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Phytoprostanes and phytofurans modulate COX-2-linked inflammation markers in LPS-stimulated THP-1 monocytes by lipidomics workflow

Inflammation is a fundamental pathophysiological process which occurs in the course of several diseases. The present work describes the capacity of phytoprostanes (PhytoPs) and phytofurans (PhytoFs) (plant oxylipins), present in plant-based of 15-keto-PGF 2 α , PGF 2 α , PGE 2 , PGF 1 α , PGDM, and PGD 2 induced by LPS. These results indicated that specific plant oxylipins can protect against inflammatory events, encouraging further investigations using plant-based foods rich in these oxylipins or enriched extracts, to identify specific bioactivities of the diverse individual molecules, which can be useful for nutrition and health in the frame of well-defined pathophysiological processes.


Introduction
Inflammation is the first biological response of the immune system to an array of factors that disturb the homeostasis of cells and tissues, and is triggered to restore the physiological conditions [1]. In this scenario, the innate immune system plays a pivotal role in the evolution of inflammation, with its functioning mainly based on receptors that recognize conserved molecular structures of pathogens, such lipopolysaccharide (LPS). When these are recognized, the immune system initiates a variety of host responses by activating different molecular pathways involved in systemic inflammation. During this process, cells secrete cyclooxygenase (COX) products, such as prostaglandins (PGs) E 2 and F 2α (PGE 2 and PGF 2α , respectively), and their metabolites, responsible for the activation of the inflammatory cascade [2][3][4][5].
In the search for new compounds with the capacity to prevent inflammation, various studies aimed at unraveling the mechanism/s of action of secondary metabolites of higher plants. These studies revealed the valuable capability to avoid PG secretion in response to the LPS challenge through the inhibition of the enzymes involved in the production of PGs, such as phospholipase A 2 (PA2), cyclooxygenase (COX), and lipoxygenase (LOX). Therefore, they modulate the inflammatory process [6][7][8][9] due to the inhibition of specific enzymes [10][11][12], which has been associated with various health benefits.
In this regard, the presence of a new type of secondary metabolite, belonging to the family of plant oxylipins, and represented by phytoprostanes (PhytoPs) and phytofurans (PhytoFs) has been reported in plant-based foods [13][14][15]. To the present date, these compounds have been characterized in a wide diversity of plant-based foods, namely nuts, hazelnuts, almonds, vegetable oils, cereals, olives, wine, peas, rice, several tropical fruits, cocoa, macroalgae, chocolate, pistachio, date, and especially in legumes, at different concentrations . They have been suggested to be bioactive compounds, mainly based on their structural analogy with isoprostanoids [41]. However, their capacity to modulate inflammation with respect to their ability to influence the isoprostanoid profile in a pro-inflammatory environment, has not been addressed as of yet. Hence, the pro-and anti-inflammatory activity of individual PhytoPs and PhytoFs merits to be explored, as PGs play a central role in diseases coursing with inflammation [34].
In this scenario, the present article aims to uncover the capacity of individual PhytoPs and PhytoFs to modulate the inflammatory response triggered by LPS in the THP-1 human monocytic cell line, in vitro. The inflammatory modulation foreseen will be monitored by assessing the prostanoid profile through a panel of 21 human oxylipins analyzed in cells and growth media by UHPLC-QqQ-ESI-MS/MS.

Cell lines and culture conditions
The monocytic human (THP-1) cell line (ECACC® General Cell Collection-88081201) was obtained from the European Collection of Cell Culture (ECACC, Public Health England, Porton Down, Salisbury, UK). Cells were grown in RPMI 1640, supplemented with 2 mM glutamine and 10% fetal bovine serum (FBS), in 75 cm 2 cell culture flasks to the logarithmic growth phase (0.6-1.4 × 10 6 cells/mL), at 37 • C in a humidified atmosphere containing 5% CO 2 . The passage number of the cells used in this study was between 17 and 20.

LPS-induced inflammatory model
Cells were seeded into 24-well plates at 7 × 10 5 cells/well in RPMI 1640, supplemented with 2 mM glutamine and 10% fetal bovine serum (FBS) to study the modulatory effects of PhytoPs and PhytoFs on the PG profile of the human monocytic cell line THP-1. Cells were treated with 50 μM of the individual PhytoPs and PhytoFs in triplicate (n = 3) for 24h. This concentration was selected based on previous studies that detected plant oxylipins at micromolar quantities in several food matrices, such as flax oil or pea [16,31]. In this regard, although this concentration could exceed the theoretical concentration achieved in target cells (which has not already been described), especially because of the ongoing breakdown of dietary PhytoPs and PhytoFs during gastrointestinal digestion, this experimental design set-up in the present work was utilized to understand the biological potential of the plant oxylipins under consideration. An inflammatory stimulus (100 ng/mL LPS) was then added, and cells were maintained at 37 • C and 5% CO 2 for another 24-h.

Extraction of prostaglandins from cells and culture medium
The PG profile was determined in the THP-1 cell lysates (5 × 10 5 ) and their growth medium. Once the cells and supernatants exposed to 100 ng/mL LPS from Escherichia coli 0127:B8 for 24 h were collected, 0.005% BHA (final concentration) was added, and the medium was conserved at − 80 • C until the PGs were extracted. Methanol/HCl 200 mM (0.5 mL) was added to 0.5 mL THP-1 growth medium and centrifuged at 10000×g for 5 min to precipitate the serum proteins.
After removing the growth medium, the cells were lysed by incubation with lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, containing 0.005% BHA) for 1-h, on ice. Lysates were centrifuged at 8000×g for 5 min. The supernatants were collected and stored at − 80 • C until the extraction of PGs.
Both pre-processed growth medium and cell lysate extracts were first enzymatically hydrolyzed (β-glucuronidase Type HP-2 from Helix pomatia), and PGs were extracted according to the procedure already described [48,49]. After hydrolysis, the samples were subjected to a clean-up procedure by solid-phase extraction (SPE) using Strata X-AW cartridges (100 mg/3 mL), following the method described in the literature [48]. Target compounds were eluted with 1 mL of methanol and dried using a SpeedVac concentrator. The concentration of PGs was determined in cell lysates and growth media, and described in both biological matrices and as an addition of both concentrations that constitutes the total PGs synthesized by THP-1 cells.

UHPLC-QqQ-MS/MS analyses of prostaglandins
The chromatographic resolution of PGs in cell lysates and growth medium was performed by UHPLC coupled to a 6460 QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany), using the set up previously described [48,50], and an ACQUITY BEH C 18 column (2.  (Table 1), which was performed through a mass spectrometry analysis by multiple reaction monitoring (MRM) mode, and the application of electrospray ionization (ESI) in the negative mode and the ion optics settings previously described for these compounds [48,50]. Also, the identification and quantification of the PGs monitored were performed using authentic standards of the PGs referred to in Table 1. The 8-iso prostaglandin F 2α -d 4 (contains 4 deuterium atoms at positions 3, 3 ′ , 4, and 4') was used as an internal standard because of its similar ionization pattern relative to the PGs. Data acquisition and processing were performed using the MassHunter software version B.08.00 (Agilent Technologies, Walbronn, Germany). The concentration of the PGs were calculated according to standard curves that were freshly prepared each day of analysis. Additionally, the limit of quantification (LOQ), calculated as a signal/noise ratio of 10, was set up at 8 pM, which agrees with the LOQ previously reported by Labuschagne et al. for quantifying lipid peroxidation in cellular systems [51].

Statistical analysis
All treatments and extractions were performed in triplicate (n = 3), and the data were expressed as the mean ± standard deviation (SD). Statistical tests were performed at a 5% significance level using the SPSS 24.0 software package (LEAD Technologies, Inc., Chicago, USA). Data were subjected to a one-way analysis of variance (ANOVA), confirming that the ANOVA requirements were met, especially regarding the normal distribution of the residuals and the homogeneity of variance, through the Kolmogorov-Smirnov (with Lilliefors correction) and Levene tests, respectively. When statistical differences were identified, the variables were compared using Tukey's multiple range test.

Results
As mentioned before, this present work aimed at evaluating individual PhytoPs and PhytoFs on their capacity to modulate the PGs response triggered during LPS-induced inflammation in THP-1 monocytic cells. On this aspect, the starting hypothesis was that plant oxylipins are capable of modulating the PGs profile in the course of an inflammatory process.
Presently, in association with this objective, it is well-known that monocytes and macrophages are key elements of the innate immune system and the inflammatory process [52]. Hence, the so-called "THP-1" is a human leukemia monocytic cell line, characterized by a strong correspondence with the monocytic fraction of peripheral blood mononuclear cells, which preserves a robust capacity to synthesize many molecules related to the inflammatory process [53]. Although THP-1 cells may not express matching features relative to primary monocytes and their regulatory mechanisms, they are characterized by their physiological properties, which are characteristic of primary monocytes in vivo [54]. Because of this, the THP-1 cell line has widely been used as a model to study the molecular pathways involved in the inflammatory response, and the capacity of food compounds to modulate such pathways, which are strongly linked with the course of many pathophysiological situations [7,55].

THP-1 human monocytic cell line viability
Before evaluating the capacity of PhytoPs and PhytoFs to modulate inflammation and avoid non-addressed harmful effects of these plant oxylipins on THP-1 cells during the inflammation assay, the cytotoxic effect of PhytoPs and PhytoFs against the human THP-1 cells was assessed. With this objective, an XTT assay was carried out after 24 and 48-h in the presence of decreasing concentrations of the individual PhytoPs, 9-F 1t -PhytoP, 9-epi-9-F 1t -PhytoP, ent-16-F 1t -PhytoP, ent-16-epi-16-F 1t -PhytoP, ent-9-D 1t -PhytoP, 16 The results obtained evidenced a minimal modification of the THP-1 cell viability when exposed to high concentrations of individual PhytoPs and PhytoFs. In this regard, the range of concentrations tested for the ten plant oxylipins evaluated did not decrease the viability of THP-1 cells significantly after 24-h, relative to the untreated control, except for ent- Accordingly, the limited cytotoxic effect attributable to PhytoPs and PhytoFs after 24 and 48-h could be dependent on the structural similarities with the mammal's oxylipins (isoprostanoids) already described at diverse concentrations in human cells, without a deleterious effect at homeostatic concentrations. On the other hand, the promotion of viability induced by a select group of PhytoPs and PhytoFs, mainly after a 48-h exposure for most concentrations, could be related to a diverse capacity of the diverse PhytoPs and PhytoFs to penetrate cells, according to their chemical structure [56]. Moreover, the increase in cell viability could also be influenced by the capacity of PhytoPs and PhytoFs to provide additional biochemical advantages to cells (i.e., contributing to the redox-balance in cells) [56]. In this aspect, regarding PhytoPs and PhytoFs, this hypothesis is in good agreement with previous descriptions on the cytotoxicity of PhytoPs and PhytoFs extracts obtained from Gracilaria longissima (edible red algae) by Martínez-Sánchez et al., who reported no cytotoxicity and no effect of cell proliferation nor induction of cell death of intestinal epithelial cells by PhytoPs and PhytoFs at concentrations of up to 50 ng/mL [34].

Modulation of the inflammatory response-mediated through intracellular prostaglandins by plant oxylipins after LPS-induced inflammation
Once the safe conditions of individual PhytoPs and PhytoFs regarding THP-1 cells were found, the study of their ability to modulate LPS-induced inflammation by monitoring changes in the quantitative PGs profile was initiated. Thus, the capacity to modulate the inflammatory response was monitored by assessing a panel of 21 PGs (Table 1), analyzed in cells and growth medium, and also measured as the sum of intra-and extra-cellular PGs, as both are secreted by cells and provide the complete picture of the PG response. This experimental design allowed providing further insights into the specific inflammatory modulation of individual PhytoPs and PhytoFs by evaluating changes in the intracellular and extracellular concentration of the 7 PGs found in quantifiable amounts in THP-1 cells (15-keto-PGF 2α , PGF 2α , 11β-PGF 2α , PGE 2 , PGD 2 , PGDM, and PGF 1α ). Also, this work provides new complementary information with respect to previous descriptions of the biological functions of PhytoPs and PhytoFs, focused on the cytokines profile and/or the phenotype of immune-cells through earlier works which focused on a single plant oxylipin or complete plant extracts of PhytoPs and PhytoFs [30,34,[57][58][59][60][61]. In this regard, the use of whole extracts constitutes an approach that allows retrieving valuable results, but is limited in scope. There is still a need for additional experimental inputs for a sound comparison of the anti-inflammatory contribution of the diverse individual plant oxylipins. Indeed, unraveling the biological relevance of the various individual PhytoPs and PhytoFs would allow selecting those plant-based foods that have the adequate quantitative profile for these compounds, according to the diverse pathophysiological conditions, in this case, inflammation. Because of this, it is not surprising that the resulting information on the biological functions, not just regarding plant oxylipins but other bioactive phytochemicals, remains confusing, while a sound scientific proof of the many different protective features attributed to plant-based foods is still mostly incomplete [25,62].
The concentration of the PGs was measured in cell lysates of THP-1 monocytes treated with individual PhytoPs and PhytoFs, as agents that could prevent the inflammation triggered by LPS. Six PGs (15-keto-PGF 2α , PGE 2 , PGF 1α , PGD 2 , 11β-PGF 2α , and PGDM) were quantified in control (untreated) cells, as well as in cells exposed to LPS, while in LPSstimulated cells, the presence of PGF 2α was also detected at a concentration of 27.76 ng/mL (Fig. 2).

Modulation of the inflammatory response-mediated through prostaglandins excreted in the cells' supernatant by plant oxylipins after LPS-induced inflammation
The PG profile in the culture medium of THP-1 monocytes is shown in Fig. 3. The seven PGs found in the cell lysates were also present in the supernatants.
Two out of the seven PGs detected were not found in untreated control cells (PGF 2α and its metabolite 15-keto-PGF 2α ). On the one hand, PGF 2α released to the medium was significantly decreased by 9-F 1t -PhytoP, 9-D 1t -PhytoP, and 16-B 1 -PhytoP, and ent-9(RS)-12-epi-ST-Δ10-13-PhytoF, which once added to the growth medium as a preventive treatment against the pro-inflammatory effect of LPS, mitigated the PGF 2α concentration 1.6-fold, on average, in comparison with LPS treated cells (10.263 ng/mL) (Fig. 3); and, on the other, the release of 15-keto-PGF 2α in cells treated with almost all individual PhytoPs and PhytoFs was higher than in cells exposed only to LPS, with the exception of ent-9(RS)-12-epi-ST-Δ 10 -13-PhytoF, which did not show significant differences regarding LPS-treated cells. Therefore, all experimental conditions (LPS plus individual PhytoPs or PhytoFs or LPS alone) allowed describing concentrations in the growth medium (0.021-0.056 ng/mL) that were higher than those found for untreated control cells, in which 15-keto-PGF 2α was not detected (Fig. 3).
With respect to the PGE 2 released to the medium, its concentration increase 1.8-fold as a result of LPS exposure (0.309 ng/mL), relative to untreated control cells (0.169 ng/mL) (Fig. 3). All individual PhytoPs and PhytoFs decreased the concentration of PGE 2 in the growth media, with 9-F 1t -PhytoP and 9-L 1 -PhytoP being the most efficient plant oxylipins regarding this effect, which resulted in concentrations that were even lower than the described in control cells (Fig. 3).

Understanding the role of phytoprostanes and phytofurans as modulators of the prostaglandin profile of LPS-stimulated THP-1 monocytes
Although the changes in the intra-and extra-cellular concentrations of PGs as a result of the application of individual PhytoPs and PhytoFs as preventive agents of inflammation could indicate controversial biological activities of the separate compounds, it has to be taken into consideration that both of them are the result of the THP-cells metabolism. Accordingly, this would entail that the diverse human oxylipins monitored in the present work could be characterized by distinct kinetics, not only referring to their synthesis but also their excretion. In this regard, considering the level of these oxylipins as a whole (intraand extra-cellular accumulative concentrations) would help with the understanding of the actual capacity of individual PhytoPs and PhytoFs to modulate the inflammatory response.
Thus, applying this analytical approach, it was observed that the concentration of the PGs PGE 2 and PGDM were not modified significantly by neither the pro-inflammatory stimulus applied nor by the treatment with individual PhytoPs or PhytoFs (Fig. 4). Also, with respect to the capacity of these compounds to prevent the modulation of the synthesis of PGs induced by LPS, the increase detected for the PGs 15keto-PGF 2α , PGF 1α , and 11-β-PGF 2α was not reverted by individual PhytoPs or PhytoFs. Indeed, regarding specific PhytoPs and PhytoFs, a biological activity reinforces the increasing trend recorded when applying the pro-inflammatory stimulus. Hence, this was observed for 15-keto-PGF 2α for almost all the PhytoPs and PhytoFs which, although in a non-significant manner, exhibited the capacity to increase the concentration recorded when treating THP-1 cells with LPS by 1.7-fold, on average. A similar trend was observed for PGF 1α and 11-β-PGF 2α for which individual PhytoPs and PhytoFs increased their concentration by up to 2.3 and 1.7-fold, on average, relative to LPS-treated cells (0.077 and 0.310 ng/mL, respectively). Moreover, for these two PGs, the increase was statistically significant (p < 0.01) for 9-F 1t -PhytoP (0.179 ng/ mL) and ent-16(RS)-9-epi-ST-Δ 14 -10-PhytoF (0.527 ng/mL) (Fig. 4). On the contrary, individual PhytoPs and PhytoFs were competent for mitigating the increase of specific PGs induced by LPS. In this regard, the increased induced on the PGF 2α concentration was significantly prevented by ent-16-epi-16-F 1t -PhytoP, ent-9-D 1t -PhytoP, 16-B 1 -PhytoP, ent-16(RS)-9-epi-ST-Δ 14 -10-PhytoF, and ent-16(RS)-13-epi-ST-Δ 14 -9-PhytoF, which restored values that were not significantly different relative to untreated controls (Fig. 4). Similarly, concerning PGD 2 , although most of the PhytoPs evaluated did not provide a significant mitigation of the pro-inflammatory changes induced by LPS, ent-9-D 1t -PhytoP reduced its level down to values found in the untreated control. Regarding PGD 2 , it is important to notice that again ent-16(RS)-9-epi-ST-Δ 14 -10-PhytoF reinforced the increase induced by LPS, giving rise to even significantly higher concentrations (0.133 ng/mL) (Fig. 4).
The biological activities of PhytoPs and PhytoFs have been suggested based on their structural similarity with mammal oxylipins derived from arachidonic acid (C20:4, n-6, AA) (isoprostanes and prostaglandins) that have important biological functions [63]. In this regard, to date, the bioactivity of PhytoPs and, to a lower extent, of PhytoFs, has been described mainly for their immunomodulatory power [57][58][59]61,[64][65][66][67], anti-cancer activity [68], and anti-inflammatory potential [34,61,[69][70][71][72]. However, despite these studies, the current knowledge on these compounds' biological functions is still scarce, being restricted to assessing whole plant-extracts and, in marginal cases, some individual PhytoPs (e.g., 16-E 1 -PhytoP). At the same time, there continues to be a gap of information concerning the biological interest of most PhytoPs and PhytoFs described so far.
In this scenario, the current availability of a wide range of synthesized individual PhytoPs and PhytoFs [42][43][44][45][46][47] has allowed new experimental designs that will somehow contribute to clarify the biological functions of these plant oxylipins, providing valuable information that can complete the current knowledge generated. Regarding the diverse aspects raised, one that needs to be addressed is the capacity of PhytoPs and PhytoFs to modulate the concentration of mammal isoprostanoids. This is of particular relevance because of the role of mammal isoprostanoids in an array of pathophysiological events, namely immunomodulation, vasoconstriction, platelet activation, and anti-aggregation, smooth muscle contraction of bronchi, and anti-inflammatory and apoptosis-inducing properties [73]. Therefore, describing the capacity of PhytoPs and PhytoFs to modulate the PG level during inflammation would allow for a better understanding of the current biological interest of plant oxylipins.
As for the immunomodulatory capacity already demonstrated by PhytoPs, this has been attributed to the structural analogy with human isoprostanoids (e.g., 16-E 1 -PhytoP vs PGE 2 ) (Traidl-Hoffmann et al., 2005). This structural similarity allows 16-E 1 -PhytoP to mimic the functionality of PGE 2 , thus inhibiting the production of interleukin (IL)-12 by dendritic cells and the activation of the peroxisome proliferatoractivated receptor-gamma (PPAR-γ) dependent mechanisms, that in turn cause the inhibition of the nuclear factor kappa-light-chainenhancer of activated B cell (NF-κB) [57,58]. Interestingly, these pathways are also involved in inflammation, and could indicate that the modulation of PG level achieved when exposed to a pro-inflammatory stimulus (Figs. 2 and 3) could be related to an anti-inflammatory effect of PhytoPs and PhytoFs.
Indeed, aside from the immunomodulatory competences, human isoprostanoids have also been explicitly associated to anti-inflammatory activity due to the presence of an A-, J-, or deoxy-J-ring systems in their structure [70]. Thus, the presence of these reactive groups (hydroxyl and keto groups) on the cyclopentenone ring, and additional electrophilic properties enclosed in the chemical structure of PhytoPs and PhytoFs, have prompted the evaluation of coincident bioactivities on the modulation of inflammation by these plant oxylipins. In this respect, the assessment of diverse PhytoPs on anti-inflammatory functions has allowed pointing out the 9 and 16 series of the PhytoPs A 1 , B 1 , and deoxy-J 1 , as those with the anti-inflammatory potential [69], which has been attributed to their structural analogy with the mammal's PGA 1 and deoxy-PGJ 2 [71].
In the sequence of these previous characterizations, the present work describes the capacity of individual PhytoPs and PhytoFs to modulate the PG profile in a pro-inflammatory environment in vitro, using LPSstimulated THP-1 monocytes. Inflammatory mediators derived from COX-2 (Table 1) were measured through a targeted metabolomic approach. In this regard, the PG profile of LPS-stimulated monocytes was characterized by an increased concentration of seven PGs and their metabolites (15-keto-PGF 2α , PGF 2α , 11β-PGF 2α , PGE 2 , PGD 2 , PGDM, and PGF 1α ), tentatively as a result of COX-2 up-regulation [74]. Considering to the short half-life of primary PGs, in the present study, some PG conjugates were also monitored after an enzymatic hydrolysis that breaks down the glucuronide moieties present in PGs [75], thus determining all isoprostanoids present in cells lysates and supernatants and avoiding underestimations of their concentration [48].
The infection of cells by microorganisms activates the inflammatory response due to the recognition of the LPS present in the microbial wall by receptors of immune cells [2], which results in an overproduction of inflammatory mediators. Namely, PGE 2 , which is synthesized from PGH 2 (formed from AA by COX-2), with the participation of various PG synthases [74] (Fig. 5), has versatile biological activities related to the inflammation process depending on binding to different E-prostanoid (EP) receptors (EP1, EP2, EP3, and EP4) [76], and specifically in monocytes [77]. The participation of PGE 2 in inflammation has been described as having a multifaceted role. This involves pro-inflammatory biological effects during the initial phases of the inflammatory process, including cell recruiting, activation, and proliferation, apoptosis, angiogenesis, and enhancement of immune cell surveillance, and augmenting sensory nerves to increase the pain response [78]. Besides, PGE 2 also controls mechanisms related to the resolution of inflammation in subsequent phases by acting as an immunosuppressive agent with respect to the cytokines and chemokines profiles expressed by immune cells (IL-12, IL-12p40, IFN-γ, and TNF-α, among others), as well as the expression level of their cognate receptors [79]. This anti-inflammatory effect is of special relevance in monocytes. In this cell population, PGE 2 triggers the inhibitory production of the chemokines responsible for the chemo-attraction of naïve T cells (CCL19), thus interfering with the activation of effector T cells [80].
The results obtained in the present work evidenced a multidimensional modulation of intracellular PGE 2 and release into the growth medium. Thus, in the presence of specific PhytoPs and PhytoFs (9-F 1t -PhytoP, ent-9-D 1t -PhytoP, ent-16(RS)-9-epi-ST-Δ 14 -10-PhytoF, and ent-9 (RS)-12-epi-ST-Δ 10 -13-PhytoF) in the medium, found a non-significant decrease (p > 0.05), of this PG (Fig. 2). In this respect, diverse in vivo studies have demonstrated that this effect could be due to an inhibition of COX-2, which entails the subsequent decrease of the PGE 2 concentration [74], preventing the pro-inflammatory signaling cascade triggered by this PG [57,58]. Alternatively, some PhytoPs induced a significant increase in PGE 2 in THP-1 cells (ent-16-F 1t -PhytoP and 16-B 1 -PhytoP), which suggests specific, or even reverse, activities of the diverse plant oxylipins on the PGE 2 metabolism, probably affecting the activity of PGE 2 synthase and COX-2 in many ways [81]. Also, this modulatory activity was not precisely equal when considering intracellular levels and the concentration in the growth medium as a result of PGE 2 , which could be due to the time window required for the biological activity of the diverse PhytoPs and PhytoFs, depending on the signaling route or molecular targets [82]. This result would suggest that PhytoPs and PhytoFs, through a multifaceted capacity to modulate of PGE 2 , could be involved in the modification of the blood vessels wall permeability and thus, on the formation of edemas and the infiltration of leukocytes, during the different phases of the inflammatory process [83]. Interestingly the analysis of the additive (intra-and extra-cellular concentrations of PGE 2 ) did not reproduce the significant differences observed when analyzing both fractions separately, which reinforces the idea on the relevance of the kinetics of synthesis of this PG and the effect of the separate PhytoPs and PhytoFs in specific phases of the inflammatory process.
Mammal PGE 2 , through the action of PGE 2 9-keto-reductase, is reduced to obtain PGF 2α , which in turn, can also be synthesized from PGH 2 as a result of a reduction catalyzed by PGH 2 endoperoxidereductase [84] (Fig. 5). Therefore, PGF 2α is involved in creating the inflammatory environment by influencing the synthesis and secretion of IL1β, IL6, IL8, and TNFα [85]. PGF 2α was neither detected in THP-1 cells nor released to the growth medium under control conditions, while after exposure to LPS, it was found in quantifiable concentrations in both cases (Figs. 2 and 3). The decreased concentration of PGF 2α induced by specific PhytoPs and PhytoFs (9-F 1t -PhytoP, ent-9-D 1t -PhytoP, 16-B 1 -PhytoP, 9-L 1 -PhytoP, and ent-9(RS)-12-epi-ST-Δ 10 -13-PhytoF) could be attributed to a decreased synthesis of PGE 2 according to the above referred potential mechanisms of action [81] or even to the inhibition of the enzymatic routes responsible for its formation from PGE 2 or PGH 2 upon their capacity to interact with the complex network of substrates or enzymes characterized by a high sequence homology such as the PGF synthases AKR1C2 and AKR1C3, among others [86]. This trend was even stronger when considering the PGF 2α released into the growth media, where almost all PhytoPs and PhytoFs mitigated the concentration of this PG. This is of particular relevance concerning 9-L 1 -PhytoP, which surprisingly exhibited the most potent capacity to increase the intracellular level of PGF 2α , while regarding the amount of this PG released to the growth medium, it appeared as the most potent compound in decreasing the concentration of PGF 2α in the THP-1 growth medium. This finding reinforces the idea of existing specific time windows for the separate biological action of the plant oxylipins. PGF 2α is, in turn, metabolized to 15-keto-PGF 2α (Fig. 5), which is characterized by an anti-inflammatory activity developed by woundingactivated chemical defense molecules [87]. The upregulation of the intracellular concentration of 15-keto-PGF 2α induced by specific Phy-toPs and PhytoFs (ent-16-epi-16-F 1t -PhytoP, 16-B 1 -PhytoP, and ent-9 (RS)-12-epi-ST-Δ 10 -13-PhytoF), or downregulation (ent-9-D 1t -PhytoP), contrasted with the almost constant increase of 15-keto-PGF 2α released caused by nearly all plant oxylipins, which is in good agreement with the cumulative significance of this result. Hence, this outcome strongly suggests the anti-inflammatory potential of PhytoPs and PhytoFs, according to the biological functions described for 15-keto-PGF 2α [87]. PGF 1α , a PG derived from dihomo-γ-linolenic acid, was found in THP-1 cells lysate, and the growth medium of LPS stimulated monocytes. Although neither individual PhytoPs nor PhytoFs exhibited the capacity to mitigate the increase in the intracellular concentration of PGF 1α , its release into the growth medium was significantly diminished by 9-L 1 -PhytoP, 16-B 1 -PhytoP, ent-16(RS)-13-epi-ST-Δ 14 -9-PhytoF, and ent-16 (RS)-9-epi-ST-Δ 14 -10-PhytoF (Figs. 2 and 3). However, the joint analysis of the intra-and extracellular levels performed to provide information on the total synthesis of this PG as a result of the treatments monitored, did not confirm the capacity of PhytoPs and PhytoFs to modulate its status, which again, could be related to the ability of these compounds to take part in specific phases of the inflammatory process. PGD 2 , which was also detected in THP-1 monocytes, is closely involved in the course and resolution of inflammation. In this regard, PGD 2 is synthesized from PGH 2 by prostaglandin D-synthase and metabolized into several metabolites, including 11β-PGF 2α and PGDM, which maintain the pro-and anti-inflammatory characteristics described above for PGE 2 in the diverse phases of the inflammatory process [88]. The biological interest of PGD 2 regarding inflammation is found in its capacity to activate the PPAR-γ receptor by itself and by its metabolites, thus contributing to the resolution of inflammation [88]. A similar response of PGD 2 and 11β-PGF 2α was observed in both THP-1 cells and growth medium treated with individual PhytoPs and PhytoFs as modulatory agents of the inflammation induced by LPS (Figs. 2 and  3). PGD 2 is a structural isomer of PGE 2 , and this could be responsible for the opposite response of PGD 2 and its metabolites relative to PGE 2 , within the frame of the specific PG profile and metabolism of monocytes [89], as a specific modulatory action of the separate PhytoPs and PhytoFs.

Conclusions
As natural modulators of biological processes, the phytochemical compounds present in plant-based foods could be responsible for their anti-inflammatory properties. However, at present, there is still an open discussion on their competence to improve human health, in this case, regarding inflammation. Thus, concerning the newly-described class of secondary metabolites (PhytoPs and PhytoFs), specific compounds within this family (ent-16-epi-16-F 1t -PhytoP, ent-9-D 1t -PhytoP, 16-B 1 -PhytoP, ent-16(RS)-9-epi-ST-Δ 14 -10-PhytoF, and ent-16(RS)-13-epi-ST-Δ 14 -9-PhytoF) showed, in addition to an absence of cytotoxicity on THP-1 monocytic cells, a valuable anti-inflammatory potential by modulating, in vitro, at 50 μM, the intracellular level of key PGs (15-keto-PGF 2α , PGF 2α , PGE 2 , PGF 1α , PGD 2 , and PGDM in THP-1 cells), mediators of the inflammatory process, as well as their release into the growth medium. Although this concentration could exceed the level obtained in target cells (almost unexplored) this preliminary study will enable the future discovery of the biological potential of the bioaccessible and bioavailable fraction of the target compounds, in respect to specific pathophysiological conditions. However, both intra-and extra-cellular PGs in an additive form restrict the significant capacity to modulate the quantitative PG profile to PGF 2α (ent-16-epi-16-F 1t -PhytoP, ent-9-D 1t -PhytoP, and 16-B 1 -PhytoP), PGD 2 (9-D 1t -PhytoP and ent-16(RS)-9-epi-ST-Δ 14 -10-PhytoF), and 15-epi-15-F 2t (ent-16-epi-16-F 1t -PhytoP, ent-9-D 1t -PhytoP, 16-B 1 -PhytoP, and 9-L 1 -PhytoP), suggesting that the different individual PhytoPs and PhytoFs could act in diverse phases of the inflammatory process, characterized by the diverse quantitative profile of PGs according to their custom synthesis kinetic. Indeed, according to these results, the capacity of PhytoPs and PhytoFs to modulate the synthesis and overall level of PGs in the diverse phases of the inflammatory process needs to be further explored to understand their actual relevance as modulators of this pathological event. Also, as these plant oxylipins are present in a wide variety of plant-based foods, according to current knowledge, the unraveling of their anti-inflammatory potential is of high value in order to shed some light on the relative contribution of the separate bioactive components of these foods, in vivo, that would allow selecting the most beneficial compounds according to their quantitative profile of PhytoPs and PhytoFs. The findings described in the present work, regarding the capacity of individual PhytoPs and PhytoFs to restore the homeostasis of PGs in a pro-inflammatory environment, contribute to overcoming a critical limitation of studies with vegetable extracts that do not allow identifying the specific molecule/s displaying anti-inflammatory activities. On the other hand, the identification of the individual PhytoPs and PhytoFs responsible for specific bioactivities would allow extracting conclusions on the structureactivity relationship for this family of compounds and making suggestions on the possible mechanism of action of the individual PhytoPs and PhytoFs. These outcomes demonstrate the potential anti-inflammatory effects of specific PhytoPs and PhytoFs; however, based on these results, further mechanistic in vitro studies and in vivo experiments are needed to elucidate the operative character of the bioaccessible and bioavailable fractions of these compounds, and the involvement of the diverse cytokines and chemokines that co-participate in the inflammatory response.