Natural product formulations with improved residual insect repellent/deterrent activity

Abstract

Natural products were screened for their insect repellent activity, and carrot seed essential oil gave very high activity in biting repellent/deterrent bioassays. Analysis of the oil revealed the presence of 47 compounds, mainly mono- and sesqui-terpenes. The sesquiterpene, carotol, constituted more than 75% w/w of the oil. In the initial screening, the essential oil gave high biting deterrent activity and high repellent activity comparable to DEET against both Aedes aegypti and Anophies quadrimaculatus species of mosquitoes. The active fraction mainly comprises pure carotol. The essential oil and the pure compound have a potential to be developed and used as effective repellent against mosquitoes.

Claims

1. A mosquito repellent/deterrent comprising a mosquito repellant/deterrent amount of at least one of carrot seed essential oil or carotol and mixtures thereof further comprising DEET said deterrent being either alone or in an acceptable carrier therefor, wherein, in the case of mosquitoes that are vectors of disease causing pathogenic microbes, the repellent/deterrent is additionally effective for preventing infection with such diseases.

2. The mosquito repellent/deterrent according to claim 1, further comprising undecanoic acid.

3. A method for protecting individuals from mosquito bites comprising applying to an individual a mosquito repellent/deterrent according to claim 1.

4. The method for protecting individuals from mosquito bites comprising applying to an individual a mosquito repellent/deterrent according to claim 2.

5. An insect repellent/deterrent comprising an insect repellant/deterrent amount of at least one of carrot seed essential oil or carotol and mixtures thereof and DEET, the repellent/deterrent being either alone or in an acceptable carrier therefor.

6. The insect repellant/deterrent according to claim 5, further comprising undecanoic acid.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows .sup.1H and .sup.13C nuclear magnetic resonance (NMR) spectroscopic data for carotol (CDCl.sub.3, 400 MHz).

(2) FIG. 2 shows the major components of carrot seed essential oil from GC analysis.

(3) FIG. 3 shows the biting deterrent activity of carrot seed essential oil against Ae. aegypti.

(4) FIG. 4 shows the biting deterrent activity of fractions of the carrot seed essential oil against Ae. aegypti.

(5) FIG. 5 shows the biting deterrent activity of carotol against Ae. aegypti.

(6) FIG. 6 shows the biting deterrent activity of carrot seed essential oil and carotol against An. quadrimaculatus.

(7) FIG. 7 shows the residual repellent activity of DEET, carrot seed essential oil and carotol against Aedes aegypti females in an in vitro bioassay.

(8) FIG. 8 shows the residual repellent activity of DEET, carrot seed essential oil and carotol against Aedes aegypti females in an in vitro bioassay.

(9) FIG. 9 shows the repellent activity of DEET, carrot seed essential oil and carotol against Aedes aegypti females in an in vivo “cloth patch” bioassay.

(10) FIG. 10 shows the residual repellent activity of DEET, carrot seed essential oil and carotol against Aedes aegypti females in an in vivo “cloth patch” assay.

(11) FIG. 11 shows repellent activity of DEET and carotol against Aedes albopictus females in an in vivo, “cloth patch” bioassay.

(12) FIG. 12 shows repellent activity of DEET and carotol against Anopheles quadrimaculatus females in an in vivo, “cloth patch” bioassay.

(13) FIG. 13 shows residual activity of DEET and combinations with carrot oil and carotol (F) in in vitro A&K bioassay.

(14) FIG. 14 shows residual activity of carrot oil and carotol (F) in direct skin application bioassay.

(15) FIG. 15 shows residual activity of carrot essential oil and combinations with baby oil and petroleum jelly in direct skin application bioassay.

(16) FIGS. 16A and 16B show residual activity of DEET and combinations with carrot essential oil and carotol (F) in direct skin application bioassay.

(17) FIG. 17 shows residual activity of picaridin and combinations with carrot essential oil in direct skin application bioassay.

(18) FIG. 18 shows residual activity of DEET and combinations with geranium (Pelargonium graveolens) and lemon Eucalyptus (Eucalyptus citriodora) essential oils in direct skin application bioassay.

(19) FIG. 19 shows the residual repellent activity of carrot seed essential oil and combinations of undecanoic acid and geranium essential oil in in vivo direct skin application bioassays.

DETAILED DESCRIPTION OF THE INVENTION

(20) Where the indefinite article “a” or “an” is used with respect to a statement or description of the presence of a component in the composition of this invention, it is to be understood, unless the statement or description explicitly provides to the contrary, that the use of such indefinite article does not limit the presence of the component in the composition to one in number.

(21) Although illustrative embodiments of the invention will be described in detail, it is to be understood that the present invention is not limited to those precise embodiments, and that various changes and modifications can be affected therein by one skilled in the art without departing from the scope and spirit of the invention as defined by the appended claims.

(22) 1. Materials and Methods

(23) Materials

(24) Carrot (Daucus carota), geranium (Pelargonium graveolens) and lemon Eucalyptus (Eucalyptus citiodora) 100% pure therapeutic essential oils were purchased from Edens Garden, 1322 Calle Avanzado, San Clemente, Calif. DEET and undecanoic acid were purchased from Sigma-Aldrich (St. Louis, Mo.). Picaridin was purchased from Cayman Chemical (Ann Arbor, Mich.).

(25) General Experimental Procedures

(26) Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker™ spectrometer at 400 (.sup.1H NMR) and 100 MHz (.sup.13C NMR). GC/FID analyses were carried out on a Varian™ CP-3380 gas chromatograph equipped with a Varian™ CP-8400 automatic liquid sampler, a capillary injector and flame ionization detector. The column was a 30 m×0.25 mm DB-5, 0.25μ film (J&W Scientific, Inc.). Data were recorded using a Dell® Optiplex™ GX1 computer operating with Microsoft® Windows XP® and Varian Star™ (version 6.41) workstation software. Helium was used as the carrier gas. An indicating moisture trap and an indicating oxygen trap located in the helium line from upstream to downstream, respectively, were used. Helium was used as the “make-up” gas at the detector. Hydrogen and compressed air were used as the combustion gases. The instrument parameters used for monitoring samples were: Air—30 psi (400 mL/min); Hydrogen—30 psi (30 mL/min.); column head pressure—14 psi (1.0 mL/min); split flow rate—50 mL/min; split ratio—50:1; septum purge flow rate—5 mL/min; make up gas pressure—20 psi (20 mL/min); injector temp—220° C.; detector temp—240° C.; initial oven temp—60° C.; initial temperature hold time—1 min; temperature rate—3° C./min.; final oven temperature—240° C. and final temperature hold time—4 min. A sample of 1.0 μL was injected.

(27) GC/MS analyses were carried out on a Thermo Finnigan TRACE™ MS interfaced to a TRACE™ 2000 GC equipped with an AS2000 auto sampler and a single capillary injector, and an electron impact (EI+) source was used. High purity helium was used as the carrier gas, and a high capacity oxygen trap was located in the helium line. Dell® Optiplex™ 745 workstation operating with Microsoft® Windows XP® was used. Data were collected and processed using ThermoQuest™ Xcaliber™ software (Ver. 1.2). The column was a 30 m×0.25 mm DB-5, 0.25μ film (J&W Scientific, Inc.) with a 50:1 split injection. The injector temperature was set at 220° C., and the oven temperature programmed at 60° C. for 1 minute, then ramped to 240° C. at a rate of 3° C./minute and held at the final temperature for 4 minutes. A sample of 1.0 μL was injected.

(28) Column chromatographic separations were performed on silica gel 60 (0.04-0.063 mm). TLC was performed on precoated TLC plates with silica gel 60 F254 (0.2 mm, Merck®). The solvent system used for TLC analysis was: n-hexane: EtOAc (95:5).

(29) Bioassay Guided Fractionation and Isolation of Carotol

(30) A small quantity of carrot seed oil (700 mg) was subjected to biologically guided fractionation on a Si gel column eluted with a mixture of ethyl acetate (EtOAc) and hexanes with increasing polarity of EtOAc to 5%. Seven fractions (1-7) were collected, concentrated under vacuum and submitted for biting deterrent activity. Fraction 3 showed promising biting deterrent activity. The GC/MS analysis of the active fraction indicated that this fraction contained more than 95% of carotol.

(31) Carotol was isolated as colorless oil. The .sup.1H NMR showed four methyl signals at 0.89 (d, J=6.0 Hz, Me-15), 0.97 (d, J=6.0 Hz, Me-14), 0.91 (s, Me-11), and 1.65 (s, Me-12). It also displayed an oliphenic proton at 5.29 (t, J=6.0 Hz, H-3) corresponding to carbon resonating at 122.3 ppm in the .sup.13C NMR (Table 1/FIG. 1). The .sup.13C NMR and DEPT-135 spectra revealed 15 signals corresponding to four methyl, five methylene, three methane and three quaternary carbons (Table 1/FIG. 1). The .sup.1H and .sup.13C NMR data were superimposed with those previously reported for carotol (Jasicka-Misiak et al. 2004). The chemical structure of carotol was confirmed by comparing its MS fragmentation pattern with the NIST library in the GC/MS.

(32) In order to isolate more quantity of carotol and to study the possibility of the presence of more active compounds, large scale fractionation was carried out as follows:

(33) Carrot essential oil fraction (6.0 g) was subjected to silica gel column (120 g, 5×100 cm), eluted with n-hexane/EtOAc (100:0 to 95:5). Ten fractions (1-10), 50 mL each, were collected, and similar fractions were combined based on TLC (5% EtOAc/hexanes) to give six fractions. These fractions were tested and the fractions 1-5 (4.0 g) showed good biting deterrent activity. The active fraction was re-chromatographed on Si gel CC isocratically eluted with 2% EtOAc/hexanes to yield 3.51 g of an active compound identified as carotol. Carotol was then tested against adult female mosquitoes for the biting deterrent activity. A large cage in vitro bioassay system was used to determine the repellent activity of carotol. Details of the bioassay system are given in the following section. In vivo “cloth patch” repellency bioassays were also conducted to compare the levels of repellency of carotol with DEET, which was used as a positive standard.

(34) Insects

(35) Adults of Aedes aegypti L., Aedes albopictus Skuse, and Anopheles quadrimaculatus Say used in these studies were from the laboratory colonies maintained at the Mosquito and Fly Research Unit at the Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, Fla. (Pridgeon et al. 2007). For biting deterrence and repellent bioassays, eggs were hatched and the insects were reared to the adult stage in the laboratory and maintained at 27±2° C. and 60±10% RH with a photoperiod regimen of 12:12 h (L:D). 8-18 day old adult females were used in these bioassays.

(36) Mosquito Biting Bioassays

(37) Experiments were conducted by using a six-celled in vitro Klun and Debboun (K&D) module bioassay system developed by Klun et al. (2005) for quantitative evaluation of biting deterrence. Briefly, the assay system consists of a six well reservoir with each of the 3×4 cm wells containing 6 mL of blood or feeding solution. As described by Ali et al. (2012), a feeding solution consisting of CPDA-1 and ATP was used instead of blood. Green fluorescent tracer dye (www.blacklightworld.com) was used to determine the feeding by the females. Carrot seed essential oil was initially tested to determine the biting deterrent activity. Essential oil showed strong biting deterrent activity and 7 fractions of the essential oil were further tested to identify the compound(s) responsible for this activity. The fraction, which showed the highest activity, was identified to contain more than 95% of carotol. Essential oil and carotol were further tested against Ae. aegypti and An. quadrimaculatus in this bioassay. Treatments of carrot seed essential oil at 10 μg/cm.sup.2 and carotol were tested at 10 and 5 μg/cm.sup.2, whereas DEET (97% N,N-diethyl-meta-toluamide) (Sigma Aldrich, St. Louis, Mo.) at 25 nmol/cm.sup.2 was used as positive control. All the treatments were freshly prepared in molecular biology grade 100% ethanol (Fisher Scientific Chemical Co. Fairlawn, N.J.) at the time of bioassay.

(38) The temperature of the solution in the reservoirs was maintained at 37° C. by continuously passing warm water through the reservoir using a circulatory bath. The reservoirs were covered with a layer of collagen membrane (Devro, Sandy Run, S.C.). The test compounds were randomly applied to six 4×5 cm areas of organdy cloth and positioned over the membrane-covered CPDA-1+ATP solution with a Teflon® separator placed between the treated cloth and the six-celled module to prevent the contamination of the module. A six-celled K&D module containing five female mosquitoes per cell was positioned over cloth treatments covering the six CPDA-1+ATP solution membrane wells, and trap doors were opened to expose the treatments to these females. The number of mosquitoes biting through organdy treatments in each cell was recorded after a 3 minute exposure, and mosquitoes were prodded back into the cells to check the actual feeding. Mosquitoes were squashed and the presence of green fluorescent tracer dye (or not) in the gut was used as an indicator of feeding. A replicate consisted of six treatments: four test compounds, DEET (a standard biting deterrent) and ethanol-treated organdy as solvent control applied randomly. Sets of 5 replications each with 5 females per treatment were conducted on 2-3 different days using newly-treated organdy and a new batch of females in each replication. Treatments were replicated 10-15 times. Proportion not biting (PNB) was calculated using the procedure described by Ali et al. (2012).

(39) In Vitro A&K Repellent Bioassay

(40) The A&K (Ali and Khan) bioassay system is based on the concept that the mosquitoes are attracted to warm temperatures, and this system uses warm temperature to serve as stimulus for landing and feeding. The details of the A&K bioassay systems are given in Ali et al. (2017).

(41) In summary, this in vitro system consisted of a 30×30×30 cm collapsible aluminum cage (Model 1450B, BioQuip Products, 2321 Gladwick Street, Rancho Dominguez, Calif. 90220, USA) with metal screens. On one panel of the cage, metal screen was replaced with a clear acrylic transparent sheet. This transparent panel had a 120×35 mm slit through which the blood box containing a removable feeding device was attached. The blood box had three sides covered with acrylic sheet while the front side was open to insert the feeding device. The top of the blood box had a sliding door used to expose the females to the treatment while doing the bioassay. The sliding door served to contain the females inside the cage when the feeding device was pulled out for loading the treatment. This door was slid open when the feeding device was pushed in during the bioassay to expose the females to the treatment.

(42) The feeding device had one 3×4 cm rectangular reservoir that contained the feeding solution. The feeding device was connected to the water circulator. The reservoir of the feeding device with feeding solution was covered with a chemical treated collagen sheet. To observe the landing and feeding of female mosquitoes, feeding solution in the reservoir was heated to match the human body temperature using a water circulator which was set at 37° C. The feeding solution had 3-4° C. higher temperature than the solid platform, and this increased temperature of the feeding solution became a better source of attraction and preference for the mosquitoes than the other parts of the feeding device. Collagen did not allow the movement of the liquid through it, and the sugars present in the feeding solution may not act as stimulus for landing and feeding of the mosquitoes. The blood box had an outside acrylic extension that served as a platform outside the cage to hold the feeding device while loading the treatment.

(43) The rectangular reservoir of the feeding device was filled with the CPDA 1+ATP solution having a few drops of green fluorescent traceable water-soluble dye to serve as a feeding source. A test was started when the 3×4 cm marked areas of collagen were treated with the desired chemical in a volume of 50 μL/20 cm.sup.2. The treated collagen sheet was then secured on the reservoir using a thin layer of high vacuum grease (Dow Corning Corporation, Midland, Mich. 48686). The feeding device was then pushed into the blood box and the sliding door was opened to expose females to the treatment. Mosquitoes landing and biting was observed for 1 minute, and the number of females that started to bite was recorded.

(44) Stock solutions were made in ethanol. All the dilutions were made in EtOH and applied in a volume of 50 μL covering 20 cm.sup.2 of the collagen sheet. Minimum effective dosage (MED) values in this in vitro bioassay were determined using a method described by Katritzky et al. (2010).

(45) The cage contained 200±10, 8-18 day old female mosquitoes. The number of mosquitoes landing and biting were recorded visually for 1 minute. Three or four cages were used at a time, and one treatment was evaluated in a single cage. The minimum dose at which 2 or less mosquitoes (i.e. less than or equal to 1% of 200 females in the cage) started feeding in 1 minute was the minimum effective dose (“MED”). Continuous exposure of the mosquitoes in the cage can result in reduced landing and biting, therefore, 3-4 cages were used and only one replication was completed for each chemical in a single cage. To ensure accuracy of the treatment, rectangular areas of (3×4 cm (12 cm.sup.2) or 7.5×4 cm (30 cm.sup.2) were marked on the collagen that matched the measurement of the rectangular liquid reservoirs. Treatments were applied in a volume of 50 μL (12 cm.sup.2 area) or 107 μL (30 cm.sup.2 area). Treated collagen sheets were secured on a feeding reservoir containing CPDA-1+ATP solution using a thin layer of grease (Dow Corning Corp, Midland, Mich.). The feeding device was pushed inside the blood box and the sliding door was closed to expose the females to the treatment.

(46) To ensure the normal activity (≥10% landing), control treatment was repeated after every 5 replications, and the bioassay was stopped if the response was less than the control. The data is presented as MED values with the mean percentage biting (±SEM) in parenthesis. As per the criterion, the minimum dose at which feeding was ≤1% was considered as MED. 5-10 replications were completed in a single day using a cage with 200 females.

(47) In Vivo Mosquito Repellent Assays

(48) Cloth patch bioassays were conducted by using in vivo bioassay system described by Katritzky et al. (2008, 2010), with minor modifications. Approximately 500 (±5%) mosquitoes, consisting primarily of females, were transferred into a test cage (45×45×45 cm) using an aspirator. A series of dosages of test compounds were tested to determine MED for repellency of the mosquitoes. MED refers to a concentration of the compound at which biting is less than or equal to 1% (i.e. less than or equal to 5 out of 500 females in the cage) during a 1 minute exposure time. A series of concentrations ranging between 6.25 to 100 μg/cm.sup.2 were tested in the A&K bioassay.

(49) A single test consisted of covering the hand of a volunteer with a soft-embossed long cuff poly glove (Atlantis Products, Mankato, Minn.), then by a powder-free latex glove (Diamond Grip, Microflex Corporation, Reno, Nev.). The arm was then covered with a knee high stocking (Leggs® Everyday Knee Highs, Winston-Salem N.C. 27102) to avoid the contact of treated muslin cloth with the skin. A plastic sleeve constructed of polyvinyl was then placed around the arm. Velcro strips were used to fix the plastic sleeve around the arm. About half-way between the wrist and elbow, a 4×7.5 cm slit in the plastic sleeve was used to assess mosquito landing and biting behavior. This opening permitted the attractive odors from the skin surface to emanate out and attract mosquitoes through this opening. During testing, this 30 cm.sup.2 open area was covered with a piece of treated muslin cloth. Because of constraint of man power, only one male volunteer who was provided written informed consent participated in this study. A protocol approved by the University of Mississippi Human Use Institutional Review Board (IRB protocol #15-070) was followed.

(50) Stock solutions with concentration of 80 μg/ul were prepared in ethanol. Concentrations ranging between 6.25 to 100 μg/cm.sup.2 were selected for testing based on previous experience of the inventors with the A&K bioassay. A piece of muslin cloth measuring 8×13 cm in size was used. An area of 4×7.5 cm was marked in the center of muslin cloth. Approximately 2.5×7 cm pieces of cardboard were attached by staples on the sides of the muslin cloth. Treatments were applied to the marked area of the cloth in 215 μL volume using a pipetter. After drying, the treated muslin cloth was secured on the opening of the plastic sleeve by using an adhesive tape.

(51) The test started when the arm with sleeve and treated muslin cloth was inserted into the mosquito cage. Mosquito landing and biting was observed for a period of 1 minute. Residual activity was determined by exposing the females to the treated muslin cloth at an interval of 30 minutes. The treated muslin patch was kept on the arm after the initial treatment and tested every half an hour thereafter.

(52) Testing started at the highest dose (100 μg/cm.sup.2) and the dose was lowered to reach the MED. Because the mosquitoes show reduced behavioral activity upon repeated exposure to repellent odors from the arm, after 5 successive exposures of the treatments, the caged mosquitoes were allowed a 15 min recovery period.

(53) Direct Skin Application Bioassay

(54) Direct skin application bioassays were conducted by using a powder-free latex glove (Diamond Grip, Microflex Corporation, Reno, Nev.). An opening of 3×4 cm was cut through the glove to fit on dorsal surface of the hand (FIG. 1). After wearing the glove, a wristband was used to avoid the biting on the hand near the border of the glove. Approximately, 500 (±5%), consisting primarily of females were transferred to a test cage (45×45×45 cm) using an aspirator. A series of dosages were tested to determine MED for repellency as well as residual activity against mosquitoes. A single test consisted of covering the hand of a volunteer with a powder-free latex glove and treating 12 cm.sup.2 surface area, cut through the glove, on dorsal surface of the hand. Marked skin surface area was treated with the test compound in 50 μl of total volume. Because of constrain of man power, only one male volunteer who was provided with a written informed consent participated in this study. A protocol approved by the University of Mississippi Human Use Institutional Review Board (IRB protocol #15-070) was followed.

(55) The test started when the hand treated (3×4 cm area cut through the glove on the dorsal surface) with a test compound was inserted into the mosquito cage. Mosquito landing and biting was observed for a period of 1 min. After 1 min exposure, the hand was gently shaked and the number of biting females (feeding females do not fly) was recorded. Any treatment with ≥≥5 females biting during 1 min test period was considered as “passed” whereas a treatment with ≥5 bites out of 500 mosquitoes was considered as “failed”. In case of passing the next lower and in case of failure next higher serial dose was tested to reach MED. Additionally, data on the residual repellent activity was recorded by exposing the treatments to the females after every 30 minutes until the treatment failed. Carrot essential oil, carotol and DEET and the mixtures were tested.

(56) Statistical Analysis:

(57) Data on the PNB were analyzed using SAS Proc ANOVA (SAS Institute 2007) and means were separate using Ryan-Einot-Gabriel-Welsch multiple range test. Means and standard errors of MED values were calculated by using SAS Proc Means or Microsoft Excel 2010.

(58) Results and Discussion

(59) In the initial screening, the essential oil gave high biting deterrent activity which was comparable to DEET (97% purity N,N-diethyl-meta-toluamide). Through bioassay-guided fractionation, a fraction that gave activity like the essential oil and the DEET was identified. From this active fraction, the active metabolite, carotol, was isolated by chromatographic techniques. .sup.1H and .sup.13C NMR spectroscopic data for carotol (CDCl.sub.3, 400 MHz) is given in Table 1/FIG.1.

(60) GC/FID and GC/MS analysis of carrot seed essential oil revealed the presence of 47 compounds, mainly mono- and sesquiterpenes (Table 1/FIG. 1). Carotol constituted more than 75% w/w of the essential oil. The other major compounds, as shown in Table 2/FIG. 2, were muurolene (4.86%), (z)-β-farnesene (2.9%) and diepicedrene (1.1%). There are many publications that have reported the composition of carrot oils. Ozcan and Chalchat (2007) reported carotol (66.8%), daucene (7.84%), (Z,Z)-α-faenesene (5.86%), germacrene D (2.34%), trans-α-bergamotene (2.41%) and β-selenene (2.2%) as the major contents of the carrot seed essential oil, whereas the seed oil contained carotol (30.6%), daucol (12.6%) and copaenol (0.62%) as main constituents. Maxia et al. (2009) reported only 0.7 and 1.5% of carotol in the essential oils of flowering umbels and umbels with ripe seeds of Daucus carota L. sunsp. carota collected from Portugal, whereas the essential oils from Italy had 25.1 and 2.4% carotol in similar samples. Jasicka-Misiak et al. (2004) reported 38.6% carotol in carrot seed oil purchased from Augustus Oils Ltd., London, with β-caryophyllene (10.66%), caryophyllene oxide (4.34%), α-pinene (3.94%) and farnesene (3.35%) as the other major contents. Mazzoni et al. (1999) reported carotol as the major content (73.1, 69.7 and 36.1%) in 3 commercial carrot seed essential oil samples. Flamini et al. (2014) reported 37.2, 22.0, 20.3, 24.2, 4.7, 17.5, 8.6, 6.4 and 49.8% of carotol in various samples of essential oils of seeds from 9 commercial varieties of Daucus carota L. subsp. sativus collected from various parts of Italy, whereas only 0.2% and 0% carotol was present in the flower and seed essential oils, respectively. Cu et al. (1989) reported variation in percent of carotol present in the carrot oils extracted in different solvents. They reported 13.97, 40.91, 39.06, 31.75 and 18.29% of carotol in oils extracted in methylfuran (sylvan), 1,1,2-trichloro-1,2,2-trifluoroethane (TTH), EtOH (ethanol), CH.sub.2Cl.sub.2 and hydrodistillation, respectively. Nigam and Radhakrishnan (1963) reported carotol (70%) and daucol (11.3%) as major contents of the carrot essential oil, whereas another study has reported carotol (79.5%) as a major sesquiterpene compound present in the essential oil of carrot (Gupta and Gupta 1958).

(61) The published data suggest that the quantity of the major constituents of the carrot essential oils vary in different samples. This variation could be due to the plant parts used, variety, harvest timings, geographical location as affected by the climatic factors, genetic origin and the way the samples are prepared. Therefore, the chemical composition of the essential oil is likely to vary from sample to sample that will affect the biological properties and effectiveness of the essential oils.

(62) Biting deterrent screening data of the carrot seed essential oil are given in Table 3/FIG. 3. Carrot seed essential oil at 10 μg/cm.sup.2 showed biting deterrent activity similar to DEET at 25 nmol/cm.sup.2 in the K&D bioassay. Based on this activity, this essential oil was selected for further studies. Carrot seed essential oil was fractionated into 7 fractions. These fractions were then tested for their biting deterrent activity (Table 4/FIG. 4). Screening results of these fractions indicated that the biting activity of F-3 was the highest (PNB=0.72), which was similar to DEET. Fraction F-3 was then retested to reconfirm the biting deterrent activity. A major compound identified from this fraction was carotol. This compound was further purified and tested for its activity to confirm the results of the present inventors' preliminary data (Table 5/FIG. 5). The detailed testing of carotol resulted in a proportion not biting (PNB) value (0.9) at 10 μg/cm.sup.2, similar to DEET (PNB=0.85) at 25 nmol/cm.sup.2, showing good biting deterrent activity. Biting deterrent activity of carotol at 5 μg/cm.sup.2 with a PNB value of 0.66 was slightly lower than DEET.

(63) Carrot seed essential oil and carotol were also tested against An. quadrimaculatus (Table 6/FIG. 6). Results from carrot seed essential oil and carotol with PNB values of 0.84 and 0.78, respectively, at 10 μg/cm.sup.2, gave biting deterrent activity were similar to DEET. Biting deterrence of carotol at 25 nmol/cm.sup.2 with PNB value of 0.72 was also similar to DEET at 25 nmol/cm.sup.2.

(64) In the A&K bioassay using a 12 cm.sup.2 surface area, carrot seed essential oil and carotol gave repellent activity similar to DEET. The MED value for all the three treatments was 11.7 μg/cm.sup.2 (Table 7/FIG. 7). In in vitro residual repellent activity, the essential oil and carotol gave activity similar to DEET at 46.9 μg/cm.sup.2 up to 120 minutes post treatment (Table 8/FIG. 8). At 23.4 μg/cm.sup.2, DEET showed full activity up to 120 minutes, whereas the carrot seed essential oil and carotol crossed the MED limit after 60 minutes post treatment compared to the activity of DEET at this dose. At 11.7 μg/cm.sup.2, DEET was within the range of MED values up to 30 minutes, compared to the essential oil and carotol that failed at this dose.

(65) The MED value was 25 μg/cm.sup.2 for carrot seed essential oil and carotol, whereas the MED of DEET was 12.5 μg/cm.sup.2 in cloth patch bioassay. In in vivo cloth patch bioassay, carrot seed essential oil and carotol showed repellency similar to DEET up to 120 minutes post treatment at the dosages of 50 and of 25 μg/cm.sup.2 (Table 9/FIG. 9). In residual repellent activity study, carrot essential oil and carotol gave activity like DEET at the dosages of 50 and 25 μg/cm.sup.2 up to 120 minutes post treatment (Table 10/FIG. 10). At 12.5 μg/cm.sup.2, both carrot seed essential oil and carotol failed, whereas DEET lost its activity at 30 minutes post treatment. Data from the in vivo cloth patch bioassay on DEET corroborates the findings of Ali et al. (2012), and Katritzky et al. (2010), who reported MED values of 9 μg/cm.sup.2, respectively. Results of the carrot seed essential oil and carotol are reported here for the first time.

(66) In conclusion, carrot seed essential oil and its major compound, carotol, showed very high biting deterrent activity in the high throughput bioassay. In large cage in vitro and in vivo repellent bioassays, the natural products also showed an excellent repellent activity. Similarly, in in vitro and in vivo cloth patch bioassays, both the essential oil and carotol gave excellent residual repellent activity at the dosages comparable to DEET. This is the first report on the biting deterrent and repellent activity of carrot seed essential oil and carotol against mosquitoes.

(67) 2. Direct Skin Application Bioassay.

(68) Direct skin application bioassays were conducted by using a powder-free latex glove (Diamond Grip™, Microflex Corporation, Reno, Nev.). An opening of 3×4 cm was cut through the glove to fit on the dorsal surface of the hand. After donning the glove, a wristband was used to avoid biting on the hand near the border of the glove. Approximately 500 (±5%) mosquitoes, consisting primarily of females, were transferred to a test cage (45×45×45 cm) using an aspirator. A series of dosages were tested to determine MED for repellency as well as residual activity against mosquitoes.

(69) A single test consisted of covering the hand of a volunteer with a powder-free latex glove and treating a 12 cm.sup.2 surface area, cut through the glove, on the dorsal surface of the hand. Marked skin surface area was treated with the test compound in 50 μl of total volume. Because of constraints of man power, only one male volunteer who was provided with a written informed consent participated in this study. A protocol approved by the University of Mississippi Human Use Institutional Review Board (IRB Protocol #15-070) was followed.

(70) The test started when the hand treated (3×4 cm area cut through the glove on the dorsal surface) with a test compound was inserted into the mosquito cage. Mosquito landing and biting was observed for a period of 1 min. After 1 min exposure, the hand was gently shaked and the number of biting females (feeding females do not fly) was recorded. Any treatment with ≤5 females biting during the 1 min test period was considered as “passed”, whereas a treatment with ≥5 bites out of 500 mosquitoes was considered as “failed”. In case of passing, the next lower, and in case of failure, the next higher, serial dose was tested to reach MED. Additionally, data on the residual repellent activity were recorded by exposing the treatments to the females after every 30 minutes until the treatment failed. Carrot seed essential oil, carotol, geranium and lemon Eucalyptus essential oils, DEET, picaridin and the mixtures were tested.

(71) Results and Discussion

(72) In vivo, cloth patch bioassay data against Ae. albopictus are given in Table 11/FIG. 11. A MED value of DEET and carotol was 12.5 μg/cm.sup.2. The value of DEET against Ae. albopictus was similar, whereas a MED value of carotol was lower (25 μg/cm.sup.2) than Ae. aegypti (Table 9/FIG. 9).

(73) In vivo cloth patch bioassay data against Anopheles quadrimaculatus are given in Table 12/FIG. 12. A MED value of DEET and carotol was 6.25 μg/cm.sup.2. A MED value against An. quadrimaculatus in both DEET and carotol is lower than Ae. aegypti (DEET=12.5 and carotol=25 μg/cm.sup.2, respectively) or Ae. albopictus, with MED value of 12.5 μg/cm.sup.2.

(74) In the A&K bioassay, mixtures of DEET with the essential oil or carotol showed a MED value of 12.5 μg/cm.sup.2 (6.25+6.25=12.5 μg/cm.sup.2), which is one half of the dose of individual compounds, and was significantly lower than the individual applications of DEET, carrot seed essential oil or carotol, with a MED value of 25 μg/cm.sup.2 (Table 13/FIG. 13). Amounts of the individual components in the mixture were ¼ of the MED value of individual compounds. In in vivo direct skin application bioassay, both the essential oil and carotol at 8, 12.5, 25 and 50% application rates were active, and reached to the MED values at 1.5, 1.5, 2 and 2.5-h post application, respectively (Table 14/FIG. 14).

(75) In direct skin application bioassays, mixtures of petroleum jelly and Johnson's® baby oil with the essential oil increased the residual activity by 100% at the application rate of 12.5% (Table 15/FIG. 15). These data indicated that improvement in formulations can make these natural products as effective commercial repellents.

(76) Data on the repellent activity of mixtures of DEET with carrot essential oil and carotol are given in Table 16/FIGS. 16A, 16B. These mixtures of DEET with the essential oil and carotol showed very promising results in direct skin application bioassay. All the mixtures were active, and showed a substantial increase in residual repellent activity at lower dosages when compared to various treatments used alone. DEET at 1% rate of application crossed the MED level at 1-h post application, whereas the addition of 1% carrot essential oil or carotol increased the residual repellent activity by 100%. There was a 33% increase in residual activity when the activity of 1+1=2% mixtures were compared with 2% of DEET alone. Addition of 2% essential oil or carotol in 2% DEET increased the residual repellent activity by 100% (1.5-h), whereas this increase was 25% when the mixture was compared with the MED value of 4% DEET. Mixture of 4% of the essential oil or carotol with 4% DEET increased residual activity by 175% (3.5-h) when compared with the MED value of 4% DEET, whereas the residual repellent activity of this mixture was similar to 8% application rate of DEET. Mixture of 8.3% of the essential oil or carotol with 4.2% DEET increased the residual activity by 225-250% (4.5-5-h), 30-40% (1.5-2-h) and 0%, when compared to 4%, 8% and 12.5% treatment of DEET, respectively. These data indicated a strong potential of these natural products to substantially increase the residual activity of DEET in mixtures at low concentrations.

(77) Data on the residual repellent activity of picaridin and its mixtures with carrot seed essential oil in direct skin application bioassay are given in Table 17/FIG. 17. There was 100% increase in the residual activity in a 4%+4% mixture of picaridin and carrot seed essential oil, when compared to 4% or 8% application rates of picaridin alone. However this increase in residual activity was lower as compared to DEET (Table 16/FIGS. 16A, 16B).

(78) Data on the repellent activity of mixtures of DEET with the essential oils of geranium (Pelargonium graveolens) and lemon Eucalyptus (Eucalyptus citriodora) in a direct skin application bioassay are given in Table 18/FIG. 18. The mixtures of DEET with these essential oils did not show any promising results in a direct skin application bioassay. Combinations of these essential oils with DEET did not show much increase in residual repellent activity. DEET at 2% rate of application crossed the MED level at 1-h post application, and addition of 2% of any of these two essential oils did not improve the residual repellent activity. There was unsubstantiated improvement in residual activity when 4% DEET was mixed with 4 and 8.3% of these essential oils. The data indicated that these essential oils do not have any synergistic activity in mixtures.

(79) In order to determine synergy in mixtures, two essential oils that are reported to show repellency for mixtures were selected and tested. The data did not show any improvement in residual activity when geranium or lemon Eucalyptus essential oil was mixed with DEET and undecanoic acid. However, in the mixture of carrot seed essential oil with DEET, picaridin or undecanoic acid residual activity increased, significantly.

(80) Regarding Table 19/FIG. 19, a direct skin application method was used in this study, using the methodology described above. Data on the residual repellent activity of undecanoic acid and its mixtures with geranium essential oil and carrot seed essential oil in direct skin application bioassay are given in Table 19/FIG. 19. Undecanoic acid at 12.5% crossed MED level at 1.5-h post application, whereas geranium essential oil at 12.5% lasted for 1-h. Mixture of undecanoic acid with geranium essential oil at 12.5% (6.25+6.25=12.5%) did not improve the residual repellant activity. Mixture of undecanoic acid with carrot seed essential oil at 12.5% (6.25+6.25=12.5%) increased residual activity by 125% when compared to undecanoic acid alone at 12.5%. However, there was no increase in residual activity when the mixture of undecanoic acid with carrot seed essential oil at 25% (12.5+12.5=25%) was compared with a mixture of 12.5%. These data indicated a synergistic activity in the mixture of undecanoic acid with carrot seed essential oil.

(81) Summary

(82) In conclusion, carrot seed essential oil and its major compound, carotol, showed excellent biting deterrent activity in the high throughput bioassay. In the in vitro A&K bioassay and an in vivo repellent bioassay, these natural products showed excellent repellent activity. Similarly, in in vitro and in vivo cloth patch bioassays, both the essential oil and carotol gave very promising residual repellent activity at dosages comparable to DEET. The data on the mixtures of DEET with the carrot seed essential oil and carotol showed extended residual repellency when compared to essential oil, carotol or DEET alone. Repellency of the mixtures in direct skin application is very promising, and indicated that mixtures of these natural products with DEET can decrease the amount of DEET required for efficacy and increase residual repellent activity.

(83) A synergistic effect of carrot seed essential oil was also observed in mixtures with picaridin. Synergy with DEET and picaridin is a unique characteristic of carrot seed essential oil and carotol which is not present in other active essential oils like geranium and lemon Eucalyptus.

(84) This is the first report on the biting deterrent and repellent activity of carrot seed essential oil and carotol against mosquitoes. These data strongly suggest that synergy of carrot seed essential oil and carotol in mixtures with DEET or picaridin presents a strong potential of these natural products to be developed as commercial repellents against mosquitoes. Increase in residual repellency when these products were mixed with petroleum jelly and mineral oil as carriers indicated a potential for these natural products to be used as commercial natural repellents in standard or optimized formulations.

(85) A major commercial advantage of carrot essential oil is that it is commercially available, biodegradable, and safe. Carrot essential oil is on the Food and Drug Administration's GRAS list and is used commercially in cosmetic formulations. Carotol, which is the major component of the essential oil, is likely to be safe for application to the skin and can be economically extracted from the essential oil.

(86) The above description is only representative of illustrative embodiments and examples. For the convenience of the reader, the above description has focused on a limited number of representative examples of all possible embodiments, examples that teach the principles of the invention. The description has not attempted to exhaustively enumerate all possible variations or even combinations of those variations described. That alternate embodiments may not have been presented for a specific portion of the invention, or that further undescribed alternate embodiments may be available for a portion, is not to be considered a disclaimer of those alternate embodiments. One of ordinary skill will appreciate that many of those undescribed embodiments involve differences in technology and materials rather than differences in the application of the principles of the invention. Accordingly, the invention is not intended to be limited to less than the scope set forth in the following claims and equivalents.

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