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Ellagitannins modulate the inflammatory response of human neutrophils ex vivo.


Ellagitannin-rich plant materials are commonly used in the traditional medicine as anti-inflammatory, antioxidant and antimicrobial agents (Evans 2009; Lipinska et al. 2014; Piwowarski et al. 2014b). Most of current applications of tannin-rich plant drugs are associated with external use. Infusions containing high concentration of ellagitannins prepared from oak bark (Quercus spp.), wood avens rhizome (Geum urbanum L), common agrimony herb (Agrimonia eupatoria L.) or tormentil rhizome (Potentilla erecta (L.) Raeusch.) are often used topically in skin diseases or as therapeutic rinses and mouthwashes in order to threat bacterial infection and reduce the inflammation (Kumar et al. 2015; Menkovic et al. 2011; Tomczyk and Latte 2009). The tannin-rich extracts like Punica granatum pericarp extract or Quercus alba bark extract are also used as active ingredients in herbal toothpastes used in everyday hygiene of oral cavity. Tannic acid which consists of hydrolysable tannins, mainly pentagalloylglucose (penta-O-galloyl-[beta]-D-glucose -1) and its derivatives, was used as a major ingredient of toothpaste used in the treatment of teeth and to prevent tooth decay (Hirota et al. 1986). Herbal mixtures containing oak bark and/or tormentil rhizome are often used in gums inflammations (Salam et al. 2015; Tomczyk and Latte 2009). The observed effect of ellagitannin-rich plant extract is often connected with non-specific astringent, antimicrobial or hemostatic properties of this group of compounds (Evans 2009). The contribution of pure ellagitannins to the anti-inflammatory activity of plant extracts is still not resolved.

Neutrophils, which belong to the polymorphonuclear cell family (PMNs) are the most abundant group of white cells in human blood. They are a crucial part of the innate immune system. Neutrophils are mobilized from bone marrow and migrate to the place of infection where they are responsible for inflammatory response of the host. Neutrophils can be activated by exogenous factors like components of bacterial wall (lipopolysaccharide (LPS)) or endogenous factor like interleukin 8 (IL-8). Activated neutrophils generate huge amounts of reactive oxygen species (ROS) including superoxide ion ([O.sub.2.sup.-]), hydrogen peroxide and hypochlorous acid (HCIO). They also secrete several cytokines (TNF-[alpha], IL-8, IL-1[beta]) and extracellular matrix degrading enzymes such as elastase and matrix metalloproteinases 2 and--9 (MMP-2 and 9). Those factors are mainly responsible for neutrophils migration and microbial destruction (Cascao et al. 2009; Mocsai 2013; Tintinger et al. 2013; Witko-Sarsat et al. 2000).

Dental caries, periodontal and gingival tissues diseases are the main dental pathologies affecting humankind. These conditions are caused by plaque formed by Gram-negative bacteria (Rosas-Pinon et al. 2012). It has been shown that the mucosal efflux of neutrophils plays an essential role in the generation and progression of inflammatory response of the host against bacteria (Galgut et al. 2001). On the other hand the chronic over-activation of neutrophils results in host tissue damage and is believed to be responsible for the development of destructive phases of periodontal and gingival diseases (Galgut et al. 2001). The hyperactivated neutrophils are responsible for the host tissue damage and instead of host defense create condition favorable for pathogenic bacteria to grow and invade (Nussbaum and Shapira 2011; Scott and Krauss 2012).

Previous research on the bioactivity of ellgitannins and ellagitannin-rich extracts revealed that the observed anti-inflammatory effect might be explained by the specific inhibition of some pro-inflammatory enzymes or inhibition of molecular signaling path responsible for the initiation and progression of inflammation rather than through non-specific interactions. Some monomeric and dimeric ellagitannins were proven to inhibit hyaluronidase and metalloproteinase-2/-9 activity (Lee et al. 1993; Tanimura et al. 2005). Oenothein B isolated from Epilobium angustifolium, which is a dimeric macrocydic ellagitannins was proven to inhibit hyaluronidase and elastase activity as well as was being able to inhibit the production or secretion of some pro-inflammatory cytokines in in vitro studies (Kiss et al. 2011; Schmid et al. 2012). Similar properties were proven for monomeric and dimeric ellagitannins purified from Lythrum salicaria (Piwowarski and Kiss 2015). In contrast Hrenn et al. (2006) showed that dimeric ellagitannin agrimoniin (which is a dominating polyphenol in Agrimoniae herba and Tormentillae rhizoma) and monomeric pedunculagin (present in Geum urbanum, Agrimoniae herba and Tormentillae rhizoma) caused the direct inhibition of neutrophil elastase but did not influence the release of this proteolitic enzyme from stimulated cells. Other studies indicated that coriariin A, agrimoniin but not their precursor PGG were able modulate the inflammatory response of PBMC through the elevation of TNF-[alpha] levels (Feldman et al. 1999). Immunostimulatory effects were also observed by Kolodziej et al. (2001) for monomeric, dimeric and trimeric ellagitannins using RAW 264.7 macrophages cell line.

The aim of the present study the study was to establish if ellagitannins and their precursor--pentagalloylglucose (1) can modulate the inflammatory response of ex-vivo stimulated neutrophils. Basing on the results obtained for ellagitannins with different molecular structures the contribution of this class of polyphenols to the anti-inflammatory activity of plant extracts used in the prevention and treatment of oral cavity diseases is discussed.

Materials and methods


Camptothecin (98% purity), luminol, f-MLP (formyl-met-leuphenylalanine), SAAVNA (N-succinyl-alanine-alanine-valinine-pnitroanilide), cytochalasin B, TMB (3,3',5, 5'-tetramethylbenzidine) liquid substrate system, Hanks' balanced salt solution (HSSB), L-glutamine, fetal bovine serum (FBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and RPMI1640 medium were purchased from Sigma-Aldrich GmbH (Steinheim, Germany). LPS (lipopolysacharide) was purchased from Merck Millipore (Billerica, MA, USA). Curcumin (C) and quercetin (Q) (>95% purity) were purchased from Carl Roth (Karlsruhe, Germany). Propidium iodide was purchased from BD Biosciences (San Diego, CA, USA). All substances used were of > 95% purity. Phosphate-buffered saline (PBS) was purchased from Gibco (Carlsbad, CA, USA).

Ellagitannins and PGG

Compounds used in the present study were isolated previously from chosen plant materials. Penta-O-galloyl-/6-D-glucose (1) was isolated from defatted seed of Oenothera paradoxa Hudziok (Kiss et al. 2008). 1-O-galloyl-4,6-(S)-HHDP-[beta]-D-glucose (2), pedunculagin (3), stachyurin (4), casuarinin (5), stenophyllanin A (8), gemin G (10) and gemin A (11) were isolated from root of Geum urbanum L. (Piwowarski et al. 2014a). Vescalagin (6), castalagin (7), salicarinin A (13) and salicarinin B (14) were purified from aerial parts of Lythrum salicaria L. (Piwowarski and Kiss 2013). Oenothein B (9) was isolated from aerial parts of Oenothera hoelsheri Renner ex Rostanski (Granica and Kiss 2012). Agrimoniin (12) was obtained from aerial parts of Agrimonia eupatoria L. (Granica et al. 2013). All compounds used were of > 95% HPLC-DAD-MS purity. Before each experiment compounds were dissolved in deionized water or in 70% ethanol (v/v) in the case of PGG and then diluted to obtain stock solutions of 400 [micro]M and filtrated through 0.22 [micro]m sterile syringe filter (Carl Roth). Compounds were stored in the darkness at 4[degrees]C and were used for no longer than 72 h. All compounds were stable in experimental conditions.

Isolation of human neutrophils

The buffy coat was prepared from peripheral venous blood collected from healthy human donors (20-35 years old) at the Warsaw Blood Donation Center. All Donors declared that they were nonsmokers and did not take any medications. They were confirmed to be healthy and all tests carried out showed values within a normal range. Neutrophils were isolated using a standard method by dextran sedimentation and centrifugation in a Ficoll Hypaque gradient (Boyum 1968). The purity of neutrophils preparation was over 97%. After isolation, cells were suspended in HBSS, PBS or culture medium and maintained at 4[degrees]C before use.

Evaluation of ROS production by human neutrophils

The ROS production by f-MLP-stimulated neutrophils was determined using luminol-dependent chemiluminescence. The concentrations of compounds used in the experiment were 1, 5 and 20 [micro]M. Following isolation, cells were suspended in HBSS. Cell suspension (3.0 x [10.sup.5]) was incubated with 50 [micro]l of the samples with tested compounds in the proper concentration and luminol in a 96-well plate. ROS production was initiated by the addition of f-MLP (0.1 [micro]g/ml) to obtain a total volume of 200 [micro]l/well. Changes in chemiluminescence were measured over a 40 min period at intervals of 2 min in a microplate reader (BioTek). The values obtained in the point corresponding to the maximum of chemiluminescence were taken for calculations. Background chemiluminescence produced by non-stimulated cells was also determined. As a positive control, quercetin was used at concentration of 10 [micro]M. The percentage of ROS production was calculated in comparison to the control without investigated compounds, taking into account chemiluminescence emission inhibited by tested compounds.

Evaluation of elastase release by human neutrophils

Neutrophil elastase release was determined using SAAVNA as a substrate, and p-nitrophenol was measured spectrophotometrically according to modified assay of Liou et al. (2013). 100 [micro]l of cell suspension (2 x [10.sup.6]) was preincubated with 25 [micro]l of each compound at final concentrations of 1,5 and 20 [micro]M for 15 min at 37[degrees]C and then stimulated with cytochalasin B (0.6 [micro]g/ml) and f-MLP (1 [micro]g/ml) for 15 min. The neutrophils were centrifuged (2000 rpm; 10 min; 4[degrees]C). After the addition of 50 [micro]l of SAAVNA solution (1.6 mg/ml) to 100 [micro]l of supernatant, the extent of p-nitrophenol was measured spectrophotometrically for 1 h at intervals of 20 min, at 412 nm using a microplate reader (BioTek). The effect on elastase release was calculated as the percentage of released enzyme in comparison with stimulated control without tested extract. Quercetin at the concentration of 20 [micro]M was used as a positive control according to Kanashiro et al. (2007).

Evaluation of IL-8, TNF-[alpha] and MMP-9 production

Neutrophils (2 x [10.sup.5]/ml) were cultured in 24-well plates in RPMI 1640 medium with 10% FBS, 10 mM HEPES, and 2 mM L-glutamine in the absence or presence of LPS (100 ng/ml) for 24 h at 37[degrees]C with 5% C[O.sub.2] in the absence or presence of test compounds at final concentrations of 1, 5 and 20 [micro]M added to 1 ml of cell suspension 1 h before stimulation. After 24 h the neutrophils were collected and centrifuged (2000 rpm; 10 min; 4[degrees]C). The amount of released cytokines into cell supernatants was measured by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA or R&D Systems, Minneapolis, MN, USA). Quercetin (50 [micro]M) was used as a positive control for IL-8 and TNF-[alpha] release experiments and curcumin at the concentration of 20 [micro]M was used in MMP-9 release assay.

Expression of TLR-4 receptor

Influence of tested compounds at a concentration of 20 [micro]M on the surface expression TLR-4 receptor on neutrophils was determined using flow cytometry. After 24 h incubation with compounds and LPS, the neutrophils were harvested and centrifuged (2000 rpm; 10 min; 4[degrees]C). After removing supernatants, cells were re-suspended in 100 [micro]l PBS buffer. Neutrophils were marked with monoclonal antibodies against TLR-4-(FITC)-conjugate (R&D Systems, Minneapolis, MN, USA) and incubated for 30 min at 4[degrees]C in the dark. Cells were analyzed by flow cytometry using FACSCalibur and data from 10000 events were recorded. Quercetin (50 [micro]M) was used as a positive control.

Evaluation of neutrophils' viability and apoptosis

Neutrophils' viability and apoptosis was determined by staining with propidium iodide (PI) and Annexin V-FITC using Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) following the manufacturer's instructions. Neutrophils (2 x [10.sup.6]/ml) were cultured in a 24-well plate in RPMI 1640 medium (as above) in the absence or presence investigated compounds at a final concentration 20 [micro]M added 1 h before the stimulation with LPS (100 ng/ml). Cells were collected, centrifuged (2000 rpm; 10 min; 4[degrees]C) and resuspended in 100 [micro]l of binding buffer. Next 5 [micro]l of Annexin and 5 [micro]l 1 of propidium iodide solutions were added. The mixture was vortexed and incubated for 15 min at room temperature in the darkness. Then, 400 [micro]l of cold binding buffer was added and the cells were analyzed by flow cytometry (FACSCalibur) within 1 h after labeling and data from 10,000 events were recorded. Before each experiment compensation (using untreated cells, cells with Annexin V and cells with iodide propidium) was performed and quadrants have been set. Quercetin at a concentration of 50 [micro]M was used as a positive control.

Statistical analysis

The results were expressed as a mean [+ or -] SEM of the indicated number of experiments. Statistical significance of differences between the mean values was calculated using one-way analysis of variance (ANOVA) with Tukey's post-hoc test or Dunnet's post-hoc test. All analyses were performed using STATISTICA software v. 10.0 PL (Stat-Soft, Poland). Statistical significance was set at * P < 0.05.


Effect on ROS production

All investigated ellagitannins decreased the production of ROS from f-MLP/cytochalazin B stimulated neutrophils (Fig. 1). Compounds were active at all three investigated concentrations. The observed effect was concentration-dependent. The decrease of ROS production at the concentration of 5 [micro]M was comparable for all tested compounds (production down to 21.7 [+ or -] 0.4 for 1 to 41.6 [+ or -] 1.7% for 3) to the positive control--quercetin at the concentration of 10 [micro]M (production 28.3 [+ or -] 4.3%). At the concentration of 20 [micro]M acted significantly stronger (ROS production reduced to 2.7 [+ or -] 0.3% for 10 to 6.4 [+ or -] 0.3% for 9).

Effect on elastase release

In the elastase release assay half of investigated compounds displayed no activity (Fig. 2). Only 1 and dimeric ellagitannins except from 9 were able to inhibit the release of elastase from human neutrophils. Penta-O-galloyl-[beta]-D-glucose (1) was active at concentrations of 5 and 20 [micro]M and the observed effect was moderate (elastase release down to 75.7 [+ or -] 3.8% and 65.7 [+ or -] 6.3%, respectively) and significantly weaker than observed for the positive control--quercetin (50 [micro]M, production decreased to 35.2 [+ or -] 6.6%). Stenophyllanin A (8), which is a complex dimeric tannin, agrimoniin (12) and gemin A (11) were active only at the concentration of 20 [micro]M and the inhibition of elastase release was comparable to the positive control (production down to 47.9 [+ or -] 5.4%, 53.5 [+ or -] 7.0% and 39.6 [+ or -] 6.8%, respectively). Three compounds 10,13 and 14 displayed inhibitory activity at all tested concentration in the concentration-dependent manner. The activity of 13 and 14 was weaker (1 [micro]M and 5 [micro]M) or comparable (20 [micro]M) to the positive control. Gemin G (10) at the concentration of 20 [micro]M was the only compound, which inhibited the elastase release stronger (release down to 21.9 [+ or -] 4.3%) than quercetin.

Effect on 1L-8 production

Investigated compounds had a diverse effect on the production of IL-8 by LPS stimulated cells (Fig. 3). Casuarinin (5) was the only compound active at all tasted concentrations and the observed effect was concentration-dependent (production down to 44.9 [+ or -] 4.9-66.0 [+ or -] 8.0%). Other monomeric ellagitannins such as 3, 4, 7 and 13 were also active but only at concentrations of 5 and 20 [micro]M and the observed effect was weaker (production decreased to 45.2 [+ or -] 6.0 75.0 [+ or -] 10.0%) than in the case of positive control quercetin (production down to 23.8 [+ or -] 2.7%). Ellagitanins precursors 1 and 2 were also active at 5 and 20 [micro]M. Penta-O-galloyl-[beta]-D-glucose (1) at 20 [micro]M was the most potent compound and decreased the production of IL-8 down to 18.7 [+ or -] 4.2%, thus its activity was stronger than observed for quercetin. In the case of dimeric tannins 6 was active at 5 and 20 [micro]M (production 49.4 [+ or -] 0.4% and 41.4 [+ or -] 0.2%, respectively). Oenothein B (9), gemin G (10), gemin A (11) and agrimoniin (12) were able to decrease the production of IL-8 only at the highest used concentration (20 [micro]M). Weak inhibitory effect was also observed for 13 and 14 used at concentrations of 5 and 20 [micro]M (production down to 59.2 [+ or -] 7.4 79.8 [+ or -] 4.9%).

Effect on MMP-9 production

Most of tested compounds did not influence the MMP-9 production by neutrophils (Fig. 4). Only three dimeric ellagitannins--11, 12 and 14 at the concentration of 20 [micro]M were able to decrease the production level of MMP-9 (production decreased to 68.7 [+ or -] 3.8, 79.8 [+ or -] 3.3%, and 82.6 [+ or -] 1.4% respectively). In the case of 12 and 14 effect was weaker than positive control curcumin at 20 [micro]M, which caused the production decrease down to 63.8 [+ or -] 4.7%. Gemin A (11) at 20 [micro]M displayed the effect similar to curcumin.

Effect on TNF-[alpha] production.

Penta-O-galloyl-[beta]-D-glucose (1) was the only compound that was able to decrease the production of TNF-[alpha] by LPS stimulated neutrophils at concentration of 5 and 20 [micro]M (production down to 57.5 [+ or -] 7.3% and 48.8 [+ or -] 5.2%, respectively) (Fig. 5). None of tested compound was active at the lowest concentration (1 [micro]M). Some of monomeric ellagitannins including 4, 5, 7 and 8 at the concentration of 20 [micro]M caused significant induction of TNF-[alpha] production by neutrophils (Fig. 5). The observed increase was found to be statistically significant (P < 0.05). The effect was from 171.1 [+ or -] 5.2% for 6 to 252.4 [+ or -] 16.0% for 7. The overproduction of TNF-[alpha] by cells was even stronger for dimeric tannins. In the case of 13 and 14 the increase in TNF-[alpha] production was observed only at the concentration of 20 [micro]M (190.0 [+ or -] 15.2% and 181.2 [+ or -] 7.4%, respectively). Other compounds including 8,9,10,11 and 12 induced the TNF-[alpha] production at concentrations of 5 and 20 [micro]M (Fig. 5). The strongest effect was observed for 9 at 5 and 20 [micro]M and 10 at 20 [micro]M (production increased to 305.3 [+ or -] 4.8%, 316.0 [+ or -] 7.5% and 309.8 [+ or -] 27.5%, respectively). Quercetin at the concentration of 50 [micro]M, which was used as a positive control, decreased the production of TNF-[alpha] down to 77.4 [+ or -] 2.7%.

Effect on the surface expression of TLR-4 receptor

The surface expression of TLR-4 receptor on human neutrophils was evaluated by flow cytometery method. All tannins were tested at the concentration of 20 [micro]M. The decrease in surface expression ofTLR-4 was observed for all monomeric tannins and two dimers (8 and 11). The decrease was statistically significant (P < 0.05). The effect was similar for all active compounds and varied from 76.6 [+ or -] 3.3% for 2 to 89.3 [+ or -] 1.6% for 10 (Fig. 6). On the other hand 9 displayed statistically significant induction of surface expression of investigated receptor (expression up to 111.0 [+ or -] 1.2% compared to stimulated control). The rest of ellagitannins remained inactive in the tested model. Positive control--quercetin at the concentration of 50 [micro]M decreased the TLR-4 level down to 68.1 [+ or -] 3.4%.

Influence on neutrophils' viability and apoptosis

The viability and apoptosis of neutrophils after 24 h incubation with compounds at the concentration of 20 [micro]M was examined using PI and Annexin V staining using flow cytometry. The results show that tested compounds were not cytotoxic and did not cause significant increase of neutrophils necrosis (Fig. 7). The percentage of necrotic cells detected in the assay varied from 0.9 [+ or -] 0.7% for 6 to 3.8 [+ or -] 0.8% 12 compared to 1.8 [+ or -] 1.0% for ST and 1.0 [+ or -] 1.3% for NST control. It was observed that after incubation with monomeric ellagitannins, 1 and 2 the percentage of cells in early apoptosis increased compared to ST control (Fig. 7). The highest percentage of early apoptotic cells was detected after the treatment with 6 (50.6 [+ or -] 4.0%) and the lowest level was confirmed for 5 (41.5 [+ or -] 11.6%). However the effect was weaker than for the positive control--querectin (50 [micro]M, 62.0 [+ or -] 11.7%). In contrast to monomeric tannins compounds with dimeric structures caused a significant increase in the number of late apoptotic neutrophils (Fig. 7). The strongest effect was observed for 12 (49.7 [+ or -] 10.0%) and lowest value of late apoptotic cells after incubation with dimeric tannins was confirmed in the case of complex dimer 8 (21.7 [+ or -] 8.3%).


Human neutrophils, when activated by exogenous factors like bacterial LPS are responsible for the generation of first line host-dependent defence against pathogens. They induce and sustain inflammatory response through the production and release of pro-inflammatory enzymes and cytokines. It was shown that human neutrophils are engaged in the early pathogenesis of oral cavity infections. They play a crucial role in the destruction of bacterial biofilm created on teeth and gums (Vitkov et al. 2009). In the case of prolonged bacterial infections, LPS-overactivated neutrophils are responsible for the chronic inflammatory response, which often leads to the destruction of healthy tissues of the host (Kinane 2000). It is known that plant polyphenols including ellagitannins may modulate the response of human immune system (Jancinova et al. 2012; Kolodziej et al. 2001; Veres 2012). Tannins are dominating polyphenols in plant remedies like wood avens, oak bark, tormentil rhizome or tannic acid popularly used in the prevention and in the treatment of oral cavity inflammations (Konig et al. 1994; Kumar et al. 2015; Piwowarski et al. 2014a; Tomczyk and Latte 2009). In the present study for the first time we studied the influence of chosen ellagitannins and their precursor (penta-O-galloyl-/J-D-glucose) on the inflammatory response of human neutrophils in the context of oral cavity diseases. The results showed that all compounds were able to decrease the ROS levels in cultures of stimulated neutrophils. However due to known antioxidant properties of polyphenols, the direct scavenging activities are potent to be responsible for observed effects, as well as can interfere with applied chemiluminescence-based detection method. No significant differences or trends in the potency of tested compounds were observed that could be connected with their chemical structure. Dimeric compounds and ellagitannin precursor 1 were exclusively able to inhibit the release of elastase. None of monomeric compounds decrease the level of this enzyme. Interestingly similar but less spectacular result was obtained for the second proteolytic enzyme (MMP9) investigated in the present study. The production of MMP-9 was inhibited after the treatment of cells with some dimeric ellagitannins (11, 12 and 14). Gemin A (11) (the most abundant polyphenol occurring in extracts from Geum urbanum rhizome) was the most potent inhibitor of MMP-9 displaying similar activity as positive control. IL-8 of the most important pro-inflammatory cytokines produced by activated neutrophils. The present study revealed that monomeric tannins displayed stronger inhibitory effect than compounds possessing dimeric structure. The strong inhibitory effect was observed for pedunculagin (3), casuarinin (4), vescalagin (6) and castalagin (7), which can be found in high quantities in plant remedies prepared from oak bark (Nonaka et al. 1985). The most potent IL-8 inhibitor was penta-O-galloyl-^-D-glucose (1) that is the major compound of tannic acid used for the production of some herbal toothpastes. Penta-O-galloyl-[beta]-D-glucose (1) was the only compound able to inhibit the production of TNF-[alpha]. In contrast ellagitannins especially dimeric compounds significantly induced the production of this cytokine. The observed effect stays with the agreement with previous studies indicating the immunostimulatory activity of ellagitannins (Kolodziej et al. 2001). TNF-[alpha] if present in high concentration, is any important factor regulating the number of viable and activated neutrophils through the induction of their apoptosis (Luo and Loison 2008). The flow cytometry analysis of the influence of tested compounds on neutrophil apoptosis showed that the increase in TNF-[alpha] levels correlates with the increased number of apoptotic cells. The observed effect should be considered as beneficial especially in the treatment of chronic inflammation states within oral cavity, when the number of activated neutrophils is elevated and the regulation of apoptosis might be impaired. The induction of neutrophils apoptosis may protect the host tissues from the destruction caused by proteolytic enzymes and cytokines produced by over-activated cells. Toll like receptor 4 (TLR-4) is considered as the major molecular target for bacterial LPS. The activation of TLR signaling plays a crucial role in the innate immune system response. However the over-production of pro-inflammatory factors due to chronic stimulation of TLRs may lead to the tissue destruction of the host. The analysis of the surface expression of TLR-4 receptor showed 1, tested monomeric ellagitannins and 10 were able to significantly decrease the number of TLR4 receptor units on the surface of activated neutrophils. This effect might be of great benefit because of decreased neutrophil sensitivity to pathogenic factors in chronic inflammation. Basing on the obtained results it can be hypothesized that ellagitannins are potent to modulate molecular pathways associated with TLR-4 signaling, but drawing a precise conclusion requires conducting more intrinsic studies.

In conclusion the present study proved that penta-O-galloyl-[beta]-D-glucose and ellagitaninns are able to modulate the inflammatory response of human neutrophils. Some differences in the activity between monomeric and dimeric compounds were observed and described. The results show that ellagotannins should be considered as compounds responsible for the beneficial effect of ellagitannin-rich remedies. The immunomodulatory activity of tested compounds in human neutrophils model justifies the usage of tannin-rich plant material as effective agents in the treatment of oral cavity inflammations. Despite observed differences between activities of tested compounds, obtained results do not provide full explanation, which particular structural features of ellagitannins determine their bioactivity as well as do not explain what would be the outcome if their complex mixture (such as in plant extracts) is used.


Article history:

Received 18 August 2015

Revised 28 September 2015

Accepted 1 October 2015

Conflict of interest

The authors confirm that there are no conflicts of interests associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.


The project was financially supported by Polish National Science Center research Grant PRELUDIUM-DEC-2012/07/N/NZ7/01136.


Boyum, A., 1968. A one-stage procedure for isolation of granulocytes and lymphocytes from human blood. General sedimentation properties of white blood cells in a 1 g gravity field. Scand. J. Clin. Lab. Investig. 97, 51-76.

Cascao, R., Rosario, H.S., Fonseca, J.E., 2009. Neutrophils: warriors and commanders in immune mediated inflammatory diseases. Acta Reumatol. Port. 34, 313-326.

Evans, W.C., 2009. Trease and Evans Pharmacognosy. Elsevier, New York.

Feldman, K.S., Sahasrabudhe. K., Smith, R.S., Scheuchenzuber, W.J., 1999. Immunostimulation by plant polyphenols: A relationship between tumor necrosis factor-alpha production and tannin structure. Bioorg. Med. Chem. Lett. 9, 985-990.

Galgut. P., Galgut, N., Dowsett, S.A., Kowolik, M.J., 2001. Periodontics: Current Concepts Ant Treatment Strategies. Martin Dunitz Ltd., London.

Granica, S., Kiss, A.K., 2012. Secondary metabolites from aerial parts of Oenothera hoelscheri Renner ex Rostanski. Biochem. Syst. Ecol. 44, 44-47.

Granica, S., Krupa, K., Klebowska, A., Kiss, A.K., 2013. Development and validation of HPLC-DAD-CAD-MS3 method for qualitative and quantitative standardization of polyphenols in Agrimoniae eupatoriae herba (Ph. Eur). J. Pharm. Biomed. Anal. 86, 112-122.

Hirota, K., Akahane, S., Tomioka, K., 1986. Compositions Applied in the Mouth. Google Patents.

Hrenn, A., Steinbrecher, T., Labahn, A., Schwager, J., Schempp, C.M., Merfort, L, 2006. Plant phenolics inhibit neutrophil eiastase. Planta Med. 72, 1127-1131.

Jancinova, V., Perecko, T., Harmatha, J., Nosal, R., Drabikova, K., 2012. Decreased activity and accelerated apoptosis of neutrophils in the presence of natural polyphenols. Interdiscip. Toxicol. 5, 59-64.

Kanashiro, A., Souza, J.G., Kabeya, L.M., Azzolini, A.E.C.S., Lucisano-Valim, Y.M., 2007. Eiastase release by stimulated neutrophils inhibited by flavonoids: Importance of the catechol group. Z. Naturforsch. C 62, 357-361.

Kinane, D.F., 2000. Regulators of tissue destruction and homeostasis as diagnostic aids in periodontology. Periodontol 2000 24, 215-225.

Kiss, A.K., Bazylko, A., Filipek, A., Granica, S., Jaszewska, E., Kiarszys, U., Kosmider, A., Piwowarski, J., 2011. Oenothein B's contribution to the anti-inflammatory and antioxidant activity of Epilobium sp. Phytomedicine 18, 557-560.

Kiss, A.K., Derwinska, M., Dawidowska, A., Naruszewicz, M., 2008. Novel biological properties of Oenothera paradoxa defatted seed extracts: Effects on metallopeptidase activity.J. Agric. Food Chem. 56, 7845-7852.

Kolodziej, H., Kayser, O., Kiderlen, A.F., Ito, H., Hatano, T., Yoshida, T., Foo, L.Y., 2001. Antileishmanial activity of hydrolyzable tannins and their modulatory effects on nitric oxide and tumour necrosis factor-alpha release in macrophages in vitro. Planta Med. 67, 825-832.

KSnig, M., Scholz, E., Flartmann, R., Lehmann, W., Rimpler, H., 1994. Ellagitannins and complex tannins from Quercus petraea Bark. J. Nat. Prod. 57, 1411-1415.

Kumar, K., Sharma, Y.P., Manhas, R.K., Bhatia, FL, 2015. Ethnomedicinal plants of Shankaracharya Hill, Srinagar, J & K, India. J. Ethnopharm. 170, 255-274.

Lee, J., Lee, S.H., Min, K.R., Lee, K.S., Ro, J.S., Ryu, J.C., Kim, Y., 1993. Inhibitory effects of hydrolyzable tannins on Ca2+-activated hyaluronidase [2], Planta Med. 59, 381-382.

Liou, J.R., El-Shazly, M., Du, Y.C., Tseng, C.N., Hwang, T.L., Chuang, Y.L.. Hsu, Y.M.. Hsieh, P.W., Wu, C.C., Chen, S.L., Hou, M.F., Chang, F.R., Wu, Y.C., 2013, 1,5-Diphenylpent-3-en-1-ynes and methyl naphthalene carboxylates from Lawsonia inermis and their anti-inflammatory activity. Phytochemistry 88, 67-73.

Lipinska, L., Klewicka, E., Sdjka, M., 2014. Structure, occurrence and biological activity of ellagitannins: a general review. A. Acta Sci. Pol., Technol. Aliment. 13, 289-299.

Luo, H.R., Loison, F., 2008. Constitutive neutrophil apoptosis: mechanisms and regulation. Am. J. Hematol. 83, 288-295.

Menkovit, N., Savikin, K., Tasic, S., Zdunic, G., SteSevic, D., Milosavljevit, S., Vincek, D., 2011. Ethnobotanical study on traditional uses of wild medicinal plants in Prokletije Mountains (Montenegro). J. Ethnopharm. 133, 97-107.

Mocsai, A., 2013. Diverse novel functions of neutrophils in immunity, inflammation, and beyond. J. Exp. Med. 210, 1283-1299.

Nonaka, G.-i., Nishimura, H., Nishioka, I., 1985. Tannins and related compounds. Part 26. Isolation and structures of stenophyllanins A, B, and C, novel tannins from Quercus stenophylla. J. Chem. Soc. Perkin Trans. 1, 163-172.

Nussbaum, G., Shapira, L., 2011. How has neutrophil research improved our understanding of periodontal pathogenesis? J. Clin. Periodontol 38, 49-59.

Piwowarski, J.P., Granica, S., Kosifiski, M., Kiss, A.K., 2014. Secondary metabolites from roots of Ceum urbanum L Biochem. Syst. Ecol. 53, 46-50.

Piwowarski, J.P., Granica, S., Zwierzynska, M., Stefanska, J., Schopohl, P., Melzig, M.F., Kiss, A.K., 2014. Role of human gut microbiota metabolism in the anti-inflammatory effect of traditionally used ellagitannin-rich plant materials. J. Ethnopharm. 155, 801-809.

Piwowarski, J.P., Kiss, A.K., 2013. C-glucosidic Ellagitannins from Lythri herba (European Pharmacopoeia): Chromatographic Profile and Structure Determination. Phytochem. Anal. 24, 336-348.

Piwowarski, J.P., Kiss, A.K., 2015. Contribution of C-glucosidic ellagitannins to Lythrum salicaria L influence on pro-inflammatory functions of human neutrophils. J. Nat. Med. 69, 100-110.

Rosas-Pinon, Y., Mejia, A., Diaz-Ruiz, G., Aguilar, M.I., Sanchez-Nieto, S., Rivero-Cruz, J.F., 2012. Ethnobotanical survey and antibacterial activity of plants used in the Aidplane region of Mexico for the treatment of oral cavity infections. J. Ethnopharmacol. 141, 860-865.

Salam, R., Sarker, B.K., Haq, M.R., Khojon, J.U., 2015. Antimicrobial activity of medicinal plant for oral health and hygiene. Int J. Nat. Soc. Sci. 2, 1-12.

Schmid, D., Gruber, M., Piskaty, C., Woehs, F., Renner, A., Nagy, Z., Kaltenboeck, A., Wasserscheid, T., Bazylko, A., Kiss, A.K., Moeslinger, T., 2012. Inhibition of NF-kappa B-dependent cytokine and inducible nitric oxide synthesis by the Macrocyclic Ellagitannin Oenothein B in TLR-stimulated RAW 264.7 macrophages. J. Nat. Prod. 75, 870-875.

Scott, D.A., Krauss, J., 2012. Neutrophils in periodontal inflammation. Front. Oral Biol. 15, 56-83.

Tanimura, S., Kadomoto, R., Tanaka, T., Zhang, Y.J., Kouno, I., Kohno, M., 2005. Suppression of tumor cell invasiveness by hydrolyzable tannins (plant polyphenols) via the inhibition of matrix metalloproteinase-2/-9 activity. Biochem. Biophys. Res. Commun. 330, 1306-1313.

Tintinger, G.R., Anderson, R., Feldman, C., 2013. Pharmacological approaches to regulate neutrophil activity. Sem. Immunopathol. 35, 395-409.

Tomczyk, M., Latt S, K.P., 2009. Potentilla-A review of its phytochemical and pharmacological profile. J. Ethnopharm. 122, 184-204.

Veres, B., 2012. Anti-inflammatory role of natural polyphenols and their degradation products. In: Fernandez, R. (Ed.). Severe Sepsis and Septic Shock--Understanding a Serious Killer. InTech..

Vitkov, L, Klappacher, M., Hannig, M., Krautgartner, W.D., 2009. Extracellular neutrophil traps in periodontitis. J. Periodontal Res. 44, 664-672.

Witko-Sarsat, V., Rieu, P., Descamps-Latscha, B., Lesavre, P., Halbwachs-Mecarelli, L, 2000. Neutrophils: molecules, functions and pathophysiological aspects. Lab. Invest. 80, 617-653.

Sebastian Granica, Jakub P. Piwowarski *, Anna K. Kiss

Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-2097 Warsaw, Poland

* Corresponding author. Tel./fax: +48 22 572 09 85.

E-mail address: (J.P. Piwowarski).
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Author:Granica, Sebastian; Piwowarski, Jakub P.; Kiss, Anna K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EXPO
Date:Dec 15, 2015
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