Susceptibility of pathogenic bacteria and fungi to essential oils of wildDaucus carota

The compositions of the essential oils of wild Daucus carota L. (Apiaceae) from the ripe fruits, unripe fruits, flowers, root, leaves, and stem, collected in Serbia, were analyzed by GC and GC/MS. The oils from all samples contained α-pinene (7.05–51.23%) and sabinene (2.68–36.69%) as major constituents. The other dominant compound in ripe and unripe fruits essential oil was α-muurolene (8.23–10.97%). Antibacterial and antifungal properties of these oils, against eight bacterial and eight fungal strains, were tested by a microdilution technique. The most prominent biological activity was achieved by isolated essential oils of ripe and unripe fruits of D. carota.

Keywords: Antibacterial activity; antifungal activity; Daucus carota; essential oils; microdilution method

Daucus L. (Apiaceae) includes about 60 species (1972) growing in Europe, Africa, West Asia, and also a few of them in Australia and North America ([15]). The wild ancestors of the carrot are likely to have come from Afghanistan, which remains the center of diversity of Daucus carota L., the wild carrot, sometimes called Queen Anne's lace ([12]).

D. carota has been reported to have chemical constituents belonging to polyacetylenes ([23]), essential oils ([19]), flavonoids ([11]), and phenylpropanoids ([25]). A new daucane-type sesquiterpene alcohol, trans- dauc-8-en-4β-ol, has been isolated and characterized from carrot seed oil, and also five known sesquiterpene compounds: trans-dauca-8,11-diene; dauca-5,8-diene; acora-4,9-diene; acora-4,10-diene; (E)-β-10,11-dihydro-10,11-epoxyfarnesene; and (E)-methylisoeugenol, were identified as new reported constituents in carrot seed oil ([24]).

The sesquiterpenes found in D. carota, such as carotol, β-caryophyllene, and caryophyllene oxide, exhibited fungicidal and herbicidal activity ([16]). Essential oil of D. carota seeds has been used for therapeutic purposes based on a variety of activities such as stimulant, antiseptic, tonic, diuretic, hepatic, anthelmetic, smooth muscle relaxant and some other properties ([21]). Hypotensive, cardiac and CNS depressant ([6]) properties of D. carota seed essential oil have been reported. Carrots showed protective activity for the alleviation of chloroform-induced hepatocellular injury in the mouse ([5]). Activity of D. carota acetone, ethanol, hexane, and methanol extracts exhibited significant activity against Culex annulirostris (Skuse) ([31]).

Carrot seed oil is widely used as a flavoring agent in food products and perfumery since it blends very well in all kinds of perfumes. Moreover, the oils of some varieties were proved to have antibacterial ([19]) and fungicidal activities ([3]; [10]). Daucus carota, the taxonomic source of carrot seed oil, has been divided into 12 interrelated and interhybridized subspecies ([34]). However, the material of commercial carrot oil is generally the fruits (seeds) of the root vegetable carrot of mixed cultivars of Daucus carota ssp. sativus (Hoffm.) Archang. All D. carota subspecies possess essential oils, so their composition has been examined as a potential commercial source ([22]). The chemical composition of cultivated carrot seed oil has been the subject of frequent research (Benecke et al., 1987; [8]; [26]; [22]; [24]) and the oils of wild carrot collected from many habitats worldwide have also been previously examined ([27]; [30]).

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms ([2]).

This study determined chemical composition and investigated the antimicrobial activity of essential oils isolated from the flowers, stem, leaves, root, unripe and ripe seeds of wild D. carota.

Plant material (ripe fruits, l; unripe fruits, 2; flowers, 3; root, 4; leaves, 5; stem, 6) of wild Daucus carota was collected during July-September in 2007, by Prof. Dr. Dragoljub Grubišić in Belgrade, Serbia. The material was dried at room temperature. A voucher specimen (No. MJADG07) has been deposited at the Institute for Biological Research Siniša Stanković.

Essential oils were isolated by hydrodistillation in a Clevenger-type apparatus for 3 h. The obtained essential oils were stored at +4°C until further analyses. Qualitative and quantitative analyses of the essential oils were performed using GC and GC/MS. The GC analysis of the oils was carried out on a GC HP-5890 II apparatus, equipped with split-splitless injector, attached to a HP-5 column (25 m × 0.32 mm, 0.52 μm film thickness) and fitted to FID. Carrier gas was H2 (1 mL/min). One μL of sample solution in ethanol (0.2%) was injected in split mode (1:30) at 250°C. Detector temperature was 300°C (FID), while column temperature was linearly programmed from 40–260°C, 4°C/min. As for GC/MS analysis, a Hewlet-Packard, G 1800C Series II GCD model and a HP-5MS column (30 m × 0.25 mm × 0.25 μm) were used. Mass spectra were recorded in EI regime (70 eV), in m/z range 40–400. Transfer line was heated at 260°C.

Identification of the individual essential oil components was accomplished by comparison of retention times with standard substances and by matching mass spectral data with MS libraries (NIST and Wiley 275.l) using a computer search and literature ([1]). For the purpose of quantitative analysis, area percentagess obtained by FID were used as base.

For the bioassays, five Gram(2) bacterial species: Enterobacter cloacae (clinical isolate), Salmonella typhimurium (ATCC 13311), Listeria monocytogenes (NCTC 7973), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35210) and three Gram(+) bacterial species: Staphylococcus aureus (ATCC 6538), Micrococcus flavus (ATCC 9341), Bacillus cereus (clinical isolate) and eight fungi: Fusarium sporotrichoides (ITM 946), Fulvia fulvum (TK 5318), Trichoderma viride (IAM 5061), Penicillium ochrochloron (ATCC 9112), Penicillium funiculosum (ATCC 36839), Aspergillus ochraceus (ATCC 12066), Aspergillus flavus (ATCC 9643) and Aspergillus fumigatus (ATCC 9142) were used. All of the organisms tested were from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research Siniša Stanković, Belgrade, Serbia. The micromycetes were maintained on malt agar (MA), bacteria on Mueller-Hinton agar (MH) and cultures were stored at +4°C and subcultured once a month ([7]).

In order to investigate the antimicrobial activity of the isolated essential oils, the modified microdilution technique was used ([14]; [9]). Bacterial species were cultured overnight at 37ºC in Tryptic soy broth (TSB) medium. The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The fungal and bacterial cell suspension was adjusted with sterile saline to a concentration of 1.0 3 105 units in a final volume of 100 μL per well. The inocula were stored at +4°C for further use. Dilutions of the inocula were cultured on solid MH for bacteria and solid MA for fungi to verify the absence of contamination and to check the validity of the inoculum.

Minimum inhibitory concentrations (MICs) determination was performed by a serial dilution technique using 96-well microtiter plates. The investigated essential oils were added in broth TSB medium (bacteria)/broth malt medium (fungi) with inoculum. The microplates were incubated for 48 h at 37°C for bacteria, or 72 h at 28°C for fungi, respectively. The lowest concentrations without visible growth (at the binocular microscope) were defined as MICs.

The minimum bactericidal (MBCs) and minimum fungicidal concentrations (MFCs) were determined by serial subcultivation of a 2 μL into microtiter plates containing 100 μL of broth per well and further incubation for 48 h at 37°C or 72 h at 28°C, respectively. The lowest concentration with no visible growth was defined as MBC/MFC, indicating 99.5% killing of the original inoculum.

Streptomycin was used a reference antibiotic while bifonazole and ketoconazole were used as positive controls for antifungal activity (0.1–2 mg/mL).

The results of the chemical analysis of wild Daucus carota essential oils from different plant parts are presented in Table 1. A total of 48 compounds were identified in D. carota essential oil of ripe fruits, 60 in unripe fruits, 48 in flowers, 50 in root, 60 in leaves and 63 compounds were detected in stem essential oil. Ripe fruit essential oil was opulent in sabinene (27.16%), α-pinene (21.30%), α-muurolene (8.23%), β-caryophyllene (6.82%) and α-ylangene (5.21%). The most abundant constituent in unripe fruit essential oil was α-muurolene (10.97%) followed by sabinene (10.67), caryophyllene oxide (7.70%), α-amorphene (7.57%), α-pinene (7.05%), carotol (6.15%), dimenone (5.28%), and α-ylangene (4.88%). Essential oil from flowers was luxuriant in the content of bicyclic monoterpene α-pinene (51.23%), ensued by limonene (9.59%), sabinene (8.62%) and β-myrcene (7.18%). Sabinene (36.39%), α-pinene (24.56%), limonene (6.53%), and β-pinene (5.39%) were the main immanent molecules in the root essential oil. Leaf essential oil consisted most of α-pinene (30.83%), limonene (8.60%), trans-phytol (6.97%), β-myrcene (5.60%), and germacrene D (4.56%). Although most of the constituents were identified in the stem essential oil, they consisted of just 68.75% of the total oil contents, represented by α-pinene (18.53%), α-bisabolol (6.02%), and limonene (5.74%).

Table 1.  Chemical composition of Daucus carota essential oils (EO).

4 *KI: Kovats index on DB-5 column; 1 ripe fruits EO 2 unripe fruits EO; 3 flowers EO; 4 root EO; 5 leaves EO; 6 stem.

Most of the D. carota oils from different regions belong to the sabinene chemotype. All investigated samples of the oils with the large amount of sabinene contained marked quantities of α-pinene ([22]). The essential oil of wild Daucus carota L. from Corsica consisted chiefly of phenylpropanoids (45.9%) and monoterpene hydrocarbons (38.6%), with (E)-methylisoeugenol (33.0%), α-pinene (24.9%) and elemicin (11.4%) as major components ([13]). Forty components were identified in carrot umbel oils. The oils of D. carota L. ssp. sativus were dominated by monoterpene hydrocarbons (66–85%) represented mainly by α-pinene (40–46%) and myrcene (12–24%). [32] showed that oils obtained from different parts of wild carrot plant consist mainly of monoterpene hydrocarbons (71.9–83.8%); they have not confirmed the presence of carotol, daucol and daucene - the sesquiterpenes that are specific for a chemical composition of the oils obtained from the cultivated breeds of carrot (D. carota ssp. sativus) ([4]; [27]).

In this paper, we confirmed the presence of carotol (6.15%) in unripe fruits, 1.87% in stem, 1.70% in ripe fruits, 0.86% in leaves and 0.72% in root sample. We did not notice the presence of carotol in flowers. Daucene was detected only in leaves (0.39%), while the presence of daucol was not noticed in our samples. The previous investigation showed that the most abundant sesquiterpene constituents in carrot oil are caryrophyllene (4.6–13.2%) and carotol (1.2–6.1%) ([20]). About 50 monoterpenoids, sesquiterpenoids, and propenylbenzenes were found in leaf essential oil of two carrot varieties. The essential oil compositions, especially percentage of sabinene and limonene, were significantly different between the varieties ([17]).

The antibacterial effect of D. carota oils is presented in Table 2. The most prominent activity in the microdilution test was against Bacillus cereus (MIC of 5.0–50.0 μL/mL; MBC of 10.0–50.0 μL/mL) for all oils tested. In particular, ripe (MIC 5.0–25.0 μL/mL; MBC 10.0–25.0 μL/mL) and unripe fruits (MIC and MBC 10.0–25.0 μL/mL) essential oils exhibited the strongest antibacterial effect in correlation to the other isolated oils. Root essential oil showed inhibition at 5.0–150.0 μL/mL and bactericidal effect was achieved at 10.0–200.0 μL/mL. Essential oil from stem showed MIC at 25.0–250.0 μL/mL and MBC at 50.0–300.0 μL/mL. Leaf oil exhibited MIC and MBC at 25.0–350.0 μL/mL, while flower oil demonstrated the lowest antibacterial activity with MIC and MBC at 50.0–400.0 μL/mL. It is evident that the activity of oils decline in specific order: ripe fruits > unripe fruits > root > stem > leaves > flowers essential oils. All the essential oils were more effective than the commercial drug streptomycin against Pseudomonas aeruginosa and Escherichia coli.

Table 2.  Antibacterial activity of Daucus carota essential oils, μL/mL (EO).

5 #Minimum inhibitory concentration; ##Minimum bactericidal concentration; 1, ripe fruits EO; 2, unripe fruits EO; 3, flowers EO; 4, root EO; 5, leaves EO; 6, stem EO.

Fungi were more sensitive then bacteria to the effect of the essential oils. Essential oils from all parts of D. carota exhibited a fungistatic and fungicidal effect. The most sensitive species was Fulvia fulvum with MIC of 2.0–100.0 μL/mL and MFC of 5.0–100.0 μL/mL. Trichoderma viride (MIC of 25.0–100.0 μL/mL and MFC of 25.0–150.0 μL/mL) and Aspergillus ochraceus (MIC of 10.0–150.0 μL/mL and MFC of 25.0–150.0 μL/mL) were the most resistant species. As in the case of the antibacterial effect, the essential oil of unripe fruits manifested the strongest antifungal potential followed by the ripe fruit oil, root oil, stem oil, leaf oil and flower oil. Investigated essential oils were more efficient than the commercial drug bifonazole and much more active than ketoconazol (Table 3).

Table 3.  Antifungal activity of Daucus carota essential oils, μl/ml (EO).

6 #Minimum inhibitory concentration, ##Minimum fungicidal concentration; 1 ripe fruits EO, 2 unripe fruits EO, 3 flowers EO, 4 root EO, 5 leaves EO, 6 stem EO.

The oils of D. carota L. ssp. sativa, with a high percentage of neryl acetate (25.0%) showed a weak antibacterial effect as compared with the oil of D. carota L. ssp. carota ([18]). The effects of the essential oils of carrot on the proliferation of 21 pathogenic strains were examined in vitro. In preliminary screening by the disc diffusion method, 15 and 18 strains of the pathogens were susceptible to carrot oil. The minimum inhibitory concentration (MIC) value of the oil ranged from less than 0.97 μL/mL to more than 125 μL/mL towards the tested strains ([28]). The essential oil of wild D. carota L. obtained from aerial parts at the end of the flowering stage was reported to have antimicrobial potential against the human enteropathogen Campylobacter jejuni. (E)-Methylisoeugenol and elemicin were the molecules found responsible for the antibacterial activity ([29]).

The results of our investigation indicated that oils from unripe and ripe fruit of wild Daucus carota were the most promising with a wide activity spectrum against all pathogens tested. This highly selective activity of essential oils investigated towards pathogenic bacterial and fungal strains provides an eminent evidence of their in vitro efficacy. As a conclusion, results revealed a good antimicrobial potential of the essential oils isolated from different part of wild carrot from Serbia.

The authors are grateful to the Ministry of Science of Serbia for financial support (Grants No. 143031 and 143041).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.