Exploring Potential Antifungal Agents from Artemisia annua L., Using In-vitro and In-silico approaches.

Abstract

Fungal infections pose a significant global health burden, with drug resistance severely limiting treatment options. While Artemisia annua L., has been widely studied for its antifungal properties in various regions, there is however limited research on the antifungal potential of A. annua L., cultivated in Malawi. Given that environmental factors influence the plant’s phytochemical biosynthesis, composition and yield, its therapeutic activity may differ from those reported elsewhere. This study aimed to evaluate the antifungal potential of A. annua L. grown in Malawi through in-vitro assays and explore the molecular interactions and pharmacokinetic profiles of literature-reported bioactive compounds from A. annua using in-silico approaches. Antifungal efficacy of the plant extracts was evaluated through in-vitro susceptibility testing using the disk diffusion method. Qualitative phytochemical analysis was employed to identify the presence of key bioactive groups. In parallel, molecular docking studies were carried out in PyRx to examine interactions between literature-sourced compounds and selected fungal targets (CYP51 and AFSQS). Additionally, the pharmacokinetic properties, toxicity, and drug-likeness of five top-performing compounds were predicted using ADMET lab 2.0. Aqueous extracts of A. annua L. demonstrated stronger antifungal activity than ethanolic extracts against Candida spp. and Cryptococcus neoformans in disk diffusion assays. Phytochemical tests confirmed the presence of key bioactive classes. Literature-sourced flavonoids showed strong binding affinities to fungal targets CYP51 and AFSQS, indicating promising molecular interactions relevant to antifungal therapy. ADMET profiling further supported the suitability of these compounds, highlighting good drug-likeness, minimal predicted toxicity, and acceptable pharmacokinetic features, despite limited absorption potential. All five compounds were predicted to localize to mitochondria, suggesting a possible intracellular mode of antifungal action. Together, these findings support the therapeutic relevance of A. annua derived flavonoids as promising leads for antifungal drug development.

Keywords

Fungal infections, drug resistance, Artemisia annua L., antifungal drug development, natural antifungal agents

Introduction

4.2 Phytochemical screening

ADMET Profiles of selected compounds from A.annua L.,

DISCUSION

The rise of drug-resistant fungal pathogens is a growing health challenge that calls for new and effective treatments [46]. Current antifungal agents, such as azoles and polyenes, often face issues like toxicity, side effects, and the development of resistant strains [47]. These problems highlight the need for alternative solutions, including biologically active compounds found in medicinal plants[48]. These plant-derived phytochemicals offer potential antifungal and antibacterial activities and could play a key role in overcoming antimicrobial resistance [49]. The current study explored potential antifungal agents from Artemisia annua L. that would work against some biological targets in selected fungal pathogens using in-vitro and in-silico approaches. 

5.2 Antifungal Activities of Artemisia annua L., Plant Extracts

Artemisia annua L. has demonstrated antifungal activity against Candida spp. and C. neoformans, indicating its potential as a source of promising antifungal agents. The descriptive statistics in Table 2 highlight the zone of inhibition for the ethanoic and aqueous extracts from Artemisia annua L., across varying concentrations (100mg/ml, 50mg/ml, 25mg/ml, and 10mg/ml) against Candida spp. and Cryptococcus neoformans, with ketoconazole as a control. Both aqueous and ethanolic extracts showed antifungal activity against Candida spp and cryptococcus neoformans. These results are supported by several studies that have shown that artemisia annua’s  aqueous and ethanolic extracts have antifungal activity[50,51,47,52]. However, a study conducted in Pakistan by  Ikram et al., (2015) reported no antifungal activity of Artemisia annua L. on Candida albicans [53]. This suggest that the antifungal activity of Artemisia annua L. may be influenced by some factors.  For example, Nageeb et al., (2014) reported that Artemisia annua’s chemical composition is influenced by the habitat in which it grows consequently affecting its medicinal efficacy.

It has been reported that the choice of extraction solvent significantly impacts the extraction efficiency and the bioactivity of the isolated compounds[54]. This study reports that aqueous extracts consistently showed higher mean inhibition zones compared to the ethanoic extracts, particularly at higher concentrations. For example, the aqueous extract demonstrated higher antifungal activity, with mean inhibition zones of 17.83 mm and 19.5 mm for Candida spp. and C. neoformans, respectively, at 100 mg/ml.  The multiple comparisons provide pairwise analyses of the treatments, offering detailed insights into their relative efficacy. The aqueous extract consistently showed significantly greater antifungal activity than the ethanoic extract (mean difference = 2.08, p < 0.05). The enhanced antifungal activity of the aqueous extract may be linked to its ability to extract polar compounds like saponins identified exclusively in this extract which are known for their strong bioactivity [55]. These findings align with Oniha et al., (2021) who observed that aqueous extracts often retain a higher concentration of water-soluble bioactive compounds, enhancing their antifungal potency.

Azole antifungal agents are essential in treating fungal infections. Ketoconazole, used as the standard, consistently showed the highest inhibition zones, averaging 19.33 mm and 18.5 mm for Candida spp. and C. neoformans, respectively and significantly outperformed both extracts. Aqueous extracts were  less effective than ketoconazole (mean difference = -3.31, p < 0.05) and the mean difference between ketoconazole and the ethanoic extract was 5.40 (p < 0.05), emphasizing the superior efficacy of synthetic antifungal agents. Its effectiveness underscores its established role as a benchmark in antifungal studies [57,58]. However, the promising performance of the aqueous extract, particularly at higher concentrations, suggests its potential as a natural alternative, especially in cases where synthetic drug resistance is a concern[49,56] and in regions with limited access to synthetic drugs. These findings are consistent with studies indicating the potency of aqueous extracts for antifungal activity[47,57,59] These results also highlight the necessity of further exploring the phytochemical composition of the extracts to identify specific compounds responsible for their antifungal activity. Isolating and optimization of these compounds can enhance the efficacy of plant-derived treatments.

The observed standard deviations across the treatments indicate low variability, reflecting the reproducibility and reliability of the experimental procedures. This consistency aligns with the principles of reproducibility in scientific research, which emphasize the importance of obtaining consistent results under similar conditions [60]. Furthermore, the dose-dependent nature of antifungal efficacy demonstrated in this study highlights the potential of plant-derived extracts as effective therapeutic agents. These findings are consistent with broader research into the antimicrobial potential of plant extracts, which have shown promising results in combating fungal pathogens [61] [62]. The integration of such extracts into therapeutic applications offers a valuable avenue for addressing antimicrobial resistance [63].

Artemisia annua L., has been reported to contain a diversity of bioactive compounds [64,65]. In this study the qualitative phytochemical analysis of aqueous and ethanolic extracts of Artemisia annua  reveals a rich diversity of bioactive compounds. Both extracts contained a broad spectrum of phytochemical groups, including phenolics, glycosides, terpenoids, alkaloids, flavonoids, and tannins, all of which are known for their significant therapeutic properties, such as antioxidant, anti-inflammatory, and antimicrobial activities[64,66,67]. Notably, saponins were detected only in the aqueous extract, reflecting the differential solubility of compounds depending on the solvent used. This may suggest that aqueous extraction is more effective for isolating saponins, which are widely recognized for their antifungal properties [68,69,70]. However, a study conducted in Pakistan by  Ikram et al., (2015) reported the presence of saponins in ethanolic extracts. This can be supported by a study by Nageeb et al., (2014) who reported that Artemisia annua’s chemical composition is influenced by the habitat. The consistent presence of phenolics, glycosides, terpenoids, alkaloids, and flavonoids in both extracts highlights the pharmacological potential of Artemisia annua, particularly for its antifungal efficacy, which aligns with previous studies emphasizing their roles in microbial inhibition [71,72]. The presence of tannins further enhances the medicinal profile of these extracts, contributing to their observed bioactivity against fungal pathogens. Overall, these findings underline the importance of solvent selection in maximizing the recovery of bioactive compounds, particularly for applications in antifungal drug discovery and development.

The exploration of antifungal agents derived from Artemisia annua L., represents a growing area of interest in natural product-based drug discovery. This study focused on computational screening of 60 phytochemical compounds, ultimately selecting potential antifungal agents based on molecular docking results against two fungal target proteins: CYP51 and AFSQS. The use of established antifungal drugs like ketoconazole (CYP51) and terbinafine (AFSQS), as positive controls provided a comparative framework to evaluate the efficacy of these selected compounds.

The molecular docking results revealed that apigenin,diosmetin,chryseriol, genkwanin, acacetin, kempferol, luteolin, tamarixetin as potential antifungal agents with with diosmetin, apigenin, chryseriol, acacetin and luteolin exhibiting excellent binding affinity, with docking scores surpassing terbinafine, a control drug, suggesting their potential as effective antifungal agents. The success of these selected compounds can be attributed to their strong ligand-protein interactions, which are crucial for effective enzyme inhibition in fungal pathogens. Previous studies have demonstrated that flavonoid-based compounds exhibit potent antifungal properties, showing promising potential for antifungal therapy and prevention by significantly reducing fungal virulence and downregulating antifungal resistance-associated genes [73,74]. This activity is crucial as it not only weakens fungal pathogenicity but also mitigates drug resistance, thereby enhancing the efficacy of existing antifungal treatments and providing a foundation for the development of novel, plant-derived antifungal therapeutics.

Apigenin showed higher docking score against all the target proteins, with predominant hydrogen bond, Pi-Anion and Pi-Pi stacked interactions which facilitates stability within the proteins binding pockets [75]. Some studies have reported antifungal activity of apigenin and its mechanism of action. For example, it is reported that apigenin downregulates genes linked to drug resistance and ergosterol biosynthesis. It also significantly reduces biofilm formation and hyphal transition, both key virulence factors in Candida infections [73].  Futhermore, apigenin disrupts fungal cell membrane integrity, induces mitochondrial dysfunction, increases reactive oxygen species (ROS), and triggers apoptosis in Candida albicans through mitochondrial calcium signaling [76,77,78]. These results align with invitro findings that apigenin inhibits the growth of various Candida species, with minimum inhibitory concentrations (MIC) ranging from 0.10 to 0.15 mg/mL and minimum fungicidal concentrations (MFC) from 0.15 to 0.30 mg/mL [76]. These findings highlight apigenin as a promising antifungal candidate, capable of targeting multiple resistance pathways and mitigating fungal pathogenicity.

Similary, diosmetin showed high docking scores (-8.9Kcal/mole, -9.0Kcal/mol) across all the target proteins, with predominant Pi-Pi stacking, hydrogen bonding, and Pi-Anion interactions facilitating enhanced stability within the protein binding pockets. Zhao et al. (2023) provide further biological relevance for diosmetin’s antimicrobial effects, demonstrating that the flavonoid effectively suppresses microbial growth by attenuating inflammation and ferroptosis, a form of iron-dependent cell death associated with oxidative stress and metabolic disruption [79]. This suggests that diosmetin’s efficacy against fungal targets extends beyond direct enzyme inhibition, as it may also contribute to fungal cell damage by modulating oxidative stress pathways. The attenuation of inflammation-related signaling further supports its therapeutic role, as fungal infections often induce strong inflammatory responses that compromise host immune defenses.

Furthermore, chryseriol, and acacetin  similarly demonstrated significant interactions with conserved amino acid residues like TYR 229, PHE 241, ASP 233, reinforcing their potential as CYP51 inhibitors. CYP51 (14-alpha demethylase) is essential for ergosterol synthesis in fungi, a key component of the fungal cell membrane. Inhibiting CYP51 disrupts membrane integrity and leads to fungal cell death [74]. Computational and molecular docking studies have identified specific flavonoid-like compounds with strong binding affinity to CYP51, outperforming standard antifungal drugs in docking scores [74,80]. Most tested flavonoids exhibit low cytotoxicity to human cells, supporting their potential for safe therapeutic use [73]. Antifungal activity of Chryseriol has also been reported and just like other flavones, chryseriol may act by modulating key metabolic enzymes and disrupting fungal cell processes [81]. Whilst acacetin’s diverse pharmacological profile includes anti-inflammatory, anticancer, and antimicrobial effects has been reported suggesting its potential as an antifungal agent, though more targeted studies are needed [81]. Acacetin on the other hand is noted for its broad antimicrobial activity, including inhibition of microbial growth, but direct evidence of its antifungal potency is limited. Its diverse pharmacological profile includes anti-inflammatory, anticancer, and antimicrobial effects, suggesting potential as an antifungal agent, though more targeted studies are needed[82].

Apart from CYP51 inhibition, selected compounds also exhibited strong interactions with AFSQS, a key target involved in fungal metabolism. AFSQS plays a crucial role in squalene epoxidation, a key step in fungal lipid metabolism, making it a validated drug target for antifungal therapy [45]. Several flavonoids in this study like Genkwanin, Tamarixetin, and Luteolin demonstrated Pi-Pi stacking and hydrogen bonding interactions, stabilizing their binding within the AFSQS pocket. This mode of binding has previously been noted in similar molecular docking studies, where flavonoid compounds exhibited high-affinity binding to fungal squalene epoxidase enzymes, disrupting lipid biosynthesis and leading to fungal cell death [83,84]. In a similar study  molecular docking revealed strong binding affinities for apigenin driven by hydrophobic and electrostatic interactions with critical residues in the squalene epoxidase active site [85]. Although the antifungal activity of Genkwanin and Tamarixetin has not been extensively studied, their antioxidant and anti-inflammatory properties offer strong support for their potential role in fungal inhibition. By reducing oxidative stress and modulating inflammatory responses, these compounds may help weaken fungal survival mechanisms, enhance host defense, and disrupt key virulence factors, reinforcing their viability as antifungal agents. On the otherhand Luteolin inhibited growth of Candida albicans with the minimal inhibitory concentration of 37.5 µg/mL and showed ability to act as biofilm formation inhibitors [86].

A notable observation in this study was the consistent superiority of flavonoid-based compounds over steroidal phytochemicals such as Stigmasterol and Beta-Sitosterol. Although Stigmasterol with docking score; -10.3Kcal/mole and -10.4Kcal/mole for CYP51 and AFSQS repectively demonstrated strong binding affinity across CYP51 and AFSQS , its interactions were predominantly Pi-Alkyl and Van der Waals forces, which may contribute to lipid perturbation rather than direct enzyme inhibition [87]. In contrast, flavonoid compounds such as Diosmetin and Apigenin exhibited broader structural adaptability, enabling multi-target interactions across CYP51 and  AFSQS. This versatility is a crucial advantage in drug development, as compounds that engage multiple fungal targets could mitigate drug resistance mechanisms often observed in pathogenic fungi [88]

The comparison with two control drugs ketoconazole and terbinafine, further strengthened the findings. Ketoconazole, a well-established azole-based CYP51 inhibitor, exhibited a docking score of -10.2, slightly outperforming Diosmetin but with fewer interactions with critical amino acid residues. Terbinafine, an AFSQS inhibitor, recorded a docking score of -8.2, placing it below several selected flavonoids, including apigenin (-8.5) and chryseriol (-8.5), which engaged key residues more effectively. This finding aligns with studies reporting flavonoid compounds as potential alternatives to terbinafine, given their broader bioavailability and minimal toxicity . For example, apigenin, showed a potential as an antifungal agent for treating dermatophytosis in mice, with complete recovery comparable to standard drug terbinafine in 12 days[84].

The ADMET profiling of five selected phytocompounds from Artemisia annua L. Diosmetin, apigenin, chryseriol, acacetin, and luteolin revealed a consistent pattern of drug likeness and moderate pharmacokinetic and safety properties that support their candidacy as antifungal leads. All five compounds complied with Lipinski’s Rule of Five, indicating favorable oral bioavailability profiles, at least from a molecular property standpoint [89]. Acacetin exhibited the highest drug likeness (QED?=?0.756), implying strong structural suitability for drug development, while Luteolin showed the lowest (QED?=?0.601), yet still within acceptable range for natural product scaffolds. LogP values ranged from 3.19 to 3.65 across the compounds, suggesting adequate lipophilicity for membrane permeability, while logS values between –3.70 and –3.91 indicated low to moderate aqueous solubility typical for polyphenolic molecules. Importantly, hepatotoxicity predictions flagged Chryseriol with a probability of 0.06, suggesting a potential safety, whereas the remaining compounds scored 0.1, indicating low predicted hepatotoxicity and hence a safer profile for systemic application [90]. This distinction could guide structure activity relationship (SAR) refinement to improve tolerability.

Although all compounds demonstrated high predicted oral bioavailability (F30%), their HIA scores were poor, and Caco-2 permeability values were below threshold, pointing to possible absorption limitations. However, they were uniformly non-inhibitory to P-glycoprotein, reducing concerns for efflux mediated resistance. Plasma protein binding (PPB) percentages ranged from 95.60% to 97.25%, suggesting high systemic retention, while volume of distribution (VD) and fraction unbound (Fu) values were within typical bounds for moderate tissue penetration. Blood–brain barrier (BBB) penetration predictions were moderate for all compounds, which may be acceptable or even advantageous depending on intended tissue targets. All five showed non-substrate behavior for CYP3A4, paired with weak to moderate inhibition indicating manageable risks for drug–drug interactions. Toxicity markers like hERG inhibition were absent or low across the board. Skin sensitization and fish toxicity were consistently high-risk, necessitating environmental and dermal safety evaluation in future formulations.

Interestingly, all compounds were predicted to localize to mitochondria, which may suggest intracellular antifungal mechanisms involving oxidative stress or mitochondrial disruption [91]. Given the natural origin and multi-target potential of flavonoids, their profiles are consistent with previously studied scaffolds in antifungal drug development [92]. The ADMET evaluation indicates that A. annua-derived compounds, particularly acacetin chryseriol and diosmetin, possess promising drug-like properties, modest toxicity risk, and acceptable pharmacokinetics for further optimization. These findings complement the molecular docking and phytochemical screening results, reinforcing their potential as lead antifungal candidates.

CONCLUSION

This study evaluated the antifungal potential of Artemisia annua L. cultivated in Malawi and explored the pharmacological relevance of literature-reported compounds through in-silico techniques. The in-vitro assays revealed that aqueous extracts exerted stronger inhibitory effects against Candida spp. and Cryptococcus neoformans than ethanolic extracts, supporting the presence of potent, water-soluble bioactive compounds within the locally cultivated plant. Phytochemical screening confirmed the presence of multiple secondary metabolite classes which are known to exhibit broad-spectrum antifungal activity. These findings highlight not only the therapeutic value of A. annua in the context of fungal infections but also the importance of evaluating medicinal plants within their local agroecological environments, given how environmental factors influence phytochemical composition. This aspect is particularly relevant for resource-limited settings, where indigenous medicinal plants could serve as accessible and cost-effective alternatives to synthetic antifungal agents.

Complementing the in-vitro findings, the in-silico analysis of literature-reported compounds provided deeper insight into the pharmacological potential of specific flavonoids. Molecular docking revealed strong interactions between key fungal targets (CYP51 and AFSQS) and compounds such as diosmetin, apigenin, acacetin, and chryseriol, with binding affinities comparable to or exceeding those of reference drugs. The ADMET profiling of these compounds further validated their drug-likeness, low toxicity profiles, and favorable pharmacokinetic characteristics despite absorption limitations reinforcing their candidacy for further development. Notably, the predicted mitochondrial localization of all five compounds suggests a possible intracellular antifungal mechanism that warrants further exploration. Taken together, this work underscores the untapped therapeutic potential of A. annua L. cultivated in Malawi and positions these literature-derived flavonoids as promising leads for antifungal drug development. The integrative research strategy adopted here provides a model for future studies aiming to bridge local biodiversity with rational drug discovery.

RECOMMENDATION

To advance these findings, it is recommended that future studies focus on isolating and characterizing specific bioactive compounds from A. annua L. grown in Malawi, followed by targeted antifungal testing. Experimental validation of molecular docking predictions using enzyme inhibition assays against CYP51 and AFSQS is essential to confirm the functional activity of literature-identified compounds. In addition, in-vivo studies should be pursued to assess bioavailability, toxicity, and pharmacokinetic behavior. Structural optimization through molecular dynamics simulations and QSAR modeling could enhance potency and drug-likeness. Exploring synergistic effects with existing antifungals may offer strategies to combat resistance and reduce toxicity. Finally, mechanistic studies investigating gene regulation and virulence suppression by selected flavonoids would help elucidate their therapeutic potential at the molecular level. Such integrative efforts will be instrumental in transforming phytochemicals from A. annua into viable antifungal therapeutics.

CONFLICT OF INTEREST: The authors declare that they have no competing interest.

FUNDING: Funding for this research was provided by the NORHED II Grant scholarship.

AUTHOR DECLARATIONS

Ethics approval

This study did not involve experiments on human or animal subjects, and therefore did not require written consent for its execution. However, the research protocol was approved by the Malawi University of Science and Technology Ethics Committee (MUSTREC). Additionally, a formal request for the collection fungal pathogens (Candida spp., and Cryptococcus neoformans) isolates was sent to Malawi Liverpool Wellcome Trust Laboratory manager who facilitated the collection process.

Consent to participate

Not applicable. This study did not involve human participants, human data, or animal subjects.

Consent for publication

Not applicable. The manuscript does not contain any individual person’s data in any form (including images or videos).

Availability of data and materials

All data generated or analyzed during this study are included in this published article. Additional datasets are available from the corresponding author upon reasonable request.

REFERENCES

1. E. Puumala, S. Fallah, N. Robbins, and L. E. Cowen, “Advancements and challenges in antifungal therapeutic development,” Clin. Microbiol. Rev., vol. 37, no. 1, 2024, doi: 10.1128/cmr.00142-23.
2. D. J. Smith et al., “Public Health Research Priorities for Fungal Diseases: A Multidisciplinary Approach to Save Lives,” J. Fungi, vol. 9, no. 8, 2023, doi: 10.3390/jof9080820.
3. E. Rayens and K. A. Norris, “Prevalence and Healthcare Burden of Fungal Infections in the United States, 2018,” Open Forum Infect. Dis., vol. 9, no. 1, 2022, doi: 10.1093/ofid/ofab593.
4. World Health Organization, Guidelines for diagnosing, preventing and managing cryptococcal disease among adults, adolescents and children living with HIV. 2022. [Online]. Available: https://www.who.int/publications/i/item/9789240052178
5. K. Kalua, B. Zimba, and D. W. Denning, “Estimated burden of serious fungal infections in Malawi,” J. Fungi, vol. 4, no. 2, 2018, doi: 10.3390/jof4020061.
6. T. B. Tufa et al., “Disseminated cryptococcosis is a common finding among human immunodeficiency virus-infected patients with suspected sepsis and is associated with higher mortality rates,” J. Fungi, vol. 9, no. 8, p. 836, 2023.
7. T. Alemayehu, S. Ayalew, T. Buzayehu, and D. Daka, “Magnitude of Cryptococcosis among HIV patients in sub-Saharan Africa countries: a systematic review and meta-analysis,” Afr. Health Sci., vol. 20, no. 1, pp. 114–121, 2020.
8. B. Goni, I. Kida, I. Saidu, H. Yusuph, M. Brown, and B. Bakki, “Cryptococcal neorformans Antigenemia among HIV-Infected Patients in North Eastern Nigeria,” J Transm Dis Immun, vol. 1, no. 1, pp. 1–8, 2017.
9. T. K. Nyazika et al., “Epidemiology and aetiologies of cryptococcal meningitis in Africa, 1950–2017: protocol for a systematic review,” BMJ Open, vol. 8, no. 7, p. e020654, 2018.
10. R. Rajasingham et al., “Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis,” Lancet Infect. Dis., vol. 17, no. 8, pp. 873–881, 2017.
11. K. Odoj et al., “Tracking Candidemia Trends and Antifungal Resistance Patterns across Europe: An In-Depth Analysis of Surveillance Systems and Surveillance Studies,” J. Fungi, vol. 10, no. 10, p. 685, 2024.
12. P. G. Pappas, M. S. Lionakis, M. C. Arendrup, L. Ostrosky-Zeichner, and B. J. Kullberg, “Invasive candidiasis,” Nat. Rev. Dis. Prim., vol. 4, no. 1Salmanton-García, J., Cornely, O. A., Stemler, J., Bara?, A., Steinmann, J., Siváková, A., Akalin, E. H., Arikan-Akdagli, S., Loughlin, L., Toscano, C. (2024). Attributable mortality of candidemia–results from the ECMM Candida III multinational Europea, pp. 1–20, 2018.
13. J. Salmanton-García et al., “Attributable mortality of candidemia–results from the ECMM Candida III multinational European observational cohort study,” J. Infect., vol. 89, no. 3, p. 106229, 2024.
14. B. Mete et al., “Change in species distribution and antifungal susceptibility of candidemias in an intensive care unit of a university hospital (10-year experience),” Eur. J. Clin. Microbiol. Infect. Dis., vol. 40, pp. 325–333, 2021.
15. M. Hoenigl et al., “Novel antifungals and treatment approaches to tackle resistance and improve outcomes of invasive fungal disease,” Clin. Microbiol. Rev., 2024, doi: 10.1128/cmr.00074-23.
16. N. P. Wiederhold, “Antifungal resistance: current trends and future strategies to combat,” Infect. Drug Resist., vol. 10, pp. 249–259, 2017, doi: 10.2147/IDR.S124918.
17. D. Karaman, O. G?r??g?n, A. O. G?r??g?n, and H. Malyer, “Discovery of new herbal anthelmintics from Artemisia Annua L . via in silico molecular docking and in vivo extract application,” vol. 42, no. 1, pp. 198–210, 2024, doi: 10.14744/sigma.2021.00075.
18. V. O. Apeh et al., “Exploring the potential of aqueous extracts of Artemisia annua ANAMED (A3) for developing new anti-malarial agents: In vivo and silico computational approach,” Eng. Reports, no. August, pp. 1–23, 2023, doi: 10.1002/eng2.12831.
19. H. Ekiert, J. ?wi?tkowska, P. Klin, A. Rzepiela, and A. Szopa, “Artemisia annua-Importance in Traditional Medicine and Current State of Knowledge on the Chemistry, Biological Activity and Possible Applications,” Planta Med., vol. 87, no. 8, pp. 584–599, 2021, doi: 10.1055/a-1345-9528.
20. M. I. Ngwu et al., “Synergistic antifungal activity of combinations of Artemisia annua extract with fluconazole against resistant strains of Candida albicans,” Trop. J. Pharm. Res., vol. 23, no. 6, pp. 941–949, 2024, doi: 10.4314/tjpr.v23i6.4.
21. T. Ragab, A. S. Mansour, D. D. Khalaf, A. I. Elshamy, and A. E. N. G. El-gendy, “Antimicrobial effects of essential oils of Artemisia annua, Mentha longifolia, and Vitex agnus-castus and their nanoemulsions against pathogenic microbes causing cattle mastitis,” Egypt. J. Chem., vol. 66, no. 3, pp. 483–493, 2023.
22. R. Soni, G. Shankar, P. Mukhopadhyay, and V. Gupta, “A concise review on Artemisia annua L.: A major source of diverse medicinal compounds,” Ind. Crops Prod., vol. 184, p. 115072, 2022.
23. A. Septembre-Malaterre et al., “Artemisia annua, a traditional plant brought to light,” Int. J. Mol. Sci., vol. 21, no. 14, p. 4986, 2020.
24. J. Zhu et al., “Four New Compounds Obtained from Cultured Cells of Artemisia annua,” Molecules, vol. 22, no. 12, p. 2264, 2017.
25. T. Efferth, F. Herrmann, A. Tahrani, and M. Wink, “Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin,” Phytomedicine, vol. 18, no. 11, pp. 959–969, 2011, doi: 10.1016/j.phymed.2011.06.008.
26. A. Nageeb, A. Al-Tawashi, A.-H. Emwas, Z. Al-Talla, and N. Al-Rifai, “Comparison of Artemisia annua Bioactivities between Traditional Medicine and Chemical Extracts,” Curr. Bioact. Compd., vol. 9, no. 4, pp. 324–332, 2014, doi: 10.2174/157340720904140404151439.
27. A.-E. Segneanu, C. N. Marin, I. O.-F. Ghirlea, C. V. I. Feier, C. Muntean, and I. Grozescu, “Artemisia annua growing wild in Romania—A metabolite profile approach to target a drug delivery system based on magnetite nanoparticles,” Plants, vol. 10, no. 11, p. 2245, 2021.
28. J. R. C. Bacong and D. E. O. Juanico, “Predictive Chromatography of Leaf Extracts Through Encoded Environmental Forcing on Phytochemical Synthesis,” Front. Plant Sci., vol. 12, p. 613507, 2021.
29. L. A. Adjeroh, M. O. Nwachukwu, P. N. Abara, J. C. Nnokwe, J. N. Azorji, and I. O. Osinomumu, “Phytochemical Screening and Antibacteria/ Antifungi Activities of Root and Shoot Extracts of Euphorbia hirta (Asthma Weed),” Int. J. Trop. Dis. Heal., vol. 41, no. 6, pp. 1–10, 2020, doi: 10.9734/ijtdh/2020/v41i630281.
30. S. K. Attah, P. F. Ayeh-Kumi, A. A. Sittie, I. V. Oppong, and A. K. Nyarko, “Extracts of Euphorbia hirta Linn. (Euphorbiaceae) and Rauvolfia vomitoria Afzel (Apocynaceae) demonstrate activities against Onchocerca volvulus Microfilariae in vitro,” BMC Complement. Altern. Med., vol. 13, 2013, doi: 10.1186/1472-6882-13-66.
31. I. Haman, N. N. Ngwasiri, A. Adamou, E. D. Abladam, N. Y. Njintang, and D. Ndjonka, “Ethnopharmacology and anthelmintic screening of some plants used in traditional medicine in the Far-North of Cameroon,” Investig. Med. Chem. Pharmacol., vol. 7, no. 2, pp. 1–9, 2024, doi: 10.31183/imcp.2024.00095.
32. M. Baber, S. B. Hussain, and S. Nawazish-i-husain, “i l n e n s r i t i t r e l c i l n e n s r i t,” pp. 1–7, 2025.
33. A. Espinel-Ingroff and T. M. Kerkering, “Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi,” J. Clin. Microbiol., vol. 29, no. 2, pp. 393–394, 1991.
34. S. J. Doddanna, S. Patel, M. A. Sundarrao, and R. S. Veerabhadrappa, “Antimicrobial activity of plant extracts on Candida albicans?: An in vitro study,” pp. 401–405, 2013, doi: 10.4103/0970-9290.118358.
35. C. S. de Almeida et al., “No ????????????????????? ?????????????????Title,” Rev. Bras. Linguística Apl., vol. 5, no. 1, pp. 1689–1699, 2016, [Online]. Available: https://revistas.ufrj.br/index.php/rce/article/download/1659/1508%0Ahttp://hipatiapress.com/hpjournals/index.php/qre/article/view/1348%5Cnhttp://www.tandfonline.com/doi/abs/10.1080/09500799708666915%5Cnhttps://mckinseyonsociety.com/downloads/reports/Educa
36. M. E. M. Makhlof, M. M. El-Sheekh, M. M. Al-Tuwaijri, K. A. El-Tarabily, and A. Elsayed, “In vitro Assessment of Ulva lactuca Mediated Selenium Nanoparticles (USeNPs) through Elevating the Action of Ketoconazole Antibiotic against Pathogenic Yeast Species and Wound Healing Capacity through Inhibition of Cyclooxygenase (Cox-1) Activity,” Egypt. J. Bot., vol. 63, no. 3, pp. 1155–1171, 2023.
37. M. A. M. Mace et al., “3D Printable Alginate–Chitosan Hydrogel Loaded With Ketoconazole Exhibits Anticryptococcal Activity,” Biopolymers, vol. 116, no. 1, p. e23638, 2025.
38. S. Vol, “of Ar ch Ar ch ive,” vol. 24, no. 2, pp. 234–243, 2010.
39. B. B. Yimam and A. Desalew, “Phytochemical screening, antibacterial effect, and essential oil extract from the leaf of Artemisia afra against on selected pathogens,” Adv. Microbiol., vol. 12, no. 7, pp. 386–397, 2022.
40. J. R. Pillai, A. F. Wali, A. M. Al-Azzawi, R. Akhter, H. A. El-Serehy, and I. Akbar, “Phytochemical analysis and antimicrobial activity of Enicostemma littorale,” J. King Saud Univ. - Sci., vol. 32, no. 8, pp. 3279–3285, 2020, doi: 10.1016/j.jksus.2020.09.011.
41. F. Odutayo, C. Ezeamagu, T. Kabiawu, D. Aina, and G. Mensah-Agyei, “Phytochemical screening and antimicrobial activity of Chromolaena odorata leaf extract against selected microorganisms,” J. Adv. Med. Pharm. Sci., vol. 13, no. 4, pp. 1–9, 2017.
42. H. Kim and D. G. Lee, “Naringin?generated ROS promotes mitochondria?mediated apoptosis in Candida albicans,” IUBMB Life, vol. 73, no. 7, pp. 953–967, 2021.
43. N. M. O’Boyle, M. Banck, C. A. James, C. Morley, T. Vandermeersch, and G. R. Hutchison, “Open Babel: An open chemical toolbox.,” J. Cheminform., vol. 3, p. 33, Oct. 2011, doi: 10.1186/1758-2946-3-33.
44. Y. N. Ruma, M. V Keniya, J. D. A. Tyndall, and B. C. Monk, “Characterisation of Candida parapsilosis CYP51 as a Drug Target Using  Saccharomyces cerevisiae as Host.,” J. fungi (Basel, Switzerland), vol. 8, no. 1, Jan. 2022, doi: 10.3390/jof8010069.
45. S. R. Malwal et al., “A Structural and Bioinformatics Investigation of a Fungal Squalene Synthase and  Comparisons with Other Membrane Proteins.,” ACS omega, vol. 7, no. 26, pp. 22601–22612, Jul. 2022, doi: 10.1021/acsomega.2c01924.
46. G. Escolano-Casado et al., “Copper-functionalized hydroxyapatite nanoparticles to counteract fungal infections,” Surfaces and Interfaces, vol. 62, p. 106179, 2025.
47. K. S. Allemailem, “Aqueous extract of Artemisia annua shows in vitro antimicrobial activity and an in vivo chemopreventive effect in a small-cell lung cancer model,” Plants, vol. 11, no. 23, p. 3341, 2022.
48. X. Zhou, M. Zeng, F. Huang, G. Qin, Z. Song, and F. Liu, “The potential role of plant secondary metabolites on antifungal and immunomodulatory effect,” Appl. Microbiol. Biotechnol., vol. 107, no. 14, pp. 4471–4492, 2023.
49. Ž. Maželien, “Antifungal and Antibacterial Activity of Aqueous and Ethanolic Extracts of Different Rosa rugosa Parts,” 2025.
50. A. S. Johnson, I. B. Obot, and U. S. Ukpong, “Green synthesis of silver nanoparticles using Artemisia annua and Sida acuta leaves extract and their antimicrobial, antioxidant and corrosion inhibition potentials,” J. Mater. Environ. Sci, vol. 5, no. 3, pp. 899–906, 2014.
51. N. Khatoon, Y. Sharma, M. Sardar, and N. Manzoor, “Mode of action and anti-Candida activity of Artemisia annua mediated-synthesized silver nanoparticles,” J. Mycol. Med., vol. 29, no. 3, pp. 201–209, 2019.
52. H. Kim, B. G.-T. K. J. of Ecology, and  undefined 2001, “The antifungal activity of chemical substances from Artemisia annua,” The Ecological Society of Korea.
53. M. Ikram et al., “Antimicrobial activity and phytochemical screening of Artemisia annua L. and Millotus philippensis (Lam.) Mull. Arg. leaves,” Am. J. Agric. Environ. Sci., vol. 15, no. 12, pp. 2437–2441, 2015.
54. J. Lee et al., “The Influence of Solvent Choice on the Extraction of Bioactive Compounds from Asteraceae?: A Comparative Review,” pp. 1–21, 2024.
55. D.-S. Fan et al., “Beyond traditional uses: the multifaceted bioactivities of steroidal saponins from the genus Trillium and their modern applications,” Phytochem. Rev., pp. 1–62, 2025.
56. M. Oniha, A. Eni, O. Akinnola, E. A. Omonigbehin, E. F. Ahuekwe, and J. F. Olorunshola, “In vitro antifungal activity of extracts of Moringa oleifera on phytopathogenic fungi affecting Carica papaya,” Open Access Maced. J. Med. Sci., vol. 9, no. A, pp. 1081–1085, 2021.
57. C. S. Ezeonu, C. Imo, D. I. Agwaranze, A. Iruka, and A. Joseph, “Antifungal effect of aqueous and ethanolic extracts of neem leaves , stem bark and seeds on fungal rot diseases of yam and cocoyam,” Chem. Biol. Technol. Agric., pp. 1–9, 2018, doi: 10.1186/s40538-018-0130-3.
58. N. Dudhipala and A. A. Ay, “Amelioration of ketoconazole in lipid nanoparticles for enhanced antifungal  activity and bioavailability through oral administration for management of fungal infections.,” Chem. Phys. Lipids, vol. 232, p. 104953, Oct. 2020, doi: 10.1016/j.chemphyslip.2020.104953.
59. L. Lajoie, A.-S. Fabiano-Tixier, and F. Chemat, “Water as Green Solvent: Methods of Solubilisation and Extraction of Natural  Products-Past, Present and Future Solutions.,” Pharmaceuticals (Basel)., vol. 15, no. 12, Dec. 2022, doi: 10.3390/ph15121507.
60. C. Hirsch and S. Schildknecht, “In Vitro Research Reproducibility: Keeping Up High Standards.,” Front. Pharmacol., vol. 10, p. 1484, 2019, doi: 10.3389/fphar.2019.01484.
61. E. K. Amadi, “Antifungal activity of herbal plants against Candida albicans,” Int. J. Pharmacol. Clin. Res., vol. 1, no. 1, pp. 35–39, 2019, doi: 10.33545/26647613.2019.v1.i1a.6.
62. A. A. Al-fakih, M. Mansour, and S. Saif, “In vitro Antifungal Activities of Some Plant Extracts against Fungal Pathogens In vitro Antifungal Activities of Some Plant Extracts against Fungal Pathogens Causing Cutaneous Mycoses,” no. November, 2022, doi: 10.12691/ajps-10-1-6.
63. S. Y. Timothy, C. H. Wazis, R. G. Adati, and I. D. Maspalma, “Antifungal activity of aqueous and ethanolic leaf extracts of Cassia alata linn,” J. Appl. Pharm. Sci., vol. 2, no. 7, pp. 182–185, 2012, doi: 10.7324/JAPS.2012.2728.
64. E. Patricia E, C. I. Ogbonna, O. Abigail I, J. O. Ejembi, and O. J Shola, “Evaluation of the Divergent Phytochemicals Contained in Aqueous Leaves Extracts of Artemisia annua L. var chiknensis and Cannabis sativa L. Using HPLC and GC-MS Analytical Protocols,” Acta Sci. Microbiol., vol. 6, no. 5, pp. 07–14, 2023, doi: 10.31080/asmi.2023.06.1241.
65. L. Jazan and A. A. Sinai, “Comparative Phytochemical Study of Artemisia sp . in the Middle East?: A Focus on Antimicrobial Activities and GC-MS Analysis in A .,” 2024.
66. A. Rudzi?ska et al., “Phytochemicals in cancer treatment and cancer prevention—review on epidemiological data and clinical trials,” Nutrients, vol. 15, no. 8, p. 1896, 2023.
67. J. H. Doughari, Phytochemicals: extraction methods, basic structures and mode of action as potential chemotherapeutic agents. INTECH Open Access Publisher Rijeka, Croatia, 2012.
68. J. K. Tsuzuki et al., “Antifungal activity of the extracts and saponins from Sapindus saponaria L.,” An. Acad. Bras. Cienc., vol. 79, pp. 577–583, 2007.
69. A. Pikhtirova, E. Pecka-Kie?b, and F. Zigo, “Antimicrobial activity of saponin-containing plants: review,” J. dairy, Vet. Anim. Res., vol. 12, no. 2, pp. 121–127, 2023.
70. A. O. Olusola, “Katalyst Phytochemical Screening and Antifungal Potentials of Extracts of Entada Africana,” vol. 5, pp. 1–7, 2025.
71. M. E. Bordean et al., “Antibacterial and Phytochemical Screening of Artemisia Species,” Antioxidants, vol. 12, no. 3, pp. 1–17, 2023, doi: 10.3390/antiox12030596.
72. H. Harouak, J. Ibijbijen, and L. Nassiri, “Phytochemical Profile and Best Aqueous Extraction Method Between Two Species from Asteraceae Family that Are Very Effective Against Oral Diseases,” Chem. Africa, vol. 5, no. 3, pp. 543–555, 2022.
73. M. Ivanov et al., “Flavones, flavonols, and glycosylated derivatives—Impact on candida albicans growth and virulence, expression of cdr1 and erg11, cytotoxicity,” Pharmaceuticals, vol. 14, no. 1, p. 27, 2020.
74. A. Begum, S. S. Nagar, R. S. Upendra, and R. Karthik, “Discovery of Novel Antifungal Agents from Polygonatum odoratum Targeting 14-Alpha Demethylase Enzyme for the Treatment of Black Fungus (Mucormycosis),” in 2024 International Conference on Intelligent Systems and Advanced Applications (ICISAA), IEEE, 2024, pp. 1–5.
75. E. Irfan, E. Dilshad, F. Ahmad, F. N. Almajhdi, T. Hussain, and G. Abdi, “Phytoconstituents of Artemisia annua as potential inhibitors of SARS CoV2 main protease?: an in silico study,” pp. 1–19, 2024, doi: 10.1186/s12879-024-09387-w.
76. M. Ivanov et al., “Apigenin: Antifungal activity of flavonoid commonly found in beer and wine,” VII nau?no-stru?ni Simp. sa me?unarodnim u?eš?em" Pivo, Pivar. sirovine i tržište", no. Apstrakti, pp. 45–46, 2024.
77. W. Lee, E.-R. Woo, and D. G. Lee, “Effect of apigenin isolated from Aster yomena against Candida albicans: Apigenin-triggered apoptotic pathway regulated by mitochondrial calcium signaling,” J. Ethnopharmacol., vol. 231, pp. 19–28, 2019.
78. H. Lee, E.-R. Woo, and D. G. Lee, “Apigenin induces cell shrinkage in Candida albicans by membrane perturbation,” FEMS Yeast Res., vol. 18, no. 1, p. foy003, 2018.
79. L. Zhao, L. Jin, and B. Yang, “Diosmetin alleviates S. aureus-induced mastitis by inhibiting SIRT1/GPX4 mediated ferroptosis,” Life Sci., vol. 331, p. 122060, 2023.
80. S. Saha, Prinsa, and S. M. A. Kawsar, “Natural Flavonoids as Primary Amoebic Meningoencephalitis Inhibitor: Virtual Screening, Molecular Docking, MD Simulation, MMPBSA, Density Functional Theory, Principal Component, and Gibbs Free Energy Landscape Analyses,” Chem. Biodivers., vol. 22, no. 4, p. e202402521, 2025.
81. D. Barreca et al., “Citrus flavones: An update on sources, biological functions, and health promoting properties,” Plants, vol. 9, no. 3, p. 288, 2020.
82. S. Singh, P. Gupta, A. Meena, and S. Luqman, “Acacetin, a flavone with diverse therapeutic potential in cancer, inflammation, infections and other metabolic disorders,” Food Chem. Toxicol., vol. 145, p. 111708, 2020.
83. E. M. Kamel, S. I. Othman, H. A. Rudayni, A. A. Allam, and A. M. Lamsabhi, “Multi-pronged molecular insights into flavonoid-mediated inhibition of squalene epoxidase: a pathway to novel therapeutics,” RSC Adv., vol. 15, no. 5, pp. 3829–3848, 2025.
84. Y. An et al., “Construction and evaluation of molecular models: guide and design of novel SE inhibitors,” ACS Med. Chem. Lett., vol. 11, no. 6, pp. 1152–1159, 2020.
85. R. H. Elsayed et al., “Unveiling the Molecular Mechanisms of Squalene Epoxidase Inhibition by Flavonoids from Erythrina speciosa: Integrative Computational and Experimental Insights,” Rev. Bras. Farmacogn., pp. 1–20, 2025.
86. M. Ivanov et al., “Flavones, flavonols, and glycosylated derivatives—impact on candida albicans growth and virulence, expression of cdr1 and erg11, cytotoxicity,” Pharmaceuticals, vol. 14, no. 1, pp. 1–12, 2021, doi: 10.3390/ph14010027.
87. Y. Yang, X. Tang, H. Hu, X. Zhan, X. Zhang, and X. Zhang, “Molecular insight into the binding properties of marine algogenic dissolved organic matter for polybrominated diphenyl ethers and their combined effect on marine zooplankton,” Sci. Total Environ., vol. 921, p. 171131, 2024.
88. M. Zhou et al., “A dual-targeting antifungal is effective against multidrug-resistant human fungal pathogens,” Nat. Microbiol., vol. 9, no. 5, pp. 1325–1339, 2024.
89. H. Isnaini et al., “In silico test of brotowali (Tinospora crispa) as potential anticancer agent targeting mTOR on colorectal cancer,” Menara Perkeb., vol. 93, no. 1, 2025.
90. K. Bhatkhande, N. N. Vysyaraju, R. R. Alavala, and K. M. Gokhale, “Recent Advances in Drug-Likeness Screening by Using the Software and Online Tools,” Appl. Comput. Tools Drug Des. Dev., pp. 683–717, 2025.
91. Y. Zhang et al., “Antifungal effect of volatile organic compounds produced by Pseudomonas chlororaphis subsp. aureofaciens SPS-41 on oxidative stress and mitochondrial dysfunction of Ceratocystis fimbriata,” Pestic. Biochem. Physiol., vol. 173, p. 104777, 2021.
92. S. Kim et al., “PubChem in 2021: new data content and improved web interfaces.,” Nucleic Acids Res., vol. 49, no. D1, pp. D1388–D1395, Jan. 2021, doi: 10.1093/nar/gkaa971.