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Article

Biological Activities of the Essential Oil from Erigeron floribundus

1
School of Pharmacy, University of Camerino, Camerino 62032, Italy
2
Department of Clinical Sciences, Section of Biochemistry, Polytechnic University of Marche, Ancona 60131, Italy
3
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå 90187, Sweden
4
Laboratory of Medicinal Plant Biochemistry, Food Science and Nutrition, Department of Biochemistry, Faculty of Sciences, University of Dschang, PO Box 67, Dschang, Cameroon
5
School of Biosciences and Veterinary Medicine, University of Camerino, Camerino 62032, Italy
*
Authors to whom correspondence should be addressed.
Molecules 2016, 21(8), 1065; https://doi.org/10.3390/molecules21081065
Received: 12 July 2016 / Revised: 10 August 2016 / Accepted: 11 August 2016 / Published: 13 August 2016
(This article belongs to the Section Natural Products Chemistry)

Abstract

:
Erigeron floribundus (Asteraceae) is an herbaceous plant widely used in Cameroonian traditional medicine to treat various diseases of microbial and non-microbial origin. In the present study, we evaluated the in vitro biological activities displayed by the essential oil obtained from the aerial parts of E. floribundus, namely the antioxidant, antimicrobial and antiproliferative activities. Moreover, we investigated the inhibitory effects of E. floribundus essential oil on nicotinate mononucleotide adenylyltransferase (NadD), a promising new target for developing novel antibiotics, and Trypanosoma brucei, the protozoan parasite responsible for Human African trypanosomiasis. The essential oil composition was dominated by spathulenol (12.2%), caryophyllene oxide (12.4%) and limonene (8.8%). The E. floribundus oil showed a good activity against Staphylococcus aureus (inhibition zone diameter, IZD of 14 mm, minimum inhibitory concentration, MIC of 512 µg/mL). Interestingly, it inhibited the NadD enzyme from S. aureus (IC50 of 98 µg/mL), with no effects on mammalian orthologue enzymes. In addition, T. brucei proliferation was inhibited with IC50 values of 33.5 µg/mL with the essential oil and 5.6 µg/mL with the active component limonene. The essential oil exhibited strong cytotoxicity on HCT 116 colon carcinoma cells with an IC50 value of 14.89 µg/mL, and remarkable ferric reducing antioxidant power (tocopherol-equivalent antioxidant capacity, TEAC = 411.9 μmol·TE/g).

Graphical Abstract

1. Introduction

Erigeron floribundus (Kunth) Sch. Bip. (Asteraceae) is an herbaceous plant, 1.5 m in height, with pubescent, lanceolate leaves and flowers in yellowish panicles. In Cameroon, it is commonly found as a weed along roadsides, and it is widely used in folk medicine to treat angina, female infertility, AIDS, dental pain, headache and various diseases of microbial and non-microbial origin [1,2,3,4,5,6]. The leaf aqueous extract was proven to possess analgesic, anti-inflammatory and immunomodulatory activity [1,2], the dichloromethane extract inhibition against dermatophytes [7], and the leaf ethanolic and pentane extract antiplasmodial activity [8]. Despite the interest in its bioactivity, the plant has received little phytochemical investigation. With regard to the essential oil, a previous work performed on Cameroonian plants showed sesquiterpene hydrocarbons as the major essential oil constituents [9].
Given the wide use of the plant in traditional medicine, we evaluated the in vitro biological activities displayed by the essential oil obtained from the aerial parts of E. floribundus, namely the antioxidant, antiproliferative and antimicrobial activities that were investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT), and agar disc diffusion and microdilution methods, respectively.
Because essential oils represent an interesting alternative approach against the occurrence of drug resistance in many infectious bacterial pathogens [10] and fighting protozoan parasites [11], we completed the work by investigating the activity of E. floribundus essential oil on nicotinate mononucleotide adenylyltransferase (NadD) and Trypanosoma brucei. Being natural in origin, essential oils and their components are believed to be environmentally favourable and user-friendly [12]. Moreover, using innovative technologies, such as nanoencapsulation, many limits of their application in medicine (mainly low water solubility and stability, high volatility and side effects) could be overcome [13].
NadD occupies a central position in bacterial NAD+ biosynthesis and is required for both de novo and salvage routes to generate NAD+ [14,15,16]. NadD has been recognized as a promising new target for developing novel antibiotics due to its crucial role in synthesizing NAD+ [17,18,19,20]. Another attractive aspect of targeting NadD is that it is highly conserved in the overwhelming majority of bacterial species including most pathogens. Therefore, drugs developed based on the inhibition of NadD have the potential of possessing wide-spectrum antibacterial activity.
T. brucei is a protozoan parasite responsible for Human African trypanosomiasis (HAT), a neglected disease also known as African sleeping sickness. HAT threatens primarily rural populations and is fatal unless treated. To date, there are a few drugs approved, although none of them are satisfactory, due to treatment failures and toxicity, and the parenteral administration that is inappropriate in settings with poor medical infrastructure. Therefore, there is an urgent need to improve HAT treatment by enhancing the oral administration and the discovery and development of cost-effective new drugs. Drug discovery efforts are nowadays directed towards natural products and medicinal plants represent a validated source for discovery of new lead compounds and standardized herbal medicines against trypanosomiasis [21].

2. Results

2.1. Essential Oil Analysis

The composition of the essential oil hydrodistilled from the aerial parts of E. floribundus is reported in Table 1. GC-MS analysis revealed that the essential oil had a complex chemical profile. Separation of volatile components was accomplished by using both polar and apolar columns (HP-5 MS and DB-WAX, respectively). A total of eighty-five constituents were identified accounting for 78.6% of the total composition. The main fraction of the essential oil was given by sesquiterpenes (60.4%), which were represented by oxygenated compounds (38.5%) and hydrocarbons (21.9%). Main representatives of these groups were caryophyllene oxide (12.4%) and spathulenol (12.2%), and (E)-β-farnesene (5.5%) and (E)-caryophyllene (4.2%), respectively. Monoterpenes constituted a minor part of the oil (13.0%), with hydrocarbons (11.5%) as the predominant components. Among them, limonene was the most representative (8.8%).

2.2. Cytotoxicity Assessment

General cell toxic effects of E. floribundus essential oil were evaluated using the MTT assay against the following human tumour cell lines: A375 human malignant melanoma cell line, MDA–MB 231 human breast adenocarcinoma cell line, and the HCT116 human colon carcinoma cell line. All cell lines were submitted to increasing concentrations of essential oil for 72 h. The results, collected in Table 2, show that essential oil exhibited a significant growth inhibition against the examined human cancer cells and induced a concentration-dependent inhibitory effect in the dilutions ranging from 0.78–200 μg/mL. The IC50 values of the essential oil were 14.9, 20.8, and 22.5 μg/mL on HCT116, MDA–MB 231, and A375 cell lines, respectively.

2.3. Antimicrobial Activity

The essential oil of E. floribundus showed a good antimicrobial activity against Staphylococcus aureus using both disc diffusion and microdilution methods. The inhibition zone diameter (IZD) of almost 15 mm was indicative of a good activity. The corresponding minimum inhibitory concentration (MIC) was equal to 512 µg/mL (Table 3). S. aureus is Gram-positive as well as Enterococcus faecalis. The latter was also included in the panel of tested bacteria but resulted in being less susceptible to the essential oil than S. aureus (IZD ≅ 9 mm) as confirmed by the much higher MIC value (4096 µg/mL). None of the Gram-negative bacterial species, namely E. coli and P. aeruginosa, were inhibited as assessed by the disc diffusion test. Therefore, antimicrobial susceptibility by the microdilution method was not investigated. In contrast, the yeast C. albicans growth was inhibited by the essential oil (IZD = 9 mm), which showed a minimum inhibitory concentration of 512 µg/mL by the microdilution method.
In view of the appreciable activity against S. aureus, an additional series of S. aureus clinical isolates from catheter-associated infections was selected to confirm the observation and to prove the extendibility of the antimicrobial activity of the essential oil beyond the reference strain ATCC 29213. Four isolates were selected based on their proven resistance towards oxacillin, the congener of methicillin [25]. Hence, the four isolates were MRSA (methicillin-resistant Staphylococcus aureus). The MICs of essential oil against the clinical isolates were substantially superimposable to the one obtained against the reference strain, differing on average by one dilution (MIC ≅ 1024 µg/mL).

2.4. NadD Inhibition Analysis

In our effort to explore natural compounds against nicotinate mononucleotide adenylyltransferase of the NadD family [26,27], an essential enzyme of NAD biosynthesis in most bacteria, we evaluated the in vitro inhibition of E. floribundus essential oil against NadD from S. aureus. A preliminary single point inhibition test at the concentration of 80 μg/mL indicated a ~60% inhibition with no effect on the ubiquitous mammalian isoform NMNAT1 [28]. Motivated by these results and by an observed broad-spectrum antimicrobial activity based on the diffusion IZD and MIC methods (Table 3), we performed a dose-response inhibition using an oil concentration range of 5–160 μg/mL. The IC50 value of the essential oil for SaNadD was 98 ± 11 μg/mL.

2.5. Antioxidant Activity

Despite the interest on the bioactivity of E. floribundus based on its uses in Cameroonian traditional medicine, we evaluated for the first time the antioxidant properties of the essential oil. Antioxidant activity is a complex process usually occurring through several mechanisms and its evaluation is often carried out by more than one test method [29]. In this study, three antioxidant assays, namely DPPH free radical scavenging activity, ABTS radical cation scavenging activity, and ferric reducing antioxidant power (FRAP) were applied to evaluate the antioxidant properties of E. floribundus essential oil. As reported in Table 4, the antioxidant activity determined by DPPH radical scavenging ability showed a value of 16.9 ± 0.1 μmol·(Trolox equivalents, TE)/g, with an IC50 value about 250 times higher than that of the positive control Trolox. Higher antioxidant activity was detected in the ABTS radical cation scavenging activity assay, with an average TE value of 85.5 ± 3.6 μmol·TE/g, and an IC50 value about 46 times higher than that of Trolox. It is noteworthy that the essential oil appeared to act as a good reducing agent (FRAP activity, TEAC = 411.9 ± 2.40 μmol·TE/g).

2.6. Inhibition of T. brucei Proliferation

A test of the E. floribundus oil against T. brucei proliferation showed that it was active at a much lower concentration than the Balb/3T3 mouse fibroblasts used as a control (Table 5). When analyzing some of the components in the oil, we found that limonene had a six times higher activity against T. brucei than the oil itself (IC50 = 5.6 µg/mL) indicating that this is the major antitrypanosomal agent in E. floribundus oil. A calculation based on that limonene represents 8.8% of the oil and gives six times higher inhibition than the oil itself, which indicates that about half of the antitrypanosomal activity in the extract comes from limonene.

3. Discussion

Only a few papers dealing with the chemical composition of E. floribundus essential oil are available in literature. From a comparison of our data with those of Kuiate et al. [9], we notice significant differences mainly from a quantitative point of view. In that study, the sesquiterpene hydrocarbons (39.9%–74.8%) were revealed as predominant over oxygenated compounds (7.0%–13.8%). The major representatives of these groups were the same but they occurred in different amounts, i.e., (E)-caryophyllene (8.5%–20.1%), (E)-β-farnesene (14.6%–24.1%), caryophyllene oxide (0.8%–5.4%) and spathulenol (1.0%–4.1%). Similarly, therein the monoterpenes hydrocarbons constituted a small fraction (5.2%–17.3%), with limonene as the most abundant compound (2.5%–11.4%). In addition, the presence of polyacetylenic constituents such as (E)-2-lachnophyllum ester (3.4%–26.8%) was reported. We assume that the above differences may depend on chemical polymorphism of the species and different periods of collection.
This work reports on the cytotoxic activity of E. floribundus essential oil. A compound or few compounds do not emerge from the composition of essential oil, which can be responsible for the cytotoxic activity on human tumor cell lines. Caryophyllene oxide provided evidence of potent cytotoxic activity against HepG2, AGS, HeLa, SNU-1, and SNU-16 cells, with IC50 values of 3.95, 12.6, 13.55, 16.79, and 27.39 μM, respectively [30]. Spathulenol was reported weakly active on human epidermoid carcinoma (KB) and inactive on human breast cancer (BC) and human small cell lung cancer (NCIH187) cell lines [31]. In addition, (E)-caryophyllene showed weak activity on the cell lines tested, with IC50 values ranging from 43.27 to 67.14 µg/mL for MDA–MB 231 and A375, respectively [32]. Our previous study has demonstrated that limonene showed antiproliferative activity on the same cell lines as tested above with IC50 values ranging from 18.4 to 124.0 µg/mL, respectively [33]. Limonene also induces apoptosis in LS174T colon cancer cells and in a lymphoma cell line (35 µg/mL, IC50) [34,35]. Moreover, it shows antitumor activity on lung adenocarcinoma A549 (0.098 µL/mL, IC50) and hepatocarcinoma HepG2 cells (0.150 µL/mL, IC50) [36]. To our knowledge, data reporting the cytotoxic activity of (E)-β-farnesene are missing. However, the concentrations of caryophyllene oxide (12.4%), spathulenol (12.2%), limonene (8.8%), (E)-β-farnesene (5.5%) and (E)-caryophyllene (4.2%) cannot fully explain the cytotoxic activity of E. floribundus essential oil, which means that some other minor compounds contributed to the activity of the essential oil
The antimicrobial activity investigation revealed that the essential oil is mainly active against the Gram-positive bacteria. A special emphasis might be put on the results obtained with S. aureus. Both the reference strain and the methicillin-resistant clinical isolates from invasive infections were susceptible to the essential oil. Given the importance of S. aureus in human medical microbiology and food microbiology, the result is an important outcome. To our knowledge, this is the first study reporting on the antibacterial activity of the essential oil from E. floribundus. On the contrary, one single previous study investigated its antifungal activity [9]. Kuiate and coll. obtained a MIC equal to about 2 mg/mL against C. albicans, while in the present investigation the essential oil was inhibiting the yeast growth at 0.512 mg/mL. Considering the differences in composition outlined above, it can be possible to speculate on the putative constituents contributing to the anti-Candida activity. Candidates might be spathulenol and caryophyllene oxide. The hypothesis on the first is not sustained by the literature where spathulenol is described solely as possessing anti-inflammatory properties. Caryophyllene oxide was instead already investigated for its antifungal properties and is approved by the FDA as a food and cosmetic preservative [37].
On the bacterial side, a hypothesis may be done with hexadecanoic acid: its concentration in the oil (2.5%) could be compatible with its reported anti-staphylococcal activity [38].
Of note, the in vitro inhibition of NadD focusing on isolating individual components of the above oil mixtures that retain both such activities.
From radical scavenging activity assays (i.e., DPPH and ABTS, Table 4), we found that the E. floribundus essential oil displayed a weak activity. The chemical profile of the oil, with spathulenol (12.2%), caryophyllene oxide (12.4%) and limonene (8.8%) as the major components, may partially justify the low inhibition on these radicals [39,40,41]. On the other hand, the high value of reducing power indicated that the essential oil components are able to act as electron donors and reduce the oxidized intermediate of lipid peroxidation so that they can act as primary and secondary antioxidants. Further studies are required to confirm the potential of this oil as reducing agent.
To the best of our knowledge, this is the first report on trypanocidal activity of the essential oil of E. floribundus, which further confirms the medical value of the plant. With an IC50 value of 5.6 µg/mL the lipophilic hydrocarbon limonene showed a quite remarkable trypanocidal activity against T. brucei. Additionally, limonene did not show significant toxicity in Balb/3T3 mouse fibroblasts (IC50 > 100 µg/mL) and exhibited a favourable selectivity index (SI > 17.85). Its trypanocidal activity could be explained by the high degree of unsaturation (two double bonds) in its chemical structure. In addition, its exocyclic methylene group can easily react with SH groups in proteins (e.g., trypanothione synthase), thus enhancing its bioactivity by exposing the parasite to oxidative damage [42]. However, further investigations are required in order to clarify the mechanism of action of essential oil and limonene itself and to evaluate their therapeutic values for trypanosomiasis infections.

4. Materials and Methods

4.1. Plant Materials

Aerial parts of E. floribundus were harvested in Dschang, West Province of Cameroon (1450 m a.s.l.) in February 2013. The plant was identified at the Cameroon National Herbarium (Yaoundé), where a voucher specimen was deposited (5619SRF/Cam). Plant material was dried at room temperature in the shade for one week before undergoing total extraction.

4.2. Hydrodistillation

The dry aerial parts (500 g) were reduced into small pieces, then subjected to hydrodistillation in a Clevenger-type apparatus for 4 h using 6 L of deionized water. The essential oil yield (0.2%, n = 3) was estimated on a dry-weight basis (w/w). Once obtained, the oil was dried (Na2SO4), transferred into an amber glass flask, and kept at −20 °C before chemical analysis and biological experiments.

4.3. Chemicals

For identification of volatiles the following analytical standards were purchased from Sigma-Aldrich (Milan, Italy): α-pinene, β-pinene, p-cymene, limonene, γ-terpinene, linalool, trans-pinocarveol, terpinen-4-ol, α-terpineol, myrtenol, nerol, carvone, geraniol, geranial, eugenol, (E)-caryophyllene, α-humulene, (E)-β-farnesene, (E)-β-ionone, (E)-nerolidol, caryophyllene oxide, n-pentacosane, n-heptacosane; (E)-Phytol was previously isolated from Onosma echioides [43]. For retention-index (RI) determination, a mixture of hydrocarbons, ranging from octane (C8) to triacontane (C30) (Supelco, Bellefonte, PA, USA) was used and run under the experimental conditions reported below. All compounds were of analytical standard grade. Analytical-grade hexane solvent was purchased from Carlo Erba (Milan, Italy); it was successively distilled through a Vigreux column before use.

4.4. Chemical Analysis of Essential Oil

GC-MS Analysis was performed on an Agilent 6890N gas chromatograph (Santa Clara, CA, USA) coupled to a 5973N mass spectrometer using two different capillary columns: a HP-5 MS column (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1-mm film thickness; J & W Scientific (Folsom, CA, USA), and a DB-WAX column (polyethylene glycol; 30 m, 0.25 mm i.d., 0.25-mm film thickness; J & W Scientific,). The oven temperature program was the following: 5 min at 60 °C, subsequently 4 °C/min up to 220 °C, then 11 °C/min up to 280 °C, held for 15 min, for a total run of 65 min. Injector and detector temperatures were 280 °C. He was used as the carrier gas, at a flow rate of 1 mL/min. Split ratio, 1:50; acquisition mass range, m/z 29–400. All mass spectra were acquired in electron–impact (EI) mode with an ionization voltage of 70 eV. Oil samples were diluted to 1:100 in n-hexane, and the volume injected was 2 µL. Whenever possible, the essential oil constituents were identified by co-injection with authentic standards. Otherwise, the peak assignment was carried out according to the recommendations of the International Organization of the Flavour Industry (http://www.iofi.org/), i.e., by the interactive combination of chromatographic linear retention indices that were consistent with those reported in the literature [22,23,24] for apolar and polar stationary phases, and MS data consisting in computer matching with the WILEY275, NIST 08, ADAMS, FFNSC2 and home-made (based on the analysis of reference oils and commercially available standards) libraries. Quantification of essential oil components was achieved by peak-area internal normalisation without using correction factors.

4.5. Antimicrobial Activity

The essential oil of E. floribundus was tested against a panel of reference bacterial and yeast species including Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Candida albicans ATCC 24433. Extension of the investigation on S. aureus was accomplished using four additional S. aureus clinical isolates (Table 3) that were resistant to methicillin and maintained in the collection of the Microbiology Unit of the School of Pharmacy at the University of Camerino [25]. Bacterial strains were cultured overnight at 37 °C in blood agar plates. C. albicans was grown in Sabouraud Dextrose Agar. Tests by the paper disc diffusion method were performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines [44]. Briefly, a suspension of the tested microorganism (1–2 × 108 cells per mL in saline-106 per mL for Candida) was spread on the solid media plates using a sterile cotton swab. Sterile paper discs (6 mm in diameter) were placed on the surface of inoculated plates and spotted with 10 µL of either the essential oil or 1 mg of extract dissolved in DMSO. The plates were incubated 24 h at 35 ± 1 °C (48 h for C. albicans). The diameters of zone inhibition (including the 6 mm disc) were measured with a gauge. A reading of more than 6 mm indicated growth inhibition. No zone inhibition was observed using DMSO alone. Ciprofloxacin (5 μg disc) and Nystatin (100 Units disc) were used as reference antimicrobials against bacteria and fungi, respectively. Each test was repeated at least twice. Antibiotic susceptibility testing by the microdilution method was performed following the international guidelines [44]. Briefly, two-fold serial dilutions of each mixture in Cation Adjusted Mueller Hinton Broth for bacteria and Roswell Park Memorial Institute (RPMI) 1640 for Candida were set in 96-well plates starting from 2048 mg/L. An equal volume of the microbial inoculum (106 cfu/mL), obtained by direct colony suspension of an overnight culture, was added to each well of the microtiter plate containing 0.1 mL of the serially diluted test oil/extract. After incubation for 18–24 h at 35 °C, in normal atmosphere, Minimum Inhibitory Concentrations (MICs) were defined as the lowest concentration of compound able to inhibit the growth of the microorganisms. All tests were done in triplicate.

4.6. Antioxidant Activity

The antioxidant activity of E. floribundus essential oil was measured by using different radical generating systems (DPPH, ABTS and FRAP assay) to assess the free radical scavenging and reducing properties of the oil [45]. All the experiments were conducted in a 96-well microplate assay according to the method of Srinivasan et al. [46], for DPPH assay and following previously-described protocols by Re et al. [47], for measuring the total radical scavenging capacity on ABTS radical. The ferric reducing antioxidant power (FRAP assay) was carried out according to the procedure described by Müller et al. [48], by monitoring the reduction of Fe3+-tripyridyl triazine (TPTZ) to blue-coloured Fe2+-TPTZ. The ability of E. floribundus essential oil to scavenge the different radicals in all three assays was compared to Trolox used as standard. Results as tocopherol-equivalent antioxidant capacity (TEAC) were expressed in μM Trolox equivalents (TE)/g of essential oil.

4.7. Cytotoxic Activity on Tumor Cells

A375 (human malignant melanoma cells) and MDA–MB 231 cells (human breast adenocarcinoma cells) were cultured in Dulbecco′s Modified Eagle′s Medium (DMEM) with 2 mM l-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS). HCT116 cells (human colon carcinoma cells), were cultured in RPMI1640 medium with 2 mM l-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, and supplemented with 10% HI-FBS. Cells were cultured in a humidified atmosphere at 37 °C in presence of 5% CO2. The MTT assay was used as a relative measure of cell viability. Cell-viability assays were carried out as described [49]. Briefly, cells were seeded at the density of 2 × 104 cells/mL. After 24 h, samples were exposed to different concentrations of essential oil (0.78–200 μg/mL). Cells were incubated for 72 h in a humidified atmosphere of 5% CO2 at 37 °C. Cisplatin was used as the positive control (0.05–20 µg/mL). At the end of incubation, each well received 10 µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) (5 mg/mL in phosphate-buffered saline, PBS) and the plates were incubated for 4 h at 37 °C. The extent of MTT reduction was measured spectrophotometrically at 540 nm using a Titertek Multiscan microElisa (Labsystems, Helsinki, Finland. Experiments were conducted in triplicate. Cytotoxicity is expressed as the concentration of compound inhibiting cell growth by 50% (IC50). The IC50 values were determined with the GraphPad Prism 4 computer program (GraphPad Software, San Diego, CA, USA).

4.8. Enzyme Inhibition Assay

Recombinant S. aureus NadD, available in our laboratory, was used as a representative bacterial target of the NadD enzyme family. Previous reports, indeed, have proven that this target enzyme is highly conserved in bacteria [17] and small molecule inhibitors may be simultaneously active on NadDs from distant species such as S. aureus and M. tuberculosis [50]. Murine NMNAT1 was chosen as the functionally equivalent mammalian enzyme [51].
The enzyme inhibition assay setup for the bacterial enzyme and the mammalian counter target is described in detail in Vitali et al. [27]. Briefly, enzyme rates and inhibition were measured by an established endpoint assay based on the detection at 620 nm of the phosphate byproduct formed [17]. A continuous assay based on detection at 340 nm of the reduced form of nicotinamide adenin dinucleotide (NADH) formed was used as an alternate assay [52].

4.9. T. brucei and Mammalian Cell Culture

T. brucei BSFs (TC221) were cultured at 37 °C with 5% CO2 in HMI-9 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher Scientific [Gibco], Waltham, MA, USA). Mouse BALB/3T3 fibroblast (ATCC no CCL-163) were cultivated at 37 °C and 5% CO2 in Dulbecco′s modified Eagle′s medium (Sigma-Aldrich), supplemented with 10% (v/v) heat-inactivated fetal bovine serum, glutamine (0.584 g/L) and 10 mL/L 100× penicillin-streptomycin (Thermo Fisher Scientific [Gibco]) [53].

4.10. Growth Inhibition Assay on T. brucei and Balb/3T3 Cells

The essential oil was dissolved in dimethyl sulfoxide (DMSO). It was serially diluted with growth medium to concentrations ranging from 2 × 10−5 to 200 µg/mL in 96-well microtiter plates (100 µL/well). Subsequently, 100 µL of T. brucei or mammalian cell culture was added to each well (20,000 cells/well). After 48 h incubation, the plates were treated for 24 h with 20 µL of resazurine (Sigma-Aldrich). They were subsequently quantified by fluorescence (540 nm excitation and 590 nm emission) using an Infinite M200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The IC50 values were calculated by fitting the data to a log inhibitor vs. response curve (variable slope, four parameters) using the GraphPad Prism version 5.04 software.

5. Conclusions

The present work provides new insights into the phytochemical and biological properties of E. floribundus. The essential oil of this plant revealed a good potential as bacterial NadD inhibitor, thus being a promising candidate for antibiotic formulations. The exhibited trypanocidal activity, on one hand, confirmed partially the traditional use in the treatment of protozoal diseases, and on the other hand, makes it a potential candidate for standardized herbal medicines against trypanosomiasis that are highly requested in developing countries with poor medical infrastructure and difficult access to conventional medical treatments.

Acknowledgments

The authors would like to thank the University of Camerino (FAR 2014/15, Fondo di Ateneo per la Ricerca, FPI000044) for financial support. Anders Hofer also thanks the Swedish Research Council (2012-1932) and the Kempe Foundation.

Author Contributions

The contributions of the respective authors are as follows: P.C. Biapa Nya collected the plant material; F. Maggi performed hydrodistillation and supervised the GC analyses, as well as interpreted the data to prepare the manuscript; R. Petrelli and L. Cappellacci conceived the research and edited and revised the manuscript; D. Petrelli and L.A. Vitali performed the antimicrobial experiments; L. Sorci and G. Orsomando carried out experiments on enzymes; F. Ranjbarian and A. Hofer conducted the tests for antitrypanosomal activity; G. Lupidi executed the antioxidant assays; L. Quassinti and M. Bramucci evaluated the cytotoxic activity on tumor cells. All authors read and approved the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the essential oil from Erigeron floribundus are available from the authors.
Table 1. Composition of the essential oil hydrodistilled from the aerial parts of Erigeron floribundus.
Table 1. Composition of the essential oil hydrodistilled from the aerial parts of Erigeron floribundus.
N.Component 1RI Calc. 2RI Lit. 3% 4ID 5
HP-5MSDBWAXApolarPolar
1butyl methyl ketone78010887861088tr 6RI, MS
2hexanal792102480110240.1RI, MS
3(3E)-hexenol844137184413710.2RI, MS
4n-hexanol858136386313630.1RI, MS
51-(2-methyl-2-cyclopenten-1-yl)-ethanone871 trMS
6α-pinene921102693210260.2Std
7sabinene96011259691125trRI, MS
8β-pinene962111297411122.1Std
9myrcene983116798811650.1Std
102-pentyl furan983123198412310.1RI, MS
11p-cymene1017127110201272trStd
12limonene10201199102411998.8Std
13(E)-β-ocimene10421255104412550.1RI, MS
14γ-terpinene1051125310541255trStd
15terpinolene1080128310861282trRI, MS
16linalool10961555109515560.1Std
17n-nonanal1102139311001395trRI, MS
18(E)-4,8-dimethyl-1,3,7-nonatriene1112 trMS
19trans-p-mentha-2,8-dien-1-ol1114 1119 trRI, MS
20trans-pinocarveol11291656113516570.1Std
21nerol oxide1150 1154 trRI, MS
22pinocarvone11531563116015650.1RI, MS
23lavandulol11631686117016860.1RI, MS
24terpinen-4-ol11691600117416000.1Std
25trans-isocarveol118018381175 trRI, MS
26α-terpineol11821688118616900.2Std
27myrtenal11881648119516480.1RI, MS
28myrtenol11881794119417940.1Std
29β-cyclocitral1212 1217 0.1RI, MS
30nerol12231840122718380.4Std
31carvone12361744123917450.1Std
32geraniol1253185012491852trStd
33perilla aldehyde1265178312651784trRI, MS
34geranial1268173012641730trStd
35perilla alcohol12912028129020290.1RI, MS
36silphiperfol-5-ene131014211326 0.3RI, MS
37α-cubebene133814571345 0.1RI, MS
38eugenol13512164135621670.1Std
39α-copaene13621488137414880.7RI, MS
40modheph-2-ene136315161377 0.7RI, MS
41α-isocomene137015291377 0.5RI, MS
42β-cubebene1378 1379 0.2RI, MS
43β-elemene1380 1389 0.2RI, MS
44β-isocomene1387 1407 0.4RI, MS
45(Z)-caryophyllene139215701408 2.3RI, MS
46(E)-caryophyllene14031590141715854.2Std
47β-copaene141415351430 0.4RI, MS
48β-gurjunene1422 1431 0.1RI, MS
49α-trans-bergamotene14251583143215862.1RI, MS
50α-humulene14371663145216651.0Std
51geranyl acetone1447 1453 0.1RI, MS
52(E)-β-farnesene14511745145417455.5Std
53γ-muurolene146416831478 0.4RI, MS
54germacrene D14661701148417000.1RI, MS
55β-selinene1470 1489 0.1RI, MS
56ar-curcumene14731770147917691.0RI, MS
57(E)-β-ionone14761934148719360.1Std
58cis-β-guaiene1481 1492 0.2RI, MS
59γ-curcumene148116731481 0.2RI, MS
60α-muurolene148717191500 0.2RI, MS
61modhephen-8-β-ol1492 1513 0.2RI, MS
62γ-cadinene1499 1513 0.3RI, MS
63δ-cadinene15111753152217520.5RI, MS
64α-calacorene152819071544 0.1RI, MS
65(E)-nerolidol15572048156120492.1Std
66spathulenol156421271577213212.2RI, MS
67caryophyllene oxide156719771582198312.4Std
68salvial-4(14)-en-1-one157720001594 0.8RI, MS
69humulene epoxide II15912034160820401.7RI, MS
70caryophylla-4(12),8(13)-dien-5α-ol1615 1639 0.6RI, MS
71caryophylla-4(12),8(13)-dien-5β-ol1619 1639 1.0RI, MS
72tau-cadinol16262126162521251.0RI, MS
73muurola-4,10(14)-dien-1-beta-ol1637 1630 1.0RI, MS
74α-cadinol16402232165222321.0RI, MS
7514-hydroxy-9-epi-(E)-caryophyllene1656 1668 2.1RI, MS
76germacra-4(15),5,10(14)-trien-1-α-ol1669 1685 2.1RI, MS
77pentadecanal17062041170520410.6RI, MS
78β-bisabolenol 1776 1785 0.2RI, MS
7914-hydroxy-δ-cadinene1787 1788 0.3RI, MS
80neophytadiene1831 0.2MS
812-pentadecanone, 6,10,14-trimethyl-18372131183821310.3RI, MS
82hexadecanoic acid19622918195929172.5RI, MS
83(E)-phytol20942624209626220.7Std
84n-pentacosane2500250025002500trStd
85n-heptacosane2700270027002700trStd
Total identified (%) 78.6
Grouped compounds
Monoterpene hydrocarbons 11.5
Oxygenated monoterpenes 1.5
Sesquiterpene hydrocarbons 21.9
Oxygenated sesquiterpenes 38.5
Others 5.2
1 Compounds are listed in order of their elution from an HP-5MS column. Their nomenclature was in accordance with Adams [22]. 2 Linear retention index on HP-5MS and DB-Wax column, experimentally determined using homologous series of C8–C30 alkanes. 3 Linear retention index taken from Adams [22] and/or NIST08 [23] for apolar columns, and from FFNSC2 [24] and/or NIST08 for polar columns. 4 Percentage values are means of three determinations with an RSD% for the main components below 5% in all cases. 5 Identification methods: std, based on comparison with authentic compounds; MS based on comparison with Wiley. ADAMS and NIST08 MS database; RI, based on comparison of RI with those reported in ADAMS and NIST08. 6 tr, traces (mean value below 0.1%).
Table 2. In vitro growth inhibition of tumor cells by Erigeron floribundus essential oil.
Table 2. In vitro growth inhibition of tumor cells by Erigeron floribundus essential oil.
Cell Line (IC50 µg/mL) 1
A375 2MDA–MB 231 3HCT116 4
Essential oil22.520.814.9
95% C.I. 520.6–24.616.82–25.7713.10–17.00
Positive control
Cisplatin0.52.52.5
95% C.I.0.4–0.52.0–3.02.2–2.9
1 IC50 = the concentration of compound that affords a 50% reduction in cell growth (after 72 h of incubation). 2 Human malignant melanoma cell line. 3 Human breast adenocarcinoma cell line. 4 Human colon carcinoma cell line. 5 C.I. Confidence interval.
Table 3. In vitro antimicrobial activity of Erigeron floribundus essential oil.
Table 3. In vitro antimicrobial activity of Erigeron floribundus essential oil.
SpeciesStrainEssential OilReference Antibiotic 1
IZD 2 (mm)MIC 3 (µg/mL)IZD (mm)MIC (µg/mL)
S. aureusATCC 2921314.3 ± 0.651220.67 ± 1.150.25
MRSA 4 -19 1024–2048
MRSA-42 512–1024
MRSA-75 512–1024
MRSA-101 512–1024
E. faecalisATCC 292128.7 ± 0.6409624.0 ± 2.60.5–1
E. coliATCC 25922n.a. 5N.D. 619.7 ± 0.60.016
P. aeruginosaATCC 27853n.a.N.D.21.7 ± 0.60.5
C. albicansATCC 244339.0 ± 1.051216.33 ± 0.584.0
1 Ciprofloxacin for Gram-positive species, gentamicin for Gram-negative ones; nystatin for C. albicans. 2 IZD, inhibition zone diameter. Each value is an average of three independent experiments with standard errors indicated. 3 MIC; minimum inhibitory concentration. 4 MRSA, Methicillin-Resistant Staphylococcus aureus. 5 n.a., no activity. 6 N.D., not determined.
Table 4. Antioxidant activity of Erigeron floribundus essential oil.
Table 4. Antioxidant activity of Erigeron floribundus essential oil.
DPPHABTSFRAP
TEAC 1 μmol·TE/gIC50 2 μg/mLTEAC 1 μmol·TE/gIC50 2 μg/mLTEAC 1 μmol·TE/g
E. floribundus oil16.9 ± 0.1356.7 ± 7.185.5 ± 3.674.9 ± 3.2411.9 ± 2.40
Trolox 1.51 ± 0.04 1.6 ± 0.01
1 TEAC: Trolox equivalent (TE) antioxidant concentration. 2 IC50: The concentration giving a reduction of 50%. Each value is an average of three independent experiments with standard errors indicated.
Table 5. Inhibition of T. brucei and Balb/3T3 fibroblast proliferation.
Table 5. Inhibition of T. brucei and Balb/3T3 fibroblast proliferation.
T. brucei IC50 1 (μg/mL)Balb/3T3 IC50 1 (μg/mL)Selectivity Index (SI)
E. floribundus oil33.5 ± 2.7>200>5.97
Carophyllene oxide>200N.D.
Limonene5.6 ± 1.6>100>17.85
1 Each value is an average of three independent experiments with standard errors indicated.

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Petrelli, R.; Orsomando, G.; Sorci, L.; Maggi, F.; Ranjbarian, F.; Biapa Nya, P.C.; Petrelli, D.; Vitali, L.A.; Lupidi, G.; Quassinti, L.; Bramucci, M.; Hofer, A.; Cappellacci, L. Biological Activities of the Essential Oil from Erigeron floribundus. Molecules 2016, 21, 1065. https://doi.org/10.3390/molecules21081065

AMA Style

Petrelli R, Orsomando G, Sorci L, Maggi F, Ranjbarian F, Biapa Nya PC, Petrelli D, Vitali LA, Lupidi G, Quassinti L, Bramucci M, Hofer A, Cappellacci L. Biological Activities of the Essential Oil from Erigeron floribundus. Molecules. 2016; 21(8):1065. https://doi.org/10.3390/molecules21081065

Chicago/Turabian Style

Petrelli, Riccardo, Giuseppe Orsomando, Leonardo Sorci, Filippo Maggi, Farahnaz Ranjbarian, Prosper C. Biapa Nya, Dezemona Petrelli, Luca A. Vitali, Giulio Lupidi, Luana Quassinti, Massimo Bramucci, Anders Hofer, and Loredana Cappellacci. 2016. "Biological Activities of the Essential Oil from Erigeron floribundus" Molecules 21, no. 8: 1065. https://doi.org/10.3390/molecules21081065

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