Syzygium Aromaticum (Clove) Extracts Demonstrate Superior Antifungal Activity Compared to Conventional Drugs Against Clinical Oral Candida Isolates from Gharyan, Libya: A Comparative In Vitro Study

Syzygium Aromaticum (Clove) Extracts Demonstrate Superior Antifungal Activity Compared to Conventional Drugs Against Clinical Oral Candida Isolates from Gharyan, Libya: A Comparative In Vitro Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Syzygium Aromaticum (Clove) Extracts Demonstrate Superior Antifungal Activity Compared to Conventional Drugs Against Clinical Oral Candida Isolates from Gharyan, Libya: A Comparative In Vitro Study Issa Amara, Aymaan Salim Omar Alfourti, Joheni Jwely, Mohamed Al-Ryani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9233497/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The escalating challenge of antifungal resistance and the side effects associated with conventional drugs have intensified the search for natural alternatives. This study investigated the efficacy of Syzygium aromaticum (clove) extracts compared to standard antifungal agents against clinical oral Candida isolates from Libyan patients. Methods Five Candida species ( C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis ) were isolated from patients in Gharyan City, Libya. The antifungal susceptibility to Amphotericin B, Miconazole, and Nystatin was tested using the disc diffusion method. The activity of aqueous and alcoholic clove extracts (25%, 50%, 100%) and pure clove oil (Eugenol) was evaluated using a well-diffusion assay. Results Among conventional antifungals, Amphotericin B was the most effective (Mean Inhibition Zone: 27.83 ± 5.22 mm), followed by Miconazole (22.43 ± 6.50 mm) and Nystatin (20.60 ± 3.70 mm). Remarkably, clove extracts demonstrated superior activity. The 100% aqueous clove extract showed the highest overall efficacy (53.93 ± 27.82 mm), significantly outperforming all conventional drugs (p < 0.001). The 100% alcoholic extract was also highly effective (41.40 ± 9.83 mm). Clove oil showed no inhibitory activity. Efficacy was concentration-dependent and species-specific. For instance, C. glabrata was highly susceptible to aqueous extracts (90.00 mm inhibition at 100%), while C. krusei showed relative resilience, responding similarly to both clove extracts and conventional drugs. Conclusion Clove extracts, particularly aqueous preparations at high concentrations, exhibit potent and broad-spectrum antifungal activity against clinical oral Candida isolates, surpassing standard antifungals. These findings position Syzygium aromaticum as a highly promising, naturally sourced candidate for developing new therapeutic or preventive strategies against oral candidiasis, especially in regions where access to conventional medicine is limited. Mycology Clinical Pharmacology Syzygium aromaticum Clove Antifungal Oral Candidiasis Candida Eugenol Natural Products Libya Figures Figure 1 1. Introduction The human oral cavity harbors one of the most diverse microbial environments in the body, second only to the gastrointestinal tract, containing over 700 bacterial species alongside numerous fungi, viruses, and protozoa [ 1 ]. This complex ecosystem, with its warm, humid, and nutrient-rich conditions on teeth and soft tissue surfaces, provides an ideal habitat for various microorganisms that have co-evolved with humans, developing symbiotic relationships crucial to maintaining health [ 2 ]. The fungal component of this ecosystem—the oral mycobiome plays a vital role in maintaining homeostasis, with colonization beginning shortly after birth and progressing with age [ 3 , 4 ]. Among the fungal inhabitants of the oral cavity, Candida species predominate, particularly Candida albicans , followed by Cladosporium , Aureobasidium , and Saccharomycetes [ 5 ]. Under normal physiological conditions, these organisms exist as harmless commensals; however, when the oral environment is perturbed, they can transition into opportunistic pathogens, causing oral candidiasis—the most prevalent fungal infection of the oral mucosa [ 6 , 7 ]. This transition from commensal to pathogen involves complex virulence mechanisms, including the yeast-to-hyphal morphological switch, which enables tissue invasion; secretion of hydrolytic enzymes such as secreted aspartyl proteinases (SAPs) and phospholipases; and robust biofilm formation on both mucosal surfaces and prosthetic devices [ 8 – 10 ]. These biofilms not only protect fungal cells from host immune responses but also significantly reduce susceptibility to antifungal agents, often requiring concentrations many times higher than those effective against planktonic cells [ 11 , 12 ]. Oral candidiasis manifests in several clinical forms—pseudomembranous, erythematous, chronic hyperplastic, and angular cheilitis—with symptoms including white plaques, erythema, burning sensations, altered taste, and pain that can compromise nutritional intake [ 6 , 13 ]. The condition is particularly prevalent among immunocompromised individuals, including those with HIV/AIDS, cancer patients undergoing chemotherapy, organ transplant recipients, and individuals with diabetes mellitus [ 14 – 16 ]. Additional risk factors include poor oral hygiene, malnutrition, high-carbohydrate diets, xerostomia, pregnancy, corticosteroid therapy, and broad-spectrum antibiotic use [ 17 – 19 ]. The significance of oral fungal infections extends beyond local morbidity. They may serve as indicators of systemic disease, with recurrent or persistent candidiasis often signaling underlying immune dysfunction or endocrinopathies [ 20 ]. In vulnerable populations, oral lesions may represent the first manifestation of systemic mycoses, as demonstrated in a 25-year retrospective study from Brazil where oral involvement was the initial presentation in paracoccidioidomycosis, histoplasmosis, and disseminated candidiasis [ 21 ]. Furthermore, oral Candida carriage has been associated with dental caries, periodontal disease, and even systemic conditions such as metabolic dysfunction-associated fatty liver disease (MAFLD), where fungal dysbiosis correlates with elevated inflammatory markers and disease severity [ 22 – 24 ]. The current antifungal arsenal, while effective, faces significant limitations. Polyenes such as Amphotericin B and Nystatin, and azoles including Miconazole and Fluconazole, are associated with dose-limiting toxicities, adverse effects, and increasing antimicrobial resistance [ 25 , 26 ]. Amphotericin B, though broad-spectrum and fungicidal, carries substantial nephrotoxicity risk [ 27 , 28 ]. Nystatin, while safer for topical oral use, exhibits instability in light, heat, and humidity, limiting its practical application [ 29 ]. The emergence of resistant strains—particularly among non- Candida albicans (NCA) species such as C. glabrata , C. krusei , and C. tropicalis —has further complicated management [ 30 – 32 ]. These species often demonstrate intrinsic or acquired resistance to azole antifungals, form robust biofilms, and are increasingly prevalent in clinical isolates worldwide [ 33 – 35 ]. The situation is particularly challenging in resource-limited settings. In Libya, recent studies have documented high oral Candida carriage rates, with C. albicans predominating but significant proportions of NCA species including C. glabrata and C. tropicalis [ 36 ]. Among Libyan diabetic patients, angular cheilitis prevalence exceeds 35%, strongly associated with poor glycemic control and Candida colonization [ 37 ]. A local study in Musrata identified 13 Candida species from diabetic patients, underscoring the diversity of fungal pathogens in the region [ 38 ]. These epidemiological realities, combined with limited access to advanced diagnostics, empirical treatment practices, and the rising costs of conventional antifungals, necessitate exploration of alternative therapeutic approaches that are effective, affordable, and culturally acceptable. Medicinal plants have served as foundational elements of traditional medicine for centuries and represent a rich reservoir of novel bioactive compounds [ 39 ]. Among these, Syzygium aromaticum (L.) Merr. & L.M. Perry, commonly known as clove, has garnered particular attention for its potent pharmacological properties, including antimicrobial, antioxidant, anti-inflammatory, and analgesic activities [ 40 ]. The primary bioactive constituent, Eugenol (4-allyl-2-methoxyphenol), comprises 70–90% of clove essential oil and exerts antifungal effects through multiple mechanisms: disruption of fungal cell membrane integrity, inhibition of ergosterol synthesis, interference with enzymatic processes, and suppression of biofilm formation and hyphal development [ 41 – 43 ]. Additional compounds such as β-caryophyllene, eugenyl acetate, and α-humulene may contribute synergistically to its antimicrobial efficacy [ 44 ]. The antifungal potential of clove has been increasingly documented. Studies have demonstrated its efficacy against Candida albicans and NCA species, with mechanisms including membrane permeabilization, inhibition of germ tube formation, and disruption of mature biofilms [ 45 , 46 ]. Nanoemulsified clove oil formulations have shown enhanced stability, bioavailability, and sustained release, further improving antifungal activity [ 42 ]. Comparative investigations have revealed that clove extracts exhibit comparable or superior activity to conventional antifungals, with the advantage of natural origin and minimal toxicity [ 24 , 47 ]. Clove-based nanoparticles synthesized from S. aromaticum have demonstrated broad-spectrum antimicrobial, anticancer, and antioxidant properties, suggesting potential for multifaceted therapeutic applications [ 48 ]. However, despite this growing body of evidence, direct comparative data evaluating various clove preparations—aqueous extracts, alcoholic extracts, and pure oil—against a comprehensive panel of clinical oral Candida isolates alongside standard antifungal agents remain limited, particularly from North African populations. The present study addresses this gap by conducting a comparative in vitro evaluation of the efficacy of conventional antifungal drugs (Amphotericin B, Miconazole, Nystatin) and various concentrations of S. aromaticum extracts against clinically confirmed oral Candida isolates obtained from patients in the Gharyan region of Libya. By integrating species-specific susceptibility testing and statistical comparisons, this investigation aims to establish the potential of clove extracts as viable alternatives or adjuncts to conventional therapy, particularly relevant for resource-constrained healthcare settings where antifungal resistance and treatment access pose significant challenges. 2. Materials and Methods 2.1. Study Design and Setting This comparative in vitro study was designed to evaluate the antifungal efficacy of Syzygium aromaticum (clove) extracts against clinical oral Candida isolates. Sample collection was conducted among patients attending the outpatient clinics at Gharyan Central Hospital and three primary healthcare centers in the Gharyan region, Libya. All mycological analysis, including culture, identification, and antifungal susceptibility testing, was performed at the Fungi Laboratory, Faculty of Medical Technology Alriyaina, University of Zintan, Libya. 2.2. Ethical Considerations The study protocol was reviewed and approved by the Research Ethics Committee of the Faculty of Medicine, University of Gharyan (Approval No.: UOG/MED/2025/08, dated 25 January 2025). Written informed consent was obtained from all adult participants prior to sample collection. For minor participants (under 18 years of age), consent was obtained from their parents or legal guardians. All procedures were conducted in accordance with the ethical standards of the Declaration of Helsinki of 1975, as revised in 2013. 2.3. Study Population and Sample Collection Period Sample collection was carried out over a period from 2 February 2025 to 20 April 2025. A total of 140 patients attending the outpatient clinics for various medical complaints were enrolled in the study. To ensure anonymity and proper tracking throughout the laboratory procedures, each patient was assigned a unique serial number upon enrollment. 2.4. Inclusion and Exclusion Criteria Inclusion criteria: Patients of both genders, aged between 2 and 78 years, who presented with clinical signs suggestive of oral fungal infection (e.g., white plaques, erythema, burning sensation, angular cheilitis) and were willing to provide informed consent were included in the study. Exclusion criteria: Patients were excluded if they had received systemic or topical antifungal therapy within the preceding two weeks, were on immunosuppressive therapy, had a known diagnosis of HIV/AIDS or other severe immunocompromising conditions, or were unable to cooperate with the sample collection procedures. 2.5. Sample Collection Procedure Under strict aseptic conditions and following a standardized precautionary protocol to avoid contamination, samples were collected from multiple oral sites depending on the clinical presentation. These sites included carious lesions or supragingival plaque on tooth surfaces; the buccal mucosa, palate, tongue dorsum, and floor of mouth; the fitting surface of removable dentures (if present); and deep periodontal pockets using sterile curettes. For each patient, three sterile swabs were used to collect specimens from the affected sites: the first swab was used for direct microscopic examination, the second for primary culture, and the third for subculture and maintenance. 2.6. Sample Transport and Storage Immediately after collection, each swab was placed into a sterile container and maintained at 4–8°C in a chilled, insulated transport box. All samples were transported to the Fungi Laboratory within 4–6 hours of collection for immediate processing. Samples that could not be processed immediately were stored at 4°C for a maximum of 24 hours before culture. 2.7. Direct Microscopic Examination Gram Staining: Smears prepared from the first swab were heat-fixed and stained using the standard Gram staining procedure. The smears were then examined under light microscopy with a 100× oil immersion objective for the presence of budding yeast cells, pseudohyphae, and true hyphae characteristic of Candida species. Lactophenol Cotton Blue (LPCB) Staining: A second smear from the first swab was mounted in LPCB stain and examined under low power (10× and 40×) to observe fungal morphological features, including chlamydospores, blastoconidia, and pseudohyphae. 2.8. Culture Media and Isolation Primary Culture: The second sterile swab was streaked onto Petri dishes containing Sabouraud Dextrose Agar (SDA) (Oxoid, UK). The medium was supplemented with chloramphenicol (0.05 g/L) to inhibit bacterial growth and cycloheximide (0.5 g/L) to suppress saprophytic fungi, thereby promoting the selective isolation of pathogenic Candida species. Inoculated plates were incubated aerobically at 37°C for 24–48 hours. Plates showing no growth after 48 hours were re-incubated at room temperature (25–30°C) for an additional 5–7 days to allow for the growth of slow-growing fungi. Subculture: Suspected Candida colonies, identified by their creamy, smooth, and pasty appearance, were subcultured onto fresh SDA plates to obtain pure cultures. The pure isolates were then maintained on SDA slants at 4°C for subsequent species identification and antifungal susceptibility testing. 2.9. Fungal Isolates and Preparation For this study, five clinically confirmed Candida species ( C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis ) were obtained from the stock cultures preserved from the patient samples described above. For susceptibility testing, fresh subcultures were prepared on SDA and incubated at 37°C for 24-48 hours. A standardized inoculum suspension equivalent to a 0.5 McFarland standard was prepared in sterile saline for each isolate. 2.10. Conventional Antifungal Agents Commercially available antifungal discs (Liofilchem, Italy) were used: Amphotericin B (20 µg), Miconazole (10 µg), and Nystatin (100 IU). These were selected as representatives of major antifungal classes used in clinical practice. 2.11. Preparation of Clove Extracts and Oil Alcoholic Extract: Dried clove buds (50 g) were ground and macerated in 500 mL of 96% ethanol for 48 hours in a dark environment with continuous shaking. The mixture was filtered through sterile gauze and filter paper. The filtrate was concentrated using a rotary evaporator, and the resulting extract was dried in an oven at 40±2°C to obtain a powder. Aqueous Extract: The same process was repeated using 500 mL of sterile distilled water as the solvent. Extract Concentrations: Both the alcoholic and aqueous clove extracts were reconstituted to final concentrations of 25%, 50%, and 100% (w/v) using their respective solvents. Clove Oil: Commercially available Eugenol oil (DHARMA research, USA) was used directly. 2.12. Antifungal Susceptibility Testing Disc Diffusion for Conventional Drugs: The assay was performed on Mueller Hinton Agar (MHA) supplemented with 2% glucose and 0.5 µg/mL methylene blue, as recommended [8]. The standardized fungal suspension was swabbed uniformly onto the agar plates. Antifungal discs were placed on the inoculated surface, and plates were incubated at 37°C for 24-48 hours. The zones of inhibition (ZOI) were measured in millimeters (mm). All tests were performed in triplicate. Well Diffusion for Clove Extracts/Oil: After swabbing the agar plates with the fungal inoculum, wells (10 mm diameter) were punched into the agar using a sterile cork borer. Each well was filled with 50 µL of the respective clove extract concentration or pure clove oil. Plates were incubated as above, and the ZOI was measured. Tests were performed in triplicate for each isolate and concentration. 2.13. Statistical Analysis Data were analyzed using SPSS Statistics version 27. Descriptive statistics (mean ± standard deviation) were calculated. The significance of differences in the mean ZOI among different treatments and across Candida species was determined using a one-way analysis of variance (ANOVA), followed by post-hoc Tukey's HSD test for multiple comparisons. A p-value of less than 0.05 was considered statistically significant. 3. Results Efficacy of Conventional Antifungal Agents All tested Candida species were susceptible to the conventional antifungal agents, but with varying degrees of efficacy (Table 1). Amphotericin B demonstrated the strongest inhibitory effect, with a mean ZOI of 27.83 ± 5.22 mm. This was significantly greater than the ZOIs produced by Miconazole (22.43 ± 6.50 mm) and Nystatin (20.60 ± 3.70 mm) (F = 6.796, p = 0.004). Table 1. Efficacy of conventional antifungal agents against oral Candida isolates (Mean ZOI in mm ± SD). Antibiotics N Mean ± SD F value P value Amphotericin B 15 27.83 a ± 5.216 6.796 0.004 Miconazole 15 22.43 b ± 6.502 Nystatin 15 20.60 b ± 3.699 Different superscript letters (a, b) within a column indicate statistically significant differences (p < 0.05). Table (1) presents the results suggest that Amphotericin B was significantly more effective in reducing oral fungal infections compared to the other two agents. The findings provide important evidence for clinicians when selecting antifungal therapy, supporting the use of Amphotericin B as a potentially superior option in this context. Comparative Efficacy of All Antifungal Agents A comprehensive comparison of all tested agents revealed highly significant differences (F = 152.69, p < 0.001) (Table 2). The 100% aqueous clove extract was the most effective treatment overall, producing a mean ZOI of 53.93 ± 27.82 mm. This was followed by the 50% aqueous extract (43.80 ± 33.67 mm) and the 100% alcoholic extract (41.40 ± 9.83 mm). All three of these natural preparations were significantly more effective than the conventional drugs Amphotericin B, Miconazole, and Nystatin. Pure clove oil showed no measurable antifungal activity under the test conditions. Following graph displays different antifungal agents used in this study (figure 1). Table 2. Comparative efficacy of all antifungal agents and clove extracts (Mean ZOI in mm ± SD). Antifungal agents N Mean SD F value P value Amphotericin B 15 27.83 a 152.692 < 0.001 Miconazole 15 22.43 b 6.502 Nystatin 15 20.60 bd 3.699 Alcoholic clove extract 25% 15 18.27 d 8.610 Alcoholic clove extract 50% 15 29.37 a 7.005 Alcoholic clove extract 100% 15 41.40 e 9.832 Aqueous clove extract 25% 15 35.13 f 26.457 Aqueous clove extract 50% 15 43.80 e 33.671 Aqueous clove extract 100% 15 53.93 g 27.82 Clove oil 15 0.00 0.000 Different superscript letters (a-g) within a column indicate statistically significant differences (p < 0.05). Species-Specific Antifungal Response The efficacy of the agents varied considerably depending on the Candida species (Table 3). Aqueous clove extracts were exceptionally effective against C. albicans and C. glabrata , with ZOIs exceeding 80 mm at 50% and 100% concentrations. In contrast, C. krusei and C. parapsilosis were less susceptible to the aqueous extracts, with the highest ZOIs being 32.00 mm and 33.33 mm, respectively. For these species, the conventional antifungals, particularly Amphotericin B and Miconazole, showed comparable or slightly better activity than the clove extracts. C. tropicalis was most effectively inhibited by the 100% alcoholic clove extract (45.61 mm). Table 3. Species-specific response: Mean ZOI (mm) of antifungal agents against different Candida species. Candida spp. Treatment (Most Effective) Mean ZOI ± SD Treatment (Conventional) Mean ZOI ± SD C. albicans Aq. Extract 100% 83.33 ± 3.06 Amphotericin B 26.33 ± 10.61 C. glabrata Aq. Extract 100% 90.00 ± 0.00 Amphotericin B 31.00 ± 1.80 C. krusei Aq. Extract 100% 32.00 ± 2.00 Miconazole 31.17 ± 0.29 C. parapsilosis Alc. Extract 100% 43.44 ± 8.95 Amphotericin B 25.17 ± 4.81 C. tropicalis Alc. Extract 100% 45.61 ± 2.97 Amphotericin B 28.33 ± 4.54 4. Discussion The search for effective and safe antifungal agents is a persistent priority in medical mycology. Our findings demonstrate that crude extracts of Syzygium aromaticum possess remarkable antifungal activity, significantly surpassing that of commonly used conventional drugs against clinical oral Candida isolates. The superior performance of Amphotericin B over the azole (Miconazole) and the other polyene (Nystatin) is consistent with its broad-spectrum, fungicidal nature [ 9 ]. However, its clinical use is often hampered by nephrotoxicity and other side effects [ 10 ]. The most striking result of this study is the potent efficacy of clove extracts. The 100% aqueous extract emerged as the most powerful agent overall. This was an unexpected but significant finding, as alcoholic extracts are typically more efficient at extracting non-polar bioactive compounds like Eugenol [ 11 ]. The superior performance of the aqueous extract in our assay could be due to better diffusion of its constituents through the aqueous-based agar medium, leading to a larger observable zone of inhibition. Both aqueous and alcoholic extracts exhibited a clear dose-dependent response, with 100% concentrations being vastly more effective than 25% concentrations, underscoring the concentration of active antifungal components. The failure of pure clove oil (Eugenol) to produce an inhibition zone was paradoxical. This could be attributed to its high volatility and hydrophobic nature, preventing its effective diffusion from the well into the agar matrix. In clinical dental applications, Eugenol is often used in a paste form with zinc oxide, which may facilitate its release and activity, unlike the pure oil in a well-diffusion test [ 12 ]. The species-specific variation in susceptibility is clinically crucial. The high susceptibility of C. albicans and C. glabrata to aqueous clove extracts is promising, given their high clinical prevalence. In contrast, the relative resilience of C. krusei , which is intrinsically less susceptible to fluconazole [ 13 ], to both clove extracts and conventional drugs highlights its challenging nature. This suggests that while clove extracts are a powerful broad-spectrum alternative, species identification remains important for tailoring therapy, especially in recalcitrant cases. The mechanism of action is widely attributed to Eugenol, which can integrate into and disrupt the fungal cell membrane, leading to increased permeability and cell death [ 7 ]. Additionally, clove extracts are known to inhibit germ tube and biofilm formation, key virulence factors of Candida [ 14 ]. The presence of other synergistic compounds in the crude extract, such as β-caryophyllene, may further enhance its antifungal potency beyond that of pure Eugenol alone [ 15 ]. Limitations and Future Directions A limitation of this study is the use of the well-diffusion method, which, while excellent for screening, does not provide a quantitative Minimum Inhibitory Concentration (MIC). Future work should determine the MIC and Minimum Fungicidal Concentration (MFC) of these extracts. Furthermore, in vivo studies and the development of stable formulations (e.g., mouthwashes, gels) are essential next steps to translate these findings into clinical practice. 5. Conclusion This study provides compelling evidence that Syzygium aromaticum (clove) extracts, particularly aqueous extracts at high concentrations, are potent inhibitors of clinical oral Candida species, exhibiting efficacy that surpasses standard antifungal drugs. Given their natural origin, anticipated low cost, and reduced potential for side effects, clove extracts represent a highly promising alternative or adjunctive therapy for managing oral candidiasis. This is especially relevant for resource-limited settings like Libya, where access to conventional antifungals may be constrained. We recommend further phytochemical analysis and clinical trials to develop these findings into tangible therapeutic options. References Deo PN, Deshmukh R (2019) Oral microbiome: Unveiling the fundamentals. J Oral Maxillofac Pathol 23(1):122–128 Kilian M, Chapple IL, Hannig M et al (2016) The oral microbiome – an update for oral healthcare professionals. 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Curr Med Mycol 10(2):e2024008 Aldabaan N, Shaikh S, Alqahtani A et al (2024) Biological activities of nanoparticles synthesized from Syzygium aromaticum. Green Chem Lett Rev 17(1):2289456 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9233497","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":613512176,"identity":"c2ae32c0-af7e-481c-a6f7-b434155d4fce","order_by":0,"name":"Issa Amara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYDCCAxCKsY2BsYGBoQLCk2BgsCBWyxm4FgnCWhqgGglr4Tve/OwBY46NbB/74bYHH+cdlpNvYD54m4dBIrEBhxbJM8fMDRi3pRm38SS2G87cdtiYsYEt2RqfFoMbCWYSjNsOJ7ZJMLZJ8wIZzQw8ZtJ4tdx//g2o5T9Ey985QL0M/N/wa7nBA7LlAEQLY8PhxB4GHja8WiTP5JRJJG5LBvmlTbLnWLqxBDObseUcAwljXFr4jh/fJvFxm53s/PbjzyR+1FjLybc3P7zxpsJGFpcWMEhA4TGDHczgiFcLVmBPso5RMApGwSgYrgAAd6NSX9vxJHwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0780-8737","institution":"University of Zintan","correspondingAuthor":true,"prefix":"","firstName":"Issa","middleName":"","lastName":"Amara","suffix":""},{"id":613512177,"identity":"c3f395a7-14db-4ed2-8307-5649ef2d5c53","order_by":1,"name":"Aymaan Salim Omar Alfourti","email":"","orcid":"https://orcid.org/0009-0003-3078-1901","institution":"Libyan Academy for Postgraduate Studies","correspondingAuthor":false,"prefix":"","firstName":"Aymaan","middleName":"Salim Omar","lastName":"Alfourti","suffix":""},{"id":613512178,"identity":"8a0620d0-0a89-471e-9585-9f29a1911ef8","order_by":2,"name":"Joheni Jwely","email":"","orcid":"https://orcid.org/0009-0000-4972-0496","institution":"University of Zintan","correspondingAuthor":false,"prefix":"","firstName":"Joheni","middleName":"","lastName":"Jwely","suffix":""},{"id":613512179,"identity":"7e2f5771-febe-4542-b253-05ad8b8ad58f","order_by":3,"name":"Mohamed Al-Ryani","email":"","orcid":"https://orcid.org/0009-0007-7696-2125","institution":"University of Zintan","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Al-Ryani","suffix":""}],"badges":[],"createdAt":"2026-03-26 11:28:05","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9233497/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9233497/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105881460,"identity":"496c441f-0058-4949-bb03-00ec9d1334ff","added_by":"auto","created_at":"2026-04-01 06:51:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficacy of antifungal agents to inhibition of oral fungal infections.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure.png","url":"https://assets-eu.researchsquare.com/files/rs-9233497/v1/da4888224b0baa65aadebad0.png"},{"id":105905341,"identity":"eed4e016-a3d1-4fdd-baeb-dc1fee745e72","added_by":"auto","created_at":"2026-04-01 10:11:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1233158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9233497/v1/b68f06a1-6b82-46b1-a2bd-e2f3273ab88a.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSyzygium Aromaticum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(Clove) Extracts Demonstrate Superior Antifungal Activity Compared to Conventional Drugs Against Clinical Oral \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Isolates from Gharyan, Libya: A Comparative In Vitro Study\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe human oral cavity harbors one of the most diverse microbial environments in the body, second only to the gastrointestinal tract, containing over 700 bacterial species alongside numerous fungi, viruses, and protozoa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This complex ecosystem, with its warm, humid, and nutrient-rich conditions on teeth and soft tissue surfaces, provides an ideal habitat for various microorganisms that have co-evolved with humans, developing symbiotic relationships crucial to maintaining health [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The fungal component of this ecosystem\u0026mdash;the oral mycobiome plays a vital role in maintaining homeostasis, with colonization beginning shortly after birth and progressing with age [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the fungal inhabitants of the oral cavity, \u003cem\u003eCandida\u003c/em\u003e species predominate, particularly \u003cem\u003eCandida albicans\u003c/em\u003e, followed by \u003cem\u003eCladosporium\u003c/em\u003e, \u003cem\u003eAureobasidium\u003c/em\u003e, and \u003cem\u003eSaccharomycetes\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Under normal physiological conditions, these organisms exist as harmless commensals; however, when the oral environment is perturbed, they can transition into opportunistic pathogens, causing oral candidiasis\u0026mdash;the most prevalent fungal infection of the oral mucosa [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This transition from commensal to pathogen involves complex virulence mechanisms, including the yeast-to-hyphal morphological switch, which enables tissue invasion; secretion of hydrolytic enzymes such as secreted aspartyl proteinases (SAPs) and phospholipases; and robust biofilm formation on both mucosal surfaces and prosthetic devices [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These biofilms not only protect fungal cells from host immune responses but also significantly reduce susceptibility to antifungal agents, often requiring concentrations many times higher than those effective against planktonic cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOral candidiasis manifests in several clinical forms\u0026mdash;pseudomembranous, erythematous, chronic hyperplastic, and angular cheilitis\u0026mdash;with symptoms including white plaques, erythema, burning sensations, altered taste, and pain that can compromise nutritional intake [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The condition is particularly prevalent among immunocompromised individuals, including those with HIV/AIDS, cancer patients undergoing chemotherapy, organ transplant recipients, and individuals with diabetes mellitus [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additional risk factors include poor oral hygiene, malnutrition, high-carbohydrate diets, xerostomia, pregnancy, corticosteroid therapy, and broad-spectrum antibiotic use [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe significance of oral fungal infections extends beyond local morbidity. They may serve as indicators of systemic disease, with recurrent or persistent candidiasis often signaling underlying immune dysfunction or endocrinopathies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In vulnerable populations, oral lesions may represent the first manifestation of systemic mycoses, as demonstrated in a 25-year retrospective study from Brazil where oral involvement was the initial presentation in paracoccidioidomycosis, histoplasmosis, and disseminated candidiasis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, oral \u003cem\u003eCandida\u003c/em\u003e carriage has been associated with dental caries, periodontal disease, and even systemic conditions such as metabolic dysfunction-associated fatty liver disease (MAFLD), where fungal dysbiosis correlates with elevated inflammatory markers and disease severity [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current antifungal arsenal, while effective, faces significant limitations. Polyenes such as Amphotericin B and Nystatin, and azoles including Miconazole and Fluconazole, are associated with dose-limiting toxicities, adverse effects, and increasing antimicrobial resistance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Amphotericin B, though broad-spectrum and fungicidal, carries substantial nephrotoxicity risk [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Nystatin, while safer for topical oral use, exhibits instability in light, heat, and humidity, limiting its practical application [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The emergence of resistant strains\u0026mdash;particularly among non-\u003cem\u003eCandida albicans\u003c/em\u003e (NCA) species such as \u003cem\u003eC. glabrata\u003c/em\u003e, \u003cem\u003eC. krusei\u003c/em\u003e, and \u003cem\u003eC. tropicalis\u003c/em\u003e\u0026mdash;has further complicated management [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These species often demonstrate intrinsic or acquired resistance to azole antifungals, form robust biofilms, and are increasingly prevalent in clinical isolates worldwide [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe situation is particularly challenging in resource-limited settings. In Libya, recent studies have documented high oral \u003cem\u003eCandida\u003c/em\u003e carriage rates, with \u003cem\u003eC. albicans\u003c/em\u003e predominating but significant proportions of NCA species including \u003cem\u003eC. glabrata\u003c/em\u003e and \u003cem\u003eC. tropicalis\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Among Libyan diabetic patients, angular cheilitis prevalence exceeds 35%, strongly associated with poor glycemic control and \u003cem\u003eCandida\u003c/em\u003e colonization [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A local study in Musrata identified 13 \u003cem\u003eCandida\u003c/em\u003e species from diabetic patients, underscoring the diversity of fungal pathogens in the region [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These epidemiological realities, combined with limited access to advanced diagnostics, empirical treatment practices, and the rising costs of conventional antifungals, necessitate exploration of alternative therapeutic approaches that are effective, affordable, and culturally acceptable.\u003c/p\u003e \u003cp\u003eMedicinal plants have served as foundational elements of traditional medicine for centuries and represent a rich reservoir of novel bioactive compounds [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Among these, \u003cem\u003eSyzygium aromaticum\u003c/em\u003e (L.) Merr. \u0026amp; L.M. Perry, commonly known as clove, has garnered particular attention for its potent pharmacological properties, including antimicrobial, antioxidant, anti-inflammatory, and analgesic activities [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The primary bioactive constituent, Eugenol (4-allyl-2-methoxyphenol), comprises 70\u0026ndash;90% of clove essential oil and exerts antifungal effects through multiple mechanisms: disruption of fungal cell membrane integrity, inhibition of ergosterol synthesis, interference with enzymatic processes, and suppression of biofilm formation and hyphal development [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additional compounds such as β-caryophyllene, eugenyl acetate, and α-humulene may contribute synergistically to its antimicrobial efficacy [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe antifungal potential of clove has been increasingly documented. Studies have demonstrated its efficacy against \u003cem\u003eCandida albicans\u003c/em\u003e and NCA species, with mechanisms including membrane permeabilization, inhibition of germ tube formation, and disruption of mature biofilms [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Nanoemulsified clove oil formulations have shown enhanced stability, bioavailability, and sustained release, further improving antifungal activity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Comparative investigations have revealed that clove extracts exhibit comparable or superior activity to conventional antifungals, with the advantage of natural origin and minimal toxicity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Clove-based nanoparticles synthesized from \u003cem\u003eS. aromaticum\u003c/em\u003e have demonstrated broad-spectrum antimicrobial, anticancer, and antioxidant properties, suggesting potential for multifaceted therapeutic applications [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, despite this growing body of evidence, direct comparative data evaluating various clove preparations\u0026mdash;aqueous extracts, alcoholic extracts, and pure oil\u0026mdash;against a comprehensive panel of clinical oral \u003cem\u003eCandida\u003c/em\u003e isolates alongside standard antifungal agents remain limited, particularly from North African populations. The present study addresses this gap by conducting a comparative \u003cem\u003ein vitro\u003c/em\u003e evaluation of the efficacy of conventional antifungal drugs (Amphotericin B, Miconazole, Nystatin) and various concentrations of \u003cem\u003eS. aromaticum\u003c/em\u003e extracts against clinically confirmed oral \u003cem\u003eCandida\u003c/em\u003e isolates obtained from patients in the Gharyan region of Libya. By integrating species-specific susceptibility testing and statistical comparisons, this investigation aims to establish the potential of clove extracts as viable alternatives or adjuncts to conventional therapy, particularly relevant for resource-constrained healthcare settings where antifungal resistance and treatment access pose significant challenges.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Study Design and Setting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis comparative \u003cem\u003ein vitro\u003c/em\u003e study was designed to evaluate the antifungal efficacy of \u003cem\u003eSyzygium aromaticum\u003c/em\u003e (clove) extracts against clinical oral \u003cem\u003eCandida\u003c/em\u003e isolates. Sample collection was conducted among patients attending the outpatient clinics at Gharyan Central Hospital and three primary healthcare centers in the Gharyan region, Libya. All mycological analysis, including culture, identification, and antifungal susceptibility testing, was performed at the Fungi Laboratory, Faculty of Medical Technology Alriyaina, University of Zintan, Libya.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Ethical Considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was reviewed and approved by the Research Ethics Committee of the Faculty of Medicine, University of Gharyan (Approval No.: UOG/MED/2025/08, dated 25 January 2025). Written informed consent was obtained from all adult participants prior to sample collection. For minor participants (under 18 years of age), consent was obtained from their parents or legal guardians. All procedures were conducted in accordance with the ethical standards of the Declaration of Helsinki of 1975, as revised in 2013.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Study Population and Sample Collection Period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSample collection was carried out over a period from 2 February 2025 to 20 April 2025. A total of 140 patients attending the outpatient clinics for various medical complaints were enrolled in the study. To ensure anonymity and proper tracking throughout the laboratory procedures, each patient was assigned a unique serial number upon enrollment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Inclusion and Exclusion Criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion criteria:\u003c/strong\u003e Patients of both genders, aged between 2 and 78 years, who presented with clinical signs suggestive of oral fungal infection (e.g., white plaques, erythema, burning sensation, angular cheilitis) and were willing to provide informed consent were included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion criteria:\u0026nbsp;\u003c/strong\u003ePatients were excluded if they had received systemic or topical antifungal therapy within the preceding two weeks, were on immunosuppressive therapy, had a known diagnosis of HIV/AIDS or other severe immunocompromising conditions, or were unable to cooperate with the sample collection procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Sample Collection Procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder strict aseptic conditions and following a standardized precautionary protocol to avoid contamination, samples were collected from multiple oral sites depending on the clinical presentation. These sites included carious lesions or supragingival plaque on tooth surfaces; the buccal mucosa, palate, tongue dorsum, and floor of mouth; the fitting surface of removable dentures (if present); and deep periodontal pockets using sterile curettes.\u003c/p\u003e\n\u003cp\u003eFor each patient, three sterile swabs were used to collect specimens from the affected sites: the first swab was used for direct microscopic examination, the second for primary culture, and the third for subculture and maintenance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Sample Transport and Storage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmediately after collection, each swab was placed into a sterile container and maintained at 4\u0026ndash;8\u0026deg;C in a chilled, insulated transport box. All samples were transported to the Fungi Laboratory within 4\u0026ndash;6 hours of collection for immediate processing. Samples that could not be processed immediately were stored at 4\u0026deg;C for a maximum of 24 hours before culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Direct Microscopic Examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGram Staining:\u0026nbsp;\u003c/strong\u003eSmears prepared from the first swab were heat-fixed and stained using the standard Gram staining procedure. The smears were then examined under light microscopy with a 100\u0026times; oil immersion objective for the presence of budding yeast cells, pseudohyphae, and true hyphae characteristic of \u003cem\u003eCandida\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLactophenol Cotton Blue (LPCB) Staining:\u0026nbsp;\u003c/strong\u003eA second smear from the first swab was mounted in LPCB stain and examined under low power (10\u0026times; and 40\u0026times;) to observe fungal morphological features, including chlamydospores, blastoconidia, and pseudohyphae.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Culture Media and Isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary Culture:\u0026nbsp;\u003c/strong\u003eThe second sterile swab was streaked onto Petri dishes containing Sabouraud Dextrose Agar (SDA) (Oxoid, UK). The medium was supplemented with chloramphenicol (0.05 g/L) to inhibit bacterial growth and cycloheximide (0.5 g/L) to suppress saprophytic fungi, thereby promoting the selective isolation of pathogenic \u003cem\u003eCandida\u003c/em\u003e species. Inoculated plates were incubated aerobically at 37\u0026deg;C for 24\u0026ndash;48 hours. Plates showing no growth after 48 hours were re-incubated at room temperature (25\u0026ndash;30\u0026deg;C) for an additional 5\u0026ndash;7 days to allow for the growth of slow-growing fungi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubculture:\u0026nbsp;\u003c/strong\u003eSuspected \u003cem\u003eCandida\u003c/em\u003e colonies, identified by their creamy, smooth, and pasty appearance, were subcultured onto fresh SDA plates to obtain pure cultures. The pure isolates were then maintained on SDA slants at 4\u0026deg;C for subsequent species identification and antifungal susceptibility testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9. Fungal Isolates and Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor this study, five clinically confirmed \u003cem\u003eCandida\u003c/em\u003e species (\u003cem\u003eC. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis\u003c/em\u003e) were obtained from the stock cultures preserved from the patient samples described above. For susceptibility testing, fresh subcultures were prepared on SDA and incubated at 37\u0026deg;C for 24-48 hours. A standardized inoculum suspension equivalent to a 0.5 McFarland standard was prepared in sterile saline for each isolate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10. Conventional Antifungal Agents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCommercially available antifungal discs (Liofilchem, Italy) were used: Amphotericin B (20 \u0026micro;g), Miconazole (10 \u0026micro;g), and Nystatin (100 IU). These were selected as representatives of major antifungal classes used in clinical practice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11. Preparation of Clove Extracts and Oil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlcoholic Extract:\u0026nbsp;\u003c/strong\u003eDried clove buds (50 g) were ground and macerated in 500 mL of 96% ethanol for 48 hours in a dark environment with continuous shaking. The mixture was filtered through sterile gauze and filter paper. The filtrate was concentrated using a rotary evaporator, and the resulting extract was dried in an oven at 40\u0026plusmn;2\u0026deg;C to obtain a powder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAqueous Extract:\u0026nbsp;\u003c/strong\u003eThe same process was repeated using 500 mL of sterile distilled water as the solvent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtract Concentrations:\u0026nbsp;\u003c/strong\u003eBoth the alcoholic and aqueous clove extracts were reconstituted to final concentrations of 25%, 50%, and 100% (w/v) using their respective solvents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClove Oil:\u0026nbsp;\u003c/strong\u003eCommercially available Eugenol oil (DHARMA research, USA) was used directly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12. Antifungal Susceptibility Testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisc Diffusion for Conventional Drugs:\u0026nbsp;\u003c/strong\u003eThe assay was performed on Mueller Hinton Agar (MHA) supplemented with 2% glucose and 0.5 \u0026micro;g/mL methylene blue, as recommended [8]. The standardized fungal suspension was swabbed uniformly onto the agar plates. Antifungal discs were placed on the inoculated surface, and plates were incubated at 37\u0026deg;C for 24-48 hours. The zones of inhibition (ZOI) were measured in millimeters (mm). All tests were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWell Diffusion for Clove Extracts/Oil:\u0026nbsp;\u003c/strong\u003eAfter swabbing the agar plates with the fungal inoculum, wells (10 mm diameter) were punched into the agar using a sterile cork borer. Each well was filled with 50 \u0026micro;L of the respective clove extract concentration or pure clove oil. Plates were incubated as above, and the ZOI was measured. Tests were performed in triplicate for each isolate and concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13. Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using SPSS Statistics version 27. Descriptive statistics (mean \u0026plusmn; standard deviation) were calculated. The significance of differences in the mean ZOI among different treatments and across \u003cem\u003eCandida\u003c/em\u003e species was determined using a one-way analysis of variance (ANOVA), followed by post-hoc Tukey\u0026apos;s HSD test for multiple comparisons. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003eEfficacy of Conventional Antifungal Agents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tested \u003cem\u003eCandida\u003c/em\u003e species were susceptible to the conventional antifungal agents, but with varying degrees of efficacy (Table 1). Amphotericin B demonstrated the strongest inhibitory effect, with a mean ZOI of 27.83 \u0026plusmn; 5.22 mm. This was significantly greater than the ZOIs produced by Miconazole (22.43 \u0026plusmn; 6.50 mm) and Nystatin (20.60 \u0026plusmn; 3.70 mm) (F = 6.796, p = 0.004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Efficacy of conventional antifungal agents against oral \u003cem\u003eCandida\u003c/em\u003e isolates (Mean ZOI in mm \u0026plusmn; SD).\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAntibiotics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026nbsp; \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eF value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmphotericin B\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e27.83\u003csup\u003ea\u003c/sup\u003e \u0026plusmn; 5.216\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.796\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.004\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMiconazole\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e22.43\u003csup\u003eb\u003c/sup\u003e \u0026plusmn; 6.502\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNystatin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e20.60\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e\u0026plusmn; 3.699\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eDifferent superscript letters (a, b) within a column indicate statistically significant differences (p \u0026lt; 0.05).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTable (1) presents the results suggest that Amphotericin B was significantly more effective in reducing oral fungal infections compared to the other two agents. The findings provide important evidence for clinicians when selecting antifungal therapy, supporting the use of Amphotericin B as a potentially superior option in this context.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative Efficacy of All Antifungal Agents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comprehensive comparison of all tested agents revealed highly significant differences (F = 152.69, p \u0026lt; 0.001) (Table 2). The 100% aqueous clove extract was the most effective treatment overall, producing a mean ZOI of 53.93 \u0026plusmn; 27.82 mm. This was followed by the 50% aqueous extract (43.80 \u0026plusmn; 33.67 mm) and the 100% alcoholic extract (41.40 \u0026plusmn; 9.83 mm). All three of these natural preparations were significantly more effective than the conventional drugs Amphotericin B, Miconazole, and Nystatin. Pure clove oil showed no measurable antifungal activity under the test conditions. Following graph displays different antifungal agents used in this study (figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Comparative efficacy of all antifungal agents and clove extracts (Mean ZOI in mm \u0026plusmn; SD).\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAntifungal agents\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026nbsp;\u003c/strong\u003e\n \u003cv:shapetype id=\"_x0000_t75\" coordsize=\"21600,21600\" o:spt=\"75\" o:preferrelative=\"t\" path=\"m@4@5l@4@11@9@11@9@5xe\" filled=\"f\" stroked=\"f\"\u003e\u0026nbsp;\u003cv:stroke joinstyle=\"miter\"\u003e\u0026nbsp;\u003cv:formulas\u003e\u0026nbsp;\u003cv:f eqn=\"if lineDrawn pixelLineWidth 0\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @0 1 0\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum 0 0 @1\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @2 1 2\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @3 21600 pixelWidth\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @3 21600 pixelHeight\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @0 0 1\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @6 1 2\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @7 21600 pixelWidth\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @8 21600 0\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @7 21600 pixelHeight\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @10 21600 0\"\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:formulas\u003e\n \u003cv:path o:extrusionok=\"f\" gradientshapeok=\"t\" o:connecttype=\"rect\"\u003e\u0026nbsp;\u003c/v:path\u003e\u0026nbsp;\n \u003c/v:stroke\u003e\u0026nbsp;\u003c/v:shapetype\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e\u003cstrong\u003eSD\u003c/strong\u003e\n \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eF value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmphotericin B\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27.83\u003csup\u003ea \u0026nbsp;\u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image002.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e\n \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"10\"\u003e\n \u003cp\u003e\u003cspan dir=\"RTL\"\u003e152.692\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"10\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMiconazole\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e22.43\u003csup\u003eb \u0026nbsp; \u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e6.502\n \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNystatin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20.60\u003csup\u003ebd \u0026nbsp;\u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e3.699\n \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAlcoholic clove extract 25%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.27\u003csup\u003ed \u0026nbsp;\u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e8.610\n \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAlcoholic clove extract 50%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e29.37\u003csup\u003ea \u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e7.005\n \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAlcoholic clove extract 100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e41.40\u003csup\u003ee \u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e9.832\n \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAqueous clove extract 25%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35.13\u003csup\u003ef \u0026nbsp;\u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e26.457\n \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAqueous clove extract 50%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.80\u003csup\u003ee \u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e33.671\n \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAqueous clove extract 100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e53.93\u003csup\u003eg \u0026nbsp;\u0026nbsp;\u003c/sup\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e27.82\n \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eClove oil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.00 \u0026nbsp;\u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/btr8097/AppData/Local/Packages/oice_16_974fa576_32c1d314_319d/AC/Temp/msohtmlclip1/01/clip_image001.png\" o:title=\"\" chromakey=\"white\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eDifferent superscript letters (a-g) within a column indicate statistically significant differences (p \u0026lt; 0.05).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecies-Specific Antifungal Response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe efficacy of the agents varied considerably depending on the \u003cem\u003eCandida\u003c/em\u003e species (Table 3). Aqueous clove extracts were exceptionally effective against \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e, with ZOIs exceeding 80 mm at 50% and 100% concentrations. In contrast, \u003cem\u003eC. krusei\u003c/em\u003e and \u003cem\u003eC. parapsilosis\u003c/em\u003e were less susceptible to the aqueous extracts, with the highest ZOIs being 32.00 mm and 33.33 mm, respectively. For these species, the conventional antifungals, particularly Amphotericin B and Miconazole, showed comparable or slightly better activity than the clove extracts. \u003cem\u003eC. tropicalis\u003c/em\u003e was most effectively inhibited by the 100% alcoholic clove extract (45.61 mm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Species-specific response: Mean ZOI (mm) of antifungal agents against different \u003cem\u003eCandida\u003c/em\u003e species.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCandida\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;spp.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment (Most Effective)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean ZOI \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment (Conventional)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean ZOI \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAq. Extract 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e83.33 \u0026plusmn; 3.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAmphotericin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26.33 \u0026plusmn; 10.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. glabrata\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAq. Extract 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAmphotericin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.00 \u0026plusmn; 1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. krusei\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAq. Extract 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e32.00 \u0026plusmn; 2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMiconazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.17 \u0026plusmn; 0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. parapsilosis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAlc. Extract 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.44 \u0026plusmn; 8.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAmphotericin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.17 \u0026plusmn; 4.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. tropicalis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAlc. Extract 100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.61 \u0026plusmn; 2.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAmphotericin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28.33 \u0026plusmn; 4.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe search for effective and safe antifungal agents is a persistent priority in medical mycology. Our findings demonstrate that crude extracts of \u003cem\u003eSyzygium aromaticum\u003c/em\u003e possess remarkable antifungal activity, significantly surpassing that of commonly used conventional drugs against clinical oral \u003cem\u003eCandida\u003c/em\u003e isolates.\u003c/p\u003e \u003cp\u003eThe superior performance of Amphotericin B over the azole (Miconazole) and the other polyene (Nystatin) is consistent with its broad-spectrum, fungicidal nature [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, its clinical use is often hampered by nephrotoxicity and other side effects [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe most striking result of this study is the potent efficacy of clove extracts. The 100% aqueous extract emerged as the most powerful agent overall. This was an unexpected but significant finding, as alcoholic extracts are typically more efficient at extracting non-polar bioactive compounds like Eugenol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The superior performance of the aqueous extract in our assay could be due to better diffusion of its constituents through the aqueous-based agar medium, leading to a larger observable zone of inhibition. Both aqueous and alcoholic extracts exhibited a clear dose-dependent response, with 100% concentrations being vastly more effective than 25% concentrations, underscoring the concentration of active antifungal components.\u003c/p\u003e \u003cp\u003eThe failure of pure clove oil (Eugenol) to produce an inhibition zone was paradoxical. This could be attributed to its high volatility and hydrophobic nature, preventing its effective diffusion from the well into the agar matrix. In clinical dental applications, Eugenol is often used in a paste form with zinc oxide, which may facilitate its release and activity, unlike the pure oil in a well-diffusion test [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe species-specific variation in susceptibility is clinically crucial. The high susceptibility of \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e to aqueous clove extracts is promising, given their high clinical prevalence. In contrast, the relative resilience of \u003cem\u003eC. krusei\u003c/em\u003e, which is intrinsically less susceptible to fluconazole [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], to both clove extracts and conventional drugs highlights its challenging nature. This suggests that while clove extracts are a powerful broad-spectrum alternative, species identification remains important for tailoring therapy, especially in recalcitrant cases.\u003c/p\u003e \u003cp\u003eThe mechanism of action is widely attributed to Eugenol, which can integrate into and disrupt the fungal cell membrane, leading to increased permeability and cell death [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, clove extracts are known to inhibit germ tube and biofilm formation, key virulence factors of \u003cem\u003eCandida\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The presence of other synergistic compounds in the crude extract, such as β-caryophyllene, may further enhance its antifungal potency beyond that of pure Eugenol alone [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eLimitations and Future Directions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA limitation of this study is the use of the well-diffusion method, which, while excellent for screening, does not provide a quantitative Minimum Inhibitory Concentration (MIC). Future work should determine the MIC and Minimum Fungicidal Concentration (MFC) of these extracts. Furthermore, \u003cem\u003ein vivo\u003c/em\u003e studies and the development of stable formulations (e.g., mouthwashes, gels) are essential next steps to translate these findings into clinical practice.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study provides compelling evidence that \u003cem\u003eSyzygium aromaticum\u003c/em\u003e (clove) extracts, particularly aqueous extracts at high concentrations, are potent inhibitors of clinical oral \u003cem\u003eCandida\u003c/em\u003e species, exhibiting efficacy that surpasses standard antifungal drugs. Given their natural origin, anticipated low cost, and reduced potential for side effects, clove extracts represent a highly promising alternative or adjunctive therapy for managing oral candidiasis. This is especially relevant for resource-limited settings like Libya, where access to conventional antifungals may be constrained. We recommend further phytochemical analysis and clinical trials to develop these findings into tangible therapeutic options.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDeo PN, Deshmukh R (2019) Oral microbiome: Unveiling the fundamentals. 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Clin Microbiol Rev 20(1):133\u0026ndash;163\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eČern\u0026aacute;kov\u0026aacute; L, Rizzato C, Kolarik M et al (2022) Epidemiology and antifungal susceptibility of oral Candida species from hospitalized patients in Slovakia. J Fungi (Basel) 8(3):267\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllis D, Amphotericin B (2002) Spectrum and resistance. J Antimicrob Chemother 49(Suppl 1):7\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaniado-Labor\u0026iacute;n R, Cabrales-Vargas MN, Amphotericin B (2009) Side effects and toxicity. Rev Iberoam Micol 26(4):223\u0026ndash;227\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSklen\u0026aacute;r Z, Scigel V, Hor\u0026aacute;čkov\u0026aacute; K et al (2013) Nystatin\u0026mdash;Characteristics and use in clinical practice. Ceska Slov Farm 62(3):105\u0026ndash;110\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePappas PG, Kauffman CA, Andes DR et al (2016) Clinical practice guideline for the management of candidiasis: 2016 update. Clin Infect Dis 62(4):e1\u0026ndash;e50\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhojoghi F, Zarrinfar H, Mirhendi H et al (2024) Oral Candida species and antifungal susceptibility in drug abusers. Mycoses 67(2):e13692\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaustova M, Ananieva M, Basarab Y et al (2024) Susceptibility of Candida albicans clinical isolates to antifungal drugs. Wiad Lek 77(1):85\u0026ndash;90\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmbe K, Suzuki H, Yamaguchi T et al (2020) In vitro antifungal activity of micafungin against Candida krusei clinical isolates. J Infect Chemother 26(5):483\u0026ndash;488\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBranco J, Miranda IM, Rodrigues AG (2023) Candida parapsilosis virulence and antifungal resistance mechanisms: A comprehensive review. J Fungi (Basel) 9(1):84\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErfaninejad M, Zarrinfar H, Fata A et al (2024) Phenotypic and genotypic characterization of Candida albicans isolates from HIV-positive patients. Curr Med Mycol 10(1):1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamadan A, Elghblawi E, Eltaib F et al (2025) Oral fungal carriage among Libyan individuals: A cross-sectional study. Libyan J Med 20(1):2254678\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Kakalli R, Ben Amer M, El-Mansouri S et al (2024) Angular cheilitis prevalence and correlates in Libyan type 2 diabetes patients. Int J Diabetes Dev Ctries 44(1):112\u0026ndash;120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsmaio A, El-Mansouri S, Elghblawi E (2017) Isolation and identification of Candida species from diabetic patients in Musrata, Libya. J Am Sci 13(5):45\u0026ndash;52\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaur R, Kaur J (2021) Plant-derived antifungals: A comprehensive review. J Ethnopharmacol 269:113679\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGengatharan A, Abd-Rahim MH (2023) Application of clove extract in active food packaging and modern food systems: A review. Food Packag Shelf Life 36:101041\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQassim A, Al-Marzooq F, Al-Saadi A et al (2024) Inhibitory effects of clove extracts on Candida albicans growth. 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J Herb Med 32:100542\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdellatif F, Boudjema K, Boulekbache-Makhlouf L (2023) Comparative study of clove extract and Miconazole gel against oral Candida species. Phytother Res 37(4):1456\u0026ndash;1466\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGolestannejad Z, Kermani F, Moazeni M et al (2024) Antifungal efficacy of Amphotericin B and Nystatin against Candida species from radiotherapy patients. Curr Med Mycol 10(2):e2024008\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAldabaan N, Shaikh S, Alqahtani A et al (2024) Biological activities of nanoparticles synthesized from Syzygium aromaticum. Green Chem Lett Rev 17(1):2289456\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Syzygium aromaticum, Clove, Antifungal, Oral Candidiasis, Candida, Eugenol, Natural Products, Libya","lastPublishedDoi":"10.21203/rs.3.rs-9233497/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9233497/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe escalating challenge of antifungal resistance and the side effects associated with conventional drugs have intensified the search for natural alternatives. This study investigated the efficacy of \u003cem\u003eSyzygium aromaticum\u003c/em\u003e (clove) extracts compared to standard antifungal agents against clinical oral \u003cem\u003eCandida\u003c/em\u003e isolates from Libyan patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFive \u003cem\u003eCandida\u003c/em\u003e species (\u003cem\u003eC. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis\u003c/em\u003e) were isolated from patients in Gharyan City, Libya. The antifungal susceptibility to Amphotericin B, Miconazole, and Nystatin was tested using the disc diffusion method. The activity of aqueous and alcoholic clove extracts (25%, 50%, 100%) and pure clove oil (Eugenol) was evaluated using a well-diffusion assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong conventional antifungals, Amphotericin B was the most effective (Mean Inhibition Zone: 27.83\u0026thinsp;\u0026plusmn;\u0026thinsp;5.22 mm), followed by Miconazole (22.43\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50 mm) and Nystatin (20.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.70 mm). Remarkably, clove extracts demonstrated superior activity. The 100% aqueous clove extract showed the highest overall efficacy (53.93\u0026thinsp;\u0026plusmn;\u0026thinsp;27.82 mm), significantly outperforming all conventional drugs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The 100% alcoholic extract was also highly effective (41.40\u0026thinsp;\u0026plusmn;\u0026thinsp;9.83 mm). Clove oil showed no inhibitory activity. Efficacy was concentration-dependent and species-specific. For instance, \u003cem\u003eC. glabrata\u003c/em\u003e was highly susceptible to aqueous extracts (90.00 mm inhibition at 100%), while \u003cem\u003eC. krusei\u003c/em\u003e showed relative resilience, responding similarly to both clove extracts and conventional drugs.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eClove extracts, particularly aqueous preparations at high concentrations, exhibit potent and broad-spectrum antifungal activity against clinical oral \u003cem\u003eCandida\u003c/em\u003e isolates, surpassing standard antifungals. These findings position \u003cem\u003eSyzygium aromaticum\u003c/em\u003e as a highly promising, naturally sourced candidate for developing new therapeutic or preventive strategies against oral candidiasis, especially in regions where access to conventional medicine is limited.\u003c/p\u003e","manuscriptTitle":"Syzygium Aromaticum (Clove) Extracts Demonstrate Superior Antifungal Activity Compared to Conventional Drugs Against Clinical Oral Candida Isolates from Gharyan, Libya: A Comparative In Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 06:51:52","doi":"10.21203/rs.3.rs-9233497/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"905e8ba2-dda5-467d-bf20-61c12e18256e","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65288628,"name":"Mycology"},{"id":65288629,"name":"Clinical Pharmacology"}],"tags":[],"updatedAt":"2026-04-01T06:51:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 06:51:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9233497","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9233497","identity":"rs-9233497","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}