Toxicological Parameters of a Formulation Containing Cinnamaldehyde for Use in Treatment of Oral Fungal Infections: An In Vivo Study

Abstract

Objective. We aimed to define the safety and toxicity of both isolated and embedded cinnamaldehyde using a pharmaceutical formulation for the treatment of oral fungal infections in an in vivo study. Materials and Methods. Acute toxicity was assessed in studies with Galleria mellonella larvae and Danio rerio embryos (zebrafish), and genotoxicity was assessed in a mouse model. The pharmaceutical formulation (orabase ointment) containing cinnamaldehyde was evaluated for verification of both in vitro antifungal activity and toxicity in keratinized oral rat mucosa. Results. In Galleria mellonella larvae, cinnamaldehyde was not toxic up to the highest dose tested (20 mg/kg) and presented no genotoxicity up to the dose of 4 mg/kg in the model using mice. However, it was found to be toxic in zebrafish embryos up to a concentration of 0.035 μg/mL; LC₅₀ 0.311; EC₅₀ 0.097 (egg hatching delay); and 0.105 (Pericardial edema). In the orabase antifungal susceptibility test, cinnamaldehyde exhibited activity in concentrations greater than 200 μg/mL. As for safety in the animal model with rats, the orabase ointment proved to be safe for use on keratinized mucosa up to the maximum concentration tested (700 μg/mL). Conclusions. At the concentrations tested, cinnamaldehyde was not toxic in vertebrate and invertebrate animal models and did not exhibit genotoxic activity. In addition, when used in the form of an ointment in orabase, having already recognized antifungal activity, it was shown to be safe up to the highest concentration tested.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Toxicity is a major challenge in pharmaceutical drug development, and many products with health applications cause undesirable side effects [1]. Factors that influence toxicity involve the chemical structure of the molecule, the dosage, and the mode of use [2]. In vivo studies using differing investigative animal models for assessing the toxicity of new therapeutic products are essential to support research that aims at evaluating safety and efficacy in randomized controlled clinical trials in humans [3].

The worldwide frequency of fungal infections has increased in recent years, especially in immunosuppressed individuals [4]. These infections can present superficially, but they can also bring serious systemic disease [5]. The limited number of antifungal classes [6], the increased prevalence of serious infections caused by Candida spp. [7], and the increase in microbial resistance to drugs [8], often due to repeated or long-term therapies [9], are motivating factors in the search for new antifungals. Compounds derived from plants have great potential to be used as antifungal agents [10]. Cinnamaldehyde, the main component obtained from the oil of the leaves and bark of Cinnamomum zeylanicum Blume (cinnamon), is one of the most effective inhibitors of microbial growth [11]; it presents both anti-inflammatory [12] and tissue repairing activity [13].

In a previous study, we reported on the antifungal effects of cinnamaldehyde on Candida species and demonstrated its strong fungicidal activity (MICs and MFCs ranging from 18.91 μM to 37.83 μM) [14] through a mechanism of action likely related to ergosterol complexation. In addition, fungal microcultures treated with cinnamaldehyde presented impairment of fungal cell development, with observed expression of rare pseudohyphae and the absence of chlamydeoconidio, reducing fungal biofilm by 33.75% to 64.52% ($p<0.0001$) in low concentrations (151.3–378.3 M), in addition to being noncytotoxic to human keratinocytes and erythrocytes [15]. These results support further investigation, including evaluating toxicity and safety in animal models.

The literature has already demonstrated topical use of cinnamaldehyde in an in vivo model in the treatment of skin wounds resulting from Pseudomonas aeruginosa infection [16] and also in the form of an ointment tested in wound healing in diabetic mice [13]. However, its use as an ointment in orabase for topical treatment of fungal infections that affect the oral cavity has not yet been evaluated. The objective of this study was to define the safety and toxicity of cinnamaldehyde in in vivo models and define a pharmaceutical form which both is safe and contains cinnamaldehyde in its composition.

2. Materials and Methods

2.1. Outcome Measurements

The first stage of the work consisted in defining the toxicity of cinnamaldehyde in animal models with invertebrates (larvae of Galleria mellonella), vertebrates (zebrafish embryos Danio rerio), and also genotoxicity (mice). The second stage consisted of developing the orabase formulation to define its antifungal activity when tested for safety in an animal model using rats, as described in Figure 1.

[figure omitted; refer to PDF]

We are the first to study the acute toxicity of cinnamaldehyde using G. mellonella larvae. This model, in addition to being viable, low-cost, and validated [17, 24], provides answers quickly, as the larvae have a short life cycle (approximately 6 weeks), and there is no need for specialized equipment. Additionally, the immune system of this invertebrate animal is similar to that of mammals [25, 26].

In a study using G. mellonella, the authors assessed toxicities of fruit extracts. The extracts of Sageretia elegans and Byrsonima arthropoda were shown to cause acute toxicity effects in the larvae, and Spondias mombin reduced the survival of the larval population by almost 50% as compared to the vehicle ($p<0.05$) [27]. Another study evaluated the acute toxicity of Eugenia brasiliensis fruit pulp extract and found that the extract had no toxic effects on these same larvae when administered in antibiofilm concentrations ($10\times \text{MIC}$) [23]. There are no toxicity studies on cinnamaldehyde or extracts of plants that contain cinnamaldehyde, against G. mellonella larvae. Our results revealed that none of the systemic doses (0.15–20 mg/kg) resulted in larval death. This difference may be explained due to the differing parts of the plant (peel and fruit pulp extract) used in the studies. Cinnamaldehyde is extracted from the skin of species of the genus Cinnamomum, and in the other studies mentioned above, the compounds under analysis were extracted from fruit pulps. Further, the values of the doses tested differed, being calculated based on the antibiofilm value of each substance.

3.2. Toxicity in Zebrafish Embryos

In order to determine whether cinnamaldehyde was toxic to zebrafish development, its embryos were exposed to increasing concentrations of the substance. This is the first report characterizing the effects of cinnamaldehyde on the development of zebrafish.

The survival rate and malformations in zebrafish were measured, and cinnamaldehyde presented dose-dependent effects on embryo survival up to 96 hpf (Figure 3). Survival, as presented in the graph for the concentrations of 0.017, 0.035, 0.07, and 0.14 μg/mL, did not differ significantly from the negative control, but at the highest concentrations tested, the embryo’s survival decreased by 26.25% (for the concentration of 0.28 μg/mL vs. the control; $p=0.024$) and by 85% (for the concentration of 0.56 μg/mL vs. the control; $p<0.0001$).

[figure omitted; refer to PDF]

The embryotoxicity of cinnamaldehyde included mortality endpoints and malformations. Figure 4 presents an overview of the cumulative effects of cinnamaldehyde on zebrafish embryos and larvae at 96 h of exposure. The number of nonlethal and lethal outcomes worsened as concentrations increased.

[figure omitted; refer to PDF]

Table 1 presents a general panel of cinnamaldehyde lethality in relation to the zebrafish development parameters. In the morphological analysis, it was observed that cinnamaldehyde induced changes in zebrafish embryos starting at a concentration of 0.035 μg/mL (LOAEL), e.g., delays in egg hatching ($\text{E}{\text{C}}_{50}=0.097\mu \text{g}/\text{mL}$) and pericardial edema ($\text{E}{\text{C}}_{50}=0.105\mu \text{g}/\text{mL}$). The lowest concentration tested for the absence of adverse effects on the embryos (NOAEL) was 0.017 μg/mL.

Table 1

Effects of cinnamaldehyde on developmental parameters of zebrafish early stages after 96 h of exposure.

96 h: embryotoxicological adverse effects	LC50	EC50	LOAEL	NOAEL
Mortality (all lethal effects)	0.311	NA	0.14	0.07
Coagulation of viable embryos			0.14	0.07
Lack of somite formation			—	—
Lack of heartbeat			0.28	0.14
Nondetachment of the tail bud from the yolk sac			0.28	0.14
Nonlethal effects
Eye malformation	—	—	—	—
Otolith malformation	—	—	—	—
Mouth malformation	—	—	—	—
Spine malformation	—	—	—	—
Body pigmentation	—	—	—	—
Egg hatching delay	—	0.097	0.035	0.017
Yolk sac edema	—	NC	0.035	0.017
Body malformation	—	NC	0.14	0.070
Pericardial edema	—	0.105	0.035	0.017
Head edema	—	—	—	—
Blood clotting	—	—	—	—
Undersize	—	—	—	—
Developmental delay	—	NC	0.14	0.070

LOAEL: lowest observed adverse effect level in μg/mL; NOAEL: no observed adverse effect level in μg/mL; —: not observed; LC₅₀: median lethal concentration in μg/mL; EC₅₀: median effective concentration values in μg/mL; NA: not applicable; NC: not calculated.

This vertebrate animal model has been shown to be useful for toxicology, especially in drug screening and human disease studies [28]; it is a reliable, controllable, and reproducible model [29]. Yet, some authors suggest that for assessing toxicity, this experimental model is very sensitive, with the initial stages of development being commonly the most sensitive to chemical exposure [30].

Often, the toxic responses of substances are conserved beyond the zebrafish and are thus also found in toxicological studies in humans [31]. Yet, in species such as rats, mice, and guinea pigs, studies have already shown that acute cinnamaldehyde toxicity is relatively low, with LD₅₀ values (50% lethal dose) ranging from 0.6 to more than 2 g/kg [32]. In this study, it was shown that cinnamaldehyde induced mortality at the two highest concentrations tested; on the other hand, the toxic concentrations caused morphological changes such as egg hatching delay, yolk sac edema, body malformation, pericardial edema, and developmental delay (Figure 5). This may be due to differing embryo polarities, which cause the molecule to act by affinity (lipophilicity), a chemical characteristic of the molecule under analysis [33].

[figures omitted; refer to PDF]

The high mortality found at the desired concentrations may be due to small variations in the rate of embryo development, such as the variation in the number of embryos emerging from their chorions at the time of treatment (malformations occur more frequently while the embryos are in the chorion), the ability of oil components to cross the embryonic barrier [34], accumulation of blood close to the anterior portion of the yolk sac, weakening of the vessels, pericardial edema, necrotic tissue effects which interrupt the structure of the heart, or increased pressure resulting from the edema [35].

3.3. Evaluation of Genotoxicity

A 4 mg/kg single dose of the treatment sample in the animals did not induce an increase in the number of micronucleated erythrocytes in peripheral blood ($7.8\pm 0.3$), as compared to the control group ($8.6\pm 0.4$). As expected, cyclophosphamide induced a significant increase in the number of micronucleated erythrocytes ($18.4\pm 0.5$; $p<0.05$), as to the control group ($8.6\pm 0.4$) (Table 2).

Table 2

Effect of single-dose cinnamaldehyde (4 mg/kg) and cyclophosphamide (50 mg/kg) administrations on the number of micronucleated mice erythrocytes, in peripheral blood after 48 hours of treatment.

Groups	Dose (mg/kg)	No. of micronucleated cells
Control	—	$$8.6\pm 0.4$$
Cyclophosphamide	50	$18.4\pm 0.5$a
Cinnamaldehyde	4	$$7.8\pm 0.3$$

Data presented as $\text{mean}\pm \text{standard}\text{error}\text{of}\text{the}\text{mean}$ of five animals. ^a$p<0.05$ compared to the control group (12% Tween 80), analyzed by ANOVA followed by Dunnet and Tukey.

Taking these results into account, it can be inferred that cinnamaldehyde at a dose of 4 mg/kg does not present genotoxic effects on the erythroid system. However, a previous study has shown that cinnamaldehyde at doses of 850, 1700, and 2550 mg/kg is capable of inducing micronucleus formation in liver cells [36]. The genotoxicity of cinnamaldehyde has also been demonstrated in the literature in an in vitro model using a reversal assay. The authors proved that cinnamaldehyde presents no mutagenic activity in the genotoxicity test, since it did not interact with DNA to transmit and trigger mutations [37].

3.4. Fungal Susceptibility Test

In order to evaluate the therapeutic efficacy of the orabase ointment containing cinnamaldehyde, its antifungal activity was estimated. This pharmaceutical form was chosen due to its advantages in providing easier application, in spreading the product over the affected region, and for promoting adhesion at the tissue lesion site. The formulation also comes flavored, which improves product acceptance and increases patient adherence, all while acting as a mucoprotective barrier for ulcerated oral lesions [38].

In vitro and in vivo test agreement is important, since to date, in both models, and in the concentrations and doses respectively tested, cinnamaldehyde presented antifungal activity without cytotoxic effects [15] and in the invertebrate model presented toxicity with no genotoxic effects as well. Thus, the results obtained in these tests help to estimate and define orabase ointment product concentrations that may be used in further studies on safety.

For good test resolution, agar thickness and uniformity are determinants [21]. Thus, the Petri dishes used in the assay for antifungal activity were prepared in a standardized way with the same agar volume and a central hole of the same diameter. The strain was exposed to different concentrations of cinnamaldehyde (12.5, 25, 50, 100, 200, 300, 400, 500, 600, 700, and 800 μg/mL), and antifungal activity was estimated using the diameter of the inhibition zone. It was observed that the halo formed by the ointments was concentration dependent, with no statistical difference between miconazole and the ointment containing cinnamaldehyde at 200 μg/mL. Statistical differences from the positive control started at 300 μg/mL, yet there was no difference between the 300 and 700 μg/mL concentrations ($p>0.05$). Ointments at concentrations of between 12.5 and 100 μg/mL did not present antifungal activity (data not shown). According to our results, the orabase ointments containing cinnamaldehyde inhibited Candida albicans growth, a species commonly present in the oral cavity of human beings [5]. The results are presented in Figures 6 and 7.

[figure omitted; refer to PDF]

The literature had already demonstrated the in vitro effects of cinnamaldehyde (isolated) against species of the genus Candida [15]. The effect of Cinnamomum zeylanicum Blume essential oil, a product that contains a significant amount of cinnamaldehyde in its composition, has also been demonstrated in the form of a spray for treatment of oral candidiasis associated with the use of removable prosthesis in a phase II clinical trial [39]. However, the effect of cinnamaldehyde in the form of an ointment in orabase for use in the oral cavity has not yet been defined. Thus, in addition to antifungal activity, it was necessary to test the safety of this new pharmaceutical form in animals to arrive at phase I clinical trials in humans.

3.5. Toxicity Study: Rat Keratinized Oral Mucosa

The results revealed that at all concentrations tested (25, 50, 100, 200, 300, 400, 500, 600, and 700 μg/mL), the animals remained healthy, not presenting clinical and/or macroscopic evidence of inflammation. Histological analyses of palate specimens presented preservation of epidermis (Ep), keratinocytes, and basal cells without dermatopathological changes and preservation of the dermis (Dm) which presented no vascular and/or cellular changes, being compatible with normal mucosa at all doses tested. The results are presented in Figure 8.

[figures omitted; refer to PDF]

These results were expected since the literature had already demonstrated the anti-inflammatory capacity of cinnamaldehyde [11]. The activity involves induction of apoptosis; inhibition of cell proliferation in immune responses mediated by monocytes/macrophages; suppression of nitric oxide production, with positive regulation of costimulatory molecules (CD80 and CD69) and standard recognition receptors (toll-like receptor 2 (TLR2), complement receptor (CR3) [40]); and lower levels of TNF-α and IL-6 [12].

This anti-inflammatory and regenerative capacity was also tested in an in vivo model. Certain authors have tested cinnamaldehyde in topical form, demonstrating its ability to help heal wounds infected with Pseudomonas aeruginosa. This is because cinnamaldehyde induces lower levels of interleukin-17 (IL-17), vascular endothelial growth factor (VEGF), and nitric oxide [16]. Another study evaluating the effectiveness of an ointment prepared from hydroethanolic extract of Cinnamomum verum, containing 11.26% cinnamaldehyde in its composition, revealed an acceleration in wound healing in diabetic mice. This occurs due to a greater proliferation of fibroblasts, collagen deposition, reepithelialization, and keratin biosynthesis [13]. Tissue regeneration has also found to be favored through induction of peripheral vasodilation through activation of transient receptor potential of ankyrin-1 (TRPA1) in mice [41].

4. Conclusions

Cinnamaldehyde was shown to be nontoxic in vertebrate and invertebrate animal models, while also presenting no genotoxic activity. When used in the form of an ointment in orabase, with recognized antifungal activity against Candida albicans, cinnamaldehyde presented no clinical or histological evidence of inflammatory effect in animal mucosa.

Ethical Approval

All methods used in this study were approved by the Ethic Commission in Animal Use in Research (CEUA) of the Federal University of Paraiba, approval number 2342111119.

Authors’ Contributions

All of the authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Danielle da Nóbrega Alves, Rafael Xavier Martins, Elba dos Santos Ferreira, Adriano Francisco Alves, Jéssica Cabral de Andrade, Tatianne Mota Batista, Josy Goldoni Lazarini, Luana Souza Amorim, Pedro Luiz Rosalen, Davi Felipe Farias, and Ricardo Dias de Castro. The first draft of the manuscript was written by Danielle da Nóbrega Alves, and all of the authors commented on previous versions of the manuscript. All of the authors have read and approved the final manuscript.

Acknowledgments

This study was financed in party by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES), Finance Code: PROEX AUXPE-0521/2020.

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