Anti-microbial effect of Piper betle crude extract and essential oil incorporated into short-term soft lining material

Introduction

Denture stomatitis is an inflammatory lesion of oral mucosa commonly found in denture wearers. The etio­logy of denture stomatitis is multifactorial [1]. Generally, Candida species, in particular C. albicans, are the etiology of denture stomatitis, although denture plaque bacteria may be involved. Bacteria can contribute to the colonization and growth of Candida strains in the oral cavity, according to a prior study [2]. Additionally, soft tissue trauma should be properly considered when treating denture stomatitis. Since there are many contributing factors affecting denture stomatitis, there could be more than one solution required for the treatment. As a result, materials for denture lining are frequently used as an adjunctive treatment in combination with medications [1]. Previous studies demonstrated that various denture liners showed typically no effect on bacteria, but the biofilm on the denture surface could infect mucosal tissue [3, 4]. Therefore, the anti-microbial activity of soft lining materials has been still improving. Schneid reported that it is effective to incorporate chlorhexidine, clotrimazole, fluconazole, and nystatin into a soft liner as the sustained-release delivery system for the treatment of denture stomatitis. The results revealed that C. albicans growth was suppressed by all medications released from the tissue conditioner [5]. Since the overuse of antibiotics leads to a variety of side effects, including drug resistance, many researchers have been studying the novel anti-fungal agents. Consequently, numerous plants have undergone thorough research in an effort to discover more potent and harmless anti- microbial properties [6].
Piper betle (Piperaceae) is a traditional herb that contains various pharmacological properties, such as anti-microbial, anti-oxidant, anti-carcinogenic, and anti- inflammatory effects [7]. Kumpanich et al. reported that Piper betle crude extract (PBC) can be used as an additive in the denture soft liner with the optimum composition of 5% w/w against Candida albicans [8]. Clinical application of PBC is still somewhat restricted due to its dark green color and difficulty in preparation. Piper betle essential oil (PBO) is an additional extracted form that appears as a clear liquid; however, its anti- microbial effect on oral pathogens is currently unknown [8-12].

Objectives

The aim of the present study was to evaluate the anti-microbial activity of PBC and PBO incorporated into soft liner with different concentrations against C. albi­cans and S. mutans. Cytotoxicity of various additives applied into soft lining material was also inve­stigated.

Material and methods

Piper betle essential oil and crude extract

PBC was provided from the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. PBO was purchased from Thai Betel Leaf Products Co., Ltd., Bangkok, Thailand. Chemical composition of PBC and PBO are shown in Table 1.

Soft lining material

Soft lining material (GC Soft LinerTM, GC Dental Products Corp., Tokyo, Japan) was used in this study. The standard powder/liquid ratio was 1.22 g/1 g (500 µl). Its chemical compositions are listed in Table 2 [13].

Microbial culture

C. albicans (ATCC® 10231TM, Manassas, VA, USA) and S. mutans (ATCC® 25175TM, Manassas, VA, USA) were used in this study. C. albicans and S. mutans were inocu­lated into Sabouraud dextrose broth (Becton, Dickinson and company, Sparks, MD, USA) and brain heart infusion broth (Becton, Dickinson and company, Sparks, MD, USA), respectively. After 24 h, the turbidity of suspension of C. albicans and S. mutans were diluted to 1.0 McFarland unit (1.5 × 108 CFU/ml) and 0.5 McFarland unit (0.75 × 108 CFU/ml), respectively. C. albicans and S. mutans were inoculated on an agar plate using a spread plate method.

Anti-microbial activity of PBC and PBO

Anti-microbial activity of PBC and PBO were assessed by an agar disc diffusion method. Final concentrations of PBC and PBO were prepared in dimethylsulfoxide (DMSO) (Sigma-Aldrich, Inc., St. Louis, MO, USA) using serial dilution technique to obtain 0.675, 1.25, 2.5, 5, 10% w/w and v/v, respectively. DMSO was utilized as a negative control for both species. 30% v/v nystatin oral suspension, 100,000 IU/ml or 33.3 mg/ml (NYS) (Continental Pharm Co., Ltd., Bangkok, Thailand), and 5% v/v of 2% chlorhexidine gluconate (CHX) (Faculty of Dentistry, Mahidol University, Bangkok, Thailand) were used as positive controls for anti- fungal and anti-bacterial assays, respectively. 20 µl of tested solution was soaked into a 6 mm diameter paper filter disc before placing on the agar. Plates were subsequently incubated at 37oC for 48 h, and the mean value of inhibition zones was recorded. Each experimental group (n = 5) was replicated five times independently.

Anti-microbial activity of soft lining material incorporated with PBC and PBO

Anti-microbial activities of GC soft liner incorporated with PBC and PBO were assessed with the agar well diffusion method. The wells with a dimension of 6 mm in diameter and 4 mm in depth were created in each agar plate using a punch hole technique. According to a manufacturer’s recommendation, GC soft liner with 1.22 g of powder and 1 g (500 µl) of liquid was manually mixed with various amounts of PBC and PBO in a sterile beaker for 30 seconds at ambient temperature. Regarding anti-fungal assay, the final concentrations of soft liner with PBC were 2.5%, 5%, 10%, 20%, 30%, 40% w/w, and with PBO were 2.5%, 5%, 10%, 20%, 30%, 40% v/v. As for anti-bacterial assay, the final concentrations of PBC and PBO in GC soft liner were 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% w/w, and 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% v/v, respectively. Final concentration of 30% v/v nystatin oral suspension and 5% v/v of 2% chlorhexidine gluconate in GC soft liner were prepared as positive controls for anti-fungal and anti-bacterial assays, respectively. Negative control was soft lining materials without additives. The samples were added into each well and incubated at 37oC for 48 h, and the mean inhibition zone was calculated. Each experimental group (n = 5) was replicated five times independently.

Cytotoxicity test

According to ISO 10993-5, the eluting method was adapted to evaluate cytotoxicity of various additives incorporated into GC soft liner [14]. Cell viability of human gingival fibroblast (HGF) cell line (ATCC® CRL-2014, Manassas, VA, USA) was performed using the eluting extract. Specimens were prepared in dimension of 20 × 5 × 2 mm (3 cm2/ml extracted ratio) with stainless steel mold, in accordance with ISO 10993-12 [15]. GC soft liner was mixed with 5% and 10% w/w PBC, 30% v/v nystatin oral suspension, and 5% v/v of 2% chlor­hexidine gluconate. GC soft liner without additives was utilized as a control. Each side of the specimens was sterilized with UV light for 15 min. Each specimen was immersed in the 1 ml Dulbecco’s modified Eagle’s medium (DMEM) (HyCloneTM, Logan, UT, USA) with 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin-streptomycin solution, and incubated in a shaking incubator at 37°C for 24 h. The incubated culture media was collected for cell cytotoxicity test. As a negative control, culture medium without samples was also incubated in the same manner.
DMEM supplemented with 10% v/v FBS and 1% v/v penicillin-streptomycin solution was used as a culture media for HGF cells. HGF cells were cultured in 96-well plates with seeding density of 10,000 cells/well/100 µl in 5% CO2 incubator at 37°C for 24 h. Then, the media were discarded, and 200 µl aliquots of the prepared extracted solutions, including negative control media, were subsequently added into 96-well plates. The samples were incubated in 5% CO2 incubator at 37°C for 24 h. MTT assay was conducted to assess cell viability using microplate spectrophotometer (μOuantTM, BioTek instruments Inc., Winooski, VT, USA) at 570 nm. Cytotoxicity of GC soft liner combined with various additives was expressed as percentage of cell viability compared with GC soft liner without additive. Each experimental group (n = 3) was replicated three times independently.

Statistical analysis

All statistical analyses were performed using PASW Statistics software (IBM Corp., released 2019, IBM SPSS Sta­tistics for Windows, version 26.0, Armonk, NY, USA). One-way ANOVA and Tukey’s multiple comparison tests were performed to analyze the inhibition zones and percentages of cell viability. Significance level was set at α = 0.05.

Results

Anti-microbial activity of PBC and PBO on C. albicans and S. mutans

The means inhibition zone of PBC and PBO against C. albicans and S. mutans were investigated (Table 3). The inhibitory zones against C. albicans were detectable in 2.5% w/w PBC and 2.5% v/v PBO (Figures 1A, 1B), whereas the inhibitory zones of S. mutans were detectable in 2.5% w/w PBC and 10% v/v PBO (Figures 1C, 1D). As the concentration of PBC and PBO increased, the inhibition zones also increased. Furthermore, similar to the positive control (nystatin), 5% v/v PBO and 5% w/w PBC demonstrated remarkable anti-fungal activity against C. albicans.

Anti-microbial activity of GC soft liner incorporated with PBC and PBO

The anti-fungal and anti-bacterial activities of GC soft liner combined with various concentration of PBC and PBO are illustrated in Figures 2 and 3, respectively. The mean inhibition zone of PBC and PBO against C. albicans and S. mutans is shown in Table 4. GC soft liner with additive demonstrated inhibitory effect on C. albicans with 5% w/w PBC and 20% v/v PBO, while the inhibitory zone of S. mutans was detected in GC soft liner with 10% w/w PBC and 60% v/v PBO. The inhibition zones increased as the concentration of PBC and PBO also increased.

Effects of PBC incorporated into GC soft liner on cytotoxicity of HGF cell line

The results of anti-bacterial and anti-fungal tests in this present study showed that 5% and 10% w/w PBC presented anti-bacterial and anti-fungal effects. Therefore, MTT assay was conducted to investigate the cell viability of HGF cell line after exposing to soft lining materials with 5% and 10% w/w PBC, 5% v/v of 2% chlorhexidine gluconate, and 30% v/v of nystatin oral suspension comparing with soft lining materials without additives. Statistical analysis with one-way ANOVA test showed no significant difference among soft lining materials with additives, compared with no additive (Figure 4).

Discussion

Because of the overuse of conventional antibiotics, natural-derived medicines, such as many herbal plants with lower toxicity have been investigated. P. betle leaves containing 0.7-2.6% essential oils, mainly phenolic and terpenoid compounds, were examined as an anti- microbial agent [16, 17]. P. betle leaves can be processed through extraction to obtain the crude or distillation to obtain the oil. P. betle contains a variety of biologically active compounds, the concentration of which depends on the plant’s diversity, climate, harvesting time, and geographic region [18].
The compositions of 4-chromanol, eugenol, and hydroxychavicol play important roles in antiseptic and anti-microbial properties [7, 19]. Previous studies showed PBC containing 4-chromanol and eugenol, which acted as anti-candidal agents, and MIC against C. albicans ranged from 0.125 to 25 mg/ml [20-22]. Other studies found that MIC against S. mutans ranged from 1.56 to 10 mg/ml [18]. Correspondingly, MIC of PBC against C. albicans and S. mutans in this study was 2.5% w/w. Moreover, another study found that 4-chromanol within PBC contained anti-bacterial and anti-biofilm properties. Therefore, PBC had dual action in preventing and eradicating biofilm formation [23]. According to Nalina et al., PBC could damage the bacterial cell membrane and reduce acid production [24]. In addition, Kawsud et al. demonstrated that 4-chromanol was a crucial component of PBC that acted as an anti-candidal agent by preventing biofilm formation and eliminating existing biofilm [12]. Regarding PBO, previous studies have found that MIC of PBO against C. albicans was ranging from 0.078% to 1.6% v/v [9-11]. PBO’s main ingredient, hydroxychavicol, is responsible for both anti- fungal and anti-bacterial actions by altering cell membrane structure, which affects microbial membrane permeability [9, 25]. Furthermore, the eugenol in PBO and PBC has anti-bacterial effect. Compounds that react or interact with nucleophilic groups or solutes in the cell membrane and cause it to malfunction, have unspecific anti-candidal mechanisms of action [25]. On the contrary, other earlier studies found that eugenol did not exhibit anti-candidal and anti-bacterial activities [12, 23]. In this study, PBC contained 4-chromanol and eugenol, both of which have anti-bacterial and anti-candidal effects, whereas PBO only contains eugenol and some hydroxychavicol. This could explain why PBO pre­sented lower anti-bacterial and anti-candidal properties than PBC.
Polyethyl or polymethyl methacrylate resin is a major component of soft lining materials, and it is combined with a plasticizer, such as dibutyl phthalate (MW = 278.34 g/mol) or ethanol (MW = 46.07 g/mol). Because of the low molecular weight (MW) of the plasticizer, the anti-bacterial agent was gradually released from soft lining material molecules, making the soft lining material itself a drug carrier. The low molecular weight composition of PBC used in this study (Table 5) could possibly leach out and maintain its effective dose.
Regarding the mechanical properties of GC soft liner, Kumpanich et al. demonstrated that 5% w/w PBC incorporated into GC soft liner did not significantly affect the material’s gelation time and surface hardness. This 5% w/w PBC was the optimum concentration that should promote inhibitory effect against C. albicans without a dramatic change in material properties. This study also found that incorporating 10% w/w PBC into GC soft liner resulted in significantly longer gelation time and an alteration in the material’s surface hardness. The gelation time is related to the material setting and working times. As the gelation time increases, the working time also increases. Moreover, the longer gelation time also affect the hardness of GC soft liner. The study showed that the hardness of 10% w/w PBC incorporated into GC soft liner at 2-hr and 7-day incubation time was 13.44 ± 1.53 and 21.98 ± 1.40, respectively [8]. However, according to ISO:10139-1:2018 standard, the hardness of soft material should be within a 30-50 units at 2-hr incubation time, and should not exceed 60 units at 7-day incubation time, while the hardness of extra soft material should be lower than 30 units at 2-hr incubation time and 60 units at 7-days incubation time [26]. Therefore, the hardness of GC soft liner incorporating 10% w/w PBC was in accordance with ISO:10139-1:2018 standard.
Adverse reactions, including allergies and local chemical irritation, may occur from materials used in the oral cavity due to direct and indirect contacts with the oral mucosa [27]. Roy et al. [28] used murine (Ehrlich Ascites Carcinoma and Melanoma B-16 cells) and human cancer cell lines to investigate the cytotoxic effect of PBC extract. They found that the concentration of PBC had an effect on cell viability. Although PBC extract was cyto­toxic to both normal and tumor cell lines, tumor cells were more lethal than normal cells. On the other hand, Valle et al. [29] concluded that PBC was not harmful to normal human dermal fibroblasts. Previous studies demonstrated that GC soft liner was less cytotoxic than other commercial products, such as Coe Comfort and Kerr FITT [30]. Consistently, the current study found that PBC incorporated into GC soft liner had no cytoto­xicity on HGF cell line.
Therefore, denture wearers benefit from the incorporation of anti-fungal and anti-bacterial substances into soft lining material to avoid infection. As a result, it may reduce inflammation as well as injury of tissue. However, PBC has not been investigated in clinical trials due to its dark green color and difficulty in preparation. Purification of a PBC active ingredient should be examined in future research that could lead to its therapeutic application.

Conclusions

According to this in vitro study, PBC and PBO promote anti-microbial activity against C. albicans and S. mutans. However, when compared to PBC, GC soft liner combined with PBO demonstrated lower anti-bacterial and anti-candidal efficacy. The application of GC soft liner incorporated with PBC at an optimal concentration of 10% w/w showed anti-microbial activity against C. albi­cans and S. mutans, with no difference in cytoto­xicity. PBC is a natural agent that could be used to treat denture stomatitis and decrease secondary caries with no harmful effect.

Acknowledgments

The authors would like to thank Dr. Jindaporn Puri­pattanavong from Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand for providing Piper betle extract, and Dr. Widchaya Kanchanavasita for his advice on statistical analysis.

Conflict of interests

The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.   

References