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Abstract: Although medicinal herbs with fungicidal effects have been ubiquitously employed in traditional medicine, such effects of culinary herbs and spices still have to be elucidated. Therefore, it is noteworthy to determine the antifungal efficacy of some edible herbs used in Thai cuisine against sessile Candida albicans cultures, and to inquire if they can be further utilized as naturally-derived antifungals. Fourteen essential oils extracted from Thai culinary herbs and spices were tested for their antifungal activity against C. albicans using the agar disk diffusion method followed by broth micro-dilution method for the determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration. The oils with potent antifungal effects against planktonic fungi were then assessed for their effect against sessile fungus (adherent organisms and established biofilm culture). MIC of the oils against sessile C. albicans was evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide reduction assay. All selected culinary herbs and spices, except galangal, garlic, and turmeric, exhib-ited inhibitory effects on planktonic yeast cells. Cinnamon bark and sweet basil leaf essential oils exhibited potent fungicidal effect on planktonic and sessile fungus. Sessile MICs were 8-16 times higher than planktonic MICs. Consequently, both cinnamon

bark and sweet basil leaf herbal oils seem to be highly effective anti-Candida choices.(J Oral Sci 58, 365-371, 2016)

Keywords: cinnamon (Cinnamomum zealanicum); sweet basil (Ocimum basilicum); essential oil; Candida albicans; minimum inhibitory concentration (MIC); sessile minimum inhibitory concentration (sMIC).

IntroductionCandida albicans is a normal commensal fungus present on human mucosal surfaces, including the oral cavity, gastrointestinal tract, genitourinary tract, and skin, that becomes an opportunistic pathogen in immunocompro-mised or immunodeficient individuals (1,2). This fungal infection may cause superficial diseases, such as oral thrush (oral candidiasis) and vulvovaginal candidiasis, as well as systemic diseases that have high morbidity and mortality, such as candidemia and disseminated candidiasis (3-5). All infections are closely associated with the biofilm formation of Candida species (6). The community structure of these yeast cells have well-regulated metabolic processes and inter-organism communication, and act as a robust shelter that protects the interior organisms from antifungal drug attacks (7). The biofilm formed has been shown to be refractory (less susceptible) to several antifungal drugs, such as azoles (fluconazole, ketoconazole, and miconazole), polyenes (amphotericin B, nystatin, and natamycin), and echini-candins (caspofungin, micafungin, and anidulafungin), which are commonly employed as prophylactic and therapeutic agents against candidal infections (8,9).

Journal of Oral Science, Vol. 58, No. 3, 365-371, 2016

Original

Effect of essential oils prepared from Thai culinary herbs on sessile Candida albicans cultures

Ray S. Hovijitra1), Suwan Choonharuangdej2), and Theerathavaj Srithavaj1)

1)Maxillofacial Prosthodontic Unit, Department of Prosthodontics, Faculty of Dentistry, Mahidol University, Bangkok, Thailand

2)Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand

(Received December 29, 2015; Accepted March 9, 2016)

Correspondence to Dr. Suwan Choonharuangdej, Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, 6 Yothee Road, Rajathewee, Bangkok 10400, ThailandFax: +66-2-200-7804 E-mail: suwan.cho@mahidol.ac.thdoi.org/10.2334/josnusd.15-0736DN/JST.JSTAGE/josnusd/15-0736

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These available antifungal drugs are unaffordable agents in many developing countries for candidal infection management, thus alternative, effective, and afford-able antifungals have continually been elucidated and developed. Novel naturally-derived products with potent inhibitory or fungicidal activity against Candida biofilms are of clinical interest.

Plants containing phytochemicals, such as medicinal herbs with fungicidal effects, have been extensively investigated and employed in traditional medicine for years. However, relatively less attention has been paid to culinary herbs and spices used as flavoring, coloring, and preserving agents in Eastern and Western cuisine that exhibit medicinal antioxidant, antibacterial, and antifungal activities (10-14). Therefore, it is noteworthy to examine the inhibitory and fungicidal activities of selected Thai herbs and spices against the C. albicans biofilm. This will allow us to identify edible plants with impressive fungicidal effects, especially against fungal biofilms, which can be considered as safe (less side effects and toxicity), inexpensive, and locally-produced alternatives for candidiasis management.

Materials and MethodsThis study included the following culinary herbs and spices: sweet (Thai) basil (Ocimum basilicum), cinnamon (Cinnamomum verum or C. zealanicum), coconut (Cocos nucifera), fennel (Foeniculum vulgare), galangal (Alpinia galangal), garlic (Allium sativum), kaffir lime (Citrus hystrix), lemon (Citrus limon), lime (Citrus aurantifolia), orange (Citrus sinensis), pepper-mint (Mentha piperita), spearmint (Mentha spicata), and turmeric (Curcuma longa). Fourteen essential oils were extracted from different parts (leaves, bark, root/rhizome, and fruit) of the selected plants (Tropicalife Co., Ltd, Yoniso Chemicals Co., Ltd, Bangkok, Thailand). RPMI-1640 medium, morpholine propanesulfonic acid (MOPS), XTT sodium salt [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide], menadione (2-methyl-1,4-naphthoquinone), dimethyl sulfoxide (DMSO), and yeast nitrogen base were purchased from Sigma-Aldrich Life Science (Saint Louis, MO, USA). Sabouraud dextrose agar (SDA) and Mueller-Hinton agar were purchased from Becton, Dick-inson (Thailand) Ltd. (Bangkok, Thailand). C. albicans ATCC 10231 was provided by the Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand.

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against planktonic C. albicans cultureA preliminary examination of the antifungal activity of selected herbal essential oils [5%, 10% (v/v) with 1% DMSO and undiluted] was conducted using the agar disk diffusion method on Mueller-Hinton agar. Those herbs with inhibitory activity against C. albicans ATCC 10231 were then evaluated for their MIC and MFC values using the broth micro-dilution method with some modifica-tions (15). Briefly, sterile, flat-bottom, 96-well microtiter plates were used, and the tested oils and clotrimazole were dissolved with 1% DMSO (in RPMI-1640 medium buffered to pH 7.0 with 0.165 M MOPS) to yield working solutions with concentrations of 25 µL/mL [2.5% (v/v)] and 2.5 mg/mL [0.25% (w/v)], designated as tests and positive controls, respectively. Thereafter, the culinary herbs and clotrimazole solutions were two-fold diluted serially in 135 µL of RPMI-1640 medium placed in each well of the first ten columns, prior to the addition of 15 µL of fungal suspension, so as to yield a final inoculum of 1-5 × 103 cells/mL. Additionally, 1% DMSO and RPMI-1640 medium were used as diluent and growth controls, respectively. The plates were incubated at 37°C for 48 h before visual inspection for growth of the tested organisms. MIC was the lowest concentration of a certain spice or clotrimazole required to impede the fungal growth (visually clear well). Minimum fungicidal concentration (MFC), defined as the lowest concentra-tion of spice capable of eliminating the tested organisms, was evaluated by transferring 25 µL of the solution in each clear well to SDA and incubating at 37°C for 48 h. The experiment was carried out at five different time points, and all samples were tested in triplicate each time.

MIC against in vitro sessile C. albicans culturesBiofilm development exhibits two phases in vitro, namely adhesion and biofilm formation. Culinary herbs with the most impressive antifungal activity against the planktonic C. albicans were further analyzed for their antifungal effects on the adhesion and biofilm phases of sessile fungus. Sessile MIC (sMIC) was determined according to the methods described previously with some modifications (16-18).

Adhesion phaseSolutions of the two culinary herbs and spices exhibiting the highest antifungal activities and clotrimazole were prepared [with 1% DMSO in yeast nitrogen base supple-mented with 100 mM glucose (YNB-100)] in a separate 96-well microtiter plate, as described previously. The

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concentrations of the tested spices and clotrimazole ranged between 1.25% (v/v) [12.5 µL/mL] and 2.5 mg/mL, respective to their MICs. C. albicans was freshly subcultured on SDA at 37°C overnight, and a few colo-nies were transferred and grown in yeast nitrogen base supplemented with 50 mM glucose (YNB-50) overnight in a 37°C incubator on a rotary shaker at 75 rpm. The organisms were then washed twice with 20 mL of 0.1 M phosphate-buffered saline (PBS), pH7.2, prior, to being re-suspended with YNB-100 medium to yield 1 × 107 organisms/mL. Each well of columns 1-11 contained 100 µL of C. albicans suspension, while an identical volume of YNB-100 medium was placed in each well of column 12. The plate was placed in a 37°C incubator on a rotary shaker at 75 rpm for 90 min. The medium was gently aspirated and washed once with 200 µL of PBS, pH 7.2. Thereafter, 100 µL of the herbal or clotrimazole solu-tion prepared previously was transferred to each well of columns 1-10, while columns 11 and 12 were filled with 100 µL of YNB-100 medium and designated as unchal-lenged adherent cells and sterility controls, respectively. The plate was then subjected to constant agitation at 75 rpm for 24 h at 37°C. After the herbal/drug dilutions were carefully aspirated, the adherent cells were gently washed 4 times with 200 µL of PBS. The viable mass of the adherent organisms was finally determined by XTT reduction assay as follows.

XTT sodium salt was dissolved in PBS at a concen-tration of 1 mg/mL and then stored at −70°C until use. Additionally, 0.4 mM menadione solution was prepared in acetone and stored in aliquots at −70°C until use. Thereafter, the XTT solution was thawed and freshly mixed with menadione solution at a volume ratio of

5:1, and 12 µL of XTT-menadione mixed with 100 µL of PBS was added to each well. The plate was kept in the dark at 37°C for 2 h, and 80 µL of metabolic reac-tion mixture from each well was transferred to a new flat-bottom, 96-well plate and read using a colorimetric microtiter plate reader at a wavelength of 490 nm. Sessile MIC (sMIC) of the tested spices was interpolated by comparing with the unchallenged cells (equivalent to 100% of colorimetric reading). The sMIC90 and sMIC50 of the tested spices were the antifungal concentrations at which the colorimetric readings were reduced by 90% and 50%, respectively.

Biofilm phaseC. albicans suspension was prepared, plated in a 96-well microtiter plate, and incubated as described in the adhe-sion phase section. The inoculum was aspirated carefully, gently washed once with 200 µL PBS, and 200 µL of YNB-100 was immediately added to each well. The plate was then placed in a 37°C incubator with rotary shaker at 75 rpm for 24 h to establish fungal biofilm. After incubation, the established biofilm was gently washed twice with 200 µL PBS, and 100 µL of each dilution of spices or clotrimazole was added to every column, except columns 11-12 which contained 100 µL of YNB-100 and were known as unchallenged biofilm and sterility controls, respectively. After 24 h incubation, sMIC90 and sMIC50 of the tested spices and clotrimazole against the established fungal biofilm were analyzed by XTT reduction assay, as described in the adhesion phase section. This experiment was carried out at five different time points, and the samples were triplicated each time.

Table 1 Plant parts and extraction methods of the selected culinary herb essential oils tested for their antifungal activity

Culinary herbs Plant parts Extraction methodsSweet (Thai) basil (Ocimum basilicum) Leaves Steam distillationFennel (Foeniculum vulgare) Leaves Steam distillationLemon (Citrus limon) Peel Cold pressedLime (Citrus aurantifolia) Peel Cold pressedKaffir lime (Citrus hystrix) Leaves Steam distillationKaffir lime (Citrus hystrix) Peel Cold pressedOrange (Citrus sinensis) Peel Cold pressedGalangal/blue ginger (Alpinia galanga) Rhizome (root) Hydroglycolic extractionGarlic (Allium sativum) Clove Hydroglycerinated extractionTurmeric (Curcuma longa) Rhizome (root) Hydroglycerinated extractionCoconut (Cocos nucifera) Milk Non-heated, non-chemical extractionCinnamon (Cinnamomum zeylanicum) Bark Steam distillationSpearmint (Mentha spicata) Leaves Steam distillationPeppermint (Mentha piperita) Leaves Steam distillation

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ResultsMIC and MFC against planktonic C. albicans cultureAll essential oils tested in this study were extracted from different parts of the selected culinary herbs and spices using appropriate methods, as described in Table 1. The agar disk diffusion method showed that all undiluted herbal essential oils except galangal, garlic, and turmeric oils strongly inhibited the growth of C. albicans. However, at low concentrations of 5% and 10% (v/v), only seven essential oils, consisting of cinnamon bark, sweet basil leaves, orange peel, spearmint leaves, lemon peel, fennel leaves, and kaffir lime leaves still demonstrated inhibi-tion zones against the tested organisms (Table 2; Figs. 1, 2). The MIC and MFC values of these essential oils against the planktonic C. albicans were further analyzed using the broth micro-dilution method.

The anti-Candida efficacy of the herbs tested in this study was separated into three groups, namely, strong

[MIC < 0.5 µL/mL or 0.05% (v/v)], moderate [0.5 µL/mL ≤ MIC ≤ 1.0 µL/mL or 0.1% (v/v)], and weak [MIC > 1.0 µL/mL] activity. Cinnamon bark essential oil exhib-ited the strongest inhibitory effect against planktonic C. albicans, and was followed by sweet basil leaf oils. Orange peel and kaffir lime leaf oils expressed moderate antifungal activity against C. albicans, and lemon peel, spearmint leaf, and fennel leaf oils exhibited weak activity, with fennel leaf oil being the least effective one. The planktonic MIC and MFC values of the essential oils tested, ranging from the most to the least potent antifungal activity, have been summarized in Table 3. Additionally, the MIC value of clotrimazole against the tested yeast cells was 8.79 µg/mL.

MIC against in vitro sessile C. albicans culturesThere are two forms of sessile C. albicans during biofilm development, namely, adherent organisms and biofilm

Table 2 Antifungal activity of the selected culinary herb essential oils against C. albicans, determined by agar disk diffusion method

Culinary herbsInhibition zone

[mean of diameter (mm) ± standard deviation]100% 10% 5%

Cinnamon (C. zeylanicum) bark ≥90 63 ± 4.63 61 ± 3.15Sweet basil (O. basilicum) leaves ≥90 11 ± 1.47 10 ± 0.64Orange (C. sinensis) peel ≥90 9 ± 0.98 8 ± 1.72Spearmint (M. spicata) leaves ≥90 9 ± 1.51 8 ± 1.03Lemon (C. limon) peel ≥90 8 ± 1.47 7 ± 1.37Fennel (F. vulgare) leaves ≥90 8 ± 0.55 7 ± 0.75Kaffir lime (C. hystrix) leaves ≥90 7 ± 1.47 7 ± 0.81Lime (C. aurantifolia) peel ≥90 – –Kaffir lime (C. hystrix) peel ≥90 – –Coconut (C. nucifera) milk ≥90 – –Peppermint (M. piperita) leaves ≥90 – –Galangal (A. galanga) rhizome/root – NT NTGarlic (A. sativum) cloves – NT NTTurmeric (C. longa) rhizome/root – NT NT(–), no inhibition zone; NT, not tested

Fig. 1 Inhibitory efficacy of 10% (v/v) cinnamon essential oil (CI), 1% (v/v) dimethyl sulfoxide (DMSO), and 0.5% (w/v) clotrimazole (positive control) against C. albicans, determined by agar disk diffusion method.

Fig. 2 Inhibitory efficacy of 5% (v/v) essential oils extracted from coconut milk (CO), kaffir lime peel (KLP), orange peel (OR), peppermint leaves (PE), and spearmint leaves (SP) against C. albicans, determined by agar disk diffusion method.

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form. Cinnamon bark and sweet basil leaf essential oils were analyzed for their effects against both sessile forms of C. albicans, and were found to successfully eliminate both forms at concentrations higher than their MICs. The fungal biofilm was largely reduced by 90% (sMIC90) after being treated with concentrations as high as 8-16 times the MICs [0.039% (v/v) of cinnamon bark oil; 0.625% (v/v) of sweet basil leaf oil]. In contrast, after treatment with twice the MICs of these oils [0.01% (v/v); 0.078% (v/v)], the fungal biofilm was seen to decrease by 50% (sMIC50). In the adhesion phase, the adherent fungal cells were reduced by 50% (sMIC50) or up to 90% (sMIC90) after exposure to cinnamon bark and sweet basil leaf essential oils at concentrations equal to their MICs [0.005% (v/v); 0.039% (v/v)] or 4 times the MIC [0.02% (v/v); 0.156% (v/v)], respectively. The anti-sessile Candida efficacy of both essential oils has been illustrated in Table 4. Additionally, the sMIC90 of clotrimazole against adherent and biofilm forms of C. albicans were approximately 4 and 32 times their MIC (35.16 µg/mL; 281.25 µg/mL), respectively.

DiscussionThis study examined the antifungal efficacy, particularly against the Candida species, of several herbs and spices popularly used in Thai cuisine. It is important to ensure that these edible herbs or spices are safe and appropriate for medical use in human beings, and exhibit less toxicity and side effects. Out of fourteen selected spice essential oils, those extracted from lime peel, kaffir lime peel, coconut milk, and peppermint leaves exhibited relatively

less inhibitory activity, as determined by agar disk diffu-sion method. All of these less inhibitory activity oils were excluded from the MIC and MFC analysis as large amounts of such herbs are required to achieve sufficient effectiveness, and this may potentially have undesirable consequences such as high toxicity, side effects, and cost.

The MIC of clotrimazole, an antifungal used as a posi-tive control in this study, was slightly different from that reported previously (19). MIC values of the tested essen-tial oils, estimated by the broth micro-dilution method, correlated well with the inhibition zones determined by the agar disk diffusion method. The anti-planktonic Candida effects of cinnamon bark, sweet basil leaf, kaffir lime peel, lemon peel, and fennel leaf essential oils tested in this study were much more potent than those previ-ously reported (20-25), and only lemon peel essential oil exhibited effects similar to previous reports (26). These disparities further support the notion that the wide range of variation in biological properties (pharmaceutical property, etc.) of natural products, such as herbs or spices, is mainly influenced by plantation. Climatic, seasonal, and geographic conditions as well as harvest period have been reported to affect the chemical compositions of herbs, which in turn directly influence their biological properties (27,28). Extraction method is another impor-tant factor to be considered in order to produce desirable quantities, content, and active compositions of the plants. Therefore, well standardization of herbal extracts is crucial in order to obtain good quality and quantity of products with less variation in their biological properties.

The Candida genus, particularly C. albicans, can

Table 3 MICs and MFCs of the selected culinary herb essential oils against planktonic C. albicans, determined by broth micro-dilution method

Culinary herbs MIC (μL/mL) [% (v/v)] MFC (μL/mL) [% (v/v)]Cinnamon (C. zeylanicum) bark 0.049 [0.005] 0.098 [0.010]Sweet basil (O. basilicum) leaves 0.391 [0.039] 0.391 [0.039]Orange (C. sinensis) peel 0.781 [0.078] 1.563 [0.156]Kaffir lime (C. hystrix) leaves 0.781 [0.078] 1.563 [0.156]Lemon (C. limon) peel 1.563 [0.156] 3.125 [0.313]Spearmint (M. spicata) leaves 3.125 [0.313] 3.125 [0.313]Fennel (F. vulgare) leaves 6.250 [0.625] 6.250 [0.625]MIC, minimum inhibitory concentration; MFC, minimum fungicidal concentration

Table 4 Effect of cinnamon bark and sweet basil leaf essential oils on sessile C. albicans

Culinary herb essential oilssMIC90 [sMIC50] (µL/mL) [% (v/v)]

Adhesion mode Biofilm modeCinnamon (C. zeylanicum) bark 0.195 [0.049] 0.391 [0.098]

0.0195 [0.0049] 0.0391 [0.0098]Sweet basil (O. basilicum) leaves 1.562 [0.391] 6.250 [0.781]

0.1562 [0.0391] 0.625 [0.0781]sMIC, sessile MIC

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cause either mild or deadly infections in individuals with immune impairment. In addition to many virulence factors being produced by the yeast cell, its biofilm phase of growth is commonly associated with various forms of candidiasis and is directly related to therapeutic failure. The tolerance of C. albicans biofilm to available anti-fungal agents is higher in comparison to its planktonic counterpart, and this serves as a major concern during disease management (6). It is important to analyze the antifungal effects of cinnamon bark and sweet basil leaf essential oils against the sessile Candida cultures as these oils exhibited the most potent inhibitory effects on the planktonic yeast cells. Similar to MIC against planktonic C. albicans, sessile MIC (sMIC) value reflects the degree to which the tested spices attack the adherent organisms and in vitro established biofilm. In other words, the lower the sMIC values, the more powerful the attack of the spice on the sessile fungus.

MICs against the sessile fungus (sMIC), either adherent or biofilm form, of cinnamon bark, sweet basil leaf essen-tial oils, and clotrimazole were significantly higher (8 to 32 times) than their planktonic counterparts. These find-ings have reiterated the fact that sessile fungal cultures are more resistant to several antifungal agents (either synthetic or naturally-derived) than planktonic cultures (7,29,30). Many intrinsic properties of fungal biofilms such as low metabolic activity, contact-induced gene expression, presence of extracellular matrix, persister cells, and high anti-oxidative capacities exhibit major involvement in its resistance to the administrated anti-fungal agents (16). As with other fungicidal substances, cinnamon bark and sweet basil leaf essential oils were unable to directly attack the organisms embedded in the biofilm but instead had to diffuse through a strong shelter formed by complicated architectural community surrounded with extracellular matrix. Thus, relatively high concentrations of the oils may be required to achieve sufficient inhibitory or fungicidal effect on the hidden microorganisms.

Intriguingly, the cinnamon bark essential oil tested in this study expressed much more potent fungicidal effects on sessile C. albicans than previously reported by Pires and colleagues (30). This discrepancy may be the consequence of variations in the chemical composition and biological properties of the spice itself, experimental procedure employed, or strain of microorganism tested. Additionally, this study appears to be one of the few reports focusing on the antifungal efficacy of sweet basil leaf essential oil against sessile C. albicans, even though its activity is relatively less than that of cinnamon bark oil. Cinnamaldehyde and methyl chevicol were found

to be major fungitoxic components of the essential oils prepared from cinnamon bark and sweet basil leaves (22,31,32), respectively. It is possible to state that the different degrees of fungicidal efficacy against C. albi-cans biofilm culture exhibited by these two essential oils may have resulted from the variety, quality, and quantity of the active components present in the individual spice.

In summary, the selected Thai culinary herbs serve not only as food flavorings but also as antifungal agents. These findings clearly illustrate that neither biofilm nor planktonic cultures of C. albicans were able to tolerate the attack of cinnamon bark and sweet basil leaf essential oils. Even though these spice essential oils exhibited impressive in vitro anti-sessile Candida efficiency, further investigations on various issues including toxicity, sustainability, etc. are required before they can be used clinically for candidal infection management.

AcknowledgmentsThis research was fully supported by the Faculty of Dentistry, Mahidol University, especially the Department of Oral Micro-biology and Maxillofacial Prosthetic Unit. We also extend our sincere gratitude to Mr. Arthur Navarro and Ms. Thaniya Muadchiangka for manuscript editing and laboratory assistance, respectively.

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