Hrev_master Healthcare in Low-resource Settings 2023; volume 11:11754 The effect of ethanol extract of Cosmos caudatus leaves on the percentage of the cell cycle in Candida albicans culture Lidia Lushinta, Heni Suryani Department of Midwifery, Politeknik Kesehatan Kementerian Kesehatan Kalimantan Timur, Samarinda, Indonesia Abstract Candidiasis is a common fungal gynecological disease among humans. The use of antifungal agents, such as Fluconazole, has been reported to increase resistance to candidiasis by 7%. This study aimed to investigate the effect of antifungal flavonoids from Cosmos caudatus leaf extract on the cell cycle percentage in C. albicans culture. This research employed a true experimental post- test only with a control group design. The C. albicans isolate was obtained from the Microbiology Laboratory under the code C. albicans SV-1148. The isolates were cultured in Sabouraud Dextrose Agar (SDA) medium and Sabouraud Dextrose Broth (SDB). The sample group was divided into a negative control group, a positive control group with fluconazole (60 µg/ml), and a treatment group with various concentrations of ethanol extract from Kenikir leaves (C. caudatus Kunth.) - 5%, 10%, 20%, 40%, and combinations (20% ethanol extract from Kenikir leaves + 30 µg/ml Fluconazole). This study revealed a reduction in the per- centage of cell cycles in the S phase (DNA synthesis) in the treat- ment group receiving ethanol extract from Kenikir leaves (C. cau- datus Kunth.) and the combination treatment group compared to the negative control group. The study suggests that this decrease in the percentage of cell cycles results from DNA damage caused by the presence of flavonoids in Kenikir leaves (C. caudatus Kunth.). Kenikir leaves (C. caudatus Kunth.) have the potential to decrease the percentage of S-phase cell cycles (DNA synthesis) in the culture of C. albicans. This research demonstrates that Kenikir leaves (C. caudatus Kunth.) contain natural flavonoids with anti- fungal properties and have the potential to be used as an alterna- tive medicine for candidiasis in humans. It is hoped that incorpo- rating Kenikir as a food ingredient can serve as an alternative pre- vention and treatment approach for candidiasis. Introduction Fungal infections claim the lives of over 1.5 million people and afflict more than one billion individuals.1 Serious yeast infec- tions arise as a consequence of other health conditions, including asthma, AIDS, cancer, organ transplantation, and corticosteroid therapy.2 Early and accurate diagnosis permits the immediate ini- tiation of antifungal therapy.3 Nonetheless, the administration of this therapy is frequently delayed, and the unavailability of treat- ment can result in severe chronic illnesses. Recent global esti- mates have identified 700,000 cases of invasive candidiasis.4 The prevalence of candidiasis is reported to be twenty-two percent (22%), and this percentage remains consistent among adult women and female adolescents.5,6 Furthermore, it is estimated that approximately 75% of women will experience at least one can- didiasis episode in their lifetime.7 C. albicans is the species most commonly implicated in cases of candidiasis.8 Candidiasis is often associated with the production of a thick, white, cream, or yellow discharge from the vaginal canal.9 The primary and preferred treatment for Candidiasis is an antifungal drug, specifically Fluconazole.8,10 The mechanism of action of the azole class of antifungal drugs involves the disrup- tion of cell membranes by inhibiting the activity of lanosterol Correspondence: Lidia Lushinta, Politeknik Kesehatan Kementerian Kesehatan Kalimantan Timur, Samarinda, Indonesia E-mail: lidialushinta@gmail.com Key word: herbs; antifungal activity; C. caudatus; C. albicans; cell cycle. Contributions: LL Conceptualization, Data Curation, Formal Analysis, Methodology, Validation, Visualization, Writing – Original Draft, Review & Editing; HS Methodology, Visualization, Writing – Review & Editing. Conflict of interest: the authors declare no conflict of interest. Ethics approval and consent to participate: the research has received ethical approval from the Ethics Committee, Medical Faculty, Universitas Brawijaya, Indonesia based on ethical certificate 166/EC/KEPK/05/2019. During the research, the researcher pays attention to the ethical principles of information to consent, respect for human rights, beneficence and non-maleficence. Patient consent for publication: written informed consent was obtained for anonymized patient information to be published in this article. Funding: this research did not receive external funding. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Received: 11 September 2023. Accepted: 2 October 2023. Early access: 12 October 2023. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2023 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2023; 11:11754 doi:10.4081/hls.2023.11754 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affili- ated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. [Healthcare in Low-resource Settings 2023; 11:11754] [page 11] Non -co mmerc ial us e o nly 14-𝛼-demethylase, an enzyme essential for ergosterol biosynthesis.11 Prolonged use of this drug has led to resistance against Fluconazole in C. albicans, despite its high cure rate.12 According to a 2017 survey conducted by the Centers for Disease Control (CDC), resistance to Candida spp., specifically at a rate of 6.5%, was observed.13 Research on plants used in traditional medicine aims to identi- fy alternative treatments, and numerous antimicrobial properties have been identified in natural ingredients used as herbal remedies.14 According to Hayat et al. (2017), flavonoids exhibit antimicrobial activity against various strains of microorganisms, including Staphylococcus aureus.15 Flavonoids have the ability to form non-specific bonds such as hydrophobic, hydrogen, and covalent bonds with proteins. They have also been studied for their lipophilic properties, which can disrupt microbial membranes.16 Research conducted by Han et al. (2016) revealed that flavonoids inhibit the cell cycle of C. albicans in the S phase when induced by Rubus chingii.17 The synergistic activity is attributed to alterations in membrane fluidity, increased Fluconazole influx, interference with membrane-bound signaling proteins, and cell cycle arrest. Inhibition of the cell cycle in the S phase, specifically during DNA synthesis, is a response to DNA damage. Changes in the cell cycle are closely tied to DNA damage. The presence of chemical compounds in herbal extracts can induce DNA damage, leading to cell cycle arrest and apoptosis. During the S phase checkpoint, two key aspects are examined: the cell’s adequate size for division into two individual cells and the accurate duplication of DNA replication.18,19 The content of flavonoids in Kenikir leaves measures 52.2±4.06 mg per 100 grams.20 Kenikir (C. caudatus Kunth) is among the most commonly found plants in Indonesia. Research conducted by Rasdi et al. (2010) revealed that Kenikir (C. cauda- tus Kunth) can function as an antimicrobial agent against bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and C. albicans.21 It is imperative to con- duct in vitro testing for anti-Candida activity before experimenting on animals to assess the potential toxic effects of natural ingredient extracts directly on specific cell types or tissues within a controlled environment and a short timeframe.22 Furthermore, in vitro tests, as highlighted by Visvesvara and Garcia (2002), are employed to identify potential therapeutic agents. This study demonstrates the impact of antifungal flavonoids from C. caudatus leaf extract on the cell cycle percentage in C. albicans culture.23 Materials and Methods Design of the study This research employs a true experimental research design, specifically a posttest-only control group design. Manufacture of Kenikir leaf ethanol extract The process of obtaining Kenikir leaf powder extract involves two stages: maceration and evaporation. In the maceration stage, finely ground Kenikir leaves, totaling 500 grams, are placed in a glass jar. Subsequently, 1 liter of 90% ethanol is added, and the mixture is stirred for 30 minutes to ensure thorough mixing. It is then left to settle for one hour, as recommended by Aswanida (2015) and Asworo (2017).24,25 The maceration process is conduct- ed at a temperature of 55°C. Afterward, the top portion of the ethanol-solvent mixture, containing the active substance, is sepa- rated using Whatman number 2 filter paper. The remaining active substance-solvent mixture proceeds to the evaporation stage. In the evaporation stage, the filtered mixture is transferred into an evaporating flask, connected to a rotary evaporator and a water bath heater. Water is added to the water bath, and electricity is sup- plied with a temperature setting of 78.4°C. The separation of the ethanol solvent from the active substance in the evaporating flask continues until no more ethanol drips into the holding flask. Typically, this results in one flask containing approximately 900 ml of solution. The filtered solution is then further evaporated until the Kenikir leaf ethanol extract solidifies. The final yield obtained is 70 mL of Kenikir leaf ethanol extract paste. The culture of C. albicans and treatment C. albicans isolate (SV-1148) was procured from the Microbiology Laboratory of Brawijaya University in Malang, Indonesia. A smear of C. albicans (SV-1148) was obtained from a 36-year-old patient at Saiful Anwar RSSA Hospital in Malang. The C. albicans isolates were cultured on Sabouraud Dextrose Agar (SDA) medium and incubated for 48 hours at 37°C. The sample groups were divided into a negative control group, a positive con- trol group treated with fluconazole (60 µg/mL),26 and a treatment group subjected to ethanol extraction from C. caudatus leaves at concentrations of 5%, 10%, 20%, and 40%.27 Cell cycle assay C. albicans isolates (1×103 cells), cultured in Sabouraud Dextrose Broth media, were harvested. Following 24 hours of incubation, the cells were washed with PBS three times and cen- trifuged at 3500 rpm for 3 minutes. Subsequently, they were fixed with 70% ethanol for 24 hours at 4°C 28. Following the fixation, 50 μl of RNase A at a concentration of 200 μg/mL was added to the cells, and the mixture was allowed to react for 2 hours at 37°C. For DNA staining, 50 μg/mL of propidium iodide was introduced, and the mixture was incubated for 30 minutes at 4°C in the dark. The cells were then analyzed using a flow cytometer. The flow cytome- ters utilized in this research included the BD CellQuest flow cytometry machine, BD CellQuest Pro software (version 5.1 or higher), and BD™ Inits software (version 4.1 or higher). Statistical analysis All experiments were performed with four replicates as per the replication formula and sample size calculation. The data were assessed for homogeneity of variance using the Levene index and expressed as the mean ± standard deviation. The results of the nor- mality test using the Shapiro-Wilk test yielded a p>0.05, indicating that the data is normally distributed. Furthermore, the data homo- geneity test produced a p-value of 0.208 (p>0.05), indicating that the data possesses uniform variance. Based on these findings, sta- tistical analysis proceeded with a one-way ANOVA test. Significant differences were analyzed through one-way analysis of variance (ANOVA). All statistical analyses were conducted using SPSS 25.0 (SPSS Inc., Chicago, IL, USA), with a significance level of p<0.05 considered statistically significant, and p<0.01 considered highly statistically significant. Ethical approval for this research was obtained from the Ethics Committee of the Medical Faculty at Universitas Brawijaya, Indonesia, under ethical certifi- cate 166/EC/KEPK/05/2019. Throughout the research, the researcher adhered to ethical principles, including informed con- sent, respect for human rights, beneficence, and non-maleficence. [page 12] [Healthcare in Low-resource Settings 2023; 11:11754] Transforming Healthcare in Low-Resource Settings: a Multidisciplinary Approach Towards Sustainable Solutions Non -co mmerc ial us e o nly Results The administration of Kenikir leaves (C. caudatus Kunth.), ethanol extract, fluconazole, and combination dosages resulted in a decrease in the percentage of cell cycles in the C. albicans cul- ture. Specifically, the ethanol extract of Kenikir leaves (C. cauda- tus Kunth.) was effective in inhibiting the cell cycle in the S phase (DNA Synthesis). This inhibition was assessed using PI and RNAse A to detect changes in the cell cycle of C. albicans follow- ing the administration of the ethanol extract of Kenikir leaves (C. caudatus Kunth.). The results, as depicted in Figure 1, indicate that ethanol extracts of Kenikir leaves (C. caudatus Kunth.) at various concen- trations (5%, 10%, 20%, 40%), as well as in the combination group (C. caudatus Kunth. 20% + Fluconazole 30 µg/mL), effectively inhibited the cell cycle in the S phase (DNA synthesis) noted as “M2.” This inhibition is demonstrated by a reduction in the per- centage of the cell cycle in the S phase (DNA synthesis) in the C. albicans culture when compared to both the negative control and positive control groups. In Figure 2, the average percentage of inhibition of the S-phase cell cycle (DNA synthesis) in the C. albicans culture is shown to decrease in the treatment group compared to the negative control and positive control groups. The lowest percentage of inhibition of the S phase cell cycle, measuring 1.16±0.38, was observed in the treatment group with a concentration of ethanol extract of Kenikir leaves (C. caudatus Kunth.) at 20%. In contrast, the highest per- centage of S phase cell cycle inhibition was found in the negative control group, with a value of 25.4±1.61. Based on Figure 2 and the Tukey HSD test, it can be concluded that the concentration of ethanol extract of Kenikir leaves (C. caudatus Kunth.) significant- ly responsible for decreasing the percentage of the S phase cell cycle (DNA synthesis) in the C. albicans culture was a concentra- tion of 10%. This is supported by the significant difference (p<0.05) when compared to both negative controls and positive controls. Discussion The objective of this study was to assess the impact of ethanol extracts from Kenikir leaves (C. caudatus Kunth.) as antifungals on the reduction of the cell cycle percentage in C. albicans culture. Kenikir leaves (C. caudatus Kunth.) are explored as potential nat- ural alternatives for the safe and effective treatment of candidiasis. The antifungal activity of these extracts was examined at concen- trations of 5%, 10%, 20%, and 40%, as well as in combination with fluconazole (20% ethanol extract + 30 µg/mL fluconazole). Based on the study results and subsequent statistical analysis, it was observed that the administration of ethanol extracts from Kenikir leaves (C. caudatus Kunth.) at different concentrations (5%, 10%, 20%, and 40%), along with the combination treatment group (20% ethanol extract of Kenikir leaves + 30 µg/mL fluconazole), signif- icantly inhibited the cell cycle in the S phase. Notably, Kenikir leaves (C. caudatus Kunth.) were found to contain approximately 52.2 ± 4.06 mg of flavonoids per 100 grams, as documented by Andarwulan et al. (2010).29 When comparing the positive control group to the Kenikir leaves ethanol extract treatment group at a 5% concentration, no significant difference was observed in reducing the percentage of the S phase in the cell cycle. However, signifi- cant differences were noted when compared to the negative control group and the treatment groups with ethanol extract concentrations of 10%, 20%, and the combination treatment group (20% ethanol extract of Kenikir leaves + 30 µg/ml fluconazole). Among the var- ious concentrations tested, the concentration of 10% ethanol extract of Kenikir leaves (C. caudatus Kunth.) demonstrated sig- nificant effectiveness in reducing the percentage of the S phase cell cycle (DNA synthesis) in C. albicans cultures. This was supported by statistically significant differences (p<0.05) when compared to both the negative and positive control groups. Notably, the combi- nation treatment group (20% ethanol extract of Kenikir leaves + 30 µg/ml fluconazole) exhibited the lowest decline in the percentage of the cell cycle in the S phase. These findings align with prior research conducted by Jung et al. (2007) and Han et al. (2016).30,31 Han et al. (2016) demonstrat- ed inhibition of the S-phase cell cycle (DNA synthesis) in C. albi- cans when treated with a combination of Fluconazole and Rubus chingii extract, which contains flavonoids as antimicrobials. This is consistent with the biochemical compounds found in Kenikir leaves (C. caudatus Kunth.). Similarly, Jung et al. (2007) isolated resveratrol from ethyl acetate extract from grape skins, showing that resveratrol, a type of flavonoid, inhibits the S phase of the C. albicans cell cycle.32 [Healthcare in Low-resource Settings 2023; 11:11754] [page 13] Transforming Healthcare in Low-Resource Settings: a Multidisciplinary Approach Towards Sustainable Solutions Figure 1. Percentage results of C. albicans cell cycle with flowcy- tometry analysis. Figure 2. Histogram of the average percentage of C. albicans cell cycle in S. Phase. Non -co mmerc ial us e o nly This anti-Candida activity is mediated by cell entry via a trans- port system that requires the consumption of ATP. The action of resveratrol as a toxic agent in fungal cells induces several intracel- lular physiological changes, which are indicated by the inhibition of the cell cycle and the accumulation of trehalose (a class of car- bohydrates) which is synthesized as an energy source, to survive in freezing conditions and lack of water. It can be concluded that resveratrol inhibits the process of cell division and affects the growth of fungal cells.32,33 According to Iyer and Rhind (2017),34 cell cycle inhibition in the S phase (DNA synthesis) has been found in some organisms as a response to DNA damage. DNA can be damaged by extrinsic and intrinsic factors. Extrinsic factors that can damage DNA include ultraviolet light (UV, ionizing radiation (IR), and chemicals such as methyl-methane sulfonate (MMS), mitomycin C, cisplatin, pso- ralen, camptothecin (CPT), and etoposide.34 Intrinsic factor wrong one of them is reactive oxygen species (ROS) which is produced as a product of cell metabolism, which can cause oxidative damage to DNA.34 Changes in the cell cycle are closely related to DNA damage. The administration of extracts containing flavonoids can cause DNA damage in the presence of excessive accumulation of ROS, leading to cell cycle arrest and apoptosis.31 Checkpoint acti- vation is in response to faulty replication in mitosis, thus being one of the important reasons why cells lacking S phase checkpoints die. The S phase checkpoint is very important for chromosome replication in yeast.35 In the process of the cell cycle, different checkpoints ensure that cellular events occur in the correct order and time. The first checkpoint is located at the end of the G1 phase, just before entering the S phase. There is an important checkpoint before the cell cycle enters the mitotic phase (checkpoint G2 / M), similar to the G1/S transition. In the S phase (DNA synthesis), there are two aspects examined, namely the cell is sufficiently sized to divide into two individual cells, and the DNA has been duplicated correctly.36 Cell size is known to influence cell cycle regulation as a cell cycle checkpoint. Dysfunction of cell volume regulation leads to cell cycle arrest, leading to apoptosis.37 Inhibition of the cell cycle in C. albicans can cause morphological changes, namely cells that are inhibited in the G1 phase tend to be more like hyphae, while inhibition in the S, G2, and M phases leads to cells like pseudohyphae.38 In this study, whole extract from Kenikir leaves (C. caudatus Kunth.) was used, which contains flavonoids, but the total flavonoid content in the ethanol extract of Kenikir leaves (C. cau- datus Kunth.) has not been measured. Conclusions In this study, the mechanism underlying the antifungal activity of ethanol extracts from Kenikir leaves (C. caudatus Kunth.) is elu- cidated through the observed reduction in the cell cycle percent- age. This research underscores that Kenikir leaves (C. caudatus Kunth.) are rich in natural compounds known as flavonoids. Flavonoids are believed to be the primary constituents responsible for the antifungal properties exhibited by Kenikir leaves, thus hold- ing promise as a potential alternative medicine for the treatment of candidiasis in humans. References 1. Soedarsono S, Prasetiyo Y, Mertaniasih N. Fungal isolates findings of sputum samples in new and previously treated cases of pulmonary tuberculosis in dr. soetomo hospital surabaya, Indonesia. Int J Mycobacteriol 2020;9:190-4. 2. Indhi EN, Supranianondo K, Chusniati S, Legowo D, Sarudji S, Tacharina MR, et al. 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