Antifungal Activity of Bulgarian Rose Damascena Oil against Vaginitis-Causing Opportunistic Fungi

Since Bulgarian rose damascena oil is known for its anti-inflammatory, antioxidant, and antimicrobial properties, we investigated its antifungal activity against the species of Candida, which are among the most common opportunistic fungal pathogens. Our disk-diffusion assay revealed that Bulgarian rose damascena oil effectively inhibited the growth of Candida albicans along with various bacteria. The minimum inhibitory and fungicidal concentrations against Candida albicans and Candida glabrata were all 0.25%. Under our experimental conditions, Bulgarian rose damascena oil showed better inhibitory effects on Candida glabrata and Candida albicans than several popular essential oils reported to have antifungal activity other than Origanum vulgare oil. Interestingly, Bulgarian rose damascena oil showed better antifungal activity against Candida species at acidic pH and induced cell death of Candida species in the culture medium, with cell death seen in 25–35% of the cells exposed to 0.05% Bulgarian rose damascena oil. Furthermore, Bulgarian rose damascena oil inhibited the hyphal growth of Candida albicans cultured in the RPMI medium with fetal bovine serum. These findings collectively suggest that Bulgarian rose damascena oil has antifungal activity against Candida species and thus could potentially be developed in novel therapies for vaginitis-causing pathogenic fungi.


Introduction
Pathogenic fungi cause various diseases in humans and other organisms [1].Some of them cause relatively mild skin infections, while others cause serious skin infections or lifethreatening systemic infections [2].One of the most common systemic infections worldwide is caused by Candida species [3].Human Candida species inhabit various body surfaces (e.g., the oral cavity, gastrointestinal tract, vagina, and skin) of healthy persons as a commensal organism [4] and cause infections or candidiasis in superfcial mucous membranes [3].A common female disease is vaginitis, which is an infammation of the vagina caused by Candida species [5].Vulvovaginal candidiasis (VVC), which is a vaginal yeast infection caused by Candida species, is the leading cause of vaginitis [6].Of the various Candida species, only a few are opportunistic pathogens in humans [3].Candida albicans is the most common pathogen causing VVC, and other nonalbicans Candida appear to be on the increase [7,8].Of the non-albicans Candida, Candida glabrata, which was considered a relatively nonpathogenic saprophyte in the normal fora of healthy individuals [9], is regarded as the second most common cause of VVC [10].When the balance between the vaginal colonization of Candida and the host environment is disturbed, a weak immune system can allow pathogens to cause VVC [3,7].Te most widely available treatments are the local applications of nystatin or topical azoles (e.g., clotrimazole, butoconazole, and miconazole) or triazoles (e.g., fuconazole) [6,11,12].Both oral fuconazole and vaginal nystatin are considered to be particularly efective in treating VVC [6].However, the increased use of that immunosuppressive therapy together with broadspectrum antimycotic therapy leads to an increased frequency of mucosal and systemic infections caused by Candida species, particularly Candida glabrata [13].
Te paucity of efective antifungal agents and the emergence of drug resistance have prompted researchers to seek novel antifungal agents [12,14,15].Extracts of various plants, including herbs and traditional medicinal plants, have long been known to ofer antifungal activities.For instance, Acacia nilotica and Coriandrum sativum have been found to possess antifungal activities that decrease bioflm growth or inhibit the synthesis of cell wall components by yeast [16,17].Importantly, various essential oils, including Origanum vulgare, Ocimum basilicum, Lavandula, Mentha spicata, Mentha piperita, and Melaleuca alternifolia essential oils, have been shown to inhibit the fungal growth and activity of Candida albicans more efciently than the known antifungal drugs, including clotrimazole [18].
Rosa damascena is a small plant with aromatic pale pink fowers, also known as Bulgarian rose or Damask rose; it is mainly applied for its perfuming efects [19,20].However, Rosa damascena also contains favonoids, such as kaempferol, quercetin, terpenes, tannins, and vitamin C [19][20][21], and its essential oil has been used as a natural medicine to treat infammation, coughs [19,22], and digestive problems [23].Recently, Bulgarian rose damascena oil was reported to confer broad-spectrum antimicrobial activities, including antibacterial efects against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli [19,21], as well as antiviral activity [24].In the present study, we investigated the antifungal efects of Bulgarian rose damascena oil against Candida albicans and Candida glabrata.

Strains and Media.
Luria broth (LB) and potato dextrose agar (PDA) were purchased from Alpha Biosciences (Baltimore, MD, USA).Staphylococcus aureus KCTC1621, Staphylococcus epidermidis KCTC1917, and Pseudomonas aeruginosa KCTC1636 were purchased from Korean Collection for Type Cultures (KCTC, Jeongeup, Korea).Bacillus subtilis KACC14741 and Candida albicans strains KACC 30003 and KACC 30004 were purchased from Korean Agricultural Culture Collection (KACC, Joenju, Korea).Escherichia coli DH5α was purchased from RBC Bioscience (Taiwan).Candida glabrata strains KBNO6P00368 and KBNO6P00369 were obtained from Chonbuk National University Hospital (Cheongju, Korea).Te Gram-positive (Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermidis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains were cultured at 37 °C under constant shaking (200 rpm) in the complete liquid medium (LB) consisting of 10 g/L enzymatic digest of casein, 5 g/L yeast extract, and 10 g/L sodium chloride.Fungi (Candida glabrata and Candida albicans) were cultured at 37 °C under constant shaking (200 rpm) in the complete liquid medium (PDA) consisting of 4 g/L potato infusion and 20 g/L dextrose.

Determination of the Antibacterial
Activity by the Disk-Difusion Method.Te antibacterial and antifungal activities of the Bulgarian rose damascena oil against microorganisms were examined by the standard method of the European Committee on Antibiotic Susceptibility Testing (EUCAST) disk-difusion assay [25,26].After determining the bacterial count with spectrophotometric (turbidimetric) analysis by measuring absorbance at 600 nm with the microplate reader (Molecular devices, San Jose, CA, USA), microorganisms with a total number of 1 × 10 6 in 40 μL were spread onto LB or PDA plates and sterile 8-mm paper disks (Advantec, Tokyo, Japan) were placed onto the plates.All essential oils were dissolved in 70% ethanol at a concentration varying from 0 to 10% (v/v), and 40 μL of each sample was applied to discs and then the plates were incubated at 37 °C for 24 h.Te inhibition zones were measured in millimeters using a Vernier caliper.Ampicillin (50 mg/ml), fuconazole (1 mg/ ml and 2 mg/ml), and 70% ethanol were used as positive controls for bacteria and yeasts, respectively.

Determination of the Minimum Inhibitory Concentrations
(MICs) by the Microbroth-Dilution Method.Serial doubling dilutions of Bulgarian rose damascena oil with the PDA liquid medium at concentrations ranging from 0 to 0.25% were prepared.Te wells of a 96-well plate were flled with 50 μL/well of each Bulgarian rose damascena oil dilution and 50 μL/well of each Candida species with a total number of 1 × 10 3 cells/μL.Te plates were incubated for 24 h at 37 °C, and the MIC was determined by measuring absorbance at 600 nm.Te MIC endpoint was taken as the lowest concentration of Bulgarian rose damascena oil with which no growth was detected, as determined by the EUCAST diskdifusion assay [25,26].

Determination of the Minimum Fungicidal Concentrations (MFCs).
After the determination of the MIC of the Bulgarian rose damascena oil, media from each well that lacked visible fungal growth were replicated on PDA plates and incubated for 24 h at 37 °C.Te lowest concentration at which 99.9% of the fungal population was killed in PDA plates was taken as the MFC endpoint [26].

Infuence of pH on the Antifungal Activity of Bulgarian
Rose Damascena Oil.Candida species were incubated in the PDA liquid medium (pH 5.4) and 10 µL of each culture (1 × 10 3 cells/μL) was mixed with 90 µL of the PDA liquid medium with diferent pH values (pH 5.1, 5.4, 5.7, and 6.0), resulting in a fnal concentration below the MIC (0.125%) of Bulgarian rose damascena oil.After 24 h incubation, the growth of Candida species was determined based on the optical density of the samples.

2
Evidence-Based Complementary and Alternative Medicine 2.7.Trypan Blue Staining.Media containing Candida were diluted with the same volume of 0.4% trypan blue solution and incubated for 3 min at room temperature.Each sample was loaded to a hemocytometer, the cells were counted under a microscope, and the average number of stained cells was determined [27].
Each sample was mixed with an equal volume of trypan blue, and 10 μL of the mixture was transferred to the hemocytometer and viewed under light microscopy to assess hyphal formation.
2.9.Statistical Analysis.Data are presented as the mean ± SD (standard deviation) for each independent experiment.Statistical analyses were performed using an unpaired Student's t-test.A value of p < 0.05 was considered to represent a statistically signifcant diference.

Antibacterial and Antifungal Activities of Bulgarian Rose
Damascena Oil.To investigate the antibacterial and antifungal activities of Bulgarian rose damascena oil, we performed disk-difusion assays with fve strains of bacteria and one strain of fungus (Figure 1).Bulgarian rose damascena oil dissolved in 70% ethanol was applied to the bacteria strains and the formation of a clear zone was examined after 24 h; ampicillin (fnal 2 mg/ml) and 70% ethanol were used as positive controls for Gram-positive and Gram-negative bacteria (Figure 1(a)).Our results revealed that 1% Bulgarian rose damascena oil efectively inhibited the growth of Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli with inhibition zones of 10-12 mm diameter, confrming the antibacterial properties of the Bulgarian rose damascena oil.We further found that 1% Bulgarian rose damascena oil efectively inhibited Candida albicans with an inhibition zone of 13.5 mm, whereas 50 mg/ml ampicillin, an antibacterial antibiotic, did not inhibit the growth of Candida albicans (Figure 1(b)).
To examine the antifungal properties of Bulgarian rose damascena oil against Candida albicans and Candida glabrata, which are human opportunistic pathogens of VVC [3], a disk-difusion test was performed with two strains of Candida glabrata and two strains of Candida albicans (Figure 2).In the case of Candida glabrata KBNO6P00368, Bulgarian rose damascena oil induced the formation of clear zones with diameters of 8.2 ± 0.2 mm at 0.1%, 8.5 ± 0.4 mm at 0.5%, 9.5 ± 1.6 mm at 1.0%, and 12.1 ± 1.0 mm at 10%, suggesting that Bulgarian rose damascena oil inhibited the growth of Candida glabrata KBNO6P00368 in a dosedependent manner (Figure 2 Interestingly, the antifungal activity of 10% Bulgarian rose damascena oil against all the test strains was greater than the activity of 2 mg/ml fuconazole, a known antifungal drug (Figure 2(b)).

Determination of the Minimum Inhibitory Concentration and Minimum Fungicidal Concentration for Bulgarian Rose
Damascena Oil against Candida Species.To further assess the antifungal potential of Bulgarian rose damascena oil, the microdilution method was used to determine the minimum inhibitory concentrations (MICs) and the minimum fungicidal concentrations (MFCs) values (Figure 3).Te MIC value for Bulgarian rose damascena oil was 0.25% against both Candida glabrata and Candida albicans (Figure 3(a)).Tereafter, media from all Bulgarian rose damascena oiltreated wells showing no visible fungal growth were replicated on PDA plates for the determination of the MFC.Te calculated MFC was 0.25% for both Candida glabrata and Candida albicans (Figure 3(b)).

pH Dependence of the Antifungal Activity of Rosa damascena Essential
Oil.Since the antibacterial properties of natural antibacterial materials are known to depend on pH [28,29], we next investigated the antifungal activity of Bulgarian rose damascena oil in media of diferent pH values (Figure 4).Our data revealed that fungal growth was very similar in a range of pH 5.1-6.0 among the four strains of Candida species (Figure 4(a)), but the antifungal activity of the Bulgarian rose damascena oil difered across this range of pH.When applied at a concentration of 0.125%, the Bulgarian rose damascena oil more efectively inhibited the growth of all Candida species cultured on the PDA media of pH 5.1 versus the PDA media of pH 6.0 (Figure 4(b)).To further confrm this fnding, Candida species treated with Bulgarian rose damascena oil were stained with trypan blue and the dead (stained) cells were counted (Figure 5).Since 0.125% of Bulgarian rose damascena oil efectively inhibited the growth of all Candida species, to quantify cell death, we lowered the amount of Bulgarian rose damascena oil into 0.05%.In the PDA medium of pH 5.1 lacking the essential oil, we did not observe any trypan blue staining of Candida species.In contrast, around 25-35% of Candida glabrata and Candida albicans cells treated with 0.05% Bulgarian rose damascena oil exhibited trypan blue staining (Figure 5), supporting the idea that Bulgarian rose damascena oil substantially induces cell death of Candida species (Figure 5(b)).Tis efect was greatly reduced against Candida species grown in the PDA medium of pH 6.0 (Figure 5(b)), further supporting our contention that the antifungal activity of Bulgarian rose damascena oil is pH dependent.

Inhibitory Efect of Bulgarian Rose Damascena Oil on the
Hyphal Growth of Candida albicans.Candida albicans can penetrate the human epithelial and endothelial cells during the early stages of infection and cause damage through the formation of certain morphological forms, such as hyphae Evidence-Based Complementary and Alternative Medicine [30][31][32][33].Tus, we next investigated the ability of Bulgarian rose damascena oil to inhibit the formation of hyphae (Figure 6).Candida albicans cells were cultured in the RPMI medium and the formation of hyphae was examined.A small number of hyphae was formed in Candida albicans cultured in RPMI without FBS, whereas elongated hyphal forms were seen among most of the Candida albicans cells cultured in RPMI containing 10% FBS, which is a commonly used hyphal inductor.Bulgarian rose damascena oil (at both 0.05% and 0.1%) completely abolished the hyphal formation      Evidence-Based Complementary and Alternative Medicine induced by 10% FBS (Figure 6).When Candida species cultured in the RPMI medium were stained with trypan blue (Figure 7), <5% of Candida glabrata or Candida albicans cells were stained in the absence of Bulgarian rose damascena oil, and this proportion was concentration-dependently increased by treatment with Bulgarian rose damascena oil, suggesting that treatment with Bulgarian rose damascena oil could decrease the infectivity of this pathogen by decreasing hyphal formation and killing cells.

Comparative Analysis of the Antifungal Activity of
Various Essential Oils against Candida Species.Essential oils extracted from various plants are well-known as antimicrobial agents that can exhibit a broad spectrum of activities, including antifungal properties [17,18], with antifungal MIC values of 0.25∼4% (v/v) [34][35][36].We, therefore, further compared the efcacies of Bulgarian rose damascena oil and other essential oils (Origanum vulgare, Ocimum basilicum, Mentha spicata, Melaleuca alternifolia, Mentha piperita, Eucalyptus, and Lavandula) against Candida species [34][35][36] by the disk-difusion assay.Under our experimental conditions with 2% in the midrange to compare diferent essential oils, the ability of Bulgarian rose damascena oil to inhibit the tested strains of Candida glabrata and Candida albicans was comparable to those of Mentha piperita oil, whereas the other tested oils except Origanum vulgare oil showed weaker inhibitory efects (Table 1).

Discussion
Although a variety of natural products, including essential oils, have been found to possess antifungal activity [34][35][36], no previous study had examined the potential action of Bulgarian rose damascena oil against Candida albicans and Candida glabrata, which are human opportunistic pathogens that can cause VVC [3].In this study, we demonstrate that Bulgarian rose damascena oil had antifungal activity against Candida species, including Candida glabrata and Candida albicans, with the MIC and MFC of 0.25% (Figure 3).Consistently, 0.05% Bulgarian rose damascena oil induced cell death of 25-35% of Candida cells of two different species (Figure 5).As natural antimicrobial agents, essential oils exert a broad spectrum of activities, including antifungal properties [18,34,35].Origanum vulgare, Ocimum basilicum, Mentha spicata, Melaleuca alternifolia, Mentha piperita, Eucalyptus, and Lavandula essential oils have been shown to be particularly efective against fungi, with most of them exhibiting the MIC of 0.25∼4% (v/v) [34][35][36].Among the essential oils tested herein, Bulgarian rose damascena oil showed a strong inhibitory efect on all of Candida glabrata and Candida albicans that was better than those of all other tested essential oils except for Origanum vulgare oil, which showed a comparable activity (Table 1).Consistent with this, Bulgarian rose damascena oil induced cell death of Candida species in culture media (Figure 5) and inhibited the hyphal growth of Candida albicans cultured in RPMI media (Figure 7).Based on  Evidence-Based Complementary and Alternative Medicine these fndings, we suggest that Bulgarian rose damascena oil could be developed for novel therapies against human pathogenic fungi that may be applied in cosmetic formulations.For instance, ginger and lemon essential oils are key ingredients of many antiacne creams because of their antibacterial activity against Propionibacterium acnes, and lemon essential oils have an antioxidant activity that could have antiaging efects [37,38].In addition, since essential oils can deter insects, many essential oils are used as mosquito repellents [38,39].

Evidence-Based Complementary and Alternative Medicine
Natural products, such as Bulgarian rose damascena oil, ofer signifcant advantages for commercial use.For example, vaginitis is characterized by infammation of the vagina, which can lead to discharge, itching, and pain [3,7,11].One of the most common causes of vulvovaginal itching and discharge is the infection from Candida types [6].At present, various compounds (including antibiotics) are mixed together to efciently overcome the symptoms of candidiasis [6,11,12].However, the use of Bulgarian rose damascena oil could provide safe, convenient, and efective antifungal efects in this context (Table 1).Especially considering that the normal vaginal pH level is moderately acidic and the antifungal properties of Bulgarian rose damascena oil are stronger against both Candida glabrata and Candida albicans at acidic pH, Bulgarian rose damascena oil could provide alternatives therapies for vaginal candidiasis.Besides, given that essential oils are the foundation of many cosmetics [37][38][39], Bulgarian rose damascena oil could also prove very valuable in the cosmetic industry.
In summary, we herein show that Bulgarian rose damascena oil exhibits antifungal activity against Candida species that can cause vaginitis.Although further studies will be required to fully elucidate the mechanism underlying Bulgarian rose damascena oil-mediated antifungal activity, our present fndings could potentially facilitate research for new alternatives or complementary therapies against vaginal candidiasis.

Conclusion
We herein show that Bulgarian rose damascena oil exhibits antifungal activity against Candida species that can cause vaginitis.Although further studies will be required to fully elucidate the mechanism underlying Bulgarian rose damascena oil-mediated antifungal activity, our present fndings could potentially facilitate research for new alternatives or complementary therapies against vaginal candidiasis.

Figure 2 :
Figure 2: Antifungal activity of Bulgarian rose damascena oil against Candida species.Te disk-difusion analysis of Bulgarian rose damascena oil against Candida species.

Figure 3 :Figure 4 :
Figure 3: Te MIC and MFC of Bulgarian rose damascena oil against Candida species.(a) Candida species were cultured in the PDA liquid medium containing the indicated concentrations of Bulgarian rose damascena oil for 24 h and the absorbance of microtiter plates was measured at 600 nm (top panel).(b) MFC determination with replica plate of all the wells lacking fungal growth.Te MFC was determined as the lowest concentration of Bulgarian rose damascena oil at which no visible colony formation was detected (bottom panel).

Figure 5 :
Figure 5: Efect of Bulgarian rose damascena oil on the cell death of Candida species at diferent pH levels.Candida species were incubated in the PDA medium at diferent pH values with 0.05% of Bulgarian rose damascene oil for 24 h and stained.Cells were examined under a microscope.Scale bars � 5 µm (a) and counted (b).

Figure 6 :
Figure 6: Efects of Bulgarian rose damascena oil on hyphal development.Candida albicans was incubated in the RPMI medium containing 0% or 10% FBS in the presence of the indicated concentrations of Bulgarian rose damascena oil for 24 h.Bright-feld microscopic images are shown.Scale bars � 20 μm.

Figure 7 :
Figure 7: Cell death of Candida species treated with Bulgarian rose damascena oil.Candida species were incubated in the RPMI medium at 0.05% and 0.1% of Bulgarian rose damascene oil for 24 h and stained.Cells were examined under a microscope (scale bars � 5 μm (a) and counted (b)).

Table 1 :
Efcacy of Bulgarian rose damascena oil against Candida species.