In 2019 alone, the economic burden of fungal disease-related illnesses and deaths in the United States was estimated to exceed USD 24.3 billion (Kumar et al., 2022; Benedict et al., 2022). Invasive candidiasis and aspergillosis accounted for 30 % of this cost, while noninvasive candidiasis, a widespread infection, also contributed a significantly (Benedict et al., 2022).

Invasive candidiasis is a severe fungal infection that can affect various organs, often occurring in immunocompromised patients with weakened host defense mechanisms (Lass-Flörl et al., 2024). The presence of invasive candidiasis is associated with a high mortality rates, with an attributable death rate of 49 % which can rise to 98 % in patients with septic shock and delayed antifungal treatment (Thomas-Rüddel et al., 2022). The most critical form of invasive candidiasis, candidemia, represents a major cause hospital-acquired bloodstream infections, surpassing some bacterial pathogens, such as Pseudomonas aeruginosa (Timsit et al., 2020). Candida species are among the most common and serious infectious agents in intensive care units (IUCs), with candidemia cases rank fifth in IUCs in the United States and Europe, highlighting the substantial threat posed by Candida infections (Selvan et al., 2022). While Candida albicans remains the most prevalent causative species, infections caused by multidrug-resistant non-Candida albicans species, including C. parapsilosis, C. tropicalis, C. glabrata (Nakaseomyces glabratus), C. krusei and C. auris, are increasingly concerning (Mamali et al., 2022; Pfaller et al., 2019; Pappas et al., 2018). The incidence of non-Candida albicans species has been rising globally (Pappas et al., 2018; Vallabhaneni et al., 2015; Lortholary et al., 2011). Furthermore, according to the 2019 Antibiotic Resistance Threats in the United States report by the CDC (Centers for Disease Control and Prevention), drug-resistant Candida species were responsible for 34,800 infections and 1,700 deaths (Pianalto and Alspaugh, 2016; Mota Fernandes et al., 2021).

Beyond drug resistance, existing antifungal agents—including polyenes (e.g., amphotericin B), triazoles (e.g., fluconazole), and echinocandins (e.g., caspofungin)—exhibit high toxicity and a narrow therapeutic index. Their limited oral bioavailability significantly restricts treatment efficacy (Hasim and Coleman, 2019; Gómez-López, 2020). Broad-spectrum triazoles, such as posaconazole and voriconazole, face further limitations due to drug-drug interactions, variable bioavailability, acute adverse events, and emerging resistance (Miceli and Kauffman, 2015).

In addition to systemic antifungals, topical imidazole derivatives such as clotrimazole remain a mainstay for superficial candidiasis. Clotrimazole exerts fungistatic and fungicidal effects via inhibition of ergosterol biosynthesis, yet it presents notable limitations including poor systemic bioavailability, limited tissue penetration, and the need for frequent application, which can compromise patient adherence (Ivanov et al., 2022; Zhou et al., 2016). Although resistance is relatively rare, emerging cases—particularly involving non-Candida albicans species—have been reported, often associated with efflux pump overexpression and ERG11 mutations (Mendling et al., 2020; Keshwania et al., 2023). Local adverse effects such as burning and irritation may also reduce tolerability (Hajizadeh et al., 2025). While clotrimazole remains a valuable agent for localized infections, its pharmacokinetic constraints and formulation-dependent efficacy necessitate cautious use in complicated or recurrent cases (Mendling et al., 2020; Kaur and Kakkar, 2010). These challenges highlight the urgent need for novel antifungal agents with improved efficacy and safety profiles.

Hypericum empetrifolium subsp. empetrifolium Willd. (Hypericaceae) belongs to the section Coridium, which comprises six species (Robson, 1981; Royal Botanic Gardens, Kew, 2025). This shrub thrives in rocky terrains and is widely distributed at low altitudes throughout the Aegean region, particularly in southern mainland Greece and the coastal areas of western Türkiye (Trovato et al., 2001). In Türkiye, H. empetrifolium subsp. empetrifolium is locally known as “sarı piren” or “piren” (“sarı” meaning yellow), and its flower decoctions have been traditionally used for dyeing cloth in western Anatolia (Baytop, 1999). Additionally, the species has been reported to possess significant medicinal properties, including applications for kidney stones and gastric ulcers (Crockett et al., 2008), as well as for treating burn wounds. It also exhibits antispasmodic, laxative, anthelmintic, and antiseptic effects (Ozturk et al., 2013).

Our previous research, as part of an ongoing project investigating plant-derived antimicrobial natural products from the genus Hypericum, demonstrated that the ethanolic extract of the aerial parts of H. empetrifolium exhibited antifungal activity against C. parapsilosis (MIC = 4.88 μ g/mL), C. tropicalis (MIC = 19.53 μ g/mL), and C. albicans (MIC = 78.12 μ g/mL). Phytochemical analysis revealed that the ethanolic extract was rich in chlorogenic acid, isoquercitrin, malic acid, protocatechuic acid, quercetin, and fumaric acid. Additionally, several phenolic metabolites, including salicylic acid, caffeic acid, p-coumaric acid, rutin, nicotiflorin, rosmarinic acid, naringenin, apigenin, and vanillin, were identified in lower concentrations (Bo et al., 2021).

Apart from our previous findings, other research groups have also reported various bioactivities of H. empetrifolium. Couladis et al. reported the methanolic extract of the plant having cytotoxic effect in human colon carcinoma and human hepatoma cell lines (Couladis et al., 2002). Trovato and colleagues demonstrated the methanolic extract of the plant’s aerial parts exhibited a significant anti-inflammatory activity and analgesic effects in vivo writhing test (Trovato et al., 2001). Subsequently, Crockett et al. isolated two acylphloroglucinols with moderate to potent in vitro activity against COX-1, COX-2, and 5-LOX (Crockett et al., 2008). Schmidt et al. isolated phloroglucinol derivatives from the petroleum ether extract of the plant, which exhibited in vitro antiproliferative activity against human microvascular endothelial cells (HMEC-1) (Schmidt et al., 2012c; b). Additionally, phytochemical investigations of methanolic extract of Jordanian H. empetrifolium identified hypericin and hyperforin (Tawaha et al., 2010) as well as pseudohypericin, protohypericin, and adhyperfirin (Alali et al., 2009).

In this study, three samples of H. empetrifolium subsp. empetrifolium (sect. Coridium) were collected from different regions in Türkiye and their methanolic extracts were analyzed using HPLC-DAD and LC-HR/MS, respectively. Given that certain key metabolites, such as naphthodianthrones and phloroglucinols, are highly lipophilic and exhibit greater solubility in organic alcohols, methanol was selected as the extraction solvent, as previously recommended (Zeliou et al., 2020). The total phenolic and flavonoid content, antioxidant capacity (i.e., ABTS, DPPH, and CUPRAC). Additionally, antimicrobial activity was evaluated using the microdilution method to determine the antimicrobial potential of H. empetrifolium subsp. empetrifolium against standard yeast, Gram (−), and Gram (+) bacterial strains, and enzyme inhibitory activities, including anti-acetylcholinesterase, anti-butyrylcholinesterase, anti- α -Glucosidase, anti-tyrosinase, and anti-aging (anti-hyaluronidase and anti-elastase) activities, were assessed using ELISA reader-based methods. This study aimed to compare the metabolic profiles of H. empetrifolium subsp. empetrifolium growing in the wild habitats and conditions, along with their antimicrobial and other biological activities.

This study was conducted to address the urgent need for safer and more effective antifungal agents by investigating the phytochemical richness and bioactivity of H. empetrifolium subsp. empetrifolium collected from different ecological regions of Türkiye. As highlighted above, current antifungal treatments are constrained by toxicity, resistance, and poor bioavailability. In contrast, the demonstrated antifungal and enzyme inhibitory properties of the extracts underscore the therapeutic potential of this species. Notably, H. empetrifolium subsp. empetrifolium has received limited attention in the current literature. Therefore, this research not only expands the phytochemical and pharmacological knowledge of this underexplored species but also fills a critical gap in the literature by linking its diverse bioactivities with ecological variation.

Fungal infections represent a major public health challenge, with mortality rates comparable to malaria, tuberculosis, or HIV (Gow et al., 2022). Recent epidemiological studies have documented a rising incidence of candidemia in ICU settings, underscoring the need for novel antifungal therapies (Eun et al., 2020; Zhong et al., 2022). Hypericum species have attracted attention due to their antimicrobial properties, particularly against Gram (+) bacteria, and certain species have demonstrated significant antifungal properties certain fungal pathogens (Tavlı, 2024).

For example, among seven Hypericum species from the And Mountains, Hypericum garciae exhibited outstanding antifungal activity against Candida strains; its methanol extract achieved MIC 50 values of 5.04 μ g/mL against C. albicans and 4 μ g/mL against C. lusitaniae, while its chloroform extract outperformed fluconazole against C. tropicalis ( MIC 50 = 39.19 μ g/mL), likely reflecting its high content of quercetin-3-glucuronide, procyanidin B2, and epicatechin (Tocci et al., 2018b). Similarly, Hypericum hircinum subsp. majus extracts—prepared in methanol, 80 % ethanol, and water—demonstrated significant activity against both fluconazole-sensitive and -resistant Candida strains, with the methanol extract showing an MIC 50 of 53.5 μ g/mL against C. parapsilosis and notable effects against C. tropicalis, C. glabrata, C. albicans, and C. lusitaniae (Tocci et al., 2018a).

Synergistic effects have also been reported; the lipophilic fraction of Hypericum carinatum from Brazil, when combined with fluconazole, reduced MIC values by up to eight-fold against C. krusei and C. famata, although fluconazole alone was more effective against C. parapsilosis and C. neoformans (Meirelles et al., 2017). Additionally, n-hexane extracts from five Hypericum species collected in Brazil showed potent antifungal effects against opportunistic yeasts such as Cryptococcus neoformans (MIC ≤ 15.6 μ g/mL) and Rhodotorula mucilaginosa (MIC ≤ 62.5 μ g/mL), with Hypericum myrianthum notably rich in dimeric phloroglucinol derivatives like uliginosin B and japonisin A (Barros et al., 2013).

Methanol extracts from Hypericum humifusum and Hypericum perfoliatum, collected in Tunisia, were effective against C. albicans (MIC = 250 μ g/mL), with H. humifusum containing significant levels of hypericin (90 mg/g) and hyperforin (30 mg/g) (Bejaoui et al., 2017). In addition, 50 % ethanol extracts from the leaves and roots of Hypericum havvae demonstrated strong antifungal activity against several yeast strains, particularly when combined, achieving MIC values as low as 1.56 mg/mL against C. albicans and C. laurentii (Dulger and Dulger, 2016). Lastly, methanol extracts from six Hypericum species cultivated in Türkiye confirmed antifungal efficacy, with diethyl ether and chloroform extracts of Hypericum spectabile showing activity against C. albicans ATCC 10231 (MIC = 156 μ g/mL) (Ero et al., 2018b).

Our previous results indicated that the ethanolic extract obtained from the aerial parts of H. empetrifolium subsp. empetrifolium exhibited significant antifungal activity, with MIC values of 4.88 μ g/mL against C. parapsilosis, 19.53 μ g/mL against C. tropicalis, and 78.12 μ g/mL against C. albicans (Bo et al., 2021). In the present study, methanolic extracts prepared from H. empetrifolium subsp. empetrifolium samples collected from different geographical locations also exhibited anti-fungal activity against the same heast strains with the IC₅₀ values ranging from 78.12 to 156.2 μ g/mL. Their effectiveness was comparable to standard antifungal agents used in clinical practice. However, the extracts showed no significant activity against Gram (−) bacteria, which is consistent with existing literature. Interestingly, weak inhibitory activity was observed against the tested Gram (+) bacterial strains (Figure 1).

These results were not as robust as those observed in our previous study. This discrepancy may be attributed to extractant solvent differences or variations in the chemical composition of the plants, which can be influenced by differences in their growth conditions.

Although both ethanol and methanol are polar solvents commonly used in phytochemical research, their slight differences in polarity and solvent strength can lead to distinct extraction efficiencies for certain classes of secondary metabolites. Methanol, being slightly more polar than ethanol, may enhance the extraction of certain phenolic acids and low-molecular-weight flavonoids, whereas ethanol might favor more lipophilic or mid-polar metabolites (Chaves et al., 2020). In a comparative study on Hypericum perforatum, Alahmad et al. (2022) observed that while the overall compound profiles were similar across different solvents, water tended to yield higher concentrations of certain phenolic constituents compared to methanol and ethanol (Alahmad et al., 2022). On the other hand, various phloroglucinol derivatives, which have low water solubility, are known for their potent antimicrobial properties (Tavlı, 2024). This suggests that methanol may offer advantages in extracting specific bioactive metabolites, owing to its ability to extract both phenolics and phloroglucinol derivatives effectively.

This variation in solvent polarity likely contributed to the observed differences in antifungal activity between the two studies. Additionally, the chemical composition of the extracts may have been influenced not only by the choice of solvent, but also by ecological and geographical differences in the plant material, including factors such as altitude, climate, and soil conditions. These environmental variables are known to affect the biosynthesis and accumulation of bioactive metabolites in medicinal plants and could therefore play a significant role in the observed phytochemical variation and corresponding biological activities (Çirak et al., 2006; Çirak and Radusiene, 2019; Şenkal and Uskutoglu, 2021; Bálintová et al., 2019; Kurt et al., 2018; Kucharíková et al., 2016; Kladar et al., 2015; Ghasemi Pirbalouti et al., 2011; Hosni et al., 2011; Çırak et al., 2011; Maggi et al., 2010; AI-Rifaee et al., 2010; Toker, 2009; Bagdonaite et al., 2009; Çirak et al., 2008; Mártonfi et al., 2006; Božin et al., 2013).

Phytochemical analysis revealed that the ethanolic extract of H. empetrifolium subsp. empetrifolium was particularly rich in chlorogenic acid, isoquercitrin, malic acid, proto-catechuic acid, quercetin, and fumaric acid. Additionally, several other phenolic metabolites—including salicylic acid, caffeic acid, p-coumaric acid, rutin, nicotiflorin, rosmarinic acid, naringenin, apigenin, and vanillin—were identified at lower concentrations.

Similarly, chemical examination of the H. empetrifolium subsp. empetrifolium extracts revealed marked differences in their phytochemical profiles. For instance, while ascorbic acid levels were relatively similar among the samples notable variations were observed for several key flavonoids and phenolic acids. HE-3 exhibited a substantially higher concentration of (−)-epigallocatechin (285.744 ± 8.83 μ g/mL) compared to HE-1 (161.37 ± 4.986 μ g/mL) and HE-2 (74.253 ± 2.294 μ g/mL), suggesting a stronger potential for antioxidant effect. Similarly, hyperoside was present in much higher amounts in HE-3 (1.624.24 ± 56.199 μ g/mL) relative to HE-1 (150.715 ± 5.215 μ g/mL) and HE-2 (223.164 ± 7.722 μ g/mL), whereas rutin levels were considerably higher in HE-1 and HE-2 (10.009.532 ± 307.293, 11.444.962 ± 351.36 μ g/mL, respectively) than in HE-3 (648.337 ± 19.904 μ g/mL). In contrast, quercitrin was detected at an exceptionally high level in HE-3 (2.997.251 ± 113.296 μ g/mL) compared to low concentrations in HE-1 and HE-2 (5.84 ± 0.221, 6.236 ± 236 μ g/mL, respectively). Other metabolites, such as chlorogenic acid and fumaric acid, displayed moderate variability across the extracts, while some metabolites like dihydrokaempferol and certain flavonoids (e.g., chrysin, 3′-O-methyl quercetin) were either absent or present only in trace amounts in specific samples.

Reactive oxygen species (ROS), including free radicals such as superoxide ( O 2 • ), hydroxyl (HO • ), peroxyl (LOO • ), hydroperoxyl (HOO • ), and non-radical molecules like hydrogen peroxide ( H 2 O 2 ), are generated as by-products of mitochondrial respiration, enzymatic reactions, and exposure to both internal and external factors such as inflammation, exercise, ischemia-reperfusion injury, metal ions, respiratory burst, cigarette smoke, and industrial solvents (Foret et al., 2020; Neha et al., 2019). Although cells continuously produce ROS as signaling molecules involved in immune response, cell signaling, wound healing, and pathogen defense (Pandey et al., 2021; Schieber and Navdeep, 2014; Sies, 2018), excessive accumulation or inadequate antioxidant defense systems lead to oxidative stress—a key contributor to the pathogenesis of various diseases including cancer, cardiovascular diseases, neurodegenerative disorders, diabetes, rheumatoid arthritis, kidney, and ocular diseases (Foret et al., 2020; Pisoschi et al., 2021; Saddiqe et al., 2014; Pandey et al., 2021). ROS-induced oxidative damage to proteins, lipids, and nucleic acids may result in cell death, inflammation, LDL oxidation, or impaired insulin signaling, depending on the disease context. Plants have evolved complex antioxidant defense systems, including enzymatic and non-enzymatic metabolites such as ascorbic acid, glutathione, polyphenols, flavonoids, and terpenes (Llauradó Maury et al., 2020). While synthetic antioxidants like BHA, BHT, and propyl gallate have been widely used, their long-term safety has raised concerns due to associations with allergies, gastrointestinal issues, carcinogenic potential, and DNA damage in high doses (Lourenço et al., 2019). Consequently, there is a growing interest in plant-derived antioxidants as safer alternatives for applications in food, pharmaceuticals, and cosmetics (Pammi et al., 2022).

The radical scavenging capacities of the extracts were assessed using three distinct chemical methods.The results were as follows: maximum inhibition values of 82.60 % in the DPPH assay, 88.53 % in the ABTS assay, and an absorbance of 2.103 in the CUPRAC assay, all determined at a concentration of 100 μ g/mL. Notably, HE-1 showed the highest radical scavenging capacity in the ABTS assay, clearly outperforming the other samples. In contrast, HE-2 displayed slightly better free radical scavenging in the DPPH assay (82.60 % at 100 μ g/mL) compared to HE-1, while HE-3 demonstrated the most potent reducing power in the CUPRAC assay (Abs = 1.712 at 100 μ g/mL) (Figure 2). These differences likely reflect the variations in their phenolic and flavonoid contents, underlining the importance of phytochemical composition in determining antioxidant efficacy. It is important to note that these results are based solely on chemical assays and do not imply any pharmacological efficacy. Such methods are valuable analytical tools for characterizing antioxidant potential but cannot be equated with biological activity. Therefore, the findings should be interpreted as indicators of radical scavenging capacity rather than evidence of in vitro or in vivo antioxidant effects.

To date, different investigations in literature have not only highlighted differences among species but also revealed variations in the antioxidant potential of the same species (particularly H. perforatum) sourced from different regions. Among of them, a study showed that among three Hypericum species grown in Türkiye—Hypericum aviculariifolium subsp. depilatum var. depilatum, Hypericum salsugineum, and H. perforatum—the methanolic extracts exhibited DPPH radical scavenging capacity of 88.29 % , 86.88 % , and 81.21 % , respectively, at a concentration of 0.5 mg/mL (Maltaş et al., 2013). In the Balkans, H. perforatum has been reported to possess a DPPH free radical scavenging capacity with IC₅₀ value of 1.36 ± 0.05 μ g/mL (Božin et al., 2013), while in Greece, H. perforatum (DPPH; IC₅₀ = 10.45 ± 0.61 μ g/mL), Hypericum delphicum (DPPH; IC₅₀ = 12.98 ± 1.09 μ g/mL), and Hypericum olympicum (ABTS; IC₅₀ = 3.92 ± 0.73 μ g/mL) have demonstrated notable antioxidant effects (Kakouri et al., 2023). Moreover, a study from Bulgaria evaluating the antioxidant effects of H. olympicum and H. perforatum found that H. perforatum, which had the highest total tannin content (8.67 ± 0.02 g pyrogallol equivalents/100 g), exhibited radical scavenging capacities of 77.6 ± 0.5 % in the DPPH assay and 81.2 ± 0.4 % in the ABTS assay. In contrast, H. olympicum, despite its lower total flavonoid content (0.20 ± 0.03 g hyperoside equivalents/100 g), showed a strong total antioxidant effect of 89.9 ± 0.2 μ M Trolox equivalents/g (Zheleva-Dimitrova et al., 2010). The chemical tests assessing radical scavenging capacity results obtained in the present study are in agreement with these literature findings.

Studies on cholinesterase inhibitors have demonstrated that, beyond their established efficacy in Alzheimer’s disease, these agents may also exert potential effects on mood disorders related to depression and stress. Consequently, cholinesterase inhibitors are being considered for their potential role in the treatment of depression, in addition to neurodegenerative conditions (Fitzgerald et al., 2020). Based on the anticholinesterase activity assays results, H. empetrifolium subsp. empetrifolium extracts—especially HE-1—demonstrate promising potential for the treatment of these conditions. In the present study, the cholinesterase inhibitory activities of the extracts were determined as IC₅₀ values ranging from 8.16 ± 0.39 to 42.09 ± 1.48 μ g/mL for acetylcholinesterase (AChE) and from 2.46 ± 0.02 to 26.42 ± 0.86 μ g/mL for butyrylcholinesterase (BChE) (Figure 3). Notably, HE-1 exhibited the most potent inhibitory effects with an AChE IC₅₀ of 8.16 ± 0.39 μ g/mL and a BChE IC₅₀ of 2.46 ± 0.02 μ g/mL, outperforming the standard galantamine in BChE inhibition (galantamine: AChE, 8.53 ± 0.20 μ g/mL; BChE, 38.66 ± 0.49 μ g/mL). In contrast, HE-2 and HE-3 demonstrated relatively weaker activities. Not only present results but also our previous findings (Methanolic extract of H. empetrifolium subsp. empetrifolium inhibited 38.89 ± 1.07 % AChE, and 88.69 ±   0.62 % BChE at 200 μ g/mL concentration (Bo et al., 2021)) suggesting that variations in their phytochemical profiles may significantly influence their cholinesterase inhibitory potential.

On the other hands, various studies have explored the cholinesterase inhibitory effects of Hypericum species, shedding light on their potential as natural anticholinesterase agents. Bozin and colleagues evaluated the anti-acetylcholinesterase activity of extracts of H. perforatum, Hypericum maculatum subsp. immaculatum, H. olympicum, Hypericum richeri subsp. grise-bachii, and Hypericum barbatum, reporting that the H. perforatum extract exhibited the highest activity (IC₅₀ = 432.74 μ g/mL), which was attributed to its high hyperforin content (Božin et al., 2013). In another study demonstrated that methanolic extracts prepared from the aerial parts of Hypericum neurocalycinum and Hypericum malatyanum possessed notable anti-acetylcholinesterase effects, with EC₅₀ values of 2.16 mg/mL and 6.83 mg/mL, respectively, suggesting that flavonoids such as quercetin, kaempferol, and rutin might be responsible for this activity (Ero et al., 2018a). Furthermore, Ersoy and coworkers investigated the anti-cholinesterase activities of methanolic extracts from Hypericum calycinum, Hypericum confertum, and H. perforatum at a concentration of 200 μ g/mL, finding that H. calycinum exhibited the highest acetylcholinesterase inhibition (45.33 % ), while H. perforatum showed the strongest butyrylcholinesterase inhibition (82.50 % ). These extracts were rich in phenolic metabolites including chlorogenic acid, hyperoside, quercetin, rutin, and isoquercitrin (Ersoy et al., 2019). Additionally, a decoction prepared from Hypericum androsaemum, Hypericum undulatum, and H. perforatum displayed potent anti-acetylcholinesterase activity, with IC₅₀ values ranging from 0.62 ± 0.06 to 1.79 ± 0.37 μ g/mL. The major constituents of these extracts—chlorogenic acid, rutin, hyperoside, isoquercitrin, and quercetin—exhibited individual IC₅₀ values between 196 and 62 μ g/mL (Hernandez et al., 2010), further supporting the hypothesis that the anti-cholinesterase effects of Hypericum extracts result from the synergistic interactions among their bioactive metabolites.

The investigation of tyrosinase enzyme inhibitors is of considerable importance for the discovery of novel therapeutic agents targeting a variety of health issues. These inhibitors effectively suppress the activity of tyrosinase—an enzyme critical for melanin synthesis—thereby offering potential benefits against hyperpigmentation and skin discoloration. In the cosmetic industry, such compounds are widely incorporated into skin whitening and spot treatment products, as they play a pivotal role in reducing melanin production and treating hyperpigmentation disorders. Moreover, the oxidative stress-related effects associated with tyrosinase are currently under investigation for their implications in neurodegenerative diseases, particularly in conditions such as Parkinson’s disease (Baber et al., 2023; Fernandes and Kerkar, 2017). Previous studies have demonstrated that extracts from various Hypericum species exhibit significant antityrosinase activity. An extract prepared from H. androsaemum fruits—rich in phenolic metabolites such as shikimic acid and chlorogenic acid—showed notable tyrosinase inhibition (IC₅₀ = 229,1 μ g/mL) (López et al., 2016). Similarly, an extract obtained from the aerial parts of H. calycinum, enriched in chlorogenic acid, quercitrin, quinic acid, and isoquercitrin, also displayed potent antityrosinase activity (54,30 ± 0,49 % in 200 μ g/mL concentration) (Ersoy et al., 2020). Moreover, an extract of Hypericum laricifolium containing protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, kaempferol 3-O-glucuronide, quercetin, and kaempferol was reported to possess high antityrosinase effects (GuillenQuispe et al., 2017). Additionally, numerous metabolites have been evaluated for their tyrosinase inhibitory activity, with studies indicating that both simple phenolic metabolites and their polyphenolic derivatives exhibit strong antityrosinase activity (S et al., 2019).

In the present study, the tyrosinase inhibitory potential of H. empetrifolium subsp. empetrifolium extracts was further evaluated. These findings suggest that although H. empetrifoliumextracts possess antityrosinase activity, their inhibitory potency is substantially lower than that of kojic acid (Figure 4), similar to previous work (Bo et al., 2021).

α -Glucosidase inhibitors play a critical role in the management of type 2 diabetes, particularly in controlling postprandial blood glucose levels. These inhibitors slow the conversion of complex carbohydrates into simple sugars, thereby delaying glucose absorption and contributing to more stable blood sugar levels. Consequently, they hold significant potential for reducing the long-term complications associated with diabetes (Dirir et al., 2022b).

H.ypericumascyron extracts prepared with ethyl acetate and methanol inhibited α -Glucosidase with IC₅₀ values of 755.8 μ g/mL and 151.47 μ g/mL, respectively, while isolated constituents such as kaempferol and ursolic acid were particularly potent, with ursolic acid exhibiting an IC₅₀ of 1.78 μ M and kaempferol showing values of 61.83 μ M (Kang et al., 2011). In addition, studies on Hypericum attenuatum have demonstrated significant antidiabetic effects both in vitro and in vivo. A phenolic-rich extract improved hyperglycemia, dyslipidemia, and insulin resistance in KK-Ay mice, with observed benefits on hepatic steatosis and preservation of pancreatic β -cells. Moreover, an ethanol extract of H. attenuatum yielded isolated flavonoids—such as astragalin, guaijaverin, and quercetin—with IC 50 values of 33.90 ± 0.68 μ M, 17.23 ± 0.75 μ M, and 31.90 ± 0.34 μ M, respectively. These metabolites not only directly inhibited α -Glucosidase but also induced conformational changes in the enzyme, as demonstrated by circular dichroism and molecular docking studies, with certain combinations displaying synergistic inhibitory effects (Jin et al., 2021). Further supporting these findings, H. laricifolium methanol extracts exhibited strong α -Glucosidase inhibition ( IC 50 = 56.6 μ g/mL) alongside significant aldo reductase and antioxidant capacities. In this extract, quercetin and kaempferol were identified as major contributors, with quercetin showing an IC 50 value of 15.9 μ M for α -Glucosidase and kaempferol at 9.7 μ M (YN et al., 2017). Similarly, H. olympicum, after in vitro digestion simulation, demonstrated enhanced inhibition of both α -Glucosidase and α -amylase, with maximum inhibitory activities of 40.28 % and 89.11 % , respectively, and a notable suppression of advanced glycation end products (AGEs) formation (Akyuz et al., 2021). Collectively, these studies underscore the promise of Hypericum species as sources of α -Glucosidase inhibitors. Their diverse bioactive metabolites, particularly phenolics and flavonoids, not only contribute to direct enzyme inhibition but also may synergistically improve metabolic profiles, paving the way for the development of novel antidiabetic therapies.

Our findings are particularly noteworthy when compared with the existing literature, where various Hypericum species have also been reported to exhibit α -Glucosidase inhibition, albeit with less pronounced potency. The exceptional activity observed in our study suggests that the bioactive constituents within H. empetrifolium subsp. empetrifolium may offer significant therapeutic potential as novel antidiabetic agents (Figure 5). Given that acarbose is widely used in clinical settings yet has certain limitations, the potent inhibitory effects of these extracts highlight their promise as alternative or complementary treatments with the possibility of improved efficacy and reduced side effects.

Investigating inhibitors of extracellular matrix (ECM) degrading enzymes—such as collagenase, elastase, and hyaluronidase—is critical for developing effective anti-aging cosmeceuticals. These enzymes play a pivotal role in ECM remodeling; their overactivity contributes to the degradation of collagen, elastin, and hyaluronic acid, leading to loss of skin firmness, increased wrinkle formation, and other visible signs of aging. By targeting these enzymes, natural compounds can help preserve the skin’ structural integrity and mitigate age-related changes (Silva et al., 2021).

Studies on various Hypericum species have demonstrated promising inhibitory effects on these ECM-degrading enzymes, suggesting that their bioactive constituents could serve as safer and more effective alternatives to synthetic inhibitors in cosmetic formulations. This line of research is therefore essential not only for understanding the mechanisms behind skin aging but also for the development of innovative treatments that improve skin health and appearance. In studies assessing the anti-aging properties of Hypericum extracts from H. perforatum, H. calycinum, and H. confertum. H. calycinum consistently demonstrated the most potent activity. Its methanol extracts inhibited collagenase, elastase, and hyaluronidase with IC 50 values of 51.24, 55.77, and 22.17 μ g/mL, respectively. LC-MS/MS analysis revealed that H. calycinum was richer in key bioactive metabolites—such as chlorogenic acid, quercitrin, quinic acid, and isoquercitrin—potentially explaining its superior performance (Ersoy et al., 2019). In addition, H. androsaemum fruit extracts promoted fibroblast migration, non-competitively inhibited collagenase, and modulated IL-6 production in PBMCs, indicating promising applications in skin care (Antognoni et al., 2017). Extracts from H. hircinum, Hypericum origanifolium, and Hypericum lydium further exhibited notable elastase and collagenase inhibition (Mandrone et al., 2015). Together, these findings underscore the potential of Hypericum extracts, particularly from H. calycinum, as effective agents in combating skin aging, hyperpigmentation, and promoting tissue repair.

The results of present study offer valuable insights into the enzyme inhibitory properties of H. empetrifolium subsp. empetrifolium extracts. Notably, no measurable inhibition of hyaluronidase was observed for HE-1, HE-2, and HE-3 under the tested conditions. This suggests that the bioactive constituents in the HE extracts may lack the necessary affinity or concentration to effectively inhibit hyaluronidase, an enzyme critical for hyaluronic acid degradation.

In contrast, the HE extracts demonstrated moderate elastase inhibitory activity, with IC 50 values of 17.04 ± 0.18 μ g/mL, 17.12 ± 0.14 μ g/mL, and 21.79 ± 0.84 μ g/mL for HE-1, HE-2, and HE-3, respectively. However, these values were slightly higher than that of the standard inhibitor ursolic acid, which exhibited an IC 50 of 13.77 ± 0.17 μ g/mL. The moderate elastase inhibition observed suggests that while the extracts contain metabolites capable of modulating elastase activity, they are less potent compared to ursolic acid. This differential enzyme inhibitory profile may be attributed to the specific chemical composition of the extracts and their selective interactions with distinct enzyme active sites (Figure 6).

Although this study does not include primary ethnobotanical fieldwork, the selection of H. empetrifolium subsp. empetrifolium was based on its reported traditional use in the literature. The work aligns with the core principles of the Four Pillars of Best Practice in Ethnopharmacology (Heinrich et al., 2020), including proper taxonomic identification (with voucher specimen), use of pharmacologically relevant in vitro models, detailed phytochemical profiling, and transparent data reporting. The antifungal and enzyme inhibition assays were selected to reflect plausible therapeutic mechanisms relevant to the species’ ethnomedical context.

In conclusion, our study demonstrates that H. empetrifolium subsp. empetrifolium extracts exhibit significant antifungal activity against clinically relevant Candida strains, underscoring their potential as alternative therapeutic agents for fungal infections, their robust antifungal efficacy aligns with the pressing need for novel, effective antifungal agents.

Moreover, the extracts displayed potent α -Glucosidase inhibitory activity indicating promising antidiabetic potential. Importantly, the anti-cholinesterase assays revealed that the extracts, particularly HE-1, possess strong inhibitory effects against both acetylcholinesterase and butyrylcholinesterase, comparable to or better than standard inhibitors, thereby highlighting their potential in addressing neurodegenerative and mood-related disorders.

Phytochemical analyses via HPLC-DAD and LC-HR/MS further revealed notable variations in the profiles of key antioxidants, flavonoids, and phenolic metabolites among the samples. These differences, likely resulting from variations in collection locations and environmental conditions, emphasize the impact of growth conditions on the chemical composition and bioactivity of H. empetrifolium subsp. empetrifolium. Although lipophilic metabolites such as hypericin, pseudohypericin, and hyperforin were not detected, the presence of other bioactive phenolic constituents appears to underpin the diverse therapeutic properties observed.

Building upon these promising findings, future research should focus on the targeted isolation and structural elucidation of the most active constituents within H. empetrifolium subsp. empetrifolium extracts, employing advanced spectroscopic and chromatographic techniques. Moreover, standardized extraction protocols must be developed to ensure reproducibility and consistency in bioactivity, which are essential for translational applications. Given the demonstrated in vitro efficacy, comprehensive in vivo studies are crucial to assess pharmacokinetics, bioavailability, and potential toxicity profiles. In parallel, mechanistic investigations at the molecular level—particularly concerning enzyme inhibition and antifungal pathways—would provide deeper insights into the modes of action.

On the other hands, the notable chemical variations observed among samples collected from different geographical locations strongly suggest that environmental conditions significantly influence the phytochemical composition and, consequently, the bioactivity of H. empetrifolium subsp. empetrifolium. Therefore, future studies should include broader sampling from diverse habitats to identify the most favorable ecological conditions or specific locations associated with the highest concentration of bioactive metabolites. This approach would not only optimize raw material sourcing but also support conservation strategies and sustainable use of medicinal plant resources. Finally, the formulation of these extracts into biocompatible delivery systems could pave the way for novel phytopharmaceuticals and cosmeceuticals targeting fungal infections, metabolic disorders, and neurodegenerative diseases.