Chemical Composition and Biological Activity of Four Salvia Essential

Dec 22, 2014 - Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy, The University of Mississippi, University, Mississi...
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Chemical Composition and Biological Activity of Four Salvia Essential Oils and Individual Compounds against Two Species of Mosquitoes † § # † Abbas Ali,*,† Nurhayat Tabanca, Betul Demirci, Eugene K. Blythe, Zulfiqar Ali, ̇ K. Husnu Can Baser,§,⊥ and Ikhlas A. Khan†,‡,Δ †

National Center for Natural Products Research, The University of Mississippi, University, Mississippi 38677, United States Department of Pharmacognosy, Anadolu University, Faculty of Pharmacy, 26470 Eskisehir, Turkey # Coastal Research and Extension Center, Mississippi State University, South Mississippi Branch Experiment Station, Poplarville, Mississippi 39470, United States ⊥̇ Botany and Microbiology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia ‡ Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States Δ Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia §

S Supporting Information *

ABSTRACT: The chemical compositions of essential oils obtained from four species of genus Salvia were analyzed by gas chromatography with a flame ionization detector (GC-FID) and gas chromatography−mass spectrometry (GC-MS). The main compounds identified from Salvia species essential oils were as follows: 1,8-cineole (71.7%), α-pinene (5.1%), camphor (4.4%), and β-pinene (3.8%) in Salvia apiana; borneol (17.4%), β-eudesmol (10.4%), bornyl acetate (5%), and guaiol (4.8%) in Salvia elegans; bornyl acetate (11.4%), β-caryophyllene (6.5%), caryophyllene oxide (13.5%), and spathulenol (7.0%) in Salvia leucantha; α-thujene (25.8%), viridiflorol (20.4%), β-thujene (5.7%), and camphor (6.4%) in Salvia officinalis. In biting-deterrent bioassays, essential oils of S. leucantha and S. elegans at 10 μg/cm2 showed activity similar to that of DEET (97%, N, N-diethyl-mtoluamide) in two species of mosquitoes, whereas the activities of S. of f icinalis and S. apiana essential oils were lower than those of the other oils or DEET. Pure compounds β-eudesmol and guaiol showed biting-deterrent activity similar to DEET at 25 nmol/cm2, whereas the activity of 13-epi-manool, caryophyllene oxide, borneol, bornyl acetate, and β-caryophyllene was significantly lower than that of β-eudesmol, guaiol, or DEET. All essential oils showed larvicidal activity except that of S. apiana, which was inactive at the highest dose of 125 ppm against both mosquito species. On the basis of 95% CIs, all of the essential oils showed higher toxicity in Anopheles quadrimaculatus than in Aedes aegypti. The essential oil of S. leucantha with an LC50 value of 6.2 ppm showed highest toxicity in An. quadrimaculatus. KEYWORDS: Salvia species, β-eudesmol, guaiol, 13-epi-manool, biting deterrent, larvicide, Aedes aegypti, Anopheles quadrimaculatus

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INTRODUCTION

insects, low mammalian toxicity, and biodegradability in the environment.4−7 The genus Salvia L. (Lamiaceae) contains more than 750 species of herbs, subshrubs, and shrubs of worldwide distribution, with many of the species commonly used in local folk medical practices and in cosmetics.8−11 The name of the genus Salvia means “safe” or “well”, from the Latin word salvere, referring to the curative properties of the known culinary and medicinal herb Salvia officinalis L.12 Members of this genus are reported to produce many useful secondary metabolites including terpenes, phenolics, and their derivatives that have been central to the pharmacopoeias of many countries.13 The healing reputation of Salvia species can be traced back to Roman times, and a well-known sage, S. of f icinalis, is particularly known for this.14 Salvia species are

Mosquitoes are vectors for many pathogens that cause human diseases such as malaria, dengue fever, yellow fever, Rift Valley fever, and Chikungunya.1 When significant levels of transmission occur, epidemics can result in high rates of human morbidity and mortality. Insecticides from various chemical groups are the basic tools used for management of mosquito populations. Due to extensive and indiscriminate use of insecticides, mosquitoes have developed resistance against these chemicals.2 Plant essential oils have been suggested as alternative sources for insect control because they are selective, biodegrade to nontoxic products, and have minimal effects on nontarget organisms and the environment.3 The search for new strategies for natural products to control vectors of these diseases is desirable because this can help in managing resistance to synthetic insecticides and reducing the use of toxic chemicals.3 Much effort has, therefore, been focused to identify the effectiveness of plant essential oils against mosquito larvae because of their broad-spectrum activity against target © XXXX American Chemical Society

Received: October 14, 2014 Revised: December 13, 2014 Accepted: December 22, 2014

A

DOI: 10.1021/jf504976f J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Composition of the Essential Oils of Salvia Species RRIa 1014 1032 1076 1118 1159 1174 1176 1188 1203 1213 1246 1255 1266 1280 1290 1391 1393 1437 1450 1451 1452 1466 1478 1492 1497 1500 1532 1553 1586 1591 1600 1611 1612 1617 1628 1648 1668 1670 1677 1684 1687 1687 1704 1706 1719 1722 1725 1726 1740 1741 1755 1768 1773 1776 1804 1823 1838 1845 1849 1864 1868

compound tricyclene α-pinene camphene β-pinene δ-3-carene myrcene α-phellandrene α-terpinene limonene 1,8-cineole (Z)-β-ocimene γ-terpinene (E)-β-ocimene p-cymene terpinolene (Z)-3-hexenol 3-octanol α-thujone trans-linalool oxide ( f uranoid) β-thujone 1-octen-3-ol α-cubebene cis-linalool oxide ( f uranoid) cyclosativene α-copaene pentadecane camphor linalool pinocarvone bornyl acetate β-elemene terpinen-4-ol β-caryophyllene 6,9-guaiadiene aromadendrene myrtenal (Z)-β-farnesene trans-pinocarveol epi-zonarene β-guaiaene methylchavicol α-humulene γ-muurolene α-terpineol borneol cabreuva oxide-II verbenone germacrene D α-muurolene β-bisabolene bicyclogermacrene cabreuva oxide-IV δ-cadinene γ-cadinene myrtenol cabreuva oxide-VI (E)-β-damascenone trans-carveol calamenene p-cymen-8-ol (E)-geranyl acetone

S. apianab (%) 5.1 0.5 3.8 2.4 1.2 0.2 0.3 1.5 71.7 1.1 0.5 0.3 0.8 0.2

S. elegansb (%)

S. leucanthab (%)

S. of f icinalisb (%)

0.1 1.4 3.5 2.4

0.8 0.3 0.2

1.9 1.0 1.0

0.4 3.5

4.4

0.3 1.2 0.1 0.4 1.1 25.8 0.3

0.5

1.2 0.4 0.1 0.4 0.4

0.8

5.7

0.2

0.5

0.5 4.4 0.2

0.5 1.7 0.2

3.8 3.0 0.5 5.0

11.4 0.2

2.4 6.5 4.9 0.6 0.8 0.9

0.1 0.3 0.2

6.4 0.9

0.4 0.9 17.4 0.6 0.5

4.1 0.2 0.1 0.5 0.7 0.6

2.1

0.3 0.7

1.5

0.8 0.8 0.4 0.3 0.4 0.2

0.1

B

0.6 2.1 0.6 0.6 0.3 0.4 0.1 0.7 0.5 0.5

0.4 0.7

0.5 1.1

0.5 2.9 0.2

identificationc tR, MS tR, MS tR, MS tR, MS MS tR, MS tR, MS tR, MS tR, MS tR, MS MS tR, MS MS tR, MS tR, MS MS tR, MS tR, MS MS tR, MS tR, MS tR, MS MS MS tR, MS tR, MS tR, MS tR, MS tR, MS tR, MS tR, MS tR, MS tR, MS MS MS tR, MS MS tR, MS MS MS tR, MS tR, MS MS tR, MS tR, MS MS tR, MS tR, MS MS tR, MS tR, MS MS MS MS tR, MS MS MS tR, MS MS tR, MS MS

DOI: 10.1021/jf504976f J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. continued RRIa

compound

1898 1918 1941 1958 1984 1980 2008 2045 2071 2174 2080 2088 2098 2103 2104 2105 2130 2131 2144 2192 2185 2204 2209 2232 2247 2250 2256 2257 2264 2298 2300 2324 2389 2392 2500 2600 2622 2679 2700 2900 2931

1,11-oxidocalamenene β-calacorene α-calacorene (E)-β-ionone γ-calacorene furopelargone A caryophyllene oxide humulene epoxide-I humulene epoxide-II fokienol cubenol 1-epi-cubenol globulol guaiol viridiflorol furopelargone B salviadienol hexahydrofarnesyl acetone spathulenol nonanoic acid γ-eudesmol eremoligenol T-muurolol bulnesol trans-α-bergamotol α-eudesmol cadalene β-eudesmol 4,7-dimethyl-1-tetralone decanoic acid tricosane caryophylla-2(12),6(13)-dien-5α-ol (=caryophylladienol II) caryophylla-2(12),6-dien-5α-ol (=caryophyllenol I) caryophylla-2(12),6-dien-5β-ol (=Caryophyllenol II) pentacosane hexacosane phytol 13-epi-manool heptacosane nonacosane hexadecanoic acid

S. apianab (%)

S. elegansb (%)

0.4 0.5 0.6 1.7 0.2 0.5 0.5 0.8 0.5 4.8

S. leucanthab (%)

S. of f icinalisb (%)

0.2 1.4 2.1 0.4 3.2 13.5 0.6

0.8 1.1 4.5

0.6 20.4

0.6 1.3

4.2 0.4 1.5 7.0 0.5

3.5 0.7 0.3 0.7 0.5 3.7 2.6 10.4

1.2 2.6 0.6 3.2 1.1 2.4 2.7

0.1 0.2

3.7 0.6

0.3

0.2

monoterpene hydrocarbons oxygenated monoterpenes sesquiterpene hydrocarbons oxygenated sesquiterpenes alkanes fatty acids diterpenes others

17.9 77.3 3.3

total

99.0

1.8 4.4 5.7 1.8

tr

0.6

9.4 40.3 7.4 31.1

0.6 4.7

1.3 14.7 29.0 36.4 0.5 1.7 0.3 6.1

3.9 44.2 2.8 28 17 1.8 1.8

93.5

90.0

99.5

0.5

identificationc MS MS MS MS MS MS tR, MS MS MS MS MS MS MS tR, MS tR, MS, NMR MS MS MS tR, MS tR, MS MS MS MS tR, MS MS tR, MS MS tR, MS MS tR, MS tR, MS MS MS MS tR, MS tR, MS MS tR, MS, NMR tR, MS tR, MS tR, MS

a

RRI, relative retention indices calculated against n-alkanes on polar column. bPercent calculated from FID data for polar column; tr, trace (98%, 10 mg). 1 [α]25 D = +59.2 (c 1.0, CHCl3); H NMR (500 MHz, CDCl3) δ 0.99 (td, J = 13.5, 4.2 Hz, H-1a), 1.72 (H-1b), 1.46 (tt-like, J = 13.5, 3.5 Hz, H2a), 1.55 (H-2b), 1.14 (td, J = 13.5, 4.1 Hz, H-3a), 1.37 (H-3b), 1.05 (dd, J = 12.6, 2.7 Hz, H-5), 1.28 (H-6a), 1.68 (H-6b), 1.94 (td, J = 13.0, 5.2 Hz, H-7b), 2.35 (br d, J = 13.0 Hz, H-7a), 1.53 (H-9), 1.30 (H-11a), 1.51 (H-11b), 1.22 (H-12a), 1.71 (H-12b), 5.88 (dd, J = 17.4, 10.7 Hz, H-14), 5.02 (d, J = 10.7 Hz, H-15a), 5.17 (d, J = 17.4 Hz, H-15b), 1.27 (s, 3H, H-16), 4.49 (br s, H-17a), 4.79 (br s, H-17b), 0.84 (s, 3H, H-18), 0.77 (s, 3H, H-19), 0.65 (s, 3H, H-20); 13C NMR (125 MHz, CDCl3) δ 39.1 (t, C-1), 19.4 (t, C-2), 42.2 (t, C-3), 33.6 (s, C-4), 55.6 (d, C-5), 24.4 (t, C-6), 38.4 (t, C-7), 148.7 (s, C-8), 57.4 (d, C-9), 39.9 (s, C-10), 17.7 (t, C-11), 41.5 (t, C-12), 73.4 (s, C-13), 145.3 (d, C-14), 111.5 (t, C-15), 27.6 (q, C-16), 106.5 (t, C-17), 33.6 (q, C-18), 21.7 (q, C-19), 14.4 (q, C-20); EIMS, m/z 290 [M+] C20H34O, 272 (7), 257 (43), 244 (9), 229 (6), 216 (6), 203 (11), 189 (19), 177 (20), 161 (16), 147 (15), 137 (87), 139 (23), 123 (45), 121 (41), 115 (6), 109 (52), 107 (54), 95 (92), 93 (68), 66 (45), 81 (100), 79 (69), 71 (40), 69 (71), 67 (49), 55 (59), 43 (42), 41 (61). Gas Chromatography−Mass Spectrometry Analysis for Isolated Compound 13-epi-Manool. The purity of 13-epi-manool was analyzed using an HP 5890 series gas chromatograph linked to an HP 5970 mass spectrometer system using a DB-1 capillary column (20 m × 0.18 mm with 0.25 μm film thicknesses). Helium (1 mL/min) was used as carrier gas. The GC oven temperature was held at 50 °C for 2 min and then ramped to 280 °C at a rate of 5 °C/min. The scanned mass range was from m/z 40 to 550, and the ion source was operated in electron ionization (EI) mode with an electron energy set to 70 eV. Mosquito Bioassays. Insects. Ae. aegypti and An. quadrimaculatus larvae and adults used in these studies were from a laboratory colony maintained at the Mosquito and Fly Research Unit at the Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL, USA. For biting-deterrence 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% relative humidity with a photoperiod regimen of 12:12 h (light/dark). Adult females (8−13 days old) were used. For larval bioassays, the eggs were hatched and the larvae were maintained at the above temperature. Mosquito Biting Bioassays. Experiments were conducted by using a six-celled in vitro Klun and Debboun (K&D) module bioassay system developed by Klun et al.33 for quantitative evaluation of bitingdeterrent properties of candidate compounds. Briefly, the assay system consists of a six-well reservoir with each of the 3 × 4 cm wells containing 6 mL of blood. As described by Ali et al.,34 a feeding solution consisting of CPDA-1 (citrate−phosphate−dextrose−adenine) and ATP was used instead of blood. Green fluorescent tracer dye (www.blacklightword.com) was used to determine feeding by the females. Essential oils of S. leucantha, S. elegans, S. of f icinalis, S. apiana and individual compounds were tested in this study. Essential oils were applied at concentrations of 10 μg/cm2, pure compounds at 25 nmol/ cm2, and DEET (97%, N, N-diethyl-m-toluamide) (Sigma-Aldrich, St. Louis, MO, USA) at 25 nmol/cm2 as positive control. All treatments were freshly prepared in molecular biology grade 100% ethanol (Fisher Scientific Chemical Co., Fair Lawn, NJ, USA) at the time of bioassay. 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. Reservoirs were covered with a layer of collagen membrane (Devro, Sandy Run, SC, USA). 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 contamination of the module. A six-celled K&D module containing five female mosquitoes per cell was positioned over the 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 cloth treatments in each cell was recorded after a 3 min exposure, and mosquitoes were prodded back into the cells to check the actual feeding. Mosquitoes were squashed and the presence or absence of green fluorescent tracer dye in the gut was used as an indicator of feeding. A replicate consisted of six treatments: four test materials, DEET (a standard biting deterrent), and ethanol-treated organdy as solvent control applied randomly. Two sets of five replications each with five females per treatment were conducted on two different days using a newly treated organdy and a new batch of females in each replication. Treatments were replicated 10 times. Larval Bioassays. Bioassays were conducted to test essential oils of S. leucantha, S. elegans, S. of f icinalis, and S. apiana for their larvicidal activity against moquitoes by using the bioassay system described by Pridgeon et al.35 Five 1-day-old Ae. aegypti and An. quadrimaculatus larvae were added in a droplet of water to each well of 24-well plates (BD Labware, Franklin Lakes, NJ, USA) by use of a disposable 22.5 cm Pasteur pipet. Fifty microliters of larval diet (2% slurry of 1:1 beef liver powder (Now Foods, Bloomingdale, IL, USA) and brewer’s yeast (Lewis Laboratories Ltd., Westport, CT, USA) was added to each well by using a Finnpipette stepper (Thermo Fisher, Vantaa, Finland). All chemicals tested were diluted in ethanol. Eleven microliters of the test chemical was added to the labeled wells, whereas 11 μL of ethanol was added to control treatments. After the treatment application, the plates were swirled in clockwise and counterclockwise motions and front and back and side to side five times to ensure even mixing of the chemicals. Larval mortality was recorded 24 h after treatment. Larvae that showed no movement in the well after manual disturbance of the water were recorded as dead. A series of five concentrations ranging between 125 and 1.95 ppm were used in each treatment to obtain a range of mortality. Treatments were replicated 10 times for each essential oil. Statistical Analyses. Proportion not biting was calculated as described by Ali et al.34 As the K&D module bioassay system can handle only four treatments along with negative and positive controls, in order to make direct comparisons among more than four treatments and to compensate for variation in overall response among replicates, biting-deterrent activity was quantified as biting deterrence index (BDI). The BDIs were calculated using the formula

⎡ PNBi , j , k − PNBc , j , k ⎤ ⎥ [BDIi , j , k] = ⎢ ⎢⎣ PNBd , j , k − PNBc , j , k ⎥⎦ where PNBi,j,k denotes the proportion of females not biting when exposed to test compound i for replication j and day k (i = 1−4, j = 1− 5, k = 1−2), PNBc,j,k denotes the proportion of females not biting the solvent control “c” for replication j and day k (j = 1−5, k = 1−2), and PNBd,j,k denotes the proportion of females not biting in response to DEET “d”(positive control) for replication j and day k (j = 1−5, k = 1−2). This formula makes an adjustment for interday variation in response and incorporates information from the solvent control as well as the positive control. A BDI value of 0 indicates an effect similar to that of ethanol, whereas a value significantly greater than 0 indicates biting-deterrent effect relative to that of ethanol. BDI values not significantly different from 1 are statistically similar to DEET. BDI values were analyzed using the ANOVA procedure of SAS [single factor: test compound (fixed)] (SAS Institute, 2007), and means were separated using the Ryan−Einot−Gabriel−Welsch multiple-range test. To determine whether confidence intervals included the values of 0 or 1 for treatments, Scheffe’s multiple-comparison procedure was used with the CLM option in SAS. LC50 values for larvicidal data were calculated by using the PROBIT procedure of SAS. Control mortality was corrected by using Abbott’s formula.36

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RESULTS AND DISCUSSION The composition of four Salvia species was investigated in the present study. Identified compounds ranged between 90 and 99% (Table 1). S. apiana essential oil contained 1,8-cineole E

DOI: 10.1021/jf504976f J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry (71.7%) as a major constituent followed by monoterpene hydrocarbons α-pinene (5.1%), camphor (4.4%), and β-pinene (3.8%). S. elegans essential oil had high content of borneol (17.4%), β-eudesmol (10.4%), bornyl acetate (5%), and guaiol (4.8%). Major constituents of S. leucantha oil were bornyl acetate (11.4%), caryophyllene oxide (13.5%), β-caryophyllene (6.5%), and spathulenol (7.0%). S. off icinalis oil was rich in monoterpene hydrocarbon α-thujene (25.8%) and had high content of oxygenated sesquiterpene viridiflorol (20.4%). Many studies have reported the composition of essential oils of these Salvia species. Chemical composition data on the essential oil of S. apiana showed 1,8-cineole as a major compound.37,38 Mathew and Thoppil27 reported spathulenol (38.73%) and β-caryophyllene (10.32%) as the major compounds of the S. elegans essential oil in samples extracted from the plant material collected from Ooth, southern India. Data on S. elegans essential oil differ from the above study where spathulenol (1.3%) is a minor compound and βcaryophyllene is totally absent. On the other hand, the S. elegans essential oil obtained from the cultivated species from Eboli, Italy, contained cis-thujone (38.7%), δ-cadinene (11.5%), geranyl acetate (6.9%), and geraniol (6.5%).39 Data on the composition of S. leucantha essential oil corroborate the findings of Rondon et al.40 Negi et al.,41 and Upadhyaya et al.,42,43 who reported bornyl acetate as the major compound followed by sesquiterpene hydrocarbons such as β-caryophyllene, α-guaiene, β-gurjunene, cis-muurola-3,5-diene, germacrene D, and bicyclogermacrene. In earlier investigations of the Salvia essential oils, we studied S. leucantha volatiles obtained by microdistillation technique, which showed limonene (34.6%), α-pinene (16.7%), and borneol (16.5%) as the major compounds, whereas β-caryophyllene (5.6%) and caryophyllene oxide (5.4%) were the major sesquiterpenes with low content of oxygened monterpenene bornyl acetate (0.1%).44 These differences in chemical composition could be due to distillation technique used or growing conditions of the plants. The essential oil of S. of f icinalis was characterized by α-thujone (25.8%), β-thujone (5.7%), camphor (6.4%), and 1,8-cineole (4.4%) as major components, which also determine their chemotypes. The presence of thujone isomers in the essential oils is important because of their neurotoxic and hepatotoxic effects.45 Thujone is banned as a food additive in the United States and Europe. The most common consumption of thujone is in dietary supplements such as sage-flavored products, teas, and drops and alcoholic beverages.46 Oelschlagel at al.46 found that S. of f icinalis ‘Extrakta’ contained the highest levels of αthujone. They also reported low α-thujone in the essential oils of S. of f icinalis subsp. major, S. off icinalis ‘Berggarten’, and S. off icinalis ‘Aurea’. In the present study, the ‘Extrakta’ variety of S. off icinalis essential oil showed a high percentage (25.8%) of α-thujone and therefore cannot be recommended for use in the production of sage supplements. During the GC-MS analysis, the peak at RRI 2679 of the S. of ficinalis oil was not identified by the in-house “Baser Library of Essential Oil Constituents”; however, its mass spectrum was similar to that of 13-epi-manool according to the Wiley GC/MS Library and MassFinder 3 Library. To ensure its identity, this compound was isolated and characterized by 1D and 2D NMR spectroscopic data analyses (Figure 1). The chemical shift of H-17a (δ 4.50 for 13-epimanool and δ 4.46 for manool) is the key to distinguish 13S and 13R isomers.48 In our case, the 1H NMR chemical shift of H-17a was observed at δ 4.49 and could be concluded for 13epi-manool. Moreover, the specific rotation that we found in

Figure 1. Structure of 13-epi-manool.

this study {[α]25 D = +59.2 (c 1.0, CHCl3)} also supported the identity as 13-epi-manool {[α]24 D = +28 (c 1.5, CHCl3 for manool.49 The in vitro K&D system used in this study specifically quantified the mosquito biting-deterrent properties of Salvia essential oils and the pure compounds. Essential oils of S. leucantha (BDI value = 1.01) and S. elegans (BDI value = 0.87) showed biting-deterrent activity similar to that of DEET, whereas the activity of essential oils of S. off icinalis (BDI value = 0.63) and S. apiana (BDI value = 0.48) demonstrated low biting-deterrent activity against Ae. aegypti (Figure 2). The major compounds of the active oils S. elegans and S. leucantha were individually investigated. Among the pure compounds, (+)-β-eudesmol (BDI value = 0.81) and (−)-guaiol (BDI value = 0.82) showed the highest biting-deterrent activity, which was similar to that of DEET at 25 nmol/cm2, whereas the activities of (−)-borneol (BDI value = 0.69) and (−)-borneol acetate (BDI value = 0.52) were significantly lower than that of DEET. The sesquiterpenes (−)-β-caryophyllene, (−)-caryophyllene oxide, and (+)-spathulenol were high in S. leucantha oil, and we previously reported their biting-deterrent activity with BDI values of 0.54 and 0.66 against Ae. aegypti, respectively.50 Spathulenol has been reported to show good biting-deterrent activity against Ae. aegypti and An. stepheni,51 and similar response of high biting-deterrent activity was observed in our laboratory against Ae. aegypti (A. Ali, unpublished data). The oxygenated diterpene 13-epi-manool was also investigated for mosquito activity in a search of new mosquito-deterrent agents. The biting-deterrent activity of (+)-13-epi-manool (BDI = 0.75) was lower than that of (+)-β-eudesmol and (−)-guaiol against Ae. aegypti. In An. quadrimaculatus, essential oils of S. elegans (BDI value = 0.9) and S. leucantha (BDI value = 0.86) also showed higher biting-deterrent activity that was similar to that of DEET, whereas the activities of essential oils of S. of f icinalis (BDI value = 0.6) and S. apiana (BDI value = 0.56) were lower than that of DEET (Figure 3). Pure compounds (+)-β-eudesmol (BDI value = 0.82) and (−)-guaiol (BDI value = 0.82) showed activity similar to that of DEET at 25 nmol/ cm2. (+)-13-epi-Manool demonstrated higher biting-deterrent activity than (−)-caryophyllene oxide, (−)-borneol, (−)-bornyl acetate, and (−)-β-caryophyllene; however, its activity was significantly lower than those of (+)-β-eudesmol, (−)-guaiol, or DEET in both species. Previous research papers show repellent activity of essential oils of various Salvia species against different species of F

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Journal of Agricultural and Food Chemistry

Figure 2. Biting-deterrent index (BDI) values of the essential oils extracted from samples of aerial parts of four species of Salvia and individual compounds against female Ae. aegypti. Essential oils were tested at 10 μg/cm2 and the pure compounds were tested at 25 nmol/cm2. Ethanol was the solvent control, and DEET at 25 nmol/cm2 was used as positive control.

Figure 3. Biting-deterrent index (BDI) values of the essential oils extracted from samples of aerial parts of four species of Salvia and individual compounds against female Anophelese quadrimaculatus. Essential oils were tested at 10 μg/cm2, and the pure compounds were tested at 25 nmol/ cm2. Ethanol was the solvent control, and DEET at 25 nmol/cm2 was used as positive control.

mosquitoes. Conti et al.26 reported that essential oils of S. dorisiana, S. longifolia, and S. sclarea have significant but variable repellent activity against Ae. albopictus. Essential oils of S. dorisiana with protection efficacy of 91−95% and protection times of 9.2 to 92.4 min were most active, whereas protection times of essential oils of S. longifolia and S. sclarea ranged between 3.2 and 60 min and between 3.6 and 64.2 min, respectively.26 Amer and Melhorn25 reported good repellent activity of essential oils of S. sclarea against mosquito species of Ae. aegypti, An. stephensi, and Culex quinquefasciatus. Kang et

al.52 showed low activity of essential oil of S. off icinalis against Culex pipens pallens Cosquillett. Ali et al.50 reported significantly lower activity of (−)-βcaryophyllene and (−)-caryophyllene oxide than that of DEET against Ae. aegypti. Data of biting-deterrent activity of these compounds show a similar trend against An. quadrimaculatus. These data corroborate the finding of Paluch et al.,53 who reported high repellent activity of (+)-β-eudesmol against Ae. aegypti. G

DOI: 10.1021/jf504976f J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 2. Toxicity of Salvia Species Essential Oils against 1-Day-Old Larvae of Aedes aegypti and Anopheles quadrimaculatus at 24 h after Treatment Aedes aegypti essential oil S. apiana S. leucantha S. elegans S. off icinalis (−)-borneol (−)-bornyl acetate (−)-guaiol (+)-β-eudesmol (−)-β-caryophyllene (−)-caryophyllene oxide (+)-13-epi-manool

LC50, ppm (95% CI) c

NA* 29.5 (26.1−33.1) 14.4 (12.7−16.3) 56.9 (51.0−63.5) NA** NA** NA** NA** 26.0 (22.5−30.3)e 29.8 (25.7−34.7)e NA**

a

Anopheles quadrimaculatus χ

2

LC90, ppm (95% CI) NA* 51.8 (44.5−64.6) 27.2 (23.1−34.3) 92.4 (80.2−114.4) NA** NA** NA** NA** 64.8 (52.7−86.3) 74.1 (59.9−99.4) NA**

b

df

63.6 71.8 57.9

38 48 38

95.7 95.6

48 48

LC50, ppm (95% CI)

LC90, ppm (95% CI)

NA* 6.2 (5.3−7.2) 10.9 (9.4−12.8) 14.1 (12.1−16.6) NT**d NT** NT** NT** NT** NT** NT**

NA* 15.8 (12.8−21.1) 29.1 (23.0−40.4) 35.8 (28.2−50.7) NT** NT** NT** NT** NT** NT** NT**

χ2

df

92.8 91.7 77.9

48 48 48

a 95% confidence interval. bdf refers to degree of freedom. cNA (not active), showed