Introduction

Osteosarcoma is a tumor of mesenchymal origin (i.e., the tissue consists of stromal cells that can produce bone-like tissues) characterized by uncontrolled production of immature osteoid tissue1,2. Worldwide, osteosarcoma is ranked the eighth highest childhood cancer at about 2.4% of tumors diagnosed, and is more predominant among those aged 10–14 years3. During adolescence, osteosarcoma typically develops in the metaphysis of long bones close to the growth plates, accounting for 10% of tumors. Adolescent osteosarcoma is commonly associated with metastasis, especially lung metastasis4,5. Worldwide, 2–4 per million females and 3–5 per million males suffer from osteosarcoma, and it affects African American and Indigenous African males3.

Poor osteosarcoma prognosis is indicated by metastasis development, particularly lung metastasis, and drug resistance. Additionally, most patients do not exhibit significant lung metastases at the time of initial assessment, despite the presence of micro-metastases. The overall survival of osteosarcoma patients can be improved by early identification of micro-metastases using newly developed sensitive diagnostic techniques6. So, it is highly recommended to conduct more research to develop more effective treatment strategies3.

Greek sage, Salvia fruticosa Mill., syn. Salvia triloba L., is an aromatic small shrub from the mint family (Lamiaceae). It is abundant in the Eastern Mediterranean region, particularly in the mountainous regions in Lebanon, Palestine, Jordan, Egypt (Sinai), Syria, Cyprus, Türkiye, and Greece (Crete). Salvia fruticosa (S. fruticosa) leaves are rich in essential oils, including 1,8-cineole (most abundant), α- and β-thujone, and camphor, which are known for their antioxidant, antimicrobial, and anticancer activities. S. fruticosa leaves were used in folk medicine to treat common cold, diarrhea, toothaches, snake bites, enteritis, and sore throat. Essential oil of S. fruticosa possesses antimicrobial activity against a wide range of gram-positive and gram-negative bacteria. Furthermore, many studies revealed its anticancer activity against neuroblastoma, melanoma, prostate, colon, breast and ovarian cancers6,7,8,9,10,11,12,13,14.

The primary objective of this study is to provide novel insights into the bioactive compounds, including phenolics, flavonoids, and carbohydrates, present in S. fruticosa leaves. Specifically, this represents the first comprehensive investigation into the phenolic and flavonoid content extracted from S. fruticosa leaves using methanol and its targeted effects on both microorganisms and osteosarcoma cells. To achieve this, our study involved a clear sequence of investigative steps: first, chemical characterization and phytochemical analyses of the methanolic extract were conducted. Subsequently, we evaluated its biological activities, including its antimicrobial effect on both gram-positive and gram-negative microorganisms, as well as its impact on U2OS osteosarcoma cell viability, morphology, and migration. Additionally, the cytotoxic effect of the extract on human skin fibroblasts (HSF) was tested to assess its impact on normal human cells.

Methods

Plant material and extract preparation

S. fruticosa leaves were collected from Hebron city, Palestine, at the following geographical coordinates: 31°32’04.2"N 35°06’00.4"E in the Spring of 2022. Sample collection complies with relevant institutional, national, and international guidelines and legislations. Since the plant is not listed in the endangered or protected plants, no permission was needed for collection purposes. The plant morphological identification was performed by Dr. Rami Arafeh at Palestine-Korea Biotechnology Center, Palestine Polytechnic University, Hebron by referring to the flora of Palestine, Sinai, and Syria (see supplementary Fig. S1 online)15. Additionally, molecular identification was performed using a BLAST search and comparison of the DNA sequence of the internal transcribed spacers (ITS) in the ribosomal DNA (rDNA) region (NCBI GenBank # PQ804261).

Leaves were washed with tap water, followed by distilled water, and then left to air-dry at room temperature (25 ± 2 °C). The dried sample was ground into fine powder. Five grams of ground leaves were macerated in 70% methanol (PIOCHEM, Giza, Egypt) and placed for 15 min at room temperature in the ultrasonicator (Branson 2510 sonicator, model 2510DTH, Hampton, NH, USA) to extract polyphenolic compounds16. This was followed by filtration using cheesecloth; then, the filtrate was left to air-dry until complete evaporation of methanol at room temperature. After complete drying, approximately 4 g of the extract was collected in an Eppendorf tube and stored at room temperature for chemical and phytochemical analyses. In contrast, for testing the biological activity, the extract was dissolved in dimethyl sulfoxide (DMSO) (SIGMA - ALDRICH, Steinheim, Germany) to prepare a 75 mg/mL stock solution and kept at -20 °C.

Characterization of the chemical structure of S. fruticosa methanolic extract

FTIR was conducted to identify the major functional groups in the methanolic extract of S. fruticosa. Briefly, we combined a sample of the powdered S. fruticosa leaves with potassium bromide to form a 1.0 mm-sized pellet. Then the analysis was performed using a Nicolet 380 Thermogravimetric Analysis/Fourier Transform Infrared (TGA/FTIR) spectrometer over a range of wave numbers from 500 to 4000 cm–117.

Phytochemical analyses of S. fruticosa methanolic extract

Determination of total carbohydrate content (TCC)

The TCC in S. fruticosa extract was determined using the phenol–sulfuric acid method, with minor modification18. Briefly, the powdered S. fruticosa extract was dissolved in distilled water at a concentration of 1 mg/mL then 1 mL of the dissolved extract was added to 5 vol% phenol (Loba Chemie, Mumbai, India) and 5 mL concentrated sulfuric acid (Penta, Prague, Czech Republic) and left for 30 min in a water bath for cooling. At the end of the incubation period, a yellow-colored complex was formed and measured at 490 nm using Amersham Biosciences Ultrospec 3100 Pro UV/Visible Spectrophotometer. TCC quantification was determined in µg glucose equivalent/g based on Eq. (1), which was obtained from the standard glucose (PIOCHEM, Giza, Egypt) curve shown in supplementary Fig. S2A online.

$$\it Absorbance \,=\,0.00{\text{51}} \times {\text{Glucose }}{\text{ }}({\mu}g/mL)\,+\,0.0{\text{32}}$$
(1)

Determination of total phenolic content (TPC)

The TPC in S. fruticosa extract was determined by NAWAH Scientific (9 Al-Asmarat St, El Mokattam, Cairo, Egypt) using Folin–Ciocalteu colorimetric assay19. The powdered S. fruticosa extract was dissolved in 99.8 vol% methanol (PIOCHEM, Giza, Egypt) at a concentration of 3.2 mg/mL. Then, 10 µL of the dissolved extract was added to 100 µL of Folin-Ciocalteu reagent (Loba Chemie, Mumbai, India) (diluted 1: 10 vol.) in a 96-well plate. Then, 80 µL of 1 M sodium carbonate (El Nasr Pharmaceutical Chemicals Co., Cairo, Egypt) was added and incubated in the dark at room temperature for 20 min. A blue-colored complex resulted at the end of the incubation period, which was measured at 630 nm using a microplate reader (FluoStar Omega, Otenberg, Germany). TPC was expressed in µg caffeic acid equivalent/g based on Eq. (2) that was obtained from the standard caffeic acid (NAWAH Scientific, Cairo, Egypt) curve as shown in supplementary Fig. S2B online.

$$\it Absorbance\,=\,0.0025 \times Caffeic{\text{ }}acid{\text{ }}({\mu}g/mL){\text{ }}-{\text{ }}0.0967$$
(2)

Determination of total flavonoid content (TFC)

The TFC in S. fruticosa extract was determined by NAWAH Scientific (9 Al-Asmarat St, El Mokattam, Cairo, Egypt) using the aluminum chloride method, with minor modification20. The powdered extract was dissolved in 99.8 vol% methanol (PIOCHEM, Giza, Egypt) at a concentration of 3.2 mg/mL then in a 96-well plate, 15 µL of the dissolved extract was added to 175 µL of methanol followed by 30 µL of 1.25% aluminum chloride (Sigma - Aldrich, Steinheim, Germany) and finally 30 µL of 0.125 M sodium acetate (Sigma - Aldrich, Steinheim, Germany) were added and incubated for 5 min. At the end of the incubation period, a yellow-colored complex was formed that was measured at 420 nm using a microplate reader (FluoStar Omega, Otenberg, Germany). TFC was measured in µg rutin equivalent/g based on Eq. (3) that was obtained from the standard rutin (NAWAH Scientific, Cairo, Egypt) curve as shown in supplementary Fig. S2C online.

$$\it Absorbance\,=\,0.00{\text{2}} \times Rutin {\text{ }}({\mu}g/mL)-{\text{ }}0.0{\text{14}}$$
(3)

Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS)

The profiling of phenolic and flavonoid compounds in the extract of S. fruticosa was performed by the National Research Center (33 El Buhouth St, Dokki, Cairo, Egypt) using Liquid Chromatography coupled with Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). An ExionLC AC system was utilized for the separation process, while detection was carried out using a SCIEX Triple Quad 5500 + MS/MS system equipped with electrospray ionization (ESI)21. The separation was conducted on ZORBAX SB-C18 column (4.6 × 100 mm, 1.8 μm). The mobile phase consisted of two eluents: A (0.1% formic acid in water) and B (acetonitrile, LC/MS grade). The gradient elution was programmed as follows: 2% B from 0 to 1 min, transitioning from 2 to 60% B between 1 and 21 min, maintaining 60% B from 21 to 25 min, and returning to 2% B from 25.01 to 28 min. The flow rate was established at 0.8 mL/min, with an injection volume of 3 µL. For the Multiple Reaction Monitoring (MRM) analysis of the selected polyphenols, both positive and negative ionization modes were employed in a single run, with the following parameters: curtain gas set at 25 psi, ion spray voltage at 4500 V for positive mode and − 4500 V for negative mode, source temperature at 400 °C, ion source gases 1 and 2 at 55 psi, declustering potential at 50, collision energy at 25, and a collision energy spread of 10. The obtained chromatograms for the sample extract and standards are shown in supplementary Fig. S3 online, and the concentration of each phenolic and flavonoid compound was measured using Eq. (4).

$$\it {\text{Concentration}}=\frac{{{A_s}}}{{{A_{st}}}} \times {\text{}}{C_{st}}$$
(4)

where As = Area of sample, Ast = Area of standard, and Cst = standard concentration.

Examining the biological activities of S. fruticosa extract

Free radical scavenging test

The antioxidant activity of the extract was evaluated by NAWAH Scientific (9 Al-Asmarat St, El Mokattam, Cairo, Egypt) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity according to previous literature using Trolox as a positive control22. Briefly, a serial dilution of S. fruticosa extract was prepared in methanol (PIOCHEM, Giza, Egypt) to obtain concentrations of 5, 10, 20, 40, and 80 µg/mL. Then, 100 µL of freshly prepared DPPH reagent (0.1 vol% in methanol) was added to 100 µL of each extract concentration in a 96-well plate and incubated in the dark at room temperature for 30 min. The resulting yellow color intensity was measured at 540 nm using a microplate reader (FluoStar Omega, Otenberg, Germany) after blanking it with a mixture of DPPH and methanol without extract23,24. Absorbance values of the samples were evaluated against Trolox as a control. The ability of the extract to scavenge DPPH free radical was calculated using the following Eq. (5)22,24:

$$\it DPPH~scavenging~effect\left( \% \right){\text{}}=\left( {\frac{{{A_c}{\text{}} - {\text{}}{A_E}}}{{{A_c}}}} \right) \times {\text{1}}00$$
(5)

where AC is the average absorbance of the control (DPPH + methanol without extract), and AE is the average absorbance of the extract.

The extract’s ability to scavenge 50% of the free radicals is known as IC50 and this was calculated by plotting logarithm extract concentrations against their scavenging activity percentage and applying a nonlinear regression analysis using Eq. (6) (log (inhibitor) vs. normalized response –variable slope) on GraphPad Prism 9 software after normalizing the data to a scale ranging from 0 to 10025:

$$\it Y=\frac{{100}}{{\left( {1+~{{10}^{\left(( {log~IC50 - X} \right)~x~HillSlope))}}} \right)}}~~$$
(6)

where X = logarithmic concentration of the sample to be assessed (S. fruticosa extract and Trolox), Y = antioxidant activity percentage.

Statistically significant difference among the free radical scavenging effects of different extract concentrations on DPPH was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test at a 95% confidence level25.

Antimicrobial test for S. fruticosa extract

S. fruticosa antimicrobial activity was evaluated using a micro dilution method as previously described in26 with minor modifications27 towards four microorganisms: three bacteria: gram-positive Staphylococcus aureus (S. aureus) ATCC6538, gram-positive Bacillus subtilis (B. subtilis) ATCC35854, and gram-negative Escherichia coli (E. coli) ATCC8739 and one fungus: Saccharomyces cerevisiae (S. cerevisiae) ATCC9763 obtained from the American Type Culture Collection (ATCC, Virginia, USA). The minimum inhibitory concentration (MIC) of each microorganism was determined using 96-well microdilution plates with a flat bottom. Briefly, the extract was dissolved in 100% DMSO, followed by a serial dilution from 25 to 0.049 mg/mL using Mueller-Hinton broth (MHB, Oxoid, United Kingdom) (for bacteria) and Sabouraud Dextrose broth (SDB, Himedia, Mumbai, India) (for fungi). The tested microorganisms were isolated on Tryptone Soya agar (Himedia, Maharashtra, India) (for bacteria) and Sabouraud Dextrose agar (Himedia, Mumbai, India) (for fungi) after incubation at 37 °C for 24 h. A suspension for each microorganism was adjusted with sterile saline to achieve a turbidity of 0.5 McFarland (approximately 108 CFU/mL). Each suspension was diluted with media (MHB and SDB) to achieve a concentration of 5 × 105 CFU/mL. A volume of 10 µL of each microbial suspension was inoculated with the extract serial dilution and the final inoculum was 104 CFU/well. Two positive and one negative control were applied. A serial dilution (from 50 to 0.098%) of DMSO was inoculated with the same concentration of each microorganism (104 CFU/well) and used as a positive control to ensure that the extract achieved antimicrobial activity, not the DMSO. The second positive control is MHB and SDB inoculated with only the tested microorganisms, while both MHB and SDB were incubated and used as negative controls. The microdilution plates for the four microorganisms were incubated at 37 °C for 24 h. The experiment was performed in triplicate, and the MIC was recorded as the lowest concentration that inhibited microbial growth.

Cytotoxic effect of S. fruticosa extract

MTT cell viability assay

U2OS cells (kindly provided as gifts from Dr. Andreas Kakarougkas’ Lab (Department of Biology, The American University in Cairo, Egypt)) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, 1X) with stable glutamine and high glucose (SERANA, Pessin, Germany) supplemented with 10% Fetal Bovine Serum (FBS) (SERANA, Pessin, Germany) and 5 vol% PEN-STREP (Lonza, Verviers, Belgium) and incubated at 37 °C and 5% CO2 in humidified Hera cell CO2 incubator (Thermo-Fisher, USA). Regular cell splitting was performed before reaching confluency by washing the cells with Phosphate-Buffered Saline (PBS, 1X) (CORNING, Manassas, USA) and detaching them using 0.05 vol% of 1X trypsin-EDTA (SERANA, Pessin, Germany). We used an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan) to visualize the cells28,29.

U2OS cell viability after incubation with S. fruticosa methanolic extract was evaluated using the MTT assay30. Briefly, three experiments were conducted in which 5000 U2OS cells were incubated in a 96-well plate for 24 h at 37 °C in a CO2 incubator. Then, the cells were incubated with serial dilutions of S. fruticosa extract (prepared in DMEM complete medium) at concentrations of 150, 75, 37.5, 18.75, and 9.375 µg/mL for 24, 48, and 72 h. Then, 100 µL of fresh medium containing 10 vol% MTT solution (5 mg/mL) (SERVA, Heidelberg, Germany) was added to each well and incubated for 3 h. After the incubation period, the medium was replaced with 100 µL of DMSO and incubated in the dark at room temperature for 10 min to solubilize the formed formazan crystals, which have a purple color whose intensity is directly proportional to the number of metabolically active cells. The intensity was measured spectrophotometrically at 570 nm using a SPECTROstar nano microplate reader (BMG LABTECH, Ortenberg, Germany) after blanking the device with DMSO. The viability of treated cells was expressed as a percentage of the viability of untreated cells (negative control). The highest concentration of the extract in the serial dilution (150 µg/mL) contained 0.2 vol% DMSO. Therefore, this percentage of DMSO was prepared using DMEM complete medium and used as a control to test its effect on U2OS cell viability. The significance of the obtained viability percentage data was confirmed using two-way ANOVA analysis followed by Tukey’s multiple comparisons test at a 95% confidence level in GraphPad Prism 9 software. Nonlinear regression analysis on GraphPad Prism 9 software was performed to generate the dose-response curves for S. fruticosa extract after the three investigated time intervals using cell viability percentages obtained from the MTT assay plotted against logarithmic concentrations of S. fruticosa extract followed by applying Eq. (6) to calculate the IC50 which is the treatment dose required to inhibit cell growth by 50%31.

Evaluating treatment cytotoxicity on HSF cells compared to U2OS cells

To ensure the safety of the extract on normal cells, an MTT assay was conducted by the Central Laboratory in the Faculty of Science at Mansoura University (60 El Gomhouria St, Mansoura 1, Dakahlia, Egypt) on the available normal cells of HSF (NAWAH Scientific) to compare its results to U2OS15. Briefly, two experiments were conducted in which HSF and U2OS cells were seeded in 96-well plates at a density of 5,000 cells per well and incubated for 24 h using complete DMEM media. Then, the medium was discarded and replaced with the obtained IC50 of S. fruticosa extract and 4 µg/mL of cisplatin (as a reference), and the mixture was incubated at 37 °C in a CO2 incubator for 72 h. After that, the treatments were discarded, and 100 µL of DMEM complete media containing 10% MTT solution (5 mg/mL) was added to each well and incubated for 3 h. After the incubation period, the media was replaced by 100 µL of DMSO and incubated in the dark at room temperature for 10 min, followed by measuring the optical density at 570 nm using a SPECTROstar nano microplate reader (BMG LABTECH, Ortenberg, Germany) after blanking the device with DMSO. The viability of treated cells was expressed as a percentage of the untreated cells (negative control). The significance of the data was analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test at a 95% confidence level in GraphPad Prism 9 software15,30.

Treated U2OS cell morphology

U2OS cell morphology was observed under an Olympus IX70 inverted microscope at 20X magnification after treatment with S. fruticosa extract for 72 h. The morphological changes were assessed and compared with those of the untreated cells.

Scratch wound healing assay

A scratch wound healing assay was performed to evaluate U2OS cell migration ability after treatment with S. fruticosa extract32. Briefly, U2OS cells were seeded at a density of 100,000 cells per well in a 24-well plate and incubated until reaching 80% confluency. Then, the media were replaced with 100 µL of PBS. A scratch was made in the cell monolayer using a 200 µL sterile tip. Then, 300 µL of PBS were added to wash all the cell debris followed by adding the prepared treatment of S. fruticosa extract using DMEM complete media and incubation at 37 °C in a CO2 incubator until almost complete closure of wounds in the untreated wells (negative control) which, in our study, was achieved after 48 h. During the incubation period, images were taken at 0, 12, 24, and 48 h of incubation with the treatments using an Olympus IX70 inverted microscope at 20X magnification. The wound area was measured using ImageJ 1.51j8 software, and the wound confluency percentage was calculated according to Eq. (7). The significance of the obtained data was analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test at a 95% confidence level in GraphPad Prism 9 software32.

$$\it {\text{Wound confluency \% }}=\left( {\frac{{{\text{Wound area at zero time}} - {\text{Wound area at X h}}}}{{{\text{Wound area at zero time}}}}} \right){\text{x}}100{\text{}}$$
(7)

where X is the time point (interval).

Statistical analysis

All the above experiments were conducted in triplicate, and data are presented as mean ± standard deviation (SD) unless otherwise specified. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, CA, USA) as well as Microsoft Excel. A dose-response curve was generated after transforming and normalizing the obtained data using GraphPad Prism 9. ImageJ software was used to measure the wound area in the wound healing assay, and GraphPad Prism 9 was used to analyze the data. One-way or two-way ANOVA, followed by Dunnett’s or Tukey’s multiple comparisons tests, was used to determine the significant differences among different means. P values less than 0.05 were considered significant (**p < 0.01, ****p < 0.0001).

Results

Extraction yield and chemical characteristics of S. fruticosa extract

An average yield of 0.144 ± 0.008 g/g of the dry leaves was obtained from the ultrasonic extraction of S. fruticosa leaves in 70% methanol and the obtained FTIR spectrum of the extract is presented in Fig. 1.

Fig. 1
figure 1

FTIR spectrum of Salvia fruticosa extract.

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Phytochemical properties of S. fruticosa extract

Total carbohydrate, phenolic, and flavonoid content

TCC in S. fruticosa extract was estimated to be 45.63 ± 3.33 mg glucose equivalent/g of the extract. In , TPC and TFC were estimated to be 170.68 ± 6.52 mg caffeic acid equivalent and 44.71 ± 5.67 mg rutin equivalent/g of the extract, respectively (see supplementary Fig. S2 online).

Polyphenolics and flavonoids in S. fruticosa extract

Sixteen polyphenolic compounds were detected and measured in S. fruticosa methanolic extract using LC-ESI-MS/MS analysis. S. fruticosa extract consisted of nine phenolic compounds: chlorogenic acid, gallic acid, caffeic acid, kaempferol, ferulic acid, methyl gallate, coumaric acid, syringic acid, and 3.4-Dihydroxybenzoic acid in 0.12 to 1900.97 µg/g of the extract, whereas it contained seven flavonoids: rutin, hesperidin, quercetin, apigenin, luteolin, naringenin, and vanillin in 0.33 to 3137.22 µg/g of the extract. The retention times, areas, and fragmentation chromatograms for both the extract (sample) and standards are presented in supplementary Fig. S3 and Tables S1 and S2 online.

Biological activities of S. fruticosa extract

Antioxidant activity

As shown in Fig. 2a, the extract exhibits concentration-dependent free radical scavenging activity, ranging from 7.19 to 84.23% at concentrations of 5 to 80 µg/mL, respectively. Trolox also shows a concentration-dependent free radical scavenging activity ranging from 3.79 to 82.59% but at a lower concentration range (1.25 to 12.5 µg/mL), as shown in Fig. 2b. The IC50 of the extract and Trolox were determined using the extract dose-response curve, as shown in Fig. 2c and d. S. fruticosa extract showed an antioxidant capacity with an IC50 of 25.17 ± 0.304 µg/mL of DPPH compared to Trolox (IC50 = 6.352 µg/mL). Tukey’s multiple comparisons test results for the antioxidant activity of S. fruticosa extract and Trolox at various concentrations are summarized in supplementary Table S3 online.

Fig. 2
figure 2

Antioxidant activity of Salvia fruticosa extract using DPPH free radical scavenging activity assay. Antioxidant activity of S. fruticosa extract (a) and Trolox as a positive control (b). Dose-response curves against DPPH free radical for S. fruticosa extract (c) and Trolox (d). Data are representative of three experiments, and significance analysis was done using one-way ANOVA followed by Tukey’s multiple comparisons test, at a 95% confidence level on GraphPad Prism 9 (****p < 0.0001) (see supplementary Table S3 online). Data from dose-response curves represent the results of three experiments, and analysis was performed using nonlinear regression analysis in GraphPad Prism 9. IC50 values are shown as mean ± SD.

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Antimicrobial activity

The results of the MIC assay (Table 1) indicate that the extract exhibits effective antimicrobial activity, with the best MIC values observed against S. aureus, B. subtilis, and S. cerevisiae at 1.562 mg/mL, compared to the MIC value for E. coli, which was 12.5 mg/mL.

Table 1 Antimicrobial activity of Salvia fruticosa extract using minimum inhibitory concentration assay by microdilution method.
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U2OS cell viability after incubation with S. fruticosa extract and 0.2 vol% DMSO

As shown in Fig. 3a, S. fruticosa extract decreased U2OS cell viability in a dose- and time-dependent manner. A higher S. fruticosa extract concentration of 150 µg/mL decreased U2OS viability by 32%, 50%, and 96% after 24, 48, and 72 h, respectively, with p < 0.0001. Additionally, 75 µg/mL of S. fruticosa extract had no significant effect on U2OS cell viability after 24 h of incubation, but decreased cell viability by 20% (p = 0.0033) and 42% (p < 0.0001) after 48 and 72 h, respectively. On the other hand, lower S. fruticosa extract concentrations showed no significant difference in cell viability between treated and untreated cells over the three investigated time intervals. A dose-response curve for the extract was generated using MTT data, as shown in Fig. 3b. IC50 values obtained after 24, 48, and 72 h had standard error of residual plots (Sy.x) values of 26.04, 22.88, and 7.92, respectively. The IC50 had coefficient of determination (R2) values of 0.6241, 0.6991, and 0.9581, respectively, and all Hillslope values were lower than one. The lowest recorded IC50 value was 77.58 µg/mL after 72 h of incubation, followed by 80.85 µg/mL after 48 h of incubation and 87.75 µg/mL after 24 h of incubation with S. fruticosa extract. The IC50 obtained after 72 h of incubation had the lowest value, with the lowest Sy.x and the best R2 value (near 1), as summarized in supplementary Table S4 online.

To ensure that the cytotoxicity induced by different concentrations of S. fruticosa was due to its extract and not the DMSO used for its dissolving, we conducted an MTT assay to evaluate the effect of 0.2 vol% DMSO, which constituted the highest concentration of S. fruticosa extract (150 µg/mL), on cell viability after 24, 48, and 72 h of incubation with U2OS. As presented in Fig. 3c, a 0.2 vol% DMSO concentration had no significant effect on U2OS cell viability after incubation at the three time points.

Fig. 3
figure 3

Effect of Salvia fruticosa extract and 0.2 vol% DMSO on U2OS cell viability after 24, 48, and 72 h of incubation using MTT assay. (a) U2OS cell viability after incubation with S. fruticosa extract. The extract decreased viability in a dose and time-dependent manner. The highest decrease in cell viability (by 96%) was achieved after incubation with 150 µg/mL of the extract for 72 h. (b) S. fruticosa extract dose-response curves. The obtained dose-response curves had a negative slope, indicating a decrease in cell viability with increasing extract concentration. The obtained IC50 values were 87.75 ± 4.02 µg/mL (R² = 0.6241), 80.85 ± 10.65 µg/mL (R² = 0.6991), and 77.58 ± 3.47 µg/mL (R² = 0.9581) after 24, 48, and 72 h, respectively. (c) U2OS cell viability after incubation with 0.2 vol% DMSO, which constituted the highest concentration of the extract. No significant change in cell viability was observed over the three time points. All U2OS viability comparisons were made between the treated and untreated cells; data are representative of three experiments and significance analysis was done using two-way ANOVA followed by Tukey’s multiple comparisons test, except for the 0.2 vol% DMSO viability data analysis which was done using one-way ANOVA followed by Dunnett’s multiple comparisons test, at a 95% confidence level on GraphPad Prism 9 (**p < 0.01, ****p < 0.0001, ns = no significance). Data from dose-response curves represent the results of three experiments, and analysis was performed using nonlinear regression analysis in GraphPad Prism 9. IC50 values are shown as mean ± SD.

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Cytotoxicity of S. fruticosa extract on HSF cells compared to U2OS cells

In our study, we used HSF as a model for normal cells on which we could test the cytotoxicity of S. fruticosa extract IC50 compared to U2OS cells to evaluate the possible side effects that may be caused by the extract on normal cells using the MTT assay and cisplatin as a reference15,30. As shown in Fig. 4, cisplatin significantly decreased the viability of U2OS and HSF cells by approximately 27 ± 4.08% and 32 ± 4.08%, respectively. In contrast, the extract decreased U2OS cell viability by nearly 19% but had no significant effect on HSF viability.

Fig. 4
figure 4

Effect of Salvia fruticosa extract on the viability of HSF compared to U2OS cells after 72 h of incubation. The extract decreased U2OS cell viability by approximately 19% while it had no significant effect on HSF. On the other hand, 4 µg/mL of cisplatin showed a significant decrease in both U2OS and HSF by 32 ± 4.08% and 27 ± 4.08%, respectively. Comparisons were made between the treated and untreated cells; data are representative of three independent experiments, and significance analysis was done using two-way ANOVA followed by Tukey’s multiple comparisons test at a 95% confidence level on GraphPad Prism 9 (**p < 0.01, ****p < 0.0001, ns = no significance).

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U2OS cell morphology after incubation with S. fruticosa extract

As shown in Fig. 5a, a noticeable number of dead cells resulted after incubation with the extract. The cells lost their epithelial morphology and floated in the medium compared to the untreated cells (labeled by the black arrows). After washing cells with PBS to remove dead cells and debris (labeled by the yellow arrows), other images were taken to examine cell morphology as shown in Fig. 5b. The surviving cells were elongated, compared to the untreated cells due to the large space between cells caused by the induction of cell death by the extract, making the living cells stretch to reach each other. Additionally, the treatment altered the morphological characteristics of U2OS cells compared to those of the untreated cells. Treated cells with the extract showed morphological changes where cells shrank (with a spherical shape) and exhibited chromatin condensation with a characteristic necklace shape (labeled by the blue arrows) and blebbing (labeled by the purple arrows) instead of being elliptical with wider center and tapering ends as the untreated cells33,34.

Fig. 5
figure 5

Effect of Salvia fruticosa extract on U2OS osteosarcoma cell morphology. (a) Untreated and treated U2OS cells before washing with PBS. Untreated U2OS cells showed elliptical morphology and 100% confluency (labeled by the black arrows). Treated cells with the extract IC50 showed several dead cells and cell debris (labeled by the yellow arrows). (b) U2OS cell morphology after washing with PBS to remove dead cells and cell debris. Untreated U2OS cells showed an elliptical shape and 100% confluency (labeled by the black arrows). U2OS cells that were treated with the extract IC50 showed cell shrinkage & chromatin condensation (labeled by the blue arrow) and blebbing (labeled by the purple arrows). Images were captured using an inverted microscope with a 20X magnification lens.

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Effect of S. fruticosa extract on U2OS cell migration

The percentage of wound closure or confluency was determined after 12, 24, and 48 h of incubation with the treatments until almost complete closure of wounds in the untreated wells (negative control). After 12 and 24 h of incubation, S. fruticosa extract showed no significant difference in the percentage of wound confluency compared to the control (untreated cells). After 48 h of incubation, the extract showed a significant difference in the percentage of wound confluency (57 ± 8.39%) compared to the control’s wound closure percentage, which was 93 ± 8.39% as shown in Fig. 6.

Fig. 6
figure 6

Effect of Salvia fruticosa extract on U2OS osteosarcoma cell migration and wound closure over 12, 24, and 48 h of incubation. (a) Comparisons between treated and untreated cells revealed no significant difference in the wound closure between 12 and 24 h of incubation while the extract inhibited cell migration, with almost complete wound closure in untreated cells after 48 h. These results were supported by the statistical analysis in (b) where the extract’s IC50 concentration showed no significant decrease in wound closure percentage compared to the control (untreated cells) at the 12- and 24-hour time points. However, a significant decrease of nearly 43 ± 8.39% in wound closure was observed after 48 h. This suggests the extract’s potential to inhibit cell metastasis. Images were captured using an inverted microscope with a 20X magnification lens. Data represents three independent experiments, and significance analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test at a 95% confidence level in GraphPad Prism 9 (**p < 0.01, ns = no significance).

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Discussion

The main aim of this study is to extract bioactive compounds including polyphenols from S. fruticosa leaves and test their antimicrobial effect on two gram-positive (S. aureus ATCC6538 and B. subtilis ATCC35854), one gram-negative bacteria (E. coli ATCC8739), and one fungus (S. cerevisiae ATCC9763) as well as their cytotoxic effect on U2OS cells, as a model for bone cancer. Modern ultrasonic extraction using 70% methanol was performed on ground S. fruticosa leaves with an average yield of 0.144 ± 0.008 g/g of the dry leaves, followed by chemical characterization of the extract using FTIR. The obtained FTIR spectrum of S. fruticosa extract (Fig. 1) showed bands at wavelengths 3431.5, 2923.3, 2363.3, and 1625.6 that correspond to O-H stretching in alcohol, water, and polyphenols, aliphatic stretching (-CH2-) in fats, O = C = O stretching in carbon dioxide, and C = O stretching in amide 1, respectively. Additional bands also appear at 1385 and 1267.3 cm− 1 which correspond to the respective bending modes of vibration of CH2 and CH3 in fatty acids, proteins, and phosphate-bearing compounds, along with bands at 1163.5 and 1074.3 cm− 1 arising from the stretching vibration of –C–O–C glycoside ring bond, C–O stretching in COOH, and O–H bending in carbohydrates and polysaccharides35,36.

Numerous studies have demonstrated the anticancer effects of polysaccharides on various cell lines. They have been reported to induce apoptosis, DNA damage, disruption of mitochondrial membrane, and cell cycle arrest to kill cancer cells and prevent metastasis37,38,39,40. Additionally, polysaccharides were tested against a wide range of microorganisms for their antimicrobial activities, including increasing cell membrane permeability, inhibiting microorganism attachment to the host, or blocking membrane transport of nutrients essential for energy production41,42,43,44. Therefore, the phenol–sulfuric acid method was conducted to determine TCC in S. fruticosa extract, which was estimated to be 45.63 ± 3.33 mg glucose equivalent/g of the extract.

Sage is considered a rich source of phenolics that have been proven to have antimicrobial and anticancer activities TPC in S. fruticosa extract was measured using folin–ciocalteu assay and detected spectrophotometrically with an estimated value of 170.68 ± 6.52 mg caffeic acid equivalent/g of the extract. The measured percentage of total phenolic content in S. fruticosa, compared to that previously reported in Salvia species, is summarized in Table 2. According to our results, S. fruticosa is considered the second highest Salvia extract rich in phenolics with 17.07 ± 0.65%, after S. africana (35.06 ± 1.49%), compared to other 31 Salvia species with reported phenolic content percentages ranging from 0.7 to 35.06%41,42,43,63. This suggests that S. fruticosa extract may possess antimicrobial and anticancer properties, derived from its high content of phenolics, which have been previously reported to inhibit microbial growth and cancer progression.

Flavonoids have been reported to exhibit antimicrobial activity by suppressing nucleic acid synthesis, the functionality of the cytoplasmic membrane, and energy metabolism, in addition to their strong anticancer effects arising from their antioxidant, cell cycle-arresting, and induction of apoptosis and autophagy activities. They also inhibit cell proliferation and metastasis64,65,66,67,68,69,70. Therefore, we investigated the content of total flavonoids in S. fruticosa extract spectrophotometrically using the aluminum chloride method, and the estimated flavonoids were 44.71 ± 5.67 mg rutin equivalent/g of the extract. The percentage of total flavonoid content in S. fruticosa compared to the previously reported data in other 25 Salvia species is shown in Table 2. S. fruticosa contained 4.47 ± 0.57% of total flavonoids, which is lower than the highest reported flavonoid content in S. hierosolymitana by approximately 47.6% and ranked 6th in the highest content of flavonoids (after S. hierosolymitana, S. eigii, S. viridis, S. reuteriana, and S. hypoleuca) compared to other species with reported flavonoid content percentages ranging from 0.12 to 52.06%56,57,64,65,66,67,68,69,63,71. This indicates that S. fruticosa methanolic extract could also have good antimicrobial and anticancer properties due to its flavonoid content.

As the previous analyses indicated that S. fruticosa extract is rich in phenolics and flavonoids, we identified the major compounds from the two classes that are present in the extract using LC-ESI-MS/MS technique. As shown in Table 3, S. fruticosa extract constitutes eight phenolic compounds: chlorogenic acid, gallic acid, caffeic acid, kaempferol, ferulic acid, methyl gallate, coumaric acid, and 3.4-Dihydroxybenzoic acid while it comprises seven flavonoids: rutin, hesperidin, quercetin, apigenin, luteolin, naringenin, and vanillin in a concentration range from 0.12 to 3137.22 µg/g. According to previous studies, the detected phenolics and flavonoids were reported to be potential antimicrobial and anticancer agents72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111.

The concentrations of specific phenolics and flavonoids detected in S. fruticosa extract compared to extracts from other Salvia species are also reported in Table 3. Naringenin is the most abundant polyphenolic compound present in S. fruticosa methanolic extract, with a concentration of 3137.22 µg/g. It has also been reported in four other Salvia species, including S. verticillata, S. viridis, S. eigii, and S. hierosolymitana, in the range of 36.25–418.59 µg/g. Chlorogenic acid was also detected in S. fruticosa extract at 1900.97 µg/g, ranking it third among Salvia species rich in chlorogenic acid, after S. viridis and S. officinalis, with reported chlorogenic acid concentrations ranging from 159 to 6619.93 µg/g in eleven Salvia species. Caffeic acid and luteolin were also reported to have respective concentrations of 654.91 and 532.34 µg/g of S. fruticosa extract, rendering S. fruticosa the second Salvia rich in caffeic acid and luteolin after S. tementosa Miller. As for 3,4-dihydroxybenzoic acid, it was detected at 179.23 µg/g of the S. fruticosa extract. However, this has not been reported in other Salvia species. Apigenin was also quantified in S. fruticosa extract at 170.80 µg/g, compared to the previously reported concentrations in six Salvia species, which ranged from 13.48 to 208 µg/g. Other phenolics and flavonoids, namely p-coumaric, syringic, ferulic acids, quercetin, gallic acid, and rutin were measured at 40.14, 34.22, 21.43, 2.59, 2.24, and 0.33 µg/g of S. fruticosa extract, respectively and these are the lowest reported concentrations for these compounds in Salvia species. Hesperidin was detected at 6.45 µg/g of S. fruticosa extract, while its concentration in five Salvia species ranged from 0.0005 to 1002 µg/g. Vanillin and methyl gallate were quantified at 13.23 and 0.12 µg/g of S. fruticosa extract, respectively, but were not reported in other Salvia species. On the other hand, ellagic acid, cinnamic acid, catechin, daidzein, and myricetin were not detected in S. fruticosa methanolic extract58,61,62,112,113,114.

As the correlation between total phenolic and flavonoid content and antioxidant activity is not yet confirmed, a DPPH free radical scavenging activity assay was performed to determine the antioxidant activity of the extract23,24,115. Since a lower IC50 value indicates a higher ability of the extract to scavenge free radicals and consequently potent antioxidant activity, our extract showed a good antioxidant capacity with an IC50 of 25.17 ± 0.304 µg/mL of DPPH (Fig. 2). Thus, S. fruticosa extract ranked the 9th most potent antioxidant—after S. hydrangea, S. ceratophylla, S. africana, S. verticillate, S. mexicana, S. macrosiphon, S. ahendica, S. chloroleuca—among the determined IC50 of 26 Salvia species, ranging from 5.3 to 557.4 µg/mL as summarized in Table 256,57,58,59,60,116,117,118,119. This indicates that the extract can scavenge free radicals due to its electron- and/or hydrogen atom-donating ability and consequently could have the ability to inhibit the initiation and/or progression of free radical-mediated chain reaction and thus inhibit cancer progression through preventing DNA damage, decreasing of free radical-mediated chain reaction and thus inhibiting cancer mutagenesis and reducing abnormal cell division progression through preventing DNA damage, decreasing mutagenesis and reducing abnormal cell division120,121,122.

Table 2 Comparison of total phenolics, total flavonoids, and antioxidant activity (represented by DPPH IC50) of Salvia fruticosa (current study) to previously reported Salvia species.
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Table 3 Reported concentrations of phenolics and flavonoids in different Salvia species compared to our liquid chromatography-electrospray ionization–tandem mass spectrometry (LC-ESI- MS/MS) study on Salvia fruticosa extract.
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As the extract is a rich source of carbohydrates, phenolics, and flavonoids that have been demonstrated to exhibit good antimicrobial activity, the antimicrobial activity of the methanolic extract of S. fruticosa was examined against microorganisms causing foodborne diseases and human infections, including S. aureus, B. subtilis, E. coli, and S. cerevisiae. S. aureus and E. coli are known for their multidrug resistance and association with nosocomial infections26. So, the need to find better antimicrobial alternatives is urgent. MIC by the microdilution method was performed to evaluate the antimicrobial activity of S. fruticosa extract. The MIC values were 1.562 mg/mL for S. aureus, B. subtilis, and S. cerevisiae, and 12.5 mg/mL for E. coli. Previous studies were conducted to evaluate the antimicrobial activities of different species of Salvia on similar strains of S. aureus and E. coli, and a comparison between the results of the current study and the previous ones is summarized in Table 4123,124,125,126,127,128. The methanolic extract exhibited good antimicrobial activity against S. aureus (MIC = 1.562 mg/mL) within the reported antimicrobial range of S. fruticosa evaluated in previous studies (MIC 0.2–3.42 mg/mL). The methanolic extract also exhibited better antimicrobial activity against S. aureus compared to other extracts of S. officinalis, S. lavandulifolia, and S. sclarea (MIC ranging from 2.31 to 12.5 mg/mL). Additionally, S. fruticosa water and ethanolic extracts were also reported to exhibit higher antimicrobial activity against E. coli with MICs ranging from 0.0024 to 0.625 mg/mL relative to S. fruticosa methanolic extract in the current study (MIC = 12.5 mg/mL)123,124,125,126,127,128. These findings suggest the potential antimicrobial activity of the methanolic extract of S. fruticosa against gram-positive, gram-negative bacteria, and fungi, likely due to its substantial content of carbohydrates, phenolics, and flavonoids.

Table 4 Comparison of the antimicrobial activity of Salvia fruticosa extract of the current study to other reported Salvia species using the minimum inhibitory concentration assay.
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Being rich in polyphenolic compounds, which exhibit potent antioxidant activity and can potentially act as anticancer agents. S. fruticosa methanolic extract was investigated for its cytotoxicity on U2OS osteosarcoma cells using the MTT assay. The extract inhibited U2OS cell proliferation with an IC50 of 87.75 ± 4.02, 80.85 ± 10.65, and 77.58 ± 3.47 µg/mL after 24, 48, and 72 h of incubation, respectively, as shown in Fig. 3 and supplementary Table S3 online. To ensure that the cytotoxicity was due to the extract itself and not due to DMSO that was used to dissolve the extract, another MTT assay was performed on 0.2 vol% DMSO constituting the highest concentration of the extract and there was no significant effect on U2OS viability as illustrated in Fig. 3c. In a previous study, it was reported that the acetone extract of S. fruticosa leaves decreased U2OS cell viability but with a lower IC50 (30.21 ± 1.18 µg/mL) than the methanolic extract after 48 h of incubation which indicates that the acetone extract of S. fruticosa had a higher cytotoxicity effect on U2OS cells than the methanolic extract15.

In our study, we aimed to investigate the cytotoxic effect of the extract compared to cisplatin (as a reference) on normal human cells versus cancerous ones, with the goal of evaluating potential side effects. So, we used HSF as a model for normal cells15. Fortunately, the extract exhibited no significant effect on HSF, while decreasing U2OS viability by approximately 19% compared to 4 µg/mL of cisplatin, which inhibited both U2OS and HSF by 32 ± 4.08% and 27 ± 4.08%, respectively, as shown in Fig. 4. indicating the potential use of the extract as a treatment for osteosarcoma without affecting normal body cells.

Additionally, we assessed the effect of S. fruticosa extract on U2OS cell morphology using a 20X magnification lens of the inverted microscope. The extract showed a change in U2OS cell morphology from being elliptical with a wider center and tapering ends to becoming shrunk and exhibiting chromatin condensation with a characteristic necklace shape and blebbing as illustrated in Fig. 533,34.

Finally, in our study, we evaluated the effect of the extract on U2OS cell migration, an indicator of metastasis, by performing a scratch wound healing assay. After 48 h of incubation, the S. fruticosa extract showed a significant difference in the percentage of wound confluency, 57 ± 8.39% compared to the control at 93 ± 8.39%, indicating that the treatment may affect the genetic markers involved in cell proliferation, migration, and possibly metastasis, as shown in Fig. 6.

While this study provides valuable insights into the bioactive compounds and therapeutic potential of S. fruticosa methanolic extract, it is important to acknowledge certain limitations. Primarily, the current investigation is based on in vitro experiments, which, although crucial for initial screening and mechanistic understanding, may not fully capture the complex biological interactions occurring in vivo. Therefore, the observed antimicrobial and cytotoxic effects require further validation using animal models to evaluate efficacy, pharmacokinetics, and potential toxicity in a living system. Additionally, the scratch wound healing assay was performed without prior cell starvation, meaning the results reflect a combined effect of both cell proliferation and migration. Future research should aim to isolate and characterize the individual bioactive compounds responsible for the observed activities, moving beyond the crude extract to identify specific lead compounds for drug development. Moreover, a deeper investigation into the molecular mechanisms underlying the extract’s effects on osteosarcoma cells, such as its influence on signaling pathways, cell cycle progression, and apoptosis, would enhance our understanding. Finally, developing formulations more suitable for in vivo applications would be a critical step toward translating these promising in vitro findings into clinical relevance.

Conclusion

S. fruticosa leaves are rich sources of phenolic and flavonoid compounds, which have been previously reported to possess strong antimicrobial and anticancer effects. The methanolic extract exhibited good antimicrobial activity against both gram-positive and gram-negative bacteria, as well as fungi. It also demonstrated a powerful free radical scavenging activity. It showed inhibition of U2OS osteosarcoma cell migration while having no significant effect on normal HSF cells which indicated that the methanolic extract of S. fruticosa leaves could be a future potential treatment of different microbial infections in addition to osteosarcoma because of its high cytotoxicity and potential inhibition of U2OS cell metastasis while maintaining safety on normal cells.