Phytotherapy represents a science-based medical practice that use of plants either to treat disease or as health-promoting agents. It has been gaining importance in health field based specially in its anti-microbiological, anti-inflammatory, antioxidant, and antitumor properties [1
]. In dentistry, several phytotherapeutic agents like curcumin, bromelain, chamomilla, and baicalin among others have been used especially in inflammatory conditions because they can modulate the inflammatory process, reduce pain, and promote faster wound healing with excellent clinical response [2
Oxidative stress is a significant process in oral mucosal disease pathogenesis [8
]. It represents the imbalance between the production of free radicals and the ability of the body to eliminate these reactive species. Reactive oxygen species (ROS) are the most important free radicals generated with biological beneficial effects at low level and normal conditions. However, higher concentrations of ROS become prejudicial to the organism causing cellular damage [10
], resulting in changes in cellular metabolism, mitochondrial activity, and damage on proteins, lipids, and DNA. ROS generation is counterbalanced by the action of antioxidant mechanisms act as free radical scavengers and neutralize excess of ROS. The antioxidant system comprises several enzymatic and non-enzymatic components such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, carotenoids, phenolic acids, flavonoids, tannins, and others [9
]. Some studies have shown the beneficial role of phytotherapic formulations against oxidative stress-related human diseases, including periodontal diseases; however, the mechanism involved in the positive response is not completely understood [12
The stilbene piceatannol (3,3′,4,5′-tetrahydroxy-trans-stilbene), a resveratrol (trans-3,5,4′-trihydroxyestilbene) metabolite was first isolated from the plant Euphorbia lagasacae Spreng 1821 (WCSP, 2018) in 1984 [14
] and has been found in different sources such as grapes, peanuts, sugar cane, blueberries, and passion fruit seeds [15
]. Piceatannol has potent biological activities, including antioxidant, anti-cancer, anti-inflammatory, and anti-obesity properties. Interestingly, in comparison with red grapes, a major source of piceatannol in the human diet, the passion fruit representative sample contained 1000 to 2000 times more piceatannol in fresh matter terms. The passion fruit appears therefore as a new and promising source of piceatannol, which can be used as material for the production of nutraceuticals. Recent studies showed the piceatannol superiority over the resveratrol as antioxidant and cytoprotector [16
] due to its higher capacity of chelators/sequestrants reactive oxygen species (ROS) [17
]. Maternal supplementation with piceatannol is neuroprotective in rat neonatal hypoxia-ischemia [18
]. Piceatannol affects peripheral clock gene expression and may prevent circadian disturbance [19
Periodontal diseases usually refer to common inflammatory disorders, known as gingivitis and periodontitis, that involve a multifactorial interaction among microbial, host immunological response, and environmental modulating factors. High levels of nuclear factor-kappa B (NF-κB), transglutaminase 2 (TG2) and several inflammatory mediators, including IL-1, IL-6, TNF-α, and prostaglandin E2 are produced in periodontal diseases [20
]. These processes result in the destruction of the tissues surrounding and supporting the teeth, bone resorption, and tooth loss [21
]. Accumulating evidence has indicated a close connection between oxidative stresses in the pathogenesis of periodontal diseases. Inflammatory conditions that affect the periodontal ligament have been associated to an increase in ROS production that results in periodontal ligament destruction, bone resorption activity by stimulation of osteoclastogenesis, and decreased differentiation of osteoblasts [22
]. In addition, periodontal treatment reduces inflammation and may be beneficial for periodontitis patients’ systemic and local oxidative stress control [23
Thus, there is a therapeutic need to increase the defenses in the periodontal tissues and various compounds with antioxidant action are being tested [26
]. Adjunctive local phytotherapy has been used in periodontal diseases and has been associated with significant improvement in periodontal clinical parameters (PPD and clinical attachment level) of patients [27
]. In this way, resveratrol has demonstrated an antioxidant effect on gingival fibroblasts and in prevention of periodontal diseases progression in pre-clinical studies [28
]. Piceatannol is superior to resveratrol concerning the antioxidant and cytoprotecting effects [16
]. Therefore, the present study aimed to evaluate the piceatannol cytoprotection in human periodontal ligament fibroblasts under oxidative stress.
3. Results and Discussion
Phytotherapeutic formulations have been used as adjunctive therapy with significant improvement in periodontal diseases [27
]. Here we evaluate the effect of Piceatannol in periodontal diseases. Piceatannol is a naturally phenolic compound found in a variety of plant sources including grapes, rhubarb, peanuts, sugarcane, white tea, and the seeds of passion fruit (Passiflora edulis
). Its effects have been investigated extensively in the past decade. Several biological functions have been reported as antioxidative, anti-inflammatory, anticancer, antidiabetic, cardioprotective, neuroprotective, and immunomodulatory properties [42
]. The beneficial effects of piceatannol as antioxidant agent on periodontal diseases were not previously described.
Initially, in the present study piceatannol was obtained from yellow passion fruit seeds (Passiflora edulis
). The final concentration of 0.0425 g of piceatannol was found per gram of dried yellow passion fruit seeds. This high concentration is in agreement with a recently reported study (0.0368 g/g dried seeds) [43
]. Purity of piceatannol in purified and dried extract (PDE) achieved 66.4%. After, we tested the piceatannol antioxidant capacity by the standardized method oxygen radical absorbance capacity (ORAC) assay and the results indicated that piceatannol analyzed had 6739 µmol Trolox equivalent/mL of a 1 mM piceatannol confirming the high antioxidant capacity of this stilbene. Once the antioxidant capacity of the piceatannol was, the ability for it to protect against oxidative stress was tested in the fibroblast of the human periodontal ligament cells.
Fibroblasts of the human periodontal ligament are an important cell population responsible for the maintenance of the periodontal ligament integrity and consequent fixation of the dental element to the alveolar bone [44
]. The periodontal ligament is a loose connective tissue formed by several cellular populations, the fibroblast being the most numerous, and also containing a large amount of collagen fibers produced by it immersed in the extracellular matrix [45
]. To exclude the possibility of the cells isolated in this study were periodontal ligament stem cells, immunophenotyping was performed for mesenchymal stem cell markers. The data of flow cytometry analysis demonstrated a low percentage of cells positive for the surface markers CD73 (41.1%), CD90 (27.9%), and CD105 (22.1%), while only 0.18% of cells were positive for CD45, CD34, CD11b, CD19, and HLA-DR (Figure 1
). To confirm that the cells were fibroblasts, indirect immunofluorescence was performed to stain target antigens in this cell population. Vimentin and fibronectin proteins underwent immunostaining confirming the cells were fibroblasts. There was no labeling for cytokeratin, which excluded the possibility of being epithelial cell origin (Figure 2
In order to simulate fibroblast injury by inducing oxidative stress to simulate periodontal disease, we chose hydrogen peroxide (H2
) that has been reported in other studies as capable to easily translocate cell membranes and generate hydroxyl radicals [46
]. In our study, hPLF were exposed to 200 µM H2
for 1 h and injuries related to this exposure were evaluated after combined maintenance of these cells in a solution containing piceatannol at different concentrations. It is important to note that the carrier substance used to dissolve piceatannol (methanol 20%) was tested in all parameters (viability and general metabolism status, membrane integrity and amount of ATP, TEAC and reduced glutathione) at maximum volume necessary to prepare our most concentrated exposure solution (20 µM). No statistical difference was observed when compared to the control.
As expected, hPLF exposed to H2
had a significant decrease of cell viability in 32 ± 12% when compared to control (Figure 3
a). This condition was used to evaluate whether piceatannol in different concentrations (0.1–20 μM) could protect cell from death and act directly on cell viability. We showed that piceatannol at concentrations of 1, 5, and 10 μM maintained cell viability of hPLF in 48.5% (±6.6), 58.3% (±7.2), and 60%(±13.9), respectively (Figure 3
a), indicating a direct effect on cell protection and confirming previous study under the same range concentration (1–10 μM) in the presence of amyloid β-peptides (Aβ) as a inducer of toxicity [47
]. In this study, authors have showed that piceatannol had the ability to increase cell viability up to 60% and 79% when compared to control group. In this way, piceatannol was one of the most effective of stilbenes tested on PC12 cells.
On the other hand, the concentrations of 0.1 µM and 20 μM of piceatannol showed significant decrease of cell viability related to control, but no difference to H2
exposed cells. One possible explanation could be the dual effect of piceatannol. The pro- and antioxidant activity of piceatannol has already been tested in several models of cell culture [48
] suggesting that the piceatannol effect depends on the type of cell, its metabolic activity and phase of the cell cycle analyzed [44
]. In a study with leukemic cells, piceatannol showed potent antioxidant capacity against DNA damage. Its protective activity was 58–58.4% between 1.25 and 5 μM with pro-antioxidant effect between 10 and 50 μM [49
]. The same was observed on astrogliomas cell culture model, with pro-antioxidant effect at concentrations above 20 μM and a cytoprotective capacity at lower concentrations (5 and 10 μM) [48
]. In another study, the piceatannol effect on restoring the endothelial cell activity was evaluated under high-glucose oxidative stress. Interestingly, the piceatannol at 0.01 μM and 0.1 μM did not induce cell death for a 24 h exposure period and was also able to improve DDAH activity and thiol concentration [50
]. These differences between 0.1 μM PIC effect could be explained by the difference on oxidative stress induction by peroxide and high-glucose, but additional studies are required to clarify that.
We also checked the piceatannol-induced effect on the general metabolism status of hPLF. The enzymatic reduction of MTT to formazan served to assess the general metabolism status at the same concentration of H2
and piceatannol used in cell viability assay. Our result showed that the metabolism at 1, 5, and 10 μM of piceatannol remained similar to the control while only 0.1 μM caused a higher metabolism compared to control and H2
b). The metabolism could be closely linked to cell viability, proliferation, and oxidative stress [51
]. In this way, in order to understand how piceatannol could affect hPLF the concentrations of 0.1 and 1 μM piceatannol were chosen to continue the study analysis.
We next checked the cell membrane integrity and ATP production (Figure 4
a). Regarding membrane integrity, our results showed no difference among groups, suggesting that cell death observed in our first test likely occurred by apoptosis since the protease used in this assay is an indicative of cell death by necrosis. Besides that, piceatannol did not protect cells from mitochondrial damage as measured by decrease of ATP production in exposed H2
and piceatannol groups. This can be explained by the ability of stilbenes to inhibit the ATPase activity of ATP synthase, an enzyme that catalyzes the synthesis of ATP molecules by the oxidative phosphorylation process [52
In order to evaluate biochemistry parameters related to antioxidant defenses in the hPLF under oxidative stress conditions, Trolox equivalent antioxidant capacity (TEAC) and reduced glutathione (GSH) levels were assessed. The concentration of 0.1 μM of piceatannol showed the highest TEAC value, whereas the concentration of 1 μM was not statistically different in relation to the control and peroxide groups (Figure 4
The results of GSH, the most important non-enzymatic cellular antioxidant defense [53
] were similar to the results of the TEAC (Figure 5
). A study showed that dose-dependent piceatannol increases the concentration of GSH in keratinocytes but without any induction of injury [53
]. In another study, the intracellular level of GSH was evaluated on melanoma cell (B16 cells). Authors have showed a concentration-dependent increase of GSH levels by 5, 10 and 50 µM [54
]. This increase could be possible a result from directly action of piceatannol on enzymes that synthesize or suppress GSH degradation. In the present study, the increasing GSH at 0.1 μM indicates that the cell is activating more mechanisms of damage control, which is not enough to maintain cell viability. On the other hand, the fact that the concentration of 1 μM was not different from peroxide, suggests that the maintenance of the cellular viability promoted by piceatannol does not depend on the action of GSH and probably another antioxidant route.
Herbal extracts from natural products are considered potential candidates for the treatment of chronic periodontitis, substantially increasing the number of in vitro and in vivo studies related to the efficacy of medicinal plants with known anti-inflammatory and antibacterial actions [55
]. In our study, the effects of piceatannol on hPLF under mimetic effect of inflammatory conditions was concentration-dependent. It was possible to observe the modulation of antioxidant defenses at the concentration of 0.1 μM piceatannol through the increase of TEAC and GSH levels, even though it was not able to maintain cell viability. However, 1 μM of piceatannol was able to maintain cell viability, but did not modulated the antioxidant response. Further studies should be conducted to better understand the mechanisms of antioxidant action of piceatannol in periodontal ligament fibroblasts. We suggest that other parameters of oxidative stress should be analyzed beside the cellular physiology through maintaining the collagen production capacity of the fibroblasts.