Lam. (Tropaeolaceae) is a summer-growing species, native to South America, specifically Uruguay, Northern Argentina and Southern Brazil. It is used as an ornamental, food and medicinal plant. Its flowers make it known and appreciated throughout the world as an ornamental plant, but they are also locally consumed as part of salads [1
] and used as an antidiabetic drug [2
While the flowers of T. pentaphyllum
have their applications, the most known and consumed part of the plant is its tubers. These can reach up to 1.5 kg in weight in due time, and are commonly known in south of Brazil as “crem” or “batata-crem” (crem-potato) and also “raiz-amarga” (bitter-root) [3
]. “Crem” is a word derived from the slavic “chren”, a common denomination of the roots of Armoracia rusticana
(horseradish) in Eastern Europe. T. pentaphyllum
’s tubers and horseradish are commonly mistaken, as both are used as spices, prepared in the same way, as homemade pickles with red wine vinegar [4
]. T. pentaphyllum
’s tubers have similar organoleptic characteristics to those of the horseradish, and its consumption became popular during the 19th century with the settling of immigrants from Europe in the southern region of Brazil.
In traditional medicine, the consumption of the tubers is indicated to prevent and aid in the treatment of flu and scurvy. The decoction of the tubers is recommended as an option for the treatment of dermatosis and dermatological affections [5
]. To the best of our knowledge, there is no scientific evidence corroborating this use, nor there are any studies demonstrating the presence of compounds related to these properties.
Bacterial and fungal infections represent part of the known and reported dermatological afflictions. Their treatments can be lengthy and expensive, requiring topical and parenteral medication, and as with other microbial infections, the microorganisms responsible can develop resistance mechanisms [6
]. Therefore, the aim of the present study was to evaluate the popular use and the potential of T. pentaphyllum
tubers, through an assessment of its in vitro
antimicrobial activity and an investigation of the major constituents. For that, phytochemical analysis and antimicrobial evaluation against a series of microorganisms were conducted with the hydroalcoholic extract, its derived fractions and the essential oil obtained from the tubers.
Overall the antimicrobial activity was strong for the EO and the CfF, with MICs below the cutoff point for promising activity (i.e.
, lower than 200 µg/mL) [7
]. In addition to the bacteriostatic effect demonstrated by the low MIC values of the aforementioned extracts, bactericidal activity was also verified, albeit at higher concentrations. Since mostly of the current antibiotics are growth inhibitors, this bactericidal effect can be a relevant improvement, which also highlights a difference between mechanisms of action, indicating some direct action against the bacteria cell instead of protein synthesis inhibition mechanism (i.e
., the mechanism of azithromycin) [8
The antibacterial activity presented for the EO is due to its chemical composition. As presented in Figure 1
, the GC-MS analysis revealed that the major component of the oil is BITC. While it is not a new compound to the genus, as it was already identified in Tropaeolum majus
], this is the first report of its presence in T. pentaphyllum
. The occurrence of BITC in T. pentaphyllum
tubers explains their pronounced organoleptic properties, the reason behind its use as a condiment.
The antibacterial activity of standard BITC was tested (Table 1
), with results highly corresponding those of the EO. This is in accordance with previous publications, which demonstrated BITC bactericidal activity against a range of Gram-negative bacteria, while the tested Gram-positive bacteria were less susceptible [10
]. Kim and Lee [11
] demonstrated BITC activity against some harmful intestinal bacteria such as Clostridium difficile
and E. coli
, while not inhibiting other intestinal bacteria, such as Bifidobacterium
spp. and Lactobacillus acidophilus
, indicating that T. pentaphyllum
tubers consumption could have a similar effect against intestinal bacteria. Dias et al.
] showed that BITC is a stronger growth inhibitor against methicillin-resistant Staphylococcus aureus
(MRSA) than allyl and 2-phenylethyl isothiocyanate, with MICs ranging from 2.9 to 110 µg/mL against several MRSA strains, the closest comparison that can be made with our results is that both BITC and EO had a 40 µg/mL MIC against a standard non-MRSA strain in our work.
Antimicrobial mechanisms of action of isothiocyanates (and BITC as well) are not well established, nonetheless several modes of action are proposed such as effects on membranes, inhibition of regulatory systems (quorum sensing), inhibition of respiratory enzymes, induction of heat-shock response, oxidative stress and stringent response [13
]. Studies specifically with BITC demonstrated that it can cause the loss of membrane integrity, conversely to what was observed for ampicillin (a reference antibiotic) [10
], and it was also the most potent inhibitor of the quorum sensing system CviIR on Chromobacterium violaceum
when compared to allyl isothiocyanate and 2-phenylethyl isothiocyanate [14
]. Other mechanisms of action verified with BITC were the induction of a heat-shock-like response, reduction of O2
consumption and protein aggregation on Campylobacter jejuni
While the EO verified antibacterial activity is solely due to BITC, it is not the case for the CfF, which is a mixture of low polarity compounds as seen in Table 4
. BITC is in fact part of this mixture, and it is followed in the chromatographic run by three structurally related compounds: 2-phenylacetamide, N
-benzylacetamide and benzylcarbamide. To this moment it is not clear if these compounds are of natural occurrence, or derived from BITC, during the processing and extraction of the tubers. Similar amides were already found in plant extracts [17
]. On the contrary, there is some evidence on isothiocyanate reaction products in aqueous medium, including the formation of elemental sulfur [18
] a minor bacterial growth inhibitor [20
], which may be related to the observed effect, since it was encountered in the CfF at 31.61 min.
Additionally contributing to the demonstrated antibacterial activity of CfF are fatty acids and their esters. They comprise the bulk of the fraction and possess antimicrobial activity [21
]. Recently Tamokou et al.
] purified a fraction from the ethyl acetate extract of Albizia adianthifolias
stem bark containing only oleic and palmitic acid, two of the major constituents of the CfF, and it was active against E. faecalis
and S. aureus
, presenting MICs of 400 µg/mL and 200 µg/mL, respectively, which is less pronounced inhibitory activity than that exerted by CfF against the same bacteria (MICs of 80 and 40 µg/mL, respectively). In our study, we highlight the potent effect of the combined compounds of the fraction, rather than the action of individual constituents.
Not only was the antibacterial activity strong, but antifungal activity (Table 2
and Table 3
) was also remarkable, observed for the EO, standard BITC and CfF, for both fungal forms. T. rubrum
), one of the major causes of dermatophytosis, was highly sensitive to both BITC and CfF, with a MIC value of 2.5 µg/mL, almost the same value obtained for the tested antifungal agent, itraconazole (MIC of 2.5 µg/mL). However, itraconazole is a fungistatic agent, whereas BITC and CfF also exerted a fungicidal effect, presenting MFCs of 40 µg/mL and 320 µg/mL, respectively. Another common causative agent of dermatophytosis, M. canis
, was also very sensitive to BITC and CfF (MICs of 20 µg/mL). These data corroborates the widespread use of the tubers’ decoction to treat dermatophytosis [5
]. Other filamentous fungi that causes cutaneous and subcutaneous infections, such as F. pedrosoi
(chromoblastomycosis causative agent) and S. schenckii
(sporotrichosis causative agent) were also sensitive to both BITC and CfF, while Aspergillus
spp. presented mixed results, A. flavus
and A. niger
clinical isolates were resistant to CfF (MIC of 1280 µg/mL and with no observed fungicidal activity at the same concentration).
Antifungal activity of isothiocyanates against few fungi species was reported by Drobnica et al.
] who showed that BITC is in general 3.6 time more potent than allyl isothiocyanate. Equivalent results can be seen, as the authors obtained a MIC of 26.86 µg/mL against filamentous fungus A. niger
after four days of incubation, while we observed a MIC of 160 µg/mL for our clinical isolate of A. niger
, with two days of incubation. More recently Manici et al.
] showed isothiocyanates have fungitoxic activity against plant pathogenic fungi, and the proposed mechanisms of action were inactivation of intracellular enzymes by breakdown of disulfide bonds, inhibition of metabolic enzymes by thiocyanate radical (indicated as degradation product of isothiocyanates) and uncoupler action on oxidative phosphorylation.
While the antifungal activity of BITC is more established and explains the EO antifungal properties, the CfF presents a different situation, where activity can be ascribed to the whole set of compounds, instead of selected ones. Elemental sulfur (RT of 31.61 min), at 1.62% of the CfF, can play a major role in the observed antifungal activity. In fact, it is regarded as the oldest of all pesticides, with well-established antifungal action, whether in its inorganic or organic forms [25
There are no reports in the literature regarding the activity of the amides of the CfF composition (eluted between 17.49 min and 19.80 min). However there are some indications that they can present some inhibitory properties. Antimicrobial activity of the ethyl acetate extract from the algae Trichodesmium erythraeum
], containing, among other compounds, 2-phenylacetamide (benezeneacetamide) at 17.48%, inhibited the growth of some fungi such as T. rubrum
and Trichophyton simii
, with MICs of 500 µg/mL and 16.2 µg/mL. CfF was stronger against T. rubrum
, with a MIC of 2.5 µg/mL, and 4.37% of 2-phenylacetamide in its composition, suggesting that these amides do not play a prominent role in the antifungal activity of the CfF, albeit a synergistic contribution is possible.
In addition to the BITC and elemental sulfur contribution to the observed antifungal activity, the fatty acids and its esters, which comprise the bulk of the CfF, may also have an important role. Extracts and fractions containing fatty acids have been reported to possess at least fungistatic activity. The results of Tamokou et al.
] for a purified mixture of oleic and palmitic acid obtained from ethyl acetate extract of Albizia adianthifolia
were active against different Candida
sp. and C. neoformans
, with MICs ranging from 100 µg/mL to 400 µg/mL, higher values than those observed for CfF against a wide range of tested yeasts.
Palmitic and oleic acid, major constituents of CfF (6.75% and 10.25%, respectively), are among the fatty acids reviewed for their antifungal properties [27
]. A proposed possible mechanism of antifungal action of fatty acids indicates that they can alter fungal membrane fluidity, causing cell membrane disorganization and leakage of vital components, eventually leading to cell disintegration [28
The importance of fatty acids in the plant, and in the extract, may not be restricted only to the antimicrobial activity, but also for their action as penetration enhancers, a supporting role in the utilization of the plant against skin infections. Fatty acids and their esters can penetrate the skin, but in addition they could also promote the penetration of other active compounds such as the hydrophobic BITC, the amides and sulfur, to deeper layers of the skin, by disruption and alteration of the stratum corneum lipid strucure [29
]. An extract from the algae Botryococcus braunii
rich in palmitic and oleic acid [31
], fatty acids we identified in the CfF, enhanced the skin penetration of flurbiprofen, and while the extract were less effective than the purified fatty acids, it was also less irritating to the skin. Another work [32
] demonstrated that fatty acids can penetrate and accumulate to different degrees in the skin, and, in addition, the authors have shown that oleic acid significantly enhanced tolnaftate penetration.
To the moment, of the compounds encountered in the EO and CfF of the tubers from T. pentaphyllum, only the fatty acids have an established relation with the skin. We have not found specific studies concerning BITC, amides and elemental sulfur. Their hydrophobic, and low molecular weight structures put them among candidates for skin penetration and enhancement by combined use along with fatty acids, although experimentation with these compounds is necessary to a better understanding of the behavior towards skin.