Natural Compounds with Antimicrobial Activities in Oral Candida Infections during Head and Neck Radiotherapy

Candida spp. is a symbiont of the oral microbiota but in some cases can change its status from commensal to a pathogen causing microbial imbalance (dysbiosis) which results in oral diseases [1]. Candida is the principal fungal pathogen causing infections (candidiasis) overall during radiotherapy [1]. Indeed, they are common complications of head and neck radiation therapy (RT) and can result in pain, dysgeusia, anorexia, malnutrition, and esophageal or systemic dissemination [1]. The development of oropharyngeal candidiasis (OPC) can occur for an imbalance between fungal virulence factors and host defense is altered [1]. For example, people who have dry mouths are more susceptible because the flow of saliva across mucosal surfaces may eliminate yeasts during swallowing, which may be facilitated by the binding of Candida spp. to salivary mucins and proteoglycans [2]. Among Candida species, Candida albicans is involved in most of clinical oropharyngeal infections [2]. The prevention and treatment of the patients affected by this infection and the increased resistance to the available antifungal drugs related to Candida spp. infections demand the development of novel and more effective antifungal agents with new sources – like medicinal plants – with no side effects or toxicity. Recently, a severe threat is represented by the development of fungal infections caused by Candida albicans, Candida glabrata, and the newly found multiresistant species Candida auris with resistance to azoles and echinocandins, and the implication to manage invasive and noninvasive candidiasis worldwide [3]. Hence, novel molecules are being discovered and evaluated for their capacity to control Candida spp. growth and, in the future, will be used for the treatment of oral candidiasis, overall, during radiotherapy [2]. This review aims to discuss the antifungal activity of plant natural compounds against Candida spp., which could be effective for the management of high-risk patients during and soon after radiotherapy.

Candida is a commensal yeast of the oral cavity and may be observed in about 50% of the oropharyngeal region of healthy and non-immunocompromised individuals [4]. C. albicans is the main Candida species in the oral mycobiota, followed by C. glabrata, Candida parapsilosis, Candida tropicalis, and Candida krusei [5, 6].

The frequency of oral mycosis has remarkably increased globally, mainly with the COVID-19 outbreak [7]. Oral candidiasis is the most common fungal infection of the oral cavity, and it is categorized among the superficial mycoses [8].

Candida may establish infection in case of the oral microbial flora imbalance and immunocompromised patients, i.e., patients who are undergoing radiotherapy and/or chemotherapy treatments for cancer, patients on immunosuppressive, antibiotics or anticholinergic therapy, patients with immunodeficiency virus infections, AIDS, and diabetics [9]. Pathological factors – such as diabetes mellitus, endocrinopathies, immunosuppressive conditions, and malignancies – and individual factors – like smoking, nutritional disorders – add to the abovementioned factors for oral infection occurrence [7, 10].

In a very current context, Salehi et al. [11] stated that COVID-19 patients at high risk who have respiratory distress syndrome and use immunosuppressant drugs or corticosteroids are most likely to develop oral candidiasis. Salehi and others studied hospitalized COVID-19 patients who developed OPC to identify Candida species and C. albicans has been confirmed to be the most frequent type of organism [12].

Among Candida species, C. albicans seems to be the most frequent species detected from oral candidiasis lesions (95%), followed by C. parapsilosis, C. krusei, C. tropicalis, C. glabrata, Candida dubliniensis [8, 13]. The diagnostic approach to oral mycotic conditions is focused on clinical, microbiological, cytological, and histopathological examinations of the oral lesioned tissues, which usually appear as white papular lesions that were removed by a piece of gauze, revealing an underlying erythema [14].

Candida may be cytologically and histologically detected without a clinical oral condition in healthy individuals as a normal commensal. For this reason, it is essential to point out that a positive culture for Candida from an oral sample with no clinical presentation should eliminate the diagnosis of oral candidiasis [8].

The microbiological investigation is conducted when clinical diagnosis needs to be confirmed, when it is necessary to identify the species of Candida and their drug susceptibility and to establish a differential diagnosis with other diseases [4]. Identification of Candida sp. can be made using microscopic analyses, culture-based methods, and biochemical, proteomic analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and molecular techniques, like polymerase chain reaction-based tests [7].

Microscopic examination can be made using 10% potassium hydroxide on fresh samples, which dissolves the epithelial cells and leaves Candida intact. It also can be obtained from imprints of the lesion samples to perform conventional Giemsa or PAS staining, or rapid techniques such as gram staining. In addition, the filamentation test or germ tube test can be used to identify C. albicans. The identification of germ tubes and chlamydoconidia is indicative of infection produced by C. albicans and/or C. dubliniensis, while their absence is indicative of probable infection due to non-C. albicans species [4].

Microbiological cultures can be performed with different media. Sabouraud dextrose agar is the culture medium of choice to isolate fungal species. To inhibit bacterial growth, chloramphenicol or gentamicin is usually added to render medium selective inhibiting most bacterial species, while cycloheximide can be added to inhibit other fungi. Whitish cottony colonies are observed following incubation (24–48 h) [4].

Chromogenic media detect the activity of certain yeast enzymes through the specific hydrolysis of a chromogenic substrate in the presence of an enzyme indicator. Some chromogenic media used for the identification of Candida spp. are as follows: CHROMagar Candida^®, where C. albicans grows forming smooth green colonies, C. tropicalis forms smooth blue colonies, and C. krusei forms rough pink colonies; CHROMagar Candida Plus and HiCrome C. auris MDR selective agar (HAMA) [15, 16].

Biochemical methods include enzyme and nutrient assimilation methods and techniques combining enzymatic and nutrient assimilation tests. Some biochemical tests are as follows: Auxacolor^®, Uni-Yeast-Tek^®, API 20 C AUX^® and API 32ID, Galeria ID32C^®, VITEK^® 2 ID, Rapid Yeast Plus System^®, and Fongiscreen 4H^® [4].

Molecular techniques offer high sensitivity and specificity. These techniques include real-time polymerase chain reaction, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry [5, 7].

Colonization by Candida spp. is commonly found in cancer patients, with C. albicans being the predominant species in patients who undergo head and neck radiotherapy [17‒20]. Leung and colleagues [17] (2000) found that 42% of irradiate patients enrolled in their study were diagnosed as having oral candidiasis. These individuals survived nasopharyngeal cancer after identical radiotherapy protocols, and they were recruited 6 or more months posttreatment from the Department of Clinical Oncology, Queen Mary Hospital, the University of Hong Kong (Table 1).

To evaluate yeast presence, oral samples were obtained from patients rinsing their mouths for 60 s with 10 mL of sterile phosphate-buffered saline, then collected into a sterile container and immediately transported to the laboratory for processing. Among them, 72% tested positive for C. albicans, 27% had a positive diagnosis for C. tropicalis, and only 2 patients presented with C. parapsilosis infection. Candida famata was the least detected species, with a 3% rate Table 1. Additionally, the oral rinse samples of irradiated individuals yielded a mean of at least one yeast species as opposed to less than 0.5 yeast species recovered from the control group.

The oral rinse sampling was repeated on 17 patients 3 months later. During this period, the patients followed specific oral hygiene protocols, including topical antifungal therapy for those with clinically evident oral candidiasis. The percentage of patients with oral candidiasis decreased at the second recall from 42% to 23.5%. However, what is most interesting to note is that in one case C. albicans was replaced by C. tropicalis [17] (Table 1).

Differently, in a study conducted by Dahiya and colleagues (2003), a lower percentage of patients with OPC was found, while 26 (70.3%) of 37 patients displayed only Candida carriage state. Twenty percent of the oropharyngeal infections of the patients enrolled in this study was caused by non-albicans Candida species. One of the patients had C. albicans and C. dubliniensis infection at the same time. So, the total infections by non-albicans Candida species were 8%. The other two non-albicans Candida species detected in this study were C. tropicalis (1 patient, 2.7%) and Candida rugosa (1 patient, 2.7%). Actually, ten different species of yeast have been found, but only four were capable of producing infection [21] (Table 1).

In the same year, a cross-sectional study, conducted in the Department of Clinical Oncology, Queen Mary Hospital, the University of Hong Kong, showed that only 15% of patients (oral rinses) were infected with Candida species, with a significantly higher prevalence among nasopharyngeal carcinoma survivor patients (11.5%) than new patients (4%) [26].

Fifty-nine percent prevalence of C. albicans was found at the end of the second and third radiotherapy session in a study conducted in 2004 by Belazi and colleagues [18], in the Department of Oral Medicine and Maxillofacial Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece. They identified 23 different C. albicans strains among head and neck patients receiving radiotherapy. Moreover, non-albicans species were identified with a lower rate: C. glabrata in 3/39 patients (8%), followed by C. kefyr in 2/39 patients (5%), C. tropicalis in 1/39 patients (3%), and C. krusei in 1/39 patients (3%) [18] (Table 1).

In a prospective pilot study conducted by Jham and Colleagues [22] (2007) in a Brazilian population, it was found a correlation between the colonization rates of the patients and the outbreak of infection (Table 1). Among 21 patients, 42.9% were already colonized before radiotherapy, only with C. albicans: baseline colonization was higher (60%) in patients who developed infection, in comparison to noninfected patients (30%). This indicates that baseline colonization status could be a predictive factor of higher risk for developing Candida infection during RT [22]. The patients were screened weekly during this study. It turned out that, during RT, colonization increased to 57% (mean session of development 14th session). C. albicans was found in 23.8% of the patients, while non-albicans species in 33%. Particularly, they were found C. tropicalis, C. krusei, and Candida lusitaniae, with a percentage rate of 14.3, 9.5, and 9.5, respectively [22] (Table 1). By the end of RT, 71.4% of the patients had Candida colonization and the only species found were non-albicans species. The percentage of C. albicans was zero. The presence of C. tropicalis increased to 19%, while colonization rate by C. lusitanieae became 14.3%. C. krusei maintained the same rate. Other species, not found at baseline and during radiotherapy, were identified at the end of treatment: C. kefyr (9.5%) and C. parapsilosis (19%) [22] (Table 1). Nearly half of the patients (48%) completed RT without developing clinical infection. Three of them were already colonized before RT treatment, and by the end of RT, 60% of non-infected patients had Candida species in their saliva, exclusively non-albicans species: 20% C. lusitaniae, 10% C. tropicalis, C. krusei, C. kefyr, C. parapsilosis [22]. The results of the species identification in the saliva of infected patients showed a significant prevalence of C. tropicalis (36.3%), followed by C. parapsilosis (27.7%) and C. krusei, and C. kefyr (9%) [22].

Karbach and colleagues, in 2012, found a higher prevalence of Candida isolates in patients 2 after head and neck radiation [23]. Patients have been screened at the Department for Oral and Maxillofacial Surgery, University Medical Center of the Johannes Gutenberg-University of Mainz, Mainz, Germany. Sampling was carried out using 2 mL sodium chloride to rinse the mouth for 20 s. Candida species colonized 83% of patients. They found 47 different Candida isolates: 45% was C. albicans and the remaining rate was non-albicans species, like C. krusei, C. parapsilosis, C. zelanoydes (Table 1). Among the 53 patients, 41 (93%) have been colonized with only one species, while the remaining 3 patients (7%) presented colonization by two different species at the same time: C. krusei and Saccharomyces cerevisiae and C. parapsilosis and Saccharomyces cerevisiae in 2 patients and Candida zeylanoides and C. albicans in a patient [23]. Comparing patients who underwent radiotherapy and chemoradiotherapy for head and neck cancer in Spanish radiation oncology units, no significant difference was found (p = 0.6757). Among 49 patients treated with radiotherapy only and both radiotherapy and chemotherapy, C. albicans was found to be the predominant yeast (69%), and only 15 patients were colonized with non-albicans Candida species (31%). The non-albicans Candida species detected were C. krusei (2%), C. dubliniensis [24], C. parapsilosis (4.1%), C. tropicalis (8.2%), and C. glabrata (14.3%) [19] (Table 1). Mañas and colleagues found that colonization with C. albicans better correlates with the onset of oral and pharyngeal infections among these patients (p = 0.0273) [19].

Panghal and colleagues [24] conducted a prospective cohort analysis in the Radiotherapy Unit of the Regional Cancer Institute, Pt. B.D. Sharma University of Health Sciences, Rohtak, Haryana, included 186 patients, of which only 62 (33%) underwent radiotherapy for head and neck cancer. Samples have been obtained from oral swabs or blood collection. The results agree with the other studies and confirm that C. albicans is the most prevalent and significant yeast isolated from the oral cavity in patients treated with radiotherapy at the head and neck level: it constitutes almost 50% of all pathogens detected (Table 1). In patients’ blood, C. albicans has been found at 24% rate [24].

Twenty-two C. albicans strains were detected among 60 Argentine patients in 2012, confirming C. albicans as the most prevalent Candida species in the oral cavity of patients undergoing head and neck radiotherapy. Indeed, non-albicans Candida species were found in lower percentage: C. tropicalis (13 strains), C. parapsilosis (six strains), C. krusei (three isolates), C. dubliniensis (one isolate) (Table 1) [25]. An Italian survey conducted between April and May 2013 among major Italian radiotherapy centers estimated that OPC occurred in 30–40% of patients with head and neck cancer [27].

Ahadian and colleagues found an increase in the percentage of patients with oral candidiasis by 100 percent during and at the end of radiotherapy treatment. None of the 144 patients enrolled in this analytical cross-sectional study exhibited Candida infections during the examination before starting the treatment. However, as early as the second week of radiotherapy, every patient had oral candidiasis, which persisted until the end of the study [28]. Patients who underwent oral management appear to be less affected (25%) by oral candidiasis, according to Kawashita and colleagues [14].

One of the most recent studies found that fungal colonization was discovered, analyzing oral mucosa and tongue swabs, in 39.5%, 65.9%, and 57.7% of patients before RT, during RT, and at the end of RT, respectively. Chitaparanux and colleagues detected 136 colonized specimens in all positive colonized patients and identified ten fungal species. The majority of fungal species were C. albicans in 112 specimens of all collected oral swabs, which were also the most common species found at every period, i.e., before RT, during RT, end of RT. The second most common species was C. tropicalis. For 34 patients who had positive fungal colonization before starting RT, we found that 11 patients (32.3%) subsequently developed clinical disease [20] (Table 1). Moreover, n this paper the authors underlined that there is an underestimation of clinical oral candidiasis before and throughout the course of radiotherapy from using only clinical sign and symptoms and, for this reason, an accurate oral examination is crucial in these patients before starting the therapy.

Oral candidiasis increased from 9.1% in an intermediate evaluation to 15.2% in a final evaluation among 66 patients undergoing head and neck radiotherapy at the Mario Penna Institute (Belo Horizonte, Minas Gerais, Brazil) [29]. At baseline, 44.7% of oral swabs tested positive for Candida colonization in a study conducted by Rupe and colleagues [30] at Gemelli Advanced Radiation Therapy (ART), Fondazione Policlinico Universitario A. Gemelli-IRCSS.

Around 65 % of patients treated with radiotherapy were found positive for oral candidiasis at the Institute of Medical Sciences (IMS) and SUM Hospital, Odisha, India. Several Candida species were detected from the oral swab collected: C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, C. dubliniensis [31].

International guidelines suggest treating candidiasis locally using specific molecules with low risk of drug interactions and low risk of fungal resistance as nonspecific antiseptic solutions are generally ineffective in this treatment [32]. For mild onset of the disease, clotrimazole tablets are recommended (10 mg 5 times daily) or mucoadhesive buccal tablet of miconazole (50 mg, applied locally to the mucosal surface over the canine fossa once daily for 7–14 days) [32] or nystatin (NYS) suspension (100,000 U/mL) 4–6 mL 4 times daily, or 1–2 NYS pastilles (200,000 U each) 4 times daily, for 7–14 days [32]. For moderate to severe disease, oral fluconazole is indicated, 100–200 mg daily for 7–14 days [32]. If patients have fluconazole-refractory disease, it has recommended itraconazole solution (200 mg once daily) or posaconazole suspension (400 mg twice daily for 3 days then 400 mg daily, for up to 28 days) [32]. Alternatively voriconazole (200 mg twice daily) or AmB deoxycholate oral suspension (100 mg/mL 4 times daily) can be prescribed [32]. Intravenous echinocandin (caspofungin: 70-mg loading dose, then 50 mg daily; micafungin: 100 mg daily; or anidulafungin: 200-mg loading dose, then 100 mg daily) or intravenous AmB deoxycholate (0.3 mg/kg daily) can be used as well [32]. For patients with recurrent infections, fluconazole (100 mg, 3 times weekly) is recommended [32]. Instead, for HIV-infected patients, antiretroviral therapy is strongly recommended [32].

With globalization, antifungal resistance represents a serious emerging public health concern. Therapeutic options for treating multidrug-resistant fungi are limited and new antifungal strategies are urgently needed [33]. The major problem is the absence of drugs that are active against emerging antifungal resistance mechanisms, such as echinocandin and azole resistance mediated by FKS and CYP mutations, commonly used for the difficult-to-treat yeasts or molds carrying intrinsic antifungal resistance. The optimal features of antifungal agents should be fungicidal and should provide options for combination antifungal use, with a favorable pharmacokinetic/pharmacodynamics profile. Moreover, agents should present a few drug-drug interactions and be administered via both the oral and iv routes [34]. Until recently, there has been no new class of antifungal drugs discovered in over 40 years, and no new class brought into practice since the echinocandins in 2006 [35].

New antifungal drugs have been identified due to the large number of libraries for initial screening, repurposing of old drugs (with some previously identified/used for other indications repurposed to treat fungal infections) [36], and advances in understanding cell metabolic pathways [35]. There are now several new antifungal classes under clinical trials, which include agents that target fungal cell wall synthesis, fungal cell membrane metabolic pathways, inhibit the function of mitochondria, and drugs targeting cell signaling pathways [37].

The resistance to the available antifungal drugs related to Candida spp. infections increased as well as drug interactions, urging the development of innovative and more effective agents with antifungal action. To date, novel molecules are being discovered and assessed for their capacity to control Candida spp. growth and, probably in the future, will be used to treat oral candidiasis overall during radiotherapy [38]. Among them, aldehydes, hydrazones, and hydrazines act against fungal membranes [38]. Instead, N-acyldiamines have demonstrated moderate activity against C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis [38]. Here, the antifungal activity of isolated compounds, extracts, and essential oils (EO) of plant origin will be discussed comprehensively.

• a

  Isolated compounds refer to chemical components separated from the plant material through various extraction and purification processes to obtain single molecules or specific classes of molecules.

• b

  Extracts are mixtures containing various chemical components with potential synergistic effects resulting from the presence of multiple compounds; extracts are obtained from a plant source through different extraction methods and solvents.

• c

  Finally, EOs are concentrated hydrophobic liquids consisting of complex mixtures of low molecular weight compounds extracted from plants by steam distillation, cold pressing, and various solvents.

Punica granatum L. [Punicaceae] – commonly known as pomegranate – is a fruit belonging to Punicaceae family which produces and accumulates many bioactive compounds such as anthocyanidins, hydrobenzoic acids, hydroxycinnamic acids, alkaloids, tannins, and flavonoids [39, 40]. Pomegranate peel extracts possess active antifungal compounds such as flavones, flavonones, and other flavanols possessing anti-inflammatory, antimutagenic, and antifungal activity [39, 41]. Among them, PGN is the primary active component of pomegranate extracts with high antifungal activity against C. albicans in vitro [42, 43] and on rat models [41]. It possesses many tannins that have an antimicrobial action, for example inhibiting extracellular microbial enzymes; depriving of substrates and metal ions necessary for microbial growth; acting directly on microbial metabolism through the inhibition of oxidative phosphorylation; complexing with proteins and polysaccharides leading to cell death and precipitating membrane [43]. Da Silva et al. [43] demonstrated that the combination of NYS with PNG increased antifungal efficacy against Candida compared with the effect of the compounds tested alone. Combinations of PCG (6.25 μg/mL) with NYS (3.9 μg/mL) and 5 PCG (12.5 μg/mL) with NYS (1.95 μg/mL) were more effective since they reduced the minimum inhibitory concentration (MIC)-50 of PCG (50 μg/mL) by 8 and 4 times, respectively, increased the fungal inhibition [43]. Moreover, Lavaee et al. [44] compared the antifungal activities of aqueous, ethanolic, and methanolic extracts of the bark and root of P. granatum with fluconazole and nystatin on oral Candida strains. They concluded that the bark and root extracts exhibited antifungal activities and inhibitory effects against C. albicans and C. glabrata. Methanolic and ethanolic extracts of this plant were more effective against the investigated fungal agents than the aqueous extracts. Kumar et al. [45] compared P. granatum peel extracts with clotrimazole to assess if this extract could be used as an adequate natural substitute for synthetic antifungal drugs. In their study, they showed that P. granatum peel extract is an effective antifungal agent against C. albicans and its antifungal efficacy approximates that of standard clotrimazole (p < 0.05) [45] (Table 2). Magnolol (5.5′-diallyl-2.2′-dihydroxybiphenyl) is a polyphenolic compound, isolated from the stem bark of a traditional Chinese herbal medicine Magnolia officinalis [99], which has multiple pharmacological properties such as anti-inflammatory, anti-oxidative, antibacterial and antifungal activities [99, 100]. Sun and its colleagues showed significant antifungal activity of magnolol against C. albicans in the concentration range of 16–32 µg/mL because the compound has inhibitory effects on adhesion, yeast-hyphal transition, and biofilm formation by C. albicans (p < 0.01) [101] (Table 2).

Among polyphenols, we cannot fail to mention curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), which is a yellow polyphenolic substance also known as diferuloylmethane [64]. Its molecular structure is similar to other bioactive nonvolatile curcuminoids, such as dimethoxycurcumin and bisdemethoxycurcumin, which differ only in the number of methoxy groups on their aromatic rings. It is extracted from the rhizomes of Curcuma longa Linn (Zingiberaceae family) and has long been known as an Indian spice with potent health benefits [64]. This compound has been reported for the treatment of diseases of the oral site, such as OPC and Candida-related denture stomatitis [102]. Lee et al. [46] assessed the role of curcumin in C. albicans treatment using a doxycycline-mediated heat shock protein 90 (HSP90) strain and an HSP90-overexpressing strain of C. albicans. They demonstrated that curcumin can lead to the downregulation of CDR1 and the inhibition of efflux pump activity of CDR1. So, curcumin could impair cell growth, stress responses, and efflux pump activity of C. albicans (p < 0.05) [46] (Table 2). Lichochalcone-A is another natural polyphenol with protective activities, especially antimicrobials and anti-biofilm formation [50]. Saleem et al. [50] investigated the antifungal activity of lichochalcone-A against C. albicans biofilms both in vitro and in vivo, exploring its ability to alter critical virulence factors responsible of the pathogenicity of C. albicans. Their investigation showed that lichochalcone-A (at the concentration of 625 μm and 1,500 μm) significantly decreased the proteinases and phospholipases (p < 0.05) activity of C. albicans, suggesting that one possible antifungal mechanism involves the inhibition of the protease secretions. Moreover, these enzymes secreted by C. albicans are often associated with tissue degradation, hyphal formation, and host invasion, which are critical factors linked to the pathogenicity of C. albicans [50] (Table 2).

Another promising plant, belonging to the Zingiberaceae family, with anti-Candida activity, is ginger (Zingiber officinale) [47‒49]. Ginger rhizome is abundant in several compounds, such as phenols and terpenes. The phenolic compounds are mainly gingerols, shogaols, and paradols. The most abundant terpenes are β-bisabolene, α-farnesene, α-curcumene, and zingiberene [103]. Žitek and colleagues [104] used a combined ginger-hemp extract to verify its antifungal and antibacterial activity finding that the minimum inhibitory concentration of C. albicans was 78.3 μg/mL, while it was 156.5 μg/mL for the Gram-positive bacterium S. aureus and 625.2 μg/mL for the Gram-negative bacterium E. coli.

Interestingly, the lectins present in Cicer arietinum (chickpea) [51], a legume that belongs to the Fabaceae family [51] seem to exert an antifungal activity. Kumar et al. [51] studied the properties of C-25 lectin, a protein contained in this legume, using the Agar diffusion method against human pathogenic fungi such as C. parapsilosis, C. krusei and C. tropicalis. They concluded that C-25 acts primarily on the cell wall of Candida spp., by disrupting the cell wall and distorting the cellular morphologies (Table 2).

Nerol is a natural monoterpene compound extracted from rutaceous plants, such as orange, fingered citron, or honey-suckle [52]. Recently, it has been found to have significant antifungal activity [53] and is more effective than benzalkonium in the control of C. albicans [54], contrasting the mycelial growth of C. albicans cell membrane (p < 0.05) [52]. Wang et al. [52] demonstrated that nerol is effective against C. albicans infection and has a positive effect on OPC. Based on their results, they hypothesized that nerol destroys the cell membrane of C. albicans, which leads to apoptosis, and induces the increase of Ca²⁺ and ROS in C. albicans cells, thus causing mitochondrial dysfunction and damage the permeability of the cell membrane, through the acidification of external liquids, by inhibiting hydrogen ion efflux too [52] (Table 2).

Among rutaceous plants, also grapefruit contains anti-Candida compounds. Tsutsumi-Arai and co-workers studied the antifungal activity of grapefruit seed extract on four Candida species causing oral candidiasis, including C. albicans, proving an interesting fungicidal effect [53]. MIC and minimum fungicidal concentration (MFC) were, respectively, 19.5 μg/mL and 39 μg/mL, and the ratio MFC/MIC of grapefruit seed extract (equal to 2) demonstrated a fungicidal effect against C. albicans [53]. The scanning electron microscope images of the treated Candida cells showed various cell damage patterns, such as cell wall damage, leakage of cell contents, and cell lysis. The viability test showed grapefruit seed extract induced mortality in almost all Candida cells; conversely EpiOral^™ human buccal mucosa tissue model, treated with GSE, presented more than 90% cell viability. The grapefruit peel is rich in EOs, lipophilic liquid volatile substances with aromatic properties. The peel extract contains active substances, such as terpenes, hydrocarbons, sesquiterpenes, alcohols, aldehydes, and esters. It showed positive inhibition zones assay results, suggesting good antifungal activity against C. albicans [56, 57] (Table 2). Prasad and colleagues investigated the effect of a grapefruit leaf extract against Candida species [58]. The results showed that this EO extract was more active on C. albicans and C. krusei than the commercially available Amphotericin B [58] (Table 2).

Cuminum cyminum (Apiaceae) is an annual herbaceous plant with fruits, each one containing green seeds, which have aromatic characteristics. Its fruits have applications in the treatment of diarrhea and toothache [59]. Salvadora persica shrub has been used traditionally in folk medicine for dental hygiene and for the treatment of periodontal diseases [59]. Naeini and colleagues investigated the in vitro efficacy of an alcoholic extract from S. persica and the EO of C. cyminum as antifungal agents for candidiasis [59]. They showed that C. cyminum affects the regulation and function of the membrane-bound enzymes altering the synthesis of many cell wall polysaccharide components (i.e., β-glucan, chitin, and mannan) and altering the cell growth and morphogenesis too [59]. Moreover, some EOs can cause extensive cellular damage at much lower concentrations, probably due to better penetration and contact [59]. They concluded that C. cyminum EO and S. persica alcoholic extract had strong to moderate activity against different pathogenic Candida species and they could be alternative substances for fungi control in addition to conventional agents [59] (Table 2).

Another novel compound is the spice cinnamon which is obtained from the inner bark of several trees in the genus Cinnamomum, which itself includes approximately 250 plant species [60]. The most common species are Cinnamomum cassia (Chinese cinnamon, commonly called Cassia) and C. verum (also called C. zeylanicum, commonly known as true cinnamon). These two species contain different percentages of cinnamaldehyde [60], which can inhibit the growth of bacteria, molds, and yeasts, and the toxin production by their microorganisms [60]. Regarding fungi, Cinnamaldehyde showed high antimycotic activity against Candida spp. in vitro, with both fungistatic and fungicidal properties [61]. Veilleux et al. [62] evaluated the effects of two cinnamon fractions in vitro demonstrating their ability to attenuate the growth, the biofilm formation, and the adherence properties of C. albicans (p < 0.01) [62].

Propolis is also a natural substance active against oral microorganisms associated with dental caries and periodontal diseases; it exerts a significant action against Candida infections. Propolis’ main target appears to be the microbial cell wall [63]. Stähli et al. [63] have also demonstrated, in in vitro experiments, that propolis retards biofilm formation and, when used in high concentrations, dissolves existing biofilms (p < 0.01) (Table 2).

Simonetti et al. [65] for the first time, evaluated in vitro anti-Candida activity of a grape seed extract from wine and table cultivars of Vitis vinifera L. in an experimental murine model of vaginal candidiasis [64, 65]. They demonstrated that dried grape seed extracts obtained from the cultivars M. Palieri, Italia, Red Globe, and Negroamaro, showed the highest content of flavan-3-ols with a polymerization degree ≥4 and the best antifungal activity against C. albicans (p < 0.01) [65] (Table 2). In addition, grape seed extract from V. vinifera varieties Pinot noir and Pinot meunier showed anti-Candida activity with MIC values of 0.39 and 50 mg/mL, respectively [66]. Grape seed extract showed high antifungal activity even when encapsulated in titanium oxide nanoparticles [105]. Other anti-Candida phenolic compounds contained in V. vinifera are two monomeric stilbenes, resveratrol and its derivative, pterostilbene. Red grapes are the main dietary sources of resveratrol and pterostilbene is represented in blueberries and grapes. Resveratrol and pterostilbene exhibit several activities such as antioxidant, neuroprotective, anticancer, cardioprotective, anti-aging, and anti-inflammatory activities [106]. Pterostilbene has more potent pharmacological properties than resveratrol due to its chemical structure, which makes it more lipophilic, enhancing its membrane permeability, bioavailability, and biological potency [106]. Resveratrol, antifungal, and fungicidal activity on C. albicans has been demonstrated at the concentration of 10–20 μg/mL and confirmed by scanning electron microscopy [67]. The anti-biofilm effect of both resveratrol and pterostilbene was confirmed, as both the compounds are able to disrupt preformed biofilms of C. albicans and to inhibit a possible new formation [68, 69, 107] (Table 2).

Hypericum perforatum L. (Hypericaceae) has been one of the most investigated medicinal plants due to its antidepressant activity, although it is studied for a broad range of other biological activities including wound-healing, anti-inflammatory, antibacterial, antifungal, and antiviral activities [108]. The roots of H. perforatum produce and accumulate xanthones, bioactive polyphenols, in small amounts, which increase in vitro-regenerated roots [109]. Xanthones showed high anti-Candida activity that increases with increasing concentration. Since xanthones have high bioactivity related to their concentration, there are several studies on biotechnological strategies aimed at stimulating their production in adventitious root cultures of H. perforatum [70‒85] (Table 2). Badiali and colleagues stimulated xanthone production in root cultures using chitosan oligosaccharides (COSs) at different concentrations, leading to a 12-fold increase in cultures treated with 400 mg/L of COS [85]. The methanolic extract obtained from H. perforatum root cultures, treated with the highest COS concentration, had the greatest antifungal activity with MIC-50 value of 32 μg/mL [85].

Several (14) mongolian endemic plants were analyzed for their antifungal activity. In particular, the methanolic extracts were tested against C. albicans, C. krusei, C. parapsilosis, C. tropicalis, C. glabrata, Microsporum gypseum, Trichophyton mentagrophytes, and Malassezia furfur, in order to identify the plant species active against fungi responsible for cutaneous infections [86]. Among the species examined, only methanolic extracts obtained at room temperature from Stellaria dichotoma L., Scutellaria scordifolia L., Aquilegia sibirica Fisch. Et Schrenk, and Hyoscyamus niger L. showed antifungal activity. More specifically, S. scordifolia L., showed the best activity against Candida spp., M. furfur and dermatophytes, with geometric mean (GM) MIC50 values of 22 µg/mL, 64 µg/mL, and 32 µg/mL, respectively. Also, S. dichotoma L., A. sibirica Fisch. Et Schrenk and H. niger L. showed a lower antifungal activity. All the other extracts registered GM MIC50 values >256 µg/mL. The antifungal activity of the active species may be explained by the presence of some flavones that inhibit the growth of fungi. They were characterized by Giordani and colleagues with HPTLC analytical technique, which detected the prominent presence of luteolin and apigenin in S. scordifolia extracts, rutin in S. dichotoma and H. niger L. extracts and also chlorogenic acid and caffeic acid in the extracts of S. dichotoma L. and A. sibirica Fisch [110].

Although poorly studied, Kiwi peels also represent an interesting source of relevant biomolecules [111]. They contain dietary fibers and other metabolites such as polyphenols, carotenoids, chlorophylls, and aroma compounds [87]. Because of the presence of these active compounds, kiwi peel extracts have been tested in vitro for their possible anti-Candida activity. Results by Cairone and colleagues showed an interesting activity against C. albicans and C. glabrata. The strongest activities were shown by the ethyl acetate extracts coming from this selected cultivar, which presented MIC50 values of 4 μg/mL against three C. glabrata strains and 8 μg/mL against one C. albicans strain [87] (Table 2).

Amaranth seeds are known to biosynthesize large amounts of lipids, proteins, carbohydrates, and dietary fibers as well as other important components such as squalene [112]. De Vita and colleagues studied the antifungal activity of the a-polar fraction of Amaranthus cruentus seeds, obtained by extracting supercritical CO₂ [88]. Triacylglycerols and squalene were found as the main components after the phytochemical analysis of the crude extract. The amaranth seed extract was found to be inactive alone against C. albicans, while an interesting synergistic activity was found when tested together with terbinafine, a common antifungal drug that interferes with ergosterol biosynthesis in fungi [88]. The association showed fungicidal activity against C. albicans strains at a concentration of 500 μg/mL and 32 μg/mL for amaranth seed extract and terbinafine, respectively. This result suggests the possible application of amaranth oil as a vehicle preparing formulations with terbinafine for topical use [88] (Table 2).

Fierascu and colleagues recently reviewed the polyphenolic content and the health potential of Fragaria spp. and strawberries were evaluated as anticancer, anti-inflammatory, anti-obesity, and chemoprotective agents, as well as for their potential in antimicrobial, anti-allergenic, and antidiabetic applications [113]. Phenolic extracts of strawberries were also reported to inhibit C. albicans growth [114]. In a study conducted by Cairone and colleagues, Clery strawberry samples showed in vitro antifungal activity against C. albicans strains, with GM MIC50 values ranging from 19.36 μg/mL to 59.82 μg/mL and MIC90 values ranging from 68.13 μg/mL to 162.09 μg/mL. In addition, 80% of biofilm onset was inhibited with pasteurized and homogenized extract at a concentration of 500 μg/mL [89] (Table 2).

The antifungal properties of the EOs of mint (Mentha suaveolens), thyme (Coridothymus capitatus), oregano (Origanum hirtum), and rosemary (Rosmarinus officinalis) were investigated against four species of Candida by Spagnoletti and colleagues [90]. The antimicrobial activity of EOs against C. albicans, C. glabrata, C. krusei, and C. tropicalis was from 760 ± 290–3,120 ± 0.0 μg/mL, expressed as minimal inhibitory concentration mean. In particular, for C. albicans, all values were lower compared to the values of other Candida spp., and all EOs showed similar antimicrobial activity. The most active EOs were EOs of mint and thyme, showing a minimal fungicidal concentration mean value of 1,560 μg/mL [90] (Table 2). This aspect led to the consideration of thymol and carvacrol as the molecules responsible for the observed antifungal activity. Moreover, it can also be suggested that phytocomplexes containing thymol and carvacrol could reasonably give positive and significant responses in anti-Candida assays [115].

Carvacrol was investigated for its antifungal and antibacterial effects [116], and different studies demonstrated that this molecule is significantly active against C. albicans, and also impairs the growth of different morphological forms, such as yeast, hyphae, and even the most resistant forms of biofilm [117]. Extracts from Lycium barbarum (Goji) Fruit European Cultivars contain interesting secondary metabolites, such as gallic acid, chlorogenic acid, catechin, sinapinic acid, rutin, and carvacrol. Mocan and colleagues [91] explored the antifungal properties of the hydroalcoholic goji berries extract. Polish samples showed the best inhibitory activity with MIC50 values of 138 μg/mL and 186 μg/mL against some C. albicans strains [91]. Due to the presence in these extracts of rutin and carvacrol, well-recognized as antimicrobial agents, they decided to investigate their activity alone against the same strains and found that rutin was almost inactive with MIC values ≥64 μg/mL. Conversely, carvacrol showed a potent inhibitory activity against C. albicans with MIC50 ranging from 0.125 μg/mL to 0.25 μg/mL [91].

C. albicans biofilm can colonize mucosal surfaces such as those coating the oral and vaginal epithelia, as well as implanted medical devices such as prosthetics, heart valves, and catheters, which can seed systemic infections in humans [118]. According to the results obtained by Ourabah and colleagues, Freaxinus angustifolia leaves and bark, as well as Clematis flammula leaves extracts, could be a promising source of drugs against muco-cutaneous infections caused by C. albicans biofilm [92]. Indeed, they found antifungal activity of the ethanolic extracts against both C. albicans planktonic cells and biofilm. In particular, F. angustifolia leaf extract inhibited the biofilm formation by about 70% at the concentration of 1,000 μg/mL [92]. Minimal Biofilm Inhibition Concentration was 250 and 500 μg/mL for F. angustifolia leaves extract and F. angustifolia bark extract respectively, while C. flammula showed a lower activity against C. albicans biofilm (lower than 50%) at 1,000 μg/mL [92]. The higher inhibition level of biofilm formation shown by F. angustifolia extracts can be explained by higher amounts of polyphenols compared with that of C. flammula [92].

Laurus nobilis showed a solid antifungal activity, comparable to the one of nystatin, with a deleterious effect against C. albicans. This capacity is probably due to monoterpenes and sesquiterpenes. The EO inhibits biofilm formation and adhesion with MIC and MFC values that ranged from 250 to 500 μg/mL but increased in the presence of sorbitol (osmotic protector) and ergosterol. These data indicated an affinity for the ergosterol and the synergic activity affecting cell wall biosynthesis and membrane ionic permeability. After being applied for 1 min, every eighth, for 24 h and 48 h, L. nobilis EO showed a reduction for biofilm, with no significant difference compared to nystatin [93].

Tea tree oil (TTO) is produced as a distillate of leaves of Melaleuca alternifolia shrub and includes monoterpenes, sesquiterpenes, and their related alcohols [119]. It has been demonstrated a significant antifungal activity of TTO against several different clinical isolates of C. albicans, other Candida species and C. neoformans [94]. Moreover, in an animal model of vaginal candidiasis, it has been shown that TTO treatment is efficacious in resolving experimental Candida infection, with both fluconazole-susceptible and -resistant isolates [94]. Among the several constituents of TTO, terpinen-4-ol (T-4-ol) seems to be the most active antimicrobial compound of this oil with a MIC that was higher both in susceptible and in resistant strains of C. Albicans compared to TTO [95]. In an animal model of vaginal candidosis, T-4-ol was able to control C. albicans vaginal infections with both fluconazole-susceptible and -resistant isolates [95]. TTO (17.92 mg/mL) and T-4-ol (8.86 mg/mL) were also effective against C. albicans biofilm formation controlling in vitro biofilm proliferation [96]. Therefore, Ramage et al. [97] in 2012 evaluated the impact of TTO-based compounds on inflammation, using IL-8 as a biomarker, confirming in vitro that TTO, especially its component terpinen-4-ol (T-4-ol), exhibits strong antimicrobial properties against fungal biofilms. They isolated 100 clinical strains of C. albicans, 30 of which were from patients who received palliative care for advanced cancer. Transcript and protein analysis showed a reduction of IL-8 when treated with TTO and T-4-ol [97]. One of the main treatments for vaginal candidiasis is the use of vaginal probiotics, so Di Vito et al. [98] evaluated the in vitro microbicidal activity of vaginal suppositories (VS) containing TTO (TTO-VS) toward Candida spp. and vaginal probiotics (Bifidobacterium animalis subsp. lactis (DSM 10140) and Lactobacillus spp. (Lactobacillus casei R-215 and Lactobacillus acidophilus R-52). Fortunately, TTO-VS exhibits, in vitro, a selective fungicidal action, slightly affecting only the B. animalis strain growth belonging to the vaginal microbiota [98].

The frequency of oral mycosis has remarkably increased globally because of the abuse of antibiotic agents, immunosuppressive drugs, and immunodeficiency in humans, so it urges the development of innovative and more effective agents with antifungal action. In conclusion, in our review we offer a summary of natural products with antifungal properties (especially against Candida spp.) and effective in the prevention of radiotherapy candidiasis. As demonstrated experimentally, these substances are natural anti-inflammatory and antimicrobial compounds that can reduce the growth of Candida spp., for example by inhibiting enzymes essential to the pathogen. Although several studies have been conducted in vitro and in animal models, unfortunately it is not possible to identify recommended doses (e.g., in mg/day) for the treatment of radiotherapy-induced oral candidiasis. A limitation of the current work is the small number of preclinical and clinical studies performed in the current Literature. Further studies are needed to understand the rationale for the correlation between radiotherapy and candidiasis and the role of natural substances in the treatment of these particularly disabling side effects for cancer patients.

The authors have no conflicts of interest to declare.

This manuscript collects the bibliographic research carried out within the research project financed by the Lazio Region (Italy) n. PROT. A0375-2020-36590 – “Gruppi di ricerca 2020” – POR FESR Lazio 2014-2020 – Azione 1.2.1– CUP B85F21004080002.

Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: E.I., G.B., A.A., M.P.L.G., and C.B. Drafting the work: E.I., A.A., G.S., G.B., M.B., E.B., and C.B. Revising it critically for important intellectual content: G.G., G.P., M.C., L.D.G., and M.P.L.G.

Elena Imperia and Graziana Bonincontro contributed equally to this work.