Cistus x incanus L. is a Mediterranean evergreen shrub used in folk medicine for the treatment of inflammatory disorders but the underlying mechanisms are not fully understood. We therefore investigated the anti-inflammatory effects of an ethyl acetate fraction (EAF) from C. x incanus L. leaves on lipopolysaccharide (LPS) activated RAW 264.7 macrophages. HPLC analysis revealed myricetin and quercetin derivatives to be the major compounds in EAF; EAF up to 1 µM of total phenolic content, was not cytotoxic and inhibited the mRNA expression of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) (p < 0.05) and the production of prostaglandins E2 (PGE2) (p < 0.05). Meanwhile, EAF triggered the mRNA expression of interleukin-10 (IL-10) and elicited the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), as well as the expression of its main target gene, heme oxygenase-1 (HO-1) (p < 0.05). These data indicate that EAF attenuates experimental inflammation via the inhibition of proinflammatory mediators and at least in part, by the activation of Nrf2/HO-1 pathway. These effects are likely due to myricetin and quercetin derivatives but the role of other, less abundant components cannot be excluded. Further studies to confirm the relevance of our findings in animal models and to highlight the relative contribution of each component to the anti-inflammatory activity of EAF should be conducted.
Cistus x incanus L. (Pink Rockrose, syn. C. creticus) is a hybrid between C. albidus and C. crispus, a species of shrubby plant of the Cistaceae family, commonly used in the Mediterranean folk medicine for its anti-inflammatory and skin protective properties , , , .
Recently, different biological activities have been demonstrated for the leaf extracts of this species and have provided scientific evidence to their traditional utilizations. In particular, antioxidant , , , antiviral  and antimicrobial properties  have been described in experimental models.
Currently, C. incanus is considered a medicinal plant and the dried leaves are used as herbal infusions (“Cistus tea”) ,  and dietary supplements . In addition, the herbal extract CYSTUS052® (Dr. Pandalis Urheimische Medizin GmbH und Co. KG, Germany) has given promising results as anti-HIV agent  and in the treatment of infections of the upper respiratory tract . All these commercial products are especially promoted for their high polyphenolic content . Indeed, leaves of C. incanus are rich in proanthocyanidins and flavonols with strong antioxidant activity , , .
In a cell-free system, we reported that the ethyl acetate fraction (EAF) of C. incanus, enriched in flavonols, was the most effective in terms of radical scavenging activity compared to water fractions enriched in proanthocyanidins . However, its potential anti-inflammatory effects have not been yet investigated.
Indeed, besides their antioxidant activity, plant-derived phenols may modulate several important components involved in inflammatory pathways and are considered potential complementary sources of bioactive agents for pharmaceutical purposes .
Numerous single phenolic compounds such as resveratrol, quercetin and myricetin derivatives have shown anti-inflammatory activities , , , . In particular, the ability of polyphenols to reduce inflammation is considered due to their ability to, first, act as antioxidant, second, interfere with oxidative stress signaling and, finally, for their capacity to suppress proinflammatory signaling transductions .
However, several evidences also support the hypothesis that the use of phytocomplexes, such as leaf organic extracts has some advantages over the single active ingredients because of the synergism between the different components and the array of biological activities attributable to each different compound .
Therefore, the present study was designed to analyze the phytochemical composition of the EAF, to evaluate its anti-inflammatory activity and the underlying molecular mechanisms in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, an experimental model of inflammation.
2 Material and methods
2.1 Preparation of EAF and quantification of polyphenols by HPLC-DAD
C. incanus leaves were collected from plants growing on seashore dunes in Southern Tuscany (42° 46°N, 10° 53′E) in July 2017 by CB and AG and identified by FF and Prof. Bussotti (Department of Agriculture, Environment, Food and Forestry [DAGRI]) , University of Florence, Italy.
Harvested plant material was frozen in liquid nitrogen and stored at −80 °C. EAF was obtained following the same extraction procedure reported by Gori et al. (2016) . In this protocol, EAF was obtained from the crude ethanolic extract of C. incanus leaves through a liquid–liquid partitioning with ethyl acetate and water. EAF was then analyzed with a Perkin Elmer Flexar liquid chromatograph equipped with a quaternary 200Q/410 pump and a LC 200 diode array detector (Perkin Elmer Bradford, CT, USA). Quantification of the single phenolic compounds was directly performed by HPLC-DAD. In particular, six individual compounds, i.e. gallic acid, epicatechin, myricetin 3-O-rhamnoside, quercetin 3-O-rhamnoside and rutin, were quantified with their own four-point regression standard curves. Calibration of epicatechin was performed at 280 nm using epicatechin as reference compound. Calibration of myricetin and kaempferol derivatives was performed at 350 nm using myricetin 3-O-rhamnoside and kaempferol 7-O-glucoside as reference compounds, respectively. Analytical separation was performed on a reversed-phase Waters Nova-Pak C18 column (4.9 × 250 mm, 4 µm) (Water Milford, MA, USA), operating at 30 °C. The mobile phase consisted of 1% aqueous formic acid (solvent A) and 1% formic acid in acetonitrile (solvent B). The elution gradient consisted of 2% B isocratic for 10 min, from 2 to 98% B linear for 30 min, 98% B isocratic for 7 min, then starting condition during 5 min to re-equilibrate the column. The flow rate was 0.6 mL/min and the injection volume was 10 µL.
2.2 Cell cultures
RAW 264.7, a murine macrophage cell line obtained from American Type Culture Collection (Rockville, USA), was cultured in Dulbecco’s modified eagle medium (DMEM) (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Carlo Erba reagents, Milan, Italy), 1% L-glutamine (Carlo Erba reagents, Milan, Italy) and 1% Penicillin/Streptomycin (Carlo Erba reagents, Milan, Italy) at 37 °C in an atmosphere containing 5% CO2.
2.3 Cell viability
RAW 264.7 cells were seeded in 96-well plates at a density of 5 × 103 cells/well in 100 µL of medium added of 10% FBS. After 24 h incubation at 37 °C in 5% CO2, EAF (0.125–25 µg/mL) was added to the wells and incubated for 24 h at 37 °C in 5% CO2. Cell viability was assessed by the colorimetric method based on [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] (Promega Corporation, Madison, USA). The optical density of the chromogenic product was measured at 490 nm. Data were expressed as percentage of viable cells compared to untreated cells.
2.4 LPS-induced inflammation in RAW 264.7
RAW 264.7 cells were seeded in a 24-well microplate at a density of 1 × 105 cells/well for 24 h. The cells were then exposed to LPS (1 μg/mL) (Sigma Aldrich, Milan, Italy) alone or in the presence of EAF at concentration ranging from 0.25 to 1 µM of total phenolic content. Indomethacin 10 µM and celecoxib 3 µM (Sigma Aldrich, Milan, Italy), were used as reference anti-inflammatory drugs (positive controls). After 18 h at 37 °C, cell lysates were collected for gene expression analyses and supernatants for nitric oxide (NO) and prostaglandins E2 (PGE2) determination.
2.5 Determination of NO production
NO release was measured with Griess reagent (1% [w/v] sulfanilamide and 0.1% [w/v] N-[1-naphthyl] ethylenediamine hydrochloride in 2.5% [v/v] phosphoric acid) . 100 µL of the cell supernatant was mixed with an equal volume of Griess reagent and incubated at room temperature (RT) for 30 min. The absorbance was measured at 540 nm using a VICTOR 3 Wallac 1421 (Perkin Elmer, Ramsey, USA) and NO production calculated with reference to a standard curve obtained with NaNO2.
2.6 PGE2 determination
PGE2 levels were measured in the supernatants, using an ELISA kit (Cayman, Ann Arbor, USA) according to the manufacturer’s specifications.
2.7 Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from RAW 264.7 using the NucleoSpin® RNA (Macherey-Nagel, Bethlehem, USA). For first-strand cDNA synthesis, 100 ng of total RNA from each sample was reverse-transcribed using the Revert Aid RT Kit (Thermo Scientific, Waltham, USA). Primers were designed on the basis of the mouse GenBank sequences (Supplementary Table 1). For each target gene, the relative amount of mRNA in the samples was calculated as the ratio of each gene to ribosomal protein large P1 (RPLP-1) mRNA .
2.8 Immunocytofluorescence for nuclear factor erythroid 2-related factor 2 (Nrf2) localization
RAW 264.7 cells were grown in poly-D-lysine-coated glass dishes for 24 h, then treated with LPS (1 μg/mL), and EAF 0.5 µg/mL for 18 h. Cells were then fixed with cold 4% (w/v) paraformaldehyde for 20 min, rehydrated in PBS for 15 min and permeabilized in 0.1% (w/v) TritonX-100 at RT for 10 min. After being washed with PBS, the cells were blocked for unspecific fluorescence with 3% bovine serum albumin (BSA) for 1 h and then incubated with Rabbit anti-nuclear factor erythroid 2-related factor 2 (Nrf2) polyclonal antibody C-20 (1:300) (Santa Cruz Biotechnology, Dallas, USA) at 4 °C overnight followed by the fluorescent secondary antibodies AlexaFluor 594 goat anti-rabbit (1:333) (Invitrogen, San Giuliano Milanese, Italy). Cells were also counterstained with 4′,6-diamidin-2-phenylindole (DAPI) to show the nuclear translocation .
2.9 Image acquisition and analysis
Microscopic analysis was performed with an Olympus BX63 microscope equipped with a Metal Halide Lamp (Prior Scientific Instruments Ltd., Cambridge, United Kingdom) and a digital camera Olympus XM 10 (Olympus, Milan, Italy). A total of 10 photomicrographs were randomly taken for each sample (average number of cells/field: 100; average number of analyzed cells in total: 1000) and the number of cells with Nrf2 nuclear translocation was counted using ImageJ 1.33 image analysis software (http://rsb.info.nih.gov/ij).
2.10 Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons and expressed as means ± standard error (SEM) of four independent experiments. All analyses were carried out using GraphPad Prism 7.0 (GraphPad Software, San Diego, USA). A p-value >0.05 was considered significant.
3.1 Qualitative and quantitative characterization of polyphenolic compounds in EAF
The HPLC characterization of EAF, according to our previous investigation , revealed the presence of four classes of polyphenols: gallic acid, catechin derivatives, proanthocianidins and flavonols (myricetin derivatives, quercetin derivatives and kaempferol derivatives) (Table 1). The identification of single compounds was performed comparing the UV–VIS spectra and the retention time with those of authentic standards. Overall, our results showed that EAF was rich in total phenolic content (about 83 mM) and that myricitrin and rutin were the most abundant compounds (Table 1).
|Gallic acid||1.10 ± 0.09|
|(−)-Gallocatechin and (−)-epigallocatechin||2.19 ± 0.26|
|Proanthocyanidin polymers||6.34 ± 0.81|
|Myricetin-3-O-hexoside||0.10 ± 0.01|
|Myricetin 3-O-rhamnoside (Myricitrin)||27.0 ± 1.94|
|Other Myricetin derivatives||1.86 ± 0.19|
|Total myricetin derivates||28.96 ± 0.21|
|Quercetin-3-O-pentoside||3.80 ± 0.23|
|Quercetin 3-O-rhamnoside (Quercitrin)||6.70 ± 0.56|
|Other quercetin derivatives||14.6 ± 1.23|
|Quercetin 3-O-rutinoside (Rutin)||19.0 ± 1.45|
|Total quercetin derivates||44.10 ± 0.30|
|Kaempferol 3-O-rutinoside||0.08 ± 0.01|
|Kaempferol-3-(3′′,6′′-dicoumaroyl)-glucose||0.11 ± 0.01|
Data are mean ± SD of three replicates.
3.2 Effect of EAF on RAW 264.7 cell viability
Exposure of RAW 264.7 to different EAF concentrations ranging from 10 to 50 µM of total phenols for 24 h resulted in a concentration-dependent reduction of cell viability, whereas concentrations from 0.25 to 1 µM were not toxic (data not shown). Based on these results, concentrations of 0.25, 0.5 and 1 µM were chosen as the nontoxic range for further anti-inflammatory analysis.
3.3 Effects of EAF on LPS-induced NO production
LPS treatment resulted in a significant increase in NO production that was concentration dependently reduced by EAF. In particular, EAF at 1 µM of total phenolic content, reduced NO production by about 50% compared with LPS (p < 0.001) and it exerted slight, but significant effects also at 0.5 and 0.25 µM (p < 0.05 and p < 0.01 respectively). Celecoxib, a selective inhibitor of COX-2, markedly reduced NO production by more than 70% (p < 0.001). On the contrary, the effect of indomethacin was lower than that of celecoxib and comparable to that of EAF 0.25 µM (Figure 1).
3.4 Effect of EAF on LPS-induced expression of proinflammatory cytokines and enzymes
As shown in Table 2, LPS, after 18 h of treatment markedly induced the mRNA expression of IL-6 and reduced that of interleukin-10 (IL-10) compared to control cells. These effects were significantly counteracted by EAF 1 µM (p < 0.001) but to a lower extent compared to indomethacin 10 µM and celecoxib 3 µM. Moreover, compared to LPS alone, both the anti-inflammatory drugs, indomethacin and celecoxib, used as positive controls, significantly reduced the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) whereas EAF 1 µM had no effect. EAF at lower concentrations did not exert any significant effect (data not shown).
|Control||LPS 1 µg/mL||Indomethacin 10 µM||Celecoxib 3 µM||EAF 1 µM|
|iNOS||0.16 ± 0.16**||0.85 ± 0.25||0.54 ± 0.01*||0.37 ± 0.04**||0.68 ± 0.22|
|IL-6||0.23 ± 0.02***||0.74 ± 0.03||0.07 ± 0.01***||0.06 ± 0.01***||0.42 ± 0.02***|
|IL-10||0.42 ± 0.03**||0.26 ± 0.01||0.66 ± 0.02***||0.71 ± 0.03***||0.50 ± 0.01***|
|TNFα||0.23 ± 0.04**||0.74 ± 0.05||0.21 ± 0.01***||0.20 ± 0.03***||0.71 ± 0.06|
|IL-1β||0.36 ± 0.01***||0.99 ± 0.05||0.29 ± 0.01***||0.31 ± 0.02***||0.96 ± 0.01|
Data are expressed as mean ± SEM of four replicates; ***p < 0.001; **p < 0.01; *p < 0.05 vs LPS by one-way ANOVA and Dunnett’s multiple comparisons test.
3.5 Effect of EAF on LPS-induced COX-2 expression and PGE2 production
LPS treatment significantly increased the mRNA expression of COX-2 and PGE2 production compared to control cells (p < 0.001); upon treatment with EAF at 1 µM, COX-2 expression and PGE2 production were significantly attenuated (p < 0.001) even if to a lower extent compared to indomethacin and celecoxib (Figure 2, Panel A and B). EAF at lower concentrations (0.5 and 0.25 µM), did not exert any significant effect either on COX-2 expression and PGE2 production.
3.6 Effects of EAF on nuclear translocation of Nrf2 and HO-1 mRNA expression
In cells exposed to LPS, the percentage of cells positive for nuclear Nrf2 staining was significantly reduced compared to control cells (p < 0.01) (Figure 3, panels A, B and D). On the contrary, the treatment with EAF 1 µM restored nuclear Nrf2 expression to levels similar of those of the control cells (p < 0.01) (Figure 3, panel C, and D).
To further explore the effects of Nrf2 nuclear translocation, changes in the expression of its main downstream target gene heme oxygenase-1 (HO-1) were investigated. As shown in Figure 3 panel E, the mRNA expression of HO-1 was significantly reduced in LPS treated cells compared to control cells (p < 0.01); EAF 1 µM restored the mRNA expression of HO-1 at levels even above those of the control cells, and similar to indomethacin and celecoxib.
Recent evidence suggests that the biological activities of flavonoids go beyond their ability to counteract oxidative stress, being able to regulate signal transduction pathways, transcription factors, gene expression and microRNA , .
Our results demonstrate that EAF inhibited the LPS-induced production of PGE2 by suppressing the transcription of COX-2. EAF also reduced the mRNA expression of the proinflammatory cytokine IL-6 and elicited that of the anti-inflammatory cytokine IL-10.
These beneficial effects were accompanied by the activation of the transcription factor Nrf2 that plays an important role in cellular defense against oxidative stress  and inflammation . The activation of Nrf2 by EAF, resulted in the transcriptional upregulation of HO-1, one of its main target genes, also involved in the inhibition of LPS-induced NO production . This is consistent with the positive effect of EAF against LPS-induced production of NO and may explain that, although we did not observe significant effects of EAF on iNOS expression, the transcriptional control of HO-1 may have played a role. EAF also reduced the expression of IL-6 and this effect may be mediated by Nrf2 activation since in in vitro and in vivo models of inflammation, Kobayashi et al.  reported that the transcriptional control of IL-6 is regulated by Nrf2.
It is also important to note that although EAF was less effective in reducing proinflammatory mediators compared to celecoxib and indomethacin, its effects were detected at a lower concentration compared to the reference anti-inflammatory drugs and involved similar pathways beyond the inhibition of COX-2 and PGE2: both celecoxib and indomethacin in fact, were reported to activate NRF2/HO-1 pathway , , .
Plants from Mediterranean-type ecosystems are challenged by environmental stressors such as drought, high solar irradiance and high temperature. These conditions induce the activation of antioxidant defense systems including the biosynthesis of antioxidant phenols. As other plants from Mediterranean area , C. x incanus L leaves contain high concentrations of polyphenols and the EAF, in particular, results enriched in flavonols and exhibit a high antiradical activity .
Our phytochemical characterization demonstrated that myricetin and quercetin derivatives were the main components of EAF and several data support their anti-inflammatory activity; myricitrin, a myricetin glycoside (myricetin-3-O-rhamnoside), (25 μg/mL) inhibited advanced glycation end products (AGE)-induced inflammation and oxidative stress in cardiomyocytes via the regulation of nuclear factor-κB (NF-κB) and Nrf2/HO-1 signaling . Myricitrin (100–400 μg/mL) also inhibited NO production, IL-6 and TNFα but failed to reduce PGE2 in RAW 264.7 . In an in vivo model of cisplatin-induced inflammatory response, myricetin, dose-dependently, attenuated the production of markers of inflammation such as NF-kB, IL-6 and TNF-a, and restored Nrf-2 levels .
The other main component of EAF is rutin, the glycosidic form of quercetin. Rutin 200 µM suppressed palmitic acid-triggered production of ROS and TNFα in RAW 264.7  and at concentrations of 2.5–10 μg/mL, suppressed IL-6 production via down regulation of NF-κB, COX-2 and iNOS in LPS and IFN-γ-stimulated RAW 264.7 . The effects of quercetin and its derivatives on inflammation have been also largely investigated in preclinical models as reviewed in , . For instance, in LPS-stimulated RAW 264.7, quercetin 12.5 µM reduced NO and IL-6 production and inhibited iNOS expression and NF-κB activation ; similarly, quercetin-3-O-(200-gallate)-a-L-rhamnopyranoside (0.2-2 µg/mL), a quercetin derivative, in RAW 264.7 stimulated with peptidoglycan, reduced NO production and COX-2 and iNOS expression .
Since myricetin and quercetin derivatives are the most abundant compounds found in EAF extract, we hypothesized that they are likely responsible for the effects observed in our study; however, it is important to note that although literature data support their anti-inflammatory activity, when myricitrin and rutin were tested as single compounds, the anti-inflammatory effects were observed at much higher concentrations (in the range of 50–400 µM for myricitrin and in the range of 3–16 µM for rutin) compared to those employed in the present study.
We in fact, selected concentrations that did not affect cell viability and that are likely reachable in vivo, due to the low bioavailability of these compounds. In our study, myricetrin and rutin, representing 34.6 and 23.5% of the EAF phenolic components, had in fact a final concentrations of 156 ng/mL (0.336 µM) and 106 ng/mL (0.174 µM), respectively. It is possible that the anti-inflammatory effects of EAF depend on additive effects or synergism between these two main compounds but the contribution of other components of the EAF phytocomplex, cannot be excluded.
Interestingly, the beneficial health effects of C. incanus extracts have been demonstrated in humans in two clinical studies: 7 days treatment with tablets containing C. incanus (CYSTUS052®) extract (approximately 220 mg of polyphenols per day) decreased the symptoms of influenza and the levels of the inflammatory marker C-reactive protein in patients with upper respiratory tract infections .
The supplementation with C. incanus herbal tea enriched in myricetin, quercetin and isoquercetin exceeding (exceeding 800 μg/g dw) for 12 weeks, reduced oxidative stress markers and improved the lipid profile in healthy adults .
In conclusion, we demonstrated that nontoxic concentrations of EAF total phenolic content, reduces experimental inflammation via the inhibition of proinflammatory mediators and, at least in part, by the activation of Nrf2/HO-1 pathway. Although the in vivo bioactivity of EAF and the relative contribution of each component should be evaluated, these results provide the scientific support of its potential anti-inflammatory effects.
Funding source: University of Florence
Funding source: MIUR
Award Identifier / Grant number: FOE-2019
Funding source: CNR
Award Identifier / Grant number: DSB.AD004.271
This research was supported by the University of Florence (Fondi di Ateneo) and by funds from the Italian MIUR to CNR, project NUTR-AGE (FOE-2019, DSB.AD004.271).
Author contributions: CL designed the experiments; CL and EB analyzed and interpreted the data. MD and LC performed the experiments. AG, CB and FF prepared and characterized the extract. All authors read and approved the final manuscript.
Research funding: This study was supported by the University of Florence and funds from the Italian MIUR to CNR project NUTR-AGE (FOE-2019, DSB.AD004.271)
Conflict of interest: The authors show no conflict of interests.
Supplementary Table 1.
|Gene||Primer forward||Primer reverse||Base pair|
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