreports/ (CPT) as independent variable or response variable. Analysis of variance (ANOVA) was performed using the same software to obtain the most effective formulation.

Preparation of cream.
Phase inversion temperature method was applied for the preparation of EO-based mosquito repellent cream (EO-MRC). About 50 g cream sample was prepared in order to get enough for performing the various qualitative and quantitative assay. The oil phase (phase B) was prepared by dissolving the oil soluble excipients, except phase A (mosquito repellent active ingredients) under mild heating at 200 rpm in a hot magnetic plate stirrer (Magnetic Stirrer IKA RCT basic) and heated to 65 °C. The aqueous phase was prepared by mixing various aqueous soluble ingredients (phase C) under gentle heating and stirring. Temperature of the aqueous phase was raised to 65 °C. Phase A was gently added to the oil phase at a stirring speed of 200 rpm and 55 ± 2 °C. The mixture was then emulsified by adding phase C slowly and kept for 1 h at a stirring rate of 800 rpm and 60 ± 2 °C. The formulated EO-MRC was then kept for natural cooling.
Efficacy assessment. CPT of the developed cream (EO-MRC) formulation was carried out by arm in cage bioassay. 1 mL EO-MRC was applied to ≈ 600 cm 2 area of the forearm skin between the wrist and elbow and 1 mL of the 12% N, N-di ethyl benzamide (DEBA) based marketed cream (DBMC) was compared on the other arm. Two mosquito cages (size: 40 × 40 × 40 cm) each containing 200-250 non-blood-fed female Ae. Albopictus were used. One cage is designated for testing the EO-MRC and the other for the positive control (DBMC). During testing, hands were protected by surgical gloves for which the mosquitoes cannot bite while the volunteer avoids movement of the arm. EO-MRC and DBMC treated arms were exposed for 3 min at 30 min intervals to determine landing and/or probing activity. A single landing or probing of mosquito within a 3 min test interval concludes the test. CPT was calculated as the time (min) required for the first mosquito landing or probing after repellent application to the treated area. The median CPT and confidence intervals were estimated from the Kaplan-Meier Survival Function 46 . Efficacy was correlated with DEBA based marketed cream (DBMC). The inclusion of the specific commercial product DBMC is for comparison and does not constitute any recommendations.
Characterization. Gas chromatography-mass spectroscopy (GC-MS). Qualitative study. Different chemical components in fourteen EOs and the selected blend were identified by a GC-MS system of Agilent Technologies (5301 Stevens Creek Blvd. Santa Clara, CA 95051, United States). Test sample concentration of 500 μg/mL was prepared in GC grade acetone. A sample volume of 1 μL was introduced into the injector held at 250 °C. Oven temperature of 40-300 °C was programmed at 20 °C/min. Helium was used as carrier gas at flow rate 1 mL/ min. The injector and detector temperature were set at 250 °C and 230 °C (quad) and 150 °C (core) respectively 37 . Standard C7-C30 saturated alkanes were purchased from Sigma Aldrich Chemicals Co., St. Louis, USA. Retention indices (RI) of the identified components were determined for identification of the detected components.
% Assay by GC-MS study. Calibration samples of eugenol and citronellol were prepared by dissolving an appropriate amount in GC grade acetone to get concentrations of 62.5 μg/mL, 125 μg/mL, 250 μg/mL and 500 μg/mL. Test samples of EO-MRC, clove oil and citronella oil were prepared by dissolving a required amount in acetone to quantify the EO components in the final formulation. A sample volume of 1 μL was introduced into the injector as described in 'Qualitative study' section.
Physicochemical parameters. Physical parameters of the EO-MRC and placebo formulations were determined in order to establish aesthetic compliance and consumer acceptability. To determine the viscosity, a programmable viscometer was used (Model: DV2T, Ametek Brookfield, Middleboro, MA, USA); combined with software Rheo3000, version 1.2.2019.1 [R]. Sample volume was fixed at 30 g and viscosities were determined at 10 rpm for 40 s at room temperature using a T-Bar spindle (B-92) (Helipath spindle set, Brookfield Engineering Labs. Inc). Density was determined by using a pycnometer. pH of EO-MRC was checked by using digital pH meter (Labman Scientific instruments, Tamil Nadu, India).
Spread ability of EO-MRC was determined as per the method reported earlier by Sabale 65 . In brief, 1 g of EO-MRC was placed on 1 cm 2 pre-marked circular area on the glass slide (7.5 cm × 2.5 cm). EO-MRC was compressed using another glass slide placed from edge to centre of primary slide. 200 g of commercial weight was placed on the set up and allowed the gel to spread for the period of 1 min. The spread diameter was calculated with the aid of graph paper and spread ability was evaluated using formula expressed as Eq. (3): where, m is the commercial weight placed on the setup; l is the length of cream spread; and t is the time.
Safety assessment. Cytotoxicity by MTT assay. The reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell proliferation. The yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. With the help of spectrophotometric means, the resulting intracellular purple formazan can be quantified. The assay measures the cell proliferation rate and conversely, when metabolic events cause apoptosis or necrosis, the reduction in cell viability 66 .
Cells cultured in T-25 flasks were trypsinized and aspirated into a 5 mL centrifuge tube. Cell pellet was obtained by centrifugation at 3000 rpm. The cell count was adjusted, using DMEM HG medium, such that 200 μL www.nature.com/scientificreports/ of suspension contained approximately 10,000 cells. To each well of the 96 well microtiter plate, 200 μL of the cell suspension was added and the plate was incubated at 37 ℃ and 5% CO 2 atmosphere for 24 h. After 24 h, the spent medium was aspirated. 200 μL of different test concentrations viz. 62 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/ mL, and 1000 µg/mL, of EO-MRC were added to the respective wells. The plate was then incubated at 37 °C and 5% CO 2 atmosphere for 24 h. The plate was removed from the incubator and the drug containing media was aspirated. 200 μL of medium containing10% MTT reagent was then added to each well to get a final concentration of 0.5 mg/mL and the plate was incubated at 37 ℃ and 5% CO 2 atmosphere for 3 h. Without disturbing the crystals formed in the wells, culture medium was completely removed. 100 μL of solubilisation solution (DMSO) was added to each well and the plate was then gently shake in a rocking shaker (ROCKYMAX™, Tarsons, Kolkata, India) to solubilize the formed formazan. The absorbance was measured at a wavelength of 570 nm and also at 630 nm using a microplate reader. The percentage growth inhibition was calculated and concentration of EO-MRC needed to inhibit cell growth by 50% (IC 50 ) was generated from the dose-response curve for the cell line. Acute dermal irritation study. Acute dermal irritation study was conducted on healthy New Zealand albino rabbits following the OECD test guidelines 404 68 . Approximately 24 h before the test, fur was removed from the dorsal area of the trunk. 0.5 g EO-MRC, was directly applied to the skin and after 4 h exposure period, residual EO-MRC was removed by using water without disturbing the integrity of the epidermis and examined for signs of erythema and oedema, at 60 min, and then at 24 h, 48 h and 72 h after EO-MRC removal. Dermal reactions are graded and recorded according to the grades in the Table 8. As per the method described by Banerjee et al. 69 ; primary irritation index (PII) was calculated. Further, we have followed the Draize method of classification for PII scoring as non-irritant (if PII < 0.5), slightly irritant (if PII < 2), moderately irritant (if PII ≤ 2-5), and severely irritant (if PII > 5) 70 and then mean irritation score per time point was calculated. The mean scores at day 1, day 2 and day 3 were then summed up and followed the equation to obtain the PII.
where, Xa is the mean score of erythema formation; Xb is the mean score of edema formation; t_1 is the day 1; t_2 is the day 2; t_3 is the day 3.
Repeated dose dermal toxicity study. As per the OECD guideline 410 71 , the repeated dose dermal toxicity of the EO-MRC was carried out for a period of 21 days. Healthy Wistar rats were housed in 2 different groups (control, and EO-MRC treated) and each group contained 06 animals after initial acclimatization for at least 5 days prior to the study. Briefly, dorsal fur was removed using sterile surgeon hair removal blade (49-20 mm) and 4.0 × 4.0 dorsal area was treated with placebo and test substances for at least a 6 h/day on a 5 day/week basis, for 21 days. An observation with respect to body weight and feed consumption was monitored on a daily basis 72 .
Acute eye irritation. For this experiment, OECD TG 405, acute eye irritation testing procedure was carried out on young and healthy rabbits 50 . 50 mg EO-MRC was placed to the conjunctival sac for 5 s by gently pulling the lower lid of the right eyeball; where, left eye was served as control. Capsaicin, an ocular irritant was used as negative control. The eye of the animal was not washed for the next 24 h following installation of capsaicin. Observations for any ocular lesions at specific intervals of 1 h, 24 h, and 48 h were evaluated using slit lamp microscope (Haagstreit type AIA-11, Appaswamy) 73 .

Non-target toxicity testing. Acute toxicity test on zebrafish (Danio rerio). The acute toxicity test on
Danio rerio (D. rerio) was carried out in accordance with the guidelines of the Organization for Economic Cooperation and Development (Test no. 203, Acute Immobilization Test) 74 . Seven neonate D. rerio were exposed in each test vessel and three replicates were tested, for a total of 21 D. rerio per treatment group. D. rerio individuals were placed in containers with 250 mL of pure water, and EO-MRC was diluted with acetone and mixed into the water in dosages corresponding to the concentrations 200, 100 and 50 mg/L. The experiment was divided into three groups. The first two groups were formed by D. rerio individuals, exposed to the action of EO-MRC and negative control deltamethrin (DLM) for a period of 24 h 75 . D. rerio individuals in the third group was served as control.
The physical and chemical conditions of the acute toxicity tests were as follows: pH ranging from 7.2 to 7.6; constant temperature of 25 ± 1 °C; electrical conductivity around 160 μS/cm; dissolved oxygen above 3 mg/L. The number of dead D. rerio in the three replicates was counted and used to determine the LC 50  www.nature.com/scientificreports/ Acetylcholinesterase (AChE) activity assay. In this study, transfluthrin-1.6% (TNSF) and EO-MRC were exposed to different rat groups (n = 6) for 21 days. Control animals received no exposure. After completion of 24 h from the last exposure, all animals were humanely sacrificed by cervical dislocation. Brain tissue samples were collected and homogenized using 0.1 M phosphate buffer, pH 7.5, followed by centrifugation at 14,000 rpm for 5 min. Cleared supernatants were used for assay, as per the procedure described in the technical bulletin of Acetylcholinesterase activity assay kit (Sigma-Aldrich, St. Louis, MO 63103 USA; Catalog Number: MAK119).
Protein estimation in mosquito head part. Head part of female Ae. albopictus (n = 100 in each group) previously exposed to EO-MRV (essential oil-based mosquito repellent vaporizer), EO-MRC, TNSF and control (un-treated) were isolated and freshly homogenized in RIPA buffer and centrifuged at 12,000 rpm for 15 min. Collected supernatants were kept at − 80 °C for further utilization. As per the instructions mentioned in the Biorad DC Protein assay protocol (Bio-Rad, Hercules, CA), protein concentrations in the tissue samples were determined. Working reagents and protein standard dilutions were prepared to construct the protein standard curve. 5 μL of standards and tissue samples were placed in appropriate wells, followed by 25 μL and 200 μL of working reagents A and B respectively. The absorbance was read at 750 nm after 15 min using a microplate reader (Spinco Biotech Pvt. Ltd., India). From the results of this assay, the amount of protein in each test sample equivalent to 50 μg standard protein was calculated for further loading in the blotting process.
Western blotting. Required amount of test samples was mixed with an equal amount of the Laemmli Buffer and used for blotting. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels were prepared from the 10% acrylamide resolving gel (TGX Stain-FreeTM FastCastTM Acrylamide Kit, Bio-Rad, CA, US) as per manufacturer's instructions. Standard protein markers (Precision Plus, Kaleidoscope, Biorad) and previously prepared tissue samples were introduced into different wells of the casted SDS-PAGE gels. The electrophoresis was carried out in a Mini-PROTEAN® Tetra System and PowerPacTM HC electrophoresis power supply system (Bio-Rad, CA, US). After electrophoresis, the protein embedded gel was transferred at 15 mV (15 min) to a nitrocellulose membrane in the Trans Blot Turbo machine. The protein bands embedded membrane was then transferred to a fresh petri plate and incubated on a rocking shaker (Rockymax, Tarsons, Kolkata, India) for 1 h in a blocking solution consisting of Tris buffer saline (TBS) with 0.1% Tween 20 and 5% bovine serum albumin (BSA). After 1 h, the blocking solution was discarded and the membrane washed three times (3 min/ wash) with TBST solution. The TBST solution was completely pipetted out and without drying the membranes, specifically diluted primary antibodies were added to the membranes. The membranes were probed separately with anti-OBP2A and TRPV1 and β-actin primary antibodies (diluted in 5% BSA in Tris-buffered saline, pH 7.4) and incubated overnight at 4 °C. The next day, the membrane was washed in TBST 3 times (5 min/wash), followed by 1 h incubation with horseradish peroxidase (HRP) linked secondary antibodies (diluted in 5% BSA in Tris-buffered saline, pH 7.4, (details mentioned in Supplementary Table S5) on rocking shaker at 4 °C. After incubation, freshly prepared ECL substrate was added to the membranes and immediately visualized and interpreted in G: Box Chemi-XRQ gel doc system (Syngene, United Kingdom). The intensity of bands was calculated with the SynGene GeneTools (SynGene Laboratories, Cambridge, United Kingdom). Results are representative of three independent experiments. All WB band quantification steps were performed using custom ImageJ (National Institutes of Health; http:// rsb. info. nih. gov/ ij/) script.

In silico screening of the EO components. Molecular docking and MM-PBSA (Molecular Mechanics
Poisson-Boltzmann Surface Area) based binding free energy calculation. Molecular docking study was carried out to find the component(s) of the EOs which have a tendency to bind with the target(s) associated with repellent activity. The components of EOs used in the formulation were identified from the GC-MS analysis. Then the SMILE id of the compounds was retrieved from PubChem database (https:// pubch em. ncbi. nlm. nih. gov/) and loaded to the Discovery Studio 2020 molecular modelling software (DS 2020) (Dassault BIOVIA, San Diego, USA) 76 . The 3D structures of the compounds were generated and energy minimization of the compounds was performed according to standard protocol of DS 2020 using smart minimizer method 77 . The target proteins odorant binding protein (OBP) for Aedes species (PDB: 3K1E) and for Anopheles species (PDB: 3QME), and TRPV1 for rat (PDB: 5IS0) were downloaded from protein data bank (www. rcsb. org) 78 . The target proteins were cleaned, prepared and then energy minimized using the 'smart minimizer' method for 2000 steps with energy RMSD gradient 0.01 kcal/mol 77 . After that the binding sites for docking study were selected using the information given in Protein Data Bank for active sites of the selected targets. The active sites for OBP of Aedes species  Fig. S4). Then docking study was carried out using simulation-based docking protocol CDocker of DS 2020 which provides comparatively more accurate information regarding the binding of a compound in the active site 79 . The best docking poses generated were also analysed to observe the different non-bonding interactions took place between the target proteins and compounds. The MM-PBSA based binding free energy provides accurate information regarding the thermodynamic stability of protein-ligand complex in real physiological conditions 80,81 . Hence the best poses of the compounds selected from docking were further analysed to calculate the binding free energies (ΔG) using MM-PBSA method.

Conclusion
The present study investigates the repellent activity of a non-toxic EO-MRC formulation against mosquitoes, as per the international standards. EO-MRC tested herein, has successfully passed the efficacy study against Ae. albopictus and preclinical toxicity on different laboratory animals as per OECD test guidelines. The developed product provides effective protection up to 228 min, against Ae. albopictus without producing any health hazards in preclinical settings. Thus, the overall study substantiates the development of EO based, longer lasting, nontoxic topical cream formulation to strategically minimize mosquito bites and to decrease the mosquito-borne disease incidents. However, this study is not conclusive, further study on repellency testing using different strains of mosquito, with different bioassay procedures like tunnel bioassay, y-tube olfactometer etc., should be carried out. Non-target toxicity testing, stability testing or self-life assessment of the EO-MRC is essential for more conclusive results. In future, multiple emulsion and/or liposomal cream formulation could be designed for controlled release of anti-mosquito formulations in exposed skin to attain maximum protection time. This would reduce the dosing frequency in outdoor conditions. Study on expression of more odour responsive protein available in the mosquito head part might contribute towards the scientific community in understanding more detailed mechanism of repellent chemical. The present report will benefit research students, formulation scientists and pharmaceutical companies to develop effective, safe and longer lasting herbal mosquito repellents that could be applicable to travellers, the military in jungle operations or the general public in mosquito endemic areas to get protection from mosquitoes.