The plant kingdom is a cornucopia of biologically active metabolites which in different forms have been applied in medicine since ancient times. Mounting evidence suggests that a high-polyphenol diet is associated with a decreased risk of a variety of disorders including obesity (Guo et al., 2017) (which leads to metabolic syndrome and atherosclerosis), diabetes (Palma-Duran et al., 2017; Pham et al., 2019), cardiovascular diseases (Yamagata, 2019), and cancers (Miyata et al., 2019; Gu et al., 2020). Therefore, especially plant polyphenols have raised attention in the scientific field engaged in the search for antioxidative and anti-inflammatory compounds which could be applied as a supplementary treatment in multiple diseases. Also, the biotechnological strategies (mainly plant cell cultures) aimed at the production of polyphenols with required features (like anti-cancer properties) are of great importance, as recently discussed by Bulgakov et al. (2018). (Poly)phenolic compounds are structurally characterized by benzene (or another aromatic ring) with one or more attached hydroxyl/phenol groups. This diverse class comprises, among others, phenolic acids (e.g., caffeic, rosmarinic, salvianolic, and lithospermic acids, also known as caffetannins), flavonoids (e.g., eriocitrin, diosmin, or hesperidin), anthocyanins, lignans, stilbenes, tannins, coumarins, and hydroquinone derivatives (e.g., arbutin). Anti-cancer functions of these plant-derived compounds have been recently addressed in some review studies (Sharma et al., 2018; Swamy et al., 2018; Hazafa et al., 2020; Lachance et al., 2020; Saeedi et al., 2021) with reference to their modulatory effects on hallmarks of cancer (the major characteristics enabling neoplastic cells to evade the host organism defense systems) (Lachance et al., 2020). A rich source of polyphenolic molecules are plants accounted to Lamiaceae (or mint family; the biggest family in the order Lamiales) encompassing over 230 genera with above 7,000 species (Zhao et al., 2021). Health-beneficial properties of Lamiaceae metabolites have been recently reviewed in some publications (Michel et al., 2020; Patrignani et al., 2021). The first team has addressed the latest data on the protective actions of essential oils and phenolic extracts against oxidative, inflammatory, neoplastic, microbial, and neurodegenerative processes and the latter has discussed the cardiovascular protective role of Lamiaceae plants extracts and biologically-active compounds. Additionally, the antifungal properties of Lamiaceae-derived essential oils have been reviewed by Karpiński et al. (2020). Generally, in comparison with essential oils, less scientific information is available on the aqueous extracts and/or isolated polyphenols originating from Lamiaceae plants.

In the present work, the pro-apoptotic potential of polyphenolic metabolites derived from four Lamiaceae species; common thyme (Thymus vulgaris L.; Tv), wild thyme (Thymus serpyllum L.; Ts), sweet marjoram (Origanum majorana L.; Om), and peppermint (Mentha × piperita L.; Mp), against human leukemia cells was estimated. These plants are broadly applied in many national cuisines (including Polish gastronomy), being popular spices and herbal teas, as well as are accounted as medicinal herbs (enlisted in the Community herbal monographs of the Committee on Herbal Medicinal Products in the European Medicines Agency). They have been applied in the treatment of a variety of ailments, focusing on gastrointestinal and respiratory tract disorders in European countries including Poland (European Medicines Agency 2013; European Medicines Agency 2016; European Medicines Agency 2020; Bruneton et al., 1999; McKay et al., 2006; Mahendran et al., 2020; Jarić et al., 2015. Apart from the abundance of essential oils being responsible for multiple beneficial activities of these herbs, they contain polyphenolic compounds including caffeic acid (CA), rosmarinic (RA), luteolin-7-O-glucuronide (Lgr), luteolin-7-O-rutinoside (Lr), and eriodictyol-7-O-rutinoside (Er) (present in all four investigated species), and arbutin (Ab) (detected in sweet marjoram and common thyme), and lithospermic acid (LA) (in wild thyme and peppermint) (Fecka and Turek 2007 and Fecka and Turek (2008). The exact chemical composition in terms of common thyme and peppermint polyphenols identified using LC-MS has been described in recent publications by Bodalska et al. (2020) and Bodalska et al. (2021). An extensive discussion on the activity and composition of sweet marjoram is presented in Bouyahya et al. (2021) and Taamalli et al. (2015).

The aim of this study was to evaluate the impact of dried aqueous extracts from common thyme (Thymus vulgaris L.) (ExTv), wild thyme (Thymus serpyllum L.) (ExTs), sweet marjoram (Origanum majorana L.) (ExOm), and peppermint (Mentha × piperita L.) (ExMp), and polyphenolic compounds: CA, RA, LA, Lgr, Lr, Er, and Ab on unstimulated Jurkat cells, in comparison with their effect on staurosporine stimulated cells, with respect to their influence on cell death via apoptosis/necrosis. Moreover, the observed effects were estimated in accordance with the percentage composition of (poly)phenols, and in comparison with isolated compounds.

From the observed here effects on Jurkat cells, the most pronounced was pro-apoptotic action of polyphenolic mixtures extracted from Thymus serpyllum L. and Origanum majorana L., as well as enhancement of staurosporine-induced apoptosis observed for caffeic acid and to the lesser extent for rosmarinic acid. In our previous work, the same extracts and polyphenols had been tested on human breast cancer cell lines resistant and non-resistant to Adriamycin (MCF-7/wt and MCF-7/Adr, respectively) with an MTT assay (Berdowska et al., 2013). Agreeably, the highest cytotoxicity against MCF-7/Adr had been observed in the case of ExOm and ExTs. However, in that previous study, ExTv had demonstrated comparable cytotoxicity in comparison with ExTs, whereas in the present experiment ExTv, in contrast to ExTs, did not show the proapoptotic effect on the Jurkat cell line, neither modulatory impact on staurosporine treated cells. The lack of effect on Jurkat cells treated or non-treated with staurosporine was also observed in the case of ExMp. This finding is closer to our previous observation when ExMp had exerted an only mild cytotoxic impact on MCF-7/Adr cells, in comparison with much higher ExOm toxicity. However, all four extracts in the previous study (at concentrations of up to 250 μg/ml) had increased the viability of MCF-7/wt cells (the regular breast cancer cell line, non-resistant to antibiotics). Therefore, no definite conclusions can be drawn on the influence of the tested extracts on these two cancer types. This is due to the fact that different cell lines and experimental procedures have been applied in this and the former studies. As the analysis of (poly)phenolic metabolites of the studied extracts demonstrated, illustrated here in Figure 1; Table 2, ExMp is the most abundant in (poly)phenolic compounds (PPs reaching 47%), whereas ExOm contains only around 18% PPs. Additionally, ExMp is the richest in flavonoid glycosides (Er, Lr, and Lgr) when compared with the remaining three extracts, and shows an especially high concentration of Er reaching 27%, whereas in the other extracts it does not exceed 1%. Reversely, ExMp contains the least caffeic acid and its esters (CA + RA + LA), the subfraction most abundant in ExTs containing the greatest amounts of RA and LA. Since both CA and RA demonstrated the strongest pro-apoptotic activity in the present study, it might be hypothesized that this is CA-RA (∼ 20%) subfraction that mostly contributes to the ExTs pro-apoptotic effect. However, if this was the case, we would also have expected a pro-apoptotic action exerted by ExTv, whereas comparable to ExTs pattern was actually observed for ExOm containing lower phenolic acids percentage (like in ExMp PAs subfraction). The lack of ExMp effect on Jurkat cells apoptosis may be associated with the somewhat opposite actions of Er and Lr; whereas pure Er demonstrated anti-apoptotic action, pure Lr tended to stimulate apoptosis of Jurkat cells. Therefore, when present in the ExMp mixture they might have mutually abolished their actions giving a resultant in the shape of no effect at all. On the other hand, when Lr and Er had been tested on breast cancer cell lines, Er demonstrated a greater level of toxicity against both MCF-7/wt and MCF-7/Adr than Lr. The most toxic against both MCF-7 cell lines had been RA and LA, whereas in the present study the strongest pro-apoptotic impact was observed in the case of CA, followed by RA, whereas LA did not affect Jurkat cells apoptosis. Ab being the least cytotoxic against breast cancer cells, exhibited no impact on Jurkat cells, similarly to LA.

The comparison of biological activities exerted by plant extracts (containing mixtures of compounds) with actions of isolated substances is a problematic issue. On one hand, there is a strong tendency aimed at optimization of techniques to isolate/produce the desired compound of the highest possible degree of purity; precisely described structurally and functionally, which after further testing gives rise to the generation of a medicinal drug. On the other hand, it seems that in many cases the purification process leads to the weakening of desired biological effects. Therefore, the strategy associated with the assessment of metabolites properties in mixtures should be regarded as an alternative approach in the search for the natural substances which can be applied in many areas including adjuvant therapies. Such an approach has been lately presented in the experimental work of Ouedrhiri et al. (2021), who aimed at the optimization of the mixture design technology to achieve the best antioxidative effect of essential oil fractions from chosen Lamiaceae herbs. In both studies on human breast cancer and human leukemia cells, the most beneficial effects have been observed for polyphenolic mixtures; the greater cytotoxic impact of the studied extracts on the cells with developed multidrug resistance in the first case (Berdowska et al., 2013), and the most prominent anti-apoptotic impact of ExTs and ExOm, when compared with the effect of pure polyphenols. Nevertheless, as discussed above, likewise in the previous study (Berdowska et al., 2013), the impact of extracts did not directly reflect their (poly)phenolic composition, which might be explained by different types of interactions among the constituents in the mixtures (additive, synergistic, contradictory, enforcing, or attenuating one another activities, etc.) but also by the interference of unidentified earlier compounds. For example, the pro-apoptotic effect of ExOm may be partially ascribed (except for RA-CA subfraction), to vicenin-2 (not detected in the remaining extracts) whose anti-cancer properties have been demonstrated in some studies (Nagaprashantha et al., 2011; Li et al., 2021).

Stronger biological effects of mixtures of compounds have been also observed in some other studies (Morré et al., 2003; Seeram et al., 2005; Adams et al., 2006). Reversely, since some of the tested here compounds such as flavonoid glycosides (Er, Lr, Lgr) belong to complex molecules, they may undergo hydrolysis in the experimental conditions of cell cultures. Therefore, the observed on the cell lines effects might be associated partially with their released products. Such a phenomenon has been recognized in the experiments on ellagitannin (punicalagin) by Larrosa et al. (2006).

Among the studied phenolic compounds, the most thoroughly investigated, in regard to their health-beneficial properties, are probably caffeic and rosmarinic acids, recently addressed in some review studies. (Habtemariam et al., 2017; Colica et al., 2018; Ngo et al., 2018; Swamy et al., 2018; Elufioye and Habtemariam, 2019; Espíndola et al., 2019; Ghasemzadeh Rahbardar et al., 2020; Luo et al., 2020; Silva et al., 2020; de Araújo et al., 2021; Ekeuku et al., 2021; Hitl et al., 2021; Khan et al., 2021; Muhammad Abdul Kadar et al., 2021). For example, CA and its more studied derivative caffeic acid phenethyl ester demonstrate anti-atherosclerotic as well as blood pressure decreasing properties, thus showing potential for cardiovascular disease treatment (Silva and Lopes, 2020). CA putative protection against metabolic syndrome and associated disorders such as insulin resistance and diabetes has been discussed by Muhammad Abdul Kadar (2021). Additionally, CA due to its bone remodeling activities is a promising agent in bone diseases therapies (Ekeuku et al., 2021). An application of CA and its derivatives in infectious disorders has been discussed by Khan et al. (2021), and CA anti-cancer activities (with respect to hepatocarcinoma) have been considered by Espíndola et al. (2019). Anti-inflammatory properties of RA have been addressed by Luo et al. (2020), where its capacity to attenuate arthritis, colitis, dermatitis and similar conditions has been discussed. The hepatoprotective properties of RA have been addressed by Elufioye and Habtemariam (2019). Moreover, like in the case of CA, also RA is a promising compound in protection against diabetes (Ngo et al., 2018), as well as neoplasia developments (Swamy et al., 2018). Among the mentioned above CA and RA health-beneficial properties, their well-recognized functions are anti-cancer activities. They have been demonstrated to inhibit neoplastic cell development via different mechanisms, including the stimulation of cell death through pro-apoptotic actions. Pro-apoptotic effects of CA and RA on Jurkat cells were also observed in the present study, where both compounds enhanced staurosporine-induced apoptosis.

The limitation of this study is the narrow concentration range of the evaluated extracts/compounds which was conditioned by technical constraints of the experimental protocol. A sufficient number of Jurkat cells in each sample had to be achieved, as well as repetitions of experiments to reach statistical significance were necessary. Two experimental patterns conducted in parallel (with and without staurosporine preincubation) doubled the number of occupied wells in each 24-well plate. All of these characteristics technically restricted the number of the available wells; therefore to perform each experiment in triplicate, only three different concentration levels of the studied substances were tested

The strength of this study is the novelty in the evaluation of the impact of specifically characterized dried aqueous extracts from Mp, Ts, Tv, and Om on Jurkat cells with respect to their apoptosis modulatory properties, as well as the comparison of their effects with their (poly)phenolic components. The presented here experimental model is unique with respect to the comparison of the actions of single molecules with their properties in mixtures, as well as the applied cell line. Concluding from the literature search, there have been only a few experiments investigating the effects studied in this work substances on Jurkat cells apoptosis. On the other hand, a variety of experimental designs makes it difficult to compare the obtained here results with the data from other teams. For example, when it comes to Mentha genus species, their anti-cancer properties have been demonstrated both in in vitro studies and clinical trials (Anwar et al., 2019). However, various mint species (Mentha piperita, Mentha spicata, Mentha arvensis, Mentha longifolia), different cell lines (Hela—human malignant cervix carcinoma, Hep2—human laryngeal carcinoma, Vero—green African monkey kidney), disparate formulations in extract preparations (e.g., alcoholic vs. aqueous), and different tests and protocols applied for the estimation of cytotoxic/antiproliferative potential, causes drawing unequivocal conclusions really problematic. Whereas in the present study, no apoptosis-modulatory impact was observed when ExMp was tested on Jurkat cells, in the study by Jain et al. (2011), peppermint extracts demonstrated proapoptotic activity in the same cell line and with the application of the same test. Nevertheless, much stronger effects were obtained by the authors when chloroform or ethyl acetate was used for extract preparations, in comparison with water extract, but what is more, the authors evaluated extracts within different concentration ranges. In the present study 0.01–0.1 mg/ml concentrations were tested, whereas in Jain’s experiments the concentration range was much higher (1–100 mg/ml). Therefore, it might be suggested that, despite the observed here lack of effect for low concentrations of peppermint aqueous extracts, if a different extraction procedure and greater concentrations had been applied, then a pro-apoptotic effect might have been observed. The common thyme extract effects on Jurkat cells have been assessed in another study (Esmaeilbeig et al., 2015), in which the authors found the cytotoxic impact of Tv on the cells, which seems to stand in opposition to the present experimental finding. However, the authors did not evaluate the level of apoptosis but the viability of the cells with the MTT assay. Additionally, they incubated the cells with Tv extract for 48 h (whereas in this study the incubation time was up to 20 h), and they prepared methanol, not aqueous Tv extract. Therefore, this putative discrepancy mostly derives from the differences in the experimental protocol. Sweet marjoram ethanol extract has been tested on Jurkat cells by Abdel-Massih et al. (2010), who observed cytotoxic and pro-apoptotic impact, similar to the present study. However, the authors applied longer incubation times (48 and 96 h), as well as greater concentrations. Nevertheless, the extracts derived from Origanum majorana L. seem to demonstrate the most promising anti-cancer effects in comparison with the remaining three Lamiaceae species, which had been also observed in our previous experiments on human breast cancer cells (Berdowska et al., 2013).

When it comes to polyphenolic compounds studied in the present experiments, rosmarinic acid has previously demonstrated pro-apoptotic activity on Jurkat cells (Hur et al., 2004; Kolettas et al., 2006). Hur et al. (2004) observed that RA (at 30 µM concentration) induced apoptosis in 37.8–53.6% of Jurkat cells after 24 h incubation, and in 98.3% of the cells after 48 h incubation. The authors reported the mitochondrial pathway involvement in the mechanism of apoptosis induction because it was associated with mitochondrial membrane depolarization and the release of cytochrome C, and the participation of caspases 3 and 8. Accordingly, Kolettas et al. (2006) demonstrated both dose-dependent inhibition of Jurkat cells proliferation and induction of apoptosis by RA. The authors showed RA-stimulated downregulation of cyclin D3 and p21Cip1/Waf1 and upregulation of p27Kip1, which suggested the cell growth arrest at the G1/S phase. Additionally, they observed that the induction of apoptosis by RA correlated with the suppression of Bcl-2. Therefore, RA seems to be able to stimulate apoptosis of Jurkat cells via the reduction of anti-apoptotic Bcl-2 proteins, thus activating the mitochondrial pathway. In the present study, RA demonstrated a tendency to stimulate apoptosis in Jurkat cells, but this effect reached statistical significance only at 90 micromolar concentration in the cells treated with staurosporine, hence reinforcing the pro-apoptotic impact of staurosporine by increasing the number of apoptotic cells by 7%. The weaker pro-apoptotic effect of RA observed here when compared to Hur et al. (2004) may be ascribed to the shorter incubation time; in this study, it was 20 h, whereas in the Hur et al. (2004) experiments the incubation time was longer reaching 24 and 48 h. Since the authors observed a much greater effect after 48 h, it might be suggested that RA requires more than 20 h to exert its pro-apoptotic effect.

In conclusion, it might be hypothesized that these are rather (poly)phenolic extracts that demonstrate required properties against pathological cells than purified compounds. In the present experiment the most beneficial pro-apoptotic actions against human leukemia Jurkat cells have been observed in the case of dried aqueous extracts from Thymus serpyllum L. and Origanum majorana L. Less evident pro-apoptotic effects were demonstrated for purified (poly)phenolic compounds, namely caffeic and rosmarinic acids, which enhanced especially staurosporine-induced apoptosis. Nevertheless, due to the abovementioned limitation of the present study, if longer incubation time and greater concentrations of the tested compounds had been applied, stronger effects might have been observed. Therefore, the present experiments may be considered rather as a pilot study to encourage their continuation with the aim to gain more definite conclusions.