Bioactive fatty acids and anticancer potential of leaf extracts from Stachys byzantina K. KochAbstract
Stachys byzantina is a plant species of culinary and ethnomedicinal relevance, recognized for its versatility and as a source of essential micronutrients. This study aimed to determine the chemical composition and fatty acid profile of the hexane leaf extract, as well as evaluate the anticancer activity of the hexane, ethanolic, hydroethanolic, acetone, and hydroacetone extracts of Stachys byzantina. The fatty acid profile was determined using gas chromatography, and characterization was carried out according to quality and identity indices of crude oil, including iodine, acidity, and peroxide values. Anticancer activity was assessed through the Sulforhodamine B cytotoxicity assay. The results revealed that the hexane extract was predominantly composed of polyunsaturated fatty acids, such as α-linolenic and linoleic acids, along with the saturated fatty acid palmitic acid. . UV–Visible spectrophotometric analysis indicated the presence of tocopherols, carotenoids, and chlorophyll in the hexane extract. Regarding anticancer activity, the hexane extract demonstrated efficacy against glioma, breast adenocarcinoma, and murine melanoma cells, while the hydroethanolic and hydroacetone extracts were active against murine melanoma cells. Importantly, the tested extracts showed no cytotoxic effects on normal human umbilical cord cells or murine fibroblasts, suggesting that their consumption can be considered safe.
Keywords:
vegetable oils; Lamiaceae; bioactive compounds; polyunsaturated fatty acids; cytotoxicity; nutraceutical potential; phytochemistry
Resumo
Stachys byzantina é uma planta que tem destaque tanto na culinária quanto na medicina tradicional, evidenciando sua versatilidade e benefícios, como fornecimento de importantes micronutrientes. A pesquisa objetivou determinar a composição química, o perfil de ácidos graxos do extrato hexânico das folhas e avaliar a atividade anticâncer dos extratos hexânico, etanólico, hidroetanólico, acetônico e hidroacetônico da Stachys byzantina. O perfil de ácidos graxos foi determinado por análise cromatográfica gasosa. A sua caracterização foi feita conforme os índices de qualidade e identidade do óleo bruto: índices de iodo, de acidez e de peróxido. Para avaliação da atividade anticâncer foi utilizado o ensaio de citotoxicidade com Sulforodamina B. Os resultados obtidos demostraram que a composição predominante do extrato hexânico consiste em ácidos graxos poli-insaturados, como o α-linolênico e ácido linoleico, além do ácido graxo saturado palmítico. Na análise espectrométrica UV/Visível o extrato hexânico indicou a presença de tocoferóis, carotenoides e clorofila. Na atividade anticâncer, o extrato hexânico demonstrou-se efetivo nas células de glioma, adenocarcinoma de mama e melanoma murino. Os extratos hidroetanólico e hidroacetônico apresentaram atividade nas células de melanoma murino. Os extratos testados não apresentaram efeito citotóxico nas células normais de cordão umbilical e fibroblasto murino, evidenciando que seu consumo pode ser considerado seguro.
Palavras-chave:
óleos vegetais; Lamiaceae; compostos bioativos; ácidos graxos poliinsaturados; citotoxicidade; potencial nutracêutico; fitoquímica
1. Introduction
Stachys byzantina K. Koch, popularly known as "peixinho-da-horta" or "pulmonária", is native to Turkey, Asia, and the Caucasus. In Brazil, it is cultivated in the South and Southeast regions. This is a perennial herb, reaching approximately 40 cm in height with simple, thick leaves that are 5 to 14 cm long (Kinupp and Lorenzi, 2014). It contains vitamin A, phenolic compounds, and minerals such as iron and calcium (Silva et al., 2024). Owing to this composition, it exhibits high antioxidant potential (Stegăruș et al., 2021; Silva et al., 2024), as well as antimicrobial activity (Benedec et al., 2023; Kiashi et al., 2021).
This species is little known as an edible plant outside Brazil and is classified in the country as a Non-Conventional Edible Plant (NCEP). NCEPs are plants or plant parts such as roots, tubers, bulbs, rhizomes, stems, leaves, shoots, flowers, fruits, or seeds, which are not popularly consumed or even unknown as food by the majority of the population. Among these plants, some species stand out not only for their nutritional potential but also for their lipid composition.
Currently, there is an emphasis on the quality of lipid intake, with a preference for replacing saturated fatty acids with unsaturated fatty acids for a healthier diet (Schwingshackl et al., 2022).
Therefore, it is important to investigate the presence and proportions of fatty acids in Stachys byzantina leaves since there is evidence that consuming these compounds can have several beneficial effects on physiological and metabolic aspects. Therefore, the objective of the present study was to determine the chemical composition, fatty acid profile, and potential anticancer activity of Stachys byzantina leaf extracts.
2. Methods
2.1. Leaf collection and sample preparation
The leaves of S. byzantina were collected from the Flor do Cerrado garden in Campo Grande, Mato Grosso do Sul, Brazil (20°31'10.373" S, 54°44'56.987" W) in August 2021. The specimen was deposited in the Herbarium of the Federal University of Mato Grosso do Sul, Brazil, with CGMS identification number 81583.
The leaves were collected, mixed, and washed with distilled water, then immediately dried in a forced-air oven at 40 °C for 48 hours. The dried sample was ground in a TEC-631 laboratory mill (Tecnal®) with a mortar and pestle, then placed in an airtight amber glass vial and frozen at -18 °C for later analysis.
2.2. Proximate composition
The centesimal composition was performed on the ground leaves according to the standards of the Adolfo Lutz Institute – IAL (2008) and the Association of Official Analytical Chemists – AOAC (2005). The samples were analyzed for moisture content using the gravimetric method in an oven at 105 °C until constant weight; fixed mineral residue using the gravimetric method with calcination in a muffle furnace at 550 °C; proteins using the classical Micro-Kjeldahl method; and lipids using the Soxhlet method (IAL, 2008). Total carbohydrates (Nifext fraction) were determined by difference from the other fractions (AOAC, 2005).
2.3. Oil preparation and fatty acid profile
To determine the fatty acid profile, the oil was extracted by static maceration using hexane as a solvent. The supernatant formed was collected every 72 hours into an amber vial until it became colorless.
It was subsequently dried in an evaporator (Rotary Evaporator R-3, BUCHI) at 35-40 °C and the sample was again placed in an amber airtight glass bottle and stored in a freezer at -18 °C for later analysis (ANVISA, 2019; Bittencourt-Junior et al., 2012; Lima and Coelho, 2013).
The fatty acids were esterified according to the method suggested by Maia and Rodriguez-Amaya (1993). The fatty acid methyl esters were analyzed by gas chromatography (GC 201, Shimadzu, Kyoto, Japan) to obtain their peaks.
Individual peaks of fatty acid methyl esters (FAMEs) were identified by comparing their relative retention times with the 37-FAME standard (Supelco C22, 99% pure). In addition, we used the index of atherogenicity and the index of thrombogenicity (Ulbricht and Southgate, 1991).
2.4. Quality of oil from Stachys byzantina leaves
The oil extracted from the leaves of S. byzantina was characterized by iodine value, acid value, and peroxide value. The iodine value (method 330/IV) and acid value (method 325/IV) were quantified according to the analytical standards of the Adolfo Lutz Institute (IAL, 2008), while the peroxide value (Cd 8-53 method) was determined according to the analytical standards of the American Oil Chemists' Society (AOCS, 1990).
2.5. Optical analysis: UV-Visible
S. byzantina oil was diluted in HPLC-grade hexane (99.9% spectroscopic grade) at concentrations of 0.02 g/L, 0.35 g/L, and 1.03 g/L. UV/Visible absorptions were performed using a Lambda 265 UV/Vis spectrophotometer (Perkin Elmer, Waltham, MA, USA), and spectra were collected in the 200-800 nm range. For UV/Vis absorption measurements, the diluted oil was placed in a four-sided polished quartz cuvette with a 10 mm path length.
2.6. Evaluation of the in vitro anticancer activity of Stachys byzantina oil
Cell proliferation was determined using the colorimetric method with sulforhodamine B (SRB) (Sigma, USA). Using the concentration-response curve for cell line, GI50 (concentration causing 50% cell growth inhibition) was determined by nonlinear regression analysis (sigmoidal fitting) using the program Origin 6.0 (OriginLab Corporation, Northampton, MA, USA).
The hexane extract or crude oil (HES), ethanolic extract (EES), dichloromethane extract (DME), hydroethanolic extract (70:30) (HEES), and hydroketone extract (20:80) (HAES) were tested against tumor cell lines from the American Type Culture Collection (ATCC, Manassas, VA, USA). The hexane extract was evaluated for GI50 (concentration that inhibits 50% of cell growth) in neoplastic cell lines of murine melanoma (B16-F10 ATCC – CRL-6475), breast carcinoma (MCF7 ATCC – HTB-22), kidney carcinoma (786-0 ATCC – CRL-1932), glioma (U251 - CVCL_0021) and normal murine fibroblast cells (NIH/3T3 – ATCC® CRL-1658) to determine the Selectivity Index (corresponds to the division of the compound GI50 in NIH-3T3 immortalized cells and compound GI50 in tumor cell line) with doxorubicin as a positive control (Houghton et al., 2007; Cândido et al., 2022).
3. Results and Discussion
3.1. Proximate composition
The results of the proximate composition of S. byzantina leaves are expressed in Table 1.
The high moisture content (85.40%) observed in the leaves highlights the importance of consuming or processing them fresh. Vegetables such as lettuce (Latuca sativa Linnaeus) and cilantro (Coriandrum sativum Linnaeus) also have a high moisture content, 94.85% and 89.89%, respectively (Lima et al., 2024).
The carbohydrate content found in the dried leaves was 66.23%, a value close to the 66.60% found by Silva (Silva et al., 2024) in leaves of the same species collected in Paraná, Brazil, eight months after planting and dried at room temperature. However, the ash (9.28%), protein (16.24%), and lipid (6.23%) contents found in the dried leaves were slightly different from those found by the same authors, who described values of 6.83, 11.86, and 2.13% for ash, protein, and lipid, respectively, performing the same analyses. This variation may possibly occur according to the time of leaf harvest and the soil used for cultivation.
3.2. Oil yield and fatty acid profile
The crude oil yield obtained from the fresh leaves of S. byzantina was 1.7%, indicating a relatively low lipid content. Nevertheless, subsequent characterization demonstrated that 48% of the oil volume was composed of fatty acids, underscoring the nutritional relevance of this fraction.
The fatty acid profile (Table 2) revealed a clear predominance of unsaturated fatty acids (ΣUFAs = 78.36%), of which polyunsaturated fatty acids (PUFAs) accounted for the major fraction (73.97%). Within this group, α-linolenic acid (C18:3n3; 59.31%) and linoleic acid (C18:2n6c; 13.38%) were the most abundant, together contributing more than 70% of the total fatty acids. These compounds are essential fatty acids, requiring dietary intake since they are not synthesized endogenously (Lunn and Theobald, 2006). The predominance of PUFAs aligns with findings in other Stachys species, such as S. lavandulifolia (79.8%) and S. iberica (82.9%) (Kilic et al., 2017), reinforcing that the genus is consistently rich in unsaturated fatty acids.
The biological importance of PUFAs lies in their structural role in membrane phospholipids and as precursors of lipid mediators (Dyall et al., 2022). Omega-3 fatty acids such as α-linolenic acid are particularly associated with cardiovascular protection, cognitive improvement, and regulation of inflammatory responses (Rajaram, 2014; Wang et al., 2024). Moreover, PUFA-derived metabolites, including resolvins and protectins, exert potent anti-inflammatory effects by reducing the activation of nuclear factor-kB and lowering the production of cytokines such as interleukin-6 and TNF-α (Siriwardhana et al., 2012). Similarly, linoleic acid (omega-6) plays versatile roles in energy metabolism, lipid esterification, and membrane fluidity (Whelan and Fritsche, 2013), although excessive conversion into arachidonic acid may contribute to inflammatory disorders (Choque et al., 2014).
Monounsaturated fatty acids (MUFAs) comprised 4.40%, with oleic acid (C18:1n9c; 3.43%) as the major representative. Oleic acid is widely recognized for its cardioprotective and anti-inflammatory properties and is a hallmark of healthy diets such as the Mediterranean diet. Minor MUFAs, such as palmitoleic (C16:1; 0.46%), erucic (C22:1n9; 0.50%), and nervonic acid (C24:1n9; 0.89%), although present in small proportions, expand the biochemical diversity of the oil and may contribute to specific bioactivities, such as nervous system modulation.
Saturated fatty acids (SFAs) accounted for 20.73%, a proportion higher than those reported in S. iberica (10.5%) and S. lavandulifolia (10.0%) (Kilic et al., 2017), and also considerably higher than that found in traditionally consumed leafy vegetables, such as arugula (Eruca sativa Mill; 7.74%) (El-Nwehy et al., 2023). The predominant SFA was palmitic acid (C16:0; 12.25%), followed by stearic acid (2.15%). Although palmitic acid is the most common fatty acid in the human body, acquired both exogenously and via de novo lipogenesis (Carta et al., 2017), its excessive intake is associated with hypercholesterolemia, activation of inflammatory pathways, and increased cardiometabolic risk (Mason et al., 2009; Korbecki and Bajdak-Rusinek, 2019). However, emerging evidence suggests that the deleterious effects of SFAs depend on their dietary source, matrix, and overall dietary context (Perna and Hewlings, 2023).
In addition to the fatty acid distribution, nutritional quality indices provide insight into the health implications of the lipid fraction. The atherogenic index (0.20) and thrombogenic index (0.08) were remarkably low, while the hypocholesterolemic/hypercholesterolemic (h/H) ratio (5.77) was highly favorable. These values indicate that S. byzantina oil has a lipid profile with low inflammatory potential and high nutritional quality, consistent with cardioprotective and metabolic benefits (Kmiecik et al., 2023).
Taken together, the fatty acid composition of S. byzantina leaves demonstrates a strong predominance of essential polyunsaturated fatty acids, particularly α-linolenic acid, combined with favorable lipid indices. Despite the relatively high proportion of palmitic acid compared with related Stachys species, the overall profile suggests a nutritionally beneficial oil with promising functional applications, especially in the context of cardiovascular health, metabolic regulation, and anti-inflammatory effects.
3.3. Quality of oil from Stachys byzantina leaves
The physicochemical analysis of the crude oil extracted from Stachys byzantina leaves revealed important quality parameters that provide insight into its chemical stability and potential suitability for human consumption (Table 3). Quality indices such as iodine, acidity, and peroxide values are widely recognized as key indicators for assessing the stability and degradation status of edible oils (Yang et al., 2024). The iodine index reflects the degree of unsaturation in triglycerides, while the acidity index indicates the presence of free fatty acids generated through hydrolysis, oxidation, or fermentation. The peroxide index, in turn, is a primary measure of lipid oxidation and rancidity (IAL, 2008; Al-Bachir and Koudsi, 2021; Asadi and Farahmandfar, 2020).
The iodine value of S. byzantina oil was 190.36 g I2 100 g−1, a value considerably higher than those reported for most conventional edible oils and above the reference values described in the Codex Alimentarius by Food and Agriculture Organization (FAO, 1999). Such a high iodine number is consistent with oils rich in polyunsaturated fatty acids (PUFAs), corroborating the fatty acid profile previously determined for this species, where α-linolenic and linoleic acids predominated. While this composition reinforces the nutritional relevance of the oil due to its abundance of essential fatty acids, it also indicates increased susceptibility to oxidative degradation compared to oils dominated by saturated or monounsaturated fatty acids (Silva et al., 2022).
The acidity index was 13.08 mg KOH g−1, markedly higher than the Codex Alimentarius limit of 4.0 mg KOH g−1 for cold-pressed and unrefined oils (FAO, 1999). Elevated acidity levels signal hydrolytic degradation of triglycerides, leading to free fatty acid accumulation, which may be accelerated by enzymatic lipase activity or by exposure to heat, moisture, and light during extraction or storage. Such degradation negatively impacts oil quality, reducing its sensory acceptance and stability.
The peroxide index reached 1097.45 mEq kg−1, far exceeding the Codex Alimentarius threshold of ≤15 mEq kg−1 for edible oils (FAO, 1999). Peroxide values this elevated indicate advanced lipid oxidation, reflecting extensive hydroperoxide accumulation typical of rancid oils. These findings demonstrate that, despite the high nutritional potential of the oil due to its PUFA composition, its oxidative stability is extremely poor. This instability is expected for PUFA-rich matrices, given their higher reactivity to oxygen, temperature fluctuations, and light exposure.
When compared to commonly consumed oils such as olive and soybean, the values obtained for S. byzantina oil are substantially higher. For unheated olive oil, Geng et al. (2023) reported iodine, acidity, and peroxide values of 113.55 g I2 100 g−1, 1.80 mg KOH g−1, and 0.026 mEq kg−1, respectively. After only one minute of microwave heating at high power, these values decreased or slightly increased to 100.46 g I2 100 g−1, 3.29 mg KOH g−1, and 0.043 mEq kg−1, respectively, remaining well below those observed in S. byzantina. Similarly, unheated soybean oil presented iodine, acidity, and peroxide values of 114.42 g I2 100 g−1, 1.46 mg KOH g−1, and 0.059 mEq kg−1, respectively, which after heating shifted to 105.12 g I2 100 g−1, 2.51 mg KOH g−1, and 0.065 mEq kg−1 (Geng et al., 2023). These comparative data emphasize that the oil from S. byzantina has a significantly higher unsaturation degree but also suffers from accelerated hydrolytic and oxidative degradation.
Taken together, these results demonstrate that although S. byzantina oil is characterized by a favorable fatty acid composition, rich in essential PUFAs, its high acidity and peroxide values do not comply with international standards for edible oils, rendering it unsuitable for direct human consumption in its crude state. These findings highlight the need for improved extraction and preservation strategies, such as cold pressing under inert atmosphere, addition of natural antioxidants, and appropriate refining processes, to minimize lipid oxidation and free fatty acid accumulation. With such adjustments, S. byzantina oil could become a viable source of bioactive fatty acids for nutritional or functional applications.
3.4. Optical molecular analysis: UV-Vis
The UV–Vis spectra of Stachys byzantina leaf oil, recorded at concentrations of 0.02 g/L, 0.34 g/L, and 1.03 g/L in hexane, exhibited characteristic absorption bands associated with natural pigments and antioxidants (Figure 1). In the 200–300 nm region, strong absorption was detected, mainly attributed to π→π* transitions of conjugated double bonds, consistent with tocopherols and other phenolic constituents. A shoulder around 320–350 nm was observed, particularly at intermediate concentration, suggesting the presence of carotenoids with extended conjugation systems. In the visible region (400–500 nm), a prominent band at approximately 430–450 nm was identified, corresponding to carotenoids such as lutein and β-carotene. At longer wavelengths, distinct bands appeared near 660–670 nm and around 700 nm, characteristic of chlorophylls a and b. The intensity of these signals increased proportionally with concentration, in accordance with Beer–Lambert’s law, with the 1.03 g/L spectrum showing the most pronounced absorption. These spectral features confirm the presence of tocopherols, carotenoids, and chlorophylls in the oil, corroborating its biochemical composition and reinforcing its potential as a natural source of antioxidant compounds.
Optical molecular analysis used Stachys byzantina leaves oil diluted in hexane HPLC 99.9% at 0.02g/L, 0.34g/L e 1.03g/L. UV-Visible absorption spectrum wavelength collected between 200 and 800 nm on three different concentrations.
The biological relevance of these compounds has been widely documented. Tocopherols, a group of vitamin E derivatives supplied through vegetable oils, oilseeds, and nuts (Szewczyk et al., 2021), are represented by the absorption band between 200 and 300 nm (Smyk, 2021), detected in the 0.02 g/L dilution (Figure 1). Among them, α-tocopherol is selectively retained in human tissues and acts as a peroxyl radical scavenger, protecting polyunsaturated fatty acids in membranes and lipoproteins and preventing lipid peroxidation (Niki and Traber, 2012).
Carotenoids, responsible for the yellow, orange, and red colors of many foods, were evident in the spectrum at 0.34 g/L, with maximum absorption between 400 and 518 nm, consistent with their three-peak profile (Rodríguez-Amaya and Kimura, 2004). These pigments are natural antioxidants linked to a reduced risk of chronic diseases (González-Peña et al., 2023). Provitamin A carotenoids such as β-carotene, α-carotene, γ-carotene, and β-cryptoxanthin are essential for visual health, acting as photoprotectors for the retina and lens and mitigating oxidative damage (Johra et al., 2020). Additionally, lutein, zeaxanthin, and lycopene contribute to free radical elimination and protection against inflammation and degenerative disorders (González-Peña et al., 2023). Since humans cannot synthesize carotenoids, dietary intake, particularly through green leafy vegetables and colorful fruits, remains the only source of these bioactive molecules (Hacke et al., 2023).
Finally, chlorophylls, which exhibited strong absorption peaks at 660–670 nm and 700 nm, were especially intense at 1.03 g/L (Figure 1). These pigments not only define the green color of plants but also provide multiple biological activities, including antioxidant, anti-inflammatory, anticancer, antiobesity, antimutagenic, and antigenotoxic effects (Martins et al., 2023). Their absorption maximum around 652 nm (Palta, 1990) was consistent with the spectral signatures observed in this study.
Taken together, the spectral and functional characterization confirms that the oil from S. byzantina leaves is a natural source of tocopherols, carotenoids, and chlorophylls, compounds with recognized antioxidant activity and potential health benefits.
3.5. Evaluation of in vitro anticancer activity of Stachys byzantina oil and selectivity index
The parameter used to determine anticancer activity was the GI50, defined as the concentration required to inhibit 50% of cell growth. In this study, GI50 values above 250 µg/mL were considered indicative of inactivity against neoplastic cell lines and absence of cytotoxicity toward normal cells. The results of the assays conducted on neoplastic and non-neoplastic cell lines are summarized in Tables 4 and 5.
Growth inhibition values (GI50) of the hexane extract (crude oil) from S. byzantina in neoplastic cell lines of murine melanoma, breast adenocarcinoma, kidney, glioma, and normal murine fibroblast cells.
Growth inhibition values (GI50) of Stachys byzantina extracts in murine melanoma cell lines, prostate adenocarcinoma, human umbilical cord cells and normal murine fibroblast cells.
The hexane extract (HE), corresponding to the crude oil of Stachys byzantina, demonstrated selective cytotoxic activity (Table 4). The most pronounced effect was observed against U-251 glioma cells, with a GI50 of 38.29 µg/mL, followed by MCF-7 breast adenocarcinoma (59.08 µg/mL) and B16-F10 murine melanoma cells (78.08 µg/mL). In contrast, the extract was inactive against 786-0 kidney carcinoma (GI50 = 278.37 µg/mL). Importantly, no cytotoxic effect was detected in NIH/3T3 normal murine fibroblasts (GI50 > 250 µg/mL), reinforcing the selectivity of the extract toward tumor cells.
The hydroethanolic (HEE) and hydroacetonic (HAE) extracts also demonstrated significant activity, but selectively against B16-F10 melanoma cells, with GI50 values of 31.61 µg/mL and 25.66 µg/mL, respectively (Table 5). Both extracts were inactive against PC-3 prostate adenocarcinoma, HUVEC umbilical cord cells, and NIH/3T3 fibroblasts (GI50 > 250 µg/mL), confirming their tumor-specific activity. Notably, the HAE displayed a selectivity index approximately 10-fold higher for B16-F10 melanoma cells compared to normal fibroblasts, highlighting its potential as a candidate for further pharmacological studies.
As expected, doxorubicin, used as a positive control, showed potent cytotoxicity across all tumor lines, with GI50 values ranging from 0.02 to 0.32 µg/mL, but also exhibited significant toxicity toward normal cells. Compared with this reference drug, the S. byzantina extracts exhibited lower potency but greater selectivity, an important criterion for the identification of safer natural antineoplastic agents.
Comparative analysis with previous studies supports the activity observed. Khanavi et al. (2012) reported GI50 values above 308.7 µg/mL for methanolic extracts of several Stachys species, indicating weak activity and corroborating the higher cytotoxicity of the extracts tested in this study. Similarly, Háznagy-Radnai et al. (2008) evaluated hydromethanolic extracts of S. byzantina leaves in MCF-7 breast adenocarcinoma cells, obtaining 39.85% inhibition at 10 µg/mL. In comparison, the hexane extract in the present study achieved 50% inhibition at 59.08 µg/mL, demonstrating superior potency. Additionally, Jassbi et al. (2014) reported that the dichloromethane extract of S. byzantina exhibited an IC50 of 131.0 µg/mL against MCF-7, a value approximately twice as high as that of the hexane extract tested here, suggesting greater efficiency of the latter.
In agreement, Erdogan et al. (2013) investigated the cytotoxicity of the essential oil of Stachys rupestris against PC-3 (prostate) and MCF-7 (breast adenocarcinoma) cell lines, with IC50 values of 57.9 µg/mL and 41.3 µg/mL, respectively. The activity profile observed in the present work for the hexane extract of S. byzantina in MCF-7 cells (GI50 = 59.08 µg/mL) is comparable to these results, strengthening the evidence of the antitumor potential of Stachys species.
In sum, these results demonstrate that the oil and polar extracts of S. byzantina possess selective cytotoxic activity, particularly against glioma, breast adenocarcinoma, and melanoma cell lines, while showing minimal or no effects on normal fibroblasts and umbilical cord cells. This profile supports the hypothesis that S. byzantina is a promising source of bioactive metabolites with potential applications in anticancer drug development.
4. Conclusion
The present study provides the first integrated characterization of the nutritional composition, lipid profile, physicochemical quality, antioxidant compounds, and anticancer activity of Stachys byzantina leaves. Proximate analysis revealed high moisture content and expressive levels of carbohydrates, proteins, and lipids, confirming their nutritional value. The fatty acid profile indicated a predominance of polyunsaturated fatty acids, particularly α-linolenic and linoleic acids, associated with favorable lipid quality indices. However, physicochemical parameters, including elevated acidity and peroxide values, demonstrated poor oxidative stability of the crude oil, underscoring the need for improved extraction and storage strategies. UV–Vis analysis confirmed the presence of tocopherols, carotenoids, and chlorophylls, reinforcing the antioxidant potential of the oil. Biological assays demonstrated selective cytotoxicity of the hexane, hydroethanolic, and hydroacetonic extracts, particularly against glioma, breast adenocarcinoma, and melanoma cells, without affecting normal fibroblasts or umbilical cord cells. Collectively, these findings highlight S. byzantina as a promising source of bioactive compounds with nutritional and therapeutic relevance.
Acknowledgements
The authors thank for the support of the Graduate Program in Health and Development in the Central-West Region of Brazil, Federal University of Mato Grosso do Sul (UFMS), the Laboratory of Physical Chemistry of Foods (UFMS) and the Laboratory PRONABio (UFMS). The authors also thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brazil (CAPES) and the Brazilian Research Council (CNPq) for research grants. Funding: This research was funded by the UFMS and the CAPES — Ordinance 2016/2018.
Data Availability Statement
Todo o conjunto de dados que dá suporte aos resultados deste estudo foi incluído no artigo.
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