Phytomedicine 65 (2019) 153090

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Phytomedicine
journal homepage: www.elsevier.com/locate/phymed

Original Article

Combined extracts of Echinacea angustifolia DC. and Zingiber officinale Roscoe T

in softgel capsules: Pharmacokinetics and immunomodulatory effects
assessed by gene expression profiling
Stefano Dall'Acquaa, Iztok Grabnarb, Roberto Verardoc, Enio Klaricc, Luigi Marchionnid,
Eddie Luidy-Imadad,e, Stefania Sutf, Chiara Agostinisg, Roberta Bullah, Beatrice Perissuttii,
⁎
Dario Voinovichi,
a
Department of Pharmaceutical Sciences, University of Padova, Via F. Marzolo 5, 35131, Padova, Italy
b
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, SI-1000 Ljubljana, Slovenia
c
National Laboratory of the Interuniversity Consortium for Biotechnology, Area Science Park – Padriciano 99, 34149, Trieste, Italy
d
Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
e
Department of Biochemistry and Immunology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
f
Department of Agronomy, Food, Natural Resources, Animals and the Environment, Viale dell'Università 16 - 35020 Legnaro, Pd, Italy
g
Institute for Maternal and Child Health, IRCCS Burlo Garofolo, via dell'Istria 65/1, 34143, Trieste, Italy
h
Department of Life Sciences, University of Trieste, via Valerio, 28, 34127, Trieste, Italy
i
Department of Chemical and Pharmaceutical Sciences, University of Trieste, P.le Europa 1, 34127 Trieste, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Background and objective: Echinacea angustifolia DC. and Zingiber officinale Roscoe are two natural products with
Combination documented immunomodulatory activity, both able to modulate the expression of important immune-related
Echinacea angustifolia DC genes. Thus, their use in combination seems to be particularly promising. In this context, we have considered the
Zingiber officinale Roscoe oral supplementation of a highly standardized lipophilic extract combining both above-mentioned phytocom-
Lipophilic extract
plexes, formulated in attractive softgel capsules, with two objectives: on the one hand to study oral pharma-
Pharmacokinetics
cokinetic of main active extracts’ components and on the other hand to examine the immunomodulation and
Gene expression analysis
anti-inflammatory properties by gene expression profiling.
Methods: Softgel capsules containing a combination of E. angustifolia DC. and Z. officinale Roscoe (5 mg and
25 mg, respectively) were given by oral administration to 10 healthy volunteers. The plasma concentrations of
dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide (tetraene) for E. angustifolia DC., 6-gingerol and 6-shogaol
(free and glucuronide) for Z. officinale Roscoe were determined by LC-MS analysis, and the pharmacokinetic
analysis was performed. To understand the functional mechanisms responsible for the documented health
benefits, we also examined the overall transcriptional remodeling induced in the peripheral blood mononuclear
cells and performed an integrative functional analysis on the generated gene expression.
Results: All bioactive components were absorbed very rapidly, and their tmax were detected in plasma from
30 min to 1.40 h. The peak concentrations of tetraene, 6-gingerol, 6-shogaol and their glucuronide metabolites
were 14.74, 5.66, 9.25, 29.2 and 22.24 ng/ml, respectively. Integrated analysis performed on the generated gene
expression data highlighted immunomodulatory and anti-inflammatory effects similar to those exerted by

Abbreviations: ALDOA, Aldolase A; CAT, Correspondence-At-Top; CD, cluster of differentiation; cDNA, complementary DNA; CHD, coronary heart disease; CL/F,
apparent clearance; cRNA, complementary RNA; DEFA, alpha-defensin; FDR, False Discovery Rate; FGS, Functional Gene Sets; GEO, Gene Expression Omnibus;
GSEA, Gene Set Enrichment Analysis; GSE, Gene Expression Series; HNP, human neutrophil peptides; IFN-γ, interferon gamma; IL, interleukin; KEEG, Kyoto
Encyclopedia of Genes and Genomes; LEx, combined lipophilic extracts of Echinacea angustifolia DC. and Zingiber officinale Roscoe; MA-plot, application of a
Bland–Altman plot for visual representation of genomic data; MDA, melanoma differentiation associated gene; MsigDB, Broad Institute Molecular Signature
Database; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; PBMCs, peripheral blood mononuclear cells; PCA, principal components’ analysis;
PDZ, acronym of the first letters of the first three proteins discovered to share the domain: Post synaptic density protein, Drosophila disc large tumor suppressor and
Zonula occludens-1 protein; PMN, polymorphonuclear neutrophils; PPM1B, Protein phosphatase 1B; PRDX, Peroxiredoxin; qRT-PCR, quantitative Real Time poly-
merase chain reaction; RORA, Retinoic Acid Receptor-Related Orphan Receptor Alpha; RV, reservoir volume; SDCBP, syndecan-binding protein; Th, T helper; TNF-α,
tumor necrosis factor
⁎
Corresponding author.
E-mail address: vojnovic@units.it (D. Voinovich).

https://doi.org/10.1016/j.phymed.2019.153090
Received 14 December 2018; Received in revised form 10 September 2019; Accepted 15 September 2019
0944-7113/ © 2019 Elsevier GmbH. All rights reserved.
S. Dall'Acqua, et al.
hydrocortisone.
Conclusion: These data demonstrated that the bioactive ingredients are highly and rapidly absorbed from softgel
capsules containing the combination of the above-mentioned lipophilic extracts, providing evidence to support
their immunomodulatory and anti-inflammatory properties. These data also help in defining the mechanistic
pathways underlying the health benefits of these plant-derived bioactive compounds.
Introduction
Combination therapy, a treatment modality that combines two or
more therapeutic agents, is rational and fashionable; lot of drugs are
coadministered to enhance efficacy and tolerability, avoiding multiple
administrations and improving patient compliance (EMA/CHMP/
158268/2017). This is a marketing trend also in the field of food sup-
plements (Länger and Kubelka, 2001). Even though several papers re-
port simultaneous administration of plant extracts/compounds and
conventional drugs (e.g. antibiotics, chemotherapics, etc.)
(Hemalswarya and Doble, 2006; Samoilova et al., 2014; Trill et al.,
2017), quite surprisingly in the face of the market demand, only a few
of those deal with combination therapies of plants extracts/compounds
(Wagner and Ulrich-Merzenich, 2009; Mazumder et al., 2018;
Rondanelli et al., 2017). A further steadily growing market need in the
nutraceutical field is the quality of the plant extracts/compounds, to-
gether with the need of innovative formulations (Kaur, 2016).
In this scenario, to address the above-mentioned trends, we have
considered a highly standardized combined lipophilic extract of
Echinacea angustifolia DC. and Zingiber officinale Roscoe (below indicated
E. Angustifolia and Z. officinale) formulated in attractive softgel capsules.
These two natural products have documented immunomodulatory
activities that are able to modulate the expression of important im-
mune-related genes (Altamirano-Dimas et al., 2007; Dall'Acqua et al.,
2015;
Grzanna et al., 2005; Randolph et al., 2003; Wang et al., 2006;
Yu et al., 2011); hence their use in combination seems to be particularly
promising. Indeed, a recent clinical trial provided evidence of the fea-
sibility of their use in combination for the treatment of inflammation
and chronic pain: patients testified improvement of the symptoms and
functions (Rondanelli et al., 2017).
As for the formulation, since we already demonstrated the effect of
the drug delivery system on the pharmacokinetics of natural products,
and in particular on Echinacea alkamides (Dall'Acqua et al., 2015),
softgel capsules were chosen for this study since they had proved to be
the most bioavailable in our previous study.
Given that the content of active ingredients of both extracts was
precisely known, we were able to monitor simultaneously their plasma
profiles after oral administration in healthy volunteers, and to carry out
the pharmacokinetic analysis, also in comparison to available phar-
macokinetic literature data of E. angustifolia lipophilic extract alone
(Dall'Acqua et al., 2015). As for the Z. officinale, in most clinical trials
dry extracts have been considered with a non-measured content of ac-
tive ingredients, as already pointed out by Yu and coworkers (Yu et al.,
2011), and very limited studies have been dedicated to its pharmaco-
kinetics as yet (Yu et al., 2011; Zick et al., 2008). Thus, this study re-
presents a very remarkable contribution to the nutraceutical field.
A second purpose of the paper was the study of the im-
munomodulation of this combined supplementation, once more fa-
cilitated by the highly standardized combined extract and carried out
by means of gene expression profiling. In this context, the rise of
genomics techniques such as whole genome transcriptome analyses
enabled the measurements of whole genome effects in immune cells
(Kussmann et al., 2008; Ovesná et al., 2008). Many studies have focused
on the immunomodulatory activities that such bioactive molecules or
extracts exert on specific cell types, including lymphocytes and per-
ipheral blood mononuclear cells (PBMCs) isolated and in vitro cultured
(Denzler et al., 2010; van Breda et al. 2014; Yan et al., 2013). Since
2
Phytomedicine 65 (2019) 153090
PBMCs transcriptional profiling is a useful tool to define the im-
munomodulatory effects of different dietary interventions, we in-
vestigated the transcriptional effects on PBMCs gene expression in
healthy volunteers after oral administration of combined E. angustifolia
and Z. officinale lipophilic extracts in softgel capsules. In order to de-
termine the underlying functional mechanisms that correlate to re-
ported health benefits of the combined lipophilic extracts, an in-
tegrative functional analysis was performed on the generated gene
expression matrices.
Materials and methods
Softgel capsules (Indena, Milan, Italy), weighing 380 mg and con-
taining LEx. Each capsule contained 25 mg of Z. officinale lipophilic
extract (2,75 mg 6-gingerol, 0,75 mg 6-shogaol) and 5 mg of E. angu-
stifolia lipophilic extract enriched in alkamides (0,5 mg of dodeca-
2E,4E,8Z,10E/Z-tetraenoic isobutylamide). LC-MS-MS analysis was
performed to determine the content of above mentioned actives; the
results are reported in paragraph “LC-MS-MS analysis”.
Reagents
Acetonitrile (HPLC grade, Rotisolv) and Tris buffer (Pufferan) were
from Carl Roth GmbH+Co. (Karlsruhe, Germany). To obtain the pur-
ified water for HPLC analysis was used a Barnstead (easy pure RF)
compact ultrapure water purification system. Acetonitrile, formic acid,
hexane, ethyl acetate and methanol were from BDH (Liverpool, UK).
Tris buffer (Tris-d11, 99% D), benzanilide (98%), 6-gingerol, 6-shogaol,
pelargonic acid vanillylamide and β-glucuronidase (Type IX-A from
Escherichia coli) were purchased from Sigma-Aldrich (Milan, Italy),
dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide was obtained from
Phytolab gmb (Vestenbergsgreuth, Germany).
Study design
10 healthy volunteers, 6 males and 4 females, between 30 and 55
years of age, participated in this single blind study. All the participants
were in good health, with normal liver function and without diagnosed
allergy to either Zingiberaceae or Compositae plants. In addition, vo-
lunteers were not taking any chronic medications, must avoid smoke
and be fasted from all food and drink except water, for 12 h prior to
administration. All subjects gave their written consent to participate in
this study. Two softgel capsules were orally administered, equivalent to
5,5 mg of 6-gingerol, 1,5 mg of 6-shogaol and 1 mg of dodeca-
2E,4E,8Z,10E/Z-tetraenoic isobutylamides. Blood samples (2 × 5 ml)
were drawn from participants at baseline and at 15, 30, 45, 60, 90, 120,
180, 240, and 300 min. Plasma was separated immediately from blood
samples by centrifugation and stored frozen at −80 °C for analysis. The
participants in the study refrained from smoking, eating and drinking
until the last blood sample was taken. The study protocol was approved
by the University of Trieste Human Research Ethics Committee.
Extraction of dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamides
The plasma samples were extracted using a solid-phase extraction
technique as previously described (Woelkart et al., 2005; Guiotto et al.,
2008). Briefly, to 2 ml of plasma, 2 ml of Tris buffer and 100 μl of
benzanilide solution (10 μg/ml), used as internal standard, were added
and vortexed for 1 min. Subsequently, samples were centrifuged for
S. Dall'Acqua, et al.
10 min at 3200 rpm using Eppendorf centrifuge 5810R (Hamburg,
Germany). The supernatant was applied onto C18, 100 mg Isolute col-
umns from International Sorbent Technology (Mid Glamorgan, UK),
pre-treated with 1 reservoir volume (RV) acetonitrile followed by 1 RV
water. The C18 cartridges were placed on a VacMaster sample proces-
sing station and subsequently washed with 1 RV of water under va-
cuum. The dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides were
eluted with 2 ml acetonitrile. The eluents were evaporated under a
stream of nitrogen at 40 °C using TurboVab LV vaporator (Zymark,
Hopkinton, MA, USA), the dry residue was dissolved in 100 µl acet-
onitrile: water (6:4) and 20 μl were used for Liquid chromato-
graphy–mass spectrometry (LC/MS).
Extraction of 6-gingerol and 6-shogaol
Plasma samples (1 ml) were transferred to microcentrifuge tubes,
then spiked with 10 μl of internal standard pelargonic acid vanillyla-
mide working solution (10 μg/ml) and extracted with 4 ml ethyl
acetate/hexane (1:1, v/v). Tubes were vortexed for 20 min at high
speed and centrifuged for 15 min at 3000 rpm. The upper organic layer
was removed from the tubes, streamed into glass vials, and dried under
a stream of argon. The samples were re-suspended in 50 μl of methanol
for HPLC, vortexed at high speed and placed into autosampler vials for
LC-ESI-TQ-MS/MS analysis, liquid chromatography triple quadrupole
tandem mass spectrometry (Yu et al., 2011).
Enzymatic hydrolysis of 6-gingerol and 6-shogaol conjugates
Plasma samples (1 ml) were incubated with 200 μl of β-glucur-
onidase solution (5 mg/ml) at 37 °C for 3 h, according to the method of
Yu and coworkers (Yu et al., 2011). The samples were then mixed with
10 μl of internal standard PAV working solution (10 μg/ml) and ex-
tracted through the procedure given above.
LC-MS-MS analysis
Alkamides concentrations in samples were determined by LC/MS
analysis as previously described by Woelkart et al., 2005, using LC-ESI-
IT-MS/MS (liquid chromatography/electrospray ionization tandem
mass spectrometry) composed of Varian 212 binary chromatograph,
equipped with a Prostar 430 thermostatic autosampler and Varian 500
ion trap mass spectrometer with Varian Workstation software. Gradient
Fig. 1. Representative HPLC-DAD chromatogram of the used product; peaks assigned to the active constituents highlighted in the study are reported.
3
Phytomedicine 65 (2019) 153090
elution was used using acetonitrile with 0.1% formic acid and water
with 0.1% formic acid 40/60 (v/v) to 80/20 (v/v) in 13 min. As sta-
tionary phase a Zorbax SB-C18 Narrow Bore 2.1 × 150 mm 3.5 μm
column from Agilent Technologies was used at a flow rate of 200 µl/
min. For MS detection ESI interface operating in positive Ion mode was
used. The source voltage was set at 4.8 kV, vaporizer temperature at
300 °C, nebulizer gas pressure was at 15 psi and helium gas was used for
collision. For SRM (single reaction monitoring) mass transition m/z
248 → m/z 149 was used for quantification of dodeca-2E,4E,8Z,10E/Z-
tetraenoic isobutylamides. The analytical procedure was validated by
analysis of quality control samples. Calibration curves were obtained by
plotting the dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamides/I.S.
(Benzanilide) peak area versus the known spiked plasma concentration
of the analyte. The column was a Agilent XDB C-18 2.1 × 150 mm, the
mobile phases were water 1% Formic acid (A) and Acetonitrile (B).
Gradient started with 70% A and kept isocratic for 1 min. It then
reached 100% B after 10 min and kept isocratic for 14 min. Varian 320
TQD MS spectrometer was used as detector. The following transitions
were used for quantification purposes, namely 6-glucuronyl-sogaol
(375.5→177.5), 6-glucuronyl gingerol (471.5→177.5), 6-diglcucyronyl
shogaol (471.5→177.5), 6-shogaol (277.1→137.5), nonivamide
(294.4→137.3), 6-gingerol (295.5→177.5), 10-gingerol (351→177.1).
Spectrometer was working in positive ion mode, capillary was set at
5400 V, frying gas at 285 °C, nebulizer pressure at 50 psi and drying gas
pressure at 25 psi. Limit of detection was at 0.1 ng ml−1. Linearity was
obtained in the range of 0.1–110 ng ml−1.The amounts of 6-gingerol, 6-
shogaol and dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide in softgel
capsules were determined using the above mentioned LC-DAD-ESI-MS
method. A representative chromatogram is reported in Fig. 1. The dose
(corresponding to 2 softgel capsules) contained 5.51 ± 0.02 mg of 6-
gingerol, 1.53 ± 0.02 mg of 6-shogaol and 1.00 ± 0.02 mg of dodeca-
2E,4E,8Z,10E/Z-tetraenoic isobutylamide (mean ± S.D., n = 3)
Pharmacokinetics analysis
Non-compartmental pharmacokinetic analysis of dodeca-
2E,4E,8Z,10E/Z-tetraenoic isobutylamide, 6-gingerol, 6-shogaol, 6-
gingerol glucuronide, and 6-shogaol glucuronide was performed using
WinNonlin Version 2.1 software from Pharsight Corporation (Mountain
View, CA). Maximum plasma concentration (Cmax) and time (tmax) were
S. Dall'Acqua, et al.
reported as observed, when obtained. Terminal half-life (t1/2) was
calculated as t1/2 = ln2 / λz, where λz is the terminal slope of the
plasma concentration profile on the semi-log plot calculated by linear
regression. The area under the concentration-time curve (AUC) was
calculated using the linear trapezoidal method and extrapolated to in-
finity (AUCinf), as AUC + Clast/λz, where Clast is the predicted con-
centration at the last quantified time point. Mean residence time (MRT)
was calculated as AUMCinf/AUCinf, where AUMCinf is the area under the
first moment curve. Since fractions of absorbed dose (F) cannot be es-
timated based exclusively on post-extravascular administration data,
apparent clearance (CL/F), calculated as Dose/AUCinf, and apparent
volume of distribution based on terminal phase (Vz/F), calculated as
CL/F/λz, are reported.
Dose normalized AUClast (AUC till the last quantified time point)
values from the current study were compared to the AUClast values from
our previous pharmacokinetic study with Echinacea lipophilic extract
formulated as softgel capsules (Dall'Acqua et al., 2015) and the phar-
macokinetic parameters of active ingredients of dry Ginger extract from
a study by Yu and co-workers (Yu et al., 2011) using independent
samples t-test.
Microarray data generation
Total RNA was extracted using the RNeasy Mini Kit (Qiagen), ac-
cording to the manufacturer's protocol. Synthesis of cDNA and bioti-
nylated cRNA (from 500 ng total RNA) was performed using the
Illumina TotalPrep RNA Amplification Kit (Ambion), according to the
manufacturer's protocol. Quality assessment and quantitation of total
RNA and cRNAs were performed with Agilent RNA kits on a
Bioanalyzer 2100 System (Agilent). Hybridization of cRNAs (750 ng)
was carried out using Illumina Human 48 K gene chips (Human HT-12
v4 Expression BeadChip). Array chip washing was performed in High
Temp Wash Buffer (Illumina) at 55 °C for 10 min, followed by staining
using streptavidin-Cy3 dyes (Amersham Biosciences). Hybridized ar-
rays were stained and scanned in a BeadStation 500 System (Illumina).
Microarray data pre-processing and analysis of differential expression
Raw expression data were extracted using the Illumina
GenomeStudio software (GenomeStudio V2011.1) and all further ana-
lyses were performed using state-of-the-art ‘lumi’ (Lin et al., 2008) and
‘limma’ software packages available through the R/Bioconductor pro-
ject. Briefly, after routine quality assessment (i.e., PCA plots, hier-
archical clustering plots, MA-plots, and box plots), raw expression data
were run through quantile normalization without background correc-
tion. A generalized linear model approach, coupled with empirical
Bayes moderation of standard errors (Smyth, 2004) was used for
identifying differentially expressed genes upon treatment taking into
account sample matching status. Correction for multiple testing was
performed using the Benjamini–Hochberg method (Benjamini and
Hochberg 1995).
To compare the oral administration effects of the softgel capsules
containing LEx to other studies of interest, we analyzed, using the same
approach described above, the following publicly available datasets:
GSE14368, GSE34145 (Kolehmainen et al., 2012) GSE40838
(Boisson et al., 2012) and GSE67255 (Olnes et al., 2016). Normalized
gene expression data were obtained from the National Center for Bio-
technology Information (NCBI) Gene Expression Omnibus (GEO) data-
base and treatment groups were compared as described in the original
manuscripts or the GEO record as available. Correspondence-At-Top
plots (CAT-plots) were used to compare the agreement between ranked
vectors of differential gene expression across studies (Ross et al., 2011).
Gene set enrichment analysis (GSEA)
For the identification of enriched pathways and biological pro-
cesses, Gene Set Enrichment Analysis (GSEA) (Liberzon et al., 2011)
was performed with 1000 permutations and weighted scoring. The
enrichment were analyzed in several collections from Broad Institute
4
Phytomedicine 65 (2019) 153090
Molecular Signature Database (MsigDB) (Liberzon et al., 2011). In the
present study we focused on: a) chemical and genetic perturbations,
signaling pathways from KEGG (Kanehisa et al., 2006), Reactome
(Croft et al., 2014), and Biocarta (http://www.genecarta.com); b) bio-
logical processes from Gene Ontology (Ashburner et al., 2000); c) cis-
regulatory motifs – corresponding to genes sharing microRNA seed
sequences or transcription factor binding sites (Xie et al., 2005) and d)
manually curated Functional Gene Sets (FGS) corresponding to funda-
mental biological processes (‘Hallmarks’ FGS), immunological expres-
sion profiles, and oncogenic signatures derived from peer reviewed
manuscripts. Enrichment across contrasts and studies was compared
using Correspondence At Top (CAT) plots and the log10-transformed
FDR as previously described (Marchionni et al., 2017).
Gene expression validation by qRT-PCR
Total RNA was extracted using RNeasy Mini Kit (QIAGEN) ac-
cording to manufacturer's instructions. The purity of RNA samples was
estimated with the Nanodrop spectrophotometer (ThermoFisher
Scientific) using the ratio of absorbance values at 260 nm and 280 nm.
500 ng of total RNA were retrotranscribed with the QuantiTect Reverse
Transcription Kits (QIAGEN). For each sample SYBR® green real-time
qPCR was performed in triplicate using the SYBR® Green PCR Master
Mix (ThermoFisher Scientific) on the StepOnePlus Real-Time PCR
SYstem (ThermoFisher Scientific). The amplification protocol included
a melting curve dissociation step to confirm the inexistence of non-
specific amplification products. Gene expression data of the selected
target genes were normalized to the housekeeping gene β-actin. The
analysis was performed using the comparative Ct method (2−ΔΔCt) and
the fold change was calculated from normalized Ct values.
Results
Pharmacokinetic study
The first part of the study aimed to assess the pharmacokinetics of
the active ingredients of a highly standardized product combining 2
lipophilic extracts (E. angustifolia and Z. officinale) (hereafter also re-
ferred to as LEx) in softgel capsules. The major components, including
dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamides, 6-gingerol, 6-sho-
gaol and two conjugate metabolites, 6-gingerol glucuronide, and 6-
shogaol glucuronide, were quantified in all plasma samples at pre-
determined time points up to 4 h after administration. Mean plasma
concentration profiles are shown in Fig. 2. Results of the non-com-
partmental pharmacokinetic analysis are summarized in Table 1.
Transcriptional remodeling of PBMCs following lipophilic extracts of E.
angustifolia and Z. officinale administration
Differentially expressed genes. Peripheral blood mononuclear cells
(PBMCs), primarily composed of T-lymphocytes, B-lymphocytes, NK
cells, monocytes, macrophages and dendritic cells, are often considered
a standard in studying many aspects of immunological responses,
including the inflammatory response. To investigate the
transcriptional modulatory effect of oral administration of softgel
capsules containing LEx, we performed genome-wide transcriptional
profiling study on PBMCs isolated from 3 individuals involved in the
study. Based on plasma profiles (AUClast), three healthy volunteers were
picked, having the lowest, highest and middle AUClast values. Blood was
drawn at baseline (T0) and 24 h after softgel capsule administration
(T24), and total RNA from the isolated PMBCs was used in the DNA
microarray-based differential gene expression analysis study. Timing of
blood collection was chosen based on previous results (Dall'Acqua et al.,
2015). Based on the gene expression matrices generated, we compared
the matched variation of gene expression between PBMCs at T0 and
T24. The normalized expression values included for differential
expression analysis were filtered by the thresholds of FDR ≤ 0.05.
Using this criterion, 542 unique transcripts were found to be
S. Dall'Acqua, et al. Phytomedicine 65 (2019) 153090

Table 1
Plasma pharmacokinetic parameters of active ingredients of combined lipo-
philic extracts of Echinacea angustifolia DC. and Zingiber officinale Roscoe in 10
healthy volunteers following oral administration of lipophilic Echinacea extract
formulated as soft gelatin capsules (dose 5.51 mg of 6-gingerol, 1.53 mg of 6-
shogaol and 1.00 mg of dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide).
dodeca-2E,4E,8Z,10E/ 6-gingerol 6-shogaol
Z-tetraenoic (5.5 mg dose) (1.5 mg dose)
isobutylamides (1 mg
dose)

Mean SD Mean SD Mean SD

Free
Tmax (h) 0.53 0.34 0.58 0.24 1.35 0.70
Cmax (ng/ml) 14.74 10.55 5.66 1.75 9.25 1.76
AUClast (ng h/ml) 21.59 12.35 8.40 3.82 18.6 5.3
t1/2_Lambda_z (h) 0.841 0.406 3.32 2.40 2.65 1.11
AUCinf (ng h/ml) 22.42 12.01 10.75 4.77 31.6 9.0
AUClast/D (ng h/ 21.59 12.01 1.527 0.867 12.40 5.976
ml/mg)
Vz/F (l) 79.7 58.43 3438 4056 190.9 92.3
Cl/F (l/h) 61.17 38.37 627.2 352.2 50.9 13.9
MRTinf (h) 2.1 0.59 3.30 1.65 4.47 1.36
Glucuronide
Tmax (h) / / 1.40 0.80 0.875 0.680
Cmax (ng/ml) / / 29.2 10.6 22.24 2.78
AUClast (ng h/ml) / / 67.9 27.3 55.3 10.7
t1/2_Lambda_z (h) / / 1.84 0.89 3.49 1.68
AUCinf (ng h/ml) / / 83.3 29.0 112 39
AUClast/D (ng h/ / / 67.9 5.28 74.82 36.86
ml/mg)
Vz/F (l) / / 202.3 124.6 67.0 24.6
Cl/F (l/h) / / 76.9 37.6 15.3 6.6
MRTinf (h) / / 3.30 1.17 5.59 2.24

Validation of microarray results by Rt-qPCR analysis. Seven of the most

up-regulated (PPM1B, RORA) and down-regulated genes (ALDOA,
DEFA1/DEFA3, PRDX2, PRDX3, SDCBP) were chosen from the
generated list of statistically significant differentially expressed genes
(Supplementary Table S2) for validation by RT-qPCR. Fig. 3 shows the
overall agreement between the expression analysis results obtained by
the microarray technology and the RT-qPCR (shown at a single-patient
level).

Integrative functional analysis. In our analysis, we identified 542

Fig. 2. Mean ( ± SD) plasma concentration profiles of the active ingredients of
Echinacea angustifolia DC. and Zingiber officinale Roscoe combined lipophilic
extracts in 10 volunteers following oral administration as softgel capsules (dose
5.51 mg of 6-gingerol, 1.53 mg of 6-shogaol and 1.00 mg of dodeca-
2E,4E,8Z,10E/Z-tetraenoic isobutylamide).
differentially expressed in PBMCs after the lipophilic extracts
administration, subdivided in one transcriptional module of 293 up-
regulated genes and one of 249 down-regulated genes (see
Supplementary Table S1-A and B for a selected list of the most
differentially expressed genes). Interestingly, among the most down-
regulated genes in PBMCs isolated from the subjects, the bioinformatics
analysis highlighted the α-defensins, DEFA1, DEFA1B and DEFA3.
differentially expressed genes upon oral administration of the softgel
capsules. Barring study GSE67255 (Olnes et al., 2016), which evaluated
the effects of hydrocortisone treatment in a time-course, none of the
remaining analyzed public datasets yielded significantly expressed
genes (data not shown). We therefore benchmarked our differential
expression analysis results against this study alone. First, we assessed
the overall agreement between studies for the contrasts included in our
linear model analyses. To this end, we used CAT plots, an approach
developed for comparing lists of differentially expressed genes by
focusing at the top and the bottom of ranked lists. Overall, CAT-plots
revealed that softgel capsules' administration effects resemble those of
early hydrocortisone response (4 h), especially at the highest dosage
(250 mg). Indeed, the down-regulated genes were highly similar and
the observed agreement exceeded what would be expected by chance
alone (see Supplementary Figure S1, light blue curve A). On the
contrary, the similarity between genes down-regulated by softgel
capsule administration with low dosage hydrocortisone (50 mg) at a
late time point (8 h) did not exceed what would be expected by chance
alone (see Supplementary Figure S1, red curve D). Similar results were
also obtained for the up-regulated genes (See Supplementary Figure
S2).
Among genes differentially expressed across both the administration
of the lipophilic extracts and early 250 mg hydrocortisone treatments,
there were genes related to immune response, anti-inflammatory

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S. Dall'Acqua, et al. Phytomedicine 65 (2019) 153090

Fig. 3. Validation of selected genes differentially expressed following administration of the lipophilic extracts at two distinct time points (T0 and T24). Boxplots show
expression levels change between paired samples (3 distinct donors) analyzed by microarray (Upper Panel) and by RT-qPCR (Lower Panel). Expression levels are
expressed as normalized log2 expression intensities for microarrays and as normalized (to ACTB housekeeping gene) Ct values for RT-qPCR. Individual matching T0
and T24 measurements for each donor are connected. Our validation experiments confirmed that ALDOA, DEFA1/DEFA3, PRDX2, PRDX3, and SDCBP are down-
regulated upon treatment, while PPM1B and RORA are up-regulated. Primers pairs used for RT-qPCR are listed in Table S2).

activity, response to growth factors and hormones, and non-sense
mediated RNA decay (see heatmap gene intersection depicted in Fig. 4).
Next, we compared the agreement between studies at the levels of
Functional Gene Sets (FGS). To this end, we first performed GSEA and
then we identified the FGS significantly enriched in both studies. GSEA
was performed to capture biological processes relevant in the in-
vestigated contrasts and to test whether differential gene expression
programs associated with LEx administration were similar to other
pharmacological treatments. Specifically, the GSEA approach was used
to compute a p-value to test the hypothesis that genes contained in a
Functional Gene Set (FGS), defined by a functional annotation, tend to
be more enriched in the extremes of a list (i.e. highly ranked in a list
ordered by a functional measurement). In the present study we tested
whether the collection of FGS from MSigDB were significantly enriched
in the selected treatments. To achieve this, we ranked individual genes
by their moderate t-statistics derived from the differential expression
analysis and corrected the resulting enrichment p-values for multiple
hypothesis testing using the Benjamini and Hochberg method. This
analysis reveals that both these treatments modulate immune related
processes, such as the regulation of interleukin-8 biosynthesis, the re-
sponse to interferon alpha and gamma, and the response of CD4 T-
lymphocytes to Interleukin-4 (see GSEA heatmap in Fig. 5, and Tables
S3-11 and related Figures S3-9 in Supplementary information).
It is worth noting that, although PBMCs represent a heterogeneous
mixture of cell types, we were able to highlight an overall transcrip-
tional remodeling induced by the administered lipophilic extract, sug-
gesting that the application of transcriptome analyses in a human
dietary intervention study represents a promising approach to unveil
mechanistic pathways linked to phytochemical-induced health effects.
However, further studies are needed to better understand the exerted
effects by using more homogeneous samples, i.e. specific PBMCs sub-
populations. Nevertheless, our findings suggest that the oral adminis-
tration of softgel capsules containing a LEx might have an anti-in-
flammatory effect, similar at a transcriptional level, to hydrocortisone's.
Discussion
In this study a highly standardized combined extract of E. angusti-
folia and Z. officinale, in a modern formulation such as softgel capsules
was administered in healthy subjects with the aim of studying both the
pharmacokinetics of the main active components (and their conjugate
metabolites) of the two extracts and the immunomodulatory effects by
gene expression profiling.
In the first part of the research, plasma concentration of dodeca-
2E,4E,8Z,10E/Z-tetraenoic isobutylamides, 6-gingerol, 6-shogaol, 6-
gingerol glucuronide, and 6-shogaol glucuronide were measured up to
4 h after administration. In agreement with the high lipophilicity, do-
deca-2E,4E,8Z,10E/Z-tetraenoic isobutylamides, 6-gingerol and 6-sho-
gaol are absorbed very rapidly and are detected in plasma as early as
the first sampling time, i.e., after 15 min.
Plasma concentration profile of dodeca-2E,4E,8Z,10E/Z-tetraenoic
isobutylamides observed in the current study after supplementation of
LEx resulted very similar to the plasma concentration profile of dodeca-
2E,4E,8Z,10E/Z-tetraenoic isobutylamides described in our previous
study when pure Echinacea extract has been orally administered in
softgel capsules (Dall'Acqua et al., 2015). These data indicated the ir-
relevance of Ginger extract on pharmacokinetic parameters of iso-
butylamides, also confirmed by comparing AUClast and AUCinf para-
meters across the two studies (p = 0.316 and p = 0.490, respectively).
Since maximum concentration of 6-gingerol and 6-gingerol glucur-
onide in plasma were observed after 30 min and after 2 h, respectively,
we hypothesized that the 6-gingerol reaches plasma mainly unmodified

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S. Dall'Acqua, et al. Phytomedicine 65 (2019) 153090

Fig. 4. (Heatmap gene intersection) – Differential gene expression levels of lipophilic extracts administration (LEx) and 250 mg hydrocortisone after 4 h treatment in
contrast with control. Most genes found differentially expressed across both treatments were regulated in similar intensity and direction, indicating similar properties
of LEx and hydrocortisone (i.e. immune response modulation and anti-inflammatory activity). Color intensity and direction of differential expression are represented
by t-score derived from generalized linear models.

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S. Dall'Acqua, et al. Phytomedicine 65 (2019) 153090

Fig. 5. (Gene Set Enrichment Analysis, GSEA) – Heat-maps visualizing significantly enriched functional gene sets (FGS) as determined by Analysis of Functional
Annotation (AFA) performed on expression profiles associated with LEx and Hydrocortisone treatment at different concentrations. Rows represent selected FGS
linked to immunomodulation, indicating that LEx treatment is most similar to the effects of Hydrocortisone treatment at 250 mg after 4 h. The color scales correspond
to the absolute adjusted p-values obtained from our analysis after negative base 10 logarithmic transformations and direction attribution (i.e., the number on the
color scale increases with decreasing FDR, negative values represent down regulated FGS and vice-versa). FGS were selected from different collections to capture
signaling pathways and biological themes modulated by treatment. The databases analyzed were collected from MSigDB. Complete tables and related figures with
results from enrichment test are reported in Table S3-11 and Figures S2-8.

and that 6-gingerol glucuronide is formed by systemic metabolism. The
conjugate metabolite, 6-shogaol glucuronide, appears more rapidly and
its plasma concentration profile presents the same concentration profile
of free 6-shogaol, indicating that the conversion to the glucuronide is
very rapid and that 6-shogaol glucuronide is pre-systemically formed.
The secondary peak, at 2 h, is related to interconversion of 6-gingerol to
6-shogaol. Such interconversion had been previously reported in si-
mulated gastric fluid (Bhattarai et al., 2007).
Systemic elimination of all active components was also rapid with a
terminal half-life ranging from 0.841 ± 0.406 h for dodeca-
2E,4E,8Z,10E/Z-tetraenoic isobutylamides to 3.49 ± 1.68 h for 6-
shogaol glucuronide. These data indicate that there is no accumulation
of dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamides, 6-gingerol, 6-
shogaol and their glucuronides with the recommended once daily
dosing.
As for the pharmacokinetic analysis of Z. officinale, up to date lit-
erature data on pharmacokinetics of ginger active ingredients are very
scarce, therefore, no proper comparison with published papers can be
performed. The only paper worth of mention is by Yu and co-authors
(Yu et al., 2011), where the pharmacokinetic parameter for 6-gingerol
glucuronide, 6-shogaol, and 6-shogaol glucuronide are reported. Ac-
cordingly, we compared those results with ours. The two studies display
several differences, namely, a much higher dose level of ginger active
ingredients by Yu et al. compared to our study (52.8 mg vs. 5.5 mg for
6-gingerol and 45 mg vs. 1.5 mg for 6-shogaol). Likewise, Yu et al. used
dry ginger extracts, while - as mentioned before- we chose a lipophilic
extract, which, in a previous study, had proved a promising choice to
improve bioavailability of natural active ingredients in
(Dall'Acqua et al., 2015). Dose normalized AUClast for 6-shogaol and 6-
shogaol glucuronide determined in our study (12.40 ng h/ml/mg and

8
S. Dall'Acqua, et al.
36.87 ng h/ml/mg, respectively) were significantly higher than the
values reported by Yu and coauthors (Yu et al., 2011) (0.53 ng h/ml/
mg, and 2.44 ng h/ml/mg, respectively). For 6-gingerol glucuronide,
however, there is no difference (14.02 ng h/ml/mg vs. 12.35 ng h/ml/
mg,). The reasons behind the difference in oral bioavailability may be
quite a few. The simultaneous presence of dodeca-2E,4E,8Z,10E/Z-
tetraenoic isobutylamides can improve the bioavailability of lipophilic
compounds such as 6-shogaol and 6-gingerol through the formation of
micelles, as previously attested (Raduner et al., 2006). To verify this
hypothesis a pharmacokinetic study after oral administration of in-
dividual Z. officinale lipophilic extract is ongoing.
A further explanation of the difference in AUClast may also be the
nonlinear pharmacokinetics (less than proportional increase in AUC
with the increase of the dose).
In conclusion of the first part of the research, we can affirm that
these data testify the advantages in terms of pharmacokinetic perfor-
mance of the supplementation of softgel capsules containing the lipo-
philic standardized extracts of E. angustifolia and Z. officinale in com-
bination.
A further goal of this study was the investigation of the transcrip-
tional modulatory effect of oral administration of these softgel capsules
(containing LEx). To this aim, we performed genome-wide transcrip-
tional profiling study on PBMCs isolated from the blood of three healthy
voluntaries. We analyzed only three of ten healthy volunteers treated
with softgel capsules, selecting subjects who proved poor, medium and
high absorption (based on AUClast values). This rationale owes to pre-
viously published results testifying that the immunomodulatory effect
of tetraene was not related to its content in the formulation. In fact, in
an earlier study, the effects of the three different tetraene doses on the
decrement of a given cytokine did not differ in a significant way, nei-
ther were they related to AUC (Guiotto et al., 2008).
More than 500 transcripts were found to be differentially expressed
in PBMCs after the lipophilic extracts administration, 293 up-regulated
genes and one of 249 down-regulated genes. Thanks to the validation of
microarray results by RT-qPCR analysis, we confirmed the up-regula-
tion of mRNA expression of PPM1B and RORA and the down-regulation
of ALDOA, DEFA1/DEFA3, PRDX2, PRDX3 and SDCBP gene expression.
As described in Fig. 6, all these genes can cooperate for the reduction of
inflammation.
Interestingly, our micro-array data, also confirmed by the RT-qPCR
validation, highlighted the α-defensins, DEFA1, DEFA1B and DEFA3
down-regulation. Human α-defensins, also called human neutrophil
peptides (HNP-1 to 4), are particularly abundant in polymorphonuclear
neutrophils (PMNs), certain macrophage populations, and Paneth cells
of the small intestine (Cunliffe, 2003). These genes codify for cysteine-
rich cationic polypeptides, produced both constitutively and/or in re-
sponse to microbial products or pro-inflammatory cytokines, during
septic inflammation and acute coronary vascular disorders. Multiple
studies have reported that HNP-1–3 can produce both beneficial and
pathologic effects. Interestingly, intraperitoneal administration of HNP-
1 to mice with dextran sulfate sodium-induced colitis begets more se-
vere colitis with higher colonic cytokine expression compared to con-
trols, thus suggesting a potential pro-inflammatory role for HNP-1 in
ulcerative colitis (Hashimoto et al., 2012). Authors also suggested that
patients with ulcerative colitis are likely to benefit from reducing high
expression levels of HNP-1–3. In this context, it has been demonstrated
that HNPs utilize the signaling pathways downstream of P2 receptors to
stimulate human lung and intestinal epithelial cells to produce the pro-
inflammatory cytokine IL-8 (Ibusuki et al., 2015). It has been recently
suggested that increased α-defensin expression is a potential in-
flammatory biomarker that may predict the risk of coronary heart
disease (CHD) development in hyperlipidemia patients (Maneerat et al.,
2017). Among the most down-regulated gene transcripts, we also
highlighted the Syndecan-binding protein (SDCBP) or syntenin, also
known as MDA-9 (melanoma differentiation associated gene-9), first
discovered in melanoma cells (Jiao et al., 1998). This scaffold protein
9
Phytomedicine 65 (2019) 153090
contains two PDZ domains and participates in diverse biological pro-
cesses (Jiao et al., 1998). Previous studies have identified SDCBP as a
TNF-α- and IFN-γ inducible gene (Stier et al., 2000), and it was recently
demonstrated that IL-6 induce SDCBP expression at both mRNA and
protein levels (Cao et al., 2017). Intriguingly, the Fisher's group has
highlighted a novel role of mda-9/syntenin in tumor-promoting in-
flammation and immune suppression (Das et al., 2016).
All these modulations, as summarized by Fig. 6, concurred to shift
the leukocytes towards an anti-inflammatory phenotype.
One of the most significant and interesting findings of this study has
emerged from the integrative functional analysis, which revealed that
the effect of LEx is similar to that of early hydrocortisone response,
especially at the highest hydrocortisone dosage (250 mg). On the con-
trary, down-regulated genes by softgel capsules or by the hydro-
cortisone lowest dose (50 mg) administration were not significantly
similar at 8 h (last time point).
Previous studies demonstrated that corticosteroids exert several
effects on immunity cells, including depletion of monocytes, dendritic
cells, and CD4+ CD8+ thymocytes by apoptosis, and mobilization of
natural killer cells and regulatory T cells (Leung and Bloom, 2003). In
vitro studies also showed that corticosteroids down-modulate several
inflammatory cytokines (Da Silva, 1999) and influence intracellular
cytokine expression in T cells, resulting in an increased Th2/Th1 ratio
(Segerer et al., 2009). In vitro and animal studies demonstrate that NF-
κB exerts anti-apoptotic effects through signaling, by way of down-
stream effectors. One putative mechanism by which corticosteroids
suppress immunity is down-regulation of the NF-κB signaling, an effect
that enhances apoptosis. Olnes and coworkers demonstrated in 2016
(Olnes et al., 2016) that NF-κB signaling was down-regulated as early as
one h in PBMCs after systemic hydrocortisone administration, with
peak inhibition occurring at 4–8 h and both doses exerting a similar
magnitude of inhibition.
As we previously have demonstrated, the anti-inflammatory effect
of dodeca-2E, 4Edienoic acid isobutylamide is explicated by the down-
regulation, in terms of both gene and protein expression, of IL-6 and
TNF-α as well as of IL-8 cytokines, a major player in systemic in-
flammation (Dall'Acqua et al., 2015). In this study, the regulation of
biosynthesis of IL-8 also emerged from the selected immunologic sig-
nature analysis, a confirmation of previous data.
Conclusion
We can conclude that the treatment of healthy volunteers with the
combination of E. angustifolia and Z. officinale, titled in isobutylamides
and gingerols, in softgel capsules, provides several advantages from the
pharmacokinetic and therapeutic points of view. Due to the individual
diffused use of these extracts as health promoting products and con-
sidering the actual lack of data supporting their effectiveness in com-
bination, the data contained in the present study provide novel in-
formation supporting the co-administration of these natural products.
The experimental evidence showed that the combined extracts allows a
Fig. 6. Modulation concurring to shift the leukocytes towards an anti-in-
flammatory phenotype.
S. Dall'Acqua, et al.
high and rapid absorption of the bioactive ingredients and exerts im-
munomodulatory and anti-inflammatory effects, at least at the tran-
scriptional level, similarly to those exerted by hydrocortisone. The aim
of our study was to perform a pilot trial to assess the effects of si-
multaneous supplementation of both extracts. As a result, the research
has several limitations such as the small number of subjects considered
in the PK study, the even smaller number of subjects in the genomic
study or the single individual tested dose of active components.
Nevertheless, not only do our preliminary data support the previously
published observations regarding the pharmacokinetic and im-
munomodulatory effect of E. angustifolia, but also, thus far, this is the
first study pertaining with the combination of E. angustifolia and Z.
officinale. Inasmuch as the manuscript considers both pharmacokinetic
issues and immunomodulatory effects assessed by gene expression
profiling, it is arguably an important starting point for further in-
vestigations.
Conflict of interest
The authors declare that there are no conflicts of interest associated
with this publication and there has been no significant financial support
for this work that could have influenced its outcome.
Acknowledgments
We thank the Indena S.p.A. (Milan, Italy) for providing softgel
capsules containing combined extracts of Echinacea angustifolia DC. and
Zingiber officinale Roscoe.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.phymed.2019.153090.
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