The fabrication and assessment of mosquito repellent cream for outdoor protection

Chemicals and reagents

EOs of basil (Ocimum basilicum L.), bergamot (Citrus bergamia Risso & Poit), camphor [Cinnamomum camphora (L.) J. Presl.], cinnamon (Cinnamomum zeylanicum Blume), citronella [Cymbopogon nardus (L.) Rendle], clove (Eugenia caryophyllus Wight), eucalyptus (Eucalyptus globulus Labill.), jasmine (Jasminum officinale L.), lavender (Lavandula angustifolia Mill.), lemon grass [Cymbopogan citratus (DC.) Stapf], mentha (Mentha piperita L.), rosemary (Rosmarinus officinalis L.), patchouli (Pogostemon patchouli Benth), and wild turmeric (Curcuma aromatica Salisb.) were procured from Talent Technologies (Talent Technologies, Kanpur, India). Acetylcholinesterase (AChE) activity assay kit, Anti-OBP2A antibody, ELISA kits, 1,1-diphenyl-2-picrylhydrazyl (DPPH), radioimmunoprecipitation (RIPA) buffer and phosphate buffer saline (PBS) were purchased from Sigma Aldrich (Sigma Aldrich Chemical Co., St. Luis, USA). TRPV1 antibody was purchased from Santa Cruz (Santa Cruz, California, USA). 1-chloro-2,4-dinitrobenzene (CDNB) was purchased from Cayman (Cayman Chemical Company, Michigan, USA). Human normal lung cell line (L-132) was obtained from the National Centre for Cell Sciences (NCCS), Pune, India. High performance liquid chromatography (HPLC) grade acetone was purchased from Merck (Merck Pvt. Ltd., Mumbai, India). All other chemicals used were of the highest analytical grade available.

Test insects

5–7 days old adult female Ae. albopictus mosquitoes were housed at the laboratory insectary, Division of Pharmaceutical Technology, Defence Research Laboratory, Tezpur, Assam, India. Mosquitoes were reared by maintaining temperature at 27 ± 2 °C, relative humidity: 75 ± 5% RH and 14L:10D h of light–dark alternative cycles in standard-sized wooden cages (75 cm × 60 cm × 60 cm) with a sleeve opening on one side as described previously⁶³. 10% sucrose solution ad libitum were provided for nourishment. Before testing, the mosquitoes were starved for 24 h.

Screening of EOs

Dose response study was performed to evaluate the best oils among the fourteen EOs. This study was approved (approval number: 032/2021TMCH, 28/08/2018) by the Institutional Human Ethical Committee (IHEC), of the Tezpur Medical College & Hospital (TMCH), Tezpur, Assam, India, and all experiments were performed in accordance with relevant guidelines and regulations. Five volunteers are chosen, not allergic to mosquito bite and all volunteers provided written informed consent. A volunteer’s thigh was marked according to the door opening hole of the K&D module as described by Klun and Debboun⁶⁴. It is made of Plexiglas and the base of the rectangular cage (26 cm × 5 cm × 5 cm) has six holes, each with rectangular 3 × 4 cm holes that are opened and closed by a sliding door (Supplementary Fig. S8: Provide the photograph of K&D module). The flexor region of the forearms of a human volunteer was outlined with four rectangular (3 cm × 4 cm) test areas. A volume of 25 µL of each concentration of the EOs in soybean oil (40, 4 and 0.4 µg/cm²) and 25 µL of the soybean oil (diluent) as control was applied to the marked areas. After air drying for 5 min, a K&D module with matching cut outs in its floor was placed over the treated areas, containing five nulliparous 5–7 days old female mosquitoes in each hole. The doors of the cells were opened and the number of mosquitoes biting in each cell was recorded within a 2 min exposure, after which the doors were closed. After completion of each observation, mosquitoes were freed by opening cells of the K&D module in a sleeved screened cage. For each test, fresh sets of mosquitoes are used. Five replications for each test were carried out. The efficacy of EOs were determined by the percentage repellency against mosquitoes, using the formula or Eq. (2) described by WHO⁴⁶.

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.

Fourier transform-infra red spectroscopy (FT-IR)

Study of chemical compatibility for each formulation ingredients are necessary. All formulation ingredients possess specific value of vibrational frequency and have varied functional groups in their chemical structures. For compatibility study, each EOs, excipients to be used in cream formulation, and their physical mixture was placed one by one over the sample plate of the FT-IR instrument (Bruker, ALPHA, Billerica, MA, USA). The covering probe was placed over the sample and IR spectra was obtained over a wavelength of 2.5–25 μm at room temperature. Functional groups possessed by each individual ingredient should be identical in their physical mixture which confirms their compatibility³⁷.

Thermogravimetric analysis (TGA)

The thermal behaviour of citronella oil, clove oil, lemon grass oil, their mixture and EO-MRC were evaluated using a thermal analyser (TG 209 F1 Libra^®, NETZSCH-Gerätebau GmbH, 95100 Selb, Germany). Approximately about 10 mg sample weight was placed in the crucible each time. Nitrogen was used as a shielding gas. Heating program was fixed as 30–600 °C at a rate of 10 °C/min.

Formulation development and optimization

For optimization, a 17-run, 3-factor, 3-level Box-Behnken design (BBD) was utilized. A second order polynomial model was constructed by quadratic response surface methodology (RSM) using Design-Expert software (Version 6.0.8, Stat-Ease Inc., USA). Total seventeen formulations were obtained using EO concentrations as dependent variables against complete protection time (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 cm² 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 Function⁴⁶.

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) respectively³⁷. 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 Sabale⁶⁵. In brief, 1 g of EO-MRC was placed on 1 cm² 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)-2,5-diphenyltetrazolium 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 viability⁶⁶.

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 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% CO₂ 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% CO₂ 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% CO₂ 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% (IC₅₀) was generated from the dose–response curve for the cell line.

Animals and ethics statement

All experimenting protocols using animal were performed according to the “Principles of Laboratory Animal care” (NIH publication 85–23, revised 1985) and approved by the Institutional Animal Ethical Committee (IAEC) of Defence Research Laboratory (DRL), Tezpur, Assam, India (approval no. CPCSEA/DRL/Protocol no. 3, 20/06/2018). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals⁶⁷. All efforts were made during the study period to minimize the suffering of animals and to reduce the number of animals used.

5–8 weeks old, about 210–250 g of male healthy adult Wistar rats (Rattus norvegicus) and young and healthy New Zealand albino rabbits (Oryctolagus cuniculus) were obtained from the institutional animal housing facility and allowed to acclimatize for 7 days prior to the study. Standard food and purified water ad libitum were provided in clean and hygienic condition at 22–25 ℃, 40–70% RH with 12 h light–dark cycles.

Acute dermal irritation study

Acute dermal irritation study was conducted on healthy New Zealand albino rabbits following the OECD test guidelines 404⁶⁸. 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 410⁷¹, 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 basis⁷².

Acute eye irritation

For this experiment, OECD TG 405, acute eye irritation testing procedure was carried out on young and healthy rabbits⁵⁰. 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 h⁷⁵. 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 LC₅₀ for 24 h of exposure.

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://pubchem.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 method⁷⁷. 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/mol⁷⁷. 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 was X: 13.63, Y: 40.63, Z: 24.92 and radius 9.8 Å; for OBP of Anopheles species was X: 20.17, Y: 31.50, Z: 32.09 and radius 7.7 Å; for TRPV1 of rats was X: 107.77, Y: 92.80, Z: 103.16 and radius 9.3 Å (Supplementary 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 site⁷⁹. 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.

Source: Ecology - nature.com