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Volatiles of roots of wild-growing and cultivated Armoracia macrocarpa and their antimicrobial activity, in comparison to horseradish, A. rusticana
Industrial Crops & Products 109 (2017) 398–403
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Research paper
Volatiles of roots of wild-growing and cultivated Armoracia macrocarpa and their antimicrobial activity, in comparison to horseradish, A. rusticana
MARK
⁎
Silvana Petrovića, , Milica Drobaca, Ljuboš Ušjaka, Vladimir Filipovićb, Marina Milenkovićc, Marjan Niketićd a
Department of Pharmacognosy, University of Belgrade—Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia Institute for Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia c Department of Microbiology and Immunology, University of Belgrade—Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia d Natural History Museum, Njegoševa 51, 11000 Belgrade, Serbia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Armoracia macrocarpa Armoracia rusticana Root volatiles GC-FID and GC–MS Antibacterial activity Anticandidal activity
The plants of the genus Armoracia P. Gaertn., B. Mey. & Scherb. (Brassicaceae) contain glucosinolates, which volatile hydrolysis products are known for their antimicrobial activity. In this work, the composition and antimicrobial activity of the root volatiles of wild-growing Armoracia macrocarpa (Waldst. & Kit.) Kit. ex Baumg., obtained by hydrodistillation, were analysed for the first time. These results were compared with those of widely used horseradish, A. rusticana P. Gaertn., B. Mey. & Scherb. Additionally, the possibility of the propagation of A. macrocarpa from the roots, and impact of its cultivation on the composition and antimicrobial activity of the root volatiles were investigated. The GC-FID and GC–MS analysis revealed that all A. macrocarpa root volatile fractions were dominated by berteroin (55.0–59.0%) and lesquerellin (34.1–36.4%), and were significantly different from horseradish root volatile fraction. In microdilution method, A. macrocarpa volatile fractions exhibited weak/no antibacterial activity, while their effect against standard strain (MICs = 4.8–9.3 μg/mL) and clinical isolates (MICs = 25–119 μg/mL) of Candida albicans was significant. Horseradish volatiles exhibited better antibacterial and slightly weaker anticandidal activity. Armoracia macrocarpa represents a new source of raw materials with potential use in pharmaceutical and food industries, as well as in cookery. Preliminary results indicated the possibility of its cultivation, which is required for its preservation and sustainable usage.
1. Introduction The genus Armoracia P. Gaertn., B. Mey. & Scherb. (Brassicaceae) includes three species distributed on wet places in the temperate zone of Eurasia, from Central Europe to Siberia. Two of them, A. rusticana P. Gaertn., B. Mey. & Scherb. and A. macrocarpa (Waldst. & Kit.) Kit. ex Baumg. are recorded for the European flora, while the third (A. sisymbrioides (DC.) N. Busch ex Ganesh) is native to Siberia (Sampliner and Miller, 2009). Horseradish, A. rusticana is naturalized in the many parts of the world, where it is often cultivated for its tick and fleshy roots. It was earlier believed that this species originates from some regions of Eastern Europe (Heywood and Zohary, 1995), but its natural populations have not been found until now (Sampliner and Miller, 2009). Because of their delicious intense pungency, horseradish roots are used as a condiment for the different types of meat, and are added to pickled vegetables and some bakery products. Besides providing piquant flavour, this spice also protects food from spoiling. Moreover, horseradish ⁎
roots were reputed to be a folk medicinal herb throughout history. They were used to treat various illnesses, e.g. infections, pain associated with rheumatism and sciatica, cardiovascular and respiratory conditions, scurvy, to improve digestion and relieve colic, and also as diuretic and aphrodisiac (Agneta et al., 2013; Herz et al., 2017; Nguyen et al., 2013). The main volatile constituents of horseradish roots, responsible for its sharp aroma and flavour, are sulphur-containing compounds − isothiocyanates. These are the products of myrosinase-catalysed glucosinolates degradation, which occurs after the crushing of material. The most abundant volatile of horseradish root is allyl isothiocyanate, followed by 2-phenylethyl isothiocyanate. Accordingly, uncrushed material is rich in glucosinolates, mostly sinigrin (yielding allyl isothiocyanate), followed by gluconasturtiin (yielding 2-phenylethyl isothiocyanate) (Agneta et al., 2013; Depree et al., 1999; Vaughn and Berhow, 2005). Some other secondary metabolites, such as flavonoids (kaempferol derivatives) and caffeic acid were also identified in the roots of horseradish (Herz et al., 2017). Numerous studies dealing with
Corresponding author. E-mail addresses: [email protected] (S. Petrović), [email protected] (M. Drobac), [email protected] (L. Ušjak), vfi[email protected] (V. Filipović), [email protected] (M. Milenković), [email protected] (M. Niketić). http://dx.doi.org/10.1016/j.indcrop.2017.08.056 Received 11 May 2017; Received in revised form 20 July 2017; Accepted 29 August 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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were also collected in order to investigate their germination ability. The testing of quality, germination energy and total germination was done in Petri dishes on filter paper at 20 °C. The analysis was done for each variant with four replicates with 100 seeds per repetition. The counting of germinated seeds was carried out in accordance with international rulebook on the quality of the seeds of agricultural plants from 1999 (ISTA-RULES, 1999). The fresh roots of horseradish, A. rusticana were purchased from a local market.
the pharmacological activities of the different extracts of horseradish root and/or its isolated compounds were undertaken in recent years, and it was demonstrated that they possess antimicrobial, chemopreventive, anti-inflammatory, gastroprotective and hypocholesterolaemic activities, justifying some of the traditional uses of this plant (Agneta et al., 2013; Herz et al., 2017; Nguyen et al., 2013; Saladino et al., 2016). Moreover, there are commercial products containing allyl isothiocyanate, intended for the treatment of infections or for food preservation (Agneta et al., 2013; Dufour et al., 2015). The other Armoracia species have distinctly smaller roots and the data about their volatiles and bioactivity are absent. Armoracia macrocarpa (Danubial horseradish) is a rare and endangered endemic species that is native to Europe and grows in the marshes of the Central and Eastern Danube Basin (Pannonian and Wallachian Plain: Slovakia, Hungary, Croatia, Serbia, Bulgaria and Romania) (Sampliner and Miller, 2009; Stevanović et al., 2011). It is morphologically similar to A. rusticana, but has less rigid leaves and larger (10–15 mm) obovate to ellipsoid siliculae with more seeds (Ball, 1993). Miller, in Krupnick (2010), supposed a closely phylogenetic relationship between A. macrocarpa and A. rusticana, and according to the author, A. rusticana may have been domesticated from A. macrocarpa in Hungary or Ukraine. Armoracia macrocarpa is classified as Data Deficient species in the IUCN Red List of Threatened Species and also listed in the Annex I of the Convention on the conservation of European wildlife and natural habitats (Bern Convention). It also has different estimation, conservation and threat status at the national levels (Stevanović et al., 2011). According to some limited data, the roots of A. macrocarpa are used as a spice for pickles (Dénes et al., 2012). Pharmaceutical and food industries follow current trend to value the natural and renewable resources and constantly search for those with advantageous properties (Saladino et al., 2016). Accordingly, the aims of this study were to analyse the volatiles of wild-growing A. macrocarpa roots for the first time, as well as to evaluate the possibility of its propagation and cultivation and their impact on the volatiles composition. Additional aim of this work was to estimate the antimicrobial activity of the volatile fractions of the roots of wild-growing and cultivated A. macrocarpa against eight bacteria (standard strains) and yeast Candida albicans (standard strain and 20 clinical isolates). The composition and antimicrobial activity of A. macrocarpa root volatiles were compared with those obtained for the root volatiles of commonly used horseradish.
2.2. Isolation of volatiles Freshly collected roots were washed with clean water, peeled, cut in small pieces, ground (in electric grinder) and hydrodistilled using Clevenger-type apparatus for 3 h. Obtained volatile fractions were dried over anhydrous sodium sulphate and kept at 4 °C, in dark, in sealed glass vials until analysis. The fresh roots of wild-growing A. macrocarpa yielded 0.19% (w/w), the roots of cultivated plants propagated via the root divisions yielded 0.04% (w/w), and the roots of cultivated plants propagated via the root cuttings yielded 0.07% (w/w) of the volatile fractions. Fresh horseradish, A. rusticana roots afforded 0.14% (w/w) of the volatiles. 2.3. GC-FID and GC–MS analysis The GC-FID and GC–MS analysis of the volatiles was performed on an Agilent 6890N Gas Chromatograph equipped with a split/splitless injector (200 °C), a FID detector and a capillary column (HP-5MS, 30 m × 0.25 mm, 0.25 μm film thickness), and coupled with an Agilent 5975C MS Detector operating in the EI mode at 70 eV. The carrier gas was He, flow 1.0 mL/min. The oven temperature was programmed linearly, increasing from 60 to 280 °C at 3 °C/min. The FID and MSD transfer line temperatures were 300 and 250 °C, respectively. Split ratio was 1:10 and the injected volume was 1 μL of 1.5% solutions of the volatile fractions in hexane (GC grade). The linear retention indices (RIs) of the volatiles were determined in relation to the homologue series of n-alkanes (C8-C40) (Fluka, Buchs, Switzerland) ran under the same operating conditions. The identification of the compounds was based on the comparison of their RIs, retention times (Rt) and mass spectra to those from the NIST/NBS 05 and Wiley (8th edition) libraries, NIST Chemistry WebBook and the literature (Al-Gendy and Lockwood, 2003; Kameoka, 1986; Kjær et al., 1963; Vaughn and Berhow, 2005). Relative percentages of the compounds were calculated based on the peak areas from the FID data (Brahmi et al., 2016; Zhang et al., 2017).
2. Materials and methods 2.1. Plant material Armoracia macrocarpa was collected on 13 September 2015, in the vicinity of Belgrade (Serbia). Voucher specimen was deposited in the Herbarium of the Natural History Museum, Belgrade (BEO) under collector number 20150901. The material was identified by Dr. Marjan Niketić, curator/botanist of the BEO. One part of freshly collected roots was used for the investigation of its volatiles, and the other part for vegetative propagation in Pančevo at the research location of the Institute for Medicinal Plant Research “Dr Josif Pančić” from Belgrade. The soil on which the researches were conducted belongs to the type − black humus-gley soil. This land has the following agro-chemical characteristics: pH value of 5.4, humus content 2.3%, total nitrogen content 0.14%, the content of P2O5 3.6 mg/100 g soil and the content of K2O 36.2 mg/100 g soil. Armoracia macrocarpa was propagated via the root divisions and the root cuttings. A year after, in the autumn growing season (on 23 November 2016), two samples of the roots of cultivated A. macrocarpa were collected: one sample of the plants propagated by the root divisions, and the other of the plants propagated by the root cuttings. The volatiles of both these root samples were also investigated. The physiologically mature seeds of wild-growing A. macrocarpa
2.4. Antimicrobial activity The antimicrobial activity was tested against nine laboratory control strains: the Gram-positive bacteria Staphylococcus aureus (ATCC 6538), S. epidermidis (ATCC 12228), Bacillus subtilis (ATCC 6633) and Enterococcus faecalis (ATCC 29212), the Gram-negative bacteria Escherichia coli (ATCC 10536), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 9027) and Salmonella abony (NCTC 6017), and the yeast Candida albicans (ATCC 10231). Additionally, the activity was tested against 20 clinical isolates of C. albicans, six of them isolated from throat swabs, two from tongue swabs, two from nasal swabs, one from skin swab, two from stool, three from cervical swabs and four from vaginal swabs. These isolates were obtained from Poliklinika Beo-Lab plus (Belgrade, Serbia). In order to determine the minimum inhibitory concentrations (MICs) of the tested volatile fractions, a broth microdilution method was used according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2016) with some modifications (Petrović et al., 2017). The tests were performed in Müller-Hinton broth for bacterial strains and in Sabouraud dextrose broth for C. albicans. Twofold serial dilutions of Armoracia volatile 399
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thiocyanates (Table 1). The GC-FID chromatograms of these fractions are shown in Fig. 1. The volatiles of one wild-growing and two cultivated samples of A. macrocarpa roots were qualitatively and quantitatively very similar, indicating that the cultivation and the method of propagation did not influence their composition. Namely, A. macrocarpa root volatile fractions were dominated by methylthioalkyl isothiocyanates, mostly berteroin, i.e. 5-methylthiopentyl isothiocyanate (55.0–59.0%), and lesquerellin, i.e. 6-methylthiohexyl isothiocyanate (34.1–36.4%). Armoracia rusticana root volatile fraction was dominated by allyl isothiocyanate (56.3%) and 2-phenylethyl isothiocyanate (37.3%), and this result is consistent with previously published data (Agneta et al., 2013; Depree et al., 1999; Nguyen et al., 2013). It should be emphasized that the volatile fractions of the roots of A. macrocarpa and A. rusticana significantly differed regarding their composition. The dominant volatiles of A. macrocarpa, berteroin and lesquerellin, were not detected in the investigated sample of A. rusticana root. These findings are in agreement with previous studies on horseradish volatiles, in which berteroin and lesquerellin were mostly not detected (Depree et al., 1999), or were identified in minor quantities (0.2 and 0.1 μg/g, respectively) (Grob and Matile, 1980). Likewise, allyl isothiocyanate, the main volatile of A. rusticana, was not detected in the investigated samples of A. macrocarpa. Similarly, 2-phenylethyl isothiocyanate, the second most abundant volatile of A. rusticana, was present in A. macrocarpa volatile fractions only in low quantities (3.0–3.5%). Accordingly, significant differences in the odour and taste of A. macrocarpa and A. rusticana roots could be explained by notable differences in their volatiles composition. While allyl isothiocyanate is strongly pungent (Agneta et al., 2013), the pungency of berteroin and lesquerellin is much less intensive (Masuda et al., 1996; Uchida et al., 2012). Regarding other cruciferous spices/vegetables, both berteroin and lesquerellin, dominant A. macrocarpa volatiles, were present in the volatile fractions of Japanese horseradish, i.e. wasabi, Wasabia japonica (Miq.) Matsum., but in much lower quantities (0.36 and 2.49% in the root, and 0.58 and 0.98% in the rhizome) (Kumagai et al., 1994). Berteroin was also detected in the volatile fractions of radish, Raphanus sativus L., rucola, Eruca sativa Mill., and broccoli, Brassica oleracea L.
fractions in 1% dimethylsulfoxide (BioScience-Grade, Carl Roth, Karlsruhe, Germany) (100 μL) were prepared in 96-well microtitre plates. Final concentrations of the volatile fractions were in the range of 137.5–1100 μg/mL for bacteria, and 4.8–119 μg/mL for C. albicans. Overnight broth cultures of each strain were prepared in a final concentration of 2 × 106 CFU/mL for bacteria, and 2 × 105 CFU/mL for C. albicans, and added to the wells (100 μL). Microbial growth was determined after incubation at 37 °C for 24 h (for bacteria), and at 26 °C for 48 h (for C. albicans). As a growth indicator, 2,3,5-triphenyl-2Htetrazolium chloride (TTC) (0.01 g/mL; Sigma-Aldrich, St. Louis, MO, USA) was used in final concentration of 0.05%. The MIC was defined as the lowest concentration of the sample at which the microorganism does not demonstrate visible growth. The MICs of standard antibiotics meropenem, amikacin and ceftriaxone (against bacteria) and amphotericin B (against C. albicans) (Sigma-Aldrich, St. Louis, MO, USA) were determined in parallel experiments. All the tests were performed in duplicate. Two negative growth controls (which contained only corresponding medium) were included for each microbial strain.
3. Results and discussion 3.1. Volatiles of Armoracia macrocarpa and A. rusticana roots By GC-FID and GC–MS, hydrodistilled volatile fractions of the roots of wild-growing A. macrocarpa, as well as of two samples of the roots of cultivated A. macrocarpa (one sample originated from the plants propagated via root divisions and the other from the plants propagated via root cuttings) were investigated. It should be noted that the propagation from the seeds was not possible. Namely, the seeds absorbed water from the germinating bed, but did not germinate. The absence of germination was confirmed by observing of the cross-section of the seeds. In this work, the composition of the volatiles of commercial A. rusticana roots was also analysed, in order to compare these two Armoracia species. Chemical analysis revealed that the volatile fractions of the roots of both investigated Armoracia species consisted of glucosinolate-hydrolysis products, isothiocyanates as dominant, followed by nitriles and/or Table 1 Composition of volatile fractions of investigated Armoracia roots (%). No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
a b c d e
Rta
5.39 5.84 6.91 8.44 11.15 15.47 18.72 19.76 21.73 21.90 23.94 24.18 26.26 26.89 28.23 31.68 35.51 36.82 39.25
RIb
865 887 928 979 1056 1164 1241 1265 1311 1315 1364 1369 1419 1436 1469 1557 1659 1695 1765
Compound
A. macrocarpa
Allyl thiocyanate Allyl isothiocyanate sec-Butyl isothiocyanate 3-Butenyl isothiocyanate 3-Methylbutyl isothiocyanate 4-Methylpentyl isothiocyanate Benzenepropane nitrile Heptyl isothiocyanate 3-Methylthiopropyl isothiocyanate 6-Methylthiohexanonitrile Benzyl isothiocyanate Octyl isothiocyanate 7-Methylthioheptanonitrile 4-Methylthiobutyl isothiocyanate 2-Phenylethyl isothiocyanate 5-Methylthiopentyl isothiocyanate (berteroin) 6-Methylthiohexyl isothiocyanate (lesquerellin) 4-Phenylbutyl isothiocyanate 7-Methylthioheptyl isothiocyanate Total identified
Rt − Retention times (min) on HP-5MS column. RI − Retention indices on HP-5MS column relative to C8-C40 n-alkanes. Relative area percentage of the compounds obtained from the FID area percent data. tr − Trace (< 0.05%). Cultivated 1–propagated by root divisions; Cultivated 2–propagated by root cuttings.
400
A. rusticana e
e
Collected in wild
Cultivated 1
Cultivated 2
– – – – – 0.2 trd tr tr 0.3 – tr 0.6 1.0 3.0 59.0 34.4 1.1 0.4 100.0
– – – – – 0.3 – tr – – – 0.1 – 1.5 3.2 58.3 34.1 2.3 0.3 100.0
– – – – – 0.2 – tr – tr – tr 0.2 1.5 3.5 55.0 36.4 2.9 0.4 100.0
5.0c 56.3 0.7 0.2 tr – tr – 0.1 – 0.1 – – – 37.3 – – 0.1 – 99.8
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Fig 1. The GC-FID chromatograms of the root volatile fractions of A. macrocarpa collected in wild (a), propagated by root divisions (b) and propagated by root cuttings (c), as well as of A. rusticana (d). The compounds that are dominant in any of the investigated fractions are numbered: allyl isothiocyanate (1), Rt = 5.84 min; 2-phenylethyl isothiocyanate (2), Rt = 28.23 min; berteroin (3), Rt = 31.68 min; lesquerellin (4), Rt = 35.51 min.
var. italica Plenck (Blažević and Mastelić 2009; Jirovetz et al., 2002; Matich et al., 2015), and lesquerellin in the volatile fraction of sweet alyssum, Lobularia maritima (L.) Desv. (Vaughn and Berhow, 2005). Health promoting benefits of berteroin and lesquerellin were demonstrated in a number of previous studies. Both of them were shown to be cancer chemopreventive agents, and exhibited antiplatelet properties and activity against Helicobacter pylori (Kumagai et al., 1994; Lamy et al., 2009; Shin et al., 2004; Yano et al., 2000). Additionally, berteroin showed anti-inflammatory (Jung et al., 2014), and lesquerellin anti-allergic effect (Yamada-Kato et al., 2012).
(MICs = 265–795 μg/mL). Such broad antibacterial spectrum could be attributed to its main volatile components, allyl isothiocyanate and 2phenylethyl isothiocyanate, and also to some of its minor glucosinolatehydrolysis products (e.g. 3-butenyl and benzyl isothiocyanate, and allyl thiocyanate). Several possible mechanisms underlying antibacterial activity of these compounds are suggested, e.g. breaking down of enzyme SeS bridges, obstruction of adenosine triphosphate (ATP) synthesis, oxidative stress-mediated DNA damage and inhibition of some cell growth and proliferation regulators (Masuda et al., 1999; Nguyen et al., 2013; Saladino et al., 2016).
3.2. Antimicrobial activity
3.2.2. Anticandidal activity Candida albicans is a yeast that normally inhabits gastrointestinal tract, mouth, vagina and skin. It can cause infection known as candidiasis, mainly in patients with compromised immune defences and in those with changed normal microbiota, e.g. after the use of antibiotics (Pommerville, 2011). Armoracia macrocarpa root volatile fractions exhibited strong activity (MICs = 4.8–9.3 μg/mL) against the ATCC strain of C. albicans, comparable to the one of amphotericin B (MIC = 3.75 μg/mL). The activity of A. rusticana volatile fraction
3.2.1. Antibacterial activity The antibacterial activity of A. macrocarpa and A. rusticana root volatile fractions was significantly different (Table 2). Regarding A. macrocarpa volatile fractions, they were active only against Bacillus subtilis and Escherichia coli (MICs = 805–1100 μg/mL). On the other hand, being consistent with previous studies (Nguyen et al., 2013), A. rusticana volatile fraction was effective against all the tested bacteria
Table 2 Antibacterial activity (minimal inhibitory concentrations, MICs) of volatile fractions of investigated Armoracia roots and antibiotics (μg/mL). Bacteria
Staphylococcus aureus S. epidermidis Bacillus subtilis Enterococcus faecalis Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella abony a b
A. macrocarpa
A. rusticana
Collected in wild
Cultivated 1
> 1100 > 1100 805 > 1100 805 > 1100 > 1100 > 1100
> 1100 > 1100 915 > 1100 1100 > 1100 > 1100 > 1100
a
a
Cultivated 2 > 1110 > 1110 1110 > 1110 1110 > 1110 > 1110 > 1110
Cultivated 1–propagated by root divisions; Cultivated 2–propagated by root cuttings. n.t. − not tested.
401
265 265 265 265 265 795 530 530
Referent antibiotics Meropenem
Amikacin
Ceftriaxone
0.12 < 0.05 1.80 2.00 0.12 2.50 0.75 2.50
5.00 0.50 n.t.b n.t. 0.75 5.00 2.50 4.60
0.12 0.12 0.05 n.t. 0.12 0.75 n.t. 2.50
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phylogenetically and morphologically related, A. macrocarpa and A. rusticana were shown to be phytochemically significantly different in regard to the composition of their root volatile fractions, resulting in more prominent anticandidal activity of those of A. macrocarpa.
Table 3 MICs of volatile fractions of investigated Armoracia roots and amphotericin B (Amph B) against clinical isolates and ATCC strain of Candida albicans (μg/mL). C. albicans
A. macrocarpa
Clinical isolates
Collected in wild
Cultivated 1a
Cultivated 2a
throat throat throat throat throat throat tongue tongue nose nose skin stool stool cervix cervix cervix vagina vagina vagina vagina ATCC strain
25 100 25 25 25 25 50 50 100 25 25 50 25 50 25 25 100 50 50 25 7.8
28 28 28 28 28 28 28 28 57 28 28 28 28 28 28 28 57 28 57 28 4.8
59 30 59 30 30 30 30 30 30 30 59 30 30 30 30 30 119 59 59 30 9.3
a
A. rusticana
Amph B
Conflict of interest > 192 96 48 96 48 96 48 48 192 48 48 96 96 48 192 96 192 96 192 > 192 28.7
The authors declare no financial or other conflict of interest.
3.75 0.57 0.76 1.88 1.88 3.75 0.94 1.88 0.94 1.88 3.75 2.82 1.88 1.88 1.88 1.88 1.88 1.88 3.75 0.94 3.75
Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia [grant numbers 173021 and III46006]. We are grateful to Dr Mirjana Kovačević (Poliklinika Beo-Lab plus, Belgrade, Serbia) for providing clinical isolates of C. albicans. References Agneta, R., Möllers, C., Rivelli, A.R., 2013. Horseradish (Armoracia rusticana), a neglected medical and condiment species with a relevant glucosinolate profile: a review. Genet. Resour. Crop Evol. 60 (7), 1923–1943. Al-Gendy, A.A., Lockwood, G.B., 2003. GC–MS analysis of volatile hydrolysis products from glucosinolates in Farsetia aegyptia var. ovalis. Flavour Frag. J. 18 (2), 148–152. Ball, P.W., 1993. Armoracia. In: Tutin, T.G., Burges, A., Chater, A.O., Edmondson, J.R., Heywood, V.H., Moore, D.M., Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.), Flora Europaea 1, second ed. Cambridge University Press, Cambridge p. 346. Bertóti, R., Vasas, G., Gonda, S., Nguyen, N.M., Szőke, É., Jakab, Á., Pócsi, I., Emri, T., 2016. Glutathione protects Candida albicans against horseradish volatile oil. J. Basic Microb. 56 (10), 1071–1079. Blažević, I., Mastelić, J., 2009. Glucosinolate degradation products and other bound and free volatiles in the leaves and roots of radish (Raphanus sativus L.). Food Chem. 113 (1), 96–102. Brahmi, F., Abdenour, A., Bruno, M., Silvia, P., Alessandra, P., Danilo, F., Drifa, Y.G., Fahmi, E.M., Khodir, M., Mohamed, C., 2016. Chemical composition and in vitro antimicrobial: insecticidal and antioxidant activities of the essential oils of Mentha pulegium L. and Mentha rotundifolia (L.) Huds growing in Algeria. Ind. Crop. Prod. 88, 96–105. CLSI, 2016. Performance Standards for Antimicrobial Susceptibility Testing, 26th Informational Supplement. Approved Standard. CLSI Document M100-S. Clinical and Laboratory Standards Institute, Wayne, PA. Dénes, A., Papp, N., Babai, D., Czúcz, B., Molnár, Z., 2012. Wild plants used for food by Hungarian ethnic groups living in the Carpathian Basin. Acta Soc. Bot. Pol. 81 (4), 381–396. Depree, J.A., Howard, T.M., Savage, G.P., 1999. Flavour and pharmaceutical properties of the volatile sulphur compounds of wasabi (Wasabia japonica). Food Res. Int. 31 (5), 329–337. Dufour, V., Stahl, M., Baysse, C., 2015. The antibacterial properties of isothiocyanates. Microbiol. SGM 161 (2), 229–243. Grob, K., Matile, P., 1980. Capillary GC of glucosinolate-derived horseradish constituents. Phytochemistry 19 (8), 1789–1793. Herz, C., Tran, H.T.T., Márton, M.R., Maul, R., Baldermann, S., Schreiner, M., Lamy, E., 2017. Evaluation of an aqueous extract from horseradish root (Armoracia rusticana radix) against lipopolysaccharide-induced cellular inflammation reaction. Evid-Based Complement. Altern. Med. 2017 (12), 1–10. Heywood, V.H., Zohary, D., 1995. A catalogue of the wild relatives of cultivated plants native to Europe. Fl. Medit. 5, 375–415. ISTA-RULES, 1999. International Seed Testing Association − Proposals for the International Rules for Seed Testing. ISTA, Bassersdorf, Switzerland. Jirovetz, L., Smith, D., Buchbauer, G., 2002. Aroma compound analysis of Eruca sativa (Brassicaceae) SPME headspace leaf samples using GC, GC–MS, and olfactometry. J. Agr. Food Chem. 16, 4643–4646. Jung, Y.J., Jung, J.I., Cho, H.J., Choi, M.S., Sung, M.K., Yu, R., Kang, Y.H., Park, J.H.Y., 2014. Berteroin present in cruciferous vegetables exerts potent anti-inflammatory properties in murine macrophages and mouse skin. Int. J. Mol. Sci. 15 (11), 20686–20705. Kameoka, H., 1986. GC–MS method for volatile flavor components of foods. In: Linskens, H.F., Jackson, J.F. (Eds.), Gas Chromatography/Mass Spectrometry. Springer-Verlag, Berlin Heidelberg, pp. 254–276. Kjær, A., Ohashi, M., Wilson, J.M., Djerassi, C., 1963. Mass spectra of isothiocyanates. Acta Chem. Scand. 17 (8), 2143–2154. Krupnick, G.A., 2010. Food, glorious food. Plant Press 13 (4), 1–18. Kumagai, H., Kashima, N., Seki, T., Sakurai, H., Ishii, K., Ariga, T., 1994. Analysis of volatile components in essential oil of upland wasabi and their inhibitory effects on platelet aggregation. Biosci. Biotechnol. Biochem. 58 (12), 2131–2135. Lamy, E., Crößmann, C., Saeed, A., Schreiner, P.R., Kotke, M., Mersch-Sundermann, V., 2009. Three structurally homologous isothiocyanates exert “Janus” characteristics in human HepG2 cells. Environ. Mol. Mutagen. 50 (3), 164–170. Masuda, H., Harada, Y., Tanaka, K., Nakajima, M., Tabeta, H., 1996. Characteristic odorants of wasabi (Wasabia japonica Matum), Japanese horseradish, in comparison
Cultivated 1–propagated by root divisions; Cultivated 2–propagated by root cuttings.
against this strain was also significant (MIC = 28.7 μg/mL). Since the antimicrobial activity at concentrations bellow 100 μg/mL is very interesting (Ríos and Recio, 2005), the anticandidal activity of the root volatile fractions of the investigated Armoracia species was also tested on 20C. albicans clinical isolates, obtained from throat, tongue, nasal, skin, cervical and vaginal swabs, and stool (Table 3). Armoracia macrocarpa fraction exhibited significant activity against all the tested clinical isolates (MICs = 25–119 μg/mL), while A. rusticana fraction showed somewhat weaker effect against the most of the tested isolates (MICs = 48–192 μg/mL). In some earlier investigations, the anticandidal effect of A. rusticana root volatile fraction was also demonstrated, such as the activity of its dominant alkyl/aryl isothiocyanates (Bertóti et al., 2016; Saladino et al., 2016). It was also demonstrated that methylthioalkyl isothiocyanates berteroin, lesquerellin, as well as 7-methylthioheptyl isothiocyanate possessed comparable or even higher anticandidal activity than alkyl/aryl isothiocyanates (Masuda et al., 1999). These findings are in agreement with the results obtained in the present study, which showed that the volatile fractions of A. macrocarpa exhibited somewhat stronger effect than the one of A. rusticana. Demonstrated selective antimicrobial activity of A. macrocarpa root volatile fractions is very interesting because of its potential application in the treatment of candidiasis caused by antibacterial therapy.
4. Conclusions Results obtained in this study showed that the roots of A. macrocarpa represent new raw material which can be used in cookery and food industry. Their less intensive pungency in comparison to horseradish roots widens the range of the consumers of Armoracia originating condiments. Based on the strong anticandidal activity of the root volatiles, A. macrocarpa can be valuable for pharmaceutical industry as well. Demonstrated preliminary successful vegetative propagation and cultivation of A. macrocarpa is of particular importance not only for the possible preservation of this rare and endangered species, but also for establishing it as a potential renewable natural resource, which can be rationally exploited in the future. Additionally, although closely 402
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Brassicaceae) and its wild relatives (Armoracia spp.): reproductive biology and local uses in their native ranges. Econ. Bot. 63 (3), 303–313. Shin, I.S., Masuda, H., Naohide, K., 2004. Bactericidal activity of wasabi (Wasabia japonica) against Helicobacter pylori. Int. J. Food Microbiol. 94 (3), 255–261. Stevanović, V., Vörösváry, G., Eliáš, P., Strajeru, S., 2011. Armoracia macrocarpa. The IUCN Red List of Threatened Species 2011: e.T165173A5986073. Uchida, K., Miura, Y., Nagai, M., Tominaga, M., 2012. Isothiocyanates from Wasabia japonica activate transient receptor potential ankyrin 1 channel. Chem. Senses 37 (9), 809–818. Vaughn, S.F., Berhow, M.A., 2005. Glucosinolate hydrolysis products from various plant sources: pH effects, isolation, and purification. Ind. Crop. Prod. 21 (2), 193–202. Yamada-Kato, T., Nagai, M., Ohnishi, M., Yoshida, K., 2012. Inhibitory effects of wasabi isothiocyanates on chemical mediator release in RBL-2H3 rat basophilic leukemia cells. J. Nutr. Sci. Vitaminol. 58 (4), 303–307. Yano, T., Yajima, S., Virgona, N., Yano, Y., Otani, S., Kumagai, H., Sakurai, H., Kishimoto, M., Ichikawa, T., 2000. The effect of 6-methylthiohexyl isothiocyanate isolated from Wasabia japonica (wasabi) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-buatnone-induced lung tumorigenesis in mice. Cancer Lett. 155 (2), 115–120. Zhang, L., Yang, Z., Wei, J., Su, P., Pan, W., Zheng, X., Zhang, K., Lin, L., Tang, J., Fang, Y., Du, Z., 2017. Essential oil composition and bioactivity variation in wild-growing populations of Curcuma phaeocaulis Valeton collected from China. Ind. Crop. Prod. 103, 274–282.
with those of horseradish (Armoracia rusticana). In: Takeoka, G.R., Teranishi, R., Williams, P.J., Kobayashi, A. (Eds.), Biotechnology for Improved Foods and Flavors. American Chemical Society, Washington DC, pp. 67–78. Masuda, H., Harada, Y., Inoue, T., Kishimoto, N., Tano, T., 1999. Wasabi, japanese horseradish, and horseradish. In: Shahidi, F., Ho, C.-T. (Eds.), Flavor Chemistry of Ethnic Foods. Springer, US New York, pp. 85–96. Matich, A.J., McKenzie, M.J., Lill, R.E., McGhie, T.K., Chen, R.K.Y., Rowan, D.D., 2015. Distribution of selenoglucosinolates and their metabolites in Brassica treated with sodium selenate. J. Agr. Food Chem. 63 (7), 1896–1905. Nguyen, N.M., Gonda, S., Vasas, G., 2013. A review on the phytochemical composition and potential medicinal uses of horseradish (Armoracia rusticana) root. Food Rev. Int. 29 (3), 261–275. Petrović, S., Ušjak, L., Milenković, M., Arsenijević, J., Drobac, M., Drndarević, A., Niketić, M., 2017. Thymus dacicus as a new source of antioxidant and antimicrobial metabolites. J. Funct. Foods 28, 114–121. Pommerville, J.C., 2011. Alcamo’s Fundamentals of Microbiology, ninth ed. Jones and Bartlett Publishers, Sudbury. Ríos, J.L., Recio, M.C., 2005. Medicinal plants and antimicrobial activity. J. Ethnopharmacol. 100, 80–84. Saladino, F., Bordin, K., Luciano, F.B., Franzón, M.F., Mañes, J., Meca, G., 2016. Antimicrobial activity of the glucosinolates. In: Mérillon, J.-M., Ramawat, K.G. (Eds.), Glucosinolates. Springer International Publishing AG, Basel, pp. 1–26. Sampliner, D., Miller, A., 2009. Ethnobotany of horseradish (Armoracia rusticana,
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Research paper
Volatiles of roots of wild-growing and cultivated Armoracia macrocarpa and their antimicrobial activity, in comparison to horseradish, A. rusticana
MARK
⁎
Silvana Petrovića, , Milica Drobaca, Ljuboš Ušjaka, Vladimir Filipovićb, Marina Milenkovićc, Marjan Niketićd a
Department of Pharmacognosy, University of Belgrade—Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia Institute for Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia c Department of Microbiology and Immunology, University of Belgrade—Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia d Natural History Museum, Njegoševa 51, 11000 Belgrade, Serbia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Armoracia macrocarpa Armoracia rusticana Root volatiles GC-FID and GC–MS Antibacterial activity Anticandidal activity
The plants of the genus Armoracia P. Gaertn., B. Mey. & Scherb. (Brassicaceae) contain glucosinolates, which volatile hydrolysis products are known for their antimicrobial activity. In this work, the composition and antimicrobial activity of the root volatiles of wild-growing Armoracia macrocarpa (Waldst. & Kit.) Kit. ex Baumg., obtained by hydrodistillation, were analysed for the first time. These results were compared with those of widely used horseradish, A. rusticana P. Gaertn., B. Mey. & Scherb. Additionally, the possibility of the propagation of A. macrocarpa from the roots, and impact of its cultivation on the composition and antimicrobial activity of the root volatiles were investigated. The GC-FID and GC–MS analysis revealed that all A. macrocarpa root volatile fractions were dominated by berteroin (55.0–59.0%) and lesquerellin (34.1–36.4%), and were significantly different from horseradish root volatile fraction. In microdilution method, A. macrocarpa volatile fractions exhibited weak/no antibacterial activity, while their effect against standard strain (MICs = 4.8–9.3 μg/mL) and clinical isolates (MICs = 25–119 μg/mL) of Candida albicans was significant. Horseradish volatiles exhibited better antibacterial and slightly weaker anticandidal activity. Armoracia macrocarpa represents a new source of raw materials with potential use in pharmaceutical and food industries, as well as in cookery. Preliminary results indicated the possibility of its cultivation, which is required for its preservation and sustainable usage.
1. Introduction The genus Armoracia P. Gaertn., B. Mey. & Scherb. (Brassicaceae) includes three species distributed on wet places in the temperate zone of Eurasia, from Central Europe to Siberia. Two of them, A. rusticana P. Gaertn., B. Mey. & Scherb. and A. macrocarpa (Waldst. & Kit.) Kit. ex Baumg. are recorded for the European flora, while the third (A. sisymbrioides (DC.) N. Busch ex Ganesh) is native to Siberia (Sampliner and Miller, 2009). Horseradish, A. rusticana is naturalized in the many parts of the world, where it is often cultivated for its tick and fleshy roots. It was earlier believed that this species originates from some regions of Eastern Europe (Heywood and Zohary, 1995), but its natural populations have not been found until now (Sampliner and Miller, 2009). Because of their delicious intense pungency, horseradish roots are used as a condiment for the different types of meat, and are added to pickled vegetables and some bakery products. Besides providing piquant flavour, this spice also protects food from spoiling. Moreover, horseradish ⁎
roots were reputed to be a folk medicinal herb throughout history. They were used to treat various illnesses, e.g. infections, pain associated with rheumatism and sciatica, cardiovascular and respiratory conditions, scurvy, to improve digestion and relieve colic, and also as diuretic and aphrodisiac (Agneta et al., 2013; Herz et al., 2017; Nguyen et al., 2013). The main volatile constituents of horseradish roots, responsible for its sharp aroma and flavour, are sulphur-containing compounds − isothiocyanates. These are the products of myrosinase-catalysed glucosinolates degradation, which occurs after the crushing of material. The most abundant volatile of horseradish root is allyl isothiocyanate, followed by 2-phenylethyl isothiocyanate. Accordingly, uncrushed material is rich in glucosinolates, mostly sinigrin (yielding allyl isothiocyanate), followed by gluconasturtiin (yielding 2-phenylethyl isothiocyanate) (Agneta et al., 2013; Depree et al., 1999; Vaughn and Berhow, 2005). Some other secondary metabolites, such as flavonoids (kaempferol derivatives) and caffeic acid were also identified in the roots of horseradish (Herz et al., 2017). Numerous studies dealing with
Corresponding author. E-mail addresses: [email protected] (S. Petrović), [email protected] (M. Drobac), [email protected] (L. Ušjak), vfi[email protected] (V. Filipović), [email protected] (M. Milenković), [email protected] (M. Niketić). http://dx.doi.org/10.1016/j.indcrop.2017.08.056 Received 11 May 2017; Received in revised form 20 July 2017; Accepted 29 August 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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were also collected in order to investigate their germination ability. The testing of quality, germination energy and total germination was done in Petri dishes on filter paper at 20 °C. The analysis was done for each variant with four replicates with 100 seeds per repetition. The counting of germinated seeds was carried out in accordance with international rulebook on the quality of the seeds of agricultural plants from 1999 (ISTA-RULES, 1999). The fresh roots of horseradish, A. rusticana were purchased from a local market.
the pharmacological activities of the different extracts of horseradish root and/or its isolated compounds were undertaken in recent years, and it was demonstrated that they possess antimicrobial, chemopreventive, anti-inflammatory, gastroprotective and hypocholesterolaemic activities, justifying some of the traditional uses of this plant (Agneta et al., 2013; Herz et al., 2017; Nguyen et al., 2013; Saladino et al., 2016). Moreover, there are commercial products containing allyl isothiocyanate, intended for the treatment of infections or for food preservation (Agneta et al., 2013; Dufour et al., 2015). The other Armoracia species have distinctly smaller roots and the data about their volatiles and bioactivity are absent. Armoracia macrocarpa (Danubial horseradish) is a rare and endangered endemic species that is native to Europe and grows in the marshes of the Central and Eastern Danube Basin (Pannonian and Wallachian Plain: Slovakia, Hungary, Croatia, Serbia, Bulgaria and Romania) (Sampliner and Miller, 2009; Stevanović et al., 2011). It is morphologically similar to A. rusticana, but has less rigid leaves and larger (10–15 mm) obovate to ellipsoid siliculae with more seeds (Ball, 1993). Miller, in Krupnick (2010), supposed a closely phylogenetic relationship between A. macrocarpa and A. rusticana, and according to the author, A. rusticana may have been domesticated from A. macrocarpa in Hungary or Ukraine. Armoracia macrocarpa is classified as Data Deficient species in the IUCN Red List of Threatened Species and also listed in the Annex I of the Convention on the conservation of European wildlife and natural habitats (Bern Convention). It also has different estimation, conservation and threat status at the national levels (Stevanović et al., 2011). According to some limited data, the roots of A. macrocarpa are used as a spice for pickles (Dénes et al., 2012). Pharmaceutical and food industries follow current trend to value the natural and renewable resources and constantly search for those with advantageous properties (Saladino et al., 2016). Accordingly, the aims of this study were to analyse the volatiles of wild-growing A. macrocarpa roots for the first time, as well as to evaluate the possibility of its propagation and cultivation and their impact on the volatiles composition. Additional aim of this work was to estimate the antimicrobial activity of the volatile fractions of the roots of wild-growing and cultivated A. macrocarpa against eight bacteria (standard strains) and yeast Candida albicans (standard strain and 20 clinical isolates). The composition and antimicrobial activity of A. macrocarpa root volatiles were compared with those obtained for the root volatiles of commonly used horseradish.
2.2. Isolation of volatiles Freshly collected roots were washed with clean water, peeled, cut in small pieces, ground (in electric grinder) and hydrodistilled using Clevenger-type apparatus for 3 h. Obtained volatile fractions were dried over anhydrous sodium sulphate and kept at 4 °C, in dark, in sealed glass vials until analysis. The fresh roots of wild-growing A. macrocarpa yielded 0.19% (w/w), the roots of cultivated plants propagated via the root divisions yielded 0.04% (w/w), and the roots of cultivated plants propagated via the root cuttings yielded 0.07% (w/w) of the volatile fractions. Fresh horseradish, A. rusticana roots afforded 0.14% (w/w) of the volatiles. 2.3. GC-FID and GC–MS analysis The GC-FID and GC–MS analysis of the volatiles was performed on an Agilent 6890N Gas Chromatograph equipped with a split/splitless injector (200 °C), a FID detector and a capillary column (HP-5MS, 30 m × 0.25 mm, 0.25 μm film thickness), and coupled with an Agilent 5975C MS Detector operating in the EI mode at 70 eV. The carrier gas was He, flow 1.0 mL/min. The oven temperature was programmed linearly, increasing from 60 to 280 °C at 3 °C/min. The FID and MSD transfer line temperatures were 300 and 250 °C, respectively. Split ratio was 1:10 and the injected volume was 1 μL of 1.5% solutions of the volatile fractions in hexane (GC grade). The linear retention indices (RIs) of the volatiles were determined in relation to the homologue series of n-alkanes (C8-C40) (Fluka, Buchs, Switzerland) ran under the same operating conditions. The identification of the compounds was based on the comparison of their RIs, retention times (Rt) and mass spectra to those from the NIST/NBS 05 and Wiley (8th edition) libraries, NIST Chemistry WebBook and the literature (Al-Gendy and Lockwood, 2003; Kameoka, 1986; Kjær et al., 1963; Vaughn and Berhow, 2005). Relative percentages of the compounds were calculated based on the peak areas from the FID data (Brahmi et al., 2016; Zhang et al., 2017).
2. Materials and methods 2.1. Plant material Armoracia macrocarpa was collected on 13 September 2015, in the vicinity of Belgrade (Serbia). Voucher specimen was deposited in the Herbarium of the Natural History Museum, Belgrade (BEO) under collector number 20150901. The material was identified by Dr. Marjan Niketić, curator/botanist of the BEO. One part of freshly collected roots was used for the investigation of its volatiles, and the other part for vegetative propagation in Pančevo at the research location of the Institute for Medicinal Plant Research “Dr Josif Pančić” from Belgrade. The soil on which the researches were conducted belongs to the type − black humus-gley soil. This land has the following agro-chemical characteristics: pH value of 5.4, humus content 2.3%, total nitrogen content 0.14%, the content of P2O5 3.6 mg/100 g soil and the content of K2O 36.2 mg/100 g soil. Armoracia macrocarpa was propagated via the root divisions and the root cuttings. A year after, in the autumn growing season (on 23 November 2016), two samples of the roots of cultivated A. macrocarpa were collected: one sample of the plants propagated by the root divisions, and the other of the plants propagated by the root cuttings. The volatiles of both these root samples were also investigated. The physiologically mature seeds of wild-growing A. macrocarpa
2.4. Antimicrobial activity The antimicrobial activity was tested against nine laboratory control strains: the Gram-positive bacteria Staphylococcus aureus (ATCC 6538), S. epidermidis (ATCC 12228), Bacillus subtilis (ATCC 6633) and Enterococcus faecalis (ATCC 29212), the Gram-negative bacteria Escherichia coli (ATCC 10536), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 9027) and Salmonella abony (NCTC 6017), and the yeast Candida albicans (ATCC 10231). Additionally, the activity was tested against 20 clinical isolates of C. albicans, six of them isolated from throat swabs, two from tongue swabs, two from nasal swabs, one from skin swab, two from stool, three from cervical swabs and four from vaginal swabs. These isolates were obtained from Poliklinika Beo-Lab plus (Belgrade, Serbia). In order to determine the minimum inhibitory concentrations (MICs) of the tested volatile fractions, a broth microdilution method was used according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2016) with some modifications (Petrović et al., 2017). The tests were performed in Müller-Hinton broth for bacterial strains and in Sabouraud dextrose broth for C. albicans. Twofold serial dilutions of Armoracia volatile 399
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thiocyanates (Table 1). The GC-FID chromatograms of these fractions are shown in Fig. 1. The volatiles of one wild-growing and two cultivated samples of A. macrocarpa roots were qualitatively and quantitatively very similar, indicating that the cultivation and the method of propagation did not influence their composition. Namely, A. macrocarpa root volatile fractions were dominated by methylthioalkyl isothiocyanates, mostly berteroin, i.e. 5-methylthiopentyl isothiocyanate (55.0–59.0%), and lesquerellin, i.e. 6-methylthiohexyl isothiocyanate (34.1–36.4%). Armoracia rusticana root volatile fraction was dominated by allyl isothiocyanate (56.3%) and 2-phenylethyl isothiocyanate (37.3%), and this result is consistent with previously published data (Agneta et al., 2013; Depree et al., 1999; Nguyen et al., 2013). It should be emphasized that the volatile fractions of the roots of A. macrocarpa and A. rusticana significantly differed regarding their composition. The dominant volatiles of A. macrocarpa, berteroin and lesquerellin, were not detected in the investigated sample of A. rusticana root. These findings are in agreement with previous studies on horseradish volatiles, in which berteroin and lesquerellin were mostly not detected (Depree et al., 1999), or were identified in minor quantities (0.2 and 0.1 μg/g, respectively) (Grob and Matile, 1980). Likewise, allyl isothiocyanate, the main volatile of A. rusticana, was not detected in the investigated samples of A. macrocarpa. Similarly, 2-phenylethyl isothiocyanate, the second most abundant volatile of A. rusticana, was present in A. macrocarpa volatile fractions only in low quantities (3.0–3.5%). Accordingly, significant differences in the odour and taste of A. macrocarpa and A. rusticana roots could be explained by notable differences in their volatiles composition. While allyl isothiocyanate is strongly pungent (Agneta et al., 2013), the pungency of berteroin and lesquerellin is much less intensive (Masuda et al., 1996; Uchida et al., 2012). Regarding other cruciferous spices/vegetables, both berteroin and lesquerellin, dominant A. macrocarpa volatiles, were present in the volatile fractions of Japanese horseradish, i.e. wasabi, Wasabia japonica (Miq.) Matsum., but in much lower quantities (0.36 and 2.49% in the root, and 0.58 and 0.98% in the rhizome) (Kumagai et al., 1994). Berteroin was also detected in the volatile fractions of radish, Raphanus sativus L., rucola, Eruca sativa Mill., and broccoli, Brassica oleracea L.
fractions in 1% dimethylsulfoxide (BioScience-Grade, Carl Roth, Karlsruhe, Germany) (100 μL) were prepared in 96-well microtitre plates. Final concentrations of the volatile fractions were in the range of 137.5–1100 μg/mL for bacteria, and 4.8–119 μg/mL for C. albicans. Overnight broth cultures of each strain were prepared in a final concentration of 2 × 106 CFU/mL for bacteria, and 2 × 105 CFU/mL for C. albicans, and added to the wells (100 μL). Microbial growth was determined after incubation at 37 °C for 24 h (for bacteria), and at 26 °C for 48 h (for C. albicans). As a growth indicator, 2,3,5-triphenyl-2Htetrazolium chloride (TTC) (0.01 g/mL; Sigma-Aldrich, St. Louis, MO, USA) was used in final concentration of 0.05%. The MIC was defined as the lowest concentration of the sample at which the microorganism does not demonstrate visible growth. The MICs of standard antibiotics meropenem, amikacin and ceftriaxone (against bacteria) and amphotericin B (against C. albicans) (Sigma-Aldrich, St. Louis, MO, USA) were determined in parallel experiments. All the tests were performed in duplicate. Two negative growth controls (which contained only corresponding medium) were included for each microbial strain.
3. Results and discussion 3.1. Volatiles of Armoracia macrocarpa and A. rusticana roots By GC-FID and GC–MS, hydrodistilled volatile fractions of the roots of wild-growing A. macrocarpa, as well as of two samples of the roots of cultivated A. macrocarpa (one sample originated from the plants propagated via root divisions and the other from the plants propagated via root cuttings) were investigated. It should be noted that the propagation from the seeds was not possible. Namely, the seeds absorbed water from the germinating bed, but did not germinate. The absence of germination was confirmed by observing of the cross-section of the seeds. In this work, the composition of the volatiles of commercial A. rusticana roots was also analysed, in order to compare these two Armoracia species. Chemical analysis revealed that the volatile fractions of the roots of both investigated Armoracia species consisted of glucosinolate-hydrolysis products, isothiocyanates as dominant, followed by nitriles and/or Table 1 Composition of volatile fractions of investigated Armoracia roots (%). No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
a b c d e
Rta
5.39 5.84 6.91 8.44 11.15 15.47 18.72 19.76 21.73 21.90 23.94 24.18 26.26 26.89 28.23 31.68 35.51 36.82 39.25
RIb
865 887 928 979 1056 1164 1241 1265 1311 1315 1364 1369 1419 1436 1469 1557 1659 1695 1765
Compound
A. macrocarpa
Allyl thiocyanate Allyl isothiocyanate sec-Butyl isothiocyanate 3-Butenyl isothiocyanate 3-Methylbutyl isothiocyanate 4-Methylpentyl isothiocyanate Benzenepropane nitrile Heptyl isothiocyanate 3-Methylthiopropyl isothiocyanate 6-Methylthiohexanonitrile Benzyl isothiocyanate Octyl isothiocyanate 7-Methylthioheptanonitrile 4-Methylthiobutyl isothiocyanate 2-Phenylethyl isothiocyanate 5-Methylthiopentyl isothiocyanate (berteroin) 6-Methylthiohexyl isothiocyanate (lesquerellin) 4-Phenylbutyl isothiocyanate 7-Methylthioheptyl isothiocyanate Total identified
Rt − Retention times (min) on HP-5MS column. RI − Retention indices on HP-5MS column relative to C8-C40 n-alkanes. Relative area percentage of the compounds obtained from the FID area percent data. tr − Trace (< 0.05%). Cultivated 1–propagated by root divisions; Cultivated 2–propagated by root cuttings.
400
A. rusticana e
e
Collected in wild
Cultivated 1
Cultivated 2
– – – – – 0.2 trd tr tr 0.3 – tr 0.6 1.0 3.0 59.0 34.4 1.1 0.4 100.0
– – – – – 0.3 – tr – – – 0.1 – 1.5 3.2 58.3 34.1 2.3 0.3 100.0
– – – – – 0.2 – tr – tr – tr 0.2 1.5 3.5 55.0 36.4 2.9 0.4 100.0
5.0c 56.3 0.7 0.2 tr – tr – 0.1 – 0.1 – – – 37.3 – – 0.1 – 99.8
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Fig 1. The GC-FID chromatograms of the root volatile fractions of A. macrocarpa collected in wild (a), propagated by root divisions (b) and propagated by root cuttings (c), as well as of A. rusticana (d). The compounds that are dominant in any of the investigated fractions are numbered: allyl isothiocyanate (1), Rt = 5.84 min; 2-phenylethyl isothiocyanate (2), Rt = 28.23 min; berteroin (3), Rt = 31.68 min; lesquerellin (4), Rt = 35.51 min.
var. italica Plenck (Blažević and Mastelić 2009; Jirovetz et al., 2002; Matich et al., 2015), and lesquerellin in the volatile fraction of sweet alyssum, Lobularia maritima (L.) Desv. (Vaughn and Berhow, 2005). Health promoting benefits of berteroin and lesquerellin were demonstrated in a number of previous studies. Both of them were shown to be cancer chemopreventive agents, and exhibited antiplatelet properties and activity against Helicobacter pylori (Kumagai et al., 1994; Lamy et al., 2009; Shin et al., 2004; Yano et al., 2000). Additionally, berteroin showed anti-inflammatory (Jung et al., 2014), and lesquerellin anti-allergic effect (Yamada-Kato et al., 2012).
(MICs = 265–795 μg/mL). Such broad antibacterial spectrum could be attributed to its main volatile components, allyl isothiocyanate and 2phenylethyl isothiocyanate, and also to some of its minor glucosinolatehydrolysis products (e.g. 3-butenyl and benzyl isothiocyanate, and allyl thiocyanate). Several possible mechanisms underlying antibacterial activity of these compounds are suggested, e.g. breaking down of enzyme SeS bridges, obstruction of adenosine triphosphate (ATP) synthesis, oxidative stress-mediated DNA damage and inhibition of some cell growth and proliferation regulators (Masuda et al., 1999; Nguyen et al., 2013; Saladino et al., 2016).
3.2. Antimicrobial activity
3.2.2. Anticandidal activity Candida albicans is a yeast that normally inhabits gastrointestinal tract, mouth, vagina and skin. It can cause infection known as candidiasis, mainly in patients with compromised immune defences and in those with changed normal microbiota, e.g. after the use of antibiotics (Pommerville, 2011). Armoracia macrocarpa root volatile fractions exhibited strong activity (MICs = 4.8–9.3 μg/mL) against the ATCC strain of C. albicans, comparable to the one of amphotericin B (MIC = 3.75 μg/mL). The activity of A. rusticana volatile fraction
3.2.1. Antibacterial activity The antibacterial activity of A. macrocarpa and A. rusticana root volatile fractions was significantly different (Table 2). Regarding A. macrocarpa volatile fractions, they were active only against Bacillus subtilis and Escherichia coli (MICs = 805–1100 μg/mL). On the other hand, being consistent with previous studies (Nguyen et al., 2013), A. rusticana volatile fraction was effective against all the tested bacteria
Table 2 Antibacterial activity (minimal inhibitory concentrations, MICs) of volatile fractions of investigated Armoracia roots and antibiotics (μg/mL). Bacteria
Staphylococcus aureus S. epidermidis Bacillus subtilis Enterococcus faecalis Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella abony a b
A. macrocarpa
A. rusticana
Collected in wild
Cultivated 1
> 1100 > 1100 805 > 1100 805 > 1100 > 1100 > 1100
> 1100 > 1100 915 > 1100 1100 > 1100 > 1100 > 1100
a
a
Cultivated 2 > 1110 > 1110 1110 > 1110 1110 > 1110 > 1110 > 1110
Cultivated 1–propagated by root divisions; Cultivated 2–propagated by root cuttings. n.t. − not tested.
401
265 265 265 265 265 795 530 530
Referent antibiotics Meropenem
Amikacin
Ceftriaxone
0.12 < 0.05 1.80 2.00 0.12 2.50 0.75 2.50
5.00 0.50 n.t.b n.t. 0.75 5.00 2.50 4.60
0.12 0.12 0.05 n.t. 0.12 0.75 n.t. 2.50
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phylogenetically and morphologically related, A. macrocarpa and A. rusticana were shown to be phytochemically significantly different in regard to the composition of their root volatile fractions, resulting in more prominent anticandidal activity of those of A. macrocarpa.
Table 3 MICs of volatile fractions of investigated Armoracia roots and amphotericin B (Amph B) against clinical isolates and ATCC strain of Candida albicans (μg/mL). C. albicans
A. macrocarpa
Clinical isolates
Collected in wild
Cultivated 1a
Cultivated 2a
throat throat throat throat throat throat tongue tongue nose nose skin stool stool cervix cervix cervix vagina vagina vagina vagina ATCC strain
25 100 25 25 25 25 50 50 100 25 25 50 25 50 25 25 100 50 50 25 7.8
28 28 28 28 28 28 28 28 57 28 28 28 28 28 28 28 57 28 57 28 4.8
59 30 59 30 30 30 30 30 30 30 59 30 30 30 30 30 119 59 59 30 9.3
a
A. rusticana
Amph B
Conflict of interest > 192 96 48 96 48 96 48 48 192 48 48 96 96 48 192 96 192 96 192 > 192 28.7
The authors declare no financial or other conflict of interest.
3.75 0.57 0.76 1.88 1.88 3.75 0.94 1.88 0.94 1.88 3.75 2.82 1.88 1.88 1.88 1.88 1.88 1.88 3.75 0.94 3.75
Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia [grant numbers 173021 and III46006]. We are grateful to Dr Mirjana Kovačević (Poliklinika Beo-Lab plus, Belgrade, Serbia) for providing clinical isolates of C. albicans. References Agneta, R., Möllers, C., Rivelli, A.R., 2013. Horseradish (Armoracia rusticana), a neglected medical and condiment species with a relevant glucosinolate profile: a review. Genet. Resour. Crop Evol. 60 (7), 1923–1943. Al-Gendy, A.A., Lockwood, G.B., 2003. GC–MS analysis of volatile hydrolysis products from glucosinolates in Farsetia aegyptia var. ovalis. Flavour Frag. J. 18 (2), 148–152. Ball, P.W., 1993. Armoracia. In: Tutin, T.G., Burges, A., Chater, A.O., Edmondson, J.R., Heywood, V.H., Moore, D.M., Valentine, D.H., Walters, S.M., Webb, D.A. (Eds.), Flora Europaea 1, second ed. Cambridge University Press, Cambridge p. 346. Bertóti, R., Vasas, G., Gonda, S., Nguyen, N.M., Szőke, É., Jakab, Á., Pócsi, I., Emri, T., 2016. Glutathione protects Candida albicans against horseradish volatile oil. J. Basic Microb. 56 (10), 1071–1079. Blažević, I., Mastelić, J., 2009. Glucosinolate degradation products and other bound and free volatiles in the leaves and roots of radish (Raphanus sativus L.). Food Chem. 113 (1), 96–102. Brahmi, F., Abdenour, A., Bruno, M., Silvia, P., Alessandra, P., Danilo, F., Drifa, Y.G., Fahmi, E.M., Khodir, M., Mohamed, C., 2016. Chemical composition and in vitro antimicrobial: insecticidal and antioxidant activities of the essential oils of Mentha pulegium L. and Mentha rotundifolia (L.) Huds growing in Algeria. Ind. Crop. Prod. 88, 96–105. CLSI, 2016. Performance Standards for Antimicrobial Susceptibility Testing, 26th Informational Supplement. Approved Standard. CLSI Document M100-S. Clinical and Laboratory Standards Institute, Wayne, PA. Dénes, A., Papp, N., Babai, D., Czúcz, B., Molnár, Z., 2012. Wild plants used for food by Hungarian ethnic groups living in the Carpathian Basin. Acta Soc. Bot. Pol. 81 (4), 381–396. Depree, J.A., Howard, T.M., Savage, G.P., 1999. Flavour and pharmaceutical properties of the volatile sulphur compounds of wasabi (Wasabia japonica). Food Res. Int. 31 (5), 329–337. Dufour, V., Stahl, M., Baysse, C., 2015. The antibacterial properties of isothiocyanates. Microbiol. SGM 161 (2), 229–243. Grob, K., Matile, P., 1980. Capillary GC of glucosinolate-derived horseradish constituents. Phytochemistry 19 (8), 1789–1793. Herz, C., Tran, H.T.T., Márton, M.R., Maul, R., Baldermann, S., Schreiner, M., Lamy, E., 2017. Evaluation of an aqueous extract from horseradish root (Armoracia rusticana radix) against lipopolysaccharide-induced cellular inflammation reaction. Evid-Based Complement. Altern. Med. 2017 (12), 1–10. Heywood, V.H., Zohary, D., 1995. A catalogue of the wild relatives of cultivated plants native to Europe. Fl. Medit. 5, 375–415. ISTA-RULES, 1999. International Seed Testing Association − Proposals for the International Rules for Seed Testing. ISTA, Bassersdorf, Switzerland. Jirovetz, L., Smith, D., Buchbauer, G., 2002. Aroma compound analysis of Eruca sativa (Brassicaceae) SPME headspace leaf samples using GC, GC–MS, and olfactometry. J. Agr. Food Chem. 16, 4643–4646. Jung, Y.J., Jung, J.I., Cho, H.J., Choi, M.S., Sung, M.K., Yu, R., Kang, Y.H., Park, J.H.Y., 2014. Berteroin present in cruciferous vegetables exerts potent anti-inflammatory properties in murine macrophages and mouse skin. Int. J. Mol. Sci. 15 (11), 20686–20705. Kameoka, H., 1986. GC–MS method for volatile flavor components of foods. In: Linskens, H.F., Jackson, J.F. (Eds.), Gas Chromatography/Mass Spectrometry. Springer-Verlag, Berlin Heidelberg, pp. 254–276. Kjær, A., Ohashi, M., Wilson, J.M., Djerassi, C., 1963. Mass spectra of isothiocyanates. Acta Chem. Scand. 17 (8), 2143–2154. Krupnick, G.A., 2010. Food, glorious food. Plant Press 13 (4), 1–18. Kumagai, H., Kashima, N., Seki, T., Sakurai, H., Ishii, K., Ariga, T., 1994. Analysis of volatile components in essential oil of upland wasabi and their inhibitory effects on platelet aggregation. Biosci. Biotechnol. Biochem. 58 (12), 2131–2135. Lamy, E., Crößmann, C., Saeed, A., Schreiner, P.R., Kotke, M., Mersch-Sundermann, V., 2009. Three structurally homologous isothiocyanates exert “Janus” characteristics in human HepG2 cells. Environ. Mol. Mutagen. 50 (3), 164–170. Masuda, H., Harada, Y., Tanaka, K., Nakajima, M., Tabeta, H., 1996. Characteristic odorants of wasabi (Wasabia japonica Matum), Japanese horseradish, in comparison
Cultivated 1–propagated by root divisions; Cultivated 2–propagated by root cuttings.
against this strain was also significant (MIC = 28.7 μg/mL). Since the antimicrobial activity at concentrations bellow 100 μg/mL is very interesting (Ríos and Recio, 2005), the anticandidal activity of the root volatile fractions of the investigated Armoracia species was also tested on 20C. albicans clinical isolates, obtained from throat, tongue, nasal, skin, cervical and vaginal swabs, and stool (Table 3). Armoracia macrocarpa fraction exhibited significant activity against all the tested clinical isolates (MICs = 25–119 μg/mL), while A. rusticana fraction showed somewhat weaker effect against the most of the tested isolates (MICs = 48–192 μg/mL). In some earlier investigations, the anticandidal effect of A. rusticana root volatile fraction was also demonstrated, such as the activity of its dominant alkyl/aryl isothiocyanates (Bertóti et al., 2016; Saladino et al., 2016). It was also demonstrated that methylthioalkyl isothiocyanates berteroin, lesquerellin, as well as 7-methylthioheptyl isothiocyanate possessed comparable or even higher anticandidal activity than alkyl/aryl isothiocyanates (Masuda et al., 1999). These findings are in agreement with the results obtained in the present study, which showed that the volatile fractions of A. macrocarpa exhibited somewhat stronger effect than the one of A. rusticana. Demonstrated selective antimicrobial activity of A. macrocarpa root volatile fractions is very interesting because of its potential application in the treatment of candidiasis caused by antibacterial therapy.
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