Proanthocyanidins and Flavan-3-ols in the Prevention and Treatment of Periodontitis - Immunomodulatory Effects, Animal and Clinical Studies

This paper continues the review study on antimicrobial properties relevant to the periodontal diseases. Inflammation as a major response of the periodontal tissues attacked by pathogenic microbes can significantly exacerbate the condition. However, the bidirectional activity of phytochemicals that simultaneously inhibit bacterial proliferation and proinflammatory signaling can provide a substantial alleviation of both cause and symptoms. The modulatory effects on various aspects of inflammatory and overall immune response has been covered, including confirmed and postulated mechanisms of action, structure activity relationships and molecular targets. Further, the clinical relevance of flavan-3-ols and available outcomes from clinical studies has been analyzed and discussed. Among the numerous natural sources of flavan-3-ols and proanthocyanidins the most promising are, similarly to antibacterial properties, constituents of various foods, such as fruits of Vaccinium species, tea leaves, grape seeds, and tannin-rich medicinal herbs. Despite a vast amount of in vitro and cell-based evidence of immunomodulatory there is still much less studies using animal models and only a few clinical studies. Most of the studies, regardless of the used model indicated efficiency of these phytochemicals from cranberries and other Vaccinium species and tea extracts (green or black). Other sources such as grape seeds and traditional medicinal plants, were seldom. In conclusion, the potential of flavan-3-ols and their derivatives in prevention and alleviation of periodontitis is remarkable but clinical evidence is urgently needed for issuing credible dietary recommendation and complementary treatments.


Introduction
In the previous paper, we have demonstrated that both free flavan-3-ols and oligomeric proanthocyanidins are very promising constituents for combating various bacteria involved in periodontitis pathogenesis [1]. Here, using the same systematic approach, we have selected and discussed the recent data on anti-inflammatory and immunomodulating activities of these compounds, including in vivo models and clinical studies. Search strategy as well as inclusion, exclusion criteria and data organization are described in first part of review, in which the antimicrobial activity is discussed. According to the latest concept of periodontitis etiopathology, the development of the disease requires the co-existence of dental plaque and the host's immuneinflammatory response [2]. Periodontal bacteria cause the mobilization of innate immune response (e.g. macrophages, dendritic cells, natural killer cells, neutrophils) as well adaptive immunity mechanisms (B and T lymphocytes) leading to release of pro-inflammatory molecules (e.g., interferon-gamma, interleukin-17, tumor necrosis factor, interleukin-1, interleukin-6) and enzymes (e.g., collagenases such as matrix metalloproteinases) [3] (Figure 1). By the inflammatory response body protects itself against the bacteria and their invasion inside the deeper tissues (such as bone). However, if the inflammation persists and is poorly regulated, it causes the most troublesome detrimental changes in periodontium tissue form and function such as periodontal pockets, attachment loss, gingival recessions, tooth mobility, tooth migration and tooth loss [4]. Literature provides the data about anti-inflammatory and antioxidant effects of polyphenols which include falvan-3-ols and proanthocyanidins, drawing conclusions that they can play a beneficial role in the prevention and the progress of chronic diseases related to inflammation such as diabetes, obesity, neurodegeneration, cancers, and cardiovascular diseases, among other conditions [5]. Our review focused on the activity a high potent compounds among polyphenols in relation to periodontitis-a disease closely related to the inflammatory diseases mentioned above [2].

Influence on matrix metalloproteinases (MMPs)
It was already proved that matrix metalloproteinases (MMPs) play important roles in the connective tissue destruction of the periodontal complex. Matrix metalloproteinases (MMPs) are a calcium-dependent zinc-containing endopeptidases, responsible for the tissue remodeling and degradation of the extracellular matrix (ECM), including collagens, elastins, gelatin, matrix glycoproteins, and proteoglycan [6]. Major cell types found in the periodontium, like fibroblasts, neutrophils, and macrophages release these proteolytic enzymes [7], which secreted as latent proenzymes (except membrane type (MT)-MMPs)) must be activated extracellularly or at the cell surface by tissue, plasma or bacterial proteinases. Under normal condition, MMPs play an important Tabel 1 Immunomodulatory effects of proanthocyanidins or flavan-3-ols on host cells and tissues -in vitro studies.
Active compound/ extract/fraction Cells/tissues Methods Results Author, Years Ref.
Cranberry concentrates did not reduce HGF, SAOS-2, and macrophages viability after 24 hours of exposure. Expression of proinflammatory IL-8 and IL-6 was downregulated by PACs concentrates at 50 and 100 μg/mL but expression of antiinfammatory IL-10 was upregulated at 100 μg/mL. No influence on expression of IL-1 ß was seen. Exposed LPS-stimulated macrophages to PACs significantly decreased M1 polarization and increased M2 polarization.
PA (0.1, 1, 10 μg/ml) significantly upregulated expression of osteogenesisrelated genes and proteins and ALP activity in HPDLFs compared with the control in non-inflammatory environment. PA (1 μg/ml) significantly reversed inhibition of osteogenesis-related gene and protein expression, ALP activity, and mineralization caused by TNF-α. The underlying mechanism was that PA could regulate osteogenesis of HPDLFs via suppressing nuclear factor-kappa beta (NF-κB) signal pathway. (Huang et al. 2020) [13] 2 of 35 Catechin THP-1-derived macrophages THP-1 cells were pre-treated with catechin (40 μM) and then infected with P.gingivalis. The cytokine levels (IL-1β and TNF-α) and relevant protein expression in THP-1 cells (e.g. pro-IL-1β, TLR2, TLR4, NF-κB, MAPK and others) were measured using an ELISA kits and Western blot analysis, respectively. An apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosome formation was measured by confocal laser scanning microscopy.
Catechin inhibited P. gingivalis-induced IL-1β and TNF-α secretion in THP-1 macrophages. The decreased production of IL-1β caused by catechin was due to its inhibition of pro-IL-1β expression via the downregulation of NF-κB, p38 mitogenactivated protein kinase, and Toll-like receptor signaling. In addition, catechin inhibited the activation of inflammasomes induced by P. gingivalis, but did not affect the growth of P. gingivalis.
( Lee et al. 2020) [14] Pelargonium gingival keratinocyte cell line B11, human oral epithelial cell line GMSM-K 100 ng/mL of recombinant human TNF-α. An ELISA kit was used to quantify the levels of intracellular and released IL-1β.
Caspase-1 activation and quantification as well activation of the NF-κB signaling pathway were determined by commercial assay. Gingival keratinocytes were used to evaluate the effect of tea polyphenols on the TNF-α-induced disruption of tight junction integrity which was determined by measuring transepithelial electrical resistance (TER). ELISA kit was used to quantify IL-8 secretion by oral epithelial cells.

IL-1β
secretion by TNF-α-treated macrophages (at 62.5 μg/mL all substances inhibited the secretion of IL-1β by more than 94%, except black tea-64.5%), as well as reduced the activation of caspase-1 and NF-κB activation. Green tea extract, EGCG, theaflavins, and to a lesser extent, black tea extract protected keratinocytes against the TNF-α-mediated breakdown of barrier integrity. The treatment of keratinocytes with tea polyphenols markedly mitigated the morphological changes of tight junction proteins such as zonula occludens-1 and occludin compared to cells exposed only to TNF-α. At a concentration of 62.5 μg/mL, the green tea extract, EGCG, and theaflavins reduced the secretion of IL-8 by 93.1%, 98.8% and 70.8%, respectively. A much higher concentration of black tea extract (250 μg/mL) was required to reduce the secretion of IL-8 (78%). Adherent macrophage-like cells were pre-treated with the PACs and then were stimulated LPS-A. actinomycetemcomitans.
Secreted proinflammatory cytokines (IL-1β, TNF-α, IL-6 and CXCL8) and MMPs (MMP-3 and MMP-9) were then quantified by ELISA. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to monitor NF-κB activation. MTT assay was used to determine viability of cells treatment with PACs.
The secretion of pro-inflammatory cytokines by macrophages stimulated with LPS was significantly and dose-dependently attenuated by the PACs compared to the controls. PACs at 125 μg/mL reduced the secretion of IL-1β, TNF-α, IL-6, and CXCL8 by 75.34%, 81.64%, 48.27% and 90.19%, respectively, whereas MMP-3 and MMP-9 secretion was attenuated by 93.04% and 68.78% respectively. The PACs also inhibited the activation of the NF-κB signaling pathway.
Adherent macrophage-like cells were pre-treated with the TFs and then were stimulated with P. gingivalis. Secreted pro-inflammatory cytokines (IL-1β, TNF-α, IL-6 and CXCL8) and MMPs (MMP-3, MMP-8, MMP-9) were then quantified by ELISA. Effect of theaflavins (TFs) on the activity of MMP-9 was monitored using a fluorogenic assay. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to monitor NF-κB activation. MTT assay was used to determine viability of cells treatment with TFs. The macrophage-like cells were pre-treated with the blueberry extract and then stimulated with F. nucleatum. ELISA kits were used to quantify IL-1β, IL-6, CXCL8, TNF-α, MMP-8, and MMP-9 concentrations. Activity of MMP-9 was monitored using a fluorogenic assay. The ability of the blueberry extract to inhibit the NF-κB signaling pathway in U937-3xκB cells was investigated The blueberry extract dose-dependently inhibited the activation of NF-κB induced by F. nucleatum. A pretreatment of macrophages with the blueberry extract (62.5μg/mL) inhibited the secretion of IL-1β, TNF-α, and IL-6 by 87.3, 80.7, and 28.2%, respectively. The secretion of the chemokine CXCL8 was not affected by 62.5 μg/mL of the blueberry extract, but 500, 250, or 125μg/mL of extract decreased CXCL8 secretion by 79, 57.9, and 11.2 % respectively. The secretion of MMP-8 and MMP-9 was also dosedependently inhibited as well MMP-9 activity.
Of the 41 different cytokines, chemokines, and growth factors analyzed, A. actinomycetemcomitans LPS significantly increased the secretion only G-CFS, GRO-a, IL-6, IL-8, IP-10, and MCP-1 by the 3D coculture model compared to the unstimulated control. When used individually, AC-PACs at the lowest concentration tested (25 μg/mL) significantly reduced the secretion of G-CFS (42%), GRO-a (33%), IL-8 (39%), IP-10 (72%), and MCP-1 (72%), but had no significant effect on the secretion of IL-6, while EGCG at 5 μg/mL significantly reduced the secretion of GRO-a (13%), IL-8 (34%), IP-10 (22%), and MCP-1 (70%), but had no significant effect on the secretion of G-CSF and IL-6. AC-PACs and LL-37 acted in synergy to reduce the GRO-a, G-CSF, and IL-6, and had The commercial black tea extract (with theaflavin content of 40.23%); theaflavin, theaflavin-3,3`digallate, epigallocatechin-3gallate EGCg The oral epithelial cells (OBA-9) The epithelial cells were pre-treated with the black tea extract, theaflavin, theaflavin-3,3'digallate or EGCg prior to being stimulated with A. actinomycetemcomitans LPS. ELISA assays were used to quantify the secretion of IL-8, hBD-1, hBD-2 and hBD-4 by oral epithelial cells. The Smulow-Glickman (S-G) human gingival epithelial cell line S-G cells were incubated with IL-1β in the presence or absence of NDM or inhibitors of NF-κB, (NBD) or AP-1 (SP600125), and IL-6 levels were measured by ELISA kit. Effects of NDM on IL-1β -activated NF-κB and AP-1 and phosphorylated intermediates in both pathways were measured in cell extracts via binding to specific oligonucleotides and specific sandwich ELISAs IL-1β caused a time-and dose-dependent stimulation of S-G epithelial cell IL-6 production. NDM appeared to be a stronger inhibitor of IL-1β-stimulated IL-6 production than either NBD peptide or SP600125 alone and was similar to inhibition caused by the combination of the NBD peptide and SP600125. IL-1β stimulated NF-κB and AP-1 activation, which was inhibited by NDM. However, NDM did not significantly affect IL-1β-stimulated levels of phosphorylated intermediates in the NF-κB pathway (IκBα) or the AP-1 pathway (c-Jun, ERK1/2). In both cell lines, IL-17 significantly stimulated production of IL-6 and IL-8. Nontoxic levels of NDM (5-50 μg/ml) inhibited constitutive IL-6 and IL-8 production as well their IL-17-stimulated cytokine production by epithelial cells and fibroblasts. ( Normal human gingival fibroblast cell lines (GN23, GN56, GN60) measured via binding to an oligonucleotide containing the NF-κB consensus site.
MMP-3 in AgP fibroblasts. NDM increased IL-6 in LPS-stimulated AgP fibroblast, but decreased in normal human gingival fibroblast.
RAW 264.7 mouse macrophages, RAW 264.7 mouse macrophages infected with P. gingivalis and F. nucleatum were exposed to culture media with or without NDM. The secreted form of mouse TNF-α was quantified using two-site ELISA. Macrophage functionality was investigated using a phagocytosis assay.
IL-1 β, TNF-α, IL-6 and IL-8 production by macrophages treated with the APAC (or/and LA) and stimulated by A. actinomycetemcomitans LPS was evaluated by ELISA kits. Influence of APAC (or/and LA) on MMP-9 and P. gingivalis collagenase activities was measured by fluorometric assays.

Human osteoclast precursor cells
This study investigated the effect of AC-PACs on osteoclast formation and bone resorption activity. The degree of osteoclast formation was evaluated by quantification of TRAP-positive stained multinucleated cells, while the secretion of IL-8 and MMP-2, MMP-9 was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. Cytotoxic effect of AC-PACs on osteoclastic cells was measured by the MTT assay.
AC-PACs at 10, 25 and 50 μg/mL caused a 38%, 84% and 95 % inhibition of RANKLdependent osteoclast differentiation, respectively. AC-PACs increased the secretion of IL-8 and inhibited the secretion of both MMP-2 and MMP-9 in a dose-dependent manner. AC-PACs significantly decreasing the release of collagen helical peptides suggested that can prevent bone resorption. AC-PACs did not exhibit any toxic effect on osteoclastic cells ranging from 10 to 100 μg/mL. KB cells were pre-treated with MF (10 and 100 μg/ml) and infected with P. gingivalis. The influence of MF on P. gingivalis-induced cytokine gene expression was monitored by RT-PCR and IL-6 titres by ELISA.
10 and 100 μg/ml of MF significantly decreased (upregulated by P. gingivalis) gene expression for IL-1β, IL-8 and TNF-α, but not IL-6 compare to control cells (not exposed to MF). However, pre-incubation of the KB cells with MF before exposure to P.gingivalis resulted in significant lower concentration of IL-6 in the cells than in MF-untreated control group. A-Type Cranberry Proanthocyanidins (AC-PACs) were isolated from cranberries (Vaccinium macrocarpon)

Oral epithelial cells (GMSM-K)
The epithelial cells were pretreated with increasing concentrations of AC-PACs (25 -100 μg/mL) before the stimulation with P. gingivalis. ELISA kits were used to quantify IL-6, IL-8, and chemokine (C-C motif) ligand 5 (CCL5) concentrations in the free-cell supernatants. To understand the mechanism of action of AC-PACs, their effect NF-κB p65 activation was investigated.
AC-PACs significantly decreased the secretion of IL-8 and CCL5 at all concentrations tested in a dose-dependent manner, where 100 μg/mL reduced secretion of IL-8 and CCL5 by more than 80%. Decreased secretion of IL-8 and CCL5 was not related to loss of cell viability. AC-PACs did not affect the secretion of IL-6. A pretreatment of epithelial cells with 50 μg/mL of AC-PACs prior to the stimulation with P. gingivalis significantly decreased the P. gingivalis-induced activity of NF-κB p65 to 91% (2) the catalytic activity of recombinant MMP-1 and MMP-9, (3) the expression of 5 protein kinases and the activity of nuclear factor-kappa B (NF-κB) p65 in macrophages stimulated with LPS -using commercial kits. Determination of cytotoxicity was using MTT assay.
from stimulated macrophages at all concentrations tested (25,50, and 100 μg/mL), whereas production of MMP-3 reduced significantly only at the highest 100 μg/mL and MMP-1, MMP-9 at 50, and 100 μg/mL of AC-PACs. The catalytic activity of MMP-1 and MMP-9 was also inhibited. The inhibition of MMP production was associated with reduced phosphorylation of key intracellular kinases and the inhibition of NF-κB p65 activity.
MMP-3 and MMP-9 production by HGF-1 and macrophages treated with the cranberry fraction and then stimulated with LPS from A. actinomycetemcomitans was measured by ELISA kits. MMP-3, MMP-9 and elastase activities in the presence of the cranberry fraction were evaluated using colorimetric or fluorogenic substrates. The changes in expression and phosphorylation state of fibroblast intracellular signaling proteins induced by LPS and the cranberry fraction were characterized by antibody microarrays. MTT assay for cells.
MMP-3 and MMP-9 production by macrophages after treated with the cranberry fraction and stimulated with LPS were inhibited significnatly ,in a dosedependent manner, similary MMP-3 production by fibroblast. However, MMP-9 response after stimulated LPS wasn't observed. Cranbery fraction wasn't toxic towards fibroblast and macrophages. Cranberry fraction inhibited fibroblast intracellular signaling proteins, a phenomenon that may lead to a downregulation of activating protein-1 activity. Cells were preincubated with non-toxic concentrations of GA (4 μg/ml), EGCG (0.5 μg/ml), or GSE (4 μg/ml) and stimulated with LPS of A. actinomycetemcomitans, F. nucleatum. iNOS expression was evaluated by immunoblotting, NO production was quantified using the colorimetric Griess assay, whereas ROS production was measured with the fluorescent 123dihydrorhodamine dye.
GSE as well EGCG strongly decreased NO and ROS production and iNOS expression by LPS-stimulated macrophages. GA also revealed a strong inhibitory effect on NO production without affecting iNOS expression but slightly increasing ROS production. GTP and 5 catechins were tested for their ability to inhibit matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities, measured by fluorescence and by gelatin or casein zymography. In addition, the activation of proMMP-2 by the lectin Con A was determined following exposure to GTP.
IC50 values for the inhibition of MMP-2 and MMP-9 activities was 10 μg/ml and 0.6 μg/ml for GTP, 95 μM and 28 μM for ECG and 6 μM and 0.3 μM for EGCG, respectively. MMP-12 was inhibited more than 60% by 1 μM of ECG or EGCG. MMP-2, MMP-9 and MMP-12 activities were unaffected by C, EC and EGC. The activation of MMP-2 by Con A was reduced by 50% at 17.5 μg/ml of GTP and was almost completely inhibited at 35 μg/ml. Among catechins (at 100 μM), only EGCG inhibited the activation of MMP-2 by Con A. The activation of proMMP-2 by EGCG was inhibited in a dose-dependent manner. EGCG at 25 μM completely abolished the activation of proMMP-2 by Con A.  Gingival crevicular fluid (GCF) collected from periodontitis patients; purified collagenase;in vitro study Ethyl acetate fraction and 6 isolated catechines were tested for their ability to inhibit purified collagenase activities using collagenase of Clostridium histolyticum and supernatant of Porphyromonas gingivalis as well collagenase activity in GCF. Collagenase activity was determined using a commercially available kit.
(Makim ura et al. 1993) [44] Particularly, the n-butanol fraction from the Ulmus macrocarpa Hance bark, defined as elm extract (contain 20% of procyanidins) and the mixture of procyanidin oligomers (composed of 3 to 12 flavan-3-ol monomers, an average molecular weight of 1518) isolated from elm extract in range 100-1,000 μg/ml exhibited inhibitory effects on the MMPs, present in gingival crevicular fluid (GCF) of adult periodontitis patients (mainly, MMP-8 and MMP-9) and on the pro and active forms of MMP-2 (from the conditioned media of cultured periodontal ligament (PDL) cells treated with a periodontopathogen, Treponema lecithinolyticum) [10]. The inhibition of enzyme activity by procyanidin oligomers was more effective than by the elm extract, with IC50 values 25 and 33 μg/ml for GCF collagenases (mostly MMP-8 and MMP-9) and MMP-2, respectively. Moreover, elm extract and procyanidin oligomers inhibited proteolytic enzymes of two periopathogens, T. denticola and P. gingivalis responsible for degradation of the interstitial and basement membrane collagens as well as activating of matrix metalloproteinases e.g. MMP-8, MMP-9 or MMP-1, MMP-3 and MMP-9 [10].

Influence on bone tissue resorption
Yun et al. [42] reported an inhibitory effect of (-)-epigallocatechin gallate (EGCG) (20 μM) on the gene expression of MMP-9 in osteoblasts and on the formation of osteoclasts, what suggested that EGCG may prevent the alveolar bone resorption that occurs in periodontal diseases leading to teeth loss. Importantly, in the periodontal disease an enhanced osetoclastogenesis can occur due to the presence of the of inflammatory cytokines that stimulates osteoclast proliferation or promotes the differentiation of progenitor cells. Mature osteoclasts that derive from hematopoietic monocyte/macrophage precursors under the action of RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) mediate the destruction of the alveolar bone by attaching to the bone surface and promoting mineral dissolution. The demineralized bone matrix is later degraded by proteases such as cathepsin K and metalloproteinases (MMPs) [32]. Tanabe et al. [32] showed that A-type cranberry proanthocyanidins (AC-PACs) have influence the osteoclast formation and bone resorption activity. In a range of 10-100 μg/ml, AC-PACs inhibited RANKL-dependent osteoclast differentiation, as well as secretion of both MMP-2 and MMP-9 but secretion of IL-8 was increased. IL-8 from normal human bone marrow stromal cells inhibits the bone resorbing activity of osteoclasts. [45] Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 November 2020 doi:10.20944/preprints202011.0671.v1 Huang et al. [13] reported that proanthocyanidins (PA) may contribute to bone generation in inflammatory microenvironment via suppressing NF-κB signaling pathway and therefore may be a potential inducer of periodontal bone regeneration. In this study an effect of PA on osteogenic differentiation of human periodontal ligament fibroblasts (PDLFs) with or without TNF-α stimulation was tested and the underlying mechanism was explored. The assumption was that PDLFs are capable of differentiating into osteoblasts, but pro-inflammatory cytokines like TNF-α inhibit this process. Osteogenic differentiation-and mineralization-associated markers were detected by qRT-PCR, alizarin red S staining, and alkaline phosphatase (ALP) activity assay. In result, PA in low concentration (0.1, 1, 10 μg/ml) significantly upregulated expression of osteogenesis-related genes and proteins and ALP activity in PDLFs compared with the control. However, PA at higher concentrations of 30 and 50 μg/ml significantly suppressed the ALP activity of PDLFs. For the rest assay, authors used only lower concentration of PA -(0.1, 1, 10 μg/ml). PA in concentration of 1μg/ml significantly reversed inhibition of osteogenesis-related gene and protein expression, ALP activity, and mineralization caused by TNF-α. The authors also suggesteded that PA may reverse TNF-α inhibited osteogenic differentiation via NF-κB signaling pathway.
These authors used commercial proanthocyanidins claimed to possess an untypical for proanthocyanidins structure (figure 1) and with molecular weight =594.52. The supplier's website states that proanthocyanidins have been isolated from grapes (the fruits of Vitis vinifera L.)

Influence on cytokines
The overproduction and secretion of inflammatory cytokines by resident and immune cells modulate the progression and severity of periodontitis. Increase of such proinflammatory cytokines as: IL-1α, IL-1β, TNF-α, IL-6, and IL-17 were shown in patients with acute or chronic periodontitis [46]. More specifically, TNF-α is present at high levels in gingival crevicular fluid (GCF) and in diseased periodontal tissues, where it is positively correlated with MMPs and RANKL expression. Human and animal studies confirmed that TNF-α plays a central role in inflammatory reaction, alveolar bone resorption, and the loss of connective tissue attachment. Moreover, TNF-α up-regulates the production of other pro-inflammatory innate immunity cytokines, such as IL-1β and IL-6 associated with inflammatory cell migration and osteoclastogenesis [46]. IL-1β plays an important role in the pathogenesis of periodontitis also by regulation of the IL-6 production in a variety of cell types, including fibroblasts and epithelial cells [26]. Similarly to bacterial LPS, cellular response to cytokines or chemokines (e.g. IL-1β) can be mediated via signaling cascades, including NF-κB and MAPK/AP-1 pathways, which lead to gene expression of certain proteins (for example IL-6). There are more and more studies proving that proanthocyanidins and flavan-3-ols inhibit the secretion of cytokines by influencing NF-κB and MAPK/AP-1 activation (table 1), [14], [13], [18], [19], [20], [22], [37], [26], [28]. Many studies have shown inhibition of production and / or secretion of inflammatory cytokines by proanthocyanidins. Bodet et al. [40] demonstrated that proanthocyanidin-enriched cranberry fraction at concentrations 25-50 μg/ml, significantly inhibited the IL-6, IL-8, and PGE 2 production by gingival fibroblasts stimulated with the Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) . The most spectacular inhibitory effect was seen towards IL-8 that belongs to chemokines (CXCL8) also known as neutrophil chemotactic factor. It directs the migration of polymorphonuclear leukocytes, monocytes, and macrophages to the site of infection. Increased level of IL-8 was observed in the gingival crevicular fluid of inflamed periodontal sites [40]. PGE 2 is another proinflammatory molecule involved in destructive process in periodontal disease. It is secreted in response to proinflammatory cytokines, periodontopathogens and LPS. The cranberry fraction significantly inhibited PGE2-response even at low tested concentration-25 μg/ml, and reduced COX 2 protein expression-the enzyme involved in PGE2 production. Moreover, cranberry fraction influence on the phosphorylation and expression of various intracellular proteins (Jun, Fos, MKK3, MKK6, Rac1, Mnk1) which are implicated in cytokine production. Bodet et al. concluded that cranberry fraction may act especially via a downregulation of AP-1 activity [40]. Feldman and Grenier [31] showed an inhibitory effect of 25 or 50 μg/ml of A-type cranberry proanthocyanidins (APAC) on TNF-α, IL-6, and IL-8 secretion in a macrophage model. The 50 μg/ml concentration of APAC reduced the LPSinduced secretion of TNF-α, IL-6 and IL-8, by about 50%, but had not influence on IL-1 β. A significant reduction in IL-1 β secretion was seen when ACPAC was used together with Licochalcone A (chalcone, not proanthocyanidin). Further studies on proanthocyanidins, in the predominant amount on A-type cranberry proanthocyanidins, prove their influence on the secretion and production of interleukins, as well as provided explanation of molecular mechanisms responsible for this activity [12], [17], [23], [26], [27], [28] [29], [36] (Table 1).
Galarraga-Vinueza et al. [12], revealed that cranberry concentrate from capsules (Uriach-Aquilea OTC) containing 130 mg A-Type PACs significantly decreased M1 polarization and increased M2 polarization in LPS-stimulated macrophages. M1 phenotype of macrophage are activated by bacteria sub-products like lipopolysaccharides (LPS) and are associated with the secretion of pro-inflammatory cytokines (e.g. IL-1ß, IL-6, IL-8), whereas a M2 phenotype of macrophages are activated by alternative ways and are associated with the secretion of antiinflammatory cytokines (e.g. IL-10) and growth factors which enhance tissue repair. Galarraga-Vinueza et al. [12] confirmed the effect of A-Type PACs (50 and 100 μg/mL) on cytokine expressionproinflammatory cytokines: IL-8 and IL-6 were significantly downregulated in LPS-stimulated macrophages and A-Type PACs, whereas an anti-inflammatory IL-10 was upregulated. No influence on expression of IL-1 ß was seen. Lagha et all. [17] showed that fraction of proanthocyanidins (PACs) from cranberries at a concentration of 15.625-125 μg/mL markedly reduced cytotoxicity of leukotoxin on macrophages and significantly reduced (by about 80-90% at 15.625 and more than 98% at 125 μg/mL) release of caspase-1, IL-1β, and IL-18 from LtxA-induced macrophages. Leukotoxin (LtxA), released by A. actinomycetemcomitans is one of the important virulence factor playing a critical role in the pathogenic process of localized aggressive periodontitis (LAP). LtxA affects immune cells by activates pyroptosis of monocytes and macrophages and inducing the release of pro-inflammatory cytokines. Pyroptosis in turn, is inflammatory form of programmed cell death, involves the activation of caspase-1, which in turn covert of pro-IL-1β and pro-IL-18 to the biologically active forms. Pyroptosis in macrophages leads to the formation of pores in the plasma membrane which allow secretion of IL-1β and IL-18, cytokines known as damage-associated molecular patterns (DAMPs) and contribute to the progression of periodontitis by increasing cell migration and osteoclastogenesis [47], [48]. Moreover, PACs reduced the expression of CIAS and P2X7 genes (increase by LtxA, in macrophages) by about 30-45%, similarly for a range 15.625-125 μg/mL [17]. This is important because the activation of P2X7 receptors and CIAS leads to the rapid formation of membrane pores and to the release of IL-1β and IL-18. Lastly, cranberry PACs blocked the binding of leukotoxin (LtxA) to macrophages as well reduced ROS and superoxide production in LtxA-induced macrophages.
Cranberry proanthocyanidins (PACs) can have differently affect interleukines secretion/production, depending on a cell type. In LPS-stimulated normal human gingival fibroblast, cranberry non-dialyzable material (NDM) rich in proanthocyanidins decreased level of IL-6, what is consistent with other studies, but NDM significantly increased IL-6 in LPS-stimulated human gingival fibroblast cell line derived from a patient with aggressive periodontitis (AgP fibroblasts) [28]. This increasing level of IL-6 occurred only in the presence of LPS; NDM alone did not significantly increase constitutive IL-6 production. Simultaneously, NDM inhibited NF-κB activity (increased by LPS treatment) in AgP fibroblasts what suggested involvement of other mechanisms of IL-6 regulation in these cells.
Influence of proanthocyanidins and flavan-3-ols from other source than cranberries on the secretion and production of interleukins was also demonstrated in several studies (table 1). Jekabsone et al. [15] reported that Pelargonium sidoides root extract (PSRE) and especially proanthocyanidin fraction from PSRE (PACN) possess strong antibacterial (against Aggregatibacter actinomycetemcomitans), anti-inflammatory and gingival tissue protecting properties under periodontitis-mimicking conditions. The cells (gingival fibroblast, bone marrow-derived macrophages (BMDM) or human peripheral blood mononuclear cells (PBMCs) were stimulated using LPS (and IFNγ for BMDM) and treated with 50 and 100 ug/mL of PSRE or PACN. The extracts protected human gingival fibroblast from A. actinomycetemcomitans infection, decreased LPS-induced release of IL-8 and prostaglandin E2 from gingival fibroblasts and IL-6 from leukocytes, blocked expression IL-1β, iNOS and COX-2 but not TNF-α. Stronger anti-inflammatory activity of proanthocyanidin fraction (PACN) than root extract (PSRE) was associated with higher amounts of prodelphinidins. The study also reported that PSRE and PACN (100 μg/mL) blocked the surface presentation of CD80 and CD86 (surface markers of proinflammatory M1 phenotype) in LPS+IFNγtreated macrophages, whereas PACN was characterized by stronger activity. These results indicate that both PACN and PSRE are potent in preventing macrophage conversion to proinflamatory M1 phenotype under exposure to LPS.
Low concentrations (7.9-62.5 μg/mL) of green and black tea extracts as well as their flavan-3-ols (epigallocatechin-3-gallate, theaflavins) have influence on production and secretion proinflammatory cytokines. They attenuate the gingival epithelial barrier dysfunction caused by TNF-α and modulate the inflammatory host response. They inhibited the activation of NF-κB and caspase-1 as well as reduced IL-1β secretion by macrophages (at 62.5 μg/mL by more than 94%, except black tea-64.5%), and secretion IL-8 (only black tea required higher concentration than 62.5 μg/mL for more than 70% inhibition) by oral epithelial cells stimulated with recombinant TNF-α [16]. The green tea extract showed higher activity than black tea extract. Other studies have confirmed the inhibitory effect of flavan-3-ols obtained from tea on the secretion of pro-inflammatory cytokines from LPS stimulated macrophages as well from cytokines-stimulated gingival cells (table 1), [14], [23], [24], [35]. Ben Lagha et al. [20] presented consistent results in which they proved inhibitory effect theaflavins (TFs) from black tea on the secretion of pro-inflammatory cytokines from P. gingivalis-treated macrophages and on the activation of the NF-κB signaling pathway (table 1). Lombardo Bedran et al. [25], [24] in studies on green and black tea and their main galloylated flavan-3-ols revealed the ability of these compounds to induce human beta-defensin (hBD) secretion in gingival epithelial cells. Human betadefensins (hBDs) are antimicrobial peptides secreted by gingival epithelium in response to periopathogens. They interact with the bacterial cell membrane, leading to pore formation and finally to the lysis of major periopathogens. Evidence indicated that level of hBDs are higher in healthy gingival tissues than in diseased gingival tissues and that some periodontopathogenic bacteria, such as P. gingivalis, are capable to down-regulate hBD expression by epithelial cells and/or to inactivate hBDs by proteolytic cleavage [25]. Both green and black teas and their galloylated flavan-3-ols stimulated secretion of hBDs and increased expression of the hBD gene in gingival epithelial cells as well as prevented the degradation of hBD1 and hBD2 by P. gingivalis. Again, the tested nongalloylated flavan-3-ols -theaflavins did not induce secretion of significant amounts of hBDs by oral epithelial cells.
In addition to the above mentioned, there is a couple of other well studied sources of proanthocyanidins with proven anti-inflammatory activities linked with periodontitis.
Proanthocanydin-enriched extract from Myrothamnus flabellifolia, plants traditionally used for treatment of gingivitis and periodontitis in South Africa, decreased gene expression of IL-1β, IL-8 and TNF-α, and level of IL-6 in KB cells, pre-incubated with MF (10 and 100 μg/ml) and infected with P. gingivalis [33].

Influence on reactive oxygen species (ROS) production
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) production by immune cells stimulated by periopathogens is an imortant factor in pathogenesis of periodontitis [41]. Their overproduction can lead to oxidative damage to gingival tissue, periodontal ligament, and alveolar bone. Study of Houde et al. [41] showed that the stimulation of macrophages with LPS of A. actinomycetemcomitans and F. nucleatum induces increased NO and ROS release. However, macrophages pretreated with non-toxic concentrations of grape seed extract (GSE) containing 95% oligomeric proanthocyanidins significantly inhibited free radical generation by inhibiting the production of the proinflammatory mediators NO and ROS and by modulating iNOS protein expression.

In vivo studies reporting influence proanthocyanidins or flavan-3-ols on periodontitis in animal models
Toker et al. [49] presented results which indicated that grape seed proanthocyanidin extract (GSPE) can decrease periodontal inflammation and alveolar bone loss via decreasing MMP-8 and HIF-1α levels and increase osteoblastic activity in diabetic rats with experimental periodontitis (Table  6 of 35 2). 100 and 200 mg/kg doses of grape seed proanthocyanidin extract (GSPE) administered by oral gavage to rats with induced diabetes (D) and periodontitis (P) significantly decreased alveolar bone loss, inflammatory cell numbers, MMP-8 and HIF-1α levels compared to rats with D+P but without GSPE. Moreover, the osteoblast number increased significantly in the GSPE groups compared to the P and D+P groups.
Cai et al. [50] study indicated that epigallocatechin-3-gallate (EGCG) alleviates Porphyromonas gingivalis-induced periodontitis in mice. The mice orally inoculated with P. gingivalis in PBS, received sterile food and drinking water with 0.02% solution of EGCG from 8 weeks to 15 weeks. EGCG significantly reduced alveolar bone resorption as well as decreased the high expressions (caused by P. gingivalis infection) of inflammatory cytokines and other mediators both in serum and in gingival tissue (details in the table 2) what is consist with previous study of Lee y et al. [38], in which EGCG suppressed the progression of apical periodontitis, by diminishing Cyr61 expression (a potential osteolytic mediator) in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.
Cho et al. [51] observed decreased interleukin (IL)-6 and tumor necrosis factor (TNF) expression in rats tissue orally fed EGCG compare to group without EGCG. Downregulation of TNF and IL-6 expression by EGCG led to a decrease in osteoclast number and activity, which resulted in reduced bone loss. They also noticed reduced collagen destruction in EGCG group. Similar results achieved Lee at al. [14] studying catechin, another of major flavan-3-ols in green tea. They showed that catechin reduced the level of alveolar bone loss in a P. gingivalis-induced periodontitis mouse model. In turn, Polak et al. [29] showed that cranberry non-dialyzable material (NDM) consumption by mice infected by P. gingivalis and F. nucleatum attenuated the alveolar bone loss compared to the mice with infection but without NDM treatment. Moreover, in subcutaneous chamber model of inflammation, NDM alone reduced tumor necrosis factor-α (TNF-α) levels induced by the mixed infection. In vivo studies were supported by in vitro study (Table 1). Oral administration of commercial grape seed proanthocyanidins (PC) [52] to rats with experimentally induced periodontitis (EP) revealed that PC enhanced the host resistance and inhibited the oxidative stress. In serum, proanthocyanidins (PC) significantly decreased reactive oxygen species, lipid peroxides, lysosomal enzymes, acute phase proteins and they increased antioxidant levels. Histopathological evidence of experimental periodontitis without PC showed cellular infiltration of inflammatory cells whereas the proanthocyanidin treated groups demonstrated only scattered inflammatory cells. Tabel 2 In vivo studies reporting influence proanthocyanidins or flavan-3-ols on periodontitis in animal model.

Active compound/ extract/fraction
Animal model Methods Results

Author, Years
Ref.

Catechin
Six-week-old C57BL laboratory mice were divided into four groups: untreated normal control group, P. gingivalis-infected group P. gingivalis-infected + catechin group, catechin only-treated group.
In the P. gingivalis+catechin and catechin group, catechin (40 mg/kg body weight) was administrated orally to the mice 30 minutes before the P. gingivalis injection for 2 weeks, and subsequently every 2 days for an additional 2 weeks. The mice were euthanized and evaluated on day 49. For quantitative analysis of alveolar bone loss, themaxilla was examined using a microcomputed tomography (micro-CT) system.
In the P. gingivalis+catechin group (3)  Epigallocatechin-3-gallate (EGCG) 20 four-week-old Wistar rats divided on 2 groups: the rats (10 per each group) were given intraperitoneal injections of EGCG (80 mg/kg) or the rats (10 per each group) were given intraperitoneal injections of normal saline (NS, as control) on a daily basis until death. The animals were killed 20 days after induced apical periodontitis.
The jaws were dissected and radiographs were taken. Cyr61 and CCL2 were measured using immunohistochemistry assays.
Radiographs showed that administration of EGCG significantly attenuated periapical bone resorption compared with the control. Image analysis revealed that EGCG suppressed periapical osteolysis by an average of 57.2% in experimental model. In EGCG group, the numbers of Cyr61-synthesizing osteoblasts and infiltrating macrophages were also decreased. EGCG also markedly diminished the numbers of CCL2producing osteoblasts. Statistical analysis revealed a lower percentage of Cyr61positive osteoblasts in the EGCG-treated group (21.3%), compared with that in the control group (62.1%).  Two evaluation visits were performed on days 14 and 21 of the study for an oral clinical examination and to register the Silness and Löe index, the gingival bleeding index, the Turesky plaque index, the inflammatory crevicular fluid study (IL6), and changes in the brightness of the gingiva.
The Silness and Löe gingival index was higher in the control group than in the experimental group. The bleeding was lower in the experimental group versus the control group. In contrast to the above results, the amount of dental plaque was slightly higher (33%) in the experimental group versus in the control group. No significant differences between the study group and the control group was seen in brightness of the gingiva. Statistically significant differences in level of IL-6 were found at the baseline between the experimental group and the control group and in the subsequent visits. However, in experimental group level of IL-6 was lower. Thermo-reversible sustained-release system incorporated with green tea extract .
A controlled, split-mouth singleevaluator masked study was conducted to evaluate the effect of green tea extract as a sustainedrelease system in patients with chronic periodontitis (CP). 30 patients, each with 2 sites (test and control) having probing depths (PDs) of ≥4 mm were selected.
Green tea and placebo gels were placed at test and control sites as Assessment of gingival index (GI), pocket depth (PD), and relative clinical attachment levels (rCALs) was done at baseline and at 4 weeks.
When the comparison of means of GI, PD, and rCAL was done between baseline values and at the end of 4 weeks within the test group, and control group all the parameters were lowered and statistically highly significant. The test group showed significantly better results when compared with controls. Adjunctive local drug therapy with thermoreversible green tea gel has revealed to reduce pockets and inflammation during the 4 weeks of the clinical trial in patients with CP. Hydroxypropylcellulose strips containing green tea catechin (Taiyo Kagaku, Yokkaichi, Mie, Japan) 6 volunteers with advanced periodontitis, but with no systemic disorders. From each volunteer two pockets were selected; one for administration of the test agent and the other for placebo. Strips were applied in pockets in patients once a week for 8 weeks.
The subjects were divided randomly into the scaled group (3 subjects) non-scaled group (3 subjects) were applied in pockets in patients The clinical (pocket depth (PD)), enzymatic (peptidase activities) and microbiological effects (the proportion of black-pigmented, Gram-negative anaerobic rods (BPR) of the catechin )were determined.
The PD and the BPR were markedly decreased in the catechin group with mechanical treatment at week 8 compared to baseline. The peptidase activities in the gingival fluid were maintained at lower levels during the experimental period in the test sites with catechin, while it reached 70% of that at baseline in the placebo sites.

Conclusions
Among the numerous in vitro studies (34) on the immunomodulatory effect of proanthocyanidins or flavan-3-ols on the host cells, most concern the tea leaves extract and its compounds-catechines with presence of the galloyl moiety as the most active, as well as of A-type proanthocyanidins from fruits of Vaccinium species. Other sources of proanthocyanidins such as grape seeds and traditional medicinal plants, were seldom. The in vitro studies proved their immunomodulatory activity, among others by influencing on immune cell regulation, proinflammatory cytokines' synthesis and gene expression as well as by radical scavenging and inhibition of certain enzymes. They modulate NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and mitogen-activated protein Kinase (MAPk) pathways. Despite these promising results there is still much less studies using animal models (7) and only a few clinical studies (3). In conclusion, the potential of flavan-3-ols and their derivatives in prevention and alleviation of periodontitis is remarkable but clinical evidence is urgently needed for issuing credible dietary recommendation and complementary treatments