PMIO 2017; 4(02): e52-e58
DOI: 10.1055/s-0043-106857
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Biological Activity of Bunium persicum Essential Oil from Western Himalaya

Iris Stappen1, Nurhayat Tabanca2, 3, Abbas Ali2, David E. Wedge4, Jürgen Wanner5, Velizar Gochev6, Vikas Jaitak7, Brij Lal8, Vijay K. Kaul9, Erich Schmidt1, Leopold Jirovetz1
  • 1Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria
  • 2National Center for Natural Products Research, The University of Mississippi, University, MS, USA
  • 3Department of Entomology and Nematology, Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA
  • 4US Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, University of Mississippi, University, MS, USA
  • 5Kurt Kitzing Co., Wallerstein, Germany
  • 6Department of Biochemistry and Microbiology, “Paisii Hilendarski” – University of Plovdiv, Bulgaria
  • 7Biodiversity Division, CSIR – Institute of Himalayan Bioresource Technology, Palampur, India
  • 8Centre for Chemical and Pharmaceutical Sciences, Central University of Punjab, Bathinda, India
  • 9Ex-Scientist of CSIR – Institute of Himalayan Bioresource Technology, Palampur, H.P., India
Further Information

Correspondence

Dr. Iris Stappen
Department of Pharmaceutical Chemistry
University of Vienna
Althanstrasse 14
1090 Vienna
Austria   
Phone: +43/1/4277 555 52   
Fax: +43/1/4277 9 551   

Publication History

received 25 November 2016
revised 08 March 2017

accepted 09 March 2017

Publication Date:
14 July 2017 (online)

 

Abstract

The essential oil of Bunium persicum collected from cultivated sources in the cold desert area of Lahaul-Spiti is described for its antimicrobial, larvicidal, and biting deterrent activities. Additionally, odor characterization is given. The chemical composition of the essential oil was analyzed by simultaneous GC-MS and GC-FID. γ-Terpinene and p-cymene were found to be the major compounds. Antibacterial testing by an agar dilution assay revealed low activity of the oil against all tested bacteria. Antifungal activity was evaluated against Candida albicans as well as three species of the strawberry anthracnose causing plant pathogen Colletotrichum. Bunium persicum essential oil demonstrated antifungal activity against all four pathogens. Biting deterrent activity against Aedes aegypti was greater than the solvent control, but significantly lower than DEET. The essential oil of B. persicum exhibited larvicidal activity with an LC50 value of 58.6 ppm against Ae. aegypti larvae.


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Introduction

Many important pathogens causing severe diseases are transmitted to humans by insects. Malaria, dengue, yellow fever, and the West Nile virus are considered the most important mosquito-borne diseases in Asia, Africa, and South America [1]. However, due to global warming, these infectious pathogens spread to new areas threatening even more people and challenging academic and industrial communities [2]. Although many methods have been developed for mosquito control, the most effective method is the use of pesticides. Besides several synthetic pesticides that are available on the market, plant extracts and essential oils (EOs) have become important to research and industry due to their safety to the environment and non-target organisms [3]. The synergistic effect that has been found between some EOs and synthetic larvicides [1] would additionally offer an opportunity to develop more effective chemical control agents that are more valuable and less toxic to the environment.

Not only has the increasing presence of mosquitoes caused concern in the population, but also resistances against well-established substances have become challenging. Because of widespread indiscriminate use of antibiotics and agricultural pesticides since the 1950s, the development of chemical resistance in many bacteria, fungi, and insects, including mosquitoes, is frequent. Additionally, the use of synthetic food preservatives to prevent the growth of food-borne and spoilage bacteria and fungi is being criticized by customers because of their growing concerns over food safety. Therefore, the search for new natural antibiotics, fungicides, pest management agents, and food preservatives that have activity against multiple target sites and a low chance for the development of chemical resistance has led to an increased interest in EOs. Various studies, summed up and described in detail in a recent review by Reyes-Jurado et al. [4], have confirmed the activity of some EOs against many microbes.

In the Himalayan region, many plants and their EOs are economically important and have a history in traditional medicinal use, particularly by the populations living in rural areas where little medical care is available. Bunium persicum (Boiss.) B. Fedtsch. (Apiaceae), commonly known as black caraway or black zira, is an aromatic perennial native to the Indian subcontinent (India, Pakistan) and western to middle Asia. Bunium seeds are generally black in color, develop a characteristic pleasant aroma, and are widely used as condiments and flavoring agents in local foods [5] [6]. Seeds and the EO are well known for their digestive, anticonvulsive, diuretic, and anthelmintic effects [7] [8]. Due to the wide scope of pharmacological effects, the plants were extensively collected and sold by the Himachal Pradesh people. This led to a highly threatened population status for B. persicum. Subsequent attempts at cultivation of the plant demonstrated difficulties in germination because the seeds possess a deep dormancy mechanism. Various techniques were established to meet the complex dormancy requirements suitable for B. persicum germination [9]. Today the plants are being successfully cultivated by local farmers in the cold desert of Lahaul-Spiti, an area also with suitable farming conditions for black caraway.

In continuation of our studies on screening EOs from medicinal plants growing wild [10] [11] or cultivated in the western Himalayan region [12], the main purpose of the present investigation was to examine the EO of B. persicum. This species was collected from cultivated sources in Lahaul-Spiti at an altitude of 3 500 m, and tested for antimicrobial activities, mosquito biting deterrence, and larvicidal effect against Aedes aegypti.


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Results and Discussion

B. persicum EO was characterized by olfactory evaluation as aromatic spicy, green herbal, and cumin-like. The results of the quantitative and qualitative oil analyses by simultaneous GC-MS and GC-FID using 2 different columns are listed in [Table 1]. Thirty-one compounds were identified from B. persicum EO, accounting for 97.7–97.9% of the total oil. γ-Terpinene was present in the highest amount (40.2–40.4%), followed by p-cymene (25.8%), cumin aldehyde (12.8–12.9%), p-mentha-1,4-dien-7-al (9.1–9.2%), and p-mentha-1,3-dien-7-al (4.5–4.7%) ([Table 1]). Several investigations of B. persicum oil samples from plants collected or cultivated in Iran are described in the literature. All showed the same main components, but with different concentrations, e.g., the aromatic monoterpene p-cymene occurred at much lower amounts (5–16%) in the Iranian samples [7] [13] [14] [15] [16], whereas cumin aldehyde was higher (24%) compared to our sample from India [13] [17]. An older investigation on Bunium fruits collected in Tajikistan reported p-mentha-1,4-dien-7-al (29.0%), γ-terpinene (25.7%), β-pinene (15.6%), and cumin aldehyde (11.7%) as major components [18]. These findings once more demonstrate the influence of climatic and local factors on EO composition [7] [19] [20] and possibly the difference between cultivated and wild growing species [21]. Therefore, plants from different origins or genetic backgrounds should be studied for their chemical composition in order to be able to conclude or link to their biological activities.

Table 1 Chemical composition of B. persicum EO (in % peak area) determined by GC-FID and GC-MS.

No.

Compound

RI#

%

RI##

%

1

cumene

928

tr.

1151

tr.

2

α-thujene

931

0.3

1018

0.3

3

α-pinene

941

0.1

1015

0.2

4

sabinene

980

0.7

1109

0.7

5

β-pinene

986

0.1

1100

0.1

6

myrcene

992

0.7

1142

0.7

7

δ-3-carene

1018

0.1

1134

0.1

8

α-terpinene

1023

0.2

1163

0.2

9

p-cymene

1032

25.8

1252

25.8

10

limonene

1036

0.3

1184

0.3

11

β-phellandrene

1038

0.2

1196

0.2

12

1,8-cineole

1039

0.3

1202

0.2

13

(E)-β-ocimene

1049

tr.

1216

tr.

14

γ-terpinene

1067

40.4

1233

40.2

15

cis-sabinene hydrate

1070

tr.

1444

tr.

16

terpinolene

1096

0.3

1261

0.4

17

fenchone

1097

0.3

1376

0.3

18

linalool

1102

tr.

1523

tr.

19

trans-sabinene hydrate

1106

tr.

1530

tr.

20

fenchol

1124

0.1

1555

0.1

21

terpinen-4-ol

1188

0.2

1579

0.2

22

p-cymen-8-ol

1192

0.1

1820

0.1

23

α-terpineol

1201

0.1

1675

0.1

24

p-menth-3-en-7al

1203

0.5

1540

0.4

25

(E,E)-nona-2,4-dienal

1209

tr.

1549

0.1

26

neral

1243

tr.

1661

0.1

27

carvone

1251

tr.

1708

0.1

28

cumin aldehyde

1254

12.9

1751

12.8

29

p-mentha-1,3-dien-7-al

1299

4.7

1767

4.5

30

p-mentha-1,4-dien-7-al

1304

9.1

1772

9.2

31

cuminic acid

1374

0.3

2350

0.4

Sum

97.9

97.7

#50 m×0.25 mm×1.0 μm SE-54; ##60 m×0.25 mm×0.25 μm CW20M

In the context of screening B. persicum EO for antimicrobial activity, the oil was tested against the Gram-positive bacterium Staphylococcus aureus, and Gram-negative bacteria Escherichia coli, Salmonella abony, and Pseudomonas aeruginosa. Growth inhibition testing of Candica albicans was performed in a liquid agar broth-microdilution assay [10]. Minimum inhibitory concentrations (MICs) in μg/mL are given in [Table 2].

Table 2 Antimicrobial activity of B. persicum EO (agar dilution assay, MIC given in μg/mL).

Tested compounds

Test microorganism

S. aureus
ATCC 6538

E. coli
ATCC 25922

S. abony
ATCC 6017

P. aeruginosa
ATCC 27853

C. albicans
ATCC 10231

B. persicum EO

4000

2000

2000

8000

1000

Ciprofloxacin

0.25

0.15

0.25

1

Cefazolin

0.50

2

2

4

Amphotericin B

0.25

Fluconazole

0.25

B. persicum EO showed medium to low antimicrobial activity against all tested strains with MICs between 1 000 and 8 000 μg/mL ([Table 2]). P. aeruginosa was the least sensitive bacterium, while C. albicans proved to be the only strain sensitive to the tested oil. Essential oils generally show greater antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria, which is probably related to the bacterial cell membrane structure [4] [22]. Literature reports on antibacterial activity of B. persicum EO show diverse results. An investigation of Iranian samples attributed very high antibacterial activity against E. coli, P. aeruginosa (Gram-negative bacilli), and S. aureus (Gram-positive cocci) to the oil, reporting MIC values around 15 μg/mL [23]. In another Iranian study, B. persicum EO was found to be inactive against E. coli [24]. Results of Oroojalian et al. [25] on Iranian B. persicum EO showed an antimicrobial effect with an MIC value of 1 500 μg/mL against E. coli and were comparable to our outcome. Activity against S. aureus (750 μg/mL) reported in the same publication was higher. The use of different strains and testing methods is the most probable explanation for these varied findings, but there are differences in EO origin and chemical composition that may play a role. In general, p-cymene and γ-terpene, the main components of B. persicum EO, are regarded as monoterpenes with low antimicrobial activity [26] [27]. These two compounds accounted for 66% of the oil tested in the present study. Investigations on pure γ-terpinene against E. coli, S. aureus, and P. aeruginosa revealed no activity against any of these bacterial species [26] [28]. In another study, p-cymene showed growth inhibition zones between 17–21 mm against C. albicans and between 9.6–11 mm against S. aureus, depending on the specific strains [29]. The authors concluded that none of the single compounds tested were as active as the whole EO. Therefore, the effect of an EO seems to be related to trace compounds or potential synergistic effects of various compound combinations. Using methods and conditions described by Mann et al. [25] [26] and Longbottom et al. [30], p-cymene and γ-terpinene have demonstrated activity against P. aeruginosa.

B. persicum EO was evaluated for its antifungal activity against the strawberry anthracnose, causing fungal plant pathogens Colletotrichum acutatum, Colletotrichum fragariae, and Colletotrichum gloeosporioides. Testing was performed with two concentrations of EO at 80 μg and 160 μg applications against three Colletotrichum species in the direct overlay bioautography assay ([Table 3]). At the highest tested concentration of 160 μg/spot, B. persicum EO demonstrated better activity against all three Colletotrichum species with clear zones of 9.0–10.0 mm. A recent study showed growth inhibition activity against the soilborne phytopathogenetic fungus Fusarium osysporum [31]. In another investigation, high antifungal activity of B. persicum EO against the same fungus was determined. The authors attributed this high inhibitory activity to cumin aldehyde and p-cymene [32]. In our previous antifungal studies, we demonstrated that EOs rich in non-oxygenated mono- or sesquiterpenes did not show good antifungal activity [33] [34]. Therefore, the antifungal effect of B. persicum oil might either be correlated to p-mentha-1,4-dien-7-al, p-mentha-1,3-dien-7-al, or cumin aldehyde in accordance with [32], or attributable to possible synergistic effects of one or more compounds present in the oil. Further research is needed to conduct bioassay-guided fractionation to separate and isolate the active compounds against Colletotrichum species.

Table 3 Antifungal activity of B. persicum EO against 3 Colletotrichum species using direct bioautography assays.

Tested compounds

Mean fungal growth inhibition§ (mm)±SD

C. acutatum

C. fragariae

C. gloeosporioides

80 μg/spot

160 μg/spot

80 μg/spot

160 μg/spot

80 μg/spot

160 μg/spot

B. persicum EO

5.0±0.0

9.5±0.7

5.5±0.7

10.0±0.0

5.0±0.0

9.0±0.0

Standard fungicides*

Benomyl

Diffuse

21.5±0.7

Diffuse

Captan

12.0±0.0

18.5±0.7

19.5±0.7

Cyprodinil

Diffuse

Diffuse

Diffuse

Azoxystrobin

Diffuse

24.5±1.4

Diffuse

§Mean inhibitory zones±SD; *Technical grade of internal standards. Technical grade agrochemical fungicides (without formulation) were applied 2 μL from the 2 mm concentration

Working with whole EOs is difficult because they are a complex oily matrix of chemistry, and meaningful biological conclusions will require specific bioassay methods and approaches. Direct bioauthograph with fungal pathogents should identify the number of active compounds in the EO. Purification and subsequent testing of single entity chemicals at µmolar concentrations in a dose-response format using liquid microdilution broth assays may help determine effects on spore germination and mycelial growth, and may suggest potential structure activity relationships.

Mosquito biting deterrent activity was determined against the yellow fever mosquito, Ae. aegypti. B. persicum EO showed activity greater than the solvent control (ethanol), but it was significantly less active than DEET, a standard biting deterrent, which was used as a positive control ([Fig. 1]). Toxicity of B. persicum EO was determined in mosquito larvicidal bioassays [35]. B. persicum EO showed medium to low activity against 1-d old Ae. aegypti larvae with an LD50 of 58.6 ppm. The LD50 value of permethrin, which was used as apositive control, was 0.0034 ppm. Cheng et al. [36] reported that essential oils showing LC50 values<50 ppm can be considered as very active, whereas results>100 ppm indicated an inactive EO. Making a valid scientific conclusion of results from the literature is difficult because of the variability in testing methods and conditions used by different authors. Since there was no universally accepted guideline to assess larvicidal activity, the use of positive and negative controls is necessary to compare results and make conclusions. Recently, the EO of B. persicum, collected in the Kerman region in Iran, was investigated for its larvicidal effects against Anopheles stephensi and Culex pipiens larvae. Results indicated high toxicity against adult insects with LC50 values of 27.72 and 20.61 ppm after 24 h, respectively [17]. However, we report for the first time in this paper on the mosquito biting deterrent and larvicidal activity of B. persicum EO against Ae. aegypti. Studies using larvicidal activity to drive bioassay-guided fractionation should determine the active compounds. This method will also help determine the number of active compounds in the EO.

Zoom Image
Fig. 1 Proportion of not biting values of B. persicum EO at 10 µg/cm2 against female Ae. aegypti. DEET at 4.8 µg/cm2 was used as a positive control. Ethanol was used as a solvent control.

In conclusion, it appears that the EO of B. persicum from cultivated sources from western Himalaya had antifungal activity against Colletotrichum species, and future studies need to be conducted with active components using 1D-TLC bioautography to identify active individual compounds. The active compounds could be subsequently evaluated in 96-well microdilution broth assays against a broader range of important plant pathogenic fungi. The larvicidal activity of B. persicum EO against Ae. aegypti could be considered as medium to high. Additionally, the oil exhibited good biting deterrent activity. Based on these results, Himalayan black caraway oil (B. persicum) may be a promising potential source for new naturally derived agrochemicals or biopesticides. This research needs to be further investigated to study single entity active molecules and possible synergistic effects of chemical combinations.


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Materials and Methods

Plant material and isolation procedure

B. persicum, cultivated by local farmers, was harvested from Lauhul-spiti’a cold desert area of western Himalaya, at an altitude of 3 500 m, in October/November 2009. The samples were identified by the taxonomist (Brij Lal) of the Institute of Himalayan Bioresource Technology (IHBT Palampur, India). A voucher specimen (PLP 2845) was deposited at the Herbarium-PLP of the IHBT Palampur. Black seeds were air-dried at room temperature (about 25°C) in the shade. One kilogram of seed material was hydrodistilled in a clevenger-type apparatus as described in [12], yielding light yellow colored oil (0.52% on dry wt. basis).


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Essential oil analysis

Using a Finnigan ThermoQuest TraceGC with 2 split/splitless injectors, a FID detector, and a Finnigan Automass quadrupole mass spectrometer, GC-MS-FID analyses were carried out in one instrument on a 50 m×0.25 mm×1.0 μm SE-52 fused silica column (CS Chromatographie Service) and a 60 m×0.25 mm×0.25 μm CW20M (J&W Scientific) column, respectively. Splitting of the column effluent was done with a quartz Y connector, one outlet connected to the MS interface via a short (ca. 20 cm) 0.1 mm ID fused silica capillary flow restrictor. The other outlet was attached to a 1 m×0.25 mm deactivated fused silica capillary as a transfer line to the FID.

The use of this configuration resulted in an FID and an MS chromatogram with identical chromatographic separation and nearly the same retention times, thus facilitating peak assignments in the FID chromatogram with respect to the MS. The carrier gas was helium 5.0 with a constant flow rate of 1.5 mL/min, the injector temperature was 230°C, FID detector temperature 250°C, GC-MS interface heating 250°C, ion source 150°C, EI mode at 70 eV, scan range 40–500 amu, and scan rate of 500 µs. The following temperature program was used: 60°C for 1 min isotherm, then increased at a rate of 3°C/min to 230°C. Identification of the compounds was performed as described in [10].


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Olfactory evaluation

For olfactory evaluation, one droplet of B. persicum EO was applied onto commercially available paper blotters. Each sample was examined by a panel consisting of a professional perfumer and 2 aroma chemists over 90 min to control odor progression. Odor descriptions were compared to our own database of referenced aroma compounds.


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Antimicrobial testing

The antimicrobial effects of B. persicum EO were tested against the Gram-negative bacteria E. coli (ATCC 25922), S. abony (ATCC 6017), and P. aeruginosa (ATCC 27853) and the Gram-positive bacterium S. aureus (ATCC 6538), as well as C. albicans (ATCC 10231). The strains were purchased from the National Reference Laboratory of Mycology at the National Center of Infectious and Parasitic Diseases, Sofia; Department of Microbiology and Immunology at the Medical University of Plovdiv and Clinical Laboratory Chronolab Ltd., Plovdiv. They were deposited in the Microbial Culture Collection of the Department of Biochemistry and Microbiology (University of Plovdiv, Bulgaria) and bacterial strains were stored on Nutritional Agar (NA, HiMedia Laboratories Ltd.). The fungal strain was stored on Sabouraud Dextrose Agar (SDA, HiMedia Laboratories Ltd.). Antimicrobial activity of Bunium EO was evaluated by broth microdilution tests described earlier [10]. Minimal inhibition concentration (MIC) was defined as the lowest concentration of EO that resulted in an absorbance reduction of >90% compared to the observed absorbance of control samples without EO. For positive controls, standard antibacterial antibiotic HiComb™ MIC test strips of Ciprofloxacin and Cefazolin and antifungal HiComb™ MIC test strips of Amphotericin B and Fluconazole (HiMedia Laboratories Ltd., 100% purity) were evaluated. All tests were performed in duplicate.


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Antifungal testing

Pathogen production and bioautography procedures of Stappen et al. [10] were used to evaluate antifungal activity against fungal plant pathogens. The sensitivity of each fungal species to each test compound was determined by comparing the sizes of the inhibitory zones. Bioautography experiments were performed multiple times using both dose- and non-dose-response formats. Fungicide technical grade standards benomyl (>98%), cyprodinil (>98%), azoxystrobin (>98%), and captan (>98%; Chem Service, Inc.) were used as controls at 2 mM in 2 µL of 95% ethanol. B. persicum EO was spotted with 80 and 160 μg/spot in hexane. To detect biological activity directly on the TLC plate, silica gel plates were sprayed with one of the 3 spore suspensions adjusted to a final concentration of 3.0×105 conidia/mL with liquid potato dextrose broth (PDB, Difco) and 0.1% Tween-80. Using a 50-mL chromatographic sprayer, each TLC plate with a fluorescent indicator (250 μm, silica gel GF Uniplate; Analtech, Inc.) was sprayed lightly (to a damp appearance) 3 times with the conidial suspension. Inoculated plates were then placed in a 30×13×7.5 cm moisture chamber (39°C, 100% relative humidity; Pioneer Plastics, Inc.) and incubated in a growth chamber at 24±1°C and a 12-h photoperiod under 60±5 μmols·m−2 s−1 light. The inhibition of fungal growth was measured 4 days after treatment. The sensitivity of each fungal species to each test compound was determined by comparing the size of the inhibitory zones on the TLC plate.


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Mosquito biting testing

Mosquitoes: Ae. aegypti larvae 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, Florida, using standard rearing practices [37]. For biting deterrence bioassays, pupae were maintained in the laboratory at 27±2°C and 60±10% RH in a photoperiod regimen of 12:12 h (L:D). For larvicidal bioassays, the eggs were hatched and the larvae were maintained at the above temperature.

Mosquito biting bioassays: Experiments were conducted by using a 6-celled in vitro Klun and Debboun (K&D) module bioassay system developed by Klun et al. [38] for quantitative evaluation of biting deterrent properties of candidate compounds. Briefly, the assay system consists of a 6-well reservoir with each of the 3×4 cm wells containing 6 mL of blood. As described by Ali et al. [39], a feeding solution consisting of CPDA-1 and ATP was used instead of blood. A green fluorescent tracer dye (www.blacklightworld.com) was used to determine the feeding by the females. Treatments of the EO of B. persicum were applied at 10 µg/cm2, and DEET (97%, N, N-diethyl-meta-toluamide; Sigma Aldrich) at 25 nmol/cm2 was used as a positive control, while ethanol served as a solvent control. All treatments were conducted as described in [10].


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Larvicidal bioassays

Bioassays were conducted to test the EO of B. persicum for their larvicidal activity against Ae. aegypti by using the bioassay system described by Pridgeon et al. [40]. Further methods and statistical analyses are described in [35]. DMSO was used as a solvent to prepare the treatments and was also used as a negative control. Permethrin (95.7%; Chem Service, Inc.) was used as a positive control.


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Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgements

This study was supported in part by USDA/ARS grant No. 56-6402-1-612 and the Deployed War-Fighter Protection research program grant funded by the U.S. Department of Defense through the Armed Forces Pest Management Board. We thank Ms. J. L. Robertson and Ms. R. Pace for their help in the antifungal bioassays and Dr. James J. Becnel, Mosquito and Fly Research Unit, Center for Medical, Agricultural and Veterinary Entomology (CMAVE), USDA-ARS, Gainesville, Florida, USA, for supplying Ae. aegypti eggs. Authors Vijay K. Kaul, Brij Lal, and Vikas Jaitak are thankful to the Director CSIR-IHBT Palampur for providing necessary facilities to carry out the plant survey/collection of plant materials as well as for hydrodistillation and analytical work. We would also like to thank Alden S. Estep and William Reid, USDA-ARS, CMAVE, Gainesville, Florida USA, for internal peer review of the manuscript and their suggestions.


Correspondence

Dr. Iris Stappen
Department of Pharmaceutical Chemistry
University of Vienna
Althanstrasse 14
1090 Vienna
Austria   
Phone: +43/1/4277 555 52   
Fax: +43/1/4277 9 551   


Zoom Image
Fig. 1 Proportion of not biting values of B. persicum EO at 10 µg/cm2 against female Ae. aegypti. DEET at 4.8 µg/cm2 was used as a positive control. Ethanol was used as a solvent control.