Evaluation of the Therapeutic Potential and Safety Profile of Six Salvia Species Native to Türkiye

Abstract

The genus Salvia L. represents one of the most pharmacologically significant groups within the Lamiaceae family. This study investigates the phytochemical profiles and biological activities of six Salvia species native to Türkiye (S. dorystaechas B.T.Drew, S. sclarea L., S. glutinosa L., S. tomentosa Mill., S. argentea L., and S. aethiopis L.) to scientifically validate their extensive use in Turkish traditional medicine. Phytochemical characterization was performed using Liquid Chromatography–High-Resolution Mass Spectrometry (LC-HRMS), while biological potential was evaluated through antioxidant (DPPH), antimicrobial (MIC), and cytotoxicity (MTT on NIH/3T3 cells) assays. Among the taxa, S. dorystaechas exhibited the most potent antioxidant activity, with IC₅₀ values of 0.090 mg/mL (infusion) and 0.072 mg/mL (ethanolic), which strongly correlated with high total phenolic contents (111.50 and 125.55 mg GAE/g, respectively). This species may also serve as a potential source of bioactive compounds. Antimicrobial screenings against pathogenic bacteria and Candida spp. demonstrated modest inhibitory effects, with MIC values ranging from 625 to >5000 µg/mL. Safety profiling indicated that the ethanolic extract of S. tomentosa showed the lowest cytotoxicity (IC₅₀ 562.37 ± 49.50 µg/mL) among the tested samples, which nonetheless indicates a relatively narrow therapeutic window. LC-HRMS profiling revealed the presence of flavonoids and phenolic diterpenes, such as carnosol and rosmanol, providing a chemical rationale for the observed moderate activities. Consequently, rather than direct systemic pharmacological agents, these findings suggest that the studied Salvia species could serve as preliminary botanical sources for the isolation of specific secondary metabolites or for restricted topical applications.

1. Introduction

Globally, the Lamiaceae family represents a major botanical group, encompassing over 8000 species across 232 genera [1]. Türkiye stands as a critical genetic reservoir for this family, hosting 48 genera and 782 taxa, with a remarkable endemism rate of 44% [2]. Among these, the genus Salvia L. is particularly prominent, ranking second in Türkiye’s Lamiaceae flora with 107 taxa. Notably, approximately 10% of the world’s Salvia species are found in Türkiye, and more than half of these (54%) are endemic. This high level of biodiversity and endemism underscores Türkiye’s status as a vital evolutionary hub and offers unique opportunities for both taxonomic research and pharmacological exploration [1,2].

The Lamiaceae family includes plants used for a wide variety of purposes, including medicinal, food, spice, cosmetic, pharmaceutical, and ornamental purposes, from historical times to the present. In traditional medicine, these plants are frequently employed to treat gastritis, infections, dermatitis, bronchitis, and inflammatory disorders [3,4]. Ethnobotanical research in Türkiye demonstrates that Lamiaceae species are commonly used to address gastrointestinal, ear, nose, throat, and respiratory system disorders, primarily through infusion and decoction preparations [5]. According to Ertuğ et al., the most frequently used genera include Sideritis L., Salvia, Thymus L., and Origanum L., predominantly consumed as teas for conditions such as coughs, colds, and indigestion [6]. Additionally, another study identified the Salvia genus as the most represented among medicinally used Lamiaceae in Türkiye, with 37 taxa documented [5].

The therapeutic potential of major Salvia species, such as S. officinalis L., S. sclarea L., and S. fruticosa Mill., is internationally recognized and validated through their inclusion in various scientific monographs and pharmacopoeias, including the European and Turkish Pharmacopoeias [7,8,9,10,11,12].

Among natural resources, plants used for medicinal purposes exhibit various pharmacological effects due to the secondary metabolites they produce. In this context, the Lamiaceae family, which plays a significant role in essential oil production, contains numerous bioactive compounds, including terpenic compounds, flavonoids, lignans, coumarins, phenylpropanoids, iridoid glycosides, tannins, and fatty acids. The presence of these compounds confers antimicrobial, antiviral, antioxidant, anti-inflammatory, insect repellent, anthelmintic, hepatoprotective, antihypertensive, and analgesic properties on species belonging to this family [5]. Salvia species are primarily distributed across the Irano-Turanian and Mediterranean phytogeographic regions of Türkiye, exhibiting diverse morphological forms ranging from herbaceous to shrubby structures [13]. Beyond their botanical diversity, numerous Salvia taxa are deeply rooted in Turkish traditional medicine. According to a study [14], species such as S. officinalis, S. sclarea, S. multicaulis Vahl., S. aethiopis, and S. tomentosa are traditionally utilized for their sedative, antiseptic, and wound-healing properties, as well as for treating gastrointestinal disorders. Notably, S. dorystaechas holds significant ethnopharmacological value, with its leaves traditionally prepared as infusions to alleviate respiratory tract infections [15]. This therapeutic versatility is underpinned by a remarkably diverse chemical landscape; more than 150 polyphenolic compounds, including specialized phenolic acids and flavonoids, have been characterized within the genus [16]. The intricate synergy of these secondary metabolites provides the chemical rationale for the extensive biological activities and pharmacological potential associated with Salvia species [17].

The Salvia genus remains a cornerstone of pharmaceutical botany due to its diverse secondary metabolites and multi-target bioactivities. While foundational studies established the phenolic-antioxidant correlation in common species like S. officinalis [18], recent research has shifted towards unexplored endemic taxa and advanced analytical precision. Comparative characterizations have identified distinctive markers such as kaempferol in S. brachyantha (Bordz.) Pobed [19] and rutin in S. transsylvanica (Schur ex Griseb. & Schenk) Schur. [20]. Reflecting the latest trends, modern investigations now integrate high-resolution LC-MS metabolomics with chemometric tools such as PCA and hierarchical clustering to decode complex chemical scaffolds and validate the high antioxidant potential of species like S. huberi Hedge [21,22,23].

The selection of the Salvia species investigated in this study was guided by their extensive documentation in Turkish ethnomedicine. These taxa are widely utilized in traditional healing practices across Anatolia, primarily for treating respiratory infections, digestive disorders, and inflammatory conditions. To provide a comprehensive ethnobotanical context for our phytochemical and biological evaluations, the local names, utilized plant parts, preparation methods, and specific traditional medicinal indications of the studied species are summarized in

Table 1. Ethnobotanical information: local names and traditional medicinal uses of the studied Salvia species.

Species	Local Name	Part Used/Preparation	Traditional Uses	References
S. sclarea L.	Misk adaçayı, Ayıkulağı, Tüylü adaçayı, Paskulak	Aerial parts, leaves, flowers, powder, infusion, essential oil	Alleviation of symptoms in cold, cough, influenza, bronchitis, and asthma; sedative effects for gastric spasms, constipation.	[4,13,24,25]
S. argentea L.	Gümüş şalba, Boz şalba	Aerial parts, leaves, rhizomes, whole plant, powder, decoction, infusion	Respiratory, digestive, and hemostatic agent.	[4,26,27]
S. aethiopis L.	Habeş adaçayı, Yünlü adaçayı	Aerial parts, leaves, inflorescences, roots, infusion	Colds, flu, asthma, gastric spasms, stimulant, carminative, antiflatulent, reconstituent.	[4,13,24,28,29]
S. dorystaechas B.T.Drew	Dağ Çayı, Çalba çayı, Devren kekiği	Fresh/dried leaves, infusion	Treatment of colds and upper respiratory tract infections.	[24,30]
S. tomentosa Mill.	Şalba	Aerial parts, leaves, infusion	Gastrointestinal disorders, abdominal pain, colds, flu, and pharyngitis.	[4,31,32,33,34,35]
S. glutinosa L.	Oklu şalba, Yapışkan adaçayı	Aerial parts, leaves, flowers, infusion, decoction	Headaches, gastroenteritis, abdominal pain, throat infections, mouth sores, cough, sweating, burns, and wounds.	[36,37,38]

.

Despite the extensive research on the Salvia genus, there remains a need for comprehensive comparative characterization of specific endemic taxa within the Anatolian flora. This study distinguishes itself by providing high-resolution metabolite profiling of six Salvia species (S. dorystaechas, S. sclarea, S. glutinosa, S. tomentosa, S. argentea, and S. aethiopis). Beyond traditional bioactivity screenings, the present research establishes a standardized multi-target evaluation by comparing traditional infusions and via LC-HRMS, including the under-investigated endemic S. dorystaechas. ethanolic extracts, integrated with a toxicological assessment on the NIH/3T3 cell line. By bridging high-resolution analytical techniques with safety–efficacy assessments under identical experimental conditions, this approach aims to eliminate the inter-laboratory variability found in fragmented literature. This approach is intended to facilitate the scientific validation for the ethnomedicinal applications of these species and offers new insights into their potential as natural pharmacological resources.

2. Results

2.1. Extract Yields

The extraction yields of the studied Salvia species, obtained through 5% infusion and 70% ethanol maceration, are summarized in

Table 2. Extraction yields, total phenolic contents, and antioxidant activities of the studied Salvia species.

Extracts	Yield (%) (w/w)	TPC (mg GAE/g Extract) 1	DPPH IC50 (mg/mL) 2
SD-I	14.71	111.50 ± 0.51 b	0.09 ± 0.011 a
ST-I	13.89	91.60 ± 0.66 c	0.14 ± 0.024 b
SA-I	12.64	40.75 ± 0.83 f	0.35 ± 0.032 e,f
SG-I	12.03	47.70 ± 0.96 e	0.31 ± 0.037 e
SE-I	11.93	74.30 ± 0.58 d	0.16 ± 0.021 b
SS-I	10.67	40.60 ± 0.95 f	0.37 ± 0.016 g
SD-E	3.62	125.55 ± 0.88 a	0.07 ± 0.019 a
ST-E	3.44	96.65 ± 0.70 c	0.13 ± 0.026 b
SG-E	3.25	68.45 ± 0.81 d	0.26 ± 0.053 d
SA-E	3.21	41.90 ± 0.30 f	0.32 ± 0.028 e
SE-E	3.19	71.45 ± 0.20 d	0.18 ± 0.041 c
SS-E	2.97	48.45 ± 0.73 e	0.30 ± 0.034 e
Gallic Acid	-	-	0.003 ± 0.0001 *

¹: Values expressed as mg Gallic Acid Equivalents per gram of dry extract. ²: IC₅₀ represents the concentration required to inhibit 50% of DPPH radicals. *: Positive control. Values are expressed as mean ± sd (n = 3). Different superscript letters (a–g) within the same column indicate statistically significant differences (p < 0.05) based on one-way ANOVA followed by Tukey’s post hoc test. SD: S. dorystaechas, ST: S. tomentosa, SA: S. argentea, SG: S. glutinosa, SE: S. aethiopis, SS: S. sclarea; I: Infusion, E: 70% Ethanol.

. The yields for infusions (ranging from 10.67% to 14.71%) were consistently and significantly higher than those of the ethanolic extracts (2.97% to 3.62%).

Among the investigated taxa, Salvia dorystaechas provided the highest efficiency in both methods, yielding 14.71% (SD-I) and 3.62% (SD-E), respectively. The overall higher mass recovery in infusions can be attributed to the high polarity of water, which facilitates the extraction of bulk plant components, including primary metabolites such as mucilages and carbohydrates, alongside polar secondary metabolites. In contrast, the narrow and lower range observed in ethanolic extracts suggests a more selective recovery of specific secondary metabolites.

2.2. Total Phenolic Content and Antioxidant Activity

The antioxidant activity and total phenolic content (TPC) of the studied Salvia species are summarized in Table 2. According to the DPPH free radical scavenging assay, the ethanolic extracts (E) generally exhibited higher antioxidant potency, indicated by lower IC₅₀ to their respective infusion (I) counterparts across all species, except Salvia aethiopis (SE).

Parallel to the antioxidant activity, TPC values were significantly higher in the ethanolic extracts. Among the tested taxa, S. dorystaechas extracts (SD-E and SD-I) emerged as the most potent, with SD-E yielding the highest concentration of phenolic compounds (125.55 ± 0.88 mg GAE/g) and the strongest radical scavenging activity (IC₅₀: 0.07 ± 0.019 mg/mL). In contrast, the infusions of S. argentea (SA-I) and S. sclarea (SS-I) demonstrated the lowest TPC and antioxidant capacity among the samples. The extraction yields also varied notably, with infusions providing higher weight-to-weight recovery (10.67–14.71%) compared to ethanolic maceration (2.97–3.62%).

Statistical analysis revealed significant differences (p < 0.05) among the studied Salvia species in terms of both phenolic content and antioxidant capacity. According to Tukey’s post hoc test, the ethanolic extract of S. dorystaechas (SD-E) exhibited the highest total phenolic content and the most potent antioxidant activity (IC₅₀: 0.07 mg/mL), being statistically distinct from all other taxa (p < 0.05). However, a significant and strong negative correlation was observed between the total phenolic content and the DPPH radical scavenging activity (IC₅₀) of the studied Salvia extracts (r = −0.92, p < 0.001). The linear relationship between these parameters was further quantified by a regression analysis, yielding the equation y = −0.0035x + 0.4737 (R² = 0.9286) (Figure 1).

This strong correlation indicates a strong association between phenolic content and the observed antioxidant capacity. While these findings suggest that phenolic constituents contribute significantly to the redox-active profile of the investigated taxa, the overall bioactivity is likely influenced by a synergistic interplay with other identified secondary metabolites, such as phenolic diterpenoids (e.g., rosmanol and carnosic acid), which are also known for their potent radical scavenging properties.

2.3. Antimicrobial Activity

The antimicrobial activities of the Salvia species extracts were evaluated against a panel of pathogenic bacteria and fungi, including Escherichia coli, Staphylococcus aureus, Candida tropicalis, C. albicans, and C. krusei. The Minimum Inhibitory Concentration (MIC) values are presented in

Table 3. Minimum Inhibitory Concentrations (MIC) of the studied Salvia extracts and reference drugs (μg/mL).

	E. coli ATCC 8739	S. aureus ATCC 6538	C. tropicalis ATCC 750	C. albicans ATCC 90028	C.krusei ATCC 14243
SD-I	1250	1250	625	5000	5000
SA-I	>2500	>2500	5000	>5000	>5000
ST-I	>2500	1250	5000	>5000	>5000
SS-I	>2500	>2500	5000	>5000	>5000
SE-I	>2500	>2500	5000	>5000	>5000
SG-I	>2500	>2500	2500	>5000	>5000
SG-E	2500	625	625	1250	1250
SD-E	2500	>2500	5000	625	625
SA-E	1250	1250	625	1250	1250
ST-E	1250	1250	625	1250	1250
SS-E	625	1250	625	2500	2500
SE-E	2500	2500	1250	2500	2500
Ciprofloxacin	0.25>	0.125	-	-	-
Fluconazole	-	-	2	64	64

SD: S. dorystaechas, ST: S. tomentosa, SA: S. argentea, SG: S. glutinosa, SE: S. aethiopis, SS: S. sclarea; I: Infusion, E: 70% Ethanol. Activity intensity classification for crude extracts: moderate activity (MIC ≤ 1000 µg/mL) is highlighted in [green]; weak activity (1000 < MIC ≤ 2500 µg/mL) is highlighted in [yellow]; very weak/Not active (MIC > 2500 µg/mL) is highlighted in [gray].

. Overall, the ethanolic extracts exhibited significantly stronger antimicrobial potency compared to the infusions against both bacterial and fungal strains. Among the infusions, S. dorystaechas (SD-I) demonstrated moderate antibacterial activity against E. coli and S. aureus (MIC: 1250 µg/mL) and the most notable antifungal effect against C. tropicalis (MIC: 625 µg/mL). In contrast, most other infusion extracts displayed limited or no inhibitory effects, with MIC values typically ≥2500 µg/mL for bacteria and ≥5000 µg/mL for Candida species.

The ethanolic extracts demonstrated markedly lower MIC values, indicating higher potency. S. sclarea (SS-E) exhibited the most robust antibacterial effect against E. coli (MIC: 625 µg/mL). Regarding antifungal activity, SG-E, SD-E, SA-E, and ST-E showed substantial inhibitory effects against C. tropicalis with MIC values ranging between 625 and 1250 µg/mL. Notably, SD-E and SG-E also inhibited the growth of C. albicans and C. krusei at relatively low concentrations (MIC: 625–1250 µg/mL), whereas their respective infusions were largely inactive. As expected, the reference agents Ciprofloxacin and Fluconazole maintained significantly lower MIC values, validating the experimental setup.

The results suggest that the antimicrobial constituents of Salvia species, likely including non-polar terpenoids and specific phenolics, are more efficiently recovered through ethanolic extraction. These findings highlight the potential of organic Salvia extracts as sources of natural antimicrobial agents.

2.4. Cytotoxicity Activity

As a well-characterized and non-tumorigenic cell line, NIH/3T3 is frequently utilized for high-throughput in vitro toxicity screening of experimental therapeutics [39,40]. This cell line provides a robust platform for preliminary safety evaluations, particularly when investigating natural product-based therapeutic candidates.

Activity evaluations in antimicrobial activity studies in the literature are generally based on MIC values, although it is important to note that classification thresholds can vary significantly depending on the study’s scope and the standards adopted. While MICs for conventional antibiotics typically fall within the range of 0.01 to 10 µg/mL, plant extracts are generally considered to have antimicrobial potential when their MIC values range between 100 and 1000 µg/mL [41]. In the context of traditional medicine, plant extracts with MIC values below 8 mg/mL are generally regarded as biologically active. To provide a structured evaluation in this study, we adopted the criteria where extracts exhibiting MIC values below 100 µg/mL are considered to possess strong antibacterial activity, while those with MICs between 100 and 625 µg/mL show moderate activity, and values exceeding 625 µg/mL are indicative of weak or low antibacterial potential [42].

Similarly, the categorization of antioxidant potency via DPPH scavenging activity is subject to interpretation across different research contexts. In this study, IC₅₀ values are categorized based on widely cited literature as follows: very strong activity for values below 10 µg/mL, strong activity for values between 10 and 50 µg/mL, moderate activity for values between 50 and 100 µg/mL, weak activity for values between 100 and 250 µg/mL, and no activity for values above 250 µg/mL [43]. These categorized thresholds serve as a benchmark for comparing our findings with existing ethnobotanical and phytochemical data.

To evaluate the cytotoxic effects and preliminary selectivity of the promising samples, four extracts (SD-I, SD-E, ST-I, and ST-E) were selected for cytotoxicity screening on the NIH/3T3 non-tumorigenic cell line, based on their comparatively higher antioxidant capacities together with relatively more notable antimicrobial activity among the tested extracts. The IC₅₀ values determined after 24 h of exposure, together with the calculated Selectivity Index (SI) values, are summarized in

Table 4. IC₅₀ values of selected extracts in NIH/3T3 cells after 24-h exposure.

Extracts	IC50 Values (µg/mL)	MIC Range (µg/mL)	SImin	SImax
SD-I	173.82 ± 25.09	625–5000	0.03	0.28
SD-E	110.06 ± 0.31	625–5000	0.02	0.18
ST-I	408.02 ± 4.63	1250– >5000	0.08	0.33
ST-E	562.37 ± 49.50	625–1250	0.45	0.90

Three independent experiments were performed, and IC₅₀ values were calculated for each. Data are presented as mean IC₅₀ ± sd (standard deviation). SD: S. dorystaechas, ST: S. tomentosa, I: Infusion, E: 70% Ethanol. IC₅₀: Concentration required to reduce NIH/3T3 cell viability by 50%. SI_min: Calculated using the highest MIC value obtained for each extract. SI_max: Calculated using the lowest MIC value obtained for each extract.

. Since multiple MIC values were obtained for each extract against different microorganisms, both SI_min and SI_max values were calculated. Among the tested plant extracts, ST-E (S. tomentosa ethanol extract) exhibited the highest IC₅₀ value (562.37 ± 49.50 µg/mL) and the highest SI values (SI_min: 0.45; SI_max: 0.90), indicating lower cytotoxicity relative to the other tested extracts and comparatively better selectivity. In contrast, SD-E (S. dorystaechas ethanol extract), despite showing the strongest antioxidant activity in previous assays, demonstrated the highest cytotoxicity with an IC₅₀ of 110.06 ±0.31 µg/mL and lower SI values (SI_min: 0.02; SI_max: 0.18). The infusion extracts SD-I and ST-I showed moderate cytotoxic effects, with IC₅₀ values of 173.82 ±25.09 µg/mL and 408.02 ±4.63 µg/mL, respectively. Overall, the calculated SI values remained below 1, suggesting limited selectivity of the tested extracts toward microbial cells relative to mammalian cells under the evaluated experimental conditions [44].

2.5. Phytochemical Profiling

The LC-HRMS analysis of 70% ethanol (E) and infusion (I) extracts from six Salvia species identified nine key phytochemical compounds (

Table 5. Bioactive constituents detected in various Salvia extracts by LC-HRMS analysis.

Compound	Class	Formula	Rt (min)	MW	Exp. (m/z) MS(+)	Exp. (m/z) MS(−)	Detected in Extracts
Luteolin	Flavonoid	C15H10O6	1.23	286.24	287.06	285.04	SG-I, SD-E, SA-I, SA-E, ST-I, ST-E, SS-I, SS-E, SE-I, SE-E
Luteolin-7-O-glucuronide	Flavonoid	C21H18O12	1.10	462.37	463.09	-	SG-I, SD-I, SD-E, SA-I, SA-E, ST-I, ST-E, SS-I, SS-E, SE-I
Rosmarinic acid	Phenolic Acid	C18H16O8	1.27	360.31	-	359.08	SG-I, SG-E
Rosmanol	Polyphenol	C20H26O5	2.72	346.42	347.19	345.17	SD-I, SD-E
Apigenin	Flavonoid	C15H10O5	1.41	270.24	271.06	269.05	SA-I, SA-E, ST-I, ST-E, SS-I, SS-E, SE-I
Acacetin	Flavonoid	C16H12O5	2.64	284.25	285.08	283.06	SA-E, SS-I, SS-E, SE-E
Cynaroside (Luteolin-7-O-glucoside)	Flavonoid	C21H20O11	1.09	448.38	449.11	447.09	ST-I, ST-E
Carnosol	Terpenoid	C20H26O4	2.72	330.4	331.19	329.18	SG-E, SD-E, SA-E, ST-I, ST-E, SS-I, SS-E
Carnosic acid	Terpenoid	C20H28O4	3.51	332.4	333.20	-	SD-E, ST-I, ST-E

Rt: Retention time; MW: Molecular weight; Exp.: Experimental. SD: S. dorystaechas, ST: S. tomentosa, SA: S. argentea, SG: S. glutinosa, SE: S. aethiopis, SS: S. sclarea; I: Infusion, E: 70% Ethanol.

) (Supplementary Figures S1–S15). Luteolin, luteolin-7-O-glucuronide, and apigenin were the most prevalent constituents, detected in both the ethanol and infusion extracts of S. argentea (SA), S. tomentosa (ST), and S. sclarea (SS). In contrast, these compounds were only identified in the infusion extract of S. aethiopis (SE). In S. dorystaechas (SD), luteolin-7-O-glucuronide was the only flavonoid present in both extraction types.

S. tomentosa (ST) exhibited the highest chemical diversity among the studied species, with cynaroside (luteolin-7-O-glucoside) being uniquely detected in this taxon. S. dorystaechas (SD-E) was distinguished as the only species containing the polyphenol rosmanol, while rosmarinic acid was exclusively detected in S. glutinosa (SG). Regarding terpenoids, carnosol was identified in all species except S. aethiopis. Overall, the phytochemical profiling indicated that ethanol extracts were richer in terpenoids and phenolic compounds compared to the infusions.

3. Discussion

The phytochemical diversity and biological orientation of the Salvia genus are heavily influenced by the extraction solvent’s polarity and the specific genetic makeup of the taxa. In this study, 70% ethanol generally outperformed infusions in recovering both phenolic compounds and terpenoids, a phenomenon attributed to the intermediate polarity of the hydroethanolic mixture, which facilitates the dissolution of a broader range of secondary metabolites, including methylated flavonoids and diterpenoids, such as carnosol.

A direct correlation was observed between Total Phenolic Content (TPC) and DPPH radical scavenging activity, reinforcing the role of polyphenols as primary redox-active constituents in Salvia. The most potent antioxidant source was S. dorystaechas (SD), whose ethanolic extract had TPC values greater than 125 mg GAE/g. Based on the categorization by [43] the DPPH IC₅₀ values observed in our study, particularly for S. dorystaechas, fall within the ‘strong’ to ‘moderate’ activity ranges. This high activity is likely linked to the presence of rosmanol and carnosic acid, which were uniquely identified in this species. Compared to conventional antioxidants, rosmanol, a phenolic diterpene, is renowned for its exceptional capacity to donate hydrogen. Furthermore, in our study, 70% ethanol was more selective for recovering antioxidant-rich fractions, aligning with the report [45].

Our findings for S. sclarea and S. aethiopis are in partial agreement with the literature [46]; indeed, dynamic interspecific variation and modern technological platforms heavily shape the bioactivity profiles of these specific taxa. For instance, recent studies utilizing S. aethiopis and S. sclarea extracts for the green synthesis of silver nanoparticles (AgNPs) demonstrate that advanced formulation significantly enhances baseline antioxidant and anti-inflammatory pathways governed by phenolics like isoquercetin and naringenin [47]. However, the superior TPC values in our Turkish S. dorystaechas samples highlight the high-yield bioactive potential of Türkiye’s flora. These discrepancies between our results and those of the study [37] for S. glutinosa further underscore the profound impact of locality-dependent variations and genetic factors on the genus’s chemical profile. Geographic and regional variations contribute to distinct chemical profiles even within the same species; while our LC-HRMS profiling focused on other markers, recent investigations on S. glutinosa from the Artvin region revealed coumaric acid as a predominant compound, highlighting strong solvent-dependent antimicrobial potencies against strains like S. aureus that surpass commercial standards [48]. The high reducing potential of the extracts is strongly correlated with their polyphenolic architecture, as higher TPC values typically facilitate greater antioxidant activity [49]. This correlation was evident in our results, where the taxa with the highest phenolic content consistently demonstrated the lowest IC₅₀ values in DPPH assays. In the present study, although common phenolic acids frequently reported in the Salvia genus, including caffeic, ferulic, and p-coumaric acids, were targeted during LC-HRMS screening, they were not detected in the investigated extracts, possibly due to their occurrence at trace levels below the detection limits under the applied experimental conditions. Instead, rosmarinic acid, together with a diverse range of flavonoids (Table 5), represented the predominant polyphenolic constituents of the analyzed species.

Interestingly, rosmarinic acid was only found in S. glutinosa in our investigation, despite the fact that it is frequently mentioned as a primary biomarker for the Salvia genus. This implies that rather than rosmarinic acid alone, the antioxidant capacity in other species, such as S. tomentosa and S. argentea, is likely driven by a synergistic mix of flavonoids (luteolin, apigenin) and diterpenes. This multi-component synergy and phytochemical fluctuation are well-documented across the genus. Ethnobotanical surveys and in vivo trials on S. argentea validate its traditional therapeutic window, proving that its rosmarinic acid- and salvigenin-rich matrices exert robust anti-inflammatory responses by downregulating NF-κB and cytokine pathways without acute oral toxicity [26,50]. Concurrently, recent work on Salvia tomentosa Miller reinforces that this secondary metabolite density is highly organ-dependent and subject to cultivation factors, where combinations of aerial parts (stems, leaves, flowers) or wild versus cultivated matrices distinctly alter baseline free radical scavenging and antimicrobial outputs [51,52]. The lower IC₅₀ values of ethanol extracts (ranging from 0.07 to 0.32 mg/mL) compared to infusions highlight the necessity of organic solvents for maximizing the recovery of these bioactive fractions.

The antimicrobial results revealed that Salvia extracts are particularly effective against fungal pathogens, specifically Candida tropicalis. The biological potential of these extracts can be evaluated based on established potency thresholds; according to the report [42], plant extracts with MIC values between 100 and 625 µg/mL are considered to show moderate antibacterial activity. However, although the extracts demonstrated measurable antimicrobial activity, the obtained MIC values in this study generally indicated weak to moderate activity according to commonly accepted criteria for crude plant extracts. In particular, MIC values above 1000 µg/mL may limit the direct practical applicability of the crude extracts as potent antimicrobial agents. The antimicrobial activity observed in the present study should therefore be interpreted cautiously, especially considering that crude plant extracts often contain complex mixtures of active and inactive constituents that may reduce the apparent biological potency. Similar MIC ranges have been reported in previous studies investigating crude botanical extracts, where moderate antimicrobial effects were frequently associated with the presence of phenolic compounds, flavonoids, terpenoids, and other secondary metabolites. The ethanolic extracts of S. sclarea (SS-E) and S. glutinosa (SG-E) exhibited notable inhibitory effects, which can be attributed to the presence of carnosol and acacetin. Although the specific in vitro or molecular mechanisms of action were not directly investigated in the present study, the antimicrobial activity of the Salvia extracts can be theoretically attributed to the well-documented mechanisms of their major secondary metabolites, such as luteolin, rosmarinic acid, and carnosol, identified via LC-HRMS. Luteolin, a prominent flavonoid, has been reported to intercalate between the polar head groups and hydrophobic regions of bacterial cytoplasmic membranes, altering membrane fluidity and thickness, which ultimately leads to the disruption of membrane integrity and cell death [53]. Similarly, rosmarinic acid is known to exert significant antimicrobial and antibiofilm effects by inducing structural alterations in the microbial cell membrane, causing leakage of intracellular contents and reducing mitochondrial activity in fungal strains [54]. Furthermore, diterpenes like carnosol and carnosic acid have been shown to act as efflux pump modulators by dissipating the membrane potential gradient, thereby compromising bacterial survival and potentiating antimicrobial susceptibility [55]. Therefore, the limited to moderate MIC values observed in our study likely result from a synergistic or additive structural disruption of microbial membranes driven by these phytochemicals. These extracts should not be considered as primary antibiotic or antifungal agents, but rather as potential supportive phytotherapeutic agents, especially in topical applications, or as sources of secondary metabolites for further purification. However, the relatively high MIC values observed in this study indicate that additional purification and fractionation are needed to identify the specific compounds responsible for the antimicrobial activity. This relatively moderate efficacy may be attributed to the nature of the crude extracts, where the absolute concentration of potent antimicrobial markers, such as carnosic acid or carnosol, may be diluted by the presence of other non-active matrix components. Furthermore, the 70% ethanol used in this study, while excellent for recovering a broad range of polyphenols, might not have fully optimized the recovery of highly lipophilic constituents that often possess stronger antimicrobial properties. Future studies utilizing alternative extraction methods, such as supercritical fluid extraction (SFE) or microwave-assisted extraction, could potentially enhance the yield of these specific antimicrobial fractions and provide a clearer understanding of the genus’s maximal inhibitory potential.

The therapeutic index (selectivity) is a crucial component of pharmacological evaluation. As a non-tumorigenic cell line, NIH/3T3 provides a robust platform for preliminary safety evaluations of natural product-based candidates [39,40]. In our study, a critical balance between efficacy and toxicity was observed. S. dorystaechas (SD-E) exhibited substantial cytotoxicity in NIH/3T3 cells (IC₅₀: 110.06 µg/mL), although demonstrating the highest antioxidant and antimicrobial activities. These differences in cytotoxicity (Table 4) appear to be related to the specific phytochemical compositions identified (Table 5). The lower IC₅₀ value of SD-E (110.06 µg/mL) could be associated with its diterpenoid profile, notably the presence of rosmanol, carnosol, and carnosic acid. In the literature, these phenolic diterpenoids are reported to exhibit dose-dependent effects, where higher concentrations may lead to cellular stress in non-tumorigenic lines like NIH/3T3. This suggests that its bioactive compounds may lack specificity at higher concentrations.

In contrast, S. tomentosa (ST-E) emerged as a promising candidate showing a higher IC₅₀ value (562.37 µg/mL), which might be due to a different balance between its flavonoid and diterpenoid constituents, such as the presence of cynaroside. These findings suggest that specific chemical markers, rather than total phenolic content alone, play a role in the safety profile of these extracts. This favorable selectivity and balanced safety profile of S. tomentosa are supported by recent literature; for instance, metabolic profiles of S. tomentosa organs (roots, aerial parts, and inflorescences) predominated by rosmarinic and salvianolic acids demonstrated minimal cytotoxicity toward reference murine fibroblasts while simultaneously exerting potent, targeted anti-proliferative effects against human AGS gastric adenocarcinoma epithelial cells [56]. While extracts with MIC values below 1000 µg/mL are generally considered to have significant antimicrobial potential [41], the high cytotoxicity observed in some potent fractions can limit their application. Despite its moderate bioactivity, the high IC₅₀ value of ST-E on NIH/3T3 cells (562.37 ± 49.50 µg/mL) indicates a lower toxic potential compared to SD-E, aligning with the requirement for non-toxic natural therapeutics. The phytochemical profiling of this species, characterized by the presence of cynaroside (luteolin-7-O-glucoside), likely contributes to its balanced bioactivity–toxicity ratio.

Furthermore, the biological activities observed in this study provide a potential scientific basis for the traditional medicinal applications of these taxa, as summarized in Table 1. The notable antioxidant and antimicrobial potencies, particularly against fungal pathogens, align with the ethnomedicinal use of S. dorystaechas, S. tomentosa, and S. sclarea in treating diseases potentially related to microbial infections. The prevalence of specific secondary metabolites, such as carnosol and flavonoids identified via LC-HRMS, likely contributes to the therapeutic efficacy reported in Anatolian folk medicine. By exploring these centuries-old practices through modern high-resolution profiling, our findings reinforce the importance of documenting and preserving traditional knowledge as a roadmap for discovering new natural pharmacological agents. The high correlation coefficient (r = −0.92) found in our study aligns with previous reports on the Salvia genus, a strong association between the phenolic density and the free radical scavenging capacity. This relationship highlights the significant role of phenolic constituents in the potent antioxidant activity observed in S. dorystaechas. These results support the view that the rich phenolic and terpenoid profile of these species contributes to their therapeutic potential in treating oxidative-stress-related disorders.

The pharmacological potential of the Salvia genus is largely attributed to its diverse secondary metabolite profile, primarily categorized into phenolic compounds and terpenoids. Beyond their aromatic properties, these species serve as complex natural reservoirs for potent phenolics like rosmarinic acid and various diterpenoids. Current scientific literature supports traditional uses of Salvia, demonstrating that inter-species variations lead to distinct chemical fingerprints [57,58]. This extensive chemical diversity is consistent with the evolutionary success of the Salvia genus, which comprises over 1024 species globally distributed across major biodiversity hotspots. Comprehensive reviews on S. sclarea highlight its vast ethnobotanical legacy in treating digestive, menstrual, and inflammatory disorders, linking these traditional applications to an intricate reservoir of secondary metabolites, particularly oxygenated monoterpenoids like linalyl acetate and linalool, alongside diverse flavonoids and phenolic acids [59]. Our results indicate that this phytochemical composition is dynamic and influenced by several factors, including genetic phylogeny, geographic origin, the extraction methodology, and localized ecological conditions. The prevalence of specific markers, such as the unique presence of rosmanol in S. dorystaechas or cynaroside in S. tomentosa, ultimately dictates the efficacy and variability observed in the biological activity studies [60,61]. Furthermore, the higher recovery of terpenoids in ethanol extracts confirms that the choice of solvent significantly impacts the plant.

The variations observed in TPC and MIC values compared to previous reports [37,46] underscore the impact of Anatolian geography on Salvia phytochemistry. Localized ecological pressures often lead to the enhanced biosynthesis of specific secondary metabolites as defensive mechanisms, resulting in the chemical fingerprints observed in our LC-HRMS analysis. The detection of acacetin and carnosic acid primarily in ethanol extracts further supports the conclusion that the traditional use of infusions may not fully capture the genus’s entire pharmacological potential, particularly regarding non-polar terpenoids. While this study provides a high-resolution phytochemical and biological characterization of these six Salvia species, certain limitations remain. First, the biological activities were evaluated using crude extracts; hence, the potential synergistic or antagonistic interactions between individual compounds have yet to be fully elucidated through bio-guided isolation. Second, the current safety evaluations are limited to in vitro NIH/3T3 cell lines, necessitating future in vivo studies to confirm the systemic safety, pharmacokinetics, and bioavailability of these taxa.

Future research directions should involve the isolation of specific biomarkers identified in our LC-HRMS analysis, such as rosmanol, acacetin, and cynaroside, to investigate their molecular mechanisms of action. Additionally, further investigations including minimum bactericidal concentration (MBC), minimum fungicidal concentration (MFC), and transcriptomic studies would be necessary to better characterize their exact mechanistic pathways and mode of action.

4. Materials and Methods

4.1. Plant Material and Extraction

The plant specimens investigated in this study were collected from their natural habitats in Türkiye during the 2024 flowering season. The specimens were identified by the authors and prepared as herbarium materials, and are preserved in the Anadolu University Faculty of Pharmacy Herbarium (ESSE). The studied taxa and their respective accession numbers are as follows: S. dorystaechas B.T.Drew (ESSE 16433), S. sclarea L. (ESSE 16347), S. glutinosa L. (ESSE 16311), S. tomentosa Mill. (ESSE 16348), S. argentea L. (ESSE 16346), and S. aethiopis L. (ESSE 16432). Extracts were prepared from the flowering aerial parts of the plants using two different methods—5% infusion (aqueous) and 70% ethanol extraction—following the protocol described in the literature [62].

The extraction efficiency for each species was determined gravimetrically and expressed as a percentage yield (w/w) relative to the initial dried plant material. The yield was calculated using the following equation:

$$\mathrm{Yield} \left(\%\right)=\left(\mathrm{A}÷\mathrm{A}1\right)\times 100$$

A denotes the final mass of the dried extract obtained after solvent evaporation. A1 represents the initial mass of the pulverized plant material used in the extraction process.

4.2. Assessment of Total Phenolic Content and Antioxidant

4.2.1. Determination of Total Phenolic Content (TPC)

The total phenolic content of the extracts was quantified using the Folin–Ciocalteu reagent (FCR) (Merck, Darmstadt, Germany) as described in the literature [63]. To establish a standard for quantification, a linear calibration curve was constructed using gallic acid working solutions within a concentration range of 0.03 to 1 mg/mL. The absorbance of each standard and sample was recorded at 760 nm using a BioTek microplate spectrophotometer (Winooski, VT, USA). The resulting regression equation was utilized to determine the phenolic concentrations of the Salvia extracts. All results are expressed as milligrams of gallic acid equivalents per gram of extract (mg GAE/g).

4.2.2. DPPH Radical Scavenging Activity

The antioxidant capacity of the aerial part extracts was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, following the methodology detailed in [64]. The radical scavenging effect (Inhibition %) was calculated using the following equation:

$$\mathrm{Inhibition} \left(\%\right)=\left[\left(\mathrm{Acontrol}-\mathrm{Asample}\right)÷\mathrm{Acontrol}\right]\times 100$$

Acontrol is the absorbance of the control (containing all reagents except the sample), and Asample is the absorbance of the extract. The IC₅₀ values (the concentration required to inhibit 50% of DPPH radicals) were calculated through nonlinear regression analysis of the dose–response data. All extract concentrations used for IC₅₀ calculations were precisely determined based on the dry weight of the extract (mg dry extract/mL). Curve fitting and statistical modeling were performed using SigmaPlot 13.0 (Systat Software Inc., San Jose, CA, USA). To ensure reproducibility, all assays were conducted in triplicate, and the results are expressed as the mean values of independent experimental runs.

4.3. Assessment of Antimicrobial Activity

4.3.1. Microbial Strains and Media

The antimicrobial potential of the Salvia extracts was evaluated against a panel of microorganisms, including Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and the yeast strains Candida albicans, C. tropicalis, and C. krusei. Bacterial strains were cultured in Mueller–Hinton Broth (MHB, Sigma-Aldrich, Biolife Italiana, Milan, Italy), while yeasts were maintained in RPMI-1640 medium with L-glutamine, buffered to pH 7.0 with 3-[N-morpholino]-propanesulfonic acid (MOPS) (Sigma-Aldrich, St. Louis, MO, USA). Ciprofloxacin (Sigma-Aldrich, St. Louis, MO, USA) and fluconazole (Sanovel Pharmaceutical Industry, Istanbul, Türkiye) were employed as standard reference drugs.

4.3.2. Broth Microdilution Method

The Minimum Inhibitory Concentrations (MICs) were determined using the broth microdilution method, conducted in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines M7-A7 for bacteria [65] and M27-A2 for yeasts [66] with minor modifications. Extracts were tested within a concentration range of 78.125 to 5000 µg/mL, while reference drugs were prepared at concentrations ranging from 0.5 to 64 µg/mL.

Standardized inocula were prepared from overnight cultures to yield final concentrations of 10⁶ colony-forming units (CFU/mL) for bacteria and 1–2 × 10³ cells/mL for yeasts. The assays were performed in 96-well microplates. Following inoculation, the microplates were incubated for 24 h at 37 °C for bacteria and 48 h at 28 °C for yeasts. Positive growth controls (wells containing medium and inoculum without extracts) were included to ensure microbial viability.

To facilitate the visual detection of microbial growth, 20 μL of a 0.01% resazurin solution was added to each well. The MIC was defined as the lowest concentration of the extract that prevented a color change (from blue to pink) or visible turbidity, indicating the inhibition of metabolic activity. All experiments were performed in triplicate to ensure the reproducibility of the results.

4.4. Assessment of Cytotoxicity by MTT Assay

4.4.1. Cell Culture Conditions

The NIH/3T3 mouse embryonic fibroblast cell line (ATCC CRL-1658™) was utilized to evaluate the safety profile of the selected extracts. Cells were maintained in a humidified incubator at 37 °C with 5% CO₂. The growth medium consisted of high-glucose DMEM (89%), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. All assays were conducted using cells between passages 2 and 6 to ensure phenotypic stability.

4.4.2. MTT Viability Assay

The cytotoxic effects of four extracts, S. dorystaechas infusion (SD-I), S. dorystaechas ethanol (SD-E), S. tomentosa infusion (ST-I), and S. tomentosa ethanol (ST-E), based on their comparatively higher antioxidant activities, together with relatively more notable antimicrobial activity among the tested samples, and to enable comparison between infusion and ethanol extracts of the same species, were investigated in NIH/3T3 healthy cells. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, which correlates mitochondrial metabolic activity with the formation of insoluble formazan crystals [67].

The MTT assay was performed in 96-well microplates, with each concentration tested in quadruplicate, and all experiments were independently repeated three times. Stock solutions of the extracts were freshly prepared in dimethyl sulfoxide (DMSO) at the highest concentrations at which they were fully soluble, then diluted with culture medium and sterilized using a 0.22 µm filter, and further diluted with culture medium to achieve test concentrations (39.06 to 625 µg/mL). A 1% DMSO solution was used as the vehicle control. Cells were detached using trypsin–EDTA and centrifuged, and the resulting pellet was resuspended in complete medium. The viable cell count was determined by trypan blue exclusion. Cells were seeded at a density of 1 × 10⁴ cells/100 µL per well into 96-well plates and incubated for 24 h to allow attachment. Following incubation, the test compounds were added at their respective concentrations and incubated for an additional 24 h. After the treatment period, the media were aspirated, and cells were incubated with 0.5 mg/mL MTT solution for 3 h. The resulting formazan crystals were solubilized in 100 µL of DMSO, and absorbance was measured at 540 nm using a microplate reader. Cell viability was calculated using the formula:

% Cell viability = [(OD_Substance − OD_Control)/OD_Control] × 100.

OD: Optical Density.

The percent viability was determined for each concentration, and the half-maximal inhibitory concentration (IC₅₀) values were calculated using linear or non-linear curve fitting methods [68].

Selectivity Index (SI) values were calculated as the ratio of IC₅₀ values obtained in NIH/3T3 cells to MIC values determined in antimicrobial assays. SI_min and SI_max were calculated using the highest and lowest MIC values obtained for each extract, respectively [44].

4.5. Phytochemical Profiling via LC-HRMS Analysis

Phytochemical characterization of the Salvia extracts was performed using a high-resolution Shimadzu LC-MS-IT-TOF (Kyoto, Japan) system. The liquid chromatography (LC) module consisted of two LC-20AD binary pumps, a DGU-20A3R degasser, a CTO-10ASvp column oven, a SIL-20AC autosampler, and an SPD-M20A photodiode array (PDA) detector. Chromatographic separation was achieved on an Inertsil ODS-3 column (150 mm × 1.5 mm, 5 µm, C-18 packing). The mobile phase comprised acetonitrile and water with 0.1% formic acid, delivered at 0.15 mL/min. The column oven temperature for the samples sent in 3 µL injection volumes was optimized as 40 °C, CDL temperature as 200 °C, heat block temperature as 200 °C, and nebulizer gas flow as 1.5 L/min. The acquired high-resolution m/z values were evaluated by comparison with literature data and reference databases, enabling the tentative identification of phytochemical constituents.

4.6. Statistical Analysis

All experiments were conducted using three independent biological replicates, each performed in triplicate to ensure reproducibility. Results are expressed as mean ± standard deviation (sd). Before performing One-way Analysis of Variance (ANOVA), the data were evaluated for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test. Statistical differences between the Salvia species and their extracts were determined via ANOVA followed by Tukey’s post hoc test for multiple comparisons. The IC₅₀ values for antioxidant activities were calculated individually for each independent experiment using non-linear regression analysis, and the final values represent the mean of these independent runs. Pearson’s correlation coefficient (r) was calculated to evaluate the relationship between total phenolic content (TPC) and IC₅₀ values across all studied extracts. Statistical significance was set at p < 0.05. All statistical analyses were conducted using IBM SPSS Statistics for Windows, Version 24.0 (IBM Corp., Armonk, NY, USA).

5. Conclusions

This study provided a comprehensive evaluation of infusions and 70% ethanol extracts from six Salvia species with established ethnomedicinal backgrounds. Our findings demonstrate a significant correlation between antioxidant potency and total phenolic content across the studied taxa. Phytochemical characterization through LC-HRMS confirmed that the observed biological effects are associated with specific phenolic constituents and terpenoids.

Regarding antimicrobial efficacy, the results of the present study demonstrated that the investigated extracts possess weak to moderate activity against the tested microorganisms. Although the observed activity does not indicate strong antimicrobial potency at the crude extract level, the findings suggest that the extracts may contain bioactive constituents worthy of further investigation. In this context, the calculated selectivity index (SI) values provided preliminary information regarding the balance between antimicrobial activity and cytotoxicity, contributing to a more comprehensive biological evaluation of the extracts.

Among the evaluated samples, the ST-E (S. tomentosa ethanol extract) exhibited a balanced profile, combining notable bioactivity with comparatively lower cytotoxicity in NIH/3T3 cells. These results highlight the influence of solvent selection on the recovery of bioactive secondary metabolites and provide a scientific basis for the potential development of S. tomentosa as a natural therapeutic agent. However, future studies involving bioactivity-guided fractionation, isolation of active compounds, MBC/MFC determinations, and mechanistic analyses are required to better clarify the antimicrobial potential and pharmacological relevance of these extracts.

In conclusion, our findings contribute to the transition of fragmented ethnomedicinal knowledge toward a more validated pharmacological framework. By evaluating phytochemical profiles alongside biological activities and preliminary toxicological safety, this study offers a standardized approach for the evidence-based use of Salvia species. This comparative framework provides a reliable basis for future research into drug discovery and the sustainable utilization of Turkey’s endemic plant biodiversity.