Keywords
Echinacea purpurea
- Asteraceae - phenolic/carboxylic acids - inflammation - signaling pathways - human
primary macrophages
Introduction
Echinacea purpurea L. (Asteraceae family) preparations have been traditionally and highly used as an
immune booster, being recognized as safe by the World Health Organization [1 ]. E. purpurea extracts can interact with different entities of the immune system, modulating their
activity. The immunostimulatory activity of E. purpurea is related to the (i ) activation of different immune cells (e.g., macrophages, dendritic cells, CD4 and
CD8 T cells, natural killer cells, monocytes, myeloid progenitor cells, and splenocytes)
[2 ], [3 ], [4 ], [5 ], [6 ], [7 ], [8 ], [9 ], [10 ], [11 ], [12 ], [13 ], [14 ], [15 ], [16 ], [17 ], [18 ], (ii ) promotion of cytokine, chemokine, and immunoglobulin expression [19 ], [20 ], [21 ], [22 ], [23 ], [24 ], (iii ) stimulation of phagocytosis [21 ], [22 ], (iv ) increase in respiratory burst [2 ], [9 ], (v ) increase in cell proliferation (e.g., monocytes, B cells, CD4 and CD8 T cells, and
natural killer cells) [5 ], [6 ], [14 ], [17 ], [25 ], and (vi ) activation of
immune cells (e.g., monocytes, dendritic cells, and natural killer cells) migration
and mobility [25 ], [26 ]. This stimulatory effect may enhance, for instance, the surveillance to infections
[27 ] and a faster resolution of the disease cause [28 ].
The immunostimulatory activity of E. purpurea has been mainly attributed to polysaccharides [5 ], [6 ], [9 ], [11 ], [27 ]. Alkylamides and caffeic acid derivatives can also present this bioactivity [2 ], [3 ], [20 ], [21 ]. However, there are incongruences in the literature regarding extracts bioactivity.
Among different factors, the particular experimental cell procedures used in the several
studies contribute to the reported differences between E. purpurea extracts bioactivity. For example, a butanol fraction of its stems and leaves significantly
activated human-derived dendritic cells but not mouse-derived dendritic cells, suggesting
that specific biochemical and cellular effects of plant extracts may differ between
mammalian species [12 ], [26 ]. Therefore, the determination of the constituentsʼ fingerprint and the use of reliable
in vitro models that could mimic the human cell environment enable a greater accuracy in the
identification of the compounds responsible for a specific activity of plant extracts.
Due to its immunostimulatory activity, E. purpurea formulations could present high added value in the therapy of diseases where the
immune system is compromised (e.g., acquired immunodeficiencies). Particularly, the
activation of a critical immune cell type, such as macrophages, in these individuals
would benefit greatly the overall immune response [29 ]. In addition, there are several studies illustrating the immunostimulatory activity
of E. purpurea extracts, the majority of them being performed with cell lines or murine primary
macrophages [2 ], [9 ], [10 ], [18 ], [20 ], [22 ]. Indeed, to the best of our knowledge, only one study was carried out with human
primary macrophages to demonstrate the ability of a commercial E. purpurea formulation to stimulate the
production of cytokines, including interleukin (IL)-1β , IL-6, tumor necrosis factor (TNF)-α , and IL-10 [23 ]. However, its mechanism of action, as well as the active principles, were not investigated.
Therefore, in this study, we aimed to explore the effects of the extract that most
people consume as a tea, namely the aqueous extract (AE), in human primary monocyte‐derived
macrophage (hMDM) activity. Macrophages are often the first line of the bodyʼs defense
against noxious stimuli [29 ], their activation being essential for the strengthening of the immune system. Different
extracts were prepared by stirring flowers (F), leaves (L), and roots (R) of E. purpurea with water. Then, to determine which class of compounds were responsible for the
immunostimulatory activity, each AE was fractionated by semi-preparative high-performance
liquid chromatography (HPLC) into two fractions: phenolic/carboxylic acids
(F1) and alkylamides (F2) fractions. The production of the main pro-inflammatory cytokines
(IL-6, TNF-α , IL-1β ) and eicosanoids (prostaglandin – PG – E2) was first investigated. These inflammatory
mediators trigger different biochemical cascades in target immune cells, determining
the course of the immune response in a complex inflammatory process. Briefly, they
can, e.g., stimulate chemotaxis and phagocytosis, promote the vascular permeability,
and induce the cytotoxic and bactericidal activities of macrophages and neutrophils
[30 ]. Hence, the increased levels of pro-inflammatory cytokines and eicosanoids can have
a therapeutic action for individuals when the activation of the immune system is a
challenge. Then, the mechanism of action underlying the pro-inflammatory activity
was analyzed. The activation of macrophages is deeply related to the nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-κ B) and
mitogen-activated protein kinase (MAPK) signal transduction pathways [31 ], which are involved in the regulation of pro-inflammatory cytokine expression through
phosphorylation of the transcription factors [32 ]. Based on that, different inflammatory signaling pathways were investigated, namely
NF-κ B p65, extracellular signal-regulated kinase – ERK – 1/2, p38, and cyclooxygenase
– COX – 2 expression. The whole extract exhibited significantly higher immunostimulatory
activity than the phenolic/carboxylic acids fractions (F1), suggesting a complementary
effect between the two classes of compounds. To the best of our knowledge, this is
the first study demonstrating the stimulation of hMDMs by E. purpurea AE, through the activation of ERK 1/2 and p38 of the MAPK signaling pathway, as well
as by increasing COX-2 expression.
Results
[Table 1 ] presents the identified phenolic/carboxylic acid compounds and alkylamides in each
AE. Each extract exhibited different patterns of phenolic/carboxylic acids and alkylamides.
Seven phenolic/carboxylic acids were identified in AE-F and AE-L. AE-R only present
three phenolic/carboxylic acids. Sixteen and fourteen alkylamides were identified
in AE-R and AE-F, respectively. AE-L did not contain any identified alkylamide.
Table 1 Overview of the identified compounds (phenolic/carboxylic acids and alkylamides)
in aqueous E. purpurea extracts (AEs) obtained from flowers (F), leaves (L), and roots (R) by LC-HRMS.
Compounds
AE
F
L
R
Bold text corresponds to studied standards. Symbol “+” represents the presence of
compound; symbol “–” represents the absence of compound. E/Z stereochemistry is indicated
here in accordance with the literature [33 ], but it should be highlighted that without conformational NMR spectra, it is not
possible to conclusively distinguish between E and Z isomers.
Phenolic/carboxylic acids
Malic acid
+
+
+
Vanillic acid
+
+
–
Protocatechuic acid
+
+
–
Caftaric acid
+
+
–
Chlorogenic acid
–
–
–
Quinic acid
–
–
–
Vanillin
–
–
–
Caffeic acid
–
–
–
Benzoic acid
+
+
+
Cynarin
–
–
–
Echinacoside
–
–
–
p -coumaric acid
+
+
–
Chicoric acid
+
+
+
Rutin
–
–
–
Quercetin
–
–
–
Alkylamides
Dodeca-2E,4Z,10E-triene-8-ynoic acid isobutylamide
+
–
+
Dodeca-2E,4Z,10Z-triene-8-ynoic acid isobutylamide
+
–
+
Dodeca-2,4,10-triene-8-ynoic acid isobutylamide (isomer 1)
–
–
–
Dodeca-2E,4E,10Z-triene-8-ynoic acid isobutylamide
–
–
+
Dodeca-2Z,4E,10Z-triene-8-ynoic acid isobutylamide
–
–
+
Dodeca-2E,4E,10E-triene-8-ynoic acid isobutylamide
+
–
–
Undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide
+
–
+
Undeca-2E/Z-ene-8,10-diynoic acid isobutylamide
+
–
–
Undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide
–
–
+
Undeca-2E/Z,4Z/E-diene-8,10-diynoic acid 2-methylbutylamide
–
–
–
Pentadeca-2E,9Z-diene-12,14-diynoic acid 2-hydroxyisobutylamide
–
–
–
Dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide
+
–
+
Undeca-2E,4E-diene-8,10-diynoic acid isobutylamide
–
–
–
Dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide
–
–
+
Dodeca-2E-ene-8,10-diynoic acid isobutylamide
+
–
–
Trideca-2E,7Z-diene-10,12-diynoic acid isobutylamide
+
–
+
Dodeca-2,4-diene-8,10-diynoic acid 2-methylbutylamide
–
–
+
Dodeca-2Z,4Z,10Z-triene-8-ynoic acid isobutylamide
–
–
+
Trideca-2E,7Z-diene-10,12-diynoic acid 2-methylbutylamide
+
–
–
Dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide
+
–
+
Dodeca-2E,4Z,10E-triene-8-ynoic acid 2-methylbutylamide or
+
–
+
Dodeca-2E,4E,8Z-trienoic acid isobutylamide (isomer 1)
–
–
–
Dodeca-2E,4E-dienoic acid isobutylamide (isomer 1)
–
–
–
Pentadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide
–
–
+
Dodeca-2E,4E,8Z-trienoic acid isobutylamide
+
–
+
Trideca-2Z,7Z-diene-10,12-diynoic acid 2-methylbutylamide
–
–
–
Dodeca-2E,4E,8Z,10E/Z-tetraenoic acid 2-methylbutylamide
+
–
–
Hexadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide
–
–
–
Dodeca-2E,4E,8Z-trienoic acid isobutylamide (isomer 2)
–
–
–
Dodeca-2E,4E-dienoic acid isobutylamide
+
–
+
The chromatograms obtained for AE by semi-preparative HPLC are present in [Fig. 1 ]. The three AEs show a higher number of phenolic/carboxylic acids compounds than
alkylamides. AE-F displayed two individual peaks of alkylamides ([Fig. 1 a–ii ]), while AE-L did not exhibit any alkylamide peak ([Fig. 1 b–ii ]). AE-R revealed the highest amount of alkylamides, represented by an irregular peak
([Fig. 1 c–ii ]).
Fig. 1 Semi-preparative HPLC chromatograms of aqueous extracts obtained from flowers (a ), leaves (b ) and roots (c ), at 30 mg/mL (400 µL of injection volume), detected at 330 nm (phenolic/carboxylic
acids; a – i, b – i, and c – i) and 254 nm (alkylamides; a-ii, b-ii, and c-ii).
The metabolic activity and the relative DNA concentration of non-stimulated hMDMs
in the absence or presence of the E. purpurea AE and their phenolic/carboxylic acid fractions at different concentrations are presented
in [Fig. 2 ]. The cell metabolic activity and the DNA concentration were not affected in the
presence of the AE and phenolic/carboxylic acid fractions at any tested concentration,
showing results similar to those of the negative control (non-stimulated hMDMs without
treatment, [Fig. 2 ]). As expected, the positive control (10% of DMSO) significantly decreased the viability
of non-stimulated hMDMs.
Fig. 2 Metabolic activity (a ) and relative DNA concentration (b ) of hMDM cultured in the presence of 10% DMSO, LPS, and different concentrations
of the E. purpurea AEs obtained from flowers (F), leaves (L), roots (R) and their phenolic/carboxylic
acid fractions (F1) for 24 h. The dotted line represents the metabolic activity and
DNA concentration of the negative control (non-stimulated hMDMs without treatment).
Statistically significant differences are **** (p < 0.0001) in comparison to the negative control (non-stimulated hMDMs without treatment).
Data are represented as means ± SD (n = 3).
The immunostimulatory activity of AEs and their phenolic/carboxylic acid fractions
was evaluated by assessing the levels of pro-inflammatory cytokines (IL-6, IL-1β , and TNF-α ) and the eicosanoid PGE2, produced by non-stimulated hMDMs in the cell culture medium.
Non-stimulated hMDMs (negative control) produced basal amounts of IL-6 (5.9 ± 7.6 pg/mL),
TNF-α (40.5 ± 28.9 pg/mL), IL-1β (1.1 ± 0.6 PG/mL), and PGE2 (311.7 ± 47.9 pg/mL). As expected, lipopolysaccharide
(LPS)-stimulated hMDMs (positive control) significantly increased IL-6, TNF-α , and PGE2 production ([Fig. 3 ]). The production of IL-1β was also enhanced ≈ 6 times compared with the negative control, but it was not statistically
significant. When macrophages were incubated with the AE and their phenolic/carboxylic
acids fractions, a significant increase in those cytokines and the eicosanoid in the
culture medium was observed ([Fig. 3 ]), demonstrating its potential to stimulate naïve macrophages. In general, extracts
from flowers and leaves were the most powerful in the stimulation of the production
of pro-inflammatory mediators. Roots presented ≈ 1.3 times lower activity than the
other parts of the plant.
Fig. 3 IL-6 (a ), TNF-α (b ), IL-1β (c ), and PGE2 (d ) production of non-stimulated hMDMs cultured in the presence of LPS and different
concentrations of the E. purpurea AEs obtained from flowers (F), leaves (L), roots (R) and their phenolic/carboxylic
acid fractions (F1) for 24 h. Statistically significant differences are * (p < 0.0395), ** (p < 0.0047), *** (p < 0.0009), and **** (p < 0.0001) in comparison to the negative control (non-stimulated hMDMs without treatment)
for each different tested E. purpurea AE and F1 concentrations, and 1 (p < 0.0438), 2 (p < 0.0093), and 4 (p < 0.0001) in comparison with a (AE-F vs. AE-F-F1), b (AE-L vs. AE-L-F1), and c (AE-R
vs. AE-R-F1). Data are represented as means ± SD (n = 3).
AEs demonstrated an immunostimulatory profile for two important cytokines in inflammation,
IL-6 ([Fig. 3 a ]) and TNF-α ([Fig. 3 b ]). IL-6 and TNF-α production was dramatically enhanced in the presence of 250 µg/mL of AE-F in, respectively,
20 164.9 ± 10 943.3 and 892.7 ± 209.9 times higher than non-stimulated hMDMs. AE-L
demonstrated a comparable bioactivity to AE-F in stimulating IL-6 and TNF-α production (20 011.9 ± 8402.8 and 804.2 ± 154.4 times higher than non-stimulated
hMDMs, respectively). AE-R was able to induce 18 616.9 ± 9241.5 and 742.9 ± 195.3
times the IL-6 and TNF-α production, respectively. Regarding the phenolic/carboxylic acids fractions, only
AE-F-F1 was able to significantly stimulate hMDMs to produce IL-6 at 125 µg/mL (more
than 7136.1 ± 2903.0 times the negative control, [Fig. 3 a ]). Conversely, all the fractions significantly enhanced
TNF-α production ([Fig. 3 b ]). However, the efficacy of all phenolic/carboxylic acid fractions, to stimulate
IL-6 and TNF-α production, was dramatically lower in comparison with the whole extract. Comparing
all the E. purpurea extracts, AE-F and AE-L exhibited the highest immunostimulatory activity for IL-6
and TNF-α production, followed by AE-R, AE-F-F1, AE-L-F1, and AE-R-F1. Moreover, all the AE
demonstrated a similar or higher pro-inflammatory cytokine production than LPS-stimulated
hMDMs.
IL-1β production was markedly improved 41.4 ± 21.7 and 30.8 ± 12.2 times in the presence
of 250 µg/mL AE-F and AE-R, respectively. AE-L immunostimulatory activity was not
significantly demonstrated for this pro-inflammatory cytokine (15.7 ± 1.0 times).
Interestingly, AE-F-F1, at 250 µg/mL, demonstrated a superior immunostimulatory activity,
enhancing 239.6 ± 94.6 times the IL-1β production. AE-R-F1 showed a comparable bioactivity to AE-R (44.0 ± 8.3 times, 250 µg/mL).
IL-1β production by AE-L-F1 was not significantly enhanced (22.5 ± 10.1 times, 250 µg/mL).
Comparing all the E. purpurea extracts, the AE-F-F1 was the fraction that was stronger in stimulating IL-1β production, followed by AE-R-F1, AE-F, AE-R, AE-L-F1, and AE-L. Furthermore, all
the E. purpurea extracts showed higher IL-1β production than LPS-stimulated hMDMs.
PGE2 production was significantly enhanced 1.7 ± 0.1 and 1.4 ± 0.2 times in the presence
of AE-F (250 µg/mL) and AE-L (50 µg/mL), respectively. AE-R did not demonstrate statistically
significant differences in the induction of PGE2 production (1.3 ± 0.1, 125 µg/mL).
In this case, the phenolic/carboxylic acids fractions obtained from AE were more effective
for PGE2 production induction, showing an efficacy approximately 1.7 times higher
compared to non-stimulated hMDMs for all tested fractions, at 250 µg/mL. Comparing
all the E. purpurea extracts, AE-F-F1, AE-L-F1, AE-R-F1, and AE-F led to similar PGE2 production, followed
by AE-L and AE-R. All the phenolic/carboxylic acid fractions and AE-F demonstrated
a similar or higher induction of PGE2 production than LPS-stimulated macrophages.
To understand which mechanism of action was associated with the immunostimulatory
activity of the E. purpurea AE, the signaling pathways of several pro-inflammatory molecules in hMDMs were investigated
by Western blot. As AE at the highest tested concentration induced higher levels of
IL-6 and TNF-α production than their phenolic/carboxylic acids fractions (F1), only these ones were
investigated.
Non-stimulated hMDMs showed basal levels of ERK 1/2 ([Fig. 4 a1 ] and b1 ) and p38 phosphorylation ([Fig. 4 a2 ] and b2 ). The phosphorylation of ERK 1/2 and p38 was significantly enhanced in LPS-stimulated
hMDMs. All AEs, but mainly AE-L and AE-R, also demonstrated the ability to drastically
upregulate the expression of these inflammatory molecules, confirming their immunostimulatory
activity. AE-R demonstrated a strong potential to upregulate the ERK 1/2 phosphorylation,
being higher than that of LPS-stimulated hMDMs, followed by AE-L ≈ AE-F. The phosphorylation
of p38 was similar between the different E. purpurea AEs, and the bioactivity was at similar level as that of LPS-stimulated hMDMs.
Fig. 4 ERK 1/2 (a1 and b1 ), p38 (a2 and b2 ), NF-κ B p65 (a3 and b3 ), and COX-2 (a4 and b4 ) signaling pathway activation of non-stimulated hMDMs cultured in the presence of
LPS and E. purpurea aqueous extracts (AEs) obtained from flowers (F), leaves (L), and roots (R), at 250 µg/mL,
for 24 h. Statistically significant differences are * (p < 0.0457) and ** (p < 0.0078) in comparison to the negative control (non-stimulated hMDMs without treatment)
for each different tested E. purpurea AE. Data are represented as means ± SD (n = 3).
Basal levels of NF-κ B p65 were presented in non-stimulated hMDMs ([Fig. 4 a3 ] and b3 ). Interestingly, the phosphorylation of NF-κ B p65 was not altered in the presence of LPS or AE for 24 h of culture ([Fig. 4 a3 ] and b3 ).
Non-stimulated hMDMs did not present COX-2 expression, which was upregulated after
their LPS-stimulation ([Fig. 4 a4 ] and b4 ). AEs also showed a strong capacity to enhance the COX-2 expression, both AE-R and
AE-L being the most powerful extracts compared to AE-F. The AEs had a similar or higher
capacity to stimulate COX-2 expression in comparison with LPS.
Discussion
The immune system is a powerful tool resulting from the human bodyʼs evolution and
is designed to keep it healthy and functional [34 ]. A healthy immune system is fundamental for recognizing several antigens, ranging
from cancer to a common cold, and for developing and coordinating a complex defense
response to maintain and protect the body. However, in individuals with a depressed
or completely disabled immune system, an increased susceptibility to infections or
illness is observed [35 ]. Therefore, the prescription of drugs to enhance the immune system is needed, E. purpurea being a strong candidate due to its traditional use in the prevention of infections.
Indeed, the plants have been an excellent source of new and safer drugs with high
therapeutic value in the clinic [36 ].
We investigated the E. purpurea AE – the most common beverage drunk by the worldʼs population – composed by phenols/carboxylic
acids and alkylamides as a promoter of hMDM activity. To identify the class of compounds
present in AEs responsible for immunostimulatory activity, a fractionation by semi-preparative
HPLC was further employed for all the three AEs. The optimized method perfectly enabled
the fractionation of AE into phenolic/carboxylic acids (F1) and alkylamides (F2) due
to the different polarities of the compounds ([Fig. 1 ]). The bioactivity of the phenolic/carboxylic acid fractions obtained from AEs was
also biologically evaluated to make conclusions about the class of compounds that
mainly contribute to the immunostimulatory activity. Due to yield constrains, the
bioactivity of the alkylamide fraction (F2) was not evaluated.
The identification of the bioactive compounds present in AE performed by LC-HRMS are
in agreement with our previous work [33 ]. Only AEs were chemically identified since no significant biological activity was
observed in F1. AEs, exhibiting different fingerprint patterns considering the plant
part, are enriched in phenolic/carboxylic acid compounds, with small amounts of alkylamides
([Table 1 ]). Briefly, in flowers and leaves, there were seven phenolic/carboxylic acids identified,
namely malic acid, vanillic acid, protocatechuic acid, caftaric acid, benzoic acid,
p-coumaric acid, and chicoric acid. Only three of these (malic acid, benzoic acid,
and chicoric acid) were present in AE obtained from roots. The alkylamides were more
concentrated in roots (16 compounds) than in flowers (14 compounds). Dodeca-2E,4Z,10E-triene-8-ynoic
acid isobutylamide, dodeca-2E,4Z,10Z-triene-8-ynoic acid isobutylamide,
undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide, dodeca-2E,4Z-diene-8,10-diynoic
acid isobutylamide, trideca-2E,7Z-diene-10,12-diynoic acid isobutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic
acid isobutylamide, dodeca-2E,4Z,10E-triene-8-ynoic acid 2-methylbutylamide or dodeca-2E-ene-8,10-diynoic
acid 2-methylbutylamide, dodeca-2E,4E,8Z-trienoic acid isobutylamide, and dodeca-2E,4E-dienoic
acid isobutylamide were present in both roots and flowers. Dodeca-2E,4E,10Z-triene-8-ynoic
acid isobutylamide, dodeca-2Z,4E,10Z-triene-8-ynoic acid isobutylamide, undeca-2Z,4E-diene-8,10-diynoic
acid isobutylamide, dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide, dodeca-2,4-diene-8,10-diynoic
acid 2-methylbutylamide, dodeca-2Z,4Z,10Z-triene-8-ynoic acid isobutylamide, and pentadeca-2E,9Z-diene-12,14-diynoic
acid isobutylamide were only identified in roots. Conversely, dodeca-2E,4E,10E-triene-8-ynoic
acid isobutylamide, undeca-2E/Z-ene-8,10-diynoic acid isobutylamide,
dodeca-2E-ene-8,10-diynoic acid isobutylamide, trideca-2E,7Z-diene-10,12-diynoic acid
2-methylbutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid 2-methylbutylamide were
only present in flowers. The extracts obtained from leaves did not present any identified
alkylamide ([Fig. 1 ]). Analyzing the three AE, flowers exhibited the highest number of identified bioactive
compounds (21 compounds), followed by roots (19 compounds) and leaves (7 compounds).
In a first approach, we investigated the capacity of cytocompatible AEs and their
phenolic/carboxylic acid fractions to induce the overexpression of the main mediators
of the acute phase response, namely pro-inflammatory cytokines (IL-6, TNF-α , IL-1β ) and an eicosanoid (PGE2), in hMDMs [30 ]. The AE and their phenolic/carboxylic acid fractions were not cytotoxic for hMDMs
at any tested concentration, demonstrating their cytocompatibility ([Fig. 2 ]). In addition, AEs and their phenolic/carboxylic acid fractions induced the production
of IL-6 and TNF-α by hMDMs, having minimal effect in IL-1β and PGE2 ([Fig. 3 ]). IL-1β is synthesized as a precursor peptide (pro-IL-1β ) and secreted in the mature form (IL-1β ) by activated macrophages [30 ]. Accordingly, AE may not be able to activate certain signals required for the
complete maturation of IL-1β . Conversely, AE-F-F1 was incredibly efficient to activate the mature form of IL-1β ([Fig. 3 ]). Interestingly, when the AEs were separated into phenolic/carboxylic acid fractions,
in general, the immunostimulatory activity considerably decreased. All these results
highlight the possibility that a complementary effect between phenolic/carboxylic
acid compounds and alkylamides can occur, as we previously hypothesized with a human
cell line [33 ]. Therefore, the multiplicity of bioactive compounds present in the whole AE may
provide a greater immunostimulatory activity [23 ]. However, AE-L did not present any alkylamide [33 ], and its immunostimulatory activity was similar to AE-F, which led us to make conclusions
about the presence of other bioactive compounds, such as polysaccharides, that can
also be responsible for the
stimulation of hMDMs [5 ], [6 ], [9 ], [11 ], [27 ]. Flowers demonstrated an enhanced potential in hMDM activation, which may be correlated
with the highest number of identified bioactive compounds ([Fig. 3 ]). We also investigated the mechanism of action of the AE underlying the immunostimulatory
activity in hMDMs. No significant effects were observed in NF-κ B p65 phosphorylation when hMDMs were treated with AE ([Fig. 4 ]). Matthias et al. also reported similar results with the Jurkat human T cell line
treated with E. purpurea ethanolic extracts and their fractions [37 ]. The E. purpurea AE triggered the phosphorylation of the ERK 1/2 and p38 inflammatory proteins in
the MAPK signaling pathway in hMDMs ([Fig. 4 ]). Additionally, for the first time, we demonstrated the upregulation of COX-2 expression
in the presence of AE. These events suggest an activation of macrophages. However,
further research is needed to confirm direct evidence for the suggested causative
link. Sullivan et al. described comparable results, reporting an E. purpurea polysaccharide fraction that activated murine-derived macrophages to produce pro-inflammatory
cytokines (IL-6, TNF-α , and IL-12p70) through the activation of MAPK pathways (ERK, p38, and JNK) [9 ].
The efficacy of E. purpurea AEs and their phenolic/carboxylic acid fractions in the stimulation of hMDMs depends
on its phytochemical composition. All AEs demonstrated higher pro-inflammatory capacity
in comparison with their phenolic/carboxylic acid fractions, pointing to the existence
of a complementary effect between phenols/carboxylic acids and alkylamides. This study
also shows the importance of fractionating and chemically characterizing the plant
extracts to identify the therapeutic profile of different classes of compounds. Different
studies pointed out the bioavailability of echinacea constituents. Alkylamides are
interesting in the pharmaceutical field since they have been shown to have good permeability
using a Caco-2 cell monolayer [38 ], [39 ], demonstrating their potential oral bioavailability. Additionally, alkylamides were
detected in plasma and blood following an oral dose of echinacea as a tablet
formulation, in contrast to phenols [40 ], [41 ]. As the aqueous extracts are more enriched in phenols/carboxylic acids, advanced
techniques for encapsulating those bioactive compounds must be addressed to overcome
the poor bioavailability.
AEs strongly induce the production of IL-6 and TNF-α by stimulated hMDMs. However, a minimal effect in IL-1β and PGE2 concentrations was observed. Based on the presented results, these events
can be correlated with the activation of the ERK 1/2 and p38 signaling pathways and
the upregulation of COX-2 expression. Nonetheless, it is important to note that while
these associations were observed, additional research is needed to establish whether
these signaling events directly influence cytokine production and macrophage activity.
Flowers, presenting the highest number of identified bioactive compounds, demonstrated
the greatest immunostimulatory activity. Consequently, the activated macrophages can
provide an efficient defensive response against noxious stimulus, protecting the body
from infections and diseases. These promising results demonstrate the therapeutic
value of E. purpurea AEs in the activation of crucial immune cells, highlighting the potential
of this immunostimulant plant-based formulation in disorders in which the immune system
is impaired. In summary, the data presented in this proof-of-concept study provide
a basis for further investigation. Future work will include the use of pathway inhibitors,
knockdowns, and additional molecular targets to thoroughly elucidate the mechanisms
of action through which the bioactive compounds in AE extracts exert their effects.
Further studies supporting the role of AE in complex models of inflammation should
also be explored.
Materials and Methods
Reagents and chemicals
E. purpurea , commonly known as purple coneflower, was purchased from Cantinho das Aromáticas
in May 2017. The plants were immediately transferred to the soil and were allowed
to grow following a sustainable agriculture procedure (41°37′04.5″ N, 7°16′14.4″ W).
After one year of cultivation, the flowers and leaves were collected in a full bloom
phase (June and July 2018), while the roots, including rhizomes, were harvested in
autumn (October 2018). The plants were dried in the dark and stored at room temperature
(RT) protected from the light. A voucher specimen of both aerial parts (DB-16-EPT)
and roots (DB-15-EPR), identified by Prof. Alberto C. P. Dias, was deposited at the
Department of Biology, University of Minho, Portugal. Ultra-pure water was obtained
from a Milli-Q Direct Water Purification System (Milli-Q Direct 16, Millipore). Roswell
Park Memorial Institute (RPMI)-1640 media, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) buffer solution 1 M,
penicillin-streptomycin (10 000 U/mL), Dulbeccoʼs phosphate-buffered saline (DPBS),
Pierce Chromogenic Endotoxin Quant Kit, Quant-iT PicoGreen dsDNA Kit, Pierce Phosphatase
Inhibitor Mini Tablets, PageRuler Plus Prestained Protein Ladder (10 to 250 kDa),
Bolt Sample Reducing Agent, Bolt LDS Sample Buffer, Bis-Tris Bolt 8%, Bolt MES SDS
Running Buffer, and iBlot 2 Transfer Stacks (polyvinylidene fluoride, PVDF) were purchased
from Thermo Fisher Scientific. An OctoMACS separator, human CD14 microbeads, MS columns,
and human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) were
obtained from Miltenyi Biotec. AlamarBlue, Bio-Rad Protein Assay Dye Reagent Concentrate,
and Tween20 were purchased from Bio-Rad. Human IL-1β , IL-6, and TNF-α , DuoSet Enzyme-linked immunosorbent assay (ELISA) and DuoSet ELISA Ancillary Reagent
Kit 2 were purchased from R&D Systems. Ethanol, acetonitrile (ACN) HPLC grade, methanol
HPLC grade, formic acid analytical
grade, Histopaque-1077, human serum, lipopolysaccharide (LPS; Escherichia coli O26:B6), radioimmunoprecipitation assay (RIPA) buffer, complete mini protease inhibitor
cocktail tablets, bovine serum albumin (BSA), tris-base, and high-purity standards
of echinacoside (purity 100%), chicoric acid (purity 100%), caftaric acid (purity
> 97%), caffeic acid (purity 100%), chlorogenic acid (purity 100%), and cynarin (purity
> 98%) were obtained from Sigma-Aldrich. An echinacea isobutylamide standards kit,
composed of undeca-2E/Z-ene-8,10-diynoic acid isobutylamide (purity > 99%), dodeca-2E-ene-8,10-diynoic
acid isobutylamide (purity 100%), and dodeca-2E,4E-dienoic acid isobutylamide (purity
> 93%), was acquired from ChromaDex. High-purity standard dodeca-2E,4E,8Z,10E/Z-tetraenoic
acid isobutylamide (purity > 99%) was obtained from Biosynth Carbosynth. A PGE2 ELISA
Kit and rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were acquired from
Abcam. IRDye
800CW Goat anti-Rabbit IgG and IRDye 680RD Goat anti-Rabbit IgG secondary antibodies
were obtained from LI-COR Biosciences. Monoclonal rabbit NF-κ B p65, monoclonal rabbit p44/42 MAPK (ERK 1/2), monoclonal rabbit p38 MAPK, monoclonal
rabbit COX-2, monoclonal rabbit phospho-NF-κ B p65, monoclonal rabbit phospho-p38 MAPK, and monoclonal rabbit phospho-p44/42 MAPK
(ERK 1/2) were purchased from Cell Signaling. Sodium chloride (NaCl) was purchased
from PanReac AppliChem. Coffee filter paper N4 was acquired in a local supermarket.
Bioactive compounds extraction
The plant material was ground immediately before extraction. Dried flowers (F) and
leaves (L) were ground using a blender (Picadora Clássica 123 A320R1, Moulinex), while
dried roots (R) were ground using an Analytical Sieve Shaker (AS200 Digit, Retsch,
Germany). The AEs were prepared by stirring 20 g of sample in 150 mL of ultra-pure
water at RT for 24 h, as previously described by Vieira et al. [33 ]. The water was changed after 12 h of the extraction process. After extraction, the
AE was filtrated using a coffee filter paper N4. Both solutions were mixed, frozen
at − 80 °C and then freeze-dried (Lyoquest − 85 °C Plus Eco, Telstar). The lyophilized
extracts were stored at − 80 °C until further use. The AE tested negative for endotoxin
contamination by the limulus amebocyte lysate (Pierce Chromogenic Endotoxin Quant
kit).
Fractionation of the AE
The dry powder of AE was dissolved in ultra-pure water (30 mg/mL) and centrifuged
at 10 000 × g for 5 min (ScanSpeed Mini, Labogene). The collected supernatants were
injected (200 – 400 µL) in the LaChrom Merck Hitachi system equipped with a D-7000
Interface, an L-7100 Pump, an L-7200 autosampler, an L-7455 diode array detector (DAD),
and an HPLC System Manager HSMD-7000, version 3.0. The fractionation method previously
optimized by our research group was employed to fractionate AE [42 ]. Briefly, the chromatographic separation was performed on a Uptisphere WOD homemade
semi-preparative column (250 mm × 10 mm, 5 µm, interchrom, Interchim, Montluçon, France),
employing a gradient elution composed of 0.1% formic acid and ACN. The flow rate was
2 mL/min. Two fractions, phenolic/carboxylic acid (F1, 2 – 11 min) and alkylamide
(F2, 11 – 20 min) fractions, were obtained through the eluent collection. The organic
solvent was evaporated in a rotavapor
(R210 Buchi, Switzerland). Then, the fractions were freeze-dried (LyoQuest Plus Eco,
Telstar) to remove the water content. The powder obtained was stored at − 80 °C until
further use.
Chemical characterization of the AE
The chemical characterization of the AE was performed according to the liquid chromatography–high-resolution
mass spectrometry (LC-HRMS) analysis method described by us [33 ]. AEs were dissolved in ultra-pure water at 5.0 mg/mL. The AEs were centrifuged (10 000
× g, 5 min), and the supernatant was collected and injected into the UltiMate 3000
Dionex ultra-high-performance liquid chromatography (UHPLC, Thermo Scientific, Lisbon,
Portugal), coupled to an ultra-high-resolution quadrupole-quadrupole time-of-flight
(UHR-QqTOF) mass spectrometer (Impact II, Bruker). Bruker Compass DataAnalysis 5.1
software (Bruker) was used to process the LC-HRMS-acquired data. The identification
of the chemical compounds present in AE was confirmed by the matching of the retention
time (tR
, min), mass-to-charge ratio (m/z ) of the molecular ion, and MS/MS fragmentation patterns with the standards. When
the tR
and MS data did
not match with the available standards, the potential identity of the compound was
assigned by comparing the MS/MS spectra with the theoretical data MS/MS fragments
and data in the literature [33 ].
Preparation of AE and fractions solutions
Stock solutions of AE (5.1 mg/mL, F, L and R) and phenolic/carboxylic acid fractions
(60.0 mg/mL) were prepared in ultra-pure water and sterilized with a 0.22 µm filter.
The alkylamine fraction prepared from the AE was not biologically studied, since no
measurable amounts were obtained.
Aliquots of the stock solutions of each extract and phenolic/carboxylic acid fractions
were prepared and stored at − 80 °C. Then, serial dilutions were made with complete
RPMI-1640 culture medium with 2 mM glutamine supplemented with 10% human serum, 1%
penicillin/streptomycin, and 1% HEPES (cRPMI). Different final concentrations of AE
(50, 125, and 250 µg/mL) and phenolic/carboxylic fractions (125 and 250 µg/mL) were
tested in the biological studies.
Biological studies
Ethics statement
The collection of peripheral blood from healthy volunteers at Hospital of Braga, Portugal,
was approved on the 14th of December, 2018, by the Ethics Subcommittee for Life and
Health Sciences (SECVS) of the University of Minho, Portugal (No. 014/015). The principles
expressed in the Declaration of Helsinki were followed, and participants provided
signed informed consent.
Isolation and differentiation of monocytes
Monocytes were isolated from buffy coats, as previously described by Gonçalves et
al. [43 ]. Briefly, peripheral blood mononuclear cells (PBMCs) were subjected to a density
gradient centrifugation using a Histopaque-1077 solution. The PBMC ring was carefully
collected and washed twice with PBS. Then, the monocytes were isolated from the PBMCs
using positive magnetic beads separation with CD14 microbeads, according to the instructions
of the manufacturer. Isolated monocytes were resuspended in cRPMI. The monocytes (5 × 105 cells/well in adherent 24-well plates) were seeded in the presence of 20 ng/mL of
recombinant human GM-CSF and incubated at 37 °C in a humidified atmosphere at 5% CO2 for 7 days. The culture medium was replaced every 3 days, and the acquisition of
macrophage morphology was confirmed by visualization under an inverted microscope
(Axiovert 40, Zeiss).
Pro-inflammatory activity evaluation
The hMDMs were incubated with AEs and their phenolic/carboxylic acid fractions at
different concentrations for 24 h. Afterward, the culture medium was harvested (the
triplicates were mixed and homogenized) and stored aliquoted at − 80 °C until cytokines
quantification. The cells were washed with warm sterile DPBS, and the metabolic activity
and DNA quantification were determined as described in the following section. The
hMDMs cultured without treatment (only with culture medium) and stimulated with 100 ng/mL
of LPS for 24 h were used as negative and positive controls in the production of the
pro-inflammatory mediators, respectively [42 ].
Metabolic activity and DNA quantification
The metabolic activity and DNA concentration of hMDMs incubated with AEs and their
phenolic/carboxylic acid fractions were determined using an alamarBlue assay and a
fluorimetric dsDNA quantification kit, respectively, as previously described by Vieira
et al. [44 ]. The hMDMs cultured without treatment (only with culture medium) and treated with
10% DMSO were used as a negative and positive control, respectively, to access cytotoxicity.
The results of metabolic activity were expressed in percentages related to the negative
control. DNA contents were expressed in relative concentrations of the negative control.
Cytokine and eicosanoid quantification
The amounts of IL-1β , IL-6, TNF-α , and PGE2 were assayed using ELISA kits, according to the instructions of the manufacturer.
The obtained values were normalized by the respective DNA concentrations. The results
were expressed as fold change relative to the non-stimulated hMDM condition (negative
control).
Western blot analysis
To determine the expression of inflammatory proteins and phosphorylation of the signaling
pathways involving the activation of macrophages, Western blot analysis was performed.
The hMDMs (5 × 105 cells/well in 24-well plates) were cultured with AE for 24 h, at 37 °C and 5% CO2 . After that period of time, the medium was removed, and the cells were washed with
ice DPBS. Then, the cells were lysed in RIPA buffer containing a mixture of protease
and phosphatase inhibitors, at 4 °C for 30 min, under shaking. Then, samples were
collected into an Eppendorf tube and centrifuged (2000 rpm, 20 min). The supernatant
was used to determine the protein content using the Bio-Rad Protein Assay. Bolt sample-reducing
agent and bolt LDS sample buffer were added to 30 – 40 µg of protein. Then, the samples
were heated and denatured at 70 °C (20 min) and 95 °C (5 min). The centrifuged samples
(13 000 × g, 1 min) were loaded and separated on an 8% precast polyacrylamide gel
set on a Mini Gel Tank (Invitrogen, ThermoFisher Scientific). The proteins were transferred
from the gel to a PVDF membrane using the iBlot 2 Gel Transfer Device (Invitrogen,
ThermoFisher Scientific). After blocking for 30 min at RT with 5% BSA in tris-buffered
saline with Tween20 (TBST), the membranes were incubated overnight at 4 °C with the
following primary antibodies diluted in blocking solution: NF-κ B p65 (1 : 1000), p44/42 MAPK (ERK 1/2) (1 : 1000), p38 MAPK (1 : 1000), COX-2 (1 : 500),
phospho-NF-κ B p65 (1 : 1000), phospho-p38 MAPK (1 : 1000), phospho-p44/42 MAPK (ERK 1/2) (1 : 1000),
and GAPDH (1 : 10 000). The membranes were washed three times for 5 min with TBST,
and then, IRDye 800CW Goat anti-Rabbit IgG or IRDye 680RD Goat anti-Mouse IgG secondary
antibodies, both diluted in TBST (1 : 15,000), were incubated for 1 h at RT in the
dark. The Odyssey Fc Imaging System (LI-COR Inc., 2800, Nebraska, USA) was used for
image acquisition of the Western
blots using a near-infrared method set to 700 or 800 nm. The intensity of the bands
was quantified with Image Studio Lite software (LI-COR, Inc., Version 5.2.5). The
data were normalized to the housekeeping GAPDH. The phenolic/carboxylic acid fractions
obtained from AE were not further studied, since no significant immunostimulatory
activity was observed.
Statistical analysis
Results were expressed as mean ± standard deviation (SD) of three independent experiments,
with a minimum of three replicates for each condition. Statistical analyses were performed
using GraphPad Prism 8.0.1 software. Two-way analysis of variance (ANOVA) and Dunnettʼs
multiple comparison test or Sidakʼs multiple comparisons test were used for cell assays.
Differences between experimental groups were considered significant with a confidence
interval of 99%, whenever p < 0.01.
Contributorsʼ Statement
Sara F. Vieira: Conceptualization, Data curation, Formal analysis, Investigation,
Methodology, Software, Resources, Validation, Visualization, Writing – original draft.
Samuel M. Gonçalves: Methodology, Validation, Visualization, Writing – review & editing.
Virgínia M. F. Gonçalves: Methodology, Validation, Visualization, Writing – review
& editing. Maria E. Tiritan: Methodology, Validation, Visualization, Writing – review
& editing. Cristina Cunha: Methodology, Validation, Visualization, Writing – review
& editing. Agostinho Carvalho: Methodology, Validation, Visualization, Writing – review
& editing. Rui L. Reis: Funding acquisition; Project administration; Resources; Validation,
Visualization, Writing – review & editing. Helena Ferreira: Conceptualization, Methodology,
Validation, Supervision, Visualization, Writing – review & editing. Nuno M. Neves:
Conceptualization, Funding acquisition; Methodology, Project administration; Resources;
Validation, Supervision, Visualization, Writing – review & editing. All authors have
read and agreed to the published version of the manuscript.
