In Vitro Antifungal Activities of Sambucus ebulus Against Candida Species Using the Standard Protocol CLSI- M27-S3

Background: The most prevalent fungal infection in the mouth is Candidiasis, which causes various problems for patients. Chemical treatments such as nystatin are the most common methods that are locally used for the mouth that tastes bitter. The repeated use of this method 4 times a day and its preparation during the total consumption lead to patients’ dissatisfaction. Herbal treatments due to less medicinal side effects while having equal effects can be a suitable alternative to chemical treatments. Accordingly, this research focused on evaluating the effect of the herbal extract of the antifungal palm on the types of Candida and then comparing it with Nystatin. Methods: To this end, the effect of the herbal extract of antifungal palm on 4 types of Candida (i.e., albicans, glabrata, tropicalis, and parapsilosis) was investigated, and then the results were compared with that of nystatin, which is diagnosed by the broth microdilution method. The result of the study is descriptive, and minimum inhibitory concentration (MIC) in the blue palm herbal extract was studied in comparison with nystatin for 20 isolated Candida. Results: Based on the result, MIC in the palm herbal extract was (mL/mg) 0.25-0.5 while the inhibitory effect of nystatin growth in this type was 0.125-0.5 (mg/L), and the MIC of the remaining types of Candida, including tropicalis, glabrata, and parapsilosis was 1-0.5 (mg/mL), 0.5-2, and 0.5-1. Finally, the inhibitory effect of growth (MIC) in nystatin drugs in the mentioned types was 0.5-0.25 (mg/L), 0.5-1, and 0.5-0.25. Conclusion: Overall, the activity of the antifungal palm herbal extract is suitable against the examined types of Candida, and this extract may be used as a drug or mouthwash for candidiasis patients.

is applied as the standard. The bitter flavor and frequent usage of nystatin mouthwashfour times per day, along with repetitive preparations contributes to the lack of patient confidence (8). In the last decades,no report has been recorded for the treatment of patients with distinct clinical types of candidiasis and microbial tolerance to antifungal medicines (9). The study will also be beneficial for discovering newcost-effectiveantimicrobial and antifungal ingredients, which have limited complications. The usage of herbs in traditional medicine, including Sambucus ebulus, is one of the promising areas. S. ebulus, which has many beneficial effects against diseases, is wellknown among the typical plants in Balkans and Anatolia. This herb is used for the prevention of gastrointestinal inflammation, influenza, renal and pulmonary disorders, and injuries (10)(11)(12)(13)(14)(15). Using the Clinical and Laboratory Standards Institute (CLSI) M-27 A3 procedure, this research was conducted to assess the antifungal activity of the S. ebulus extract on the extracted candida strains from the oral cavity.

Fungal Strains
In general, 20 Candida strains were extracted from dental stomatitis patients aged around 40-60 years. Sterile swabs were employed to collect these samples, which werecultivated in Sabouraud dextrose agar (SDA) and incubated at35°C.

Extraction
The samples of S. ebulus leaves were prepared from the grocery shop by a botanist at the Shahid Beheshti School of Pharmacy. The maceration process was used for extraction. To this end, 100 g of S. ebulus powder was added since grinding, to twoliters of distilled water seethedand immediately removedfrom warmth. The dish was then wrapped with foil, and its components were filtered inthe laboratory with filter paper (Whatman 92 paper). Next, the dish was put on a water bath for 2 hours with dry extracts prepared following solvent evaporation (16).

Drug Sensitivity Evaluation
To evaluate the minimum inhibitory concentration (MIC) of the S. ebulus aquatic extract in four Candida strains including albicans, glabrata, tropicalis, and parapsilosis, eleven successiveextract concentrationswith 1/2 dilution have been applied,and the maximumand minimum concentrations were 20 mg/mL and 0.02 mg/ mL, respectively. Nystatin was also included in this analysis as the monitor, and the maximum and minimum concentrations at 11 consecutive doses were 32 mg/L and0.032 mg/L, respectively. All strains were cultivated in SDA media and put at 35°C for 72 hours to measure the anti-fungal effect of the S. ebulus extract. Then, 96-well plateswith a culturemedium of 1640 (Roswell Park Memorial Institute-1640)-RPMI and -MOPS (3-(N morpholino) propanesulfonic acid)and separate dilutions of the required extract were prepared as well. Next, the formulated fungal suspension was added to the plates, which were incubated at 35°C for 24-48 hours. The extent of the induced inhibition by the extract was then calculated according to the parameters of the CLSI protocol. The concentrations of antifungals hindering the growth of at least 90% and 50% of the strains and the candidastrains were identified as MIC90 and MIC50, respectively. To acquire MIC90 and MIC50, there should be at least 10 strains for each fungus. The geometric mean was also computed from the Excel-2013 software using the Geomean model, and each examination was performed twice in the present analysis.

Results
Based on the findings (Table 1), the geometric mean for S. ebulus and nystatin was 0.82 mg/mL and 0.36 mg/L for the 20 included Candida strains in this analysis. The development of the 20 isolated Candida strains from theoral cavitywas inhibited by nystatin and S. ebulus in the concentration rangeof0.125-1 mg/Land 2-5.0 mg/mL, respectively.

Candida albicans
The obtained data (Table 2) reveal that the S. ebulus extract within the range of 0.5-2 mg/mL inhibits the growth of C. albicans in nine different studied strainswhereas nystatin at the concentration of 3-10 mg/mL (0.25, 0.5, and 0.125) applieda growth inhibitory impact on C. albicans.

Candida glabrata
The findings (Table 3) suggested that the extract of S. ebulus in four strains of C. glabrata yeast at a concentration of 0.5-2 mg/mL suppressesthe proliferation of this fungus. Nystatinhasa growth inhibitory impact on Candida at concentrations of 3-10 mg/mL (0.25, 0.5, and 1).

Candida tropicalis
Based on the results (Table 4), the S. ebulusextract in four strains ofC. tropicaliswithin a range of 3-10 (0.25-0.5) mg/ mL and nystatin within a range of 0.125 to 1 mg/mL might inhibit fungal growth.

Candida parapsilosis
The Sambucus ebulus extract with the concentration of 3-10 (0.25-0.5) mg/mL and nystatin with the concentration range of 0.5mg/mL prevented the development of this fungus in 3 strains of C. parapsilosis (Table 5).

Discussion and Conclusion
Denture stomatitis is a fungal infection that is triggered by various Candida types and happens when dentures are used in patients and the quality of life represents a decrease. This lesion is typically controlled with nystatin (11). In recent years, several instances of drug tolerance have been observed in a range of bacteria and fungi, probably rendering that alternative treatments must be sought in this regard. In addition, the finding of new plant substitutes for chemical therapies was granted greater consideration in view of the appropriate results and lower complications of herbal medicines versus chemical medicines. Considering that no herbal remedy has been established as a chemical solution for the treatment of denture stomatitis, this study focused on identifying this alternative. In this study,  (17) performed an analysis through the agar diffusion process in one of the most current studies on the antifungal properties of four plants such as S. ebulus. The researchfocusedon the impactof the extract treatment of these four plantson eight fungal strains including C. albicans although no obvious substantial findings were obtained from the S. ebulus.
In this analysis, the agar diffusion approach, which is a qualitative tool, was used in comparison to our research. Further studies are recommended in this area. According to the universal standard protocol CLSI M27-A3, the microdilution approach was employed in our experiment and the findings were quantitative. In their research, Salehzadeh et al investigated the bactericidal effect of the S. ebulus extract throughdisk agar diffusionand found that all the examined bacteria were susceptible to the S. ebulus extract. Therefore, the plantextract can be regarded as a natural antibiotic candidate (18). Similarly, Schwaiger et al studied the anti-inflammatory activity of this plant and demonstrated that the S. ebulus leaf extract may suppresstumor necrosis factor,Vcam-11,and ICAM-12 in HUVEC 3. The anti-inflammatory function of this plant was documented and the origin of this impact was established to be due to abundant reserves of ursolic acid (8). Likewise, Jabbari et al evaluated the impact of the topical treatment of this plant gel on joint pain, particularly knee osteoarthritis. This randomizeddouble-blind, activecontrolled clinical trial focused on examining 106 samples. In all cases, the consequencesof four weeks of gel usage were favorable (including relief from pain). This plant   (20). In another study, Ivanova et al investigated the lipid profile enhancement and serum antioxidant potential of normalvolunteers between the ages of 20 and 58 after soaking the fruit of S. ebulus. In this analysis, significant quantities of TAC, TPC, and AC were contained in the soaked fruit of this plant, and 200 mL of the S. ebulus soaked fruit was eaten by a community of human volunteers every day for onemonth. The blood samples were obtained before and afterthe experiment, and there was a substantial drop in triglycerides, total cholesterol, and LDL-C at the end of the analysis. However, the HDL-C/LDL-C proportion represented an enhancement. The antioxidant potential of serum and total thiol concentrations increased as well. The findings showed how the lipid profile and the antioxidant capacity in humans can be improved usingthis plant (21). Ferrares et al fromthe University of Valladolid (Spain) also examined the effects of Sambucus nigra (a strainthat is highly similar to S. ebulus) and S. ebulus itself on cancer therapyand reportedpositive results in this regard. This paper provides evidence that ahemoglobin targeting cancer cell isproduced using both S. ebulus and S. nigra (22 ). The broth microdilution process, as previously explained in detail, was used for the analysis in order to improve final precision. To this end, 20 individuals with denturestomatitis were examined in this research. These samples were then defined, followed by precisely specifying what sort of candidawas hosted by each individual. It is worth remembering that the clinical studies on a specific fungus (e.g., C. albicans)have produced contradictory effects in multiple individuals, but this is also the case in the licensed medication,nystatin. The origin of this variation is the discrepancy between the host setting and the difference between the usual floras of each checked individual. Candida strains in this analysis comprisedC. albicans (n = 9), C. tropicalis (n = 4), C. glabrata (n = 4), C. parapsilosis (3 = n). Eventually, the impact of the S. ebulus extract was evaluated in comparison with nystatin. Promising results were obtained, and the anti-candidal characteristics of this plant were demonstrated as well.