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Tuesday, 25 April 2017

Antibacterial and antioxidant activity of essential oils and extracts from costmary (Tanacetum balsamita L.) and tansy (Tanacetum vulgare L.)

Industrial Crops and Products
Volume 102, August 2017, Pages 154–163


  • a Laboratory of New Herbal Products, Department of Vegetable and Medicinal Plants, Faculty of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Sciences SGGW, 159 Nowoursynowska Street, 02-776 Warsaw, Poland
  • b Division of Food Biotechnology and Microbiology, Department of Biotechnology, Microbiology and Food Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences SGGW, Warsaw, Poland
  • c Dipartimento di Scienze Biomediche, Odontoiatriche, e delle Immagini Morfologiche e Funzionali (BIOMORF), University of Messina, Messina, Italy
  • d Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (ChiBioFarAm), University of Messina, Messina, Italy
  • e Chromaleont s.r.l., a Start-up of the University of Messina, Messina, Italy
Received 28 October 2016, Revised 13 February 2017, Accepted 10 March 2017, Available online 29 March 2017

http://doi.org/10.1016/j.indcrop.2017.03.009
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Highlights

•
Essentials oils and phenolics differentiate T. balsamita and T. vulgare species.
•
Antibacterial activity of both species results mainly from essential oils.
•
Costmary indicate higher antibacterial activity than tansy.
•
Antioxidant effects of both species are related to phenolic acids.

Abstract

The aim of the presented study was to compare Tanacetum balsamita L. (costmary) and Tanacetum vulgare L. (tansy) in terms of the antibacterial and antioxidant activity of essential oils and hydroethanolic extracts in relation with their chemical profile. The investigated species differ as to the chemical composition and biological activities. The dominant compounds of essential oils, determined by GC–MS, were: β-thujone in costmary (84.43%) and trans-chrysanthenyl acetate in tansy (18.39%). Using HPLC-DAD, the chemical composition of phenolic acids and flavonoids were determined. Cichoric acid appeared to be the dominant phenolic compound in both species (3333.9 and 4311.3 mg × 100 g−1, respectively). Essential oil and extract of costmary was characterized by stronger antibacterial activity (expressed as MIC and MBC values) than tansy. In turn, tansy extract was distinguished by higher antioxidant potential (determined by FRAP and DPPH) in comparison to costmary. According to observed antibacterial and antioxidant activity, herb of tansy and costmary could be regarded as promising products for pharmaceutical and food industry as antiseptic and preservative agents.

Keywords

  • Essential oil;
  • Phenolics;
  • Antibacterial activity;
  • Antioxidant effect;
  • Tanacetum vulgare L.;
  • Tanacetum balsamita L.

1. Introduction

Plants belonging to Tanacetum genus (Asteraceae family) are widely distributed throughout the temperate zone of the northern hemisphere ( Heywood, 1976). They are perennial, herbaceous plants, native to Europe and Asia, but introduced in other parts of the world, as well ( Hassanpouraghdam et al., 2008 ;  Stojković et al., 2014). Some Tanacetum species have been known for ages as important medicinal plants, e.g. feverfew (Tanacetum parenthium L. Schultz Bip.) is listed in European Pharmacopeia as a traditional herbal remedy used for prophylaxis of migraine ( Wichtl, 2004 ;  European Pharmacopoeia 8th, 2008). Recently, special attention has been paid on two other species of this genus − tansy (Tanacetum vulgare L.) and costmary (Tanacetum balsamita L.). The herb of these plants has been used in traditional medicine as anthelmintic, antibacterial, digestive, and diuretic agent ( Blumenthal et al., 1998; Hassanpouraghdam et al., 2008 ;  Mureşan et al., 2014). The studies on the extracts obtained from these plants confirm different biological activities. Tansy exhibits antioxidant, antibacterial, antifungal, antihypertensive, diuretic, and anthelmintic properties as well as acaridical and repellent activity ( Chiasson et al., 2001; Lahlou et al., 2007; Lahlou et al., 2008; Hassanpouraghdam et al., 2009; Erecevit et al., 2011; Kumar and Tyagi, 2013; Baranauskiene et al., 2014; Stojković et al., 2014 ;  Godinho et al., 2014). Costmary reveals mainly antibacterial, antioxidant and astringed activity ( Bagci et al., 2008; Yousefzadi et al., 2009; Pukalskas et al., 2010; Benedec et al., 2016 ;  Venskutonis, 2016).
Tansy and costmary are rich in essential oils, predominantly: α-thujone, β-thujone, camphor, trans-chrysanthenyl acetate, 1,8-cineol, artemisia ketone, and carvone. The chemical composition of these essential oils is quite variable and according to the major compounds, both species create different chemotypes. Twenty-three chemotypes were distinguished within T. vulgare, while only four are already known for T. balsamita ( Gallori et al., 2001; Bylaitė et al., 2000; Lawrence, 2000; Başer et al., 2001; Keskitalo et al., 2001; Rohloff et al., 2004; Judzentiene and Mockute, 2005 ;  Hassanpouraghdam et al., 2009). Another important group of compounds, present in tansy, are nonvolatile sesquiterpene lactones, i.e. tanacetine, tatridine, tanachine, and parthenolide (Sanz and Marco, 1991). The herb of tansy and costmary also contains phenolic compounds, such as flavonoids and phenolic acids. Among flavonoids there are mainly glycosides of luteolin, apigenin and quercetin while phenolic acids are represented mainly by chlorogenic, caffeic and dicaffeoylquinic acids ( Williams et al., 1999a; Williams et al., 1999b; Marculescu et al., 2001; Nickavar et al., 2003; Pukalskas et al., 2010; Kurkina et al., 2011; Fraisse et al., 2011; Baranauskiene et al., 2014; Mureşan et al., 2015 ;  Benedec et al., 2016).
Taking into consideration previous studies, it seems that antioxidant, diuretic, and vascular activities of above mentioned species are attributed primarily to phenolics, while antibacterial and anthelmintic effects are conditioned by essential oil. There is little information available on the antibacterial properties of tansy and costmary extracts (Rauha et al., 2000 ;  Mureşan, 2015). So far, both species haven’t been compared comprehensively in terms of chemical composition and biological activity.
The aim of the present study was to compare T. vulgare and T. balsamita in terms of the antibacterial and antioxidant activity of essential oils and hydroethanolic extracts in relation with their chemical profile.

2. Material and methods

2.1. Plant samples

Plant raw materials (herb of T. vulgare and T. balsamita) were obtained from the Botanical Garden of Medicinal and Aromatic Plants (Koryciny, Poland). Seed material originated from wild growing populations located in Poland (tansy) and Turkey (costmary). Voucher specimens were deposited at herbarium of Department of Vegetable and Medicinal Plants, Warsaw University of Life Sciences, WULS-SGGW, Poland. Raw materials were collected in the full flowering stage and dried at 35 °C, in the dark.

2.2. Preparation of essential oils

The essential oil was isolated according to European Pharmacopeia 8th edition. 20 g of air-dried raw material was submitted to hydrodistillation for 3 h using Clevenger-type apparatus. Obtained essential oils were stored in dark vials, at 4 °C.

2.3. Preparation of hydroethanolic extracts

Air-dry, powdered raw material (5 g) was extracted with 50 mL of solvent (ethanol:water; 40:60, v/v) using Büchi Extraction System B-811 (Büchi Labortechnik AG, Flawil, Switzerland). Soxhlet hot extraction with twenty-five extraction cycles for 5 h and 10 min. was used. Extraction was repeated 10 times. Each extract was filtered and concentrated up to 5 mL using a rotary evaporator Büchi R200 (Büchi Labortechnik AG, Flawil, Switzerland). The obtained extracts were frozen in −80 °C for 2 days, then subjected to lyophilization (Labconco Freezone 2.5, Labconco, Kansas City, USA) for 2 days (−50 °C, 0.10 Mbar). Dry extracts were powdered in a porcelain mortar and stored in dark vials, at 4 °C.

2.4. Chemical analysis

The chemical composition of essential oil was determined using GC–MS and GC-FID (2.4.1), while the chemical composition of phenolic acids and flavonoids was determined by HPLC (2.4.2). All measurements were performed in triplicate.

2.4.1. Analysis of essential oils by GC–MS and GC-FID

Qualitative GC–MS analyses were carried out on a Shimadzu GC–MS-QP2010 gas chromatograph-mass spectrometer (Shimadzu, Milan, Italy) equipped with a Shimadzu autoinjector AOC-20is. Data were collected by GC–MS solution software (Shimadzu, Milan, Italy). The operational conditions were as follows: temperature program from 50 °C (2 min) to 250 °C (10 min) at 3 °C/min. Columns: SLB–5 ms (30 m × 0.25 mm i.d. × 0.25 μm film thickness) and Supelcowax-10 (30 m × 0.25 mm i.d. × 0.25 μm film thickness), both supplied by Supelco (PA, USA). The GC–MS-QP2010 was equipped with a split-splitless injector (250 °C). 10 μL of the essential oil was diluted in 1 mL of hexane. Injection volume 0.4 μL, in split mode (split ratio 50:1). Inlet pressure 37.7 kPa. Carrier gas: He, delivered at constant linear velocity (ū) 30 cm × s −1. Interface temperature: 250 °C. MS ionization mode: electron ionization. Detector voltage: 0.95 kV. Acquisition mass range: 40–350 u. Scan speed: 1666 amu × s −1. Acquisition mode: full scan, scan interval 0.20 s. Essential oil compounds identification was based on comparison of mass spectra from the Mass Spectral Database, as following: FFSNC 2, NIST 11, Wiley 9; and on comparison of retention indices (RI) relative to retention times of a series of n-hydrocarbons (C7-C30) with those reported in literature ( Adams, 2007 ;  Babushok et al., 2011).
For quantitative GC-FID analyses a Shimadzu GC-2010 gas chromatograph (Shimadzu, Milan, Italy) equipped with a Shimadzu autoinjector AOC-20is (Shimadzu, Milan, Italy), was exploited. Injection parameters and temperature program were the same as above reported for GC–MS analysis. Column was a Supelcowax-10 (30 m × 0.25 mm i.d. × 0.25 μm film thickness). Inlet pressure 100 kPa. Carrier gas: He, delivered at constant linear velocity (ū) 30 cm/s. FID (275 °C) gases: H2 (flow 50.0 mL × min −1); air (flow 400.0 mL × min −1); He (as make up, flow 50.0 mL × min −1). The percentage composition of the essential oils was computed by the normalization method from the GC peak areas.

2.4.2. Analysis of phenolic acids and flavonoids by HPLC

Commercially available standards (ChromaDex®, Irvine, USA) were separately dissolved with MeOH in 10 mL volumetric flask according to the ChromaDex’s Tech Tip 0003 Reference Standard Recovery and Dilution (https://www.chromadex.com/media/2126/techtip0003-recoverydilutionprocedures_nl_pw.pdf) and used as standard stock solutions. Further calibration levels (working solutions) were prepared by diluting 10, 50, 100, 200, 500, and 1000 μL of standard solutions with methanol in 10 mL volumetric flasks. The working solutions were injected (1 μL) on a column in six replicates (n = 6) to obtain a six-point calibration curve. Standard curve parameters were calculated using Microsoft Excel 14. Signal-to-noise ratio approach was used to determine Limit of Detection (LOD) (S/N of 3:1) and Limit of Quantitation (LOQ) (S/N of 10:1) (Table 1). The peak table and UV-spectra library (190–450 nm) of compounds were created.
Table 1. Validation parameters of the HPLC-DAD analysis (n = 6).
CompoundtRPrecision (CV.%)Regression equationLinearity (r2)Range (μg × mL−1)LOD (μg × mL−1)LOQ (μg × mL−1)
3-caffeoylquinic acid (chlorogenic acid)2.202.26y = 6 517.4x − 12 0170.99970.395–39.4560.0210.070
3,4-dihydroxy-cinnamic acid (caffeic acid)3.040.96y = 2 581.8x + 6 373.50.99990.998–99.8400.0300.100
4-hydroxy-3-methoxycinnamic acid (ferulic acid)6.171.84y = 4 806.6x + 6 054.70.99980.400–39.9680.0320.105
luteolin 7-O-glucoside8.902.36y = 2 022.2x − 1149.40.99970.191–19.0800.0540.181
3,4-dihydroxycinnamoyl-3-(3,4-dihydroxyphenyl) lactic acid
(rosmarinic acid)		10.300.99y = 2 017.9x + 1 100.40.99990.434–43.4020.0480.160
apigenin-7-O-glucoside (cosmosiin)11.501.28y = 2 338.6x − 1490.00.99990.195–19.5400.0440.147
luteolin 3′-methyl ether (chrysoeriol)11.701.79y = 1 406.0x + 413.50.99980.285–28.5000.0700.233
dicaffeoyltartaric acid (cichoric acids)12.001.07y = 2 729.4x − 2475.70.99990.457–45.6960.0360.120
quercetin14.201.77y = 3 230.7x − 6882.20.99980.408–40.8340.0370.125
Table options
1 g of dry extract was dissolved in 10 mL of methanol. The analyzes were performed using a Shimadzu HPLC equipped with auto sampler SIL-20A, photodiode array detector SPD-M10A VP PDA, and CLASS VP™ 7.3 chromatography software (Shimadzu, Kyoto, Japan). Separations were achieved using a 100 mm × 4.60 mm C18 reversed-phase Kinetex™ 2.6 μm column (Phenomenex, USA). A binary gradient of mobile phase A (deionized water adjusted to pH 3 with phosphoric acid, Sigma-Aldrich, Poznań, Poland) and B (ACN) was used at following conditions: 0.01 min − 12% B; 10.00 min − 55% B; 10.50 min − 55% B; 10.51 min − 12% B. The HPLC conditions were: flow rate 1.1 mL × min−1, oven temperature 35 °C, injection volume 1 μL, and total time of analysis was 15 min. Analytical data were recorded at wavelength of 322 nm for 3,4-dihydroxy-cinnamic acid (caffeic acid) and 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 325 nm for 3-caffeoylquinic acid (chlorogenic acid), 330 nm for 3,4-dihydroxycinnamoyl-3-(3,4-dihydroxyphenyl) lactic acid (rosmarinic acid) and dicaffeoyltartaric acid (cichoric acid), 336 nm for apigenin 7-O-glucoside (cosmosiin), 347 nm for luteolin 7-O-glucoside and luteolin 3′-methyl ether (chrysoeriol), and 366 nm for quercetin. The content of the determined compounds was calculated in mg × 100 g−1 of dry extract.

2.5. Antibacterial activity

2.5.1. Test microorganisms and preparation of inoculum

Reference strains originated from the American Type Culture Collection (ATCC, Manassas, VA, USA), clinical strains originated from the National Institute of Public Health-National Institute of Hygiene (NIPH-NIH, Warsaw, Poland), and the strain isolated from food originated from the Division of Milk Biotechnology (WULS-SGGW, Poland). The study used 8 strains of Gram-positive bacteria (B. cereus ATCC 11778, B. cereus WULS-SGGW 15, B. cereus WULS-SGGW X-13, B. subtilis ATCC 6633, S. aureus ATCC 25923, S. epidermidis ATCC 12228, L. monocytogenes NIPH-NIH 17/11, S. aureus NIPH-NIH A-529) and 11 strains of Gram-negative bacteria (E. aerogenes ATCC 13048, E. coli ATCC 25922, K. pneumoniae ATCC 13883, P. mirabilis ATCC 35659, S. enterica subsp. enterica serovar Enteritidis ATCC 13076, P. aeruginosa ATCC 27853 E. coli O26 NIPH-NIH 152/11, S. enterica subsp. enterica serovar Enteritidis NIPH-NIH 322/11, S. enterica subsp. enterica serovar Typhimurium NIPH-NIH 300/11, Y. enterocolitica O3 NIPH-NIH 383/11, S. sonnei NIPH-NIH s). The bacterial strains were cultured on Mueller–Hinton Agar (MHA, Merck, Darmstadt, Germany) and incubated at 37 °C for 18 h. Bacterial inocula were prepared in sterile 0.85% NaCl (w/v) solution to reach a population of approximately 108 CFU × mL−1.

2.5.2. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of essential oils and extracts were determined using the method of serial microdilutions (CLSI, 2009). Two series of dilutions were prepared in Müller-Hinton Broth (Merck, Darmstadt, Germany), for essential oils in the concentration range of 0.25–32 μL × mL−1, and for extracts in the concentration range of 0.5–64 μL × mL−1, using polystyrene 96-well plates, each of a volume of 200 μL (Primo, ScholaGene, Krakow, Poland). Test bacterial inoculum was added to each well of the plate, so that the final number in 1 mL was 5 × 105. The plates with bacteria were incubated at 37 °C for 24 h. The MIC value was defined as the lowest concentration of essential oil or extract, in which no visual growth of bacteria was noted. MIC examination of essential oils and extracts was repeated three times. MBC examination involved the transfer of 100 μL bacteria culture from each well where no growth was observed on the plates with Müller-Hinton Agar (Merck, Darmstadt, Germany). The plates were incubated at 37 °C for 24 h. Growth of colonies on the plates was verified after that incubation time. MBC was defined as the lowest concentration of essential oil or extract, which resulted in complete reduction of bacteria.
Percentage value of antibacterial activity of essential oils and extracts was determined based on MIC values (A) (Rangasamy et al., 2007)
A (%) = (100 × number of strains inhibited by the examined essential oil or extract)/(total number of tested strains).
The percentage of activity proves the complete antibacterial potency of particular plant preparations, i.e. it determines the number of bacterial strains susceptible to essential oil or extract.

2.6. Antioxidant activity

2.6.1. DPPH scavenging capacity assay

The antioxidant activity of extracts was assessed by DPPH free radical assay. The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method is an antioxidant assay based on electron transfer that produces a violet solution in ethanol (Huang et al., 2005). The measurement of the DPPH radical scavenging activity was performed according to Yen and Chen (1995), with modifications concerning the time of reaction, according to Szlachta and Małecka (2008). 0.25 g of dry hydroethanolic extract was dissolved in 5 mL of methanol. Then, 3 mL of methanol and 1 mL of DPPH methanolic solution (0.12 mg × mL−1) were added to 1 mL of examined extract. Absorbance was measured on the UV/vis Shimadzu 1700 PharmaSpec spectrophotometer (Shimadzu, Kyoto, Japan) after 10 min at 517 nm. The blind test extract was replaced by 1 mL of methanol. The antioxidant activity of extracts was calculated as I = [(AB − AA)/AB] × 100, where I is DPPH inhibition (%); AB is the absorbance of a blank sample (t = 0 min); AA is the absorption of extract solution (t = 10 min). Trolox in concentrations of 0.3–4.9 μg × mL−1 was used to estimate standard curve. The potential antioxidant activity was expressed as Trolox equivalent antioxidant capacity in μmol Trolox × g−1.

2.6.2. Ferric reducing antioxidant power assay (FRAP)

This method comprises determining the ability of antioxidants to reduce ferric ions (Fe3+) to ferrous ion (Fe2+) present in the extract. Ions Fe2+ and TPTZ (2,4,6-tris (2-pyridyl)-1,3,5-triazine) create an intense blue complex under acidic conditions (pH 3.6) (Benzie and Strain, 1996). 0.25 g of dry hydroethanolic extract was dissolved in 10 mL of methanol. The working reagent was prepared by mixing acetate buffer (300 mM, pH 3.6), a solution of 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3·6H2O at 10:1:1 (v/v/v). A total of 100 μL of each properly diluted extract solutions were prepared into tubes with 3 mL of working reagent and shaken for 30 s. After 2 h of incubation the absorbance was read at 593 nm. A series of Trolox and Fe2SO4 solutions in the concentration ranges of 0–479 μg × mL−1 and 0–1000 μg × mL−1, respectively, were used for preparing the calibration curve. The potential antioxidant activity was expressed as Trolox equivalent antioxidant capacity in μmol Trolox × g−1 extract and Fe2+ antioxidant capacity (Fe2+ μmol × g−1 extract) ( Benzie and Strain, 1996 ;  Kraujalyte et al., 2013).

2.7. Statistical analysis

Data were subjected to statistical analysis using Statgraphics® plus software. The mean values were compared by using the one way analysis of variance (ANOVA) and expressed as mean with standard deviation (SD). The differences between individual means were deemed to be significant at P < 0.05 and signed with the “*” in rows of tables.

3. Results and discussion

3.1. Essential oil content and composition

The total content of essential oil in the dry herb of costmary was at a level of 0.70 g × 100 g−1, while for tansy 1.20 g × 100 g−1 (Table 2). These results correspond with the data of other authors. According to Bylaitė et al. (2000), the total content of essential oil in costmary range from 0.31 to 1.25%, while Hassanpouraghdam et al. (2009) provide the value 0.58%. Given the tansy, Baranauskiene et al. (2014) indicate a level of 0.52%, while according to Rohloff et al. (2004), these values varied between 0.35 and 1.90%. Such differences are probably associated with the intraspecific variability of both Tanacetum species.
Table 2. The total content (g × 100 g−1) and gas chromatographic composition (% peak area) of essential oil samples.
No.CompoundRIaRI rangebRIcRI rangedCostmary
(T. balsamita)		Tansy
(T. vulgare)
1(Z)-salvenee935–848–0.13–
2(E)-salvenee947–857–0.08–
3tricyclene1002998–1029923906–931–0.04
4α-pinene10171008–1039934921–9440.041.32
5santolinatriene10241011–1063903900–914–0.34
6camphene10571043–1086950936–9590.020.67
7n-hexanal10741056–1106–––0.05
8β-pinene10991085–1130979964–9880.050.51
10sabinene11141098–1140972961–9810.031.74
11dehydrosabinene11231109–1137953–0.020.01
12α-phellandrene11551148–1186–––0.01
13α-terpinene11701154–119510181007–1026–0.34
14isobutyrate <isopentyl->11851165–119910121003–10180.06–
15limonene11901178–1219––0.080.09
16eucalyptol11991186–123110331021–10444.072.55
17(2E)-hexenal12131196–1238–––0.01
18chrysanthenone1217–11231120–1131–0.03
192-pentylfuran12241213–1249–––0.06
20γ-terpinene12381222–126610601049–10690.080.71
21(Z)-myroxidee1246––––0.06
22p-cymene12641246–129110251011–10330.610.82
232-methylbutyl-2-methyl-butyrate12751272–1305f––0.09–
24terpinolene12761261–130010871074–1097–0.17
252-methylbutyl isovalerate12891286–1334f––0.03–
26cis-pinocamphonee1321––––0.05
27artemisia ketone13451320–135810561050–1071–9.15
284,8 dimethyl−1,3,7 nonatrienee1365––––0.02
295-methyl−5-octen-2-onee1375–1037–0.74–
301,4-cineolee1384–1097–0.05–
31nonanal13921370–1414––0.02–
32yomogi alcohol13961377–1405995989–1000–2.49
33verbenonee1401–1398––0.04
34presilphiperfol-7-ene14051391–1425f13351335f–0.03
35artemisyl acetate14171390–1433f–––0.10
36α-thujone14211385–144111081099–11174.680.83
37carveole1425––––0.01
38β-thujone14421400–145211191106–112484.4314.28
39trans-sabinene hydrate14641425–147810711052–1074–0.02
40trans-verbenol1468–11381139–1148–3.07
41isocyclocitrale1477––––0.23
42silphiperfol−6-enee1496–1377––0.04
43artemisia alcohol1510–10801072–1092–3.84
44camphor15181481–153711491127 − 1155–3.03
45trans-chrysanthenyl acetate15321533–15901232––18.39
46nonenal (isomer?)15411509–1569–––0.09
47linalool15521507–156411001088–11090.200.05
48cis-p-menth-2-en-1-ol15671593–1645f11261115–11380.060.12
49pinocarvone15731545–159011651144–11670.08–
50cis-chrysanthenyl acetate15751533–1590f12581255–1267–0.65
51bornyl acetate15861550–160312851264–1297–0.27
52carvyl propionatee1598–1458––0.51
53terpinen-4-ol16091564–163011841165–11890.322.33
54pulegone16281626–166312221210–12530.08–
55(Z)-dihydrocarvone16371600–165012001196––1211–3.37
56(E)-dihydrocarvone16371600–165012061196–1211–11.02
57sabina ketone16411606–168311601147–1162–0.12
58trans-pinocarveol16651643–167111431124–11630.210.58
59carvotanacetone16701652–171612521230–12560.21–
60thujole1673–1140–0.350.04
61δ-terpineol16831655–1687––0.02–
62Z-citral (neral)16901641–1706–––0.22
63α-longipinene1699–13531337–1362–0.09
64α-terpineol17091659–172411981178–12030.070.26
65borneol17141653–172811741152–11770.051.49
66germacrene D17201676–172614841464–1493–0.25
57carvenonee1727–––0.03–
58piperitone17411689–174812571245–12660.020.42
69carvone17481699–175112461227–12650.030.50
70cis-piperitol17631675–1761––––
71cis-chrysanthenol17661751–176511651160–1168–3.93
72trans-p-menth-2-en-7-ol17761774–1821––0.03–
73α-campholenal1794–11261106–1134–0.15
74dihydro isocarveol1803–12111196–1277–0.60
75myrtenole1812
––0.080.18
76p-mentha−1,5-dien-7-ole1822––––0.14
771,6-dihydrocarveole1829––––0.48
78(E)-β-damascenone18401789–184213811370–13970.05–
79phenethyl propionatee1904–––0.04–
80ascaridolee1948–––0.05–
81β-ionone19651892–1958––0.12–
82neophytadienee1984–––0.13–
83phenylethyl−2-methyl-butyratee2000–––0.05–
84caryophyllene oxide20111936–202315851563–1595–0.72
85sabina ketonee2013–––0.16–
86verbenonee2038–1102––0.23
87artedouglasia oxide A2047–15301523f–0.16
88(7Z)-hexadecenale2059––––0.15
89(E)-nerolidol2074–15631527–1567–0.07
90cadin−4-en-10-ole2107–1658––0.21
91gleenol21192008–2054–––0.19
92cumin alcohol2142–11901178–11910.070.09
93valeranone21482107–2155 f1686––0.12
94spathulenol2161–15811562–15900.040.71
95phytonee2164–––0.14–
96eugenol22102100–2198–––0.34
97acorenone B2227–16921696f0.03–
98carvacrol22312140–2246––0.04–
99thymol2262–13031292–13040.67–
100longiverbenone22662222–2270f16501637–1694f–1.99
101β-eudesmol22732196–227216591637–16640.64–
102intermedeol2277–16681654–1677–0.90


Total



99.3498.89


Monoterpene hydrocarbons



0.926.87

Oxygenated monoterpenes



95.9084.37

Sesquiterpene hydrocarbons



–0.42

Oxygenated sesquiterpenes



0.715.08

Other compounds



1.820.51

Essential oil total content



0.701.20
a
RI − Kováts retention indices calculated on polar column Supelcowax 10.
b
RI range − range of Kováts retention indices on polar column reported by Babushok et al. (2011).
c
RI − Kováts retention indices calculated on unpolar column SLB 5 ms.
d
RI range − range of Kováts retention indices on unpolar column reported by Babushok et al. (2011) and Adams (2007).
e
− Identification on the basis of GC–MS spectra.
f
− Kováts retention indices on polar column reported in literature (www.nist.gov).
Table options
In the present work, 49 compounds were detected in the costmary essential oil, comprising 99.34% of total identified fraction. In the case of tansy, 76 compounds were identified, accounting for 98.89% of total identified fraction. The oxygenated monoterpenes fraction was a fundamental part in both analyzed essential oils, since it formed 95.90% and 84.37%, respectively. The major difference between the two investigated essential oils concerned the share of compounds. In the costmary, the dominant compound is β-thujone (84.43%), followed by α-thujone (4.68%) and eucalyptol (4.07%). Whereas, tansy essential oil was characterized by significantly less content of these compounds (14.28%; 0.83%; 2.55%, respectively) and the presence of a high amount of other oxygenated monoterpenes, such as trans-chrysanthenyl acetate as a dominant one (18.39%), (E)-dihydrocarvone (11.02%), and artemisia ketone (9.15%). The monoterpene hydrocarbons fraction comprised 6.87% of total identified fraction in tansy essential oil and 0.92% of costmary. Within this fraction, α-pinene and sabinene were present in the highest amount in tansy essential oil (1.32; 1.74%) and only in traces in costmary. Another difference between the investigated essential oils concerned the content and chemical composition of sesquiterpenes. Tansy essential oil contains 5.08% oxygenated sesquiterpenes and 0.42% sesquiterpene hydrocarbons. In the case of costmary, the oxygenated sesquiterpenes accounted for 0.71% of total identified fraction, while sesquiterpenes hydrocarbons were not detected at all. Moreover, costmary essential oil was characterized by highest share of other, e.g. aliphatic, compounds (1.82%) in comparison to tansy (0.51%) ( Table 2).
Obtained results are in good agreement with the literature data. Regarding the essential oil composition, both species are variable and able to create different chemotypes, but it seems that tansy is much more polymorphic than costmary. Based on the major compounds present in the essential oil, twenty three chemotypes have been recognized within tansy species. Such a high chemical variability may be explained by allogamy way of propagation as well as heterozygous character of this plant. The most frequent chemotypes include β-thujone, α-thujone, camphor, trans-chrysanthenyl acetate, eucalyptol, and artemisia ketone types. Some less often chemotypes are α-pinene, sabinene, dihydrocarvone, or chrysantenon types ( Lawrence, 2000; Keskitalo et al., 2001; Rohloff et al., 2004 ;  Judzentiene and Mockute, 2005). It seems that the investigated population of tansy may be classified into mixed chemotype as trans-chrysanthenyl acetate + β-thujone + (E)-dihydrocarvone. When regards the costmary, literature describes four main chemotypes: carvone, camphor, camphor + thujone, carvone +α-thujone types ( Bylaitė et al., 2000 ;  Başer et al., 2001; Gallori et al., 2001; Hassanpouraghdam et al., 2009). Other authors also have reported trans-chrysanthenol, β-thujone, bornyl acetate and pinocarvone as major compounds in costmary essential oil ( Bagci et al., 2008; Jaimand and Rezaee, 2005 ;  Yousefzadi et al., 2009). The population of costmary, the object of our investigation, was recognized as a clear β-thujone chemotype, due to the domination of this compound in the essential oil. However, it is worth noting that thujone is a strong neurotoxin and, by that, the use of thujone-rich raw material as medicine should be restricted and regulated ( EMA, 2012 ;  Pelkonen et al., 2013).

3.2. Phenolic compounds composition

The investigated Tanacetum species differed according to the content of determined phenolic acids and flavonoids. In general, costmary extract was characterized by higher content of both groups of phenolic compounds in comparison to tansy. Among the phenolic compounds, 5 acids were identified: 3,4-dihydroxy-cinnamic (caffeic), 4-hydroxy-3-methoxycinnamic (ferulic), 3-caffeoylquinic (chlorogenic), 3,4-dihydroxycinnamoyl-3-(3,4-dihydroxyphenyl) lactic (rosmarinic) and dicaffeoyltartaric (cichoric) acids ( Table 3, Fig. 1). In both the species, the dominant acid was cichoric acid (3333.9 mg × 100 g−1 in costmary and 2781.8 mg × 100 g−1 in tansy), followed by chlorogenic acid (1368.4 and 925.7 mg × 100 g−1, respectively). Tansy extract was distinguished by almost six times higher content of rosmarinic acid (294.3 mg × 100 g−1) compared to costmary (50.36 mg × 100 g−1). Moreover, this plant material was also rich in ferulic acid (199.3 mg × 100 g−1), a compound not found in costmary. All phenolic acids identified in the present study represent cinnamic acid derivatives. Caffeic and ferulic acids are free compounds, while chlorogenic, rosmarinic, and cichoric acid belong to depsides. The presence of chlorogenic, caffeic and ferulic acids in tansy and costmary herb was reported earlier ( Marculescu et al., 2001; Fraisse et al., 2011; Pukalskas et al., 2010; Baranauskiene et al., 2014; Mureşan et al., 2015 ;  Benedec et al., 2016). According to Mureşan et al. (2015) the dominant compound in tansy extract is chlorogenic acid. Results of our investigations indicate cichoric acid as predominant compound. According to Fraisse et al. (2011), tansy herb also contains other dicaffeoylquinic acid isomers, namely 3.5, 1.5, and 4.5 dicaffeoylquinic acids, while Baranauskiene et al. (2014) has listed: 3.4, and 4.5 dicaffeoylquinic acids and 4.5 caffeoylquinic acids, as well.
Table 3. Chemical composition of phenolic acids and flavonoids in extracts (mg × 100 g−1).
CompoundCostmary (T. balsamita)Tansy (T. vulgare)
Phenolic acids

3,4-dihydroxy-cinnamic acid (caffeic acid)49.14 ± 6.72110.22* ± 9.23
4-hydroxy-3-methoxycinnamic acid (ferulic acid)–199.3 ± 15.12
3-caffeoylquinic acid (chlorogenic acid)1368.4* ± 112.03925.7 ± 108.05
3,4-dihydroxycinnamoyl-3-(3,4-dihydroxyphenyl) lactic acid
(rosmarinic acid)		50.36 ± 4.85294.3* ± 19.26
dicaffeoyltartaric acid (cichoric acid)3333.9 ± 156.342781.8 ± 131.01
Total4801.84311.3

Flavonoids

quercetin54.44 ± 7.6527.43 ± 1.98
apigenin-7-O-glucoside (cosmosiin)1099.3* ± 98.97211.1 ± 18.15
luteolin 7-O-glucoside727.5 ± 45.67598.0 ± 48.13
luteolin 3′-methyl ether (chrysoeriol)472.4 ± 36.59627.4 ± 52.03
Total2353.61463.9
*
P < 0.05.
Table options
Fig. 1
Fig. 1. 
Chemical structures of identified phenolic acids and flavonoids: a) 3,4-dihydroxy-cinnamic acid (caffeic acid), b) 4-hydroxy-3-methoxycinnamic acid (ferulic acid), c) 3-caffeoylquinic acid (chlorogenic acid), d) 3,4-dihydroxycinnamoyl-3-(3,4-dihydroxyphenyl) lactic acid (rosmarinic acid), e) dicaffeoyltartaric acid (cichoric acid), f) quercetin, g) luteolin 7-O-glucoside, h) luteolin 3′-methyl ether (chrysoeriol), i) apigenin 7-O-glucoside (cosmosiin).
Figure options
In the case of flavonoids, 4 compounds were identified: quercetin, apigenin 7-O-glucoside (cosmosiin), luteolin 7-O-glucoside and luteolin 3′-methyl ether (chrysoeriol) ( Table 3, Fig. 1). Given costmary, apigenin 7-O-glucoside appeared to be the dominant compound (1099.3 mg × 100 g−1), present in five higher content when compared to tansy (211.1 mg × 100 g−1). Luteolin 7-O-glucoside and chrysoeriol were at the similar level in both the species (727.5 and 472.4 mg × 100 g−1 in costmary; 598.0 and 627.4 mg × 100 g−1 in tansy, respectively). Costmary extract contained almost twice as much quercetin than tansy (54.44 and 27.43 mg × 100 g−1, respectively). The presence of above-mentioned compounds and its derivatives in tansy and costmary herb was demonstrated earlier by other authors, however without through quantitative data ( Williams et al., 1999a; Williams et al., 1999b; Nickavar et al., 2003; Fraisse et al., 2011; Baranauskiene et al., 2014 ;  Mureşan et al., 2015).

3.3. Antibacterial activity

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values, with respect to selected pathogenic bacteria were determined in order to establish the antibacterial activity of essential oils and extracts of two Tanacetum species. The study involved serial microdilutions, which is a well suited method for screening of numerous combinations of plant preparations in relation to a variety of bacteria, and in addition is economical in terms of time and resources ( Klančnik et al., 2010).
Essential oils obtained from costmary and tansy were characterized by their different antibacterial activities on the tested strains (Table 4). Costmary essential oil inhibited the growth of all tested strains of bacteria. In general, the MIC value of costmary essential oil was within the range of 1–8 μL × mL−1, except for three bacteria, i.e. L. monocytogenes, S. sonnei, and P. aeruginosa, for which the MIC values were higher. Tansy essential oil showed no inhibitory activity with respect to some of the test strains in the examined range of concentrations (MIC > 32 μL × mL−1), particularly for Gram-negative bacteria. Tansy essential oil inhibited some Gram-positive bacteria, i.e. B. cereus, B. subtilis, and S. epidermidis, whose activity was stronger than costmary essential oil. Reference strains were more sensitive to the examined essential oils than clinical strains of the same species (S. aureus, E. coli, and Salmonella enterica). In most cases, the MBC values of costmary essential oil were equal to or twice their concentrations inhibiting the growth of bacteria. In the examined range of concentrations, tansy essential oil showed no bactericidal activity for most of the examined strains. Antibacterial activity of essential oil of T. balsamita with respect to S. aureus, E. coli, B. subtilis, and S. ser. Typhimurium bacteria was also demonstrated by other authors ( Bagci et al., 2008). Mikulášová and Vaverková (2009) found higher bacteriostatic activity of essential oil of T. vulgare against Gram-positive than Gram-negative bacteria, in a similar concentration range as in the present study (MIC 6–25 μL × mL−1).
Table 4. MIC and MBC values of essential oils and extracts.
StrainMIC (MBC)

Essential oils [μL × mL−1]
Extracts [mg ×  mL−1]

Costmary
(T. balsamita)		Tansy
(T. vulgare)		Costmary
(T. balsamita)		Tansy
(T. vulgare)
Gram- positive bacteria



B. cereus ATCC 117782 (4)1 (2)4 (4)8 (8)
B. cereus 152 (8)1 (>32)4 (4)8 (16)
B. cereus X-134 (4)1 (>32)4 (4)8 (8)
B. subtilis ATCC 66338 (8)4 (4)4 (4)16 (16)
S. aureus ATCC 259231 (2)2 (8)2 (2)4 (4)
S. aureus A-5294 (4)8 (8)2 (2)4 (4)
S. epidermidis ATCC 122281 (2)0.5 (2)1 (1)4 (8)
L. monocytogenes 17/1132 (32)>32 (nt)64 (>64)64 (>64)

Gram-negative bacteria



E. aerogenes ATCC 130488 (8)4 (8)64 (>64)>64 (nt)
E. coli ATCC 259221 (4)8 (>32)64 (64)64 (64)
E. coli O26 152/118 (8)>32 (nt)64 (64)64 (64)
K. pneumoniae ATCC 138838 (8)>32 (nt)2 (2)4 (4)
P. mirabilis ATCC 356592 (4)>32 (nt)32 (32)16 (16)
S. ser. Enteritidis ATCC 130762 (2)32 (32)8 (8)32 (32)
S. ser. Enteritidis 322/114 (4)>32 (nt)64 (64)64 (64)
S. ser. Typhimurium 300/118 (8)>32 (nt)64 (64)64 (64)
Y. enterocolitica O3 383/111 (2)1 (32)2 (4)2 (4)
S. sonnei ,,s”16 (16)>32 (nt)8 (16)8 (8)
P. aeruginosa ATCC 2785332 (>32)>32 (nt)16 (16)32 (32)
nt − no tested.
Table options
Table 5 presents the percentage of activity of essential oils obtained from Tanacetum. The results of this analysis confirmed that costmary essential oil was more active with respect to a broader spectrum of bacteria than tansy essential oil in all concentrations (except for 0.5 and 1 μL × mL−1). All (100%) of the test strains were inhibited by costmary essential oil at a concentration of 32 μg × mL−1, while tansy essential oil, at the same concentration, inhibited only 58% of the strains. The differences in antibacterial activity of both essential oils result from different chemical composition. The dominant component of costmary essential oil was β-thujone, the content of which was 84.43%. This compound was also present in the composition of tansy essential oil, but only at a concentration of 14.28%. Another dominant component of this essential oil, trans-chrysanthenyl acetate, was at a concentration of 18.39%. It can be suggested that more effective activity of costmary essential oil against Gram-negative bacteria, and on the other hand, more effective bacteriostatic activity of tansy essential oil against bacteria of Bacillus genus resulted only because of the presence of above-mentioned compounds. Rashid et al. (2013) also suggested that more potent activity of costmary essential oil resulted from the presence of its main component, i.e. β-thujone. In addition, the authors demonstrated that α-thujone acted mainly against Gram-negative bacteria (MIC 32–64 μg × mL−1), but it did not demonstrate bacteriostatic effect against B. subtilis. Considerably weaker bacteriostatic activity was demonstrated for chrysanthenyl acetate, whose MIC values were almost 10-fold higher, and were in the range of 256–512 μg × mL−1. This compound demonstrated an activity against B. subtilis, S. aureus, and S. ser. Typhi. The MIC and MBC of the extracts from costmary and tansy demonstrated bacteriostatic and bactericidal effect on the vast majority of test strains in concentrations higher than 1 mg × mL−1 (Table 4). Gram-positive strains (except for L. monocytogenes) were heavily inhibited by costmary extract in comparison to tansy extract. Gram-negative bacteria were more resistant to the activity of examined extracts (MIC 8–64 mg × mL−1) than the Gram-positive bacteria (MIC 1–16 mg × mL−1) except for K. pneumoniae and Y. enterocolitica strains (MIC 2–4 mg × mL−1). Higher sensitivity of Y. enterocolitica compared to other Gram-negative bacteria on plant extracts was also found by other authors ( Park et al., 2016). Similar relationship was also observed by Yuste and Fung (2003), who demonstrated higher sensitivity of Y. enterocolitica compared to S. aureus and S. ser. Enteritidis to the activity of powdered cinnamon in apple juices. Clinical and reference strains belonging to B. cereus, S. aureus, and E. coli species were characterized by a similar sensitivity to the examined extracts. Rauha et al. (2000) observed that methanol extract of T. vulgare was characterized by weak activity on Gram-positive bacteria, i.e. S. epidermidis, B. subtilis, and S. aureus, but was more effective with respect to E. coli.
Table 5. Percentage antibacterial activity (A%) of essential oils and extracts.
MIC
[mg × mL−1]		A (%)

Essential oils
Extracts

Costmary
(T. balsamita)		Tansy
(T. vulgare)		Costmary
(T. balsamita)		Tansy
(T. vulgare)
0.2500––
0.50500
1212600
24232265
458424726
884535847
1689536358
32100586868
64––10095
Table options
Higher antibacterial activity at lower concentrations was demonstrated for costmary extract (Table 5). At a concentration of 4 mg × mL−1, this extract inhibited 47% of the examined strains, while tansy extract limited the growth of 26% of the strains. At higher concentrations, e.g. 32 mg × mL−1, both extracts inhibited 68% of the examined bacteria.
In general Gram-negative bacteria were more resistant to investigated extracts than Gram-positive bacteria, except for K. pneumonia and Y. enterocolitica. Observed antibacterial activity of examined extracts can be related to the presence of flavonoids. A stronger inhibition of Gram-positive strains by costmary extract when compared to tansy can be associated with the higher content of detected flavonoid compounds in this extract ( Table 4, Table 2). These compounds exhibit a strong antibacterial activity expressed by functions, such as inhibition of nucleic acid synthesis, inhibition of cytoplasmic membrane function, and by disorders in the energy metabolism of bacteria cells ( Bylka et al., 2004; Cushnie and Lamb, 2005 ;  Cushnie and Lamb, 2011).

3.4. Antioxidant potential

Methods used in the present study, namely DPPH scavenging reaction and ferric reducing antioxidant power (FRAP) assay are attributed mainly to single electron transfer (SET) reaction. In the DPPH assay, such reaction results in the decrease in the absorbance of free radical species, visible as the change of color from purple to yellow. In turn, FRAP assay relies on the ability of antioxidants to reduce Fe3+ to Fe2+ in the presence of tripyridyltriazine (TPTZ), which result in the formation of an intense blue Fe2+-TPTZ complex. Obtained results indicate that the antioxidant potential is stronger in the case of tansy in comparison to costmary extract (Table 6). It seems that the antioxidant potential of both the investigated Tanacetum species might be attributed mainly to the presence of phenolic acids. Caffeic acid derivatives, especially rosmarinic acid, are known for high antioxidant activity ( Hong and Ho, 1997; Gűlçin, 2006; Sato et al., 2011; Bakota et al., 2015 ;  Muňoz-Muňoz et al., 2013). Thus, a high antioxidant potential of tansy extract could be related to the high content of caffeic, rosmarinic, and ferulic acids ( Table 2 ;  Table 6). According to Baranauskiene et al. (2014), main substances responsible for antioxidative activity of tansy water extract are mono- and dicaffeoylquinic acids, while Juan-Badaturuge et al. (2009) and mentioned it as 3.5 dicaffeoylquinic acid. Authors claim that antioxidant activity of tansy and costmary can be related not only to phenolic acids, but also to flavonoids ( Juan-Badaturuge et al., 2009; Pukalskas et al., 2010; Stojković et al., 2014 ;  Benedec et al., 2016). The antioxidant activity of these compounds depend on their structure as well as on the configuration and number of hydroxyl groups. Flavonoids with the substitution of hydroxyl group in ring B and ring C, e.g. quercetin, exhibit a strong antioxidant potential ( Heim et al., 2002 ;  Mishra et al., 2003). Nevertheless, results obtained in the present study do not indicate on the relationship between the presence of identified flavonoids and the antioxidant activity of the investigated Tanacetum extracts. This may stem from the fact that the dominant flavonoids in the extracts are in the form of glycosides, such as apigenin 7-O-glucoside and luteolin 7-O-glucoside. According to Mishra et al. (2003), flavonoid glycosides are characterized by less antioxidant activity than their aglycones.
Table 6. Results of antioxidant activity in vitro (DPPH, FRAP) of extracts.


Costmary
(T. balsamita)		Tansy
(T. vulgare)
DPPH[% RSC]86.80 ± 0.6188.18 ± 0.46

[μmol Trolox × g−1]13.59 ± 0.2113.86 ± 0.19

FRAP[Fe 2+ μmol × g−1]739.8 ± 30.481178.0* ± 3.66

[μmol Trolox × g−1]339.1 ± 17.12585.6* ± 2.05
*
P < 0.05.
Table options

4. Conclusions

The paper is the attempt at the direct, comprehensive comparison of tansy and costmary in respect of antibacterial and antioxidant activity. The dominant compounds in essential oils were: trans-chrysanthenyl acetate in tansy, and β-thujone in costmary. When regards phenolic compounds, cichoric acid appeared to be the dominant in both species. Essential oil and hydroethanolic extract of costmary was characterized by stronger antibacterial activity than tansy extracts. In turn, tansy hydroethanolic extract was distinguished by higher antioxidant potential in comparison to costmary. Thus, antibacterial activity seems to be related mainly to above mentioned essential oil components, while antioxidant potential may be attributed to phenolic compounds, such as caffeic, rosmarinic and ferulic acids. Due to observed antibacterial and antioxidant activity, herb of tansy and costmary may be considered as promising products used in pharmaceutical industry and veterinary medicine as antiseptic and in food industry as preservative agents. However, a domination of β-thujone in costmary directs this raw material rather to external usage.

Acknowledgment

Parts of this study was supported by Ministry of Agriculture and Rural Development, Poland, grant number HORre-029-28-22/14(92).

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