Abstract Oxidative stress and enzyme-mediated degradation of extracellular matrix components are closely interconnected biological processes that have attracted interest in phenolic-rich medicinal plants. Schefflera leucantha, a traditional medicinal plant, contains diverse secondary metabolites; however, information on how extraction conditions influence its phytochemical composition and associated biological activities remains limited. In this study, leaf extracts of S. leucantha were prepared by maceration using 50%, 70%, and 95% ethanol, and their phytochemical profiles were comparatively evaluated. Total phenolic and flavonoid contents were determined using colorimetric methods. Biological activities were assessed using cell-free in vitro assays, including superoxide radical scavenging, nitric oxide inhibition, and collagenase and elastase inhibitory activities. Extraction yield decreased with increasing ethanol concentration, whereas phytochemical contents and associated in vitro activities showed an increasing trend. The 95% ethanol extract contained higher levels of phenolics and flavonoids and exhibited lower IC₅₀ values across all assays compared to other extracts. Extracts obtained using 70% and 50% ethanol showed progressively lower phytochemical contents and correspondingly higher IC₅₀ values. However, all extracts showed lower activity than the corresponding reference standards. Overall, the findings indicate that ethanol concentration influences the phytochemical composition of S. leucantha leaf extracts and is associated with variations in their measured in vitro activities. These results provide preliminary comparative evidence linking extraction conditions with phytochemical profiles and in vitro bioactivity responses, which require further validation in more biologically relevant systems.

Keywords: Schefflera leucantha, Ethanol extraction, Cell-free in vitro assays, Superoxide radical scavenging, Nitric oxide inhibition, Collagenase inhibition, Elastase inhibition, Polyphenols

Citation:  Phungamnuay, M., Nukulkit, C., Kaewsoongnern, T., Ohn-on, W., Khoontawad, J., and Sriset, Y. 2026. Effect of ethanol concentration on phytochemical profiles and antioxidant, nitric oxide, and collagenase and elastase inhibitory activities of Schefflera leucantha leaf extracts.  Natural and Life Sciences Communications. 25(4): e2026076.

Graphical Abstract:

INTRODUCTION

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play central roles in oxidative stress and inflammation, processes that contribute to the progression of many chronic diseases. In particular, superoxide anion and nitric oxide are key mediators of cellular damage and inflammatory responses. When produced in excess, these reactive species can disrupt cellular homeostasis and promote oxidative injury (Piacenza et al., 2022; Kiran et al., 2023). Targeting superoxide and nitric oxide has therefore become an important strategy for modulating oxidative and inflammatory processes, and these molecules are commonly used as indicators in the evaluation of plant-derived bioactive compounds (Anwar et al., 2026).

In addition to redox imbalance, oxidative stress and inflammation are also associated with the degradation of the extracellular matrix (ECM) (Wölfle et al., 2014). Enzymes such as collagenase and elastase contribute to the breakdown of collagen and elastin, which are essential for maintaining tissue structure and function (amin Hussen et al., 2025). Their overactivity is linked to tissue degeneration and aging-related changes. As a result, inhibition of collagenase and elastase is widely used as a functional marker in screening studies, particularly in the development of anti-aging and anti-inflammatory agents for pharmaceutical and cosmetic applications (Wölfle et al., 2014; Eun et al., 2020).

Medicinal plants have long been recognized as valuable sources of bioactive compounds with diverse therapeutic properties. These plants contain a wide range of secondary metabolites, including phenolics and flavonoids, which have been widely studied for their antioxidant properties, as well as their roles in nitric oxide regulation and enzyme modulation (Nwozo et al., 2023). In recent years, increasing attention has been directed toward plant-derived compounds due to their potential applications in preventing and managing chronic diseases associated with oxidative stress and inflammation (Aggarwal et al., 2024; Muscolo et al., 2024; Sardar et al., 2024; Tüğen and Buruleanu, 2025). Additionally, several naturally occurring compounds isolated from medicinal plants have demonstrated significant biological activities in both experimental and preclinical studies. For instance, ethyl gallate has been reported to exhibit anti-hyperalgesic effects in fibromyalgia-like conditions, while quercetin has shown protective roles in various pathological conditions through its antioxidant and anti-inflammatory mechanisms (Chen et al., 2025; Gonçalves et al., 2025). Similarly, plant extracts rich in polyphenols have been shown to modulate inflammatory responses and enzyme activities, further supporting their potential as bioactive agents (Gargiulo et al., 2025).

Plant polyphenols, including phenolics and flavonoids, have been reported to modulate these biological processes through multiple mechanisms (Zhang and Tsao, 2016; Rudrapal et al., 2022). These compounds may directly scavenge reactive species, interfere with nitric oxide generation, or interact with enzyme active sites and metal cofactors involved in protease activity (Chen et al., 2020; Truong and Jeong, 2021). The combined evaluation of radical scavenging, nitric oxide inhibition, and enzyme inhibition therefore enables an integrated assessment of biological activities that are mechanistically interconnected rather than independent endpoints (Atanasov et al., 2021).

Schefflera leucantha R. Vig., a medicinal plant belonging to the family Araliaceae, has been traditionally used in Thailand for conditions associated with inflammation and tissue injury (Matsui et al., 2010; Wang et al., 2021). Phytochemical investigations have identified a range of secondary metabolites in its leaves, including phenolics, flavonoids, saponins, and triterpenoids—compound classes that have been associated with antioxidant, anti-inflammatory, and enzyme-modulating activities in other plant systems (Potduang et al., 2007; Hordyjewska et al., 2019; Wang et al., 2020). Previous studies have reported that S. leucantha leaf extracts exhibit antioxidant activity, tyrosinase inhibitory effects, and antibacterial properties, suggesting a broad spectrum of potential biological activities associated with its phytochemical constituents (Potduang et al., 2007; Matsui et al., 2010; Hordyjewska et al., 2019; Tharawatchruk et al., 2025). However, previous studies on S. leucantha have largely focused on individual bioactivities or specific applications, such as extract selection for formulation purposes. For example, Ponphaiboon et al. (2026) investigated extracts derived from S. leucantha leaves using different solvent systems, including aqueous and ethanol-based extractions, primarily to identify a suitable extract for herbal lozenge development. In that study, extraction conditions were not treated as a primary variable, and their influence on phytochemical composition and biological activities was not systematically examined. Moreover, previous studies have primarily focused on general antioxidant assays and broadly defined inflammation-related activities. However, these studies did not address specific mechanistic endpoints, such as superoxide and nitric oxide inhibition or enzyme-related processes involving collagenase and elastase. Therefore, a comprehensive understanding of how ethanol concentration influences phytochemical profiles and functionally relevant biological activities of S. leucantha leaf extracts remains limited. Importantly, no study to date has systematically linked ethanol concentration with both phytochemical variation and multiple mechanistically related biological activities in S. leucantha. This highlights a key gap in understanding how extraction conditions influence biologically relevant response profiles.

Extraction conditions, particularly solvent composition, play a critical role in determining both the yield and selectivity of phytochemicals (Mohammed et al., 2022). Among commonly used solvents, ethanol is widely applied in phytochemical extraction due to its safety, ease of removal, and ability to be combined with water to modulate solvent polarity. Variations in ethanol concentration enable selective extraction of compounds with different physicochemical properties (Plaskova and Mlcek, 2023). Thus, ethanol concentration serves as a key parameter influencing both phytochemical composition and associated biological activities. Previous studies have shown that ethanol-water mixtures can differentially extract phenolic compounds and other semi-polar constituents, thereby altering the biological response profiles of plant extracts (Dai and Mumper, 2010; Do et al., 2014; Ponphaiboon et al., 2026). Despite this, a systematic comparison of ethanol concentration in relation to both phytochemical profiles and multiple in vitro biological activities of S. leucantha has not been comprehensively investigated. 

By integrating phytochemical analysis with multiple mechanistically related biological assays, this study provides a more comprehensive evaluation than previous reports that focused on single endpoints. From an application perspective, understanding these relationships is particularly relevant for the development of plant-based functional ingredients in pharmaceutical, cosmetic, and nutraceutical products, where extraction conditions can directly influence efficacy and consistency.

Therefore, the present study aimed to comparatively evaluate the phytochemical profiles and selected in vitro biological activities of S. leucantha leaf extracts prepared using different ethanol concentrations. Biological activities were specifically assessed through inhibition of superoxide radicals and nitric oxide, as well as suppression of collagenase and elastase activities to provide a mechanistically coherent assessment of antioxidant-, inflammatory-, and enzyme-related responses. The findings are intended to serve as baseline data linking extraction conditions, phytochemical composition, and biologically relevant in vitro activities of S. leucantha leaf extracts.

MATERIALS AND METHODS

Materials

Aluminum chloride was purchased from RCI Labscan (Bangkok, Thailand). Ascorbic acid, collagenase from Clostridium histolyticum, epigallocatechin gallate, N-(1-naphthyl)ethylenediamine dihydrochloride (NED), nicotinamide adenine dinucleotide (NADH), N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA), nitroblue tetrazolium (NBT), N-succinyl-(Ala)₃-p-nitroanilide (SANA), phenazine methosulfate (PMS), porcine pancreatic elastase, quercetin, sodium nitroprusside, and sulfanilamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol and gallic acid were obtained from Merck (Darmstadt, Germany). Folin–Ciocalteu phenol reagent was purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). All other chemicals used in this study were of analytical grade and obtained from commercial sources.

Plant material and extraction

Dried leaves of S. leucantha were purchased from Thai Pattana Pharmacy (Mueang District, Loei Province, Thailand) in January 2025. As the plant material was obtained from a commercial source, the original growth conditions were not controlled; however, it was reported to originate from Loei Province, Thailand. The leaves used in this study were mature, consistent with those commonly employed in traditional medicinal use. Botanical authentication was performed by a certified botanist from the Thai Traditional Medicine Program, Faculty of Natural Resources, Rajamangala University of Technology Isan, Sakon Nakhon Campus, Thailand. The specimen was assigned the identification code SL2025-YS020, and a voucher specimen was deposited at the Herbarium of the Thai Traditional Medicine Program, Rajamangala University of Technology Isan, Sakon Nakhon Campus, for future reference.

The dried leaves of S. leucantha were ground into a fine powder. Extraction was carried out by maceration using ethanol at concentrations of 50%, 70%, and 95% (v/v). These concentrations were selected to represent solvents with different polarities, which influence the extraction efficiency of phytochemicals with varying physicochemical properties, particularly phenolic compounds (Mohammed et al., 2022; Ghaffar and Perveen, 2025). Variations in ethanol-water composition have been shown to affect both the yield and composition of extracted bioactive compounds due to polarity-dependent solubility (Do et al., 2014; Mamoona et al., 2025). In addition, this range has been commonly used in previous studies on S. leucantha and other medicinal plants to enable comparative evaluation of solvent-dependent extraction profiles (Ponphaiboon et al., 2026).      

For each extraction, the powdered plant material was mixed with solvent at a ratio of 1:10 (w/v) and macerated at room temperature for 5 days with occasional stirring. The extraction was conducted in a single cycle, and the endpoint was defined by the fixed extraction time together with stabilization of extract color (i.e., no further visible change in intensity).

The resulting extracts were filtered through filter paper and concentrated under reduced pressure using a rotary evaporator (Rotavapor^® R-300, Buchi, Flawil, Switzerland) at temperatures not exceeding 45°C. The extraction yield was calculated based on the weight of the dried starting material. The crude extracts were stored at 4°C until further analysis to minimize degradation and maintain compound stability.

Determination of phytochemical contents of S. leucantha leaf extracts

Total phenolics

The total phenolic content of S. leucantha leaf extracts was determined using the Folin-Ciocalteu assay with minor modifications (Sriset et al., 2021). Briefly, the extract was mixed with Folin-Ciocalteu reagent and incubated in the dark for 30 min. The absorbance was measured at 700 nm using a UV-Vis microplate reader (Infinite^® 200 pro, Tecan, Männedorf, Switzerland). Total phenolic content was quantified using a calibration curve constructed with gallic acid (0-400 µg/mL) as the reference standard and expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g). The calibration curve showed good linearity (R² = 0.999).

Total flavonoids

The total flavonoid content of S. leucantha leaf extracts was determined using the aluminum chloride (AlCl₃) colorimetric assay with minor modifications (Sriset et al., 2021). Briefly, the extract was mixed with a reaction mixture consisting of 10% aluminum chloride and 1 M sodium acetate (1:1, v/v). The mixture was incubated at room temperature for 30 min, and the absorbance was measured at 405 nm using a UV-Vis microplate reader. Total flavonoid content was quantified using a calibration curve of quercetin (0-400 µg/mL) as the reference standard and expressed as milligrams of quercetin equivalents per gram of dry extract (mg QE/g). The calibration curve showed good linearity (R² = 0.998).

All measurements were performed in five independent replicates (n = 5). The results are presented as mean ± standard deviation (SD).

In vitro biological activity assays of S. leucantha leaf extracts

The extract concentrations (0.01, 0.1, 1, and 10 mg/mL) were selected based on the limited information available on these biological activities of S. leucantha leaf extracts (Potduang et al., 2007; Matsui et al., 2010). A ten-fold dilution series was used to provide a broad concentration range, enabling observation of dose-dependent effects and facilitating reliable estimation of IC₅₀ values.

Evaluation of superoxide radical scavenging activity

Superoxide radical scavenging activity was assessed using the phenazine methosulfate (PMS)-nicotinamide adenine dinucleotide (NADH) system. The reaction mixture containing 468 µM NADH, 150 µM nitroblue tetrazolium (NBT), 60 µM PMS, and 20 mM phosphate-buffered saline (PBS, pH 7.4) was mixed with S. leucantha leaf extracts and incubated at room temperature for 30 min. Absorbance was measured at 560 nm using a UV-Vis microplate reader. Scavenging activity was evaluated by comparing absorbance in the presence and absence of the extract. The percentage inhibition was calculated as:

% Inhibition = [(A₀ − A₁) / A₀] × 100

where A₀ is the absorbance of the control (without extract) and A₁ is the absorbance in the presence of the extract or the positive control (ascorbic acid).

Appropriate blank controls were used, and sample blanks were prepared to correct for background absorbance and the natural color of the extracts. All measurements were corrected accordingly.

Superoxide radical scavenging activity was expressed as the half maximal inhibitory concentration (IC₅₀, mg/mL), defined as the concentration required to inhibit 50% of radical generation. IC₅₀ values were determined from dose-response curves using nonlinear regression analysis in GraphPad Prism software (San Diego, CA, USA). Ascorbic acid (0.00625-0.1 mg/mL) was used as the positive control (Sriset et al., 2021).

Evaluation of nitric oxide inhibitory activity

Nitric oxide inhibitory activity was assessed using the Griess reaction. Nitric oxide was generated from sodium nitroprusside in PBS (pH 7.4), and the resulting nitrite ions were quantified using the Griess reagent (Wisidsri et al., 2019; Can et al., 2022). The S. leucantha leaf extracts were incubated with 10 mM sodium nitroprusside at room temperature for 150 min, followed by the addition of the Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 20% glacial acetic acid, 1:1) and further incubation for 30 min at room temperature.  Absorbance was measured at 540 nm using a UV-Vis microplate reader. The inhibitory activity was evaluated by comparing the absorbance in the presence and absence of the extract. The percentage inhibition was calculated as:

% Inhibition = [(A₀ − A₁) / A₀] × 100

where A₀ is the absorbance of the control (without extract) and A₁ is the absorbance in the presence of the extract or the positive control (quercetin).

Appropriate blank controls were used, and sample blanks were prepared to correct for background absorbance and the natural color of the extracts. All measurements were corrected accordingly.

Nitric oxide inhibitory activity was expressed as the IC₅₀ value (mg/mL), defined as the concentration required to inhibit 50% of nitric oxide generation. IC₅₀ values were determined from dose-response curves using nonlinear regression analysis in GraphPad Prism software. Quercetin (0.00625-0.1 mg/mL) was used as the positive control (Sriset et al., 2021).

Evaluation of anti-collagenase activity

Anti-collagenase activity of S. leucantha leaf extracts was evaluated using collagenase from Clostridium histolyticum with N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA) as the substrate. The enzyme solution (2 U/mL) was prepared in Tricine buffer (pH 7.5) containing sodium chloride (5 M) and calcium chloride (100 mM), and dispensed into a 96-well microplate. The extract solution was added and pre-incubated at 37°C for 15 min. The reaction was initiated by adding FALGPA (1 mM). Absorbance was measured at 340 nm immediately after substrate addition and continuously monitored at 37°C for 20 min using a UV-Vis microplate reader. Collagenase activity was determined from the change in absorbance over time (ΔA), which reflects the reaction rate. Inhibitory activity was calculated by comparing the reaction rates in the presence and absence of the extract, using the following equation:

% Inhibition = [(∆A₀ − ∆A₁) / ∆A₀] × 100

where ΔA₀ represents the change in absorbance of the control (without extract) and ΔA₁ represents the change in absorbance in the presence of the extract or the positive control (epigallocatechin gallate; EGCG).

Appropriate blank controls were used, with enzyme and sample blanks accounting for background absorbance and the intrinsic color of the extracts, and all measurements were corrected accordingly.

The inhibitory activity was expressed as the IC₅₀ value (mg/mL), defined as the concentration required to inhibit 50% of collagenase activity. IC₅₀ values were obtained from dose-response curves using nonlinear regression analysis in GraphPad Prism. EGCG (0.03125-0.5 mg/mL) was used as a positive control under the same experimental conditions (Thring et al., 2009; Lertkanchanasak et al., 2024).

Evaluation of anti-elastase activity

Anti-elastase activity of S. leucantha leaf extracts was determined using porcine pancreatic elastase and N-succinyl-(Ala)3-p-nitroanilide (SANA) as the substrate. The enzyme solution (0.01 mg/mL) was prepared in 0.1 M Tris-HCl buffer (pH 8.0) and added to a 96-well microplate. The extract solution was mixed with the enzyme and pre-incubated, followed by the addition of SANA (0.8 mM) to initiate the reaction. Absorbance was measured at 410 nm immediately after substrate addition (initial absorbance), and again after incubation at 25°C for 20 min using a UV-Vis microplate reader. Elastase activity was determined based on the change in absorbance over the incubation period (ΔA). Inhibitory activity was calculated using the following equation:

% Inhibition = [(∆A₀ − ∆A₁) / ∆A₀] × 100

where ΔA₀ represents the change in absorbance of the control (without extract) and ΔA₁ represents the change in absorbance in the presence of the extract or the positive control (EGCG).

Appropriate blank controls were used, with enzyme and sample blanks accounting for background absorbance and the intrinsic color of the extracts, and all measurements were corrected accordingly.

The inhibitory activity was expressed as the IC₅₀ value (mg/mL), defined as the concentration required to inhibit 50% of elastase activity. IC₅₀ values were calculated from dose-response curves using nonlinear regression analysis in GraphPad Prism. EGCG (0.03125-0.5 mg/mL) was used as a positive control under identical experimental conditions (Thring et al., 2009; Lertkanchanasak et al., 2024).

Statistical analysis

All results are presented as mean ± SD. Data were checked for normality and homogeneity of variance prior to one-way analysis of variance (ANOVA). Tukey’s post hoc test was used for multiple comparisons. Differences were considered statistically significant at P < 0.05. In addition to statistical significance, the magnitude of differences and overall biological trends were also considered to ensure a biologically meaningful interpretation of the results. These included changes in phytochemical content and IC₅₀ values. Data analysis was conducted using IBM Statistical Product and Service Solutions (SPSS) version 29.0 (Armonk, NY, USA).

RESULTS

Physical characteristics and extraction yield of S. leucantha leaf extracts

The physical characteristics and extraction yields of S. leucantha leaf extracts prepared using different ethanol concentrations are presented in Table 1. A clear trend was observed in relation to ethanol concentration, where increasing ethanol concentration from 50% to 95% resulted in a gradual decrease in extraction yield. The 50% ethanol extract showed the highest yield, followed by the 70% extract, while the lowest yield was observed in the 95% ethanol extract. This pattern indicates that lower ethanol concentrations are associated with higher extraction yield, possibly due to the extraction of a broader range of compounds.

Differences in the physical appearance of the extracts were also observed according to ethanol concentration (Figure 1). The 50% ethanol extract appeared light brown with a greenish tone and exhibited relatively low viscosity. In contrast, the 70% and 95% ethanol extracts showed progressively darker coloration and higher viscosity, with the 95% ethanol extract appearing dark green to nearly black. These changes may reflect differences in the composition and concentration of extracted compounds. Having established the extraction yields, the next step was to evaluate the phytochemical composition of the extracts. Understanding how ethanol concentration influences extraction yield provides useful context for interpreting its effect on the levels of bioactive compounds, particularly phenolics and flavonoids.

Table 1. Physical characteristics and extraction yield of S. leucantha leaf extracts obtained by maceration with different ethanol concentrations.

Figure 1. Physical appearance of crude S. leucantha leaf extracts obtained by maceration with different ethanol concentrations: 50% ethanol extract (A), 70% ethanol extract (B), and 95% ethanol extract (C).

Phytochemical profiles of S. leucantha leaf extracts

The total phenolic and flavonoid contents of S. leucantha leaf extracts obtained using different ethanol concentrations are summarized in Table 2. A consistent trend was observed, where increasing ethanol concentration was associated with higher levels of total phenolics and flavonoids.

No significant differences in total phenolic and flavonoid contents were observed between the 50% and 70% ethanol extracts (P > 0.05). In contrast, the 95% ethanol extract showed significantly higher levels of both phenolics and flavonoids compared to the 50% and 70% extracts (P < 0.05), as indicated in Table 2. This pattern suggests that higher ethanol concentrations are associated with increased extraction of phenolic and flavonoid compounds, possibly due to improved solubility of less polar phytochemicals in more concentrated ethanol systems.

Correlation analysis further supported this relationship, demonstrating a strong positive association between ethanol concentration and phytochemical contents. Pearson correlation coefficients (r) were 0.989 for total phenolics and 0.999 for total flavonoids. These findings support the observed trend that increasing ethanol concentration is associated with greater enrichment of phenolic and flavonoid compounds.

Given the observed increase in phenolic and flavonoid contents with higher ethanol concentrations, the next logical step was to evaluate whether these phytochemical changes are associated with enhanced biological activities, particularly antioxidant, nitric oxide inhibitory, and enzyme inhibitory effects, including collagenase and elastase inhibition.

Table 2. Total phenolic and flavonoid contents of S. leucantha leaf extracts obtained using different ethanol concentrations.

Note: Values are presented as mean ± SD (n = 5). Different superscript letters (a, b) within the same column indicate significant differences (P < 0.05). GAE, gallic acid equivalents; QE, quercetin equivalents.

In vitro antioxidant and nitric oxide inhibitory activities of S. leucantha leaf extracts

The in vitro antioxidant and nitric oxide inhibitory activities of S. leucantha leaf extracts are presented in Table 3. Significant differences in both superoxide radical scavenging and nitric oxide inhibitory activities were observed among the extracts (P < 0.05). A clear trend was evident, where increasing ethanol concentration was associated with greater biological activities, as reflected by lower IC₅₀ values. The 95% ethanol extract exhibited the strongest activity, followed by the 70% extract, while the 50% ethanol extract showed the weakest activity. In comparison, the reference standards demonstrated markedly stronger activities than all tested extracts, as indicated by their substantially lower IC₅₀ values. These findings indicate that extracts obtained with higher ethanol concentrations show greater antioxidant and nitric oxide inhibitory activities, which is consistent with their higher phenolic and flavonoid contents. 

Following the antioxidant and nitric oxide inhibition assays, the next logical step was to examine the enzyme inhibitory activities of the extracts, as the potential therapeutic applications of these extracts also include their ability to modulate matrix-degrading enzymes, such as collagenase and elastase.

Table 3. Superoxide radical scavenging and nitric oxide inhibitory activities of S. leucantha leaf extracts expressed as IC₅₀ values.

Note: Values are presented as mean ± SD (n = 5). Different superscript letters (a, b, c, d) within the same column indicate significant differences (P < 0.05).

In vitro collagenase and elastase inhibitory activities of S. leucantha leaf extracts

The inhibitory activities of S. leucantha leaf extracts against collagenase and elastase are summarized in Table 4. Significant differences in enzyme inhibitory activities were observed among the extracts (P < 0.05). Enzyme inhibitory activities varied among the extracts, with the 95% ethanol extract showing the greatest inhibition for both collagenase and elastase, followed by the 70% ethanol extract, while the 50% ethanol extract exhibited the lowest activity. The reference standard EGCG showed greater inhibitory potency than all tested extracts, as indicated by its lower IC₅₀ values. These results indicate that extracts obtained with higher ethanol concentrations show greater collagenase and elastase inhibitory activities, which is consistent with their higher phenolic and flavonoid contents, as well as their antioxidant and nitric oxide inhibitory activities.

Table 4. Collagenase and elastase inhibitory activities of S. leucantha leaf extracts expressed as IC₅₀ values.

Note: Values are presented as mean ± SD (n = 5). Different superscript letters (a, b, c, d) within the same column indicate significant differences (P < 0.05).

Overall, the 95% ethanol extract showed the strongest biological activities among all extracts, as reflected by the lowest IC₅₀ values in the superoxide radical scavenging, nitric oxide inhibitory, collagenase inhibitory, and elastase inhibitory assays. In contrast, the 50% ethanol extract consistently exhibited the weakest activities, while the 70% ethanol extract showed intermediate performance. The higher activity of the 95% ethanol extract is consistent with its higher phenolic and flavonoid contents, suggesting a possible contribution of these compounds to the observed activities. Although all extracts showed lower activity than the reference standards, the results provide useful preliminary evidence for selecting suitable extraction conditions and support further investigation of the 95% ethanol extract.

DISCUSSION

This study presents a comparative evaluation of the phytochemical composition and in vitro antioxidant, nitric oxide inhibitory, and enzyme-related activities of S. leucantha leaf extracts prepared using different ethanol concentrations. By integrating radical scavenging, nitric oxide inhibition, and protease inhibition assays, the results provide a more comprehensive view of biological activities associated with oxidative stress regulation, inflammatory processes, and extracellular matrix degradation, which are key mechanisms involved in skin physiology and aging (Wölfle et al., 2014; amin Hussen et al., 2025).

An inverse relationship was observed between extraction yield and bioactivity. As ethanol concentration increased, extraction yield decreased, whereas phytochemical enrichment and biological activities increased. This pattern reflects the influence of solvent polarity on both extraction efficiency and compound selectivity. Ethanol-water mixtures span a range of polarities, with lower ethanol concentrations (e.g., 50%) being more polar due to higher water content, whereas higher concentrations (e.g., 95%) are less polar. More polar solvent systems tend to extract a broader range of hydrophilic constituents, including sugars, proteins, and other non-specific compounds, which may contribute to higher overall yield without selectively enriching bioactive molecules (Kaczorová et al., 2021). In contrast, many phenolic and flavonoid compounds exhibit moderate polarity and are more soluble in ethanol-rich systems (Sasidharan et al., 2011; Kaczorová et al., 2021; Lee et al., 2024). Accordingly, higher ethanol concentrations were associated with greater phenolic and flavonoid contents in the 95% ethanol extract despite its lower yield. This finding highlights that solvent composition influences not only the quantity but also the selectivity of extracted phytochemicals, with higher ethanol concentrations favoring compounds associated with antioxidant and enzyme inhibitory activities. Similar solvent-dependent extraction patterns have been reported in other medicinal plants, emphasizing that extraction yield alone does not necessarily reflect biological relevance (Dai and Mumper, 2010; Zhang et al., 2018; Alara et al., 2021).

Consistent with this trend, the 95% ethanol extract showed the highest levels of total phenolics and flavonoids and exhibited greater antioxidant and nitric oxide inhibitory activities. However, this relationship should be interpreted cautiously, as it is based on correlation rather than direct evidence of causation. Although phenolics and flavonoids are widely recognized for their antioxidant properties and their ability to influence nitric oxide-related pathways, the present study did not include compound isolation or mechanistic analysis to confirm their specific contributions. The concurrent inhibition of superoxide radicals and nitric oxide may therefore reflect the combined effects of multiple constituents within the extracts rather than the activity of a single compound class. Previous studies have demonstrated that phenolics and flavonoids can neutralize reactive oxygen species and modulate nitric oxide production through various mechanisms (Hunyadi, 2019; Middleton et al., 2000; Pietta, 2000; Tumilaar et al., 2024). In line with these findings, the activities observed in S. leucantha extracts may be partly associated with the modulation of oxidative and nitric oxide-related pathways (Kozlov et al., 2024; Prayoga et al., 2026). Nevertheless, it remains unclear whether these mechanisms directly explain the observed activities, and further studies are needed to clarify this.

Compared with previously reported general antioxidant activities of S. leucantha (Potduang et al., 2007; Ponphaiboon et al., 2026), the superoxide radical scavenging and nitric oxide inhibitory assays used in this study provide more specific insight into reactive species involved in oxidative and nitrosative stress. These species are biologically relevant, as they contribute to redox imbalance and are linked to pathways that regulate proteolytic enzymes involved in extracellular matrix degradation. Oxidative and nitrosative stress have been associated with the activation of matrix metalloproteinases responsible for collagen and elastin degradation (Eun et al., 2020; Manful et al., 2025; Suvedi et al., 2025). In addition, interactions between superoxide radicals and nitric oxide can generate reactive nitrogen species, such as peroxynitrite, which may further amplify oxidative damage and influence redox-sensitive signaling pathways (Wölfle et al., 2014; Piacenza et al., 2022; Choudhary et al., 2023). These processes have been linked to the upregulation of matrix metalloproteinases, potentially involving pathways such as NF-κB and MAPK. This may contribute to the degradation of structural proteins in the dermal matrix (Feng et al., 2024). In this context, the observed scavenging of superoxide radicals and inhibition of nitric oxide may be functionally related to the regulation of proteolytic enzyme activity. Consistent with this, the inhibitory effects of S. leucantha leaf extracts on collagenase and elastase align with previous reports on phenolic-rich plant extracts that modulate enzymes involved in extracellular matrix degradation (Li et al., 2022; Son et al., 2024). Several plant-derived extracts rich in polyphenols have been reported to exhibit collagenase and elastase inhibitory activities, although their potencies vary depending on phytochemical composition and extraction conditions (Csekes and Račková, 2021; Suzuki et al., 2023). Previous studies further support the antioxidant potential of S. leucantha. For example, Tharawatchruk et al. (2025) reported strong ferric reducing antioxidant power in aqueous extracts, while Potduang et al. (2007) demonstrated DPPH radical scavenging and tyrosinase inhibitory activities in ethanolic extracts. Although different assay systems were used, these findings collectively indicate that S. leucantha possesses broad antioxidant capacity influenced by extraction conditions. The present study extends this understanding by showing that ethanol concentration also affects nitric oxide inhibition and protease-related activities. However, direct quantitative comparison across studies remains limited due to differences in assay conditions and experimental design.

All extracts demonstrated inhibitory activity against both collagenase and elastase, although their effects were lower than those of the reference standard EGCG. The IC₅₀ values obtained in this study fall within ranges commonly reported for plant extracts (approximately 0.1-1.0 mg/mL), depending on species and extraction conditions (Thring et al., 2009; Zakaria et al., 2020; Lertkanchanasak et al., 2024). A similar pattern was observed in the antioxidant and nitric oxide assays, where all extracts showed lower activity than the reference standards, ascorbic acid and quercetin. These findings are consistent with the general observation that crude plant extracts often exhibit lower apparent activity than purified compounds, largely due to the lower concentration of active constituents and the complexity of extract composition (Choi and Shin, 2025).

The observed activity profiles may reflect the multi-component nature of S. leucantha leaf extracts, which contain compound with varying levels of potency. The stronger inhibition observed in extracts prepared with higher ethanol concentrations may be associated with the presence of polyphenolic compounds, which are known to interact with enzyme active sites or interfere with metal ions required for protease activity. Similar modes of action have been reported for phenolic compounds in other plant systems (Csekes and Račková, 2021; Suzuki et al., 2023; Lertkanchanasak et al., 2024; Tammasorn et al., 2024). These findings are also consistent with the traditional use of S. leucantha in wound healing and skin-related conditions (Matsui et al., 2010; Wang et al., 2021), although scientific evidence supporting these applications remains limited. Previous studies on S. leucantha have primarily focused on general antioxidant activities or single biological endpoints, with limited investigation into enzyme-related mechanisms or their relationship with extraction conditions (Potduang et al., 2007; Matsui et al., 2010; Hordyjewska et al., 2019; Tharawatchruk et al., 2025; Ponphaiboon et al., 2026). By linking solvent composition with phytochemical enrichment and mechanistically relevant assays, the present study provides a more integrated understanding of bioactivity profiles and offers new insight into the potential applications of S. leucantha extracts.

Phytochemical analyses of S. leucantha leaves have identified a diverse range of bioactive constituents, including flavonoids (e.g., quercetin and rutin), triterpenoids, saponins, sesquiterpenes, and phenylpropanoids (Potduang et al., 2007; Hordyjewska et al., 2019; Wang et al., 2020). Flavonoids are widely recognized for their antioxidant, anti-inflammatory, and enzyme inhibitory properties (Al-Khayri et al., 2022; Shavez et al., 2025), while triterpenoids and saponins have been associated with modulation of inflammatory responses and extracellular matrix stability (Liang et al., 2023; Wijesekara et al., 2024). The coexistence of these compound classes suggests that the observed biological activities may arise from additive or synergistic interactions rather than a single dominant compound. Structural features of polyphenolic compounds, particularly the presence of multiple hydroxyl groups, support their roles in radical scavenging, nitric oxide modulation, and enzyme inhibition (Rudrapal et al., 2024), which are consistent with the activities observed in this study.

Despite these promising in vitro findings, several limitations should be considered. The assays used in this study are based on simplified, cell-free systems and do not fully capture the complexity of biological processes in living systems. Important factors such as cellular uptake, metabolism, and bioavailability are not accounted for, and therefore the observed activities may not directly reflect biological effects in vivo (Rudrapal et al., 2024; Bas, 2026). In addition, IC₅₀ values obtained under these conditions represent relative inhibitory activity and may vary depending on assay design, enzyme sources, and experimental conditions. Previous studies have also shown that in vitro antioxidant and enzyme inhibitory activities do not always correlate with effects observed in cellular or in vivo models (Abdelfattah et al., 2022; Felicia et al., 2022; Blažková et al., 2025; Nguyen et al., 2026). Therefore, the findings of this study should be regarded as preliminary screening data rather than definitive evidence of biological efficacy. Future studies should focus on compound isolation, bioactivity-guided fractionation, and validation in more biologically relevant systems, including cell-based and in vivo models, to better understand the functional relevance of the observed activities. Given the traditional use of S. leucantha in wound healing and skin-related conditions, its potential roles in wound healing, anti-inflammatory activity, and antibacterial effects also warrant further study. Available data suggest relatively low acute toxicity (Witthawaskul et al., 2003; Potduang et al., 2007), although further studies are needed to establish a comprehensive safety profile, particularly through in vivo and long-term assessments.

The biological activities of S. leucantha leaf extracts, particularly their antioxidant, nitric oxide inhibitory, and enzyme inhibitory properties, indicate potential clinical relevance. These activities are closely linked to oxidative stress, inflammation, and extracellular matrix degradation, which are key processes underlying various skin-related conditions. Accordingly, the findings suggest potential applications in cosmetic and therapeutic contexts, including skin aging, wound healing, scar management, and acne-related conditions. In addition, the relationship between extraction conditions, phytochemical enrichment, and bioactivity provides useful insight for optimizing extract preparation. Overall, these findings highlight the potential of S. leucantha as a natural source of bioactive compounds for further investigation in pharmaceutical, nutraceutical, and cosmeceutical applications.

CONCLUSION

This study demonstrates that ethanol concentration influences the phytochemical composition of S. leucantha leaf extracts and is associated with differences in their in vitro biological activities. Extracts prepared with higher ethanol concentrations contained higher levels of phenolics and flavonoids and showed greater inhibition of superoxide radicals, nitric oxide production, and collagenase and elastase activities. However, these findings are based on cell-free in vitro assays and are lower than those of the reference standards; therefore, they should be interpreted with caution. In addition, as compound isolation was not performed, it is not possible to identify the specific active constituents or to establish clear relationships between phytochemical composition and the observed biological effects.

Overall, the results provide screening-level evidence describing trends in extraction conditions, phytochemical content, and associated in vitro activities in S. leucantha leaf extracts, rather than definitive evidence of biological efficacy. Further studies using cell-based systems and compound characterization are needed to better understand their biological.

ACKNOWLEDGEMENT

The authors would like to thank the Faculty of Natural Resources, Rajamangala University of Technology Isan, Sakon Nakhon Campus, for providing the facilities and equipment used in this research.

AUTHOR CONTRIBUTIONS

Montita Phungamnuay: Conceptualization (Lead), Investigation (Lead), Data Curation (Lead), Validation (Equal), Writing – Original Draft (Lead); Chatchanok Nukulkit: Data Curation (Equal), Formal Analysis (Equal), Supervision (Equal), Validation (Lead), Visualization (Equal), Writing – Review & Editing (Equal); Thanthika Kaewsoongnern: Data Curation (Equal), Formal Analysis (Equal), Validation (Equal), Visualization (Equal), Writing – Review & Editing (Supporting); Warin Ohn-on: Data Curation (Equal), Formal Analysis (Equal), Validation (Equal); Jarinya Khoontawad: Data Curation (Supporting), Formal Analysis (Equal), Validation (Equal); Yollada Sriset: Conceptualization (Lead), Formal Analysis (Lead), Investigation (Lead), Supervision (Lead), Validation (Equal), Visualization (Lead), Writing – Review & Editing (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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