2.2. SL-Containing Extracts
The rise of SLs as promising bioactive molecules is a consequence of the use of SL-rich plant extracts in traditional medicine for the treatment of various ailments over the centuries. Reports of the anti-inflammatory potential of natural extracts containing SLs can be found in many studies dedicated to different plant species from the
Asteraceae family, which is characterized by having structurally diverse SLs
[16]. Both in vitro and in vivo evidence indicates that this class of compounds can exert their effect on several inflammatory pathways (
Table 1).
Table 1. Anti-inflammatory effects of SL-containing natural extracts in different in vitro and in vivo experimental models. The affected inflammatory pathways are described, as well as the experimental outcomes observed in each study, and the extract concentration range tested. ↓—decrease; ↑—increase.
A chicory root (
Cichorium intybus L.) extract, rich in dihydrolactucin, lactucin, deoxylactucin, jacquinelin, and dihydrolactucopicrin, dose-dependently downregulated the gene expression of inducible cyclooxygenase (COX-2) (IC
50 = 117 μg/mL), inducible nitric oxide synthase (iNOS) (IC
50 = 39 μg/mL), as well as the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) (IC
50 = 48 μg/mL) and interleukin-1-beta (IL-1β) (IC
50 = 22 μg/mL) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, while no inhibitory effect on the expression of constitutive COX-1 was observed
[17]. In the same study, to elucidate which compounds were responsible for the extract activity, the authors isolated the main SLs from the chicory extract, and the guaianolides 8-deoxylactucin and dihydrolactucopicrin were revealed as the most active compounds
[17]. Similar effects were observed using the same in vitro model (LPS-stimulated macrophages) in the case of an
Artemisia leucodes L. extract enriched in the guaianolides austricin and leukomisin
[18]. These two pure compounds were also tested individually, and it was demonstrated that neither of them alone could explain the decreased gene expression of COX-2 and iNOS observed for the extract. This suggests that the overall effect of the extract probably results from the combined effects of each of the components therein, possibly with synergistic interactions
[18]. In these two studies, SL-containing extracts presented anti-inflammatory effects comparable to those of known drugs. That is the case with oral administration of the chicory extract, which demonstrated a comparable effect to that of indomethacin in a carrageenan-induced rat paw edema model, by reducing the inflammation and paw volume
[17]. The same extract displayed a prolonged effect in a collagen-induced arthritis model, by significantly reducing inflammation until 5 days after the end of the treatment
[17]. In the case of
Artemisia leucodes, oral administration of the extract itself was more effective than aspirin in reducing the swelling in a rat paw edema model, as well as reducing the immune cell infiltrate and granuloma formation in a cotton granuloma test (chronic inflammation challenge)
[18]. This underlines that, in some cases, an extract containing several compounds may produce a more potent anti-inflammatory response than one pure compound.
Similarly, the treatment of LPS-induced J774A.1 macrophages with an SL-rich fraction from
Artemisia khorassanica L. reduced nitric oxide (NO), TNF-α, IL-1β, as well as prostaglandin E
2 (PGE
2), which is one of the main products of the arachidonic acid (AA) cascade resulting from COX activity
[19]. In this case, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity was also reduced, which could justify these effects, since many pro-inflammatory genes including those coding for COX-2, iNOS, and several pro-inflammatory cytokines (such as TNF-α and IL-1β) display a binding site for NF-κB in their promoter region
[18][19]. In another study that focused on the anti-inflammatory bioactivity of
Artemisia extracts in J774A.1 macrophages, the SL composition of several
Artemisia species was evaluated by proton nuclear magnetic resonance (
1H-NMR) experiments
[20]. The authors concluded that
Artemisia species enriched in saturated SLs seemed to be more potent inhibitors of NO and PGE
2 production than those richer in unsaturated SL structures
[20].
A dichloromethane extract from
Eupatorium perfoliatum L. containing SLs was tested in RAW264.7 macrophages, and it was able to reduce both iNOS expression and NO production. The main compounds identified in the extract were isolated and investigated, revealing that the dimeric guaianolide diguaiaperfolin and the flavonoid eupafolin were the main active constituents. The extract also decreased the expression of several cytokines at both the gene and protein levels, namely the IL-1 and TNF families, as well as IL-6, which is generally produced as a response to stimulation by the previous two, and the colony-stimulating factor-3 (CSF-3), responsible for activating granulocytes. The authors suggested that the effect caused by the extract could have been in part due to eupafolin, a non-SL compound identified as one of the main active constituents of the extract
[21].
Xanthium spinosum L. methanolic extract inhibited COX-1 and 12-lipoxygenase (12-LOX) enzymatic pathways in human platelets and increased the synthesis of the anti-inflammatory eicosanoid 15-Hydroxyeicosatetraenoic acid (15(S)-HETE), which is an inhibitor of phospholipase A
2 [22]. The extract also inhibited 5-lipoxygenase (5-LOX) in rat polymorphonuclear leukocytes (PMNLs), and a 5-LOX bioguided fractionation of the extract resulted in the isolation of the known 12,8-guaianolide ziniolide
[22]. A 1 h pre-incubation with either the extract or isolated ziniolide was capable of inhibiting NF-κB signaling after a 7 h inflammatory stimulus, which may contribute to a more lasting anti-inflammatory effect. However, the more immediate inhibitory effects on eicosanoid biosynthesis, observed after a short incubation of only a few minutes, resulted from the direct interaction with the AA pathway enzymes rather than the mediation by NF-κB inhibition
[22].
Extracts prepared from the Arbo and Spanish varieties of
Arnica montana L., rich in helenalin and dihydrohelenalin esters, inhibited IL-1β and TNF-α release in peripheral blood mononuclear cells (PBMCs), as well as the deoxyribonucleic acid (DNA)-binding of the transcription factors NF-κB and the nuclear factor of activated T-cells (NFAT) in Jurkat cells, both of which are responsible for the transcription of pro-inflammatory genes
[23]. The Arbo variety was shown to be more effective than the Spanish one, a result attributed to the fact that the main SLs present in the former were helenalin-derivatives, as opposed to the predominant dihydrohelenalin-derivatives in the latter
[23]. These results underline the importance of the α-methylene-γ-lactone moiety in the bioactive potential of SLs. Besides the core structure of the SL, it was demonstrated in Jurkat T cells that the SL derivatives esterified with unsaturated acids, such as methacrylate or tiglinate, were more active than those esterified with saturated acids, such as acetate, a result that was confirmed in vivo when 11α,13-dihydrohelenalin methacrylate was shown to be more effective than 11α,13-dihydrohelenalin acetate in inhibiting the swelling in a mouse ear edema model
[23]. It is also worth mentioning that preliminary studies carried out by the authors suggested that the mechanism of NFAT inhibition is different from the one described for the known immunosuppressants tacrolimus (FK506) and cyclosporin
[23], which highlights the potential of SLs as alternative anti-inflammatory leads to circumvent the side effects of currently used drugs.
In a study comprising extracts from different
Centaurea species obtained with different solvents (
n-hexane, chloroform, or methanol), chloroform extracts were the most effective in inhibiting NF-κB and iNOS, bioactivities that were attributed to the presence of SLs, which tend to be preferentially extracted by this solvent due to their hydrophobicity
[24]. A chloroform extract from
C. athoa was highlighted as the one with the most promising anti-inflammatory potential. The extract inhibited NF-κB activity in vitro in human chondrosarcoma cells, to the same extent as the pure germacranolide parthenolide, which was considered as the positive control
[24]. Moreover, oral administration of this same extract to rats reduced the swelling in an in vivo paw edema model
[24]. A follow-up study from the same group revealed athoin, 14-
O-acetylathoin, and methyl-14-
O-acetylathoin-12-oate to be the main SLs present in
C. athoa [25].
Gao et al.
[26] suggested the oral use of the
Inula helenium L. extract, mainly composed of alantolactone and isoalantolactone, for the prevention and treatment of rheumatoid arthritis, after the promising results obtained in vitro and in vivo. In particular, the extract inhibited NF-κB and MAPKs activation in bEnd.3 mouse endothelial cells, and decreased the release of the pro-inflammatory mediator IL-1, the monocyte chemoattractant protein (MCP-1), and matrix metalloproteinase (MMP)-3 in primary synovial fibroblasts from patients, as well as IL-6 and iNOS in murine macrophages
[26]. Additionally, in rats, oral administration of the extract improved rheumatoid arthritis symptoms in both the developing and the developed phases of the disease
[26].
Oral administration of a fraction from
Arctium lappa L. enriched in the germacranolide onopordopicrin decreased colitis-associated histological damage in rats and prevented mucin layer loss, which is a common feature of inflammatory bowel diseases (IBD) responsible for a defective barrier function. Additionally, neutrophil infiltration was reduced, as well as the production of TNF-α and COX-2, whereas COX-1 was not affected
[27].
The concentrations upon which SL molecules are pharmacologically active are not well defined, and although SLs are described as poorly bioavailable
[29], the aforementioned in vivo reports show that, in many cases, orally administered SLs are pharmacologically active against inflammation. These conclusions highlight the anti-inflammatory potential of orally administered SLs, thereby reinforcing the importance of studying their pharmacokinetic (ADME—Absorption, Distribution, Metabolism, Excretion) profile of these molecules. On the other hand, a topical application was also shown to be effective in both acute and chronic inflammatory processes when a
Vernonia scorpioides L. extract, containing diacethylpiptocarphol and related hirsutinolides, was used to treat dermatitis and psoriasis in mice
[28]. The extract reduced edema, neutrophil infiltration, and epidermal hyperproliferation, possibly through the inhibition of the chemotactic cytokine IL-8 and NF-κB activity, and its effectiveness was comparable to that of dexamethasone
[28].
Although many natural extracts containing SLs show promising anti-inflammatory potential (Table 1), one must keep in mind that extracts may be complex mixtures containing several compounds from different classes that might interact with each other producing synergistic or antagonistic effects. Therefore, while extracts may be useful as adjuvant therapies, the major potential is provided by individual compounds that might be considered leads for the pharmaceutical industry. For this reason, to validate their anti-inflammatory applicability, SLs must be isolated from their natural sources and their effect must be studied further. In the following section, the relevant studies based on pure SLs are gathered and are divided by SL subclasses.
2.4. Guaianolides
Guaianolides recently isolated from
Ormenis mixta L. and characterized as 2,3-epoxy-1,4,10-trihydroxyguaian-12,6α-olide diastereoisomers, revealed an anti-inflammatory potential through the inhibition of NO release and COX-2 expression in murine macrophages treated with LPS
[49].
Dehydrocostuslactone (
Figure 6) exerts its anti-inflammatory effects through the inhibition of several inflammatory pathways. The treatment of THP-1 cells with dehydrocostuslactone showed its ability to inhibit the activation of the IL-6/STAT3 pathway. Moreover, it suggested that dehydrocostuslactone was able to interact directly with the cellular glutathione content. This interaction created intracellular oxidative stress, leading to the inhibition of STAT3 tyrosine-phosphorylation in IL-6-induced cells, resulting in a downregulation of the expression of genes involved in inflammatory processes, in particular, MCP-1, the C-X-C motif chemokine ligand 10 (CXCL10), and the intracellular adhesion molecule-1 (ICAM-1), with an EC
50 of 10 µM
[40][41]. The ability of dehydrocostuslactone to suppress the tyrosine-phosphorylation of STAT3 suggests that this compound may interfere with the functions of upstream JAK kinases, associated with a portion of the IL-6 receptor. Additionally, dehydrocostuslactone also interfered with IL-22/STAT3 in keratinocytes, resulting in the downregulation of inflammatory genes. The most sensitive genes to the action of this SL were those transcriptionally regulated by STAT3 and whose regulation is driven by the extracellular signal-regulated kinase (ERK) 1
[41].
Figure 6. Structures of addressed guaianolides. 6—Dehydrocostuslactone; 7—Micheliolide; 8—Cynaropicrin; 9—Arglabin; 10—11β,13-dihydrolactucin; 11—8-deoxylactucin.
In a dextran sulfate sodium (DSS)-induced colitis in mice, dehydrocostuslactone (20 mg/kg/day, orally administrated) reduced the quantity of inflammatory cytokines, such as TNF-α, IL-1β, MCP-1, myeloperoxidase (MPO), superoxidase dismutase (SOD), IL-6, IL-17, and IL-23, and once again downregulated the IL-6/STAT3 inflammatory signaling pathway, thus alleviating the colorectal damage caused by DSS
[40][41][50]. The decreased activity of this pathway is related to the further downregulation of other inflammatory mediators, such as iNOS and COX-2
[50].
In a different study, dehydrocostuslactone (20 µM) inhibited both the NF-κB pathway and the interferon regulatory factor 3 (IRF3) in LPS-stimulated murine macrophages RAW 264.7
[51], with both transcription factors being regulated upstream by the activation of the Toll-like receptors myeloid differentiation primary response 88 (MyD88) and Toll-interleukin-1 receptor domain-containing adapter- inducing interferon-β (TRIF)-dependent signaling pathways. By suppressing these receptors, dehydrocostuslactone downregulated NF-κB and IRF3, consequently preventing the expression of their target genes including COX-2, INF-β, and the interferon gamma-induced protein-10 (IP-10). Moreover, the treatment of LPS-challenged macrophages with dehydrocostuslactone leads to the suppression of IκBα degradation, strengthening the NF-κB inhibition
[51].
Micheliolide (
Figure 6), another guaianolide, may pose a therapeutic benefit for the treatment of neurodegenerative disorders via the inhibition of LPS-induced iNOS and COX-2 protein expression in BV2 microglial cells
[52]. The compound (10 µM) also demonstrated the ability to attenuate, at the transcriptional level, the expression of multiple pro-inflammatory mediators, namely, TNF-α, IL-6, IL-1β, COX-2, and iNOS, all the genes of which are regulated by the activation of the NF-κB transcription factor and the Akt pathway
[52]. The authors verified that micheliolide could block the NF-κB p65 subunit nuclear translocation, maintaining the transcription factor inactive in the cytosol
[52][53]. In the same study, micheliolide was shown to exert anti-inflammatory activity by inhibiting the activation of MAPKs, including JNK, p38, and ERK1/2, and phosphatidyl inositol 3-kinase (PI3K)/Akt. Both of these pathways culminate in the activation of NF-κB, which underlines the ability of this compound to inhibit this transcription factor and further downregulate the NF-κB-dependent inflammation players
[52].
In a different study, micheliolide proved its ability to decrease inflammatory cytokine production in murine macrophages RAW 264.7, human dendritic cells, and monocytes. The authors demonstrated that micheliolide inhibited the LPS-induced activation of NF-κB and the PI3K/Akt pathway
[54].
In vivo studies also demonstrated that pre-treatment with micheliolide, diluted in the drinking water of mice, five days before inflammatory stimuli with DSS, was able to reduce neutrophil and lymphocyte infiltration, attenuating the severity of colitis and the inflammatory damage to the colon tissue. The authors further verified that the administration of micheliolide strongly inhibited IL-6, TNF-α, and IL-1β expression in a murine model of DSS-induced colitis
[53].
In an acute peritonitis mouse model, micheliolide (20 mg/kg, intradermal injection) was able to reduce the secretion of IL-6, TNF-α, IL-1β, and MCP-1, resulting in a decreased inflammatory state
[54][55]. In a collagen-induced arthritis mouse model, micheliolide, administered intraperitoneally, reduced paw swelling and suppressed the degeneration of articular cartilage, whilst also decreasing the levels of several inflammatory mediators such as the tissue inhibitor of metalloproteinases (TIMP)-1, macrophage colony-stimulating factor (M-CSF), ICAM-1, and INF-γ, thereby reducing the proliferation, adhesion, and infiltration of leukocytes into the affected area
[55].
The guaianolide cynaropicrin (
Figure 6) possesses anti-inflammatory properties, strongly inhibiting TNF-α release from LPS-stimulated murine RAW 264.7 macrophages, and differentiated human macrophages (U937 cells). Aside from TNF-α inhibition, the compound was also effective in reducing the release of NO from RAW 264.7 macrophages stimulated with LPS and IFN-γ, in a dose-dependent manner
[56]. Cynaropicrin also suppressed the proliferation of CD4
+, CD8
+ T-, and B- lymphocytes
[56].
Arglabin (
Figure 6) was described as able to attenuate the overexpression of inflammatory mediators with a decrease in the mRNA levels of NF-κB-regulated genes, such as COX-2, iNOS, and IL-1β in peritoneal mouse macrophages
[57]. In vivo, arglabin (2.5 ng/g, intraperitoneal injection, twice daily for thirteen days) inhibited the nucleotide-binding oligomeriztion domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activity and significantly reduced the production of the cytokines IL-1α, IL-1β, and IL-18, leading to a reduction in the atherosclerotic lesions in apolipoprotein E (apoE)-deficient mice with an EC
50 of 10 nM
[57].
11β,13-dihydrolactucin (
Figure 6) has been revealed as possessing anti-inflammatory potential by reducing the activity of the calcineurin/calcineurin-responsive zinc finger-1 (Crz1) pathway, which is the yeast orthologue of the human calcineurin/NFAT pathway. Calcineurin is highly conserved between eukaryotes making this an optimal model for screening potential anti-inflammatory compounds
[58]. 11β,13-dihydrolactucin reduced the activation of the pathway with an IC
50 of 2.35 µM. Further analysis demonstrated that the compound inhibited the nuclear translocation of Crz1, which remained inactive in the cytosol, in the presence of inflammatory stimuli
[58].
8-deoxylactucin (
Figure 6) has been described as the most effective SL in a chicory extract. The compound exerts its anti-inflammatory activity by inhibiting COX-2 protein and further downregulating PGE
2 in human colorectal cancer cells HT29
[59].