Salvia officinalis L. extract and its new food antioxidant formulations induce apoptosis through mitochondrial/caspase pathway in leukemia L1210 cells

Salvia officinalis, L. (Lamiaceae) is one of the most widespread herbal species used in the area of human health and in the food-processing industry. Salvia and its extracts are known to be a rich source of antioxidants. As shown previously, the crude ethanolic extract of salvia (SE) exerts lower anti-oxidative properties in lard compared to the new salvia food formulations No. 1 (SF1; 32% of SE + 68% of the emulsifier Dimodan S-T) and No. 2 (SF2; 32% of SE + 68% of the emulsifier Topcithin 50). The aim of the present study was to investigate and compare the effects of the SE and its food formulations SF1 and SF2 on the toxicity and/or proliferation of L1210 leukemia cells. We found that SE and both SF1 and SF2 demonstrated different concentration- and time-dependent cytotoxic/antiproliferative cellular effects already within the first 24 h of the treatment. However, SE was nearly 10 times more effective than the new salvia food formulations SF1 and SF2. We investigated partially also the molecular mechanisms lying behind the action of SE, SF1 and SF2 induced apoptosis in our cell model. We found an apparent involvement of the mitochondrial/caspase-dependent pathway in the described processes. Nevertheless, further investigation is needed before salvia extract and its new antioxidant formulations can be included among the potential food antioxidants with protective properties against cancer.

it has been used in the treatment of various disorders, such as tuberculosis, psoriasis, and seborrhoeic eczemas.
Some members of this genus have even an economic importance because of their use as flavoring agents in perfumery and cosmetics. In food and food-processing industry, a major use is in the form of aqueous infusions as sage tea, which is sold legally either as food or medicine (Walch et al., 2011).
Antioxidant properties of compounds isolated from S. officinalis were described elsewhere (Cuvelier et al.,

Introduction
Salvia L., one of the largest genera of the family Lamiaceae (formerly Labiatae), is represented by over 1000 species, organized in five subgenera (Sclarea, Audibertia, Jungia, Leonia, and Salvia) as herbaceous, suffruticose, or shrubby perennial plants. The genus Salvia is one of the herbal plant genuses that has been widely used in traditional medicine all around the world (Tayarani-Najarana et al., 2013) due to its diverse biological activities, including antibacterial, spasmolytic, hemostatic, cytotoxic, and anticancer actions and as well as many others. Since ancient times contains fats and oils rich in polyunsaturated fatty acids that easily undergo undesirable oxidative reactions during food processing (heating) or storage (salt addition, freezing). To prevent such quality loss or to retard the effects of oxidation (Angelo et al., 1996), synthetic phenolic antioxidants (butylhydroxytoluene -BHT, butylhydroxyanisole -BHA, tert-butylhydroquinone -TBHQ) are commonly used, yet their safety has been questioned (Namiki, 1990; Barlow et al., 1990). Recent research focuses therefore also on polyphenolic antioxidants existing in some spices and herbs like salvia, but also rosemary or thyme (Shahidi & Wanasundara, 1992;Sekretar et al., 2003Sekretar et al., , 2004Sekretar et al., , 2006Mariutti et al., 2011;Wu et al., 2012). The increasing interest in replacing synthetic antioxidants by natural compounds (Willcox et al., 2004) of comparable efficacy would be beneficial not only for sustaining the food quality but it might also positively influence the treatment of various human pathologies. Imbalance between reactive oxygen species (ROS) formation and their elimination is involved in conditions like arthritis, cancer, diabetes, cardiovascular diseases, inflammations, neurological disorders, etc. (Halliwell, 1996).
Previously we found that the crude ethanolic extract of salvia (SE) exerted antioxidant activity comparable to BHT and its food antioxidant formulations SF1, SF2 containing a mixture of the emulsifier Dimodan S-T or lecithin Topcithin 50 even surpassed the significant antioxidant properties of SE and stabilized fats against oxidation (Sekretar et al., 2003(Sekretar et al., , 2006. In consistence with all the above mentioned facts, we used model murine leukemia L1210 cells to monitor the cytotoxic/antiproliferative activity and apoptosis induction by Salvia officinalis L. extract (SE) and its potent food antioxidant formulations SF1 and SF2.

Preparation of salvia extract and food formulations
Salvia officinalis from Fytopharma company (Malacky, Slovakia) was used for the extract preparation. The starting material (dry salvia leaves, 20 g) was over 4 h successively extracted in 500 ml Soxhlet extractor with 95% ethanol. Using a rotary vacuum evaporator, 3.4 g (17% yield) of yellowish powder was obtained and directly used for cytotoxic evaluation as 100% salvia extract (SE) or for the preparation of food formulation SF1 a SF2 (Sekretár et al., 2003(Sekretár et al., , 2004(Sekretár et al., , 2006. Formulation SF1 was prepared by mixing 32% of crude extract (SE) and 68% of the emulsifier Dimodan S-T. Formulation SF2 was prepared by mixing 32% of crude extract (SE) and 68% of the emulsifier Topcithin 50. Crude Salvia officinalis L. extract (SE), food antioxidant formulations SF1 and SF2 were dissolved in 100% DMSO. Final concentration of DMSO never exceeded 0.1% (v/v) in either control or treated cells.

Cell culture
Murine L1210 leukemia cells (obtained from ATCC, Rockville, MD, USA) were grown in RPMI medium supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a 5% (v/v) CO 2 incubator. All experiments were performed in Petri dishes (Ø 60 mm). The cells were plated at the density of 8 × 10 4 cells/ml, cell viability was determined by 0.4% (w/v) Trypan blue staining.

Growth inhibition assay
An inoculum, 8 × 10 4 cells/ml in the exponential phase of growth, was used. SE, SF1, SF2 were applied in the final concentration range of 0.1-500 μg/ml. The number of cells was determined by direct counting 24, 48 and 72 h after exposure. The cytotoxic effect of the crude extract and food formulations was evaluated as reported previously (Letasiova et al., 2006).

Caspase 3 activity assay
Cell lysates were prepared and caspase 3 activity was measured according to the manufacturer`s protocol (CaspACE™ Assay System Colorimetric, Promega Corporation). Briefly, 28 μg of total protein was added to the reaction mixtures containing colorimetric substrate peptides specific for caspase 3 (Ac-DEVD-pNA) at 37 °C for at least 2 h and data were collected every 2 h until the signal got stabilized. Absorbance at 405 nm was determined using microplate reader (Humareader, Wiesbaden, SRN) (Jantova et al., 2008). Protein concentration was determined by Lowry's method (Lowry et al., 1951).

Assessment of mitochondrial membrane potential
The changes in mitochondrial membrane potential were determined using DiOC6. Cells were treated with 50 (SE) and 500 (SF1, SF2) μg/ml for 30 min to detect the changes of mitochondrial membrane potential. The cells were harvested, washed and resuspended in PBS and 4 μl of DiOC6 (40 μM) was added. The cells were incubated at 37 °C for 30 min and analyzed by fluorescence microscopy (Repicky et al., 2009).

Statistical analysis
Data are presented as means of at least three independent experiments ± SD. Student´s test was used to determine statistically significant differences between the respective mean values and significant changes are highlighted (n.s. not significant; p<0.05).

Results
We evaluated the growth inhibition of L1210 cells treated with SE, SF1 and SF2 in concentrations of 0.1-500 μg/ml for 24-72 h ( Figure 1). As follows from the growth curves of L1210 cells ( Figure 1A,B,C), the highest used concentrations (50 and 75 μg/ml for SE, 300 and 500 μg/ml for   Table 1 shows the values of L1210 growth inhibitory concentrations IC 50 and IC 100 of SE, SF1 and SF2 for 24, 48 and 72 h. The values increased with the time of treatment of L1210 cells. The effectivity was highest by using the crude extract (SE), followed by the food antioxidant formulations SF2 and SF1.
As mentioned above, we observed that the highest concentrations used, i.e. 50 and 75 μg/ml for SE and 300 and 500 μg/ml for SF1 and SF2, respectively, caused massive cytotoxicity already within the first 24 h of treatment. Therefore, we evaluated the L1210 cell proliferation within a shorter treatment period with cells counted every 4 h within the final 12 h treatment ( Figure 1D). SE, SF1 and SF2 demonstrated distinct time-dependent cytotoxic/ antiproliferative effects. Four hours of cell treatment with the crude extract SE and the antioxidant formulation SF2 was sufficient to induce a cytostatic effect only in comparison to the effect of the antioxidant formulation SF1, which within the same treatment time caused already degradation of the treated cell. Finally, prolonged time of cell incubation with all salvia preparations tested caused further cell count decreases below the inoculum cell level, Concentrations displayed in μg/ml were calculated from the toxicity curves. reaching almost complete cell clearance following 12 h of treatment.
To understand the mechanism of cell proliferation inhibition by SE, SF1 and SF2, we next studied their ability to modulate the cell cycle and induction of apoptotic cell death in L1210 cells.
To study the impact on the cell cycle profile, we monitored the effect of 18.75, 25.0 and 50.0 μg/ml of SE; 150, 200 and 300 μg/ml of SF1 and 150, 300 and 500 μg/ml of SF2 in L1210 cells for 24 h (Figure 2). On the one hand, flow cytometry revealed that SE and its formulations did not have any significant effect on the cell cycle profile of L1210 cells. On the other hand, a significant sub-G 0 fraction was detected in L1210 cells treated for 24 h with concentrations of 50 (SE), 300 (SF1) and 500 (SF2) μg/ml.
Cells treated with concentrations of 50 (SE) and 500 (SF1 and SF2) μg/ml for 4, 8, 12, 24, 48 and 72 h were subjected to agarose gel electrophoresis to assess apoptotic DNA fragmentation. As indicated in Figure  3A, clear apoptotic DNA fragmentation was observed in L1210 cells treated for 24 h with 25 and 50 μg/ml of SE, 300 and 500 μg/ml of SF1 and 150-500 μg/ml of SF2. DNA fragmentation was also found in cells treated for 48 and 78 h with concentrations of 50 μg/ml of SE, 300 and 500 μg/ml of SF1 and 300 and 500 μg/ml of SF2 (data not shown). Furthermore, as indicated in Figure 3B, apoptotic DNA fragmentation was observed in L1210 cells treated with the highest concentrations tested (50 μg/ml of SE, 500 μg/ml of SF1 and SF2) already after 4, 8 as well as 12 h of treatment.
The evidence based on the observation of the apoptotic DNA fragmentation induction in L1210 cells treated with SE, SF1 and SF2 led us to the examine the activities of caspase 8 and 9 as well as caspase 3 (Figure 4). Since activation of caspases 8 and 9 precede activation of executive caspase 3, we measured first their activity. One hour of treatment of the L1210 cells with 50 μg/ml SE and 500 μg/ml SF1 and SF2, was sufficient to induce significant activation of caspase 9 ( Figure 4B). Caspase 9 activities upon treatment reached almost 60% activity of caspase 9 induced by 180 μg/ml of cisplatine, used as positive control. Under identical conditions, the activity of caspase 8 was measured and the obtained values were undistinguishable from that measured in control ( Figure 4A). Measurement of caspase 3 revealed that 1-h treatment of L1210 cells with 50 μg/ml SE and 500 μg/ml  SF1 was sufficient to substantially induce its activity, which was comparable to that of positive control. The activity of caspase 3 in L1210 cells treated with 500 μg/ml SF2 was only slightly increased compared to solvent treated cells (negative control, NC). Mitochondrial membrane potential decay is one of the accompanying processes of the intrinsic apoptotic pathway activation. To evaluate whether it changed upon treatment of L1210 cells with 50 μg/ml SE and 500 μg/ml SF1 and SF2, we monitored it by using fluorescence sensitive probe DiOC6. As shown in Figure 5, 30-min treatment with SE as well as with SF1 and SF2 caused considerable decrease of the mitochondrial membrane potential in comparison to the solvent treated control (NC).

Discussion
Salvia is a rich source of phytochemicals including flavonoids, sesquiterpenoides, diterpenoides, sesterterpenes and triterpenes. Several active components such as rosmarinic acid, royleanone, horminone, and acetyl horminone have been isolated from the roots of S. officinalis. These compounds, extracts and essential oils resulted in human colon, breast, leukemia and lung tumor reduction in vitro and reduction in oncogene transformed cells (Liu et al., 2000;Shoemaker et al., 2005;Rozalski et al., 2006;Fiore et al., 2006;Loizzo et al., 2007;Li et al., 2008;Tayarani-Najarana et al., 2013). Some authors also demonstrated protective properties of the genus Salvia. Gali-Muhtasib & Affara (2000) reported that the oil extract of the sage plant showed potent suppressive activities against tumor promotion in mouse skin and thus could be an effective chemopreventive agent against skin cancer. Vujosevic and Blagojevic (2004) reported antimutagenic properties of terpenoid fractions of Salvia officinalis in the mammalian system in vivo. Li et al. (2008) found that tanshinone IIA -an antioxidant constituent of Salvia mitiorrhiza, could serve as a protective agent in cancer prevention treatment. Celik and Isik (2008) determined the chemopreventive role of Salvia officinalis infusion on antioxidative defense systems in rats.
Natural antioxidants may act not only as direct ROS scavengers but they might also modulate enzymes involved in ROS elimination, drug metabolism or repair responses as well as working as signaling molecules in important cascades for cell survival (Lima et al., 2005(Lima et al., , 2007a. On balance, identification and study of novel substances from natural sources possessing antioxidant activities are important strategies to improve human health condition and life quality. With the aim to obtain new natural antioxidants to enhance the stability of fats and oils with anticancer properties, we prepared a crude ethanolic extract from dry leaves of Salvia officinalis (SE) and food antioxidant formulations SF1 and SF2 containing 32% of SE with respective addition of emulsifiers Dimodan S-T and Topcithin 50 (Sekretar et al., 2003(Sekretar et al., , 2004(Sekretar et al., and 2006. Previously we found that the antioxidant activity of our salvia extract was comparable to BHT, a synthetic phenolic antioxidant commonly used in fats and oils to retard oxidation. The crude extract contains dyes or heavy metals, which accelerate oxidation and so the extract is not directly suitable as food antioxidant. We concluded that safe food formulations prepared in this manner manifested significant antioxidant properties and stabilized fats against their oxidation (Sekretar et al., 2003). Krajcova et al. (2008) tested the effects of this extract and its formulations for their activity against common microbial contaminants of food and cosmetics products (Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes). The authors confirmed the hypothesis that the substances acted in food not only as antioxidants but also as antimicrobial agents.
In the present study, we evaluated the cytotoxic/ antiproliferative activity of salvia crude extract (SE) and its food antioxidant formulations SF1 and SF2 on murine leukemia L1210 cells. As follows from the growth curves of the L1210 cells (Figure 1), salvia extract and its formulations caused different cytotoxic effects depending on the concentration, time of exposure and substance composition. The highest used concentrations of the crude extract (SE) and its food formulations (SF1 and SF2) induced an acute cytotoxic effect manifested by immediate cell degeneration and lysis. This cytolytic effect was increased with prolonged time of treatment and it was proportional to the concentration used and the time of exposure.
Cytotoxic activity of Salvia officinalis extract on leukemia cells was reported by Shahneh et al. (2013 a,b). The authors found that methanolic extract caused dose dependent inhibition of cell proliferation of human leukemic monocyte cell line U937 and human acute myelocytic leukemia cell line KG-1A with IC 50 values 205.11 μg/ml (for U937 cells) and 179.00 μg/ml(for KG-1A NC SE SF1 SF2 Figure 5. In situ fl uorescent microscopy of the mitochondrial membrane potential changes induced in L1210 cells upon treatment with the sage extract (SE) and formulations SF1, SF2 in concentration 50 and 500 μg/ml, respectively. Application of 0.1% DMSO served as negative control, NC. Scale bar is 50μm. The addition of emulsifiers to the crude extract to create salvia food formulations, decreases the cytotoxicity compared to crude salvia extract (Figure 1). It is surprising, since Sekretar et al. (2003Sekretar et al. ( , 2006 found that these food formulations (SF1 and SF2) exhibited greater antioxidant potency compared to crude salvia extract (SE). This lower cytotoxicity might however be due to decreased salvia content in food formulations compared to 100% salvia extract.
The cell cycle analysis by flow cytometry (Figure 2) showed that neither SE nor its food antioxidant formulations SF1 a SF2 changed significantly the cell cycle profile of treated L1210 cells. What we observed was that the highest used concentration (50 μg/ml for SE, 500 μg/ml for SF1 and SF2) caused accumulation of cells in the sub G 1 population, which indicates induction of apoptotic cell death. This assumption was confirmed by the detection of intranucleosomal DNA fragmentation (Figure 3). The DNA fragmentation was detectable already after 4 h of treatment with all substances used in their highest concentrations tested ( Figure 3B).
The ability of salvia extracts or salvia components to induce apoptosis of different cell lines was reported by many authors (Li et al., 2000;Liu et al., 2000;Oh et al., 2002;Park et al., 2007). Apoptotic cell death of leukemia cells induced by the methanolic extract of Salvia officinalis L. in the concentration range 100-300 μg/ml was demonstrated by Shahneh et al. (2013 a,b). The authors reported that the extract evoked dose-dependent induction of apoptosis of human leukemia cells U937 and KG-1A after 24 h of treatment. In comparison to our results, we conclude that lower concentrations of SE and SF1 and SF2 as well as shorter time of exposure were needed to induce apoptosis in murine leukemia L1210 cells.
To reveal the molecular mechanism involved in SE, SF1 and SF2-induced apoptosis in L1210 cells, we studied their effects on caspase 9, 8 and 3 and mitochondrial membrane potential. As shown in Figure 4, cells treated with SE, SF1, SF2 have activated caspases 9 and subsequently caspase 3, while the mitochondrial membrane potential is decreased ( Figure 5). Caspase 8 activation was not observed, which may indicate that all substances tested induced apoptosis only by induction of the intrinsic mitochondrial pathway.
In summary, on the basis of the obtained results we can conclude that SE, SF1 and SF2 demonstrated different concentration-and time-dependent cytotoxic/ antiproliferative effects on murine leukemia L1210 cells.
The cytotoxicity of the substances tested was already detectable within the first 24 h of treatment. Crude salvia extract (SE) as well as its food formulations SF1 and SF2 induced apoptosis of L1210 cells by activation of caspases 9 and 3, as well as by mitochondrial membrane potential decay. On balance, the results clearly show that SE, SF1 and SF2 induce apoptosis of L1210 cells through the mitochondrial/caspase-dependent pathway.
Nature is a limitless source of substances that may replace synthetic ones to serve the purpose of protection against food alterations and its nutritional and quality degradation. Salvia officinalis L. can be one of such natural sources and our ethanolic extract SE and/or the new food antioxidant formulations SF1 and SF2 can be used to increase the shelf life of food, and moreover, they may serve as potential anti-leukemia agents.