Available via license: CC BY 4.0
Content may be subject to copyright.
RESEARCH ARTICLE
Humic substances from composted fennel
residues control the inflammation induced by
Helicobacter pylori infection in AGS cells
Mariavittoria Verrillo
1,2
, Paola CuomoID
2
*, Angela Michela Immacolata Montone
3
,
Davide Savy
2
, Riccardo Spaccini
1,2
, Rosanna Capparelli
1,2
*, Alessandro Piccolo
1,2
1Centro Interdipartimentale di Ricerca per la Risonanza Magnetica Nucleare per l’Ambiente,
l’Agroalimentare, ed i Nuovi Materiali (CERMANU), University of Naples “Federico II”, Portici, Italy,
2Department of Agricultural Sciences, University of Naples “Federico II”, Portici, Italy, 3Department of Food
Inspection, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Italy
*paola.cuomo@unina.it (PC); capparel@unina.it (RC)
Abstract
Helicobacter pylori (H.pylori) is a common human pathogen causing inflammation. Recent
studies have suggested a sophisticated interplay between mitochondria, innate immunity
and inflammatory response, thus proposing mitochondrial disfunction as the hallmark of
severe inflammatory disorders. In this study, humic substances isolated from composted
fennel residues (HS-FEN) were tested as potential therapeutical strategy to restore the mito-
chondrial physiology and control the inflammation associated with H.pylori infection. The
molecular features of HS-FEN were characterized by infrared spectrometry, thermochemo-
lysis-GC/MS, NMR spectroscopy, and high-performance size-exclusion chromatography
(HPSEC), which revealed the presence of aromatic polyphenolic components arranged in a
rather stable conformation. In vitro results showed antioxidant and anti-inflammatory proper-
ties of HS-FEN, that was found to increase the expression level of OPA-1 and SOD-2 genes
and in AGS cells stimulated with H.pylori culture filtrate (Hpcf) and concomitantly decrease
the expression level of Drp-1 gene and IL-12, IL-17 and G-CSF proteins. The hydrophobic
features of HS, their conformational arrangement and large content of bioactive molecules
may explain the beneficial effects of HS-FEN, that may potentially become an interesting
source of anti-inflammatory agents capable to counteract or prevent the H.pylori-related
inflammatory disorders.
Introduction
Composting is a highly ecological biotechnology for the management of organic bio-wastes
through a controlled microbial process based on an initial intensive degradative thermophilic
active step, followed by a mesophilic phase with slow biochemical modification combined with
a final stabilization of the composted materials [1,2]. Horticultural vegetables produce a signif-
icant amount of crop residues, wastes of agro-industrial transformations and additional
unmarketable products that represent an important potential source of organic matter to be
potentially reused as compost, soil amendment and fertilizer (Regulation (EU) 2019/1009)
PLOS ONE
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 1 / 21
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Verrillo M, Cuomo P, Montone AMI, Savy
D, Spaccini R, Capparelli R, et al. (2023) Humic
substances from composted fennel residues
control the inflammation induced by Helicobacter
pylori infection in AGS cells. PLoS ONE 18(3):
e0281631. https://doi.org/10.1371/journal.
pone.0281631
Editor: Andrea Mastinu, University of Brescia:
Universita degli Studi di Brescia, ITALY
Received: November 16, 2022
Accepted: January 28, 2023
Published: March 9, 2023
Copyright: ©2023 Verrillo et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: This information will
be available after acceptance.
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
[3,4]. Recycling of such residues within farms (On-farm composting) is increasingly adopted
as an economic and efficient biological method to re-employ agricultural biomasses [5]. For
example, the production of fennel, a common horticultural crop, has grown consistently
around the world and Italy is one of the major European producers, with approximately
532,000 tons.y
-1
[6]. This significant amount of fennel residues contains numerous bioactive
compounds, including polyphenols, that may be effectively reused in the medical or nutraceu-
tical fields, thus increasing the added values of these wastes [7,8]. The bioactivity of fennel
components is even enhanced after the aerobic microbial transformation during the compost-
ing process, and their isolation as humic matter from such a green compost provides an eco-
logical material to be usefully and profitably employed in other remunerative sectors [9].
In this respect, increasing attention is devoted to the development of innovative products to
reduce and mitigate the consequences of inflammatory processes in human diseases [10–12].
Inflammation is a defensive response of the host against endogenous or exogenous stimuli,
which results in the elimination of harmful signals and return to homeostasis [13]. Inflamma-
tion, as well as the oxidative burst, are essential to contain bacterial infections, by removing the
invading pathogen and favoring the host healing [14]. However, an unsuccessful host defence,
likely due to bacterial or host factors, may lead to a long-lasting inflammatory process, which
can cause tissue injury and, therefore, more severe pathological conditions.
H.pylori is a persistent bacterium, particularly able to evade host defence strategies and pro-
mote chronic infection and inflammation. The virulence of the pathogen is directly related to
its capability to produce toxins, as well as the different modalities to infect the host [15]. The
Vacuolating cytotoxin A (VacA) is the major virulence factor contributing to H.pylori infec-
tion. Through VacA, H.pylori may affect mitochondrial functions, contributing to the patho-
genicity of the infection and the related diseases by promoting inflammation, oxidative stress
and cell apoptosis [16–18]. H.pylori is recognized to be the major cause of gastric diseases and
gastric cancer. However, it may also interfere with physiological processes outside the stomach,
by inducing a persistent low-grade inflammatory state. Recent studies have reported a strict
association between H.pylori infection and neurological or cardiovascular disorders. In addi-
tion, the gut microbiome alteration, closely associated with the H.pylori-induced inflamma-
tion and the massive use of antibiotics to eradicate this microorganism, may promote
metabolic disorders such as obesity or diabetes [19].
The failure of common antimicrobial therapies makes H.pylori infection and the associ-
ated- inflammatory diseases a global emergence. In this context, natural organic derivatives,
such as humic substances (HS) could provide a significant pharmacological contribute. Based
on their heterogeneous molecular composition and flexible conformational properties, HS are
recognized to possess interesting antioxidant, antimicrobial, and anti-inflammatory properties
[20–23]. The recent advances in their applications in medical therapies make the HS a poten-
tial alternative approach to control the H.pylori-associated inflammation. Furthermore, HS
from green composts have also been recently applied as starting material in the synthesis of
specialized industrial products or innovative nanomaterials and hydrogels [24–29].
In the present study, we evaluated the antioxidant and anti-inflammatory activity of HS
derived from composted fennel residues (HS-FEN), and explored its role in reducing the H.
pylori-associated inflammation, following exposure of AGS cells to H.pylori culture filtrate.
Materials and methods
Green compost and extraction of Humic Substances
Green composts (CO) were produced in Experimental Farm of the University of Naples “Fed-
erico II” at Castel Volturno (CE), according to the relevant guidelines and regulations, as
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 2 / 21
reported in Savarese et al., 2022 [2]. Briefly, horticultural residues of fennel crop were mixed
with coffee husks at 60/38 w/w plus 2% of mature compost as a starter. The vegetable wastes
were placed in static piles with bottom-up oxygen fluxes to ensure aerobic transformation. The
composting process lasted 100 days, including the thermophilic and mesophilic phases and a
final maturation period. To extract humic substances (HS), finely ground compost (100 g) was
suspended in 0.1 mol L
−1
KOH solution and shaken for 24 h. Then, the extract was centrifuged
at 7000 rpm for 20 min and filtered through glass-wool. This extraction was repeated twice (1
h agitation step) and the resulting filtrates were combined. Total extracts, containing both
humic and fulvic acids, were acidified to pH 7.4 with 6 mol L
−1
HCl and dialysed (1 kD cut-off
Spectrapore membranes) against deionized water until the electrical conductivity was lower
than 0.5 dS m
−1
, and freeze-dried for further analysis.
Infrared and Solid-state
13
C NMR spectroscopies
Infrared spectra were recorded on a Perkin Elmer 1720-X FT-IR spectrometer (Waltham, MA,
USA), equipped with a diffuse reflectance (DRIFT) accessory, by accumulating up to 8 scans
with a resolution of 4 cm
-1
. Samples and oven dried KBr powder were pulverized and mixed in
an agate mortar right before spectra acquisition [30].
The solid state
13
C NMR CPMAS spectrum of HS-FEN was obtained by rotating the sample
placed in 4 mm zirconium rotors with Kel-F caps inside wide-bore MAS probe mounted on a
Bruker AV-300 magnet with the following acquisition parameters: 13,000 Hz of rotor spin
rate; 2 s of recycle time;
1
H-power for CP 92.16 W:
1
H 90˚ pulse 2.85 μs;
13
C power for CP 150,
4 W; 1 ms of contact time; 30 ms of acquisition time; 4000 scans. The Free Induction Decay
(FID) was converted by a 4 k zero filling and an exponential filter function with a line broaden-
ing of 100 Hz.
For the interpretation of
13
C-CPMAS-NMR spectra the overall chemical shift range is split
into six regions related to the main organic functional groups: 0–45 ppm (aliphatic-C), 45–
60 ppm (methoxyl-C and N-alkyl-C), 60–110 ppm (O-alkyl-C), 110–145 ppm (aromatic-C),
145–160 ppm (O-aryl-C), 160–190 ppm (carboxyl-C)(31)
,
(32). The relative contribution of
each carbon group was estimated by relating the intensity of the corresponding spectral inter-
val (Aiabs) to the total area (A0-190abs): Ai% = (Aiabs/A0-190abs) ×100, i = 0–45, 45–60, 60–
110, 110–145, 145–160, 160–190. (MestreNova 6.2.0 software, Mestre-lab Research, 2010).
In order to highlight the structural features of humic materials, four dimensionless indexes
were calculated from the relative abundance of specific components: O-Alkyl ratio A/OA =
[(0–45)/(60–110)]; Aromaticity index ARM = [(110–160)/(0–190)]; Hydrophobic index HB/
HI = [(0–45) + (110–160)]/(60–110) + (160–190)]; Lignin ratio LigR = [(45–60)/(145–160)]
[31,32].
Off-line pyrolysis TMAH-GC-MS
Off-line pyrolysis TMAH-GC-MS was performed as described by Verrillo et al. (2022) [33].
Briefly, HS-FEN (500 mg) was placed in a quartz boat and dampened with 1 mL of tetramethyl
ammonium hydroxide (TMAH) solution (25% in methanol). The mixture was then dried
under a stream of nitrogen and the quartz boat was introduced into a Pyrex tubular reactor
(50 cm ×3.5 cm i.d.) and heated at 400˚C for 30 min in a horizontal furnace (Barnstead Ther-
molyne). The products released by thermochemolysis were transferred by a helium flow (20
mL min
-1
) into a series of two chloroform (50 ml) traps kept in ice/salt baths [34]. The extracts
were concentrated using rotavapor and the residue was resuspended in 1 mL of chloroform in
a glass vial for GC-MS analysis. The identification of release compounds was performed with a
Perkin- Elmer GC Autosystem XL by using an RTX-5MS WCOT capillary column (Restek, 30
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 3 / 21
m×0.25 mm; film thickness, 0.25 μm), coupled to a PE Turbomass-Gold quadrupole mass
spectrometer. The chromatographic separation was carried out according to the following pro-
gram: 60˚C (1 min isothermal), rate 7˚C min
-1
to 320˚C (10 min isothermal). Helium was
applied as carrier gas at 1.60 mL min
-1
, the injector temperature was at 250˚C, the split-injec-
tion mode had a 30 mL min
-1
of split flow. Mass spectra were obtained in EI mode (70 eV),
scanning in the range 45–650 m/z, with a cycle time of 1 s. Comparison of mass spectra with
the NIST library database, previous published spectra and standard was performed for com-
pound identification.
High performance size exclusion chromatography
As described by Verrillo et al. (2022) [33], the HPSEC system consisted of a Shimadzu LC-
10-AD pump equipped with a Rheodyne rotary injector and 100-μL sample loop and a UV/
VIS detector (Perkin e Elmer LC295), set at 280 nm. A PolySep™GFC-P3000 300 X 7.80 mm
(Phenomenex, USA) was employed, and it was preceded by a PolySep GFC-P 35 X 7.80 safety
guard (Phenomenex, USA) and a 2 mm inlet filter. The elution flow rate was set to 0.6 mL
min
-1
, whereas the eluent was made of 0.1 mol L
-1
NaH
2
PO
4
solution (buffered at pH 7.0)
added with 4.6 mmol L
-1
NaN
3
. Prior to the chromatographic analyses, both mobile phase and
HS solution were filtered through 0.45 μm Millipore filter. Column calibration was carried out
by using sodium polystyrene sulfonates of known molecular masses: 123,000; 16,900 and 6780
Da. Furthermore, ferulic acid (194 Da) and catechol (110 Da) were used as low molecular
weight standards. HS-FEN was solubilised in the eluent solution at a concentration of 0.6 g L
-1
and eluted by HPSEC. In order to verify the conformational stability and molecular size distri-
bution of HS-FEN [35], the same humic solutions were then added with glacial acetic acid
(AcOH) to lower their pH to 3.5 and injected again into the HPSEC system. The correlation
between molar masses (MM) and elution volumes (EV) provided the following equation: log
MM = 0.1407 EV + 6.4077 (R
2
= 0.996). The Weight Average (Mw) and Number Average
(Mn) molecular weights and polydispersity (P) were therefore calculated. A Unipoint Gilson
Software was used to record and elaborate the chromatograms, while the calculations of Mw
and P were performed by the Origin software (v. 9.1, Originlab).
Antioxidant activity of humic extract from fennel composted vegetable
wastes
Antioxidant activity of HS-FEN was performed by ABTS assay as described elsewhere [2,9,32].
Briefly, ABTS test was performed in a spectrophotometric method based on the oxidation of 2,
20-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) by potas-
sium persulphate to form a radical cation (ABTS•+). The ABTS reagent was dissolved in dis-
tilled water up to a 7mM concentration to obtain the ABTS stock solution. The ABTS radical
cation (ABTS•+) was produced by reacting ABTS stock solution with 2.45mM potassium per-
sulfate (final concentration) and allowing the mixture to stand in the dark for 16 h before use.
Then, working solution of ABTS•+ was prepared by diluting the 10 mL of radical cation
(ABTS•+) solution with 800 mL of water/ethanol (50:50, v/v) mixture with an absorbance
between 0.75–0.80 at 734 nm using UV/vis spectrophotometer.
Solutions of humic samples were prepared at three different concentrations (25, 30,50 μg
mL
-1
) in ultrapure water. Then, 100 μl of HS at each concentration were hence added to 1.9 ml
of ABTS•+ working solution. The mixture was shaken for 2 minutes at dark to promote the
reaction between sample and radical solution and the absorbance was measured at 734 nm.
The results were expressed as Trolox Equivalent Antioxidant Capacity (TEAC) by means of a
linear calibration curve of Trolox (R
2
= 0.991).
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 4 / 21
Cell culture
Human gastric adenocarcinoma cell line AGS (ATCC, Manassas, VA, USA, #crl-1739) was
maintained in Dulbecco’s modification of Eagle’s medium, high glucose (DMEM; Microtech,
Naples, Italy), supplemented with 10% fetal bovine serum (FBS; Microtech, Naples, Italy), 1%
penicillin/streptomycin (Gibco, Waltham, MA, USA) and 1% L-glutamine (Gibco, Waltham,
MA, USA) in a humidified atmosphere at 37˚C and 5% CO
2
.
Helicobacter pylori culture filtrate preparation
Helicobacter pylori culture filtrate (Hpcf) was prepared as described by Cuomo et al. [36]. In
detail, culture filtrate of H.pylori P12 strain was prepared by culturing the bacterium on selec-
tive Columbia agar (Oxoid, Basingstoke, Hampshire, UK) supplemented with 7% (v/v) of defi-
brinated horse serum (Oxoid, Basingstoke, Hampshire, UK) and antibiotic mix (DENT;
Oxoid). Bacteria plates were incubated for 3–4 days at 37˚C in a 10% CO
2
atmosphere. After
growth, bacteria were scratched using brain heart infusion (BHI; Oxoid, Basingstoke, Hamp-
shire, UK) and measured. 2 x 10
7
bacteria were cultured in DMEM (Microtech, Naples, Italy)
supplemented with 10% FBS (Microtech, Naples, Italy) and incubated at 37˚C in a 10% CO
2
atmosphere for 24 hours. Finally, bacterial suspension was centrifuged at 10,000 g for 10 min-
utes and the supernatant was filtered by using a 0.22 μm filter.
Cell viability assay
AGS cells were split at 80–90% of confluence, seeded (2 x 10
4
) in a 96-well plate and incubated
at 37˚C in a 5% CO
2
atmosphere overnight. After cell attachment, the medium was replaced
with fresh one, serum-free, containing different concentrations of the HS-FEN (500 μg/mL,
250 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6 μg/mL) or Hpcf (Hpcf as it is or
diluted 1:2, 1:4, 1:8 with uninoculated growth broth) and cells were incubated for 24 hours.
After cell washing, medium containing 3–4,5-dimethylthiazol 2,5-diphenyltetrazolium bro-
mide solution (MTT 1:10; Merk, Darmstadt, Germany) was added to each well and cells fur-
ther incubated at 37˚C in a 5% CO
2
atmosphere for 3 hours. Finally, the medium was
removed, and the resultant formazan crystals dissolved in 200 μL of DMSO. Cellular MTT
reductase activity was determined by measuring the absorbance of DMSO extracts at 570 nm,
using an EnVision 2102 multilabel reader (PerkinElmer, Waltham, MA, USA). Results are
expressed as the percentage of MTT-reducing activity of treated vs. untreated cells, according
to the following formula:
% cell viability = [(mean absorbance of the sample)—(mean absorbance of the blank)]/
[(mean absorbance of the control)— (mean absorbance of the blank)] x 100.
Multiplex cytokine measurement
AGS cells were split at 80–90% of confluence, seeded (1 x 10
6
) in a 12-well plate and incubated
at 37˚C in a 5% CO
2
atmosphere overnight, until cell attachment. Cells were washed and
treated with: 1) Hpcf diluted 1:2 with uninoculated growth broth (1:2); 2) Hpcf diluted 1:2
with uninoculated growth broth (1:2) + HS-FEN 25 μg/mL. After 12 hours of treatment,
medium was collected and centrifugated at 10,000 ×g to remove debris and dead cells and ana-
lysed for the concentration of IL-17; IL-2 and G-CSF cytokines, by using the Bioplex Multiplex
human cytokine assay (Bio-Rad), as indicated by manufacturer’s instructions. Briefly, 96-well
plate was coated with 50 μL of microspheres (magnetic beads) covalently coupled to capture
antibodies able to detect specific cytokines (IL-12, IL-17 and G-CSF). After coating, coupled
beads were washed two times with 100 μL of wash buffer by vacuum filtration. 50 μL of
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 5 / 21
cytokine standards (at different dilutions), samples or controls were added, and the plate was
incubated for 30 minutes at room temperature on a shaker. Each standard was run in dupli-
cate, while samples or controls in triplicate. The plate was further washed three times with
100 μL of wash buffer by vacuum filtration. 25 μL of biotinylated detection antibody were then
added to each well, in order to create the sandwich complex, and the plate was incubated for
30 minutes at room temperature on a shaker. Following this second incubation, the plate was
washed as before, and 50 μL of streptavidin- phycoerythrin were added to each well. 10-minute
following incubation and after final wash, the beads were resuspended in 125 μL of assay
buffer. The 96-well plate containing the resuspended microspheres was placed in the Bio-Plex
200 System (Bio-Rad) and data analysed by Bio-Plex Manager
TM
Software. Identification and
quantification of cytokine is determined by the median fluorescence intensity of the fluores-
cent dye phycoerythrin. Phycoerythrin is excited at 635 and 532 nm to generate a report signal.
The median fluorescence intensity of the unknown sample is then converted into concentra-
tion (pg/mL) based on the known cytokine concentrations of the standard curve.
RNA extraction and gene expression measurement
Expression levels of OPA1,SOD2 and Drp-1 genes were measured by RT-PCR. AGS cells were
seeded (2 x 10
6
) in a 6-well plate and incubated at 37˚C in a 5% CO
2
atmosphere overnight,
until cell attachment. The next day, cells were incubated with Hpcf (1:2) for 3 hours in pres-
ence or not of HS-FEN 25 μg/mL. After incubation, RNA was extracted using TRIzol LS
reagent (Thermo Fisher Scientific). Briefly, cells were lysed in 1 mL of Trizol reagent and incu-
bated on ice for 5 minutes. 200 μL of chloroform were added and each sample was centrifuged
at 13,000 rpm, 4˚C for 15 minutes. The aqueous phase (upper phase) was transferred into a
new tube and 500 μL of isopropanol were added. The mixture was then vortexed, incubated on
ice for 5 minutes and centrifuged at 13,000 rpm, 4˚C for 10 minutes. Supernatant was dis-
carded and RNA pellet suspended using ethanol 75%. After centrifugation (13,000 rpm, 4˚C
for 7 minutes), supernatant was removed, and pellet eluted in 30 μL of RNase free water. The
quality and quantity of RNA was assessed using NanoDrop 2000c (Thermo Fisher Scientific).
cDNA was finally prepared using the high-capacity cDNA Reverse transcription kit (Thermo
Fisher Scientific).
Real-time PCR reactions were performed on a StepOne Real-Time PCR System (Thermo
Fisher Scientific), using Power SYBR Green PCR Master Mix (Applied Biosystem) as amplifi-
cation system [37]. Gene-specific primers are listed in S2 Table. The 2
-ΔΔCt
method was used
to determinate relative changes in target gene expression. As housekeeping gene, GAPDH was
used as internal control, to normalize data.
Statistical analysis
GraphPad Prism 6.0 (San Diego, CA, USA) was used to analyze data. Multiple comparisons,
among more than two experimental groups, were performed using One-way ANOVA fol-
lowed by Bonferroni post-hoc correction. Data were considered statistically significant when p
value was <0.05.
Results and discussion
Infrared spectroscopy
The DRIFT spectrum of HS-FEN (Fig 1)showed a broad absorption band around 3000–3500
cm
−1
derived from the overlap of the intense OH stretching vibrations in alcohols and pheno-
lic compounds and carboxylic acids. The bands at 2950–2920 cm
−1
are referred to symmetric
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 6 / 21
and asymmetric C-H stretching of methyl and methylene groups in aliphatic chains of lipid
compounds, associated with the weak bending vibration at 1440 cm
-1
[30]. The inclusion of
alkyl carboxylic groups is indicated by the doublet at 1714 cm
-1
of carboxyls, coupled with the
less intense shoulder related with C-O bending at 1220 cm
-1
. The signals at 1620 cm
-1
and
1540 cm
-1
may be related to either amide I or amide II bonds of peptides [31], or ring vibra-
tions of aromatic moieties [4,31]. The prominent band at 1367 cm
-1
is attributed to phenolic
functional groups and aryl ether bonds [38]. Finally, the bending of C-O bonds at 1040 cm
-1
suggests the presence of hydroxyl functions in carbohydrates and polysaccharides (Fig 1).
NMR spectroscopy
The
13
C-CPMAS NMR spectrum of HS-FEN (Fig 2) confirms not only the presence of both
apolar alkyl, aromatic compounds and polar components related to C-O and C-N containing
molecules, as already inferred by the IR spectrum, but also the general features proper of HS
from aerobic composts [31,39]. The structural features of HS may be also inferred by the
Fig 1. FTIR-DRIFT spectrum of humic substances from fennel green compost.
https://doi.org/10.1371/journal.pone.0281631.g001
Fig 2.
13
C-CPMAS-NMR spectrum of HS-FEN.
https://doi.org/10.1371/journal.pone.0281631.g002
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 7 / 21
dimensionless structural parameters calculated from the relative amounts of carbon distribu-
tion over intervals of the spectral range (Table 1). The value of hydrophobic index close to
unity, indicates a uniform partition of C between polar and apolar functional groups mainly
determined by the aliphatic components as suggested by the similar result found for the alkyl
ratio (Table 1). However, the ARM index suggests a significative content of aromatic mole-
cules, while the low level of LigR shows a close correlation of spectral intensities associated to
methoxyl groups (45–60 ppm) and O-aryl-C molecules (140–160 ppm), thus revealing the
prevalent contribution of lignin fragments and phenolic compounds in these spectral regions
(Table 1).
Off-line TMAH-Pyr-GC–MS
The thermochemolysis HS-FEN released methyl ethers and esters of alkyl and aryl compounds
of plant and microbial origin, identified as lignin derivatives (Lig), linear fatty acids (FAME),
nitrogen-containing (N) compounds, alicyclic lipid (e.g. sterols) and carbohydrates (S1 Fig
and S1 Table). The fact that a relatively smaller amount of carbohydrates can be inferred in the
thermochemolysis of HS-FEN (Table 2) in respect to the NMR spectrum is due to the poor
analytical efficiency of thermochemolysis in detecting polyhydroxy molecules in complex
matrices [1]. The most abundant compounds in HS-FEN pyrograms were lignin monomers,
which were classified according to the principal structures found in the lignified tissues of
higher plants: P = p-hydroxyphenyl, G = guaiacol (3-methoxy, 4-hydroxyphenyl), and
S = syringyl (3,5- dimethoxy, 4-hydroxyphenyl) [40]. The most abundant lignin derivatives
were the oxidized products of both di- and tri-methoxyphenylpropane, such as benzaldehyde
(G4 and S4), acetophenone (G5 and S5), and benzoic acid (G6 and S6). Other identified lignin
units were cis and trans isomers of 1-(3,4-dimethoxyphenyl)-2-methoxyethylene (G7 and G8)
Table 1. Relative distribution (%) of signal areas over chemical shift regions (ppm) and structural indexes
a
in
13
C CPMAS-NMR spectrum of HS.
Sample 200–160
Carboxyl-C
160–145
O-aryl-C
145–110
Aryl-C
110–60
O-Alkyl-C
60–45
CH
3
O/C-N
45–0
Alkyl-C
A/OA ARM HB/HI LigR
HS-FEN 12.4 8.5 12.2 24.9 16.1 25.9 1.0 0.4 0.9 1.9
a
A/OA = (0–45)/ (60–110);
b
ARM = [(110–160)/S(0–45) + (60–110)];
c
HB/HI: S[(0–45) + (45–60)/2+(110–160)] / S[(45–60)/2+(60–110)+ (160–190)];
d
Lig R: (45–
60) / (145–160).
https://doi.org/10.1371/journal.pone.0281631.t001
Table 2. Relative yield (%) of main thermochemolysis products a released from HS-FEN.
Thermochemolysis products
HS-FEN
%
Lignin 59.7
Ad/AlG
a
6.2
Ad/AlS
b
14.8
Aromatic (non-lignin) 7.4
N derivatives 18.7
FAME
c
10.5
Carbohydrates 0.4
Sterols 2.3
Alcohols 1.9
Alkanes 0.2
a
: Ad/AlG = [G6/G4];
b
: Ad/AlS = [S6/S4];
c
: Fame: fatty acid methyl ester.
https://doi.org/10.1371/journal.pone.0281631.t002
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 8 / 21
and 1-(3,4, 5- trimethoxyphenyl)-2-methoxyethylene (S7 and S8), as well as the enantiomers
of 1-(3,4-dimethoxyphenyl)-1,2,3-trimethoxypropane (G14 and G15) and the 1-(3,4,5-tri-
methoxyphenyl)-1,2,3-trimethoxypropane (S14 and S15). The 3,4-dimethoxyphenyl-2-prope-
noic acid (G18) compounds originated from either lignin guaiacyl units or suberin aromatic
domains. Additionally, other aromatic compounds were identified, such as phenols, methyl-
phenols, and alkyl-benzenes derivatives, which may have multiple origins (polysaccharides,
proteins, lignin, polyphenols). Besides the total distribution, the relative amounts of specific
lignin-derived compounds may provide useful indications [1]. Structural indexes informative
of the lignin decomposition stage may be calculated by dividing the area of the oxidized
3,4-dimethoxylbenzoic acid and 3,4,5-trimethoxylbenzoic acids (G6 and S6) over the corre-
sponding aldehydic forms (G4 and S4). The larger values found for the two structural parame-
ters (Table 2), suggest that the composting process produces an extensive lignin
decomposition into lignin fragments smaller in molecular size [41].
High performance size exclusion chromatography (HPSEC) of HS
The HPSEC chromatograms of HS from composted fennel wastes are shown in Fig 3, while
the relative nominal weight-averaged (Mw) and number-averaged (Mn) molecular weights are
reported in Table 3. This humic material showed a bimodal distribution of molecular sizes, as
commonly observed for HS of various origins [33,42]. The nominal Mw and Mn for peak A
were 83600 Da and 82400 Da, respectively, whereas they were 45200 and 36600 Da for peak B
(Table 3). Due to the similar Mw and Mn values, both peaks had a monodisperse nature, with
a polydispersity value equal or very close to 1.0 (Table 3).
Another HPSEC run was conducted after having treated the humic solution with acetic
acid (AcOH) to lower the pH from 7 to 3.5, in order to verify the conformational stability of
this humic sample. The addition of AcOH drives the formation of novel hydrogen bonds
among complementary oxygen-bearing compounds, which alter the weak interactions stabilis-
ing the humic suprastructures at pH 7 and induce a dispersion of the newly smaller formed
suprastructures through the HPSEC pore distribution [35,39]. Only a significant decrease in
the absorbance of peak A and a slight shift towards larger elution volumes for peak B were
observed upon AcOH treatment (Table 3,Fig 3). These changes can be thus explained with a
fragmentation of the hydrophobic components of HS which were originally associated into
nominally large molecular sizes (peak A) and were then diffused through the column towards
larger elution volumes (peak B). The substantial stability of the conformation of HS from the
green fennel compost upon variation of the solution pH, suggests that the saccharidic compo-
nents revealed by the above spectroscopic results, still maintained a macromolecular character
and were associated with smaller sized hydrophobic components. Similar results were recently
reported for other HS from composted agricultural residues which appeared to contain plant
derived lignin and cellulose not yet completely depolymerized after the composting process
[2,33,40].
Antioxidant activity of humic substances by fennel green compost
Antioxidant substances provide a significant protection against various diseases that are
related to oxidative stress generally induced by free radicals, such as reactive nitrogen species
(RNS) and reactive oxygen species (ROS) [43]. For this reason, radical scavenger activities of
phenolic components in natural molecules with their electron donors/acceptors behavior,
have been extensively discussed [44]. Humic substances were recently used for medical appli-
cation as natural antioxidants since they were shown to efficiently inactivate free radicals due
to their abundant content of reducing molecular components [33,40]. Here we found that
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 9 / 21
Fig 3. HPSEC chromatograms of HS-FEN before and after addition of acetic acid (AcOH) to lower solution pH
from 7 to 3.5.
https://doi.org/10.1371/journal.pone.0281631.g003
Table 3. Weight average (Mw) and number average (Mn) molecular weights, and polydispersity (P), as calculated
from UV-detected HPSEC chromatograms for HS-FEN, before and after addition of acetic acid (AcOH). Standard
deviation was <5%.
Sample (peak interval-mL) Mw Mn P
HS-A (4.8–6.4) 83593 82413 1.0
HS-B (6.4–13.1) 45170 36578 1.2
HS + AcOH-A (4.8–6.5) 78574 77059 1.0
HS + AcOH-B (4.8–14.1) 37206 23883 1.6
https://doi.org/10.1371/journal.pone.0281631.t003
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 10 / 21
HS-FEN revealed a significant free radical percent inhibition at the maximum concentration
tested (50.86%), though a clear dose effect was also visible (Fig 4). Similar results were obtained
by expressing the antioxidant ABTS results against the standard antioxidant Trolox as TEAC
(mmol of Trolox equivalentkg
−1
of sample), showing values such as 397.5 for HS-FEN at
50 μg mL
-1
followed, by 285 and 173 for the lower concentrations of 30 and 25 μg mL
-1
,
respectively. In line with previous studies, these data indicate a clear correlation between the
phenolic content of this humic sample (Table 1 and Fig 2) and its antioxidant capacities
[4,9,32]. Although antiradical and antioxidant activities of several humic substances have been
already discussed [45], no literature information is reported regarding specific HS isolated
from composted fennel residues, which may represent an important source of bioactive mate-
rial for the pharmaceutical industry [46]. Our results, which relate the antioxidant activity of
HS-FEN to its content of phenolic moieties (Tables 1and 2), are in agreement with other stud-
ies on phenolic extracts, in which mono- and oligo-hydroxylated benzene units are responsible
for the antioxidant properties [47], as major electron donating groups in humic samples [48].
The effect of HS-FEN on cell viability
The capability of the selected HS (HS-FEN) in affecting the metabolic activity of human gastric
adenocarcinoma cell line AGS was assayed by performing the MTT assay. Cells were incubated
with different concentrations of HS (from 500 μg mL
-1
to 6 μg mL
-1
) for 24 hours. As shown in
Fig 5, HS-FEN was found not to be toxic at concentrations lower than 50 μg mL
-1
, since cell
viability was larger than 80%, as compared to control cells. We also determined the concentra-
tion of HS-FEN responsible for 50% decrease of cell viability (CC
50
). Results showed 50% cyto-
toxic concentration at 65.93 μg mL
-1
. Concentrations of HS-FEN responsible for a significant
Fig 4. Antioxidant activity of HS-FEN at different concentration (50, 30, 25 μg mL
-1
). Vertical bars represent the
standard deviation (s.d.). Different capital letters indicate significant differences according to Tukey test (p0.05).
https://doi.org/10.1371/journal.pone.0281631.g004
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 11 / 21
reduction of cell viability (<80%) were excluded. This result, again, confirms the large content
of lignin fragments in HS-FEN (Tables 1and 2). High concentrations of lignin, in fact, are
known to show cytotoxic effects [49]. It is important to consider that usually the antioxidant
activity of molecules is expressed to cytotoxic concentrations. Interestingly, lignins show anti-
oxidant capacity to non-cytotoxic concentrations [49] (Fig 4). Therefore, these data have an
important implication on the potential clinical application of HS-FEN, suggesting that the
concentration of HS-FEN required for beneficial effects is safe for the analyzed cell line.
HS-FEN protects AGS cells from the Hpcf-induced oxidative stress and
inflammation
H.pylori is the main environmental agent responsible for chronic gastric inflammation, which
may result in chronic gastritis and gastric cancer. Although the mechanism disclosing the rela-
tionship between H.pylori infection and gastric cancer has not yet been elucidated, the imbal-
ance between cell proliferation and apoptosis could represent one of the factors contributing
to gastric mucosa damage and malignant transformation [50].
H.pylori secretes a variety of bacterial toxins and antigens that, upon recognition by gastric
epithelial cells, can impair gastric mucosa integrity, inducing cell apoptosis via stimulation of
pro-inflammatory mediators [51].
The vacuolating cytotoxin A (VacA) is the major virulence factor produced and released by
H.pylori [52]. VacA can enter different host cells and exert different functions, resulting in
alteration of cell physiology [19,52,53]. Inside the cells, VacA localizes in the mitochondria
and initiates the mobilization of cytochrome c, leading to apoptosis and oxidative stress [54].
It has been reported that the H.pylori culture filtrate (Hpcf) from VacA
+
P12 strain inhibits
AGS cell proliferation, inducing cell death by apoptosis [17]. In agreement with this finding,
Fig 5. Cell viability and CC
50
.MTT assay showing the AGS cells viability after incubation with increasing
concentrations of HS-FEN (spanning from 6 μg mL
-1
to 500 μg mL
-1
) for 24 hours. The concentration of 65.93 μg/mL
was estimated as that inhibiting 50% of cell viability (CC
50
).
https://doi.org/10.1371/journal.pone.0281631.g005
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 12 / 21
AGS cells stimulated with Hpcf from P12 strain showed a statistically significant viability
decrease, compared to control cells (Fig 6). Notably, this result was found to be dependent on
the Hpcf concentration. In fact, when eightfold diluted, Hpcf did not display cytotoxic effect
(Fig 6), since cell viability was close to that of control cells.
The pro-apoptotic activity of Hpcf was confirmed by analyzing the gene expression of both
OPA-1 and Drp-1 genes. Results revealed a significant decrease of OPA-1 gene expression and
increase of Drp-1 gene expression after stimulation with concentrated Hpcf (Fig 7A and 7B).
OPA-1, along with Drp-1 play critical roles in preserving mitochondrial morphology and
function. They are recognized as “mitochondria-shaping” proteins, responsible for mitochon-
dria dynamic regulation through a careful balance between fission and fusion events [55].
Down-regulation of OPA-1, together with up-regulation of Drp-1, favor mitochondria frag-
mentation and cristae remodeling, facilitating the release of cytochrome c triggered by apopto-
tic stimuli [56,57]. Therefore, our results suggest that Hpcf affects mitochondrial functions,
favoring the process of mitochondrial fission.
Mitochondria are the main source of reactive oxygen species (ROS). Alteration of mito-
chondria, promoted by loss of OPA-1 and/or Drp-1 accumulation, leads to excessive ROS
Fig 6. Hpcf affects gastric epithelial cell viability. MTT assay showing the AGS cells viability after incubation with
different concentrations of Hpcf for 24 hours. The assay was performed testing Hpcf as it is (Hpcf) or diluted with
uninoculated broth medium (Hpcf 1:2; Hpcf 1:4; Hpcf 1:8). One-way ANOVA followed by Bonferroni post hoc
correction was used to determinate statistically significant differences ( p<0.0001).
https://doi.org/10.1371/journal.pone.0281631.g006
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 13 / 21
production [57–59]. The evidence of the reduced expression of SOD2 gene in cells incubated
with Hpcf (Fig 7B), represents a further proof confirming the deleterious role of Hpcf by caus-
ing mitochondrial dysfunction and therefore, apoptosis and oxidative stress [60].
Interestingly, HS-FEN treatment was found to increase the expression of both OPA-1 and
SOD2 genes in cells stimulated or not with Hpcf (Fig 7A and 7C). On the contrary, HS-FEN
treatment was found to decrease the expression of Drp-1 gene in cells stimulated or not with
Hpcf, compared to Hpcf-stimulated cells (Fig 7B). Remarkably, cells treated with HS-FEN
alone also expressed high levels of Drp-1 gene, compared to untreated cells. However, the
increased levels of Drp-1, following HS-FEN treatment, were not remarkable compared to
Hpcf-stimulated cells (Fig 7B). This result may seem a contradiction, but it may find explana-
tion in the involvement of Drp-1 –as mitochondrial fission protein–also in cell proliferation
and mitosis [61].
These results suggest: 1) the protective role of HS-FEN under basal conditions and 2) the
beneficial effect of HS-FEN during H.pylori infection, by limiting mitochondrial ROS produc-
tion and preserving cells from mitochondrial dysfunction.
Recently, it has been demonstrated that OPA-1 silencing also causes IkB degradation and
NF-kB activation, promoting the expression of pro-inflammatory genes [62,63]. Consequently,
OPA-1 plays key roles in the NF-kB pathway.
Fig 7. HS-FEN subverts detrimental effects elicited by Hpcf. Cellular expression of OPA-1 (a), Drp-1 (b) and SOD2
(c) genes detected by quantitative PCR performed on RNA extracted from AGS cells cultured for 3 hours with HS-FEN
25 μg mL
-1
in presence or absence of Hpcf (1:2) stimulation. GAPDH was used as housekeeping gene to normalize all
samples. Data were represented as means ±s.d. of three independent experiments, each performed in triplicate. One-
way ANOVA followed by Bonferroni post hoc correction was used to determinate statistically significant differences
( p<0.0001).
https://doi.org/10.1371/journal.pone.0281631.g007
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 14 / 21
In line with this finding, our results showed increased expression of IL-12, IL-17 and
G-CSF cytokines in Hpcf stimulated cells. IL-17 is a pro-inflammatory cytokine playing critical
roles in the pathogenesis of chronic inflammatory diseases. It is produced during the H.pylori
infection and contributes to gastric mucosa damage, by inducing pro-inflammatory mediators
such as the granulocyte colony-stimulating factor (G-CSF), which participates to the acute
phase of inflammation [64,65]. IL-12, in turn, is a pro-inflammatory cytokine released during
infections and functions as point of connection between the innate and adaptative immunity
[66]. As expected, HS-FEN treatment was found to decrease the release of IL-12, IL-17 and
G-CSF by Hpcf (Fig 8).
Due to the complex molecular heterogeneity of HS-FEN, as demonstrated by both NMR and
IR spectra (Figs 1and 2), molecular basis of protective and beneficial effects exhibited by this
humic matter cannot be easily predicted. It seems plausible to attribute the protective effect of
HS-FEN and, more generally, of humic substances to their reducing properties (Fig 4). As
shown by spectroscopic results, in fact, HS-FEN is rich in phenolic and lignin components
(Tables 2and 3) which possess important antioxidant properties, by scavenging ROS [45,47].
Consequently, HS-FEN attenuates the impact of the oxidative stress on mitochondrial dynamics
and mitigates the inflammatory process protecting the cells from the oxidative stress [50].
However, a clear relationship between chemical structure and anti-inflammatory features
of humic substances has not yet clearly elucidated. A decreased release of IL-12, IL-17 and
G-CSF by HS- FEN treatment could find explanation in the conformational and structural
properties of humic molecules and the large content of polyphenolic or lignin components
(Tables 1and 2,Fig 2). Notably, the most reasonable explanation about the capability of
HS-FEN to suppress the expression of the Hpcf-induced cytokines could reside in the hydro-
phobic feature and conformational flexibility of this substance. The combined characteristic
can promote a surface adhesion to cell membranes and a subsequent disruption of the humic
suprastructures into smaller humic associations, from which, by interacting with eukaryotic
cells, small bioactive phenolic molecules may be concomitantly released, and determine a sig-
nificant reduction of the inflammatory response.
Fig 8. HS-FEN mitigates Hpcf-induced inflammation. Expression level of cytokines IL-12, IL-17 and G-CSF
detected in AGS culture medium. Graph reports pg of cytokine in mL of cell medium differently treated: 1) HS-FEN
25 μg mL
-1
; 2) Hpcf 1:2 for 12 hours; 3) Hpcf 1:2 + HS-FEN 25 μg mL
-1
for 12 hours. Values were normalized to basal
activity (control cells) and data were represented as means ±s.d. of three independent experiments, each performed in
triplicate. One-way ANOVA followed by Bonferroni post hoc correction was used to determinate statistically
significant differences ( p<0.001; p<0.0001).
https://doi.org/10.1371/journal.pone.0281631.g008
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 15 / 21
Limitations of study
Nevertheless, some limitations of the study must be noted. The major advantages of using
products of recycled agricultural biomasses—such as green composts—in pharmacological
therapy, consist in sustainability and renewability, as well as large availability of bioactive mol-
ecules. Among the various humic components, we attributed the antioxidant and anti-inflam-
matory role of HS-FEN to its abundant phenolic and lignin content, hypothesizing their
synergistic action, but we did not determine the individual effects of these components in miti-
gating the inflammatory response. This topic remains one of the main goals of our next
studies.
Moreover, apart from a clearer determination of the HS-FEN composition which may facil-
itate the assignment of its biological properties to individual chemical components, the poten-
tial use of HS-FEN as therapeutic drug also requires more pharmacological evidences. In
particular, the toxicological safety of HS-FEN has to be assessed, in order to uncover probable
clinical adverse effects.
Lastly, additional in vivo studies are needed to confirm the efficacy of HS-FEN, in order to
verify the potentiality of humic substances from composted green biomasses, not only in agri-
cultural sector as biostimulants [67], but also in medical field.
Conclusion
This work showed a key role of HS-FEN in favoring mitochondrial homeostasis, modulating
both the inflammation and the oxidative stress. Mitochondria are considered “metabolic
checkpoints” able to sense the metabolic status of cells. It is not surprising that when metabolic
changes–due to invading pathogens and/or inflammatory events–occurs, mitochondria alter
their functions. Thus, mitochondrial dysfunction reflects the cellular metabolic status and may
result in both oxidative stress and alteration of fission and fusion proteins [68]. In the present
study, factors critical for mitochondrial dynamics were found dysregulated upon Hpcf stimu-
lation. OPA-1 and SOD-2 genes decreased in Hpcf-stimulated cells, while Drp-1 gene
increased, suggesting the contribution of Hpcf in mitochondria fragmentation, which in turn
contributed to the unlimited production of reactive oxygen species and cell apoptosis. In addi-
tion, mitochondrial fragmentation, via OPA-1 dysregulation, also promotes the NF-kB activa-
tion and consequent inflammation [63]. This finding was confirmed, in this study, by up-
regulation of IL-17, IL-12 and G-CSF cytokines. All these alterations were reverted following
HS-FEN treatment. Therefore, HS-FEN seems to restore normal mitochondrial functions and
attenuate the H.pylori-associate inflammatory response. These effects may be attributed to: 1)
the considerable content of phenolic and lignin components in HS-FEN, 2) its hydrophobic
components, and, 3) the flexibility of its supramolecular conformation. Given this scenario,
HS-FEN could represent a valid alternative against the H.pylori-associated inflammation and
related disorders.
Supporting information
S1 Fig. Total ion chromatograms of thermochemolysis products of HS-FEN.
(TIF)
S1 Table. List of the main products released by the thermochemolysis from humic sub-
stances from fennel (HS-FEN).
(PDF)
S2 Table. Primers used for qRT-PCR.
(PDF)
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 16 / 21
S1 Dataset.
(DOCX)
Author Contributions
Conceptualization: Riccardo Spaccini, Rosanna Capparelli, Alessandro Piccolo.
Data curation: Mariavittoria Verrillo, Paola Cuomo, Angela Michela Immacolata Montone,
Davide Savy.
Methodology: Mariavittoria Verrillo, Paola Cuomo, Davide Savy.
Supervision: Rosanna Capparelli, Alessandro Piccolo.
Validation: Riccardo Spaccini, Rosanna Capparelli.
Writing – original draft: Mariavittoria Verrillo, Paola Cuomo.
Writing – review & editing: Riccardo Spaccini, Rosanna Capparelli, Alessandro Piccolo.
References
1. Spaccini R, Cozzolino V, Di Meo V, Savy D, Drosos M, Piccolo A. Bioactivity of humic substances and
water extracts from compost made by ligno-cellulose wastes from biorefinery. Sci Total Environ [Inter-
net]. 2018 Jul 25 [cited 2022 Jun 29]; 646:792–800. Available from: https://europepmc.org/article/med/
30064105.https://doi.org/10.1016/j.scitotenv.2018.07.334 PMID: 30064105
2. Savarese C, di Meo V, Cangemi S, Verrillo M, Savy D, Cozzolino V, et al. Bioactivity of two different
humic materials and their combination on plants growth as a function of their molecular properties. Plant
Soil. 2022 Mar 1; 472(1–2):509–26.
3. Pane C, Palese AM, Spaccini R, Piccolo A, Celano G, Zaccardelli M. Enhancing sustainability of a pro-
cessing tomato cultivation system by using bioactive compost teas. Sci Hortic (Amsterdam). 2016 Apr
20; 202:117–24.
4. Verrillo M, Salzano M, Cozzolino V, Spaccini R, Piccolo A. Bioactivity and antimicrobial properties of
chemically characterized compost teas from different green composts. Waste Manag [Internet]. 2020
Dec 5 [cited 2022 Jun 29]; 120:98–107. Available from: https://europepmc.org/article/med/33290882.
https://doi.org/10.1016/j.wasman.2020.11.013 PMID: 33290882
5. Maniadakis K, Lasaridi K, Manios Y, Kyriacou M, Manios T. Integrated Waste Management Through
Producers and Consumers Education: Composting of Vegetable Crop Residues for Reuse in Cultiva-
tion. http://dx.doi.org/101081/PFC-120027447 [Internet]. 2011 [cited 2022 Jun 29]; 39(1):169–83. Avail-
able from: https://www.tandfonline.com/doi/abs/10.1081/PFC-120027447.
6. Istat.it Agricoltura [Internet]. [cited 2022 Jun 29]. Available from: https://www.istat.it/it/agricoltura.
7. Ben-Othman S, Jõudu I, Bhat R, Beatriz M, Oliveira P, Alves RC. Bioactives from Agri-Food Wastes:
Present Insights and Future Challenges. Mol 2020, Vol 25, Page 510 [Internet]. 2020 Jan 24 [cited
2022 Jun 29];25(3):510. Available from: https://www.mdpi.com/1420-3049/25/3/510/htm.https://doi.
org/10.3390/molecules25030510 PMID: 31991658
8. Doria E, Boncompagni E, Marra A, Dossena M, Verri M, Buonocore D. Polyphenols Extraction From
Vegetable Wastes Using a Green and Sustainable Method. Front Sustain Food Syst. 2021 Sep 27;
5:342.
9. Verrillo M, Cozzolino V, Spaccini R, Piccolo A. Humic substances from green compost increase bioac-
tivity and antibacterial properties of essential oils in Basil leaves. Chem Biol Technol Agric [Internet].
2021 Dec 1 [cited 2022 Jun 29]; 8(1):1–14. Available from: https://chembioagro.springeropen.com/
articles/10.1186/s40538-021-00226-7.
10. Arulselvan P, Fard MT, Tan WS, Gothai S, Fakurazi S, Norhaizan ME, et al. Role of Antioxidants and
Natural Products in Inflammation. Oxid Med Cell Longev [Internet]. 2016 [cited 2022 Jun 29];2016.
Available from: https://pubmed.ncbi.nlm.nih.gov/27803762/.https://doi.org/10.1155/2016/5276130
PMID: 27803762
11. Bagnasco D, Povero M, Pradelli L, Brussino L, Rolla G, Caminati M, et al. Economic impact of mepolizu-
mab in uncontrolled severe eosinophilic asthma, in real life. World AllergyOrgan J [Internet].2021 Feb
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 17 / 21
1 [cited 2022 Jun 29];14(2). Available from: /pmc/articles/PMC7846931/. https://doi.org/10.1016/j.
waojou.2021.100509 PMID: 33598095
12. Bagnasco D, Paggiaro P, Latorre M, Folli C, Testino E, Bassi A, et al. Severe asthma: One disease and
multiple definitions. World Allergy Organ J [Internet]. 2021 Nov 1 [cited 2022 Jun 29];14(11). Available
from: https://pubmed.ncbi.nlm.nih.gov/34871335/.https://doi.org/10.1016/j.waojou.2021.100606
PMID: 34871335
13. Netea MG, Balkwill F, Chonchol M, Cominelli F, Donath MY, Giamarellos-Bourboulis EJ, et al. A guiding
map for inflammation. Nat Immunol [Internet]. 2017 Jul 19 [cited 2022 Jun 29]; 18(8):826–31. Available
from: https://pubmed.ncbi.nlm.nih.gov/28722720/.https://doi.org/10.1038/ni.3790 PMID: 28722720
14. GM B. A calculated response: control of inflammation by the innate immune system. J Clin Invest [Inter-
net]. 2008 Feb 1 [cited 2021 Aug 2]; 118(2):413–20. Available from: https://pubmed.ncbi.nlm.nih.gov/
18246191/.https://doi.org/10.1172/JCI34431 PMID: 18246191
15. Fulgione A, Papaianni M, Cuomo P, Paris D, Romano M, Tuccillo C, et al. Interaction between MyD88,
TIRAP and IL1RL1 against Helicobacter pylori infection. Sci Reports 2020 101 [Internet]. 2020 Sep 28
[cited 2021 Aug 2]; 10(1):1–13. Available from: https://www.nature.com/articles/s41598-020-72974-9.
16. Israel DA, Salama N, Arnold CN, Moss SF, Ando T, Wirth HP, et al. Helicobacter pylori strain-specific
differences in genetic content, identified by microarray, influence host inflammatory responses. J Clin
Invest [Internet]. 2001 [cited 2022 Jun 29]; 107(5):611–20. Available from: https://pubmed.ncbi.nlm.nih.
gov/11238562/.https://doi.org/10.1172/JCI11450 PMID: 11238562
17. Kuck D, Kolmerer B, Iking-Konert C, Krammer PH, Stremmel W, Rudi J. Vacuolating Cytotoxin of Heli-
cobacter pylori Induces Apoptosis in the Human Gastric Epithelial Cell Line AGS. Infect Immun [Inter-
net]. 2001 [cited 2022 Jun 29]; 69(8):5080. Available from: /pmc/articles/PMC98603/. https://doi.org/10.
1128/IAI.69.8.5080-5087.2001 PMID: 11447189
18. Kalisperati P, Spanou E, Pateras IS, Korkolopoulou P, Varvarigou A, Karavokyros I, et al. Inflammation,
DNA damage, Helicobacter pylori and gastric tumorigenesis. Front Genet. 2017 Feb 27; 8(FEB):20.
https://doi.org/10.3389/fgene.2017.00020 PMID: 28289428
19. Cover TL, Blaser MJ. Helicobacter pylori in health and disease. Gastroenterology [Internet]. 2009 [cited
2022 Jun 29]; 136(6):1863–73. Available from: https://pubmed.ncbi.nlm.nih.gov/19457415/.https://doi.
org/10.1053/j.gastro.2009.01.073 PMID: 19457415
20. Klo
¨cking R, Helbig B. Humic Substances, Medical Aspects and Applications of. Biopolym Online [Inter-
net]. 2001 Apr 26 [cited 2022 Jun 29]; Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/
3527600035.bpol1013.
21. Van Rensburg CEJ, Van Straten A, Dekker J. An in vitro investigation of the antimicrobial activity of oxi-
fulvic acid. J Antimicrob Chemother [Internet]. 2000 Nov 1 [cited 2022 Jun 29]; 46(5):853–4. Available
from: https://academic.oup.com/jac/article/46/5/853/668629.https://doi.org/10.1093/jac/46.5.853
PMID: 11062218
22. Jansen Van Rensburg CE, Naude PJ. Potassium humate inhibits complement activation and the pro-
duction of inflammatory cytokines in vitro. Inflammation [Internet]. 2009 Aug 9 [cited 2022 Jun 29]; 32
(4):270–6. Available from: https://link.springer.com/article/10.1007/s10753-009-9130-6.https://doi.org/
10.1007/s10753-009-9130-6 PMID: 19507015
23. Van Rensburg CEJ. The Antiinflammatory Properties of Humic Substances: A Mini Review. Phytother
Res [Internet]. 2015 Jun 1 [cited 2022 Jun 29]; 29(6):791–5. Available from: https://pubmed.ncbi.nlm.
nih.gov/25732236/.https://doi.org/10.1002/ptr.5319 PMID: 25732236
24. Bernstein N, Gorelick J, Zerahia R, Koch S. Impact of N, P, K, and humic acid supplementation on the
chemical profile of medical cannabis (Cannabis sativa L). Front Plant Sci. 2019 May 31; 10:736. https://
doi.org/10.3389/fpls.2019.00736 PMID: 31263470
25. Yalman V, Lac¸in NT. Development of humic acid and alginate-based wound dressing and evaluation on
inflammation. [Internet]. 2019 Oct 15 [cited 2022 Jun 29]; 34(12):705–17. Available from: https://www.
tandfonline.com/doi/abs/10.1080/10667857.2019.1619961.
26. Nuzzo A, Mazzei P, Drosos M, Piccolo A. Novel Humo-Pectic Hydrogels for Controlled Release of Agro-
products. ACS Sustain Chem Eng. 2020 Jul 13; 8(27):10079–88.
27. Vitiello G, Venezia V, Verrillo M, Nuzzo A, Houston J, Cimino S, et al. Hybrid humic acid/titanium dioxide
nanomaterials as highly effective antimicrobial agents against gram(-) pathogens and antibiotic contam-
inants in wastewater. Environ Res [Internet]. 2021 Feb 1 [cited 2022 Jun 29];193. Available from:
https://pubmed.ncbi.nlm.nih.gov/33271143/.https://doi.org/10.1016/j.envres.2020.110562 PMID:
33271143
28. Venezia V, Pota G, Silvestri B, Vitiello G, Di Donato P, Landi G, et al. A study on structural evolution of
hybrid humic Acids-SiO 2 nanostructures in pure water: Effects on physico-chemical and functional
properties. Chemosphere [Internet]. 2022 Jan 1 [cited 2022 Jun 29];287(Pt 1). Available from: https://
pubmed.ncbi.nlm.nih.gov/34454229/.
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 18 / 21
29. Venezia V, Verrillo M, Gallucci N, Di Girolamo R, Luciani G, D’Errico G, et al. NAExploiting bioderived
humic acids: a molecular combination with ZnO nanoparticles leads to nanostructured hybrid interfaces
with enhanced pro-oxidant and antibacterial activity. J Environ Chem Eng [Internet]. 2022; 11
(1):108973. Available from: https://doi.org/10.1016/j.jece.2022.108973.
30. Nuzzo A, Buurman P, Cozzolino V, Spaccini R, Piccolo A. Infrared spectra of soil organic matter under
a primary vegetation sequence. Chem Biol Technol Agric [Internet]. 2020 Dec 1 [cited 2022 Jun 29]; 7
(1):1–12. Available from: https://chembioagro.springeropen.com/articles/10.1186/s40538-019-0172-1.
31. Monda H, Cozzolino V, Vinci G, Drosos M, Savy D, Piccolo A. Molecular composition of the Humeome
extracted from different green composts and their biostimulation on early growth of maize. Plant Soil.
2018 Aug 1; 429(1–2):407–24.
32. Verrillo M, Savy D, Cangemi S, Savarese C, Cozzolino V, Piccolo A. Valorization of lignins from energy
crops and agro-industrial byproducts as antioxidant and antibacterial materials. J Sci Food Agric [Inter-
net]. 2022 May 1 [cited 2022 Jun 29]; 102(7):2885–92. Available from: https://onlinelibrary.wiley.com/
doi/full/10.1002/jsfa.11629. PMID: 34755340
33. Verrillo M, Parisi M, Savy D, Caiazzo G, Di Caprio R, Luciano MA, et al. Antiflammatory activity and
potential dermatological applications of characterized humic acids from a lignite and a green compost.
Sci Reports 2022 121 [Internet]. 2022 Feb 9 [cited 2022 Jun 29]; 12(1):1–13. Available from: https://
www.nature.com/articles/s41598-022-06251-2.https://doi.org/10.1038/s41598-022-06251-2 PMID:
35140310
34. Spaccini R, Mazzei P, Squartini A, Giannattasio M, Piccolo A. Molecular properties of a fermented
manure preparation used as field spray in biodynamic agriculture.
35. Piccolo A, Conte P, Trivellone E, Van Lagen B, Buurman P. Reduced heterogeneity of a lignite humic
acid by preparative HPSEC following interaction with an organic acid. Characterization of size-sepa-
rates by Pyr-GC-MS and 1H-NMR spectroscopy. Environ Sci Technol [Internet]. 2002 Jan 1 [cited 2022
Jun 29]; 36(1):76–84. Available from: https://pubmed.ncbi.nlm.nih.gov/11811494/.https://doi.org/10.
1021/es010981v PMID: 11811494
36. Cuomo P, Medaglia C, Allocca I, Montone AMI, Guerra F, Cabaro S, et al. Caulerpin Mitigates Helico-
bacter pylori-Induced Inflammation via Formyl Peptide Receptors. Int J Mol Sci [Internet]. 2021 Dec 1
[cited 2022 Jun 29];22(23). Available from: https://pubmed.ncbi.nlm.nih.gov/34884957/.https://doi.org/
10.3390/ijms222313154 PMID: 34884957
37. Capparelli R, Cuomo P, Papaianni M, Pagano C, Montone AMI, Ricciardelli A, et al. Bacteriophage-
Resistant Salmonella rissen: An In Vitro Mitigated Inflammatory Response. Viruses [Internet]. 2021 Dec
1 [cited 2022 Jun 29];13(12). Available from: https://pubmed.ncbi.nlm.nih.gov/34960737/.
38. Savy D, Cozzolino V, Vinci G, Nebbioso A, Piccolo A. Water-Soluble Lignins from Different Bioenergy
Crops Stimulate the Early Development of Maize (Zea mays, L.). Molecules [Internet]. 2015 Nov 5
[cited 2022 Jun 29]; 20(11):19958–70. Available from: https://pubmed.ncbi.nlm.nih.gov/26556330/.
https://doi.org/10.3390/molecules201119671 PMID: 26556330
39. S
ˇmejkalova
´D, Piccolo A. Aggregation and disaggregation of humic supramolecular assemblies by
NMR diffusion ordered spectroscopy (DOSY-NMR). Environ Sci Technol [Internet]. 2008 Feb 1 [cited
2022 Jun 29]; 42(3):699–706. Available from: https://pubs.acs.org/doi/full/10.1021/es071828p. PMID:
18323090
40. Verrillo M, Salzano M, Savy D, Di Meo V, Valentini M, Cozzolino V, et al. Antibacterial and antioxidant
properties of humic substances from composted agricultural biomasses. Chem Biol Technol Agric
[Internet]. 2022 Dec 1 [cited 2022 Jun 29]; 9(1):1–15. Available from: https://chembioagro.
springeropen.com/articles/10.1186/s40538-022-00291-6.
41. Savy D, Brostaux Y, Cozzolino V, Delaplace P, du Jardin P, Piccolo A. Quantitative Structure-Activity
Relationship of Humic-Like Biostimulants Derived From Agro-Industrial Byproducts and Energy Crops.
Front Plant Sci. 2020 May 26; 11:581. https://doi.org/10.3389/fpls.2020.00581 PMID: 32528492
42. Tamamura S, Ohashi R, Nagao S, Yamamoto M, Mizuno M. Molecular-size-distribution-dependent
aggregation of humic substances by Na(I), Ag(I), Ca(II), and Eu(III). Colloids Surfaces A Physicochem
Eng Asp. 2013 Oct 5; 434:9–15.
43. Vona R, Pallotta L, Cappelletti M, Severi C, Matarrese P. The Impact of Oxidative Stress in Human
Pathology: Focus on Gastrointestinal Disorders. Antioxidants 2021, Vol 10, Page 201 [Internet]. 2021
Jan 30 [cited 2022 Jun 29];10(2):201. Available from: https://www.mdpi.com/2076-3921/10/2/201/htm.
https://doi.org/10.3390/antiox10020201 PMID: 33573222
44. Santos-Sa
´nchez NF, Salas-Coronado R, Villanueva-Cañongo C, Herna
´ndez-Carlos B. Antioxidant
Compounds and Their Antioxidant Mechanism. Antioxidants [Internet]. 2019 Mar 22 [cited 2022 Jun
29]; Available from: undefined/state.item.id.
45. Zykova M V., Schepetkin IA, Belousov M V., Krivoshchekov S V., Logvinova LA, Bratishko KA, et al.
Physicochemical Characterization and Antioxidant Activity of Humic Acids Isolated from Peat of Various
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 19 / 21
Origins. Molecules [Internet]. 2018 [cited 2022 Jun 29];23(4). Available from: https://pubmed.ncbi.nlm.
nih.gov/29587351/.https://doi.org/10.3390/molecules23040753 PMID: 29587351
46. Rusu AG, Iva
´n H, Ortı
´z M, Granata G, Riccobene C, Napoli E, et al. Polymeric Nanocapsules Contain-
ing Fennel Essential Oil: Their Preparation, Physicochemical Characterization, Stability over Time and
in Simulated Gastrointestinal Conditions. Pharm 2022, Vol 14, Page 873 [Internet]. 2022 Apr 16 [cited
2022 Jun 29];14(4):873. Available from: https://www.mdpi.com/1999-4923/14/4/873/htm.
47. Vuolo MM, Lima VS, Maro
´stica Junior MR. Chapter 2 Phenolic Compounds Structure, Classification,
and Antioxidant Power. Bioact Compd Heal Benefits Potential Appl [Internet]. 2018 Dec 7 [cited 2022
Jun 29];33–50. Available from: https://app.dimensions.ai/details/publication/pub.1111365242.
48. Aeschbacher M, Graf C, Schwarzenbach RP, Sander M. Antioxidant properties of humic substances.
Environ Sci Technol [Internet]. 2012 May 1 [cited 2022 Jun 29]; 46(9):4916–25. Available from: https://
pubmed.ncbi.nlm.nih.gov/22463073/.https://doi.org/10.1021/es300039h PMID: 22463073
49. Ugartondo V, Mitjans M, Vinardell MP. Comparative antioxidant and cytotoxic effects of lignins from dif-
ferent sources. Bioresour Technol [Internet]. 2008 Sep [cited 2022 Jun 29]; 99(14):6683–7. Available
from: https://pubmed.ncbi.nlm.nih.gov/18187323/.https://doi.org/10.1016/j.biortech.2007.11.038
PMID: 18187323
50. Qin Z, Liu H-M, Gu L-B, Sun R-C, Wang X-D. Lignin as a Natural Antioxidant: Property-Structure Rela-
tionship and Potential Applications. React Funct Polym Vol One. 2020;65–93.
51. Lin WC, Tsai HF, Liao HJ, Tang CH, Wu YY, Hsu PI, et al. Helicobacter pylori sensitizes TNF-related
apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regula-
tion of FLIP. Cell Death Dis [Internet]. 2014 [cited 2022 Jun 29];5(3). Available from: https://pubmed.
ncbi.nlm.nih.gov/24603337/.https://doi.org/10.1038/cddis.2014.81 PMID: 24603337
52. Rassow J. Helicobacter pylori vacuolating toxin A and apoptosis. Cell Commun Signal [Internet]. 2011
Nov 1 [cited 2022 Jun 29]; 9(1):1–9. Available from: https://biosignaling.biomedcentral.com/articles/10.
1186/1478-811X-9-26.
53. Chatre L, Fernandes J, Michel V, Fiette L, Ave
´P, Arena G, et al. Helicobacter pylori targets mitochon-
drial import and components of mitochondrial DNA replication machinery through an alternative VacA-
dependent and a VacA-independent mechanisms. Sci Rep [Internet]. 2017 Dec 1 [cited 2022 Jun 29];7
(1). Available from: https://pubmed.ncbi.nlm.nih.gov/29162845/.https://doi.org/10.1038/s41598-017-
15567-3 PMID: 29162845
54. Galmiche A, Rassow J, Doye A, Cagnol S, Chambard JC, Contamin S, et al. The N-terminal 34 kDa
fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c
release. EMBO J [Internet]. 2000 Dec 1 [cited 2022 Jun 29]; 19(23):6361–70. Available from: https://
pubmed.ncbi.nlm.nih.gov/11101509/.https://doi.org/10.1093/emboj/19.23.6361 PMID: 11101509
55. Xie L, Zhou T, Xie Y, Bode AM, Cao Y. Mitochondria-Shaping Proteins and Chemotherapy. Front
Oncol. 2021 Nov 18; 11:769036. Available from: https://pubmed.ncbi.nlm.nih.gov/34868997/.https://
doi.org/10.3389/fonc.2021.769036 PMID: 34868997
56. Olichon A, Baricault L, Gas N, Guillou E, Valette A, Belenguer P, et al. Loss of OPA1 perturbates the
mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis. J
Biol Chem [Internet]. 2003 Mar 7 [cited 2022 Jun 29]; 278(10):7743–6. Available from: https://pubmed.
ncbi.nlm.nih.gov/12509422/.https://doi.org/10.1074/jbc.C200677200 PMID: 12509422
57. Frank S, Gaume B, Bergmann-Leitner ES, Leitner WW, Robert EG, Catez F, et al. The role of dynamin-
related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev Cell. 2001 Oct; 1(4):515–25.
Available from: https://pubmed.ncbi.nlm.nih.gov/11703942/.https://doi.org/10.1016/s1534-5807(01)
00055-7 PMID: 11703942
58. Guerra-Castellano A, Dı
´az-Quintana A, Pe
´rez-Mejı
´as G, Elena-Real CA, Gonza
´lez-Arzola K, Garcı
´a-
Mauriño SM, et al. Oxidative stress is tightly regulated by cytochrome c phosphorylation and respira-
some factors in mitochondria. Proc Natl Acad Sci U S A [Internet]. 2018 Jul 31 [cited 2022 Jun 29]; 115
(31):7955–60. Available from: https://pubmed.ncbi.nlm.nih.gov/30018060/.https://doi.org/10.1073/
pnas.1806833115 PMID: 30018060
59. Robert P, Nguyen PMC, Richard A, Grenier C, Chevrollier A, Munier M, et al. Protective role of the mito-
chondrial fusion protein OPA1 in hypertension. FASEB J [Internet]. 2021 Jul 1 [cited 2022 Jun 29];35
(7). Available from: https://pubmed.ncbi.nlm.nih.gov/34133045/.https://doi.org/10.1096/fj.
202000238RRR PMID: 34133045
60. Kannan K, Jain SK. Oxidative stress and apoptosis. Pathophysiol Off J Int Soc Pathophysiol [Internet].
2000 Sep [cited 2022 Jun 29]; 7(3):153–63. Available from: https://pubmed.ncbi.nlm.nih.gov/10996508/
.https://doi.org/10.1016/s0928-4680(00)00053-5 PMID: 10996508
61. Lima AR, Santos L, Correia M, Soares P, Sobrinho-Simões M, Melo M, et al. Dynamin-Related Protein
1 at the Crossroads of Cancer. Genes (Basel). 2018 Feb 21; 9(2):115. Available from: https://pubmed.
ncbi.nlm.nih.gov/29466320/.https://doi.org/10.3390/genes9020115 PMID: 29466320
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 20 / 21
62. Rodrı
´guez-Nuevo A, Dı
´az-Ramos A, Noguera ED, Dı
´az-Sa
´ez F, Duran X, Muñoz JP, et al. Mitochon-
drial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency. EMBO J [Internet]. 2018 May 15
[cited 2022 Jun 29]; 37(10):e96553. Available from: https://cris.bgu.ac.il/en/publications/mitochondrial-
dna-and-tlr9-drive-muscle-inflammation-upon-opa1-de.https://doi.org/10.15252/embj.201796553
PMID: 29632021
63. Herkenne S, Ek O, Zamberlan M, Pellattiero A, Chergova M, Chivite I, et al. Developmental and Tumor
Angiogenesis Requires the Mitochondria-Shaping Protein Opa1. Cell Metab [Internet]. 2020 May 5
[cited 2022 Jun 29]; 31(5):987–1003.e8. Available from: https://pubmed.ncbi.nlm.nih.gov/32315597/.
https://doi.org/10.1016/j.cmet.2020.04.007 PMID: 32315597
64. Serelli-Lee V, Ling KL, Ho C, Yeong LH, Lim GK, Ho B, et al. Persistent Helicobacter pylori Specific
Th17 Responses in Patients with Past H. pylori Infection Are Associated with ElevatedGastric Mucosal
IL-1β. PLoS One [Internet]. 2012 Jun 25 [cited 2022 Jun 29]; 7(6):e39199. Available from: https://
journals.plos.org/plosone/article?id=10.1371/journal.pone.0039199.
65. Zenobia C, Hajishengallis G. Basic biology and role of interleukin-17 in immunity and inflammation. Per-
iodontol 2000 [Internet]. 2015 Oct 1 [cited 2022 Jun 29]; 69(1):142–59. Available from: https://pubmed.
ncbi.nlm.nih.gov/26252407/.
66. Nielsen OH, Kirman I, Ru¨diger N, Hendel J, Vainer B. Upregulation of interleukin-12 and -17 in active
inflammatory bowel disease. Scand J Gastroenterol [Internet]. 2003 Feb 1 [cited 2022 Jun 30]; 38
(2):180–5. Available from: https://pubmed.ncbi.nlm.nih.gov/12678335/.https://doi.org/10.1080/
00365520310000672 PMID: 12678335
67. Savarese C, Cozzolino V, Verrillo M, Vinci G, De Martino A, Scopa A, et al. Combination of humic biosti-
mulants with a microbial inoculum improves lettuce productivity, nutrient uptake, and primary and sec-
ondary metabolism. Plant Soil [Internet]. 2022;285–314. Available from: https://doi.org/10.1007/
s11104-022-05634-8.
68. Vakifahmetoglu-Norberg H, Ouchida AT, Norberg E. The role of mitochondria in metabolism and cell
death. Vol. 482, Biochemical and Biophysical Research Communications. 2017. https://doi.org/10.
1016/j.bbrc.2016.11.088 PMID: 28212726
PLOS ONE
Anti-inflammatory effects of humic substances from fennel residues in Helicobacter pylori infection
PLOS ONE | https://doi.org/10.1371/journal.pone.0281631 March 9, 2023 21 / 21
