Trans-Resveratrol: From Phytonutrient Supplement, to Novel Nanotherapeutic Agent

Abstract

Trans-resveratrol (3,5,4′-trihydroxy-trans-stilbene) (RES) is a plant polyphenol that has been well documented for its anti-oxidant, antimicrobial, anti-inflammatory, and anti-aging properties. Moreover, compelling evidence presented in the abundance of pre-clinical studies using ligature-induced periodontitis models has positioned RES as a theoretically viable candidate for the reduction of the chronic inflammation, oxidative stress, and tissue destruction seen in periodontitis (PD). However, the instability of RES under physiological conditions, as well as its rapid hepatic clearance, has presented as a challenge to its ubiquitous application as an oral therapeutic in clinical practice. Fortunately, with the application of nanotechnology, the pharmacological profile of RES repositions the phytochemical from an herb-based supplement, useful as an adjunct therapy, to a stable and potent nanomedicine, demonstrating efficacy for the prevention and treatment of PD and its associated systemic diseases. This chapter explores the details of the potential for nano-RES as a viable therapeutic for PD.

1. Introduction

Periodontal Disease (PD) is a complex and multifactorial chronic systemic inflammatory condition that manifests via the interplay between dysbiosis of the normal microbiota of the oral cavity, and the dysregulation of the host immune response [1] The shift in the microbiota toward pathogenic organisms, activates host inflammation which persists if left untreated, ultimately progressing to continuous tissue destruction of the supporting tissues around the tooth. The clinical manifestations that result are bleeding gums, gradual tooth detachment and bone loss, which can be painful, and potentially result in edentulism, if left unchecked [1].

Since the global prevalence of PD of some form, is positioned at approximately 50% of the adult population, the growing concern around this approximation is particularly necessary, when one reflects upon the extensive list of immune-related systemic diseases with which PD is associated [2, 3, 4]. That is, PD has been associated with cardiovascular [5, 6, 7, 8, 9, 10, 11], gastrointestinal, respiratory, renal, hepatic, neurodegenerative, and autoimmune disease; obesity, diabetes, viral infections, adverse pregnancy outcomes and some cancers have also been reported to be associated with PD. Moreover, due to the systemic nature of immune dysregulation and pathogenic dysbiosis, the relationship between PD and many of these comorbidities is bidirectional [12].

One cannot emphasize more, the multifaceted pathogenesis of PD, which involves the interchange between the host tissues and the accompanying microbial communities of the oral cavity, and the microenvironment therein. For this reason, clarification of the details of the pathogenic mechanisms continues to unfold. Fortunately, knowledge gained from studies on the oral microbiome, has recently emerged [13, 14, 15].

In this way, understanding further details of the disease process of PD from the perspective of the oral microbiome can assist in the creation of novel preventative and therapeutic applications.

The oral microbiome (OM), which has been estimated to consist of greater than 10⁸ microbes per milliliter of saliva, is considered by many, as one of the most clinically important collections of microbial organisms in the human body [16]. Both prokaryotic and eukaryotic microorganisms make up the estimated 1000 microbial species of the OM. However, more than 700 microbes are prokaryotic, giving investigators a rationale for focusing on the bacterial taxa of the oral cavity when seeking to answer questions about the nature of oral health and disease [17, 18, 19].

Practicably, by gaining a deeper understanding of the mechanisms employed by “keystone” bacterial essential players, which are Gram negative anaerobes (e.g., red-complex pathogens, Porphyromonas gingivalis (Pg), Treponema denticola and Tannerella forsythia) in the inflammation process, one may be in a better position to produce an effective and up-to-date arsenal of remedies for the prevention, management, and resolution of PD [20].

It is worth noting that part of the survival mechanism for Gram negative bacteria is the dissemination of outer membrane vesicles containing various virulence factors which result in a distorted immunological dysregulation and tissue destruction [21, 22]. This strategy is a likely explanation for the discovery of pathogenic bacterial DNA at sites distal from the oral cavity. Further to this, Gram negative bacteria contain the virulence factor lipopolysaccharide (LPS) in their outer membrane, which acts as a constant inflammatory trigger via its pathogen-associated molecular patterning (PAMP). Interestingly, antibiotic therapy is not a straight forward solution, since dead Gram negatives also release LPS [23].

The treatment principle of PD is founded in the elimination of the pathogenic dysbiotic biofilm, whilst addressing the dysregulated immune response of the host. These treatment goals are first addressed via the mechanical removal of subgingival pathogenic dysbiotic plaque and tartar buildup, which is accomplished by the gold standard procedure, scaling and root planning (SRP). As a general rule, SRP is followed by lifelong comprehensive care, which may include subgingival irrigation with antibiotic solutions (www.NHS.uk; www.ADA.org). However, instances involving extensive tissue destruction require surgical intervention, which is more invasive and can be costly. Additionally, as a supplementary treatment, host modulation therapy (HMT) may be employed to address the dysregulation of the immune system that was triggered by dysbiosis [24].

Some phytochemicals have been identified as potential modulators of dysbiotic oral biofilms as well as mitigators of host inflammatory responses (e.g., Curcumin, Hesperidin, Silymarin, Resveratrol) [25, 26, 27]. Pharmacognosy, therefore, has been explored as a contributing modality for the mitigation of inflammatory disease.

Indeed, there is a body of evidence in the scientific literature outlining the potential of various forms of phyto-therapeutic intervention for the prevention and treatment of PD. Among these phytochemicals is the trans-isomer of the polyphenol, resveratrol (RES), which has been reported to alleviate many inflammatory conditions, including those associated with PD. RES has also demonstrated favorable synergistic effects on inflammation when combined with other bioactive plant-based compounds such as curcumin [28].

2. Attenuation of the Dysbiotic biofilm by RES

Since PD is initiated by a dysbiotic biofilm, modulation of the oral microbial ecosystem is a viable upstream therapeutic approach. Also, the reduction of pathogenic microbial load is a logical and necessary start to treatment, which is the primary intent of SRP and CC (www.NHS.uk; www.ADA.org).

Studies assessing the potential for addressing the pathogenic oral biofilm, reported that RES demonstrated antimicrobial action against PD pathogens. For example, O’Connor and colleagues conducted an in vitro study on different prokaryotic and eukaryotic microbes using the gold standard antiseptic rinse used in dentistry, chlorohexidine (CHX), as a positive control. They tested the inhibitory effect of RES on 15 different microbial species and only A. actinomycetemcomitans (Aa) and P. gingivalis (Pg) (ATCC2533277) showed sensitivity to RES. The authors also pointed out that the other 13 microbes were aerobic, suggesting that RES is specifically antimicrobial to anaerobes. In fact, Pg showed a much higher sensitivity to RES than Aa did in this study, suggesting that Pg may be especially vulnerable to RES, for reasons other than its anaerobic nature. However, since this was a study based on spectrophotometric optical density readings of broth cultures, the bacteria tested were planktonic and therefore not comparable to those participating in the biofilm seen in PD. Hence, although this study was a helpful preliminary survey, the definitive demonstration of the action of RES against Pg in vivo, was not solidified [29].

Interestingly, in 2019, Kugaji and colleagues assessed the effect of RES on an experimental biofilm both directly and indirectly. Firstly, direct assays were conducted via the determination of the minimal inhibitory and minimum bactericidal RES concentrations (MIC and MBC, respectively) applied to cultures of commercially available (e.g., ATCC 33277) and clinical strains (CS02) of Pg. Then, the calculated indices for MIC (156.25 μl/ml) and MBC (312.5 μl/ml) of RES, were applied to commercial strains of Pg to determine kill-time, adhesion to substrate, and morphological changes (determined by SEM). Next, RES was evaluated indirectly through the examination of the genetic expression of gingipains (i.e., Kgp and rgpA) and fimbriae (i.e., fimA), virulence factors from Pg that have been found to be instrumental in promoting the formation of the pathogenic biofilm in PD. The findings led the authors to conclude that RES displays antimicrobial action against keystone periodontal pathogen, Pg, as well as the ability to reduce the pathogenic biofilm indirectly, by decreasing the expression of its proteolytic virulence factors. It is also worth noting that in this study, the clinical Pg strain was more sensitive to RES than the commercially available strain. Regarding adhesion assays, the response to RES was also strain-dependent, which adds another layer of complexity regarding the in vivo translation of these findings [30].

Similarly, both antibacterial and anti-adherent action of RES on Pg (i.e., ATCC 33277) were reported by Lagha and colleagues in 2019. They determined an MIC and MBC of 250 μL/mL and 500 μL/mL, respectively, and applied a luminescence assay as an indicator of ATP production, to show the bioactivity of the bacteria in the experimental biofilm. The calculated MIC of RES in this study reduced biofilm viability by over 50% [31].

Conversely, Millhouse and colleagues reported that RES did not demonstrate any antimicrobial action, after they developed an experimental model of a periodontal biofilm, which consisted of a co-culture of epithelial and microbes. This intricate co-culture included simulated saliva, and was designed to mimic the host-biofilm interface to assess the antimicrobial and anti-inflammatory properties of Chlorohexidine (CHX), compared with those of RES [32].

Since it has been reported that Pg demonstrates significant differences in genetic expression when planktonic compared to being engaged in a multispecies biofilm [33], evolving such a model is an important step toward in vivo translation [32].

However, the results from Milhouse et al. cannot be attributed to the co-cultured biofilm model alone because the RES was suspended in water, while the other three studies, which reported antimicrobial action from RES, used ethanol or dimethyl sulfoxide (DMSO). Hence, the comparability between the studies is diminished by the fact that RES has virtually no solubility in water, while DMSO and ethanol are more successful as solvents [34], but often have unpredictable biological effects of their own, which must be appropriately controlled for.

Further to this, an in vivo study conducted by Cirano and colleagues, employed a modified ligature-induced rat model to evaluate the action of RES against key pathogens, Pg, T. forsythia (Tf) and Aa. This study applied ligatures pre-treated with the individual pathogens and evaluated the antimicrobial action of RES using RT PCR of all three microbial species, accompanied by a single daily gavage of a 10 mg/Kg dose of RES over a 30-day period. In this case, no statistically significant antimicrobial activity was demonstrated by RES. However, although the RES stock was prepared using ethanol, further dilutions were performed in water, which decreases the solubility of RES. The oral delivery route (gavage) in this study suggests that low oral bioavailability [35] and instability [34] of RES may have affected the results. Consequently, the authors recognize that for future studies, applying RES via topical/transmucosal administration in the in vivo models may overcome this limitation [36].

Although these studies cannot definitively conclude that RES demonstrates antimicrobial activity in vivo, further studies may bring to light the antimicrobial status of RES, as more information emerges about the oral microbiome and the mechanisms of the intercellular interactions that govern dysbiotic biofilm formation. What is clear from this analysis is that more consideration needs to be given to the solubility, formulation, and route of delivery of RES. Indeed, in vivo experimental designs may benefit from routes of administration of RES that bypass its low bioavailability and stability, as new models are evolved that provide more consistent and translatable data.

3. Modulation of host immune response by RES

Investigations using ligature-induced PD animal models, have consistently reported the favorable modification of many pro-inflammatory markers as well as the reduction and/or arrest of tissue destruction. Table 1 outlines the results of a collection of animal studies and the molecules affected by RES [28, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46].

Inflammatory Mediator and RES Action on Mediator	Action of Molecule	Formulation and Administration of RES	Type of Assay Used to Detect the Inflammatory Mediator/Marker	Tissue Sampled
IL-1β Decreased	Pro-inflammatory	Gavage 10 mg/Kg in 2% EtOH [37]; PO 10 mg/Kg Melinjo seed extract [38]; Gavage 20 mg/Kg [39]	RT-PCR [37]; RT-PCR [38]; RT-PCR [39]	Gingival tissue [37]; Gingival tissue [38]; Gingival tissue [39]
IL-4 Increased	Anti-inflammatory	Gavage 10 mg/Kg in Tween-80 /ddH2O [28]; Gavage 10 mg/Kg in Tween-80/ddH2O [40]; Gavage 10 mg/Kg in Tween 80/ddH2O [41]	ELISA [28]; ELISA [40]; ELISA [41]	Gingival tissue [28]; Gingival tissue /Blood [40]; Gingival tissue [41]
IL-6 Decreased	Pro-inflammatory	Into socket 50 μM in 0.1% DMSO [42]; Gavage 10 mg/kg in 2% EtOH [37]; PO 10 mg/Kg Melinjo seed extract [38]; Gavage 20 mg/Kg [39]	Immunohistochemistry [42]; RT-PCR [37]; RT-PCR [38]; RT-PCR [39]	Periodontium [42]; Gingival tissue [37]; Gingival tissue [38]; Gingival tissue [39]
IL-8 Decreased	Pro-inflammatory	Gavage 20 mg/Kg [39] diabetic mice	RT-PCR [39]	Gingival tissue [39]
IL-17 Decreased	Pro-inflammatory	Gavage 10 mg/Kg in EtOH/ddH2O [43]	ELISA [43]	Gingival tissue [43]
TNF-α Decreased	Pro-inflammatory	Into socket 50 μM in 0.1% DMSO [42]; Gavage 10 mg/kg in 2% EtOH [37]; PO 10 mg/Kg Melinjo seed extract [38]; Gavage 20 mg/Kg [39]	Immunohistochemistry [42]; RT-PCR [37]; RT-PCR [38]; Serological [39]	Gingival tissue [37, 42]; Gingival tissue [38]; Blood [39]
IFN-ɣ Decreased	Pro-inflammatory	Gavage 10 mg/Kg in Tween-80 /ddH2O [28]	ELISA [28]	Gingival tissue [28]
RF Decreased	Pro-inflammatory	Gavage 10 mg/Kg in Tween-80/ddH2O [40]	ELISA [40]	Blood [40]
SOD Increased	Antioxidant	Gingival injection/5 mg/kg in DMSO [44]; Gavage 10 mg/kg in Tween −80 [45]	Serological [44]; ELISA [45]	Blood [44]; Gingival tissue [45]
HO-1 Increased	ROS/RNS stress defense	Gingival injection/5 mg/kg in DMSO [44]	Western Blot [44]; Immunohistochemistry [44]	Periodontium [44]
COX-2 Decreased	Pro-inflammatory	Gingival injection/5 mg/kg in DMSO [44]	Immunohistochemistry [44]	Periodontium [44]
MMP-2 Decreased	Destructive protease	Gingival injection/5 mg/kg in DMSO [44]	Immunohistochemistry [44]	Periodontium [44]
MMP-9 Decreased	Destructive protease	Gingival injection/5 mg/kg in DMSO [44]	Immunohistochemistry [44]	Periodontium [44]
Nrf2 Increased	Antioxidant	Gingival injection/5 mg/kg in DMSO [44]; PO 10 mg/Kg Melinjo seed extract [38]	Western Blot [44]; Immunohistochemistry [44]; RT-PCR [38]	Periodontium [44]; Gingival tissue [38]
SIRT-1 Increased	Antioxidant	Gavage 10 mg/Kg in Tween-80/ddH2O [45]; PO 10 mg/Kg Melinjo seed extract in [38]	RT-PCR [45]; RT-PCR and Western blot [38]	Gingival tissue [45]; Gingival tissue [38]
ACCPA Decreased	Destructive protease; Antibody	Gavage 10 mg/Kg in Tween-80/ddH2O [40]	ELISA [40]	Blood and Gingival tissue [40]
NADPH-OX Decreased	Source of ROS damage	Gavage 10 mg/Kg in Tween-80/ddH2O [45]; Gavage 10 mg/Kg in Tween-80/ddH2O [46]	ELISA [45]; ELISA [46]	Gingival tissue [45]; Gingival tissue [46]
NF-κB Decreased	Pro-inflammatory Transcription Factor	PO 10 mg/Kg Melinjo seed extract [38]; Gavage 20 mg/Kg [39]	RT-PCR [38]; Western blot [39]	Gingival tissue [38, 39]
NQO-1 Increased	Anti-oxidant gene	PO 10 mg/Kg Melinjo seed extract [38]	RT-PCR [38]	Gingival tissue [38]
AMPK Induced Phosphorylation	Anti-inflammatory	PO 10 mg/Kg Melinjo seed extract [38]	Western blot [38]	Gingival tissue [38]
P38/MAPK Decreased Phosphorylation	Pro-inflammatory	PO 10 mg/Kg Melinjo seed extract [38]; Gavage 20 mg/Kg [39]	Western blot [38]; Western blot [39]	Gingival tissue [38]; Gingival tissue [39]
STAT3 Decreased Phosphorylation	Possible Inducer of Diabetic PD	Gavage 20 mg/Kg [39]	Western blot [39]	Gingival tissue [39]
p65/NF-κB Decreased Phosphorylation	Pro-inflammatory Transcription Factor	Gavage 20 mg/Kg [39]	Western blot [39]	Gingival tissue [39]
Inos Decreased	Pro-inflammatory	PO 10 mg/Kg Melinjo seed extract [38]	RT-PCR [38]	Gingival tissue [38]
8-OHdG Decreased	Oxidative stress marker	PO 10 mg/Kg Melinjo seed extract [38]	ELISA [38]	Urine [38]
Dityrosine Decreased	Oxidative stress marker	PO 10 mg/Kg Melinjo seed extract in [38]	ELISA [38]	Urine [38]
RANKL Decreased	Increases bone resorption	Gavage 10 mg/kg in 2% EtOH [37]; Gavage 10 mg/Kg in Tween-80/ddH2O [41]	RT-PCR [37]; RT-PCR [41]	Gingival tissue [37]; Gingival tissue [41]
NOx Decreased	Nitrosative stress marker	PO 10 mg/Kg Melinjo seed extract [38]	Serological [38]	Serum [38]
Nitrotyrosine Decreased	Nitrosative stress marker	PO 10 mg/Kg Melinjo seed extract in [38]	ELISA [38]	Serum [38]
RUNX-2 Decreased	Osteoblastic marker	Into socket 50 μM in 0.1% DMSO [42]	Immuno-histochemistry [42]	Periodontium [42]
OCN Increased	Osteoblastic hormone	Into socket 50 μM in 0.1% DMSO [42]	Immuno-histochemistry [42]	Periodontium [42]
Ki67 Decreased	Proliferation	Into socket 50 μM in 0.1% DMSO [42]	Immuno-histochemistry {37}	Periodontium [42]

Table 1.

Ligature-induced PD studies with the RES formulation, type of assay and tissue sampled for the inflammation-mediating molecules examined.

Moreover, several reports from in vitro studies have also reliably concluded that RES mitigates the dysregulated immune response seen in PD [47, 48]. For example, a study by Qu et al. used Porphyromonas endodontalis (Pe)LPS-challenged osteoblast-like mouse cells (MC3T3-E1) to identify the role of silent mating type information regulation 2 homolog 1 (SIRT-1) in the resolution of inflammation and mitigation of bone loss. Their model employed the transfection of the cells with SIRT-1 siRNA, as well as the use of SIRT-1 inhibitor, EX-527, to silence SIRT-1 activity, whilst RES was used as a SIRT-1 activator [49].

Notably, elevated levels of matrix metaloprteinase-13 (MMP-13) expression, as determined by RT-PCR, ELISA and Western blot, were evidenced upon induction by LPS. Furthermore, this induction was increased by knockdown of SIRT-1 activity, either by use of siRNA SIRT-1, or by the addition of the inhibitor, EX-527. However, pre-treatment with RES (50 μM, no solvent was specified) for 1 hour, significantly (p < 0.05) suppressed the mRNA expression (as determined by RT-PCR) and protein production (assayed by Western blotting and ELISA) of MMP-13 in the LPS-challenged cells. A chromatin immunoprecipitation assay (ChIP) then determined that SIRT-1 prevents the activation-stimulating binding of NFkB-p65 to the MMP-13 promoter [49], which aligns with the findings reporting RES as a SIRT-1 activator [50, 51, 52].

An early human in vitro study reported the use of RT-PCR and ELISA, to determine that RES decreased the mRNA levels of a range of cytokines regarded to be proinflammatory, including IL-1β, IL-6, IL-8, IL-12 and TNF-α, in Pg lipopolysaccharide-(LPS)-challenged human periodontal ligamental cells. The effect was reported to be both dose-(25 μM, 50 μM and 100 μM) and time-(0–72 hrs) dependent [53].

Similarly, more recent in vitro studies employing ELISA, found that RES significantly decreased IL-6 and IL-8, in human gingival fibroblasts (HGFs) that were stimulated by LPS [26, 54]. RES was also combined with the triterpene, Celastrol (CEL), and loaded into a collagen film to enhance the effectiveness of coated dental implants by Wang et al. [55]. Using SEM and histochemistry, it was determined that the RES-loaded collagen films stimulated the most proliferation of the human periodontal ligamental fibroblasts (HPLFs), whilst the CEL-loaded films, demonstrated the lowest bone marrow macrophage-mediated osteogenesis. Hence, it was suggested that using both RES and CEL in the collagen film was a promising approach to the development of an efficacious dental regenerative agent [55].

In 2020, Ashour et al., used Hesperetin (HESP) as a glyoxalase 1(Glo1) inducer to assess its modulation of the damage caused to HPLFs from high glucose. This experimental in vitro model showed that HPLFs overloaded with glucose, increased the cellular concentration of methylglyoxal (MG) and its modified proteins, which resulted in HPLF dysfunction and inability to bind to collagen-I. By adding RES (10 μM) to the HESP, the dysfunction was corrected through the attenuation of deregulated glucose metabolism [56].

An additional study compared the anti-inflammatory activity of Tetracycline (TC), Minocycline (MC), Quercetin (QU), and RES, in LPS-stimulated macrophage-like mouse cells, (RAW264.7). Upon the application of differential scanning calorimetry, and RT-PCR, the anti-inflammatory activity was determined as an index of COX-2 inhibition and it was concluded that the order from highest anti-inflammatory action to lowest was as follows: QU > RES > MC > TC [57].

4. Restoration of damaged periodontal tissues by RES

An extensive in vitro and in situ investigation by Wang et al., using human periodontal ligamental stem cells (HPLSCs) treated with TNF-α, found that RES conserved their osteo-differentiation. Here, histological analysis showed that RES preserved the formation of cell aggregates and made the cells more resilient to TNF-α—induced inhibition of alkaline phosphatase (PhoA) whilst promoting mineralization. Further to this, RT-PCR was used to show the partial restoration of mRNA expression of the osteo-differentiation drivers, OCN, RUNX2, PhoA and Collagen-1. Moreover, Western blotting showed restored values for OCN, Collagen-1, and RUNX2, an osteoblastic modulator, confirming the mRNA findings at the level of protein. The in vitro action of RES on the ontogenetic capabilities of HPLSCs originating from PD patients was also determined to be beneficial through histological analysis, RT-PCR and Western blot. Additionally, an ectopic regeneration experiment was conducted, involving transplantation of normal, PD alone, or RES-treated PD-induced HPLSC aggregates into nude mice, which allowed the researchers to assess the regenerative action of RES in situ [58].

An additional in vitro study on HPLSCs, employing histochemical analysis, RT-PCR, Western blot, as well as ELISA, reported that the osteogenic suppression induced by treatment with TNF-α was mitigated by RES. Also, the results of ERK1/2 pathway inhibition and activation assays showed that the anti-inflammatory action of RES was diminished by ERK1/2 pathway inhibition. This suggests that the mechanism employed by RES, involves activation of the ERK1/2 pathway in TNF-α-challenged HPLSCs [59].

Interestingly, Kudo et al., examined the role of SIRT1 in angiogenesis in PD by treating human umbilical vein endothelial cells (HUVECs) with E. coli LPS plus RES (i.e., a SIRT1 activator) or sirtinol (i.e., a SIRT1 inhibitor). Also, in this study, periapical granulomas were obtained from PD patients, whereas healthy individuals provided normal periodontal tissues, which were also assayed. Immunofluorescent imaging using antibodies against SIRT1 and Ki67, showed that the periapical granulomas had higher expression of both SIRT1 and Ki67, compared to healthy gingival tissue. Additionally, the quantification of the mRNA of SIRT1, VEGF (which stimulates endothelial growth and differentiation into vessel morphology) and VE-cadherin (an adhesion protein for endothelia) in the LPS-induced HUVECS indicated that the mRNA expression statistically increased for all three proteins (P = 0.0019 (SIRT-1), 0.00005(VEGF) and 0.0045 (VE-cadherin)) in the RES-treated groups compared to the group treated with sirtinol [60].

5. Mitigation of systemic conditions associated with PD by RES

Studies have been conducted integrating RA, DM, cigarette smoking or osteoporosis (OP) into their induced-PD models. For example, two studies employed a ligature-induced PD rat model which incorporated three, eight-minute exposures per day, to the equivalent of 10 cigarettes, for 37 days, and examined the effects of RES on bone loss and oxidative stress, respectively [40, 41]. In this study, they found that the gingival tissue of the RES-treated group displayed higher expression of anti-inflammatory cytokine IL-4, and the antioxidant SIRT-1 [40], suggesting that RES plays a beneficial role when the added risk factor of smoking is involved. Further to this, RT-PCR determined that the mRNA expression of the osteoclastic inducer, RANKL was diminished by RES [41] and ELISA showed that the ROS inducer, NADPH oxidase, was also significantly diminished in the RES-treated group (i.e., RES + Cigarette Smoke) whilst SOD was significantly higher (p < 0.05) (Table 1) [37].

Moreover, the administration of RES was found to decrease the TH17/TH2 cell ratio in diseased gingival tissue, suggesting that it may modulate the overstimulation of TH17 (i.e., TH lymphocytes purported be stimulated by the outer membrane protein of Pg) production, which contributes to tissue destruction [38, 61]. These results also indicate that RES may assist in the mitigation of the periodontal damage contributed to, by the modifiable risk factor, smoking [38]. Additionally, a study which assessed the effect of RES on experimental PD in diabetic mice, found that RES decreased the mRNA expression of LPS—induced inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α. In addition, the LPS-induced phosphorylation, and therefore activation, of transcription factors downstream to TLR4, p65 NF-κB, p38 MAPK, and STAT3, was also suppressed by RES, suggesting that RES works through the TLR4 signaling pathway [39]. Moreover, RES was also found to reduce alveolar bone loss and attenuate hyperglycemia [39, 56], demonstrating its potential as an attenuator of diabetic PD.

An additional study conducted by Correa and colleagues, examined the effect of RES compared to Ibuprofen on experimental PD and RA combined, using the ligature-induced PD rat model. Here, they determined by way of ELISA, that the administration RES or Ibuprofen resulted in the reduction of anti-cyclic citrullinated peptide antibody (ACCPA) in the tissues by 72% and 99%, respectively (p < 0.05), and, RES alone was reported to reduce serum rheumatoid factor (RF)(p < 0.05). In this study, RES also demonstrated sustained reduction in paw edema compared to Ibuprofen. Moreover, the RES treated group demonstrated reduced articular damage in the histological analysis, indicating its ability to modulate RA-induced damage in the context of experimental PD in rats [40].

Similarly, in an animal study, using ovariectomized rats and PD-induction via ligature installment, RES was compared to Zoledronate, a drug used to treat osteoporosis (OP). Here, ELISA showed that RES downregulated NADPH oxidase and reduced alveolar bone loss, but not as drastically as in the Zoledronate-treated group. Nonetheless, the results suggest that RES may attenuate alveolar bone loss in estrogen-deficient rats via the attenuation of NADPH oxidase, making NADPH oxidase a potential drug target for RES. Using a human in vitro neuroinflammation model, it was determined that several pathways that promote oxidative stress (via the decrease in AKT1, FOS, IKBKB, IRF1, JAK2, NFKB1PIK3RI, RELA, STAT1, TNF-α and TNFRSF1 mRNA) as well as iNOS (via the decrease in FOS, IKBKB, IRAK1, IRAK2, IRF1, JAK2, NFKB1, RELA and STAT1 mRNA) was attenuated by RES. The researchers in this study used qPCR and biochemical pathway analysis to examine 96 genes, present in human neuroblastoma cells, that were induced into inflammation via the application of LPS originating from Pg. It was concluded that RES decreased NF-κB-mediated acute inflammation pathways in human neuroblastoma cell culture. Further to this, RES was also reported to inhibit IGF-1 and Insulin receptor while activating the PTEN, PPARa/RXRa, PPAR metabolic pathways. This extensive in vitro study showed the potential for RES to address Pg-related disease, with a particular focus on the prevention of AD [62].

6. Clinical translation of RES

Two randomized clinical trials were conducted by Javid et al. (IRCT ID: IRCT2015012420765N1). One study evaluated the effect of RES on blood glucose, Insulin, Insulin resistance and periodontal markers in 43 type two DM patients aged 30–60 years old, with chronic moderate PD. This double-blind and placebo-controlled study, evaluated the impact of RES by supplementing the experimental group with 480 mg capsules containing RES, once a day, for 28 days. The subjects in the placebo group were given identical capsules containing 480 mg of starch. Although no significant differences in fasting blood glucose or TGs between the groups were evident, the RES-supplemented group showed significantly lower (p < 0.05) periodontal pocket depth, serum fasting glucose and insulin resistance [63].

The second study, also a randomized clinical trial, assessed the serum levels of inflammatory cytokines IL-6 and TNF-α, as well as total antioxidant capacity (TAC) and clinical attachment loss (CAL) in 43 patients with type two DM and PD. After 28 days of supplementation with 480 mg/d of RES, serum levels of IL-6 were significantly lower(p = 0.039), but the other parameters measured, showed no significant change compared to control [64]. The results of both studies do not fully align with those of the animal studies, which may be partly due to the formulation and posology used. That is, Javid et al., applied a 480 mg capsule of Polygonum cuspidatum reported to contain 240 mg of RES. In general, the animal studies used 10–20 mg/kg of pure RES, delivered via gavage, which would calibrate to approximately 730–1460 mg of RES in this study, considering the average mass of the human subjects in the RES-treated group (mean mass of subjects =73.8 ± 10.2 Kg) [63, 64].

Lastly, Shoukheba, conducted a six-month human study in 2020 consisting of 15 male smokers with moderate to severe (i.e., CAL >5 mm) PD, following SRP. Interestingly, RES gel (0.001% w/v final concentration) was directly applied to the testing sites (15 healthy, 15 with PD), applying the split mouth method at days 7,14, and 21. The clinical parameters, plaque index (PI), probing pocket depth (PPD), bleeding index (BI)and CAL were assessed. In addition, the gingival crevicular fluid was collected during the treatment visits to assess SOD levels. The SOD levels were higher in the RES group compared to control and although both groups showed a significant decrease (p < 0.05) in the clinical parameters, PPD and CAL, at the three-month mark, only the RES-treated group demonstrated significant improvement from baseline at 6 months (p < 0.05) [65].

Again, further studies using RES in the absence of SRP, need to be conducted to fully elucidate the role of RES in the attenuation of PD. In addition to this, since cigarette smokers have been reported to have higher susceptibility to an oral Pg infection (Zeller et al., 2014), perhaps more information may be gained by comparing the Pg load between groups [65].

RES has a long-term body of evidence reporting its low bioavailability, which is mainly due to its physicochemical properties. That is, RES is pH-, thermo-, and photo-sensitive, [66]. More importantly, RES is unstable under physiological pH (7.4) and temperature (37°C), thus diminishing the in vivo translatability [67, 68, 69].

There are a myriad of studies demonstrating the effective mitigation of the low bioavailability, stability and pharmacokinetic profile of RES via nano-formulation. In this way, RES may be potentially upgraded from a supplement to a nanomedicine.

7. Improvement of RES as a therapeutic via nanotechnology

Nanotechnology, which employs materials that measure at <100 nm in at least one dimension, has been found to improve the stability, bioavailability and activity of RES. Overall, there exist several studies exploring the action of various formulations of Nano-RES which reported improvements in stability, bioavailability and pharmacokinetics, compared to RES in bulk form [70, 71, 72, 73, 74, 75]. For example, nano-formulations employing solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs), are purported to have higher stability, and a pharmacokinetic profile indicative of sustained release. Interestingly, SLNs and NLCs have more economical scalability compared to liposomal systems [70], making them attractive options as RES carriers.

Successful production of the SLNs and NLCs may be accomplished using waxes, fats, oils, or combinations thereof, combined with surfactant, and the application of high-pressure shear homogenization [71], sonication [72] and [73], or both [74, 75]. Studies involving RES-loaded SLNs and NLCs, demonstrated higher stability compared to bulk RES, as indicated by the high zeta potentials reported (i.e., ζ > 20 mV, [74] and ζ < -20 mV, [71, 72, 73, 75]).

Furthermore, Singh and Pai reported a self-nano-emulsifying drug delivery system (SNEDDS) that enhanced the pharmacokinetic profile of RES in vivo. Here, the RES-loaded SNEDDS was administered orally to rats (at 20 mg/Kg body weight dose) before withdrawing serial aliquots of blood. The SNEDDS was designed to automatically form a nano-emulsion once in contact with the fluids of the gastrointestinal tract and spontaneous emulsification into stable (ζ = −29.76 mV), 57 nm nano-globules took place in 43 seconds after being placed in 0.1 M HCl. The authors noted that the enhanced oral availability of RES-loaded SNEDDS compared to crude RES was likely due to an increase in absorbability as well as diminished first pass clearing by the liver enzyme CYP3A4 [76].

Moreover, Al-Bishri and colleagues compared the anti-diabetic activity of a commercial nano-emulsion of RES (Life Enhancement, Petaluma, California, USA) with that of chromium picolinate in streptozocin-induced diabetic in rats. Two weeks after the induction of DM, rats in the experimental groups were orally administered either (80 μg/Kg body weight) or Nano-RES emulsion (20 μg/Kg body weight) every day for 30 days when serum levels for glucose insulin, as well as biomarkers: NO, SOD, CAT, GPX, GST and GSH were determined. Both nano-RES emulsion and Chromium picolinate demonstrated inhibition of the oxidative stress induced from hyperglycemia [77].

Further to this, explorations emphasizing the fortification process involved in functional food production, demonstrate the potential of functional foods to be administered as an effective prevention, management, and treatment of PD. For example, Ahmad and Gani, assessed the biological action of RES-fortified snacks and reported that starch nano-encapsulation improved the thermostability of RES whilst enhancing anti-diabetic and anti-obesity effects compared to bulk RES, which was determined by the percent inhibition of the enzymes: α-glucosidase and, pancreatic lipase, and cholesterol esterase, respectively [78]. Inhibition of lipid peroxidase, which was also reported, demonstrated antioxidant activity of the nano-formulated food complex. Moreover, since the nano-embedded RES-fortified designer food-snacks exhibited enhanced desired activity at physiological pH (7.4) and temperature (37°C) the formulation in this study shows promise as a functional food [78].

Similarly, Jayan et al. reported the sustained release of RES from ZEIN-encapsulated nanoparticles (NPs) under physiological conditions (i.e., pH 7.4, 37°C) [79] and casein-encapsulated RES NPs, designed by Penlava et al., were found to be stable through a continuous pH range mimicking those of the gastrointestinal compartments (i.e., pH 1.2 for 2 hours and pH 6.8 for 2–24 hours). Interestingly, the latter study also demonstrated in vivo (using rats), a ten-fold increase in oral availability of casein-nano-encapsulated RES compared to the bulk form as determined by blood plasma assays over a 24-hour period following a single oral dose of 15 mg/Kg of RES (in ddH2O and PEG) or Casein-encapsulated RES NPs [80].

Additionally, Rabbani and colleagues set out to explore the fortification of foods and reported that when enhanced with a nano-encapsulated formulation of RES, mayonnaise demonstrated extended shelf-life, as suggested by the reduction in peroxide value over a six-month period. The ability of the nano-RES food complex to neutralize DPPH free radicals was investigated and the initially high number of free radicals present in the mayonnaise nano-RES complex were attributed to the successful encapsulation (and therefore protection) of RES in the nano formulation. Additionally, characterization of the mayonnaise nano-RES complex was conducted using XRD and Fourier transform infrared (FTIR) spectroscopy and RES was found to be amorphous (via XRD), demonstrating its incorporation into the complex, and it was confirmed that the molecular structure of the drug was retained while interacting with the complex via hydrogen bonds (via FTIR spectroscopy) [81].

Additionally, a promising 2021 investigation by Berta et al., involved the design of an oral Nano-RES spray via encapsulation of crude RES in 2-hydroxypropyl- β-cyclodextrins (i.e., RES-HPβCD) and tested its action on plaque formation in children. This study demonstrated that after using the spray once per day, the RES-HPβCD plaque was significantly reduced; the spray was also found to be doubled in efficacy when compared to tooth brushing alone [82].

8. From trash to treasure: Sources of trans-RES

RES is widely known in the Western hemisphere as a product from the skin of red grapes but there are other viable sources. In fact, the most abundant source of RES is found in roots of the plant, P. cuspidatum (i.e., Japanese knotweed), which has been used for a while in the East as a Traditional Chinese Medicine (i.e., ko-jo-kon) for the treatment of ailments such as cardiovascular disease. Unfortunately, this valuable resource has been wasted in the West, where it is perceived as an invasive species to be burned and buried. Since Japanese knotweed is edible and consists of more than 90% RES, adjustments to the current perception of the plant, could benefit many, as awareness of its medicinal (and potentially economic) value has been surfacing in the western scientific literature over recent decades [83].

Commercially available micronized versions of pure RES powder have been available for purchase for some time (e.g., www.megaresveratrol.net and www.Biotivia.com). However, nano-formulations of RES for oral use (many are topical [84]) have just began to emerge within the last year (e.g., www.oic.com.vn/en/ (Vietnam) www.nanoceuticalsolutions.com (USA), and www.hiimmune.com/product/nano-resveratrol-30ml/ (UK)).

9. Conclusion

According to the body of evidence explored in this chapter, the future of Nano-RES as a viable medicine for the prevention, management and treatment of PD and its associated systemic diseases, is promising.

In addition to nanomedicines for treatment, the development of functional foods fortified with Nano-RES, which demonstrate the potential for the preventative measures, may be an important addition to our dietary regimen moving forward [85].

Further to this, to realize the full potential of Nano-RES, an initiative is needed, to encourage more clinical research on the efficacy of low-cost, phytonutrient-based, novel nano-formulations, specifically aimed to address PD.

This could result in the commercial availability of high quality, ubiquitously accessible, effective RES-based Nano-formulations, and ultimately, a step toward a healthier future population.