Use of Antioxidants in Oral Healthcare

The use of specific antioxidants in the proper combination can provide natural protection from environmental and inherent free-radical exposure. Antioxidants neutralize damaging free radicals that produce disease states. It has been suggested that the negative effects of nicotine could be reversed by antioxidants.¹ Antioxidants are available from different sources, including vitamins, minerals, enzymes, and hormones, as well as food and herbal supplements. The supplements may exist in bar, capsule, drops, liquid, powder, gel, and tablet forms. As an alternative medicine, herbal therapy is a treatment modality to remedy many medical and dental conditions. Antioxidants have also been used in combination with dried, fresh, and blended herbal paste. The majority of herbal supplements include green tea catechins,² aloe vera, star anise oil, myrrh gum, calendula extract, ammonium glycyrrhizate (from licorice root), fennel oil, and neem extracts.^3,4 Most recently, dental manufacturers and distributors have incorporated antioxidant supplements into toothpastes, mouth rinses/mouthwashes, lozenges, fluoride gels and dentifrices, oral sprays, breath fresheners, and other dental products for the control of gingival and periodontal diseases. The rapidly advancing field of dental pharmacotherapeutics has paved the way for the development of a wide array of antioxidants that have beneficial clinical effects.⁴ However, it is important to understand, given the inherent unstable nature of antioxidants, that simply adding an antioxidant to an oral preparation may not be an effective treatment.

Leaf and Berry Extracts

In addition to its antioxidant features, many other extracts have been used in in vitro studies to demonstrate antioxidative potential. A recent study concluded that the lactoferrin and black tea polyphenols had protective effects in vivo against carcinogen activation, DNA damage, cell proliferation, invasion, and angiogenesis in an experimental oral carcinogenesis model.⁵ Black tea theaflavin (TF) monomers containing TF-2A and TF-2B are pro-oxidants capable of inducing less oxidative stress in carcinoma cells than normal gingival fibroblasts.⁶

The potential effects of a grape seed proanthocyanidin extract and other commercial polyphenols (eg, monomeric polyphenols, gallic acid, and epigallocatechin 3-gallate) on the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and on the protein expression of inducible nitric oxide synthase were examined in murine macrophages stimulated with lipopolysaccharides (LPS) from periodontopathogens. The study demonstrated that proanthocyanidins have potent antioxidant properties and a good therapeutic agent in the prevention of periodontal diseases.⁷ A phytochemical is found in black raspberries, which are a rich, natural source of vitamins and minerals. Specifically, the active compounds in black raspberries include vitamins A, C, and E, folic acid, calcium, selenium, ß-carotene, ellagic acid, coumaric acid, and quercitin, besides different anthocyanins and phytosterols. Freeze-dried black raspberries prevent the growth of oral, esophageal, and colon cancer in rodents, and human trials have shown a decrease expression of molecular biomarkers of dysplasia.⁸ Extracts of black raspberries have been used to inhibit benzo(a)-pyrene-induced cell transformation of hamster embryo fibroblasts.⁹ However, the antioxidant levels of freeze-dried black raspberries are not reliable to warrant its commercial use. Considerable evidence also has revealed that flavonoids possess antioxidant and anti-inflammatory properties that reduce inflammatory molecule expression in macrophages and monocytes within the gingival connective tissues. Some flavonoids such as luteolin, quercetin, and genistein have regulated the nitric-oxide production of LPS-stimulated human gingival fibroblasts. Luteolin is involved with LPS signaling pathways, decreasing the activation of various mitogen-activated protein kinase (MAP kinase) family members, and prevents inflammatory mediator expression.¹⁰ Ferulic acid is also a component of raspberries and is active against free radicals such as ROS. Animal studies and in vitro studies suggest that ferulic acid may have direct antitumor activity against breast and liver cancer, and can help in the fight against the effect of carcinogenic compounds like benzopyrene and 4-nitroquinoline 1-oxide. Ferulic acid has pro-apoptotic effects that will damage the cancer cells.^11-14 The topical combinations of ascorbic acid, vitamin E, and ferulic acid may diminish oxidative stress and prevent the formation of thymine dimers in skin.¹⁵ Ferulic acid has a potent anti-lipid peroxidative effect as well as a capacity to modulate the condition of carcinogen-detoxifying agents and antioxidants in a 12-dimethylbenze[a]anthracene-induced hamster buccal pouch carcinogenesis model.^16,17 The inhibitory effect of ferulic acid on rat tongue carcinogenesis was due to the effects of a novel geranylated derivative, ethyl 3-(4'-geranyloxy-3'-methoxyphenyl)-2-propenoate.¹⁸ Ferulic acid is also involved in LPS-stimulated mouse macrophage-like cells (Raw 264.7 cells) wherein it significantly reduced nitric oxide (NO) production and could potentially be a useful agent to prevent cell damage caused by superoxide such as OH and NO.¹⁹

Vitamins and Supplements

Recent studies have indicated that vitamin E may have therapeutic effects in treating and preventing periodontal pathology.^20,21 Vitamin E is a powerful lipid-soluble antioxidant that is valuable in decreasing wound-healing time.²² To determine whether free radicals have an effect on the normal process of the cell cycle and whether vitamin E inhibits cell damage, normal human oral epithelial cells were treated with H2O2 in culture in the presence or absence of vitamin E.²³ H2O2 produced hydroxyl radicals that can affect the cell cycle. Organotypic cultures exposed to H2O2 showed characteristic features common among premalignant epithelial lesions, which include: nuclear hyperchromatism, loss of maturation and prominent nucleoli. Therefore, the conclusion is that vitamin E may have the potential to reduce oxidative damage caused by hydroxyl radicals.²³ Another vitamin supplement that is essential is folic acid. Low serum folate levels have been associated with an increased risk of periodontal disease in older adults as shown in a recent population-based, cross-sectional study.²⁴ The role of folic acid in combination with oral hygiene measures was investigated in a 1-year follow-up study on epileptic children treated with phenytoin. This study led to the conclusion that the combined effects of systemic folic acid and phenytoin slow down the onset and decrease the incidence and severity of phenytoin-induced gingival overgrowth.²⁵

Single Purified Antioxidant Molecules

Resveratrol was shown to have potential benefits in preventing or counteracting cellular damage, cancer, aging, and many other diseases.²⁶ Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenolic phytoalexin found in green vegetables, citrus fruit, and red grape wines.²⁷ Resveratrol acts by stimulating the forkhead transcription factor family influencing the three stages of carcinogenesis: initiation, promotion, and progression. These mechanisms include: regulation of the transcription factor NF-kB, inhibition of the cytochrome P450 isoenzyme CYP1A1, varied changes in androgenic actions, and expression and activity of cyclooxygenase enzymes.²⁸ In vitro, resveratrol prevented the proliferation of human pancreatic cancer cell lines and stimulated Fas/Fas ligand-mediated apoptosis. Resveratrol was potentially effective for treating neuronal death, neurological dysfunction, or other neurodegenerative genetic disorders such as Huntington's and Alzheimer's disease.²⁹ Phloretin is a relatively potent antioxidant against peroxynitrite and lipid peroxidation. The antioxidant pharmacophore of phloretin is 2, 6-dihydroxyacetophenome. The potent activity of this molecule is due to the stabilization of its radical via tautomerisation.³⁰ More recently, the combinations of topical antioxidants such as phloretin, vitamin C, and ferulic acid prevent many signs of premature aging and correct existing photodamage in skin.^30,31 This antioxidant has the ability to control the level of ROS throughout skin layers. Other functional activities include its capacity to prevent the mutation that occurred in skin cells and to participate in cultures with rapid cell turnover.³² Tetrahydrocurcuminoids (THC) are derived from curcuminoids extracted from the roots of Curcuma longa, commonly called turmeric root. Curcuminoids have topical antioxidant properties that can defend normal human keratinocytes from hypoxanthine/xanthine oxidase injury in in vitro experiments.33 Curcuminoids and THC could be added in antioxidant topical preparations to protect the skin against harmful agents.³⁴ Both in vitro and in vivo studies revealed that tumor promoter-induced oxidative stress treated with curcumin and two THCs showed that curcuminoids significantly repressed TPA-induced oxidative stress due to the infiltration of leukocytes into the inflammatory regions and inhibition of their activation.³⁵ In order to formulate a novel class of antioxidative compound in the same molecule, three known curcuminoids—curcumin (diferuloylmethane, U1), (4-hydroxy-3-methoxycinnamoyl)methane (U2), and bis-(4-hydroxycinnamoyl)methane (U3)—were converted to tetrahydrocurcuminoids (THU1, THU2, and THU3, respectively) by hydrogenation, and their antioxidative activity was monitored and evaluated by linoleic acid as the substrate in an ethanol/water system using the rabbit erythrocyte membrane ghost and rat liver microsome.³⁶ This study revealed that THU1 is a major metabolite of U1 in vivo system and had the strongest antioxidative activity among all curcuminoids for each assay type.³⁶ Therefore, it was concluded that THU1 must play a significant role in the antioxidative mechanism of U1 in vivo by converting U1 into THU1.³⁷

Conclusion

This review identifies novel and current therapeutic protective agents in recent studies and provides some foundation to anticipate and prepare for future challenges and opportunities. The properties of each single antioxidant such as resveratrol, ferulic acid, tetrahydrocurcuminoids, and phloretin have been tested both in vitro and in vivo in order to elucidate their role and to understand their mechanism of action. Some of these single antioxidants when applied to cells possess anti-angiogenic, anti-inflammatory, antiviral, and/or anti-tumor properties. It is possible that combined antioxidant supplements will provide greater protective effects against free-radical damage to human gingival and periodontal tissues than individual antioxidants. The increasing evidence from studies of combinations of antioxidants has raised hopes that these products can be useful in the treatment of dental pathoses.

Disclosure

The authors have received financial support from PerioSciences, Inc.

Acknowledgment

The authors would like to thank Jeanne Santa Cruz for providing editing assistance with this manuscript.

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About the Authors

Symone M. San Miguel, DMD, PhD
Postdoctoral Fellow
Biomedical Sciences
Baylor College of Dentistry
Texas A&M Health Science Center
Baylor College of Dentistry
Dallas, Texas

Lynne A. Opperman, PhD
Professor of Biomedical Sciences
Director of Technology Development
Texas A&M Health Science Center
Baylor College of Dentistry
Dallas, Texas

Edward P. Allen, DDS, PhD
Adjunct Professor
Department of Periodontics
Texas A&M Health Science Center
Baylor College of Dentistry
Dallas, Texas

Kathy K.H. Svoboda, PhD
Professor of Biomedical Sciences
Graduate Program Director
Texas A&M Health Science Center
Baylor College of Dentistry
Dallas, Texas