The Antifungal Effects of Hypericum perforatum Nanoemulsion against Candida albicans

1. INTRODUCTION

Acrylic resin is a thermoplastic or thermoset material that is usually obtained from acrylic acid, methacrylic acid, and acrylate monomers, such as butyl acrylate, or methacrylate monomers, such as methyl methacrylate [1]. However, one of the problems of acrylic resins despite the improvement of physical and chemical properties is their microbial infections [2].

Oral stomatitis is one of the common infections related to dentures (complete or partial), which is seen in the form of diffuse inflammation, mainly with Candida albicans. Its symptoms include inflammation and erythema of the oral mucosa covered by dentures [3, 4]. The incidence of denture stomatitis is higher among the elderly and women [3]. Etiological factors include improper prosthesis hygiene, continuous and nightly use of removable prostheses, accumulation of microbial plaque in artificial teeth, and bacterial and yeast contamination of the prosthesis surface. In addition, improperly fitting dentures can increase mucosal trauma and aggravate symptoms. Candida yeasts are the most common etiological agent of fungal infections of the oral cavity [5]. In addition, Candida-related prosthetic stomatitis, a mucosal inflammatory process under a removable denture, is the most common appearance of oral candidiasis among removable denture users [6].

The most common type of chronic oral candidiasis is Candida albicans, which is known as the most vital microorganism in this condition. All the mentioned factors seem to increase the ability of Candida albicans to colonize on denture and mucosal surfaces. Biofilm formation is an obvious and essential characteristic of the presence of Candida albicans in the oral cavity [7]. Candida albicans biofilms have various virulence factors that can increase their pathogenesis and are clinically problematic because some species are resistant to antifungal agents [8].

Antifungal treatment can eradicate Candida albicans infection and relieve the symptoms of stomatitis. However, if the artificial teeth are not disinfected and their cleanliness is not maintained, stomatitis may recur after stopping the antifungal treatment [9].

Recent advances in prosthetic materials focus on tools to reduce the development of adherent biofilms. The existing methods are effective in reducing the colonization of bacteria and yeast, and by observing the proper hygiene of the dentures, denture stomatitis can be reduced. Common treatment methods that have been suggested so far include the use of chemical drugs, such as nystatin, fluconazole, and amphotericin [10].

Due to the increase in antifungal resistance compared to antifungal drugs, other substances have been considered [11, 12]. Hypericum perforatum is a herbaceous plant belonging to the Clusiaceae family, which is used in traditional medicine and herbal therapy due to its antiseptic effects. It has shown antibacterial, antiviral, anti-inflammatory, and analgesic activities. Hypericum perforatum extract contains flavonoids and phenolic acids that show free radical scavenging activity [13, 14]. Hypericum perforatum extract has been shown to exert effective anti-inflammatory effects on animal models with acute inflammation [9].

Due to the creation of a resistant form of Candida albicans, the use of conventional drugs is not effective in some situations. Plants and natural bioactive compounds can be new sources of antifungal therapy. Some studies have shown herbal medicines and natural bioactive compounds to have antibacterial, antiviral, and antifungal effects [15-17]. Therefore, it is hypothesized that these natural products may have beneficial effects on pathogenic oral fungal flora, such as Candida albicans. Although the effects of medicinal plants as antifungal agents have been investigated in several studies, to the best of our knowledge, the effects of these natural products on Candida species have not yet been investigated. In general, herbs and nutrients, such as garlic, green tea, propolis, curcumin, licorice root, cinnamon, resveratrol, ginger, and berberine, have been found to be useful and can be considered in the treatment of Candida albicans in oral candidiasis. The aforementioned materials are used as an available and cheap option in an effort to prevent and treat oral diseases [18].

In recent years, the use of nanotechnology solutions has helped chemical and herbal medicines work better. Acrylics containing silver nanoparticles have shown good antifungal effects against Candida albicans [19, 20]. Various forms of nanomaterials are used for therapeutic applications. Nanoemulsions are biphasic dispersions of two immiscible liquids, water-in-oil or oil-in-water, stabilized by a biophilic (hydrophilic and lipophilic) surfactant. The use of nanoemulsions has increased significantly all over the world today, and their use in the treatment of oral stomatitis can also be investigated [19].

The aim of the present study was to investigate the antifungal effects of Hypericum perforatum nanoemulsion against Candida albicans.

2. METHODOLOGY

3. RESULTS

It was observed that both the free form and nano form of the studied extract showed good antifungal activity against Candida albicans. The minimum inhibitory concen- tration and the minimum fungicide concentration of the nanoemulsion form were lower than the non-nanoemulsion form (P<0.05). Among the studied groups, the nystatin group had the best effect (P<0.05).

Tukey's post-hoc test showed the nanoemulsion to have a significant difference with free form (P=0.001) and nystatin exhibited a significant difference from both of them (P=0.01 and P=0.0005, respectively).

Table 1 shows the results of the comparison of the MIC and the MFC between the studied groups.

4. DISCUSSION

Over the past two decades, the incidence of fungal infections has increased dramatically, and the frequent failure of existing antifungal therapies represents a major clinical problem [10]. The urgent need for new antifungal drugs has drawn special attention to the study of natural products. In patients with complete dentures, problems, including the loss of compatibility of the denture with the jaw, trauma to the jaw while eating, lack of oral hygiene and dentures, the presence of opportunistic micro-organisms, such as Candida albicans in the mouth, and stomatitis caused by dentures, are common. On the other hand, because the patients who use complete prostheses are mostly old, they may not have enough physical ability to fully observe hygiene due to other diseases, including Parkinson's [6], or because of Alzheimer's disease, they may not remember the health issues recommended to them by the doctor. Considering that the current topical treatments for candidiasis are nystatin, fluconazole, and in more severe cases, amphotericin B, issues, such as the side effects of using these drugs and the development of drug resistance, should also be considered for these types of topical treatments. Considering all the above issues, if it is possible to mix an antimicrobial substance in the acrylic during the preparation of the prosthesis, it can be said that the incidence of the mentioned problems would decrease [10].

The microorganism used in this study was Candida albicans fungus. One of the main factors in causing stomatitis caused by dentures in patients is the opportunistic fungus Candida albicans [23].

The Hypericum plant includes many species that are used in traditional medicine to treat pathological conditions, such as inflammation and infection caused by fungi. However, despite the widespread use of Hypericum-based products, the antifungal potential of this species has not yet been well investigated. Perforatum hypericum extract is a rich source of flavonols, benzoates, cinnamates, and flavan-3-ols. Therefore, its antifungal properties can be attributed to the richness of Perforatum hypericum extract in these substances [24].

It was observed that both the non-nano form and nano form of this extract showed good antifungal activity against Candida albicans. The nanoemulsion form provided better results than the non-nano form of the Perforatum hypericum oil. The minimum inhibitory concentration and the minimum fungicide concentration of the nanoemulsion form were lower than the non-nanoemulsion form (P<0.05). Among the studied groups, the nystatin group had the best effect (P<0.05).

Tuki and his colleagues evaluated five different species of Perforatum hypericum against a wide range of fungi isolated from patients with stomatitis. The studied species showed a broad antifungal activity against resistant fungi to Penicillium, Aspergillus, and Candida albicans [24]. In our study, both the non-nano form and nano form of this extract showed good antifungal activity against Candida albicans.

Najafi et al.'s study showed sodium bicarbonate to be very effective at low and medium concentrations of Candida albicans and showed a similar effect to nystatin and chlorhexidine, but at higher concentrations of Candida albicans, nystatin was found to be more effective [8]. In our study, the nystatin group exhibited the best effect among the studied groups.

Casemiro et al. showed that the addition of 5% and 2.5% of silver nanoparticles to acrylic resin exerted a high antimicrobial effect against Candida albicans [25]. Li and colleagues showed that nanosilver exhibited antifungal activity and prevented Candida albicans biofilm formation. In their study, the antifungal activity and the inhibitory effect on adhesion and biofilm formation by the prosthetic base resin containing nanosilver, especially at a higher concentration, were proven [2].

Kahramanlu et al. showed that in acrylic resins, increasing the concentration of silver nanoparticles led to a greater antimicrobial effect. Also, acrylic resins containing silver nanoparticles exhibited stronger anti- microbial properties on standard Candida albicans strain and generally less effect on hospital-isolated strains. In addition, it has been found that the more time passes after exposure of the bacterial suspension to acrylic resins containing silver nanoparticles, the less the residual antimicrobial effect is [7]. In our study, the nanoemulsion form had better results than the non-nano form.

It should be noted that the strain of Candida albicans used in this study was the standard strain. While in strains isolated from patients due to the possibility of previous exposure to the drug and the inducibility of resistance mechanisms, it is possible to observe antifungal effects at high concentrations (high MICs). Thus, due to the difference in inherent sensitivity, a significant difference in MICs of nystatin and the studied nanoemulsion was observed in this study.

Espinoza and his colleagues showed that the smaller the size of nanoparticles, the greater the antimicrobial and antifungal effects [26]. In our study, the nanoemulsion form demonstrated better results than the non-nano form of Perforatum hypericum oil. It should also be noted that Candida albicans is a Gram-positive species. The cell wall of Gram-positive species had between 3 and 20 layers of peptidoglycan more than Gram-negative species. Therefore, Gram-positive bacteria can be less sensitive to nanoparticles because their peptidoglycan layers are inactive [7].

It should also be considered that these types of nanomaterials can be used for the slow and long-term release of antifungal and antimicrobial drugs inside acrylic materials in dentistry. Soleymanijadidi et al. assessed the release pattern and the antimicrobial action of acrylic resins containing nanocurcumin. The release pattern showed a slow release pattern for all concentrations of nanocurcumin. The authors suggested that resins containing curcumin nanoparticles can be beneficial for antimicrobial purposes in dentistry applications [27].

Moreover, it could be interesting in the future to test Hypericum perforatum nanoemulsion in combination with other preventive treatments, such as ozone [28], photobiomodulation [29], and paraprobiotics [30] in order to understand their mutual effect on Candida and other fungal infections.

The limitation of this study is the use of Hypericum perforatum as an available and cheap option in an effort to prevent and treat oral diseases. Also, most of the evidence included has been based on in vitro and animal studies, so more clinical trials are needed.

CONCLUSION

The prepared nanoemulsion can be used to treat and reduce the nystatin-resistant Candida albicans strain. The long-term effects of nanoemulsion on acrylic, for example on its color change, can be investigated. It should be noted that the strain of Candida albicans used in this study was the standard strain. Therefore, in further studies, this nanoemulsion can be tested on nystatin-resistant species. It may also be possible to insert nanoemulsion into the acrylic structure and perform physicochemical and physicomechanical tests, as well as biocompatibility tests, and finally, the antifungal performance can be assessed in the laboratory, animal, and clinical stages.

REFERENCES