Original Research Paper

Natural Product Communications

Volume 17(4): 1–12
Identification of Antifungal Compounds from © The Author(s) 2022

Piper Plants Against Fusarium oxysporum: An

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DOI: 10.1177/1934578X221089995
Untargeted Metabolite Profiling-Based Approach journals.sagepub.com/home/npx

Diego Cárdenas-Laverde1, Sebastián Rincón-Aceldas1

and Ericsson Coy-Barrera1

Abstract
The phytopathogen Fusarium oxysporum produces considerable losses in economically important crops, making alternative control
measures urgently required. Piper plants are widely distributed in tropical regions, and they are also known to produce metabolites
with biological activity against infectious agents. As part of our continuous search for antifungals, 18 Piper-derived ethanolic extracts
were evaluated by their in vitro effect on F oxysporum mycelial growth inhibition. The total content of phenol and flavonoid measure-
ments and liquid chromatography-electrospray ionization-mass spectrometry analysis served as the chemical characterization of the
investigated extracts. Piper pulchrum, Piper barcoense, and Piper tuberculatum exhibited the highest mycelial growth inhibition (>74%). The
integration of chemical fingerprints and bioactivity datasets led to recognizing 4 bioactive candidates among extracts through single-
Y orthogonal partial least squares regression and univariate statistics. These candidates were 2 amides (1,3), an alkyl lactone (2), and a
prenylated benzoquinone (4), subsequently isolated and identified by nuclear magnetic resonance spectroscopy. These isolated com-
pounds exhibited reasonable antifungal activity (IC50 < 50 µM). The findings indicated that the correlative association is advanta-
geous for identifying bioactive metabolites within active extracts.

Keywords
Piperaceae, Piper, phenolics, antifungals, bioactivity, Fusarium oxysporum

Received: December 12th, 2021; Accepted: March 3rd, 2022.

Introduction classes such as flavonoids, amides, butenolides, piperolides,

lignans, phenylpropanoids, prenylated benzoic acid derivatives,
The Piperaceae family comprises many basal angiosperms in and alk(en)ylphenols, among others.7 Indeed, some of these
different habitats such as grasses, shrubs, lianas, epiphytes, compounds exhibited relevant activities against different
and medium-sized trees.1 This family is classified in the fungal pathogens, having promising potential to be used as
Piperales order along with the families Aristolochiaceae, lead compounds for antifungal development.7 Therefore, Piper
Hydnoraceae, Lactoridaceae, Piperaceae, Asaraceae, and species can be considered an excellent source of antifungals
Saururaceae.2 Within the Piperaceae family, ca. 3500 species and, consequently, several of them remain to be explored.8,9
have been described, which are widely distributed in tropical A critical current fungal-related problem concerns Fusarium
regions,3 having its center of origin in Peninsular Malaysia oxysporum, a phytopathogen that infects and negatively affects
and a broad dispersion in the American tropics.4 From this above 100 crop plants worldwide and even humans.10
plant family, the Piper genus is highly representative due to the Therefore, various strategies based on physical, biological,
high number of species (ca. 2172)3 and the exciting chemodiver-
sity and biological activities reported for several Piper plants.5 In
this regard, the bioactivities exhibited by Piper-derived end- 1
Bioorganic Chemistry Laboratory, Facultad de Ciencias Básicas y Aplicadas,
products (ie, extracts, essential oils, and isolated phytochemi- Universidad Militar Nueva Granada, Cajicá, Colombia
cals) have a broad outlook. However, the antibacterial and
antifungal activities against human and plant pathogens can Corresponding Author:
Ericsson Coy-Barrera, Bioorganic Chemistry Laboratory, Facultad de Ciencias
be highlighted.6 In the last few decades, several studies have Básicas y Aplicadas, Universidad Militar Nueva Granada, Cajicá 250247,
been oriented to scrutinize the antifungal potential of Colombia.
Piper-derived end-products, involving various metabolite Email: [email protected]

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2 Natural Product Communications

and/or chemical actions have been implemented to control this inhibition (MGI) was then measured using a microscale
fungus.11 However, chemical control is mainly used. Still, the amended medium assay and statistically associated with mass
uncontrolled and nonprotocolized exposure of fungicidal spectrometry (MS)-based chemical composition. Hence, anti-
agents in crops has generated various problematic issues, such fungal bioactive compounds were recognized and identified
as environmental and health risks and the emergence of F oxy- using this integration.
sporum resistant strains to different commercially available treat-
ments commonly used in medicine and agriculture.12 For these
reasons, it is still necessary to search for alternatives against Results and Discussion
F oxysporum, involving the most negligible impact on human
health, other surrounding species, and the environment, and Antifungal Activity of Piper-Derived Extracts
comprising the highest efficiency. The MGI percentages of the set of extracts (n = 18) obtained
An alternative route for controlling this phytopathogen is from leaves and stems of each Piper plant (n = 9), evaluated
oriented to naturally occurring compounds from plants,13 par- using an extract concentration of 0.1% (w/v), are presented
ticularly Piper species.7 Bio-guided fractionation is the most in Table 1. The investigated extracts showed antifungal activity
employed approach to identifying active components within a against F oxysporum at different levels, indicating that these Piper
bioactive plant-derived extract or fraction, but this practice is plants can be a source of antifungals.7 The MGI ranged from
time-consuming and expensive.14 Other approaches have 24.4% to 89.0%, involving an average of 50.9% and a median
recently emerged to overcome these disadvantages, such as stat- of 48.8%, whereas positive controls (mancozeb and prochloraz)
istically- based integration to recognize chemical/ exhibited MGI values of 97.3% and 93.8%, respectively. The
bioactivity-related patterns.15 This (un)targeted metabolomics- post hoc Tukey test classified the extract population through
based strategy associates both biological and chemical datasets multiple comparisons. This classification found that those
to disclose putatively bioactive compounds within plant mix- MGI averages were significantly different from each other
tures, which can be subsequently selected for isolation.16 In (p < .05). Thus, the most-active extracts were found forming
fact, we focus our interest on Piper species because, in a previous the significantly different groups A to E (MGI > 60%). These
study, 2 Piper plants were the most-active extracts within a group groups comprised extracts from Piper pulchrum leaves and
of 44 botanical extracts.17 Hence, as part of our research on nat- stems (PpL and PpS), Piper barcoense leaves and stems (PbaL
urally occurring antifungals, a set of 18 Piper-derived extracts and PbaS), Piper tuberculatum leaves and stems (PtL and PtS),
were assessed against F oxysporum. The mycelial growth and Piper hispidum stems (PhS). The most-active extracts were

Table 1. MGI, Yields, and TPC and TFC, respectively, of Piper-Derived Extracts.
# Piper plant Parta Codeb %MGIc Yield (%)d TPCe TFCf
1 Piper pulchrum S PpS 89.0 ± 0.2A 8.3 ± 0.3FG 64.5 ± 0.6O 49.2 ± 0.3K
2 Piper barcoense L PbaL 85.3 ± 0.2B 10.9 ± 0.3CD 221.8 ± 0.7E 96.8 ± 0.4H
3 Piper tuberculatum L PtL 74.8 ± 0.2C 17.9 ± 0.6A 195.6 ± 0.8G 85.9 ± 0.4I
4 P pulchrum L PpL 74.1 ± 0.2C 9.9 ± 0.5DE 320.5 ± 0.9C 145.4 ± 0.6C
5 P barcoense S PbaS 63.2 ± 0.3D 7.0 ± 0.3GH 112.3 ± 0.6L 43.2 ± 0.4L
6 Piper hispidum S PhS 63.1 ± 0.2D 11.4 ± 0.4C 51.5 ± 0.5Q 21.7 ± 0.3P
7 P tuberculatum S PtS 62.4 ± 0.3E 7.5 ± 0.2G 95.7 ± 0.6N 33.5 ± 0.3N
8 P hispidum L PhL 58.4 ± 0.2F 11.5 ± 0.5C 140.2 ± 0.6K 64 ± 0.4J
9 Piper divortans L PdL 56.4 ± 0.3G 10.2 ± 0.5CDE 180.2 ± 0.9H 102.2 ± 0.6G
10 P divortans S PdS 41.3 ± 0.2H 8.3 ± 0.3FG 160.8 ± 0.9J 29.3 ± 0.3O
11 Piper arboreum L PaL 39.9 ± 0.3I 10.5 ± 0.4CD 278.6 ± 0.9D 213.8 ± 0.8A
12 Piper bogotense L PboL 38.9 ± 0.3J 11.3 ± 0.4C 442.1 ± 1.0A 107.8 ± 0.5E
13 P arboreum S PaS 32.1 ± 0.2K 6.1 ± 0.5H 177.6 ± 0.8I 105.6 ± 0.5F
14 Piper crassinervium S PcS 30.8 ± 0.1L 7.9 ± 0.4FG 60.7 ± 0.7P 40.3 ± 0.4M
15 Piper marginatum L PmL 29.6 ± 0.2M 12.9 ± 0.7B 380.7 ± 1.0B 175.1 ± 0.7B
16 P marginatum S PmS 26.3 ± 0.2N 8.9 ± 0.3EF 18.6 ± 0.7R 9.9 ± 0.2Q
17 P bogotense S PboS 25.7 ± 0.2N 7.8 ± 0.3FG 98.8 ± 0.6M 30.6 ± 0.4O
18 P crassinervium L PcL 24.4 ± 0.2O 13.9 ± 0.7B 201.9 ± 1.0F 116.6 ± 0.6D
— Mancozeb — — 97.3 ± 0.2 — — —
— Prochloraz — — 93.8 ± 0.3 — — —
Abbreviations: MGI, mycelial growth inhibition; TPC, total phenolic content; TFC, total flavonoids content; GAE, gallic acid equivalents; DW, dry weight; QE,
quercetin equivalents.
a
Plant part used to prepare the extract: L = leaves; S = stems; bCode assigned to each extract; c%MGI = MGI expressed as a percentage. Each extract was evaluated
using a final extract concentration of 0.1% (w/v); dYield after ethanolic extraction from dry material to prepare the respective raw extract; eTPC = TPC expressed as
mg GAE/100 g DW; fTFC = TFC expressed as mg QE/100 g DW; values presented as mean ± standard deviation (n = 3). Different superscript letters indicate
significant differences according to the post hoc Tukey test (p < .05).
Cárdenas-Laverde et al 3

PpS and PbaL (MGI > 80%), whereas the least-active ones were ranges of 30 to 860 mg GAE/100 g DW and 20 to 440 mg QE/
Piper marginatum leaves and stems (PmL and PmS), Piper bogotense 100 g DW, respectively, for different types of extracts of Piper
stems (PboS), and Piper crassinervium leaves (PcL) (MGI < 30%). nigrum, Piper auritum, Piper betle, Piper betleoides, and Piper walli-
None of the investigated extracts have been previously chii.24‐26 Considering that phenolics (eg, benzoic acid derivatives
studied for bioactivity against F oxysporum mycelial growth. In and prenylated hydroquinones) and flavonoids (eg, flavones
addition, P pulchrum and P barcoense have no previous antifungal and chalcones) are often reported as antifungals,7 the linear rela-
studies. However, P tuberculatum and P hispidum have been tionship between MGI with TPC and TFC was further studied
described as a source of antifungal amides, whose bioactivity (Figure 1). However, no clear correlation was found (Pearson’s
against Cladosporium sphaerospermum by thin-layer chromatography correlation coefficient <0.3), indicating that the global set of phe-
(TLC)-mediated direct bioautography was evaluated, affording nolics or flavonoids is not associated with the inhibitory activity
minimal inhibitory amounts between 0.1 and 8.0 µg.18,19 shown by the whole set of extracts and, therefore, certain metab-
Similarly, amides from Piper arboreum were active against olites should be responsible for the observed bioactivity that
Cladosporium cladosporioides and C sphaerospermum.20 P crassinervium deserve to be explored. Thus, an liquid chromatography
against the fungi above, through a bioautography-based bio- (LC)-MS-based chemical characterization of the Piper extracts
guided fractionation, was also studied, and 3 prenylated hydro- was then performed.
quinones, a benzoic acid derivative, and 2 known flavanones
were isolated as antifungal metabolites.21,22 However, we found
Characterization Based on LC-Electrospray Ionization-MS
that P arboreum and P crassinervium exhibited low inhibitory capac-
ity on the mycelial growth of F oxysporum (MGI < 40%). On the Data of Piper Extracts
other hand, the essential oils of several Piper plants (including P The 18 extracts of Piper plants were analyzed by reverse-phase LC
tuberculatum, P hispidum, P bogotense, and P marginatum) were evalu- coupled to MS using electrospray ionization (RP-LC-ESI-MS).
ated against F oxysporum conidia through microdilution, obtaining This analysis led to expanding their chemical characterization.
minimal inhibitory concentrations ≥500 µg/mL.23 Thus, the resulting raw data were used to retrieve the entire set
of m/z features per extract and compiled into a matrix to build
Yields, and Total Phenolic and Flavonoid Contents of the feature intensity table (FIT). Once this compilation was
achieved, 4947 features were gathered from the 18 extracts, sug-
Piper-Derived Extracts gesting a considerable chemodiversity. In this sense, several fea-
The materials from the Piper plants yielded raw ethanolic extracts tures (ie, metabolites) were shared between extracts of the same
at different levels (Table 1). In general, leaves afforded higher species and extracts from different plant parts of other species,
yields than stems. In this regard, leaves of P tuberculatum, P margin- with varying intensity. In contrast, other features were found to
atum, and P bogotense exhibited the highest yields (>11%). In be unique for particular extracts. A heat map-based intuitive visu-
contrast, the lowest yields were obtained from the stems of alization is presented in Figure 2 to illustrate the mentioned
P arboreum, P barcoense, and P tuberculatum (<7.5%). No relation- MS-based chemical distribution.
ship was found between yields and MGI. On the other hand, After scaling the features to unit variance (ie, autoscaling),
extracts were also characterized by measuring their total phenolic the metabolite richness of some extracts could be evidenced.
and total flavonoid contents (TPC and TFC, respectively). The
resulting values are presented in Table 1. Leaf extracts generally
showed higher TPC and TFC than those of the stems (mean
TPC for leaves = 262.4 ± 100.9 mg gallic acid equivalents
[GAE]/100 g dry weight [DW], mean TPC for stems = 93.4
± 51.6 mg GAE/100 g DW). Hence, leaf extracts from P bogo-
tense, P marginatum, and P pulchrum exhibited the highest values
(TPC > 320 mg GAE/100 g DW). In contrast, stem extracts
from P crassinervium, P hispidum, and P marginatum showed the
lowest contents (TPC < 61 mg GAE/100 g DW).
In the case of TFC, leaf extracts also exhibited higher values
than stems (mean TFC for leaves = 123.1 ± 47.0 mg quercetin
equivalents [QE]/100 g DW, mean TFC for stems = 40.4 ±
27.1 mg QE/100 g DW). The highest TFC values were shown
by leaf extracts of P arboretum, P marginatum, and P pulchrum Figure 1. Correlation between %MGI with TPC and TFC,
(TFC > 145 mg QE/100 g DW), while stem-derived extracts respectively, of extracts from Piper plants. TPC and TFC are depicted as
green circles and blue squares, respectively. TPC is expressed as mg
from Piper divortans, P hispidum, and P marginatum showed the GAE/100 g DW and TFC as mg QE/100 g DW.
lowest values (TFC < 29.3 mg QE/100 g DW). These total con- Abbreviations: %MGI, percentage mycelial growth inhibition; TPC,
tents are in agreement with previous studies measuring TPC and total phenolic content; TFC, total flavonoid content; GAE, gallic acid
TFC for different Piper plants, whose respective values resulted in equivalents; DW, dry weight; QE, quercetin equivalents.
4 Natural Product Communications

Figure 2. Distribution of the intensity of the m/z features detected in extracts of Piper plants (n = 18) after analysis through reverse-phase liquid
chromatography coupled to mass spectrometry using electrospray ionization (RP-LC-ESI-MS). The heat map is organized by rows for each extract,
using the codes presented in Table 1. Each colored cell was associated with an autoscaled (scaled to unit variance, prior heatmap generation)
intensity of each detected m/z feature, depending on the color scale (dark red: high intensity; dark blue: low intensity).

Hence, leaves of Piper barbatum, P tuberculatum, and P crassiner- Piper plants. Thus, a first statistical analysis integrated the
vium and stems of P tuberculatum, P marginatum, and P bogotense plant chemical composition and the bioactivity of extracts
exhibited the highest number of metabolites with higher abun- through a single-Y orthogonal partial least squares (OPLS)
dance (Figure 2). However, such abundant metabolites are not regression (using MGI as continuous Y variable). The resulting
often shared between these extracts. In addition, apparent dif- statistical OPLS model, comprising one predictive score (t[1])
ferences between plant parts of the same Piper species were and one orthogonal component (to[1]), could discriminate
also evidenced, indicating tissue-specific chemical profiles extracts based on antifungal activity (Y-data) and chemical com-
observed previously in other reported Piper species, eg, Piper position (X-data), revealing a reasonable explained variance, ie,
methysticum or Piper gaudichaudianum.27,28 Remarkably, the R2X = 0.85, R2Y = 0.79, and predictability, ie, Q2Y = 0.62. In this
most-active extracts, such as PpL, PpS, PbaS, and PhS, exhibited sense, the OPLS-derived scores plot (Figure 3A) revealed the
fewer abundant metabolites, suggesting that these extracts con- respective discrimination mode of Piper extracts, explaining
tained particular metabolites possibly responsible for the activ- the variance according to the MGI activity (45.3% along t[1])
ity. In contrast, other active extracts, ie, PtL and PtS, exhibited and chemical composition (39.7% along to[1]), respectively.
abundant metabolite-rich chemical profiles. Based on these This 2-dataset pattern recognition was very relevant for our
facts, to focus the efforts on identifying antifungal compounds, purpose since the scores plot exposed those extracts grouped
it was decided to integrate statistically the MS-based chemical by chemical composition and conditioned by the MGI activity.
dataset of each extract with the respective antifungal activity. A color scale (20%-100% scaling) depicts the antifungal-
dependent discrimination between high (green) and low (red)
Detection of Antifungal Candidates From Piper Species MGI values as a key trend. In this regard, the most-active
extracts were right-side located in the scores plot, but had dis-
Through the Integration of Chemical Fingerprints
tinct profiles since they were highly dispersed, while the least-
and Bioactivity Datasets active extracts exhibited a lower dispersion. This fact suggested
The LC-MS-based chemical composition and MGI-based anti- that the most-active extracts might have particular compounds
fungal activity datasets were then integrated through supervised responsible for the observed antifungal action. Therefore, the
statistics to distinguish plausible antifungal compounds within predictive component loadings were examined using an S-plot
Cárdenas-Laverde et al 5

Figure 3. Integration of chromatographic data and Fusarium oxysporum mycelial inhibition datasets of Piper extracts by single-Y OPLS. %MGI was
used as a continuous Y-variable depicted as a color scale (green = high value; red = low value). (A) Scores plot. (B) S-plot. Extract codification is
related to the information presented in Table 1.
Abbreviations: OPLS, orthogonal partial least squares; %MGI, percentage mycelial growth inhibition.

transformation to discriminate the differential variables’ relative change (FC) threshold (X-axis) of 1.5 and a t-test threshold
importance and facilitate model interpretation. The S-plot (Y-axis) of 0.05 (p-value). In this plot, the significant features
(a p[1] × p(corr)[1] scatter plot taking the letter “S” shape for the most-active extracts were compounds 1-4 since they rep-
using Pareto and centering scaling) provided visualization of resented the significant features above the FC and p-value
both the covariance and the correlation structure among the thresholds for this categorical variable. Compound 1 was the
X-data and t[1].29 Consequently, the most relevant differences most significant metabolite, whereas compounds 2 and 3 exhib-
between least-active (p[t1] < 0) and most-active (p[t1] > 0) ited the highest FC value. Remarkably, a reasonable number of
extracts were evidenced through those features situated far features were significantly related to the least-active extracts (12
out of each wing, indicating high model influence with high reli- features), indicating their elevated abundance in such extracts
ability (Figure 2B). After this transformation, 4 features (num- without a relationship with antifungal activity. These variations
bered as 1-4 according to the p[1] value) were found to be of compounds 1-4 between categorical variables were ade-
relevant variables (p(corr) > 0.6, p[1] > 0.08) for the quately cross-validated through receiver operating characteristic
OPLS-based discrimination between most and least-active (ROC) curves (Figure 4B to E). Their area under the curve
extracts. These features showed a mass/charge ratio (m/z) in values was above 0.9, indicating suitable sensitivity and spe-
the range of 261.1 to 320.1 and retention times (Rt) between cificity of these compounds for discriminating the
15.7 and 29.9 min, involving the m/z/Rt pairs such as 320.1/ most-active extracts. The respective box plots of the normal-
19.2 (1), 261.1/22.1 (2), 274.1/15.7 (3), and 313.2/29.9 (4). ized metabolite abundance revealed such significant differ-
This LC-MS-derived information indicated that these features ences, involving a more considerable dispersion of 1-4 in
are related to intermediate-polarity and low-weight metabolites. the most antifungal extracts, but significantly different for
Compound 1 showed the greatest model influence (p[1] > 0.1) those least-active ones.
due to its high variance in the dataset, which abundantly The previous results indicated that our aim of integrating the
occurred in P pulchrum and P tuberculatum extracts. In contrast, chemical composition and the antifungal activity datasets could
compounds 2 and 3 exhibited the best reliability due to their be successfully achieved through single-Y OPLS for pattern
higher p(corr) since they were common to most-active extracts recognition. The covariance maximization of independent var-
(ie, P pulchrum, P barcoense, and P tuberculatum). Compound 4 iables (ie, discriminating features) regarding a continuous or cat-
exhibited the lowest reliability and model influence. However, egorical dependent variable (ie, antifungal activity) can be
these parameters for the selected compounds 1-4 exhibited adequately achieved by supervised methods such as OPLS or
very slight differences. Therefore, they were considered within partial least squares,30 which diverges from principal compo-
the pattern recognition as antifungal candidates that possibly nent analysis (PCA) since it cannot maximize the independent
mainly contributed to the observed MGI by extracts. variable covariance. Therefore, PCA was not employed as a
The statistical performance of this S-plot-based recognition first-line analysis, as commonly used. In addition, the relevant
of compounds 1-4 was additionally examined through classical fact about the utility of single-Y OPLS is the use of a continuous
univariate analysis. This analysis required a categorical variable variable. It is more advantageous than data-derived categorical
for the antifungal activity, and so the Piper dataset was subdi- variables because much information is lost. It is often tricky to
vided into 2 datasets, the most-active (MGI > 62%) and the identify the cutoff point and even the objective function to
least-active (MGI < 58%) extracts. Thus, the critical features define the extent of the categories.31 Consequently, it is prefer-
were selected by using a volcano plot (Figure 4A) with a fold able to keep continuous variables instead of categorizing them
6 Natural Product Communications

Figure 4. Selection of top-ranked features by volcano plot (A). FC threshold (X-axis) of 1.5 and t-test threshold (Y-axis) of 0.05 (p-value). Both FC
and p-values were log-transformed to build the plot. The red circles represent features above the threshold for each categorical variable, ie, the
most-active (MGI>62%) and the least-active (MGI<58%) extracts. This variation performance was cross-validated by ROC curves of compounds
1 (B), 2 (C), 3 (D), and 4 (E). The respective normalized abundance cutoffs were computed and charted as box plots.
Abbreviations: FC, fold change; MGI, mycelial growth inhibition; ROC, receiver operating characteristic.

to explore the integration of metabolite/bioactivity datasets.32 differentiation.34 In this regard, the targeted isolation of
In this regard, metabolite profiling has increasingly been 1-4 was conducted to corroborate their plausible antifungal
employed to associate the metabolite fingerprints and the activity.
biological activity of mixtures of natural origin. The main
advantage of this approach is the use of chemical fingerprints
as the source of independent variables to be integrated with a Isolation and Identification of Antifungal Candidates
dependent variable.33 In addition, the main challenge during The chemical fingerprints and bioactivity integration distin-
fractionation procedures is overcome since this integration guished compounds 1-4 as the most important features for
can also recognize unstable compounds.34 Therefore, once the antifungal activity on F oxysporum. However, an inherent
the bioactive candidates are statistically predicted, they restriction of chemical fingerprints and bioactivity dataset inte-
must be depurated or isolated to validate the correlative gration is the detection of false positives due to the synergistic
Cárdenas-Laverde et al 7

or antagonistic effects with other(s) extracts’ component(s) and activity against C sphaerospermum and C cladosporioides.19,20
the causal but correlative principle of this association.35 Additionally, compound 3 showed activity against Candida albi-
Therefore, to validate such a recognition, semipreparative high- cans, Candida krusei, Candida parapsilosis, and Cryptococcus neofor-
performance liquid chromatography (HPLC) was employed to mans (125 µg/mL < minimum inhibitory concentration (MIC)
purify the OPLS-based recognized compounds 1-4 from PbaL-, < 15.6 µg/mL).39 In the case of 7,8-dihydroyangonin (2), a
PtL-, and PpS-derived extracts and, subsequently, these purified methoxylated kavalactone, no previous studies about its anti-
compounds were evaluated against F oxysporum. Hence, com- fungal activity have been reported. However, the homologs,
pound 1 was obtained from PtL, while PbaL afforded com- 7,8-dihydrokawain and
pound 3, and PpS yielded compounds 2 and 4. Once isolated, demethoxyyangonin (5,6-dehydrokavain), exhibited reasonable
they were structurally elucidated by diagnostic analysis of MS antifungal activity against F oxysporum and F solani (MGI <
and nuclear magnetic resonance (NMR) data. These com- 38.7% at 50 ppm).40 Finally, (2’E,6’E)-2-farnesyl-1,4-
pounds were unequivocally identified as known metabolites, benzoquinone (4) was studied for its activity against C albi-
and their 13C NMR data were identical to those reported for cans and other pathogenic bacterial strains, but no activity was
8,9-dihydropiplartine (1),36 7,8-dihydroyangonin (2),37 4,5-dihy- detected.41
dropiperyline (3),18 and (2’E,6’E)-2-farnesyl-1,4-benzoquinone To the best of our knowledge, compounds 1-4 have no
(4).38 The structures of compounds 1-4 are presented in Figure 5. record so far on antifungal activity against Fusarium species,
The antifungal activity of the isolated compounds was then so they were evaluated for the first time in the present study
evaluated against F oxysporum, and their resulting IC50 values are against the in vitro mycelial growth of F oxysporum, employing
listed in Table 2. Compound 1 exhibited the most potent MGI their recognition by statistical integration of LC-MS-derived
(IC50 = 21.1 μM), with a comparable value to the positive chemical fingerprints and antifungal activity datasets. This fact
control prochloraz (IC50 = 17.7 µM), but ca. 2-fold lower activ- confirms that this association effectively identifies antifungals
ity to that of mancozeb (IC50 = 11.9 µM). Contrarily, com- against F oxysporum from Piper-derived products. However,
pounds 2 and 4 showed the lowest activity (IC50 = 43.8 and other active compounds could be missed due to the potential
48.6 μM, respectively), while compound 3 revealed an interme- presence of antagonistic effects among different components
diate antifungal effect among the isolated compounds (IC50 = of the extracts, so more integrative studies for recognizing the
28.6 µM). Remarkably, compounds 1-3 shared a similar presence of those compounds are therefore required.42 Such
structural profile since they are constituted by an oxygenated approaches could also lead to identifying several interesting
aromatic ring bridged by a C2-to-C4 moiety to link compounds, for instance, more active compounds that are
a 5-to-6-membered heterocycle, which is an interesting affected by antagonists and those with lower activity, but
relationship to be further investigated. In this sense, acting as synergists that contribute to the activity improve-
8,9-dihydropiplartine (1) (the most-active compound among ment.42 Finally, due to the isolated compounds 1-4 being eval-
1-3) has a similar structure to that of dihydrochalcones, such uated at different concentrations (ie, 0.16-100 ppm) than those
as asebogenin, recently detected as an antifungal compound of the parent extracts (1000 ppm or 0.1% w/v), a direct com-
against F oxysporum (IC50 = 25.6 µM) isolated from P elonga- parison regarding antifungal activity between individual com-
tum.17 Compound 1 (a 5,6-dihydropyridin-2(1H )-one-like pounds and extracts was not possible and, therefore,
amide isolated from P tuberculatum and Piper rugosum) and additional studies are required to outline if the isolated com-
4,5-dihydropiperyline (3) (a pyrrolidine-like amide isolated pounds are responsible for the antifungal action on F oxysporum
from P hispidum and P arboreum) have exhibited antifungal mycelial growth by the investigated Piper extracts.

Figure 5. Structures of isolated compounds after statistical pattern recognition.

8 Natural Product Communications

Table 2. Antifungal Activity of Isolated Compounds from Piper 96% ethanol at a constant shaking speed (120 rpm) using a
species. Heidolph Rotamax 120 platform orbital shaker (Heidolph
Compounds IC50 (µM)a CIb Instruments GmbH & Co. KG). The extraction lasted 1 week
1 21.1 18.9-23.5
with daily removal of the extract-containing solvent and
2 43.8 40.1-44.9 replacement by fresh 96% ethanol. These mixtures were sepa-
3 28.6 26.5-30.9 rated by filtration. The resulting solution was concentrated by
4 48.6 46.4-51.3 distillation under reduced pressure at 40 °C using an IKA RV
Pc 17.7 15.5-20.6 10 Control rotary evaporator (IKA® RV 10, IKA® Werke
Mc 11.9 7.7-14.2 GmbH & Co. KG) to afford the crude extracts. The resulting
a
Half-maximal inhibitory concentration (IC50 in μM) on Fusarium oxysporum extracts from each plant from each daily extraction were col-
mycelial growth. Data are expressed as best-fit value of IC50 after nonlinear lected, dried, and subsequently stored at −20 °C until use.
regression.b95% confidence interval (CI) (n = 4). cPositive controls: P =
prochloraz; M = mancozeb.
In Vitro Bioassay Against F Oxysporum
Conclusions F oxysporum LQB-03, a virulent isolate obtained from wilting
Physalis peruviana plants, was used as the assessed phytopatho-
The present study constitutes the first endeavor to evaluate the gen.43 This isolate had been preserved on Whatman paper #
inhibitory capacity of the studied Piper-derived extracts (n = 18) 1 at −20 °C and further reactivated in potato dextrose agar
on F oxysporum mycelial growth. Four Piper-derived extracts (PDA) to be used in the present antifungal assays. The in
(PpS, PbaL, PtL, and PpL) showed the best inhibitory activity vitro antifungal activity against F oxysporum was evaluated by
on F oxysporum mycelial growth (MGI > 74%) among the botan- inhibiting mycelial growth (12-well plate amended semisolid
ical extracts tested (n = 18). The observed antifungal activity of medium assay). The assay was performed using the respective
extracts exhibited no relationship with the respective TPC or monosporic culture of the fungal isolate, developed in a PDA
TFC values. On the other hand, the indirect detection of plau- medium for 8 days. A microscale amended medium protocol
sible antifungal candidates through metabolic profiling by asso- was performed following the previously reported procedure.44
ciating the fingerprints and the antifungal activity datasets led to Briefly, 12-well plates (autoclavable glass), semisolid medium
the recognition of the 4 metabolites 8,9-dihydropiplartine (1), (1.2 g of potato-dextrose broth, and 0.5 g of agar per 100
7,8-dihydroyangonin (2), 4,5-dihydropiperyline (3), and mL of distilled water) were used. The final volume (for treat-
(2′ E,6′ E)-2-farnesyl-1,4-benzoquinone (4), as the most-active ments and controls) was 200 μL per well (1 cm diameter).
compounds. The isolation of these metabolites and the subse- The concentration (ie, 0.1% w/v) was prepared by supplement-
quent evaluation of the purified compounds validated the stat- ing the fresh semisolid medium with the required amount of
istical recognition and, therefore, confirmed the effectiveness of each dry, crude extract (n = 18). A stock dispersion was
this indirect approach based on LC-MS-based profiles for the reached by a direct mixture between the semisolid medium (5
bioactive selection from Piper extracts. It offers an alternative mL) and the required extract (5 mg). This mixture was vigor-
for more rapid antifungal findings from natural sources. ously stirred until achieving a homogenous dispersion before
Indeed, this is the first report for these Piper compounds solidifying. Tween-20 (5%) was used to assist the dispersion
against F oxysporum. Further studies on active extracts or indi- of the extracts. The resulting homogeneously amended
vidual compounds containing (aryl)alkylated amide, lactones, medium was added into 3 wells of the 12-well plate.
and/or benzoquinone moieties should be conducted to Subsequently, a 1 mm agar-mycelial plug from 5 days actively
expand their potential as biological protective agents against growing cultures of F oxysporum was inoculated onto the
this phytopathogen. center of each well. Each 12-well plate was placed into a 17
mm Petri dish, under the appropriate conditions of humidity
and sterility, and sealed with plastic films. Each trial comprised
Materials and Methods a randomized design with 3 replicates for each extract com-
pared to an absolute control (untreated semisolid medium).
Plant Material and Preparation of Extracts Sportak (prochloraz) and Dithane (mancozeb) were employed
Leaves and stems of 9 Piper species (P arboreum Aubl., P barcoense as positive controls using the same concentration (ie, 0.1%
Yunck., P bogotense C.DC., P crassinervium Kunth, P divortans Trel. w/v). After inoculation and sealing, this assembly was incu-
& Yunck., P hispidum Sw., P marginatum Jacq., P pulchrum C. DC., bated at 25 °C. Once the colony on the negative control had
and P tuberculatum Jacq.) were collected in Meta, Casanare, and covered the whole well (after ca. 48 h), the mean colony area
Cundinamarca, Colombia (coordinates 3.543, −73.669; 5.190, (mm2) was measured for treated and untreated wells by process-
−72.547; 4.931, −74.008, respectively) between 2015 and ing with software Image J® using photographic records. The
2017. Voucher specimens are kept at Colombian National percent of MGI was calculated for each replicate. This calcula-
Herbarium. The selected healthy plant materials (without tion was made employing the equation: % MGI = [(absolute
visible damage) were separately air-dried and extracted using control area − treatment area)/absolute control area)] × 100.
Cárdenas-Laverde et al 9

In the case of pure compounds 1-4, the same procedure was and TFC data. Once normality was verified, analysis of variance
employed using serial dilutions of compounds to test the tests were conducted on all samples (type I error, significance
effect on F oxysporum mycelial growth at different concentra- value p < .05), followed by post hoc Tukey tests to determine
tions within the 0.16 to 100 ppm range. The %MGI was iden- the significant differences between data. These statistical tests
tically measured for every concentration of each purified were performed using software R version 3.4.1 (R
compound, and the respective half-maximal inhibitory concen- Foundation). On the other hand, the MS data obtained from
tration (IC50 in μM) was calculated from the dose-response LC-ESI-MS were preprocessed in MZmine 2.17, comprising
curves through nonlinear regression using GraphPad 5.0 peak detection, baseline correction, and deconvolution,46
(GraphPad Software). whose parameters are summarized in Table 3. The prepro-
cessed data were exported as a .csv format to build the FIT,
ie, (18 samples × 4947 features), and the data were autoscaled
TPC and TFC (unit-variance scaling) for adequate comparisons. Thus, a heat
The TPC and TFC were measured following the previous pro- map was built to intuitively visualize the autoscaled feature dis-
tocols.45 In the case of TPC, the extract solution (20 µL, 5 mg/ tribution using MetaboAnalyst 5.0 (McGill University).47 The
mL) was combined with 10% Folin-Ciocalteu reagent (40 µL) pretreated FIT was also combined with the respective antifungal
and 7.35% sodium carbonate solution (150 µL). The mixture data (ie, %MGI as a continuous variable) to assemble the inte-
was incubated under darkness at room temperature for 2 h, grated dataset. The resulting matrix was then imported into
and the absorbance of the solutions at 765 nm was then mea- SIMCA software (v 14.0) (Umetrics) to build the respective
sured. For TFC, an aliquot of ethanolic extract solution (70 models by single-Y OPLS. The obtained results were visualized
µL, 5 mg/mL) was added to a mixture comprising ethanol using the scores plot and S-plot. Finally, the pretreated FIT was
(50 µL), 10% aluminum trichloride (10 µL), and 0.1 M also used for univariate statistics to build the volcano plot (FC
sodium acetate (10 µL). Subsequently, the mixture was threshold (X-axis) of 1.5 and a t-test threshold (Y-axis) of 0.05
reacted for 40 min under darkness, and finally, its absorbance (p-value) and the cross-validation through ROC curves using
at 420 nm was measured. Three replicates were evaluated for MetaboAnalyst 5.0 (McGill University). For this, the integrated
each determination. A quantitation through a standard curve FIT dataset was subdivided into 2 sub-datasets, such as the
of gallic acid and quercetin was employed, respectively. most-active (MGI>62%) and the least-active (MGI<58%)
Therefore, the TPC was expressed as mg GAE/100 g DW, extracts to be used as categorical variables in the univariate
and TFC was expressed as mg QE/100 g DW. analysis.

High-Performance LC Coupled to MS
Metabolite profiles of test extracts were recorded on a
Purification and Identification of Antifungal Candidates 1-4
Shimadzu Prominence (Shimadzu Corporation) equipped by Semipreparative-Scale HPLC
with 2 binary pumps, an autoinjector, a photodiode array detec- Portions of the PpS, PbaL, and PtL extracts (500 mg) were
tor, and an LCMS2020 mass spectrometer detector with a single separately pretreated with solid-phase extraction (SPE)
quadrupole analyzer and ESI. Each Piper extract was dissolved Strata® C18-U cartridges (55 µm, 70 Å, 500 mg, 6 mL)
in absolute ethanol (5 mg/mL) and injected (20 µL) into the
HPLC system. The separation system consisted of a Synergi
C18 column (Phenomenex) (4.6 mm × 150 mm, 4 μm), and Table 3. Summary of MZmine Parameters Used to Preprocess the
Liquid chromatography-Mass Spectrometry (LC-MS) Data of Piper
a combination of solvent A (1% formic acid in Mili-Q H2O) Extracts.
and solvent B (1% formic acid in acetonitrile [ACN]). A gradi-
ent elution method was used as follows: 0 to 2 min 0% B, 2 to Preprocessing step Parameters
20 min 0% to 50% B, 20 to 30 min 50% B, 30 to 45 min 50% to Mass detection Centroid; MS1 noise level = 1 × 105
100% B, 45 to 50 min 100%, and 50 to 55 min 100% to 0% B, at ADAP chromatogram min group size in # scan: 5; group intensity
0.7 mL/min. The monitoring wavelength was 270 nm. Mass builder threshold: 1 × 107; min highest intensity:
1 × 107; m/z tolerance: 0.5
spectra were simultaneously acquired using ESI in the positive
Chromatogram min peak height: 5 × 105; peak duration: 0 to
ion mode (scan 100-2000 m/z). The MS parameters involved a deconvolution 2 min; baseline level: 1 × 105
voltage detector at 1.5 kV, a curved desolvation line at 250 °C, a Isotope grouping m/z tolerance: 0.5; retention time (RT)
heat block temperature of 400 °C, and a nebulization gas flow tolerance: 0.2; max charge: 2
of 1.5 L/min. Joining aligner m/z tolerance: 0.5; weight for m/z: 70; RT
tolerance: 0.2; weight for RT: 30
Peak filtering min peak in a row: 5; m/z range: 75 to 2000;
Statistical Analysis reset peak number ID: TRUE
Gap filling Intensity tolerance: 10%; m/z tolerance: 0.5;
A Shapiro-Wilk test was performed to determine the data nor- RT tolerance: 0.2
mality of the inhibition percentage, extraction yield, and TPC
10 Natural Product Communications

(Phenomenex). These SPE cartridges were previously condi- ORCID iD

tioned with methanol (6 mL) and then water (6 mL). After Ericsson Coy-Barrera https://orcid.org/0000-0002-3553-9749
loading the extracts, the cartridges were washed with water
(5 mL). The adsorbed components were eluted with methanol
(5 mL). Collected eluates containing depurated extracts were Statement of Human and Animal Rights
used for semipreparative HPLC-mediated isolation after geo- This article does not contain any studies with human or animal subjects.
metrical transfer from optimized analytical conditions to main-
tain resolution and profile. Thus, the target compounds 1-4
were isolated using a UFLC Prominence system (Shimadzu), Statement of Informed Consent
operated in a semipreparative mode, consisting of a pump There are no human subjects in this article, and informed consent is
(LC-20AD), a column oven (CTO-20AC), an ultraviolet/ not applicable.
visible detector (SPD-20AV), an autosampler (SIL-10AP), a
fraction collector (FRC-10A), and equipped with a reversed-
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