Targeting Skin Aging at Multiple Fronts: Integrated In Silico and In Vitro Evidence of Antioxidant, Anti-Protease, and UVB-Protective Effects of Vitex trifolia

Abstract

Skin aging is driven by oxidative stress, extracellular matrix degradation, and ultraviolet-B (UVB)-induced cellular injury. Plant-derived bioactives with multi-targeted protective actions offer promising avenues for cosmeceutical development. This study assessed ethanolic leaf extracts of Vitex trifolia, an Indonesian medicinal plant traditionally used for skin disorders. Phytochemical analysis showed a total phenolic content of 78.52 ± 0.01 mg GAE/g and total flavonoid content of 1.99 ± 0.02 mg QE/g. LC–HRMS profiling identified major flavonoid and phenolic acid derivatives. Antioxidant assays demonstrated strong radical-scavenging and reducing activities, with IC₅₀ values of 63.47 ± 0.24 (DPPH) and 70.13 ± 1.28 μg/mL (ABTS) and a Ferric Reducing Antioxidant Power (FRAP) value of 36.3 ± 0.18 FeSO₄ eq/100 g. Enzymatic studies confirmed potent collagenase inhibition (IC₅₀ = 27.94 ± 3.20 μg/mL) and moderate elastase inhibition, supported by molecular docking analysis. In HaCaT keratinocytes, the extract remained non-cytotoxic up to 100 μg/mL and exerted cytoprotective activity against UVB-induced damage at 12.5–50 μg/mL. The extract also downregulated UVB-induced matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-9 (MMP-9) expression up to 42% and 69%, respectively, outperforming ascorbic acid. These findings highlight V. trifolia as a multifunctional natural candidate for anti-photoaging cosmeceutical applications over single-compound antioxidants, as demonstrated by its combined antioxidant, enzyme-inhibitory, cytoprotective, and MMP-modulating activities, as well as a wider cell safety profile.

1. Introduction

Human skin represents a highly interactive barrier tissue in which genetically programmed senescence intersects with external environmental stressors to shape the aging trajectory [1,2]. Among these external stressors, ultraviolet B (UVB, 290–320 nm) is particularly deleterious, driving molecular alterations that prematurely remodel skin structure and function [3]. Upon UVB exposure, keratinocytes generate bursts of reactive oxygen species (ROS) that exceed intrinsic antioxidant capacity, resulting in oxidative injury to genomic DNA, structural proteins, and membrane lipids [4]. The long-term accumulation of such oxidative insults is clinically reflected in photoaging hallmarks such as deep wrinkles, uneven pigmentation, and a gradual decline in skin resilience [5].

Photoaging advances through a two-pronged mechanism: direct oxidative disruption of cellular macromolecules and enzymatic degradation of the extracellular matrix (ECM). ROS-driven activation of elastase leads to fragmentation of elastin fibers, while collagenase progressively dismantles collagen scaffolds—together producing dermal thinning and structural deterioration that visibly accelerate skin aging [6]. Targeting these proteases is therefore a pivotal intervention point. Polyphenol-rich phytochemicals constitute a natural defense strategy, acting dually as potent radical scavengers and as inhibitors of matrix-degrading proteases, including elastase and collagenase. Increasing evidence highlights their ability to modulate multiple molecular pathways simultaneously, establishing a rational basis for phytochemical-derived cosmeceuticals [1]. Specifically, ROS could induce the upregulation of matrix metalloprotease 1 (MMP-1) and MMP-9 through the mitogen-activated protein kinase (MAPK) pathway [7]. The upregulation of the MMP-1 and MMP-9 expressions responsible for collagen degradation leads to a loss of skin elasticity and the presence of wrinkles [8,9]. These mechanistic insights highlight the growing importance of exploring traditional medicinal plants as promising sources of multi-targeted anti-aging compounds.

The global interest in safe, natural skin-care agents has intensified research into botanicals with established ethnomedicinal applications. Vitex trifolia L. (legundi) is a widely used traditional plant recognized in various Asian systems of medicine for treating inflammatory and dermatological conditions [10,11]. V. trifolia is enriched in vitexicarpin, gallic acid, and isovitexin [12]. While these phytoconstituents are acknowledged for their antioxidant, anti-inflammatory, and enzyme-modulating actions, rigorous experimental validation of their anti-aging potential remains scarce.

Despite the accumulating literature on plant polyphenols, gaps persist in understanding the integrated bioactivity profiles of traditional species. Prior studies often focus narrowly on single antioxidant assays or isolated enzyme inhibition, without addressing the combined cellular and biochemical responses under UV-induced stress [1,13]. Moreover, indigenous species with long-standing ethnomedicinal use in Southeast Asia remain underexplored as cosmeceutical candidates [14]. Addressing these gaps requires standardized extraction, quantitative phytochemical characterization, and multi-level assays to bridge traditional claims with modern biomedical validation [15].

Here, we present a systematic investigation into the cosmeceutical potential of V. trifolia. Ethanolic extracts were prepared using ultrasonic-assisted extraction and microwave-assisted extraction (MAE), followed by phytochemical quantification, antioxidant profiling, elastase and collagenase inhibition kinetics, and UVB-induced cytoprotection in immortalized human keratinocytes, known as HaCaT, as well as gene expression analysis of MMP-1 and MMP-9. By interrogating oxidative stress cascades, protease-mediated matrix degradation, cellular photodamage, and modulation of MMP-1 and MMP-9 genes, this study provides foundational evidence for developing natural anti-aging cosmeceuticals derived from biodiversity. This study provides the first integrated evaluation of V. trifolia for cosmetic use, encompassing phytochemical characterization, antioxidant profiling, elastase and collagenase inhibition, and UVB-induced cytoprotection in HaCaT keratinocytes, providing a holistic view of their skin-protective potential.

2. Results

2.1. Extract Characterisation

The extraction of V. trifolia yielded 31.8%, suggesting a high abundance of ethanol-soluble phytochemicals suitable for further chemical and biological evaluation. The total flavonoid content (TFC) and total phenolic content (TPC) of the extract were measured and recorded in

Table 1. Total flavonoid, phenolic content, and yield of V. trifolia extract.

Total Flavonoid Content (mg QE/g Extract)	Total Phenolic Content (mg GAE/g Extract)	Yield (%)
1.99 ± 0.02	78.52 ± 0.01	31.8

QE: quercetin; GAE: gallic acid.

. The total flavonoid content (TFC) and total phenolic content (TPC) were quantified using the AlCl₃ and Folin–Ciocalteu methods, respectively (Table 1). The extract contained 1.99 ± 0.02 mg QE/g extract of flavonoids and 78.52 ± 0.01 mg gallic acid equivalent (GAE)/g extract of phenolic compounds, indicating a phytochemical profile dominated by polyphenolic constituents.

Furthermore, to further elucidate its chemical composition, an LC–HRMS analysis was performed on the ethanolic extract of V. trifolia. LC–HRMS profiling uncovered a remarkably rich metabolite landscape in V. trifolia ethanolic extract, with ten major bioactive compounds dominating the chemical profile (Figure 1). Notably, the methylated flavonoid casticin emerged as the predominant metabolite (AUC 2.38 × 10⁹), accompanied by an impressive array of glycosylated and polyhydroxylated flavonoid derivatives, modifications known to enhance bioavailability and therapeutic potential. The extract also yielded phenolic acids (4-coumaric acid and 4-hydroxybenzoic acid) with established antioxidant properties, alongside valuable terpenoids including lupeol, a pentacyclic triterpenoid with documented anti-inflammatory activity (

Table 2. Major compounds detected in Vitex trifolia through LC–MS analysis.

No	Compound	Molecular Formula	Molecular Weight (m/z)	Class	AUC (×106)
1	Casticin (NP-001928)	C19H18O8	374.099	Flavonoid	2384.32
2	(1ξ)-1,5-Anhydro-1-[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-8-yl]-D-galactitol	C21H20O11	448.100	Flavonoid	773.48
3	(2S,3S,4S,5R,6S)-6-{[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yl]oxy}-3,4,5-trihydroxyoxane-2-carboxylic acid	C21H18O12	462.079	Phenolic acid	522.29
4	5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-3,6-dimethoxy-4H-chromen-4-one	C18H16O8	360.083	Flavonoid	455.82
5	4-Coumaric acid	C9H8O3	164.047	Phenolic acid	339.04
6	(1R,3R,4R,4aS)-4-Hydroxy-3,4a,8,8-tetramethyl-4-[2-(5-oxo-2,5-dihydro-3-furanyl)ethyl]decahydro-1-naphthalenyl acetate	C22H34O5	378.239	Diterpenoid	285.99
7	(1r,3R,4s,5S)-4-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,3,5-trihydroxycyclohexane-1-carboxylic acid	C16H18O9	354.094	Phenolic acid	279.47
8	(1S,3R,4S,5R)-3,5-bis({[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy})-1,4-dihydroxycyclohexane-1-carboxylic acid	C25H24O12	516.126	Phenolic acid	273.62
9	Lupeol	C30H50O	426.385	Triterpenoid	229.73
10	4-Hydroxybenzoic acid	C7H6O3	138.032	Phenolic acid	161.62

). This exceptional concentration of bioactive polyphenols and terpenoids establishes V. trifolia as a promising source of structurally diverse natural products with potential pharmaceutical applications.

2.2. In Silico Analysis

2.2.1. Compounds Annotation and Bioactivity Prediction

Ten natural compounds were detected through LC–HRMS. Quercetin and gallic acid were annotated using PubChem and ChemSpider, along with batimastat and baicalain as collagenase and elastase standards, respectively. Their SMILES structures were used for PASS analysis, Protox-II toxicity prediction, SwissADME screening, and molecular docking. The full structural list, including IUPAC name, PubChem/ChemSpider IDs, SMILE annotations, and weblinks, is provided in Supplementary Table S1. Predictory inhibition tests were conducted on pancreatic elastase, leukocyte elastase, and general collagenase (Figure 2).

PASS predictions showed that quercetin, gallic acid, compound 5, compounds 7 and 8 (cyclohexane-derived phenolic esters), and compound 10 exhibited moderate predicted elastase inhibition (0.3 < Pa < 0.7). On the other hand, lupeol elicits weak potential for elastase inhibition. Moreover, compound 5 exhibited weak general collagenase inhibitors. All other compounds elicit significant MMP-9 inhibitor potential as detected in the PASS server, providing a computational rationale for docking.

2.2.2. Toxicity Prediction and Feasibility Analysis

Toxicity analysis through ProTox-II was performed to determine the toxicity class, LD₅₀, hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity for each natural compound based on the Globally Harmonized System (GHS) standard. Complete toxicity prediction outputs are summarized in Supplementary Table S2. Among the compounds tested, compound 10 showed no activity across all five parameters. Compounds exhibiting low toxicity included compound 1, compound 3, compound 7, and compound 8, all with an LD₅₀ of 5000 mg/kg. Overall, the toxicity levels of the compounds were considered acceptable for progression to the next step, as most exhibited low or moderate toxicity.

2.2.3. ADME and Drug-Likeness Analysis

Furthermore, a feasibility analysis was performed to investigate drug chemical behaviors that would support drug discovery. SwissADME screening showed that seven compounds, which included quercetin, gallic acid, casticin, compound 4, compound 5, compound 6, and compound 10, complied with both Lipinski’s Rule of Five and Veber’s criteria, supporting acceptable predicted oral bioavailability. Full ADME results are presented in Supplementary Table S3.

2.2.4. Molecular Docking with MMP-9 and Elastase

Molecular docking was conducted to evaluate the binding affinity of the natural compound in Vitex trifolia against each target protein. This analysis employed several reference compounds that generally interact with the target protein. Full results of the molecular docking are presented in Supplementary Table S4. Notably, compound 5 (−7.077 kcal/mol) and compound 7 (−8.108 kcal/mol) exhibited stronger binding affinity than batimastat (−2.739 kcal/mol). Some compounds bind favorably to MMP-9, but not elastase, and vice versa, suggesting target selectivity rather than nonspecific binding. While not the top MMP-9 binder, quercetin demonstrated balanced affinity toward both MMP-9 and elastase, making it a useful reference compound. Although elastase standard baicalein remained the strongest binder, several natural compounds show comparable moderate affinity, supporting elastase inhibition moderately. Overall, the results indicate differential target selectivity among the compounds and highlight several candidates with promising binding affinity toward MMP-9 and elastase.

2.2.5. Protein Ligand Complex Visualization

Visualization of the molecular-docking results was performed to examine the binding conformations and intermolecular interactions between the ligands and their respective target proteins. The interactions analyzed included the total number of contacts and the types of interactions involved. Figure 3 presents the three-dimensional binding diagrams and conformational visualizations of the best docking poses. Figure 3a,b illustrates the binding of compound 7, the compound with the highest binding affinity, with the MMP-9 target protein (PDB ID: 6ESM), while Figure 3c,d shows the binding of quercetin to elastase (PDB ID: 2FOE).

Furthermore, analysis of the protein–ligand interactions revealed that the compound 7–MMP-9 complex exhibited four hydrophobic interactions, six hydrogen bonds, and two salt bridges. In contrast, the quercetin–elastase complex predominantly formed hydrogen bond interactions, involving multiple residues within the active site. The detailed protein–ligand interactions are summarized in

Table 3. Molecular docking interactions between selected compounds and target proteins (MMP-9 and elastase).

Ligand–Target Complex	Hydrophobic Interaction	Hydrogen Bonds	Salt Bridges
Compound 7–MMP9	LEU222, VAL223, HIS226, TYR248	LEU188, ALA189, ALA191, HIS226, GLN227, MET247	HIS230, HIS236
Compound 7–Elastase	-	SER225, THR236, VAL224, PHE223, SER222, THR221, GLN200, CYS199, GLY198, SER203, GLY201,	-

.

The strong network of interactions in the best ligand–MMP9 complex suggests the ligand is firmly anchored in the catalytic cleft. This supports its favorable docking score (−8.108 kcal/mol) and shows that it could effectively block substrate access and inhibit MMP9 activity in vitro, if the binding pose is accurate. For the best ligand–elastase interaction, the many hydrogen bonds make up for the lack of salt bridges. This points to a stable binding mode, which explains quercetin’s relatively strong docking score against elastase (−6.065 kcal/mol) and supports its potential as a competitive inhibitor, though it is still slightly weaker than baicalein.

2.3. Antioxidant and Enzymatic Bioactivities

To connect the chemical composition with its functional relevance, antioxidant assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and Ferric Reducing Antioxidant Power (FRAP) were conducted and revealed robust radical-scavenging capacity (

Table 4. Antioxidant activity of Vitex trifolia extract and ascorbic acid based on DPPH, ABTS, and FRAP assay.

Sample	IC50—DPPH (μg/mL)	IC50—ABTS (μg/mL)	FRAP Assay (FeSO4 E/100 g Extract)
Ascorbic acid	5.39 ± 0.11	4.34 ± 0.08	316.04 ± 5.86
V. trifolia leaves extract	63.47 ± 0.24	70.13 ± 1.28	36.33 ± 0.18

). The extract achieved IC₅₀ values of 63.47 ± 0.24 µg/mL (DPPH) and 70.13 ± 1.28 µg/mL (ABTS), effectively neutralizing radicals via hydrogen atom transfer and single electron transfer mechanisms. FRAP analysis confirmed substantial reducing power (36.33 ± 0.18 FeSO₄ E/100 g extract). This multi-mechanistic antioxidant capacity aligns with the phenolic acids and methylated flavonoids identified by LC–HRMS, supporting V. trifolia’s cosmeceutical potential as a broad-spectrum antioxidant.

Given that oxidative stress and enzymatic degradation are interconnected mechanisms in skin aging, the inhibitory activity of V. trifolia leaf extract against elastase and collagenase, two key enzymes involved in dermal ECM degradation, was further assessed (

Table 5. Antielatase and anticollagenase activity of Vitex trifolia extract.

Sample	IC50—Antielastase (μg/mL)	IC50—Anticollagenase (μg/mL)
Quercetin	5.50 ± 0.05	N/A
1,10-phenanthroline	N/A	3.27 ± 0.15
V. trifolia leaves extract	400 ± 0.01	27.94 ± 3.20

). V. trifolia extract displayed a moderate anti-elastase activity (IC₅₀ = 400 ± 0.01 µg/mL) and a superior anti-collagenase activity (IC₅₀ = 27.94 ± 3.20 µg/mL).

2.4. Cytoprotective Activity of Extract

The cell viability of HaCaT cells was plotted against different concentrations of the standard and extracts, as shown in Figure 4. Cytotoxicity was evaluated according to ISO 10993-5, which considers a reduction in cell viability by more than 30% as indicative of cytotoxic potential [16]. Statistical analysis revealed that ascorbic acid significantly affected HaCaT cell viability starting at 50 μg/mL (Figure 4a), although cytotoxic potential was observed from 100 μg/mL. On the other hand, V. trifolia caused significant reductions in cell viability starting at 200 μg/mL, with cytotoxic effects appearing from 800 μg/mL (Figure 4b).

Data from cytotoxic assays were used as a reference for the subsequent cytoprotective assays, in which only non-toxic concentrations of ascorbic acid and extracts were used for pre-treatment. The results of each treatment are shown in Figure 5. As a positive control, ascorbic acid showed significant differences in protection at all concentrations (Figure 5a). Meanwhile, V. trifolia extracts showed a dose-dependent protective capability at concentrations from 12.5 to 200 μg/mL (Figure 5b). This indicates that the cytoprotective action of the extracts is effective within a certain concentration range. Therefore, 12.5 μg/mL was chosen as a treatment dose for the gene expression study, as it is the lowest concentration with a cytoprotective effect and no cytotoxicity for all extracts.

2.5. Analysis of MMP-1 and MMP-9 Expression

The expression of the MMP-1 and MMP-9 genes was evaluated after treating the cells with extracts and then exposing the cells to a UVB light source. UVB exposure was shown to significantly cause the upregulation of MMP-1 and MMP-9 expression in HaCaT cells (Figure 6). Interestingly, the result showed that treatment with V. trifolia reduced the MMP-9 by 51%, though the MMP-1 remained unaffected. Following treatment and UVB-exposure, V. trifolia extract and ascorbic acid significantly reduced MMP-1 expression compared to non-treated controls by 42% and 27%, respectively. Surprisingly, the extract showed a significant reduction of MMP-9 expression compared to controls after UVB exposure by 69%.

3. Discussion

3.1. Extract Characterization and Phytochemical Composition

Plants have long been explored as sources of bioactive compounds for cosmetic applications, largely because of their antioxidant, anti-aging, and skin-protective properties. These include plant extracts, fractions, and compounds [17,18]. However, before these natural products can be incorporated into formulations, their mechanisms of action and safety need to be carefully clarified [19,20]. Phenolic compounds, particularly flavonoids, are major contributors to antioxidant activity and modulation of ECM enzymes [21,22]. In addition to free-radical scavenging, phenolics can modulate enzymes such as elastase and collagenase that are involved in ECM turnover, thereby contributing to anti-aging [23]. Furthermore, their antioxidant activity can provide photoprotective effects against UVB exposure [24].

In this study, the total phenolic content of V. trifolia extract was measured (78.52 ± 0.01 mg GAE/g extract), consistent with earlier reports where V. trifolia extracts generally contained less than 100 mg GAE/g [25]. Meanwhile, the total flavonoid content of the extract was reported as 1.99 ± 0.02 mg QE/g extract. Specialized flavonoids such as casticin and vitexin have been reported in V. trifolia at various values [12,26], and vitexicarpin has been isolated from V. trifolia in phytochemical studies [27]. The differences in variations likely arise from species-specific metabolism and environmental factors. Previous studies reported that altitude can modify phenolic content up to threefold, particularly in regions with high ultraviolet (UV) intensity [28,29]. This is because phenolic compounds function as protective molecules produced in response to environmental stress, including UV radiation [30]. Therefore, ecological and environmental conditions likely contributed to the phenolic differences observed between these species. The extract composition in this study reflects a single collection timepoint. Hence, seasonal or environmental variation may affect phytochemical content, which could be explored in future studies.

3.2. Metabolomic Analysis

Building on this phytochemical foundation, our comprehensive LC–HRMS metabolomic analysis was conducted on a single, well-defined plant source using consistent extraction conditions and provides critical insights into the biochemical basis of V. trifolia’s skin-protective potential and ensures reproducibility for the mechanistic studies. The identification of the ten predominant high-abundance metabolites offers a molecular foundation for interpreting the extract’s functional activities. The dominance of casticin and several structurally related flavonoids aligns closely with previous metabolomic reports on Vitex species [12,25,31], reflecting a phytochemical profile enriched in antioxidant and anti-inflammatory constituents. Casticin is particularly noteworthy. It has been widely documented for its potent ROS-scavenging capacity, its ability to attenuate UV-induced signaling pathways, and its inhibition of MMP activation [20,26,31], consistent with the antioxidant and anti-collagenase effects observed in our assays. Beyond flavonoids, the substantial presence of phenolic acids (e.g., 4-coumaric acid, 4-hydroxybenzoic acid, and conjugated phenolic derivatives) further reinforces the extract’s redox-regulating effects, given their established hydrogen-donating, electron-transfer, and anti-protease properties [24,32,33]. Additionally, the detection of terpenoids, including a diterpenoid derivative and lupeol, suggests complementary bioactivities, as terpenoids have been reported to support skin repair, enhance barrier function, and exert anti-inflammatory effects [34,35,36]. Although these results provide a reliable basis for understanding the extract’s bioactivity, full standardization would still be required for future industrial-scale applications.

3.3. Antioxidant Activity

To contextualize these metabolomic findings, the antioxidant capacity of V. trifolia was evaluated using DPPH, ABTS, and FRAP assays, which together represent major antioxidant mechanisms involving single electron transfer (SET), hydrogen atom transfer (HAT), and ferric-reducing activity [37]. Phenolic compounds are key contributors to plant antioxidant behavior due to their radical-quenching, hydrogen-donating, and electron-transfer properties [3,33,38]. Consistent with this, V. trifolia exhibited strong radical-scavenging performance, with IC₅₀ values of 63.47 ± 0.24 μg/mL (DPPH) and 70.13 ± 1.28 μg/mL (ABTS), indicating efficient neutralization of nitrogen- and carbon-centered radicals [37]. The extract also demonstrated meaningful reducing power in the FRAP assay (36.33 ± 0.18 FeSO₄ E/100 g extract).

Differences among the assays likely reflect the solubility and physicochemical properties of the present antioxidants, as DPPH favors lipophilic molecules while ABTS accommodates a broader polarity range [38]. Even strong antioxidants such as ascorbic acid can show assay-dependent variability, displaying higher IC₅₀ values across assays. Overall, the multi-mechanistic antioxidant activity observed corresponds closely with the LC–HRMS metabolite profile, particularly the abundance of methylated flavonoids, phenolic acids, and terpenoids, supporting a synergistic redox-balancing effect that may help mitigate oxidative stress, a key driver of photoaging and matrix degradation.

It is also worth noting that ascorbic acid has often been used as a reference standard due to its well-established radical-scavenging potency [39]. Although V. trifolia extract showed lower radical-scavenging potency, this is expected for complex plant extracts, which act via multiple synergistic mechanisms beyond simple radical neutralization. This standardization helps contextualize the extract’s antioxidant profile across different assays and studies [40].

3.4. Anti-Enzymatic Activity

Building on this antioxidant potential, further assays were conducted to evaluate the extract’s ability to inhibit key matrix-degrading enzymes involved in skin aging. Elastase, a serine endoprotease, specifically degrades elastin fibers, maintaining skin elasticity. UV radiation-induced ROS upregulates elastase expression up to fourfold, promoting elastosis through abnormal elastin accumulation [41]. Meanwhile, collagenase (MMP-1), a zinc-dependent metalloproteinase, degrades type I and III dermal collagen, leading to wrinkle formation following UV exposure and oxidative stress [42].

The V. trifolia extract exhibited a moderate anti-elastase effect (IC₅₀ = 400 ± 0.01 µg/mL) and a notably strong anti-collagenase activity (IC₅₀ = 27.94 ± 3.20 µg/mL). The superior collagenase inhibition is consistent with the extract’s abundance of flavonoids, particularly casticin and related derivatives, which have been reported to chelate Zn²⁺ at the MMP catalytic site and suppress MMP-1 activity [32,43]. The observed IC₅₀ value falls within the range of established natural inhibitors such as Centella asiatica (45.3 µg/mL) and Camellia sinensis polyphenols (~30 µg/mL) [42,44], highlighting the potential of V. trifolia as a promising anti-collagenase agent for preventing UV-induced dermal collagen degradation in cosmetic applications.

Meanwhile, plant extracts demonstrated lower inhibition activity than standards. Quercetin and 1,10-phenanthroline are widely used as reference inhibitors due to their high potency in enzyme assays [42,43]. Plant extracts, by contrast, are complex mixtures in which many constituents contribute moderate inhibitory effects rather than a single high-affinity interaction, which typically yields lower IC₅₀ values in vitro. Such comparative use of potent standards also helps contextualize the extract activity within the broader literature and does not diminish the biological relevance of multi-constituent inhibition in cosmeceutical applications.

3.5. Molecular Docking Insights

Moreover, to further elucidate the molecular basis of enzyme inhibition, in silico molecular docking was conducted against MMP-9 and elastase. Several natural compounds exhibited favorable binding affinities, with some demonstrating stronger predicted interactions toward MMP-9 than the reference inhibitor. Molecular docking is a well-established computational approach for predicting ligand–protein interactions and estimating binding affinity, which has been widely used to support bioactivity hypotheses in natural product research [45]. Docking protocols help predict how molecules interact. Hydrophobic contacts keep the ligand in nonpolar pockets, which lowers the desolvation penalties and stabilizes the complex. In MMP-9, this likely means that the ligand stays bound longer and inhibits the enzyme more effectively than ligands with fewer contacts. Hydrogen bonds and salt bridges create specific interactions that help the ligand fit key active-site residues, ensuring proper orientation. In MMP-9, this likely means that the ligand stays bound longer and inhibits the enzyme more effectively than ligands with fewer contacts. These strong hydrophobic and hydrogen-bond interactions with key residues enhance inhibitor efficacy, and similar principles apply to elastase [46,47,48]. These results suggest that Vitex ligands can mimic important contacts of known inhibitors and may compete with the natural substrates of MMP-9 and elastase.

Notably, the best-performing ligand formed multiple hydrogen bonds, hydrophobic interactions, and salt bridges with key residues within the MMP-9 active site, suggesting a stable and potentially inhibitory binding mode. This is consistent with previous docking studies linking these interaction types to inhibitory potential, with hydrogen bonding and hydrophobic contacts identified as major contributors to ligand stability and binding affinity [49]. In contrast, elastase docking revealed a different interaction profile, with hydrogen bonding dominating the interactions and quercetin showing the strongest binding affinity among the tested compounds. These findings support the experimental enzyme inhibition results and indicate target-dependent selectivity among the V. trifolia constituents. These in silico findings complement the experimental enzyme inhibition results reported above and are consistent with recent reports that phytochemicals such as quercetin and related flavonoids can exhibit target-dependent binding preferences and interaction profiles across different enzymes [50].

3.6. Cytotoxicity and UVB-Induced Cytoprotection

Following the enzymatic inhibition results, the cellular safety of V. trifolia extracts was evaluated in HaCaT cells, a human keratinocyte model for epidermal studies, thus a model for topical formulations [51]. Cytotoxicity was assessed by measuring cell viability at different extract concentrations using the one-step 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, a colorimetric method in which metabolically active cells reduce tetrazolium to a soluble formazan dye [52,53]. Based on ISO 10993-5 criteria, extracts that reduce cell viability by more than 30% indicate cytotoxic potential [16]. Ascorbic acid, the positive control, expressed cytotoxic effects starting from a concentration as low as 100 μg/mL. This cytotoxicity is attributed to ascorbic acid’s ability to generate extracellular hydrogen peroxide, particularly in the presence of metal ions, leading to oxidative stress and cell death [54,55,56]. The extent of toxicity depends on the cells’ capacity to detoxify hydrogen peroxide, primarily via catalase, with cells expressing lower catalase levels (such as HaCaT) being more susceptible [55]. V. trifolia exhibited a relatively wide safety margin, remaining non-toxic at concentrations as high as 800 μg/mL, unlike that of ascorbic acid. These results are consistent with the previous safety data in other cell lines [57,58]. In this context, the broader non-cytotoxic range of V. trifolia extract highlights an advantage for topical application over ascorbic acid.

Subsequently, a cytoprotective assay was performed to determine whether the extracts could mitigate UVB-induced oxidative damage in keratinocytes, evaluating a compound’s ability to preserve cell viability and integrity under stress conditions such as UV exposure or chemical insult [59]. In this study, the cell was treated with extracts and exposed to UVB light at a 2.484 J/cm² dose for 2 h after 24 h of treatment, a dose under the range commonly used to model 40 min of midday sun exposure with a UV index of six [60]. The cytotoxic effects that were exhibited from UVB irradiation to HaCaT cells were likely caused by increases in intracellular ROS levels, resulting in oxidative stress that damages components of cells, such as proteins, DNA, and lipids [61]. This oxidative damage reduces the viability and functionality of cells. Moreover, UVB radiation promotes the production of ROS, including hydrogen peroxide (H₂O₂), hydroxyl radicals (-OH), and superoxide anion radicals (O₂⁻) [62]. These ROS are produced by UVB-induced photochemical reactions and cellular metabolic activities.

Both the extract and ascorbic acid demonstrated cytoprotective effects within specific concentration ranges. Ascorbic acid exhibited protection at a concentration range as low as 3.125–25 μg/mL, consistent with its known antioxidant activity at low doses [63,64]. However, at ≥50 μg/mL, it became pro-oxidant, producing hydrogen peroxide and ROS that induced apoptosis and reduced cell viability [63]. Meanwhile, V. trifolia extract exhibited protection starting at a concentration of 12.5 μg/mL. The activity of plant extracts is dose-dependent, with higher concentrations potentially triggering apoptosis or cell cycle arrest due to the combined effects of multiple bioactive compounds [65]. In contrast, V. trifolia extract displayed a broader protective range (12.5–50 μg/mL), consistent with reports of its ability to reduce ROS and modulate inflammatory signaling pathways [36]. The extract’s cytoprotective function is enhanced by its capacity to control inflammatory cytokines and signaling pathways, which enables it to sustain cell viability over a wider concentration range than extracts with fewer effective dosages.

Importantly, although ascorbic acid exhibited stronger cytoprotective effects at low concentrations, its protective window was narrow and rapidly transitioned to cytotoxicity at higher doses due to its pro-oxidant effects. This is a well-known advantage and disadvantage of ascorbic acid [66,67,68]. In contrast, V. trifolia extract maintained cytoprotective activity across a wider concentration range without inducing a marked loss of cell viability, indicating a more favorable balance of safety and efficacy. Future studies using reconstructed human skin or in vivo photodamage models could further validate the translational relevance of these cytoprotective findings.

3.7. Mechanistic Insights into MMP Regulation

As previously discussed, UVB exposure rapidly generates ROS in keratinocytes, resulting in oxidative stress that is a central trigger for skin damage and photoaging [69]. To explore the potential mechanism underlying these effects, we highlight a likely involvement of nuclear factor kappa-chain of B cells/mitogen-activated protein kinase (NF-κB/MAPK) signaling in regulating MMP expression. ROS can activate key intracellular signaling pathways, primarily the MAPK cascade (which includes ERK, JNK, and p38 [70,71]. By activating the MAPKs, the transcription factor complex activator protein 1 (AP-1), which is the primary regulator of MMP gene transcription, was then phosphorylated and activated [72]. AP-1 is then bound to TPA-responsive elements in the promoter region of MMP-1 and MMP-9 genes, hence upregulating their expression [71]. While the results of our in vitro antioxidant assays are consistent with the attenuation of UVB-induced oxidative stress, intracellular ROS levels were not directly quantified and were inferred from downstream functional markers.

Our results suggested that flavonoids and phenolic compounds in the extracts may act as ROS scavengers, thereby reducing the oxidative stress triggered by UVB exposure and subsequently suppressing the NF-κB/MAPK pathway activation that drives MMP gene expression. Such mechanisms align with previous findings showing that flavonoid-rich extracts inhibit UVB-induced MAPK activation and downstream MMP upregulation [70]. Consistent with this, UVB irradiation markedly elevated MMP-1 and MMP-9 expression in HaCaT cells, as expected from UVB-induced ROS stimulation of AP-1-mediated transcription [72]. Treatment with V. trifolia extract demonstrated a notable modulatory effect, in which under non-UVB conditions, it reduced basal MMP-1 expression by 42% and MMP-9 by 69%, suggesting intrinsic anti-inflammatory or matrix-regulating activity, which is consistent with reports of V. trifolia flavonoids suppressing pro-inflammatory gene expression [31]. In contrast, ascorbic acid showed only moderate suppression of MMP-1, likely due to its limited stability and tendency to act as a pro-oxidant under oxidative stress [73]. Because UVB-driven ROS strongly induces both MMP-1 and MMP-9, the substantial inhibition observed indicates that V. trifolia effectively attenuates upstream oxidative and inflammatory cascades, with its particularly strong suppression of MMP-9 highlighting its potential as a potent anti-photoaging agent [6].

3.8. Summary of Findings

From a formulation perspective, these findings also highlight an important distinction between reference antioxidants and complex botanical extracts. Ascorbic acid is widely used as a benchmark compound due to its well-established radical-scavenging potency. However, its activity largely reflects a single, highly reactive antioxidant mechanism. In contrast, we also demonstrated that V. trifolia extract integrates multiple bioactive constituents that collectively provide antioxidant, enzyme-inhibitory, and cytoprotective effects while maintaining a broader safety margin. This multi-mechanistic profile is particularly relevant for cosmeceutical applications, where sustained efficacy and cellular tolerance are critical.

Overall, the findings of this study demonstrate that V. trifolia possesses a multifaceted skin-protective potential, combining strong antioxidant activity, selective enzyme inhibition, cytoprotective effects, and suppression of UVB-induced MMP expression. Its rich flavonoid and phenolic content underlie both its ROS-scavenging capacity and its modulation of inflammatory and matrix-degrading pathways, suggesting a dual role in preventing oxidative damage and mitigating ECM breakdown. The broad safety margin observed in HaCaT cells further supports its suitability for topical application. Collectively, these results highlight V. trifolia as a promising natural ingredient for anti-photoaging cosmetic formulations, capable of protecting skin from UV-induced oxidative stress while regulating key enzymes and pathways implicated in wrinkle formation and tissue degradation. Future studies exploring formulation stability, bioavailability, and in vivo efficacy will be essential to fully realize its cosmeceutical potential.

4. Materials and Methods

4.1. Extract Preparation

Leaves of Vitex trifolia were collected from a single geographical location at a single timepoint to minimize variability due to environmental or seasonal factors. Leaves were taken in Bogor, West Java, Indonesia, and taxonomically identified by the Biopharmaca Research Center, Bogor, Indonesia (voucher specimen number BMK0171092016). The leaves were cleaned, dried in a drying cabinet at 30–40 °C for 5 days, ground into powder, and sieved. V. trifolia was extracted using MAE (Sharp Corporation, Osaka, Japan) with 96% ethanol at a 1:10 (w/v) ratio of leaf powder to solvent. The extraction was performed at low microwave power (30%) for 10 min and repeated three times, followed by filtration. The combined filtrates were concentrated using a rotary evaporator and further thickened in a water bath until a constant mass was obtained.

4.2. Determination of Total Phenolic Content (TPC)

The Folin–Ciocalteu (FC) method was used to examine the TPC of the extract. Gallic acid (GAE) was used as a standard for calibration (5 to 50 μg/mL concentration). In a 96-well plate, 15 µL each of standard or sample was mixed with 75 µL of 10% (v/v) FC Reagent (Merck, Darmstadt, Germany). After 8 min, 75 µL of 7.5% sodium bicarbonate was added. The plate was incubated for one hour at room temperature, and the absorbance was measured at 765 nm. The experiment was conducted in triplicate. Results were expressed as mg gallic acid equivalent (GAE)/g extract.

4.3. Determination of Total Flavonoid Content (TFC)

TFC was measured using the aluminum chloride colorimetric method. Quercetin was used as the standard for calibration (5 to 50 μg/mL concentration). In a 96-well plate, 20 µL of each standard or sample was mixed with 80 µL methanol. Subsequently, 6 µL of 5% sodium nitrate was added, followed by 6 µL of 10% aluminum chloride after 3 min. After an additional 3 min, 40 µL of 1 M sodium hydroxide were added. The experiment was performed in triplicate, and the absorbance was measured at 510 nm. TFC was expressed as mg quercetin equivalent (QE)/g extract.

4.4. Metabolomic Profiling by Liquid Chromatography–High Resolution Mass Spectrometry (LC–HRMS)

LC–HRMS analysis was performed using a Vanquish™ UHPLC system coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with electrospray ionization (ESI) in positive mode. Chromatographic separation was achieved on an Accucore™ Phenyl-Hexyl column (100 × 2.1 mm, 2.6 µm; Thermo Fisher Scientific, Bremen, Germany) using a mobile phase consisting of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B) at 0.3 mL·min⁻¹. The gradient program increased from 5% to 90% B over 16 min, followed by a 4 min isocratic hold at 90% B and then re-equilibration to initial conditions (total run time: 25 min). Samples (5–10 mg) were dissolved in MS-grade solvent, ultrasonicated for 30 min, and filtered through 0.22 µm polytetrafluoroethylene (PTFE) membranes before injection (3 µL). Full MS/data-dependent MS² (dd-MS²) spectra were acquired over m/z 66.7–1000 with resolving powers of 70,000 (full MS) and 17,500 (MS²) using the following parameters: spray voltage 3.30 kV, capillary temperature 320 °C, and sheath/auxiliary/sweep gas flows at 32/8/4 AU, respectively. Data processing was performed using Compound Discoverer™ 3.2 software (Thermo Fisher Scientific, Bremen, Germany), with compound annotation based on spectral matching against the mzCloud™ (https://www.mzcloud.org, accessed on 20 November 2024 and ChemSpider™ (http://www.chemspider.com, accessed on 20 November 2024) databases using a ±5 ppm mass accuracy threshold.

4.5. Molecular-Docking Study

4.5.1. Compound Annotation

Vitex trifolia natural compounds and target proteins collection Simplified Molecular Input Line Entry System (SMILES) notation from natural compounds from V. trifolia was taken from PubChem (https://pubchem.ncbi.nlm.nih.gov/, accessed 26 November 2025), based on LC–HRMS. A few general phenolics commonly found in Vitex trifolia were casticin, 4-coumaric acid, lupeol, and 4-hydroxybenzoic acid. The drug batimastat (PubChem ID: 5362422) is used as the standard ligand for collagenase inhibition, and the drug baicalein (PubChem ID: 5281605) is the standard ligand for elastase inhibition.

4.5.2. Virtual Screening

The biological activity analysis of the natural compounds included anticarcinogenic and apoptosis agonist based on probability of activity (Pa value) and probability of inactivity (Pi value) from each compound using the prediction of activity spectra for substances (PASS) ONLINE web server (http://way2drug.com/passonline/index.php, accessed 26 November 2025). This analysis was based on the relation between the structure of a compound and the activity that it has. Pa score is the probability that a compound becomes “active”. This predicts the probability that the compound that is being studied is categorized in the sub-class of active compounds. Pi score is the probability of becoming “inactive”, where this predicts the probability that a compound is included in the inactive category.

4.5.3. Target Protein Selection

Three-dimensional crystal structures of target proteins were obtained from the Protein Data Bank (https://www.rcsb.org/, accessed 26 November 2025). MMP-9 (PDB ID: 6ESM) was selected for collagenase activity, and porcine pancreatic elastase (PDB ID: 2FOE) for elastase activity. Both proteins were selected as standards for their respective bioassay enzyme activities.

4.5.4. Toxicity and Feasibility Analysis

Toxicity profiles, including LD₅₀ estimation and GHS classification of toxic compounds, were determined using ProTox-II (https://tox-new.charite.de/protox_II/, accessed 26 November 2025). A feasibility analysis was performed to calculate the physicochemistry parameters, predict the absorption distribution, metabolism, and excretion (ADME) parameters, and determine the pharmacokinetic properties. SMILE notation from the natural compounds of V. trifolia, which has been obtained from PubChem, was analyzed using the Swiss ADME web server (http://www.swissadme.ch/, accessed 26 November 2025).

4.5.5. Molecular Docking and Visualization

Docking simulations were conducted using SWISS-DOCK to evaluate binding affinities and ligand–protein interactions between the V. trifolia compounds and target proteins. Specific docking grids were applied while other parameters were set to default. The binding affinity result and the docking conformation were retrieved for further analysis. Docking conformations were analyzed using Biovia Discovery Studio Visualizer 2021 (Dassault Systèmes BIOVIA, San Diego, CA, USA) to examine the hydrogen bonding and binding orientations in both 2D and 3D views. PLIP online software and CB-Dock 2 (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index,accessed 26 November 2025; https://cadd.labshare.cn/cb-dock2/, accessed 26 November 2025) were used to visualize both proteins for the best ligand binding.

4.6. Antioxidant Assays

4.6.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay

A DPPH assay was conducted based on colorimetric reaction [74]. The DPPH reagent was made at a concentration of 0.1 mM. Extract samples were diluted into a series of concentrations (12.5 up to 800 μg/mL). Ascorbic acid was used as a positive control (3.125 to 100 μg/mL). In a 96-well plate, equal volumes of each extract or standard solution and the DPPH solution were mixed. Methanol was used as the solvent blank. The plate was incubated in the dark at room temperature for 30 min. Following incubation, absorbance was measured at 517 nm. The percentage of DPPH radical scavenging activity was calculated using the formula:

$$\% Scavenging activity = {\displaystyle \frac{(Absorbance blank - Absorbance sample)}{Absorbance blank}}\times 100\%$$

(1)

4.6.2. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) Assay

An antioxidant activity test with the ABTS method was conducted by following [74], with minor modifications. ABTS as a radical was made by mixing 5 mL of 7 mM ABTS with 88 μL of 140 mM K₂S₂O₈ solution, followed by incubation for 16 h in the dark and dilution with distilled water at a ratio of 1:15. Ascorbic acid was used as a standard (1 to 13 μg/mL concentration). Plant extracts were diluted in methanol. In a 96-well plate, 100 µL of extract or standard and 100 µL of diluted ABTS reagent were added, then incubated in the dark at room temperature for 6 min. The absorbance was measured at 745 nm. Using the linear curve obtained from Equation (1), IC₅₀ was calculated with Equation (2):

$$I{C}_{50} (\mu \mathrm{g}/\mathrm{m}\mathrm{L})= {\displaystyle \frac{50-\mathrm{c}}{Absorbance blank}}\times 100\%$$

(2)

where c stands for constant, and m stands for slope in a linear equation (y = mx + c).

4.6.3. Ferric Reducing Antioxidant Power (FRAP) Assay

Antioxidant activity test with the FRAP method was conducted by following [75], with a minor modification. The FRAP reagent was prepared by mixing 25 mL of 300 mM acetate buffer, 2.5 mL of 20 mM FeCl₃·6H₂O, and 2.5 mL of 10 mM TPTZ in 40 mM HCl (10:1:1, v/v/v). FeSO₄·7H₂O was used as a standard (75 to 200 μM concentration), while ascorbic acid (10 μg/mL) was used as the positive control. In a 96-well plate, 50 µL of plant extract or standard solution were mixed with 150 µL of FRAP reagent and incubated at 37 °C for 30 min in the dark. Absorbance was measured at 597 nm using a microplate reader. The FRAP value was determined with Equation (3):

$$FRAP value (\mu \mathrm{mol} {Fe}^{2+}/\mathrm{g} sample)= {\displaystyle \frac{x}{K}}$$

(3)

where x is the concentration equivalent from the calibration curve FeSO₄·7H₂O (µM), and K is the sample concentration (g/L).

Antioxidant activity was then expressed as FeSO₄·7H₂O equivalents (g/100 g sample) and calculated as Equation (4):

$$AA(FeSO4·7H2O Eq)= {\displaystyle \frac{FRAP\times Mr\times 100 \mathrm{g} sample}{{10}^6\mu \mathrm{m}\mathrm{o}\mathrm{l}}}$$

(4)

4.7. Antielastase Assay

Elastase inhibition was evaluated following [76] with modifications. Extracts were dissolved in the Tris-HCl buffer (100 mM, pH 8). Quercetin was used as a positive control. In a 96-well plate, sample solutions were mixed with Tris-HCl buffer and 20 µL of elastase (0.22 U/mL), followed by incubation at 25 °C for 15 min. Subsequently, 30 µL of 1.3 mM N-succinyl-(Ala)3-p-nitroanilide (SANA) substrate were added, and the mixture was incubated for 30 min at room temperature. Absorbance was measured at 405 nm using a microplate reader. Antielastase activity was calculated based on Equation (5):

$$Antielastase activity \left(\%\right)= {\displaystyle \frac{\left[\left(ABE-AB\right)-\left(ASE-AS\right)\right]}{\left(ABE-AB\right)}}\times 100\%$$

(5)

where ABE is the absorbance of blank with the enzyme, AB is the absorbance of the blank without the enzyme, ASE is the absorbance of the extract or control with the enzyme, and AS is the absorbance of the extract or control without the enzyme.

Subsequently, the concentration of the sample needed to inhibit 50% enzyme (IC₅₀) was calculated based on the calibration curve calculated using Equation (2).

4.8. Anticollagenase Assay

Anticollagenase activity was determined according to the method of [23], with modifications. The anti-collagenase activity of V. trifolia leaf extract was evaluated against the standard inhibitor 1,10-phenanthroline using a microplate reader based on the hydrolysis of the synthetic substrate FALGPA by Clostridium histolyticum collagenase (ChC). Each extract solution is first diluted with 2 µL 2% DMSO. The reaction mixture consisted of 35 µL of ChC solution (1.65 U/mL), extracts/standard sample solution, and Tris-HCl buffer (100 mM, pH 7.5) to a final volume of 200 µL. This mixture was incubated at 37 °C for 20 min. The reaction was initiated by adding 33 µL of 0.33 mM FALGPA solution. The final concentration ranges were 1–5 μg/mL for 1,10-phenanthroline and 10–50 μg/mL for V. trifolia leaf extract. The decrease in absorbance of FALGPA was monitored at 345 nm for 15 min using a microplate reader (Sigma-Aldrich, St. Louis, MO, USA). Substrate-free blanks and enzyme-free controls were included. Sample and blank readings were corrected using the enzyme-free controls. All assays were performed in triplicate. The inhibitory effect of the sample on anti-collagenase activity was calculated using the same equation as Equation (5). Subsequently, the concentration of the sample needed to inhibit 50% enzyme (IC₅₀) was calculated based on the calibration curve calculated using Equation (2).

4.9. Cell Culture

Immortalized human keratinocytes (HaCaT) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA), 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), and 1% penicillin–streptomycin (Gibco, Grand Island, NY, USA). Cells were cultured in a humidified incubator at 37 °C with a 5% CO₂ atmosphere. Upon reaching 80–90% confluency, cells were harvested with 0.25% trypsin–EDTA (Gibco, Grand Island, NY, USA).

4.10. Cytotoxicity Assay

The cytotoxic effects of Vitex trifolia extract as samples and ascorbic acid as a positive control were evaluated under ISO 10993-5 criteria using the MTS assay (Promega, Madison, WI, USA), also known as a one-step 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HaCaT cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well and incubated under standard culture conditions (37 °C, 5% CO₂). After 24 h and reaching 80–90% confluency, the culture medium was replaced with fresh media containing the test compounds at varying concentrations (12.5–1600 μg/mL). Following a 24 h incubation, the treatment media were removed and replaced with fresh media. A volume of 20 μL of MTS reagent was added to each well. The plates were incubated for 3 h in the dark (37 °C, 5% CO₂), and absorbance was measured at 490 nm using a microplate reader (TECAN, Männedorf, Switzerland). Cell viability (%) was calculated by comparing the absorbance of cells treated with extracts to that of cells without treatment.

4.11. Cytoprotective Assay Against UVB-Induced Damage

HaCaT cells were seeded and cultured as described in Section 4.7. Upon reaching 80–90% confluency, the cells were pretreated for 24 h with non-cytotoxic concentrations of V. trifolia extract or ascorbic acid (concentrations determined from Section 4.8). After pre-treatment, the medium was removed, and the cells were washed once with phosphate-buffered saline (PBS). Cells were then subjected to UVB irradiation inside a UV box at a distance of 10 cm from the light source at a dose of 2.484 J/cm² over a 2 h exposure period. Wells containing untreated (media-only) cells and wells protected from UVB using aluminum foil served as controls. Immediately following UVB exposure, the medium was replaced with fresh media containing MTS reagent (5:1 ratio), and the plate was incubated in the dark for 3 h (37 °C, 5% CO₂). Absorbance measurement and cell viability calculation followed the exact procedure detailed in Section 4.8.

4.12. Gene Expression Analysis of MMP-1 and MMP-9 Using Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

HaCaT cells were seeded, pretreated with non-cytotoxic concentrations of the extracts/ascorbic acid for 24 h, and then subjected to UVB irradiation (2.484 J/cm² for 2 h) exactly as described in Section 4.9. Following the 2 h UVB exposure, the cells were incubated for 24 h before being harvested for RNA isolation. Total RNA was extracted from the cells using the Total RNA mini kit (Geneaid, New Taipei City, Taiwan) according to the manufacturer’s protocol. RNA concentration and purity were determined using a NanoDrop Lite Plus Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) with an A260/A280 ratio criterion. Gene expression was analyzed by synthesizing cDNA from the RNA template using ReverTra Ace^TM qPCR RT Master Mix (Toyobo, Osaka, Japan). Synthesized cDNA underwent PCR amplification using SensiFAST SYBR No-ROX Kit (Meridian Bioscience, Cincinnati, OH, USA) and primers. Target genes included MMP-1 and MMP-9. The reference gene used for normalization was GAPDH. Primer sequences for all genes were obtained and purchased from Macrogen, Seoul, Republic of Korea. The primer sequences were GAPDH (forward 5′-AAGCCTGCCGGTGACTAACT-3′, reverse 5′-TCGCTCCACCTGACTTCC-3′), MMP-1 (forward 5′-CATGCTTTTCAACCAGGCCC-3′, reverse 5′-GGTACATCAAAGCCCCGAT-3′), and MMP-9 (forward 5′-TTTGACAGCGACAAGAAGT-3′, reverse 5′-CATTCACGTCCTTATGC-3′). RT-qPCR was performed on RT-PCR Rotor Gene Q (Qiagen, Hilden, Canada) using the thermal cycling conditions recorded in

Table 6. Thermal profile used for qRT-PCR amplification.

Step	Cycle	Temperature (°C)	Duration
Initial denaturation	1	95	2 min
Denaturation	40	95	5 s
Annealing		65	10 s
Extension		72	20 s

. Relative gene expression was calculated using the comparative Ct method (2^−ΔΔCt).

4.13. Data and Statistical Analysis

The data obtained from the experiment were analyzed and processed with GraphPad Prism 10. Normality was assessed using the Shapiro–Wilk test. Data that were normally distributed were statistically analyzed using a one-way analysis of variance (ANOVA). Dunnett’s post hoc test was conducted to compare multiple treatment groups to a single control group, specifically on the cytotoxic and cytoprotective assays. Meanwhile, Tukey’s post hoc test was conducted to compare all possible pairs of means, specifically in gene expression studies. The significance level of p < 0.05 was considered statistically significant.

5. Conclusions

Vitex trifolia ethanolic leaf extract demonstrates substantial cosmeceutical potential through a combination of antioxidant, enzymatic, and photoprotective activities. Its flavonoid- and phenolic-rich composition effectively scavenges UVB-induced ROS, attenuating MAPK/NF-κB signaling and leading to significant downregulation of MMP-1 and MMP-9 expression. The extract exhibits stronger collagenase inhibition relative to elastase, which is supported by the molecular docking results showing favorable interactions. Furthermore, the extract is non-cytotoxic up to 100 μg/mL and provides cytoprotection in HaCaT cells at 12.5–50 μg/mL. These multi-targeted actions suggest that V. trifolia is a promising natural ingredient for preventing extracellular matrix degradation and photoaging, supporting its safe and effective use in skin-protective cosmeceutical formulations. This multi-layered mechanism of action, which is supported by demonstrated antioxidant potency, enzyme inhibition, cytoprotection against UVB damage, and MMP modulation, may offer advantages over single-compound antioxidants in topical skin-care applications.