Health-Promoting Effects of Thymus Phenolic-Rich Extracts: Antioxidant, Anti-inflammatory and Antitumoral Properties

Thymus genus comprises numerous species that are particularly abundant in the West Mediterranean region. A growing body of evidence suggests that many of these species are a rich source of bioactive compounds, including phenolic compounds such as rosmarinic acid, salvianolic acids and luteolin glycosides, able to render them potential applications in a range of industrial fields. This review collects the most relevant studies focused on the antioxidant, anti-inflammatory and anti-cancer of phenolic-rich extracts from Thymus plants, highlighting correlations made by the authors with respect to the main phenolic players in such activities.


Introduction
Thymus is a Latin name of the plant genus, which is considered to derive from the Greek word thyo (perfume) by some authors, while others consider the Greek word thymos (courage, strength) [1]. Thymus genus belongs to Lamiaceae family and includes about 350 aromatic species that are distributed around the world, with particularly abundancy in the West Mediterranean region [2,3]. These species are perennial and are characterized as being herbaceous subshrubs or shrubs with 10 to 30 cm tall, containing small and simple leaves, ramified and prostrated branches and big clusters of pink, white, cream or violet flowers [2].
Thymus plants are claimed to be endowed of distinct health beneficial properties, including antimicrobial, antioxidant, anti-inflammatory, cardioprotective, neuroprotective, anticarcinogenic and hypoglycemic activities [4][5][6][7]. Although these benefits have been mostly associated with essential oils [8][9][10][11], nowadays, Thymus phenolic-rich extracts usually obtained with polar solvents, are an attractive target for the screening of bioactive compounds for possible industrial applications in distinct fields, including food, cosmetics or pharmaceutical industries [6,12]. Among them, counteraction of oxidative stress events is a central issue underlying the valorization of these plants.
Oxidative stress is generated when the balance between reactive species formation and their elimination is disrupted. Under this biological condition, there is an overproduction of reactive oxygen species (ROS) such as superoxide (O 2 -), hydroxyl radical ( • OH) and hydrogen peroxide (H 2 O 2 ), as well as of reactive nitrogen species (RNS) like nitric oxide (NO) and peroxynitrite (ONOO¯), overall leading to several mitochondrial and cellular damages. These events are closely associated to aging
Since Thymus vulgaris is a widespread species, its polar extracts have been explored for a range of biological effects, including antioxidant ability. Among the reported data for this species, water, methanolic and hydroalcoholic extracts of this plant origin were reported to have DPPH • EC 50 values in the range of 1.8 and 44.7 µg/mL [4,57,59]. Moreover, phenolic-rich extracts from T. vulgaris were shown to be good ABTS •+ (EC 50 = 0.7-13.8 µg/mL) [57,59] and • OH (EC 50 = 0.24 µg/mL) [57] scavenging agents ( Table 1).
Favorable DPPH • and reducing power results were also been observed by our group for decoctions of Thymus herba-barona, Thymus pseudolanuginosus, Thymus caespititius, T. pulegioides, Thymus fragantissimus and T. zygis [40,41], for which the EC 50 values were 1.4-2.0 higher than those of the reference commercial compounds. Among these six extracts, those rich in flavones (T. pulegioides and T. pseudolanuginosus) tended to have superior DPPH • scavenging ability and reducing Fe ability. In fact, the DPPH • scavenging ability of these extracts surpassed that recently described for decoction and infusion extracts of Thymus sipyleus Boiss. subsp. rosulans (Borbas) Jalas (EC 50 values of 43.5±1.02 and 87.38±1.73 µg/mL, corresponding to about 1.6-3.2-fold of the ascorbic acid). Interestingly, the authors disclosed a greater antioxidant ability of these polar solvent extracts in comparison to non-polar ones [56]. Different results were described for aqueous extracts of Thymus atlanticus, T. zygis and Thymus satureioides based on ABTS •+ scavenging assay, which corresponded to about 7.5-8.5 times those of the standard ascorbic acid [62].
In opposition to chemical assays, to our knowledge, only a few authors used cellular and/or in vivo models to screen the antioxidant potential of Thymus polar extracts (Table 1). Under this context, hydroethanolic extracts of T. citriodorus (50 µg/mL) were shown to reduce significantly the intracellular ROS formation in a potassium dichromate-stimulated HepG2 cells [52]. Moreover, Khouya et al. [63] reported the protection of T. satureioides against oxidative damage, by demonstrating that the addition of its crude extract or subsequent fractions (dichloromethane, ethyl acetate, methanol or aqueous) to erythrocyte suspensions resulted in an increase in haemolysis half-time (1.2 to 2.1-fold of potency compared to ascorbic acid). As regard to in vivo studies, it was only recently that Righi et al. [46] reported that the treatment of Swiss albinos mice with Thymus algeriensis hydroalcoholic extracts at 800 mg/kg bw resulted in an improvement of the antiradical ability of the plasma, as well as catalase and glutathione levels (at 400 and 800 mg/kg bw), paralleled by a decrement on the levels of malondialdehyde (200 and 400 mg/kg bw). In addition, El-Boshy et al. [58] demonstrated that supplementation of lead (Pb) intoxicated rats with ethanolic extracts from T. vulgaris could ameliorate antioxidant parameters and the overall toxic effects.
Notably, some authors evaluated the antiradical/antioxidant activity of Thymus polar extracts with regard to their phenolic content and to the presence of specific phenolic compounds [37,51], particularly with the major components (e.g., rosmarinic acid, caffeic acid, luteolin-O-glucoside, apigenin, naringenin) [37,43,44,50]. A recent study [51] focused the antioxidant potential of T. vulgaris and T. citriodorus, described a high correlation with their polyphenol levels with their Trolox equivalent antioxidant capacity (TEAC) (e.g., rosmarinic acid (r = 0.874), eriodictyol-glucoside (r = 0.759), luteolin-hexuronide (r = 0.734, p < 0.0157), and chrysoeriol-hexoside (r = 0.771)) [51]. Amongst phenolic compounds, rosmarinic acid is frequently the principal phenolic acid in the said extracts and it is believed to play a central role in their antioxidant abilities [37,44]. In fact, this caffeic acid dimer was shown to be sixteen times better than α-tocopherol [44] and twice better than trolox [4] in DPPH • -scavenging assay. It also exerts higher reducing power capacity (EC 50 = 2.7 µg/mL) than trolox (EC 50 = 6.6 µg/mL) [4] and higher ABTS •+ scavenging ability than BHT [44]. Despite that, according to Ertas et al. [44], this is less efficient that BHT in protecting against lipid peroxidation (EC 50 value was 1.2-fold higher in the ß-carotene bleaching inhibition assay). Moreover, rosmarinic acid derivatives together with other non-flavonoids and flavonoid compounds (in particular luteolin, apigenin and its derivatives) may also contribute to the antioxidant capacity of Thymus extracts, since they are good antiradical/antioxidants as evaluated in chemical and cellular models. Namely, luteolin and apigenin (50 µg/mL) were demonstrated to drastically decrease the levels of intracellular ROS under oxidative-stress conditions on a hepatic cellular model [52]. Moreover, luteolin was highlighted by its high ability to prevent lipid peroxidation events when compared to rosmarinic acid [4].

Anti-Inflammatory Activity
A growing body of experimental evidence suggests that plant extracts or their bioactive compounds (e.g., phenolic compounds) have potential therapeutic application in the treatment of several inflammatory diseases, through distinct mechanisms, including the inhibition of pro-inflammatory cytokines, intercellular adhesion molecules and enzymes (e.g., iNOS, COX-2, 5-LOX), as well as, transcription factors, such as NF-κB [20,[64][65][66][67], prevention of protein denaturation and stabilization of the potential of erythrocyte membrane [62] or control of the levels of radicals like NO [4,40]. Distinct authors evaluated the anti-inflammatory potential of Thymus polar extracts towards such events ( Table 2).
The capacity to inhibit key enzymes in inflammation was demonstrated for extracts of different Thymus species. Among them, diethyl ether, ethyl acetate and n-butanol extracts of Thymus camphoratus, Thymus carnosus and T. mastichina [49] and methanolic extracts of T. algeriensis and Thymus fontanesii [68] were demonstrated to possess high inhibitory capacity against 5-LOX, with the last two showing similar potency to that of the positive control zileuton, and about one third of the potency of celecoxibe, with regard to the ability to inhibit COX-2 [68]. Moreover, the anti-inflammatory activity of aqueous extracts of T. atlanticus, T. zygis and T. satureioides was evidenced because of their potency to prevent protein denaturation and cellular lysis in a dose-dependent manner [62], as shown in Table 2. Among them, the authors underlined the erythrocyte-protective effect of T. atlanticus, which was superior to that of indomethacin (IC 50 of 93.28 vs 97.83 µg/mL). Albeit the authors did not analyze the phenolic composition of the samples, they suggested that the anti-inflammatory effects were due to its high quantity of polyphenols found in these Thymus species [62].
The ability of natural compounds in scavenging the radical NO is frequently estimated through chemical assays and this is also the case of Thymus polar extracts. Among the distinct studies, one must emphasize the potential of ethanolic extracts from six Thymus species that showed EC 50 NO scavenging results 1-3 times higher than the tested standard compound (Trolox), with the best results being obtained for the T. pulegioides and Thymus longicaulis species (EC 50 of 69.8 and 71.6 µg/mL, respectively) [4]. In addition, T. herba-barona, T. pseudolanuginosus and T. caespititius decoctions, which were rich in caffeic acid derivatives and flavones, were all reported as good NO scavenging agents, from which that from T. caespititius origin had similar potency as ascorbic acid [40]. T. caespititius  T. zygis Studies on cellular models also support the hypothesis that Thymus phenolic-rich extracts possess anti-inflammatory activity. Among them, the work of Ustuner and colleagues [56] underlined the anti-inflammatory potential of T. sipyleus Boiss. subsp. rosulans (Borbas) Jalas aqueous extract through reducing the overproduction of NO and the expression of TNF-α, as attested in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Using the same model, Silva et al. [60] demonstrated that 50 µg/mL of hydroethanolic and aqueous extracts of T. zygis decreased the NO levels by~89% and 48%, respectively, while the aqueous extracts of T. carnosus Boiss. lowered the levels of this radical in a dose-dependent manner, reaching 90% inhibition for 200 µg/mL [69]. Besides, an hydroalcoholic extract of T. longicaulis (rich in rosmarinic acid) was shown to effectively inhibit COX-2 gene expression in THP-1 macrophages [42] and the aqueous extract of T. vulgaris were attested to decrease the release of the cytokine IL-8 for both H 2 O 2 or TNFα-stimulated peripheral blood lymphocytes (PBL) [71]. In addition, a commercial propylene glycol extract of T. vulgaris was reported to inhibit the production of cytokines IL-1β and TNF-α in LPS-activated RAW 264.7 macrophages after exposure to different concentrations of propylene glycol extracts [72].
Notably, some authors emphasized a straight correlation between the high levels of phenolic acids and/or flavonoids in the extracts with their antioxidant/anti-inflammatory activities [37,71]. Among non-flavonoids, rosmarinic acid (in general the major phenolic in Thymus polar extracts) and its derivatives are probably positive contributors to anti-inflammatory activities. In fact, rosmarinic acid was demonstrated to be effective (in vitro and in vivo) against the production and mRNA expression of TNF-α, IL-1β, IL-6, COX-2 and iNOS, and to downregulate proteins involved in the signaling NF-kB-pathway [73]. Moreover, ethyl rosmarinate, a structurally-modified rosmarinic acid, was claimed to be a potent inhibitor against lung inflammation, based on NO and PGE 2 inhibition and also by decreasing of mRNA and protein expression of iNOS and COX-2, as demonstrated in LPS-induced alveolar macrophages [74]. In the same line, flavonoids have also been shown to inhibit the expression of various enzyme systems, inflammatory mediators (e.g., molecules adhesion, cytokines and chemokines) and molecules involved in signal transduction pathways implicated in the inflammatory response [7,24,75]. In particular, luteolin (a common flavone in Thymus plants in general), was reported to inhibit the expression of COX-2 and TNF-α secretion in IFN-γ-stimulated RAW264.7 macrophage cells, and to inhibit the phosphorylation of signal transducer and activator of transcription (STAT)-3, a crucial transcription factor of proliferation and activation of T cells [76].
Opposing to the antioxidant capacity, in vivo anti-inflammatory potential of diverse thyme polar extracts have been reported on animal models of acute inflammation (e.g., paw edema induced by carrageenan, ear edema induced by toxics as croton oil, measurement of myeloperoxidase in leucocytes and total numbers of polymorphonuclear leukocytes (LPM)) [77][78][79]. Among the reported bibliography of Thymus plants, the work of Khouya et al. [43] underlined that the aqueous extracts of T. satureioides, T. atlanticus and T. zygis (at 900 µg/ear) could suppress croton oil-induced mice ear edema, and in addition, the last two (at 50 mg/kg) also inhibited carrageenan-induced rat paw edema, with similar potency to that of the non-steroidal anti-inflammatory drug indomethacin [43]. More recently, the same authors showed that the application of the dichloromethane and ethyl acetate fractions of T. satureioides (1 mg/ear) caused significant decreases in ear edema volume after 4 h (1.8-2.1-fold that of positive control indomethacin (600 µg/ear)), while oral treatment with a crude extract (60 mg/kg) inhibited significantly the carrageenan-induced rat paw oedema, during the first phase (1-5 h) [63]. In the same model, a methanolic fraction of Thymus praecox subsp. skorpilii var. skorpilii showed moderate anti-inflammatory effect [70]. Moreover, the intraperitoneal administration of an ethanolic extract of T. vulgaris (50 and 100 mg/kg) in a rat model of experimental autoimmune encephalomyelitis (EAE), was able to decrease the cytokines production by splenocytes (IFN-γ, IL-6, and IL-17), while the amount of TGF-ß secretion was significantly higher [80]. The administration of hydromethanolic extracts of T. algeriensis and T. fontanesii (600 mg/kg) resulted in the reduction (62% and 52%) on carrageenan-induced leukocyte migration into the peritoneal cavity in mice model, while diclofenac (20 mg/kg) and dexamethasone (2 mg/kg) caused 39% and 30% of reduction, respectively [68].
It is possible that major phenolic constituents of Thymus plants may contribute to their anti-inflammatory effects since either rosmarinic acid, caffeic acid and flavonoides were previously reported as to effectively counteract inflammatory events in vivo models [73,76,77,[81][82][83]. In particular, rosmarinic acid suppressed LPS-induced proinflammatory cytokine [73,77], inhibited the expression of COX-2 and iNOS protein in mice disease-inflammatory models [73,84], and reduced the myeloperoxidase activity compared with the LPS-treated cells, suggesting the neutrophil infiltration, which indirectly reflect the degree of inflammation [77]. Likewise, the flavone luteolin suppressed inflammatory signals including TNF-α, NF-kB, MCP-1 and IL-1β in LPS-induced cells, attenuating acute renal injury in mice [81]. In addition, the anti-inflammatory effects of luteolin on autoimmune thyroiditis mouse model was demonstrated by the reduction of infiltrating lymphocytes and downregulation of IL-6/STAT1 and STAT3 signaling pathway in thyroid glands [76]. In turn, administration of apigenin exerted a beneficial role in acute mouse pancreatitis, reducing necrosis to 100% and a significant inhibition of TNF-α expression [85].
The overall data focus on the anti-inflammatory potential of Thymus extracts and phenolic compounds as well [24], emphasize their potential useful in the development of alternative drugs for the treatment of a variety of diseases settled on oxidative and/or inflammatory events, including autoimmune diseases, diabetes, cancers and ageing.

Anticancer Activity
The antitumoral potency of Thymus extracts has been studied, underlying their potential as sources of chemotherapeutic agents, particularly in in vitro cellular models. As for drugs in general, the majority of these studies used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a first approach method to estimate cytotoxicity/growth-inhibitory activity of extracts in cancer cell lines (Table 3). This colorimetric method measures the activity of mitochondrial enzymes that reduce MTT to purple formazan, and this conversion is often used as an estimative of viable cells [30,59]. Similar principle methods are also applied by the authors, including those based on the reduction of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) [42,86]; or sulforhodamine B (SRB) assay, which is based on the ability of SRB to bind to protein components of cells under mild acidic conditions and that can be extracted under basic conditions, given an estimation of cell mass (extrapolated to growth activity) [87,88]. In general, literature data is presented as IC 50 or GI 50 values (inhibition concentration or grow inhibition, respectively), which both correspond to sample concentration able to inhibit 50% of the cell grow.
The antitumoral activity of the most widespread Thymus species, namely T. vulgaris and/or Thymus serpyllum, were previously screened. Commercial aqueous extracts from these two plant species were described to exhibit cytotoxic ability towards adriamycin-resistant human breast cancer cell line (MCF-7/Adr) [89]. Amongst representative compounds of the extracts (caffeic acid, rosmarinic acid, lithospermic acid, luteolin-7-O-glucuronide, luteolin-7-O-rutinoside and eriodictiol-7-O-rutinoside), rosmarinic acid and lithospermic acid were demonstrated to be the most active [89]. Notably, plant extracts (including T. vulgaris and T. serpyllum) proved to be more toxic against the cancer resistant cells than the analyzed phenolics (that exhibited greater toxicity against the non-resistant cells), suggesting the existence of synergistic or additive effects between the extracts' components [89] (Table 3). T. citriodorus Moreover, methanolic extracts of Thymus schimperi, containing high amounts of the flavone luteolin and derivatives, required doses of 50-100 µg/mL to inhibit the growth of human gastric adenocarcinoma AGS cells by 50% (IC 50 = 88 µg/mL) [91]. In addition, Galasso et al. [42] reported that T. longicaulis extracts, containing high amounts of quercetin-O-hexoside, luteolin-O-hexoside and salvianolic acid K, caused a decrease of cell viability on breast cancer MDA-MB-23, colon carcinoma HCT-116 and lung fibroblast MRC-5 cells, equal to or even higher than those observed for vinblastine, a known anti-cancer agent [25]. Besides, Esmaeili-Mahani et al. [30] reported that polar extracts of Thymus caramanicus (at 150-200 µg/mL), could reduce the viability of human breast adenocarcinoma MCF-7 for about 60% and interestingly, its concomitant treatment (40 and 80 µg/mL extract) with the vincristine effectively potentiated the effect of the anticancer drug. Aqueous extracts of T. satureioides, mainly composed of rosmarinic acid and of luteolin glucoside, exhibited an IC 50 value of 37.5 µg/mL in the same cell line [43]. The cytotoxicity capacity was also demonstrated for methanolic extracts of T. serpyllum, which caused a viability decrement of two human breast cancer cell lines (MCF-7 and MDAMB-231) after 72 h exposure (IC 50 of 509 and 276 µg/mL, respectively), disclosing less anti-proliferation ability in human normal breast cells (MCF-10A), which further proved the anticancer property of this extract [92]. Polar extracts obtained from T. carnosus Boiss. were also tested for their cytotoxicity in MCF-7, BT-474 and RAW 264.7 cells using Alamar Blues assay, with best results being obtained for the hydroethanolic extracts rich in salvianolic acids and rosmarinic acid [69]. T. zygis extracts, containing similar phenolic composition, displayed promising results in Caco-2 and HepG2 cells (IC 50 = 85.01 and 82.19 µg/mL, respectively), especially at 48 h of exposure [60]. The same group reported high cytotoxic ability of aqueous decoction and hydroethanolic extracts from different Thymus species towards Caco-2, namely T. pulegioides (IC 50 of 82.25 and 105.44 µg/mL respectively) [61] and T. citriodorus (IC 50 of 159.40 and 114.6 µg/mL respectively) [51], albeit both were less active against HepG2 cells [51]. In addition, aqueous decoction/hydroethanolic extracts of T. mastichina (rich in salvianolic acid derivatives) required low doses to inhibit the same cells growth by 50% (IC 50 of 95.65 and 51.30 µg/mL towards Caco-2; 285.03 and 180.10 µg/mL towards HepG2 cells, respectively) [54]. Interestingly, T. vulgaris, the most studied and widely used thyme species worldwide, showed the lowest cytotoxic activity against Caco-2 and HepG2 cells after 48 h, for aqueous decoction (IC 50 values of 376.8 and > 500 µg/mL, respectively) and hydroethanolic extracts (IC 50 values of 442.45 and 254.25 µg/mL, respectively) [51].
In addition, Gordo et al. [88] reported that the dichloromethane and ethanol extracts of the aerial parts of T. mastichina were able to inhibit the viability of the HCT colon cancer cell line (IC 50 value of 2.8 µg/mL and 12.0 µg/mL, respectively). Moreover, in this study, the authors also described that ursolic acid and a mixture (1:1) of oleanolic acid and ursolic acid, isolated in the chromatographic procedures, exhibited high cytotoxicity (IC 50 of 6.8 and 2.8 µg/mL, respectively), suggesting that phenolic compounds maybe related to the protective effect of this thyme extract against colon cancer cells [88]. Discrepant results were reported recently for an ethanolic extract of T. algeriensis Boiss. and Reut. since this was ineffective in suppressing cancer cell growth (required a concentration greater than 10 000 µg/mL for achieving 50% reduction of cancer cell lines survival) [45]. In turn, other study using a methanolic extract from the same species, rich in gallic and vanillic acids, reported an effective protection against in vitro Multiple Myeloma cell line (U266) proliferation in a dose-dependent manner [90].
The DNA repair ability is very important, as DNA damage can lead to carcinogenesis if replication proceeds without a proper repair. The DNA-protective effect of plant extracts against DNA strand breaks induced by the oxidant agents H 2 O 2 and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) has been performed by the comet assay, based in single cell gel electrophoresis [59,93]. Kozics et al. reported the protective effects of ethanolic extract of T. vulgaris (rich in rosmarinic acid) against DNA damage induced by strong oxidants, achieved the highest DNA-protective capacity at concentrations of 0.5 and 0.1 mg/mL, respectively [59].
Important effects of Thymus extracts in key mechanisms of tumor formation/progression (e.g., apoptosis and angiogenesis) have also been attested, both in in vitro and in vivo models.
Although less studied than essential oils [8], phenolic-rich extracts from Thymus have been evaluated for effects on apoptosis of proliferative cells by cycle analysis, mostly through the flow cytometry method [94]. Some studies also investigated the apoptosis induction of cancer cells through activation of the caspase cascade or by disruption in the balance between pro-and antiapoptotic Bcl-2 family members, after exposure to thyme polar extracts. For example, Al-Menhali et al. [95] registered moderate apoptotic capacity of aqueous extracts of T. vulgaris, which were shown to increase the activity of caspase 3/7 on human colon carcinoma cells HCT116 in a concentration-dependent manner (3.5%, 36.1% and 54.7% increase in the activity of caspase 3/7 in cells treated with 0.2, 0.4, or 0.6 mg/mL, respectively) [95]. In addition, Esmaeili-Mahani et al. [30] highlighted the anticancer activity of T. caramanicus hydroalcoholic extracts, based on their ability to induce apoptosis in human breast cancer cells MCF-7 through the increment of caspase-3 and Bax and decreasing of Bcl-2 expression. These T. caramanicus extracts (200-250 µg/mL) also potentiated the cytotoxicity of vincristine, which was accompanied by an increase in the levels of cleaved caspase-3 [30]. Moreover, treatment of MDA-MB-231 breast cancer cells for 72 h with a methanolic extract of T. serpyllum gradually increased the caspase 3/7 enzyme activity (increased 1.6-, 2.2-, and 3-fold at 10, 250, and 500 µg/mL of extract, respectively) [92]. Less promising results were reported by Caprioli and coworkers [94] for the apoptotic effect of an ethanolic extract of Thymus lanceolatus (rich in rosmarinic acid), which required a concentration of 500 µg/mL for a significant increase in programmed cell death of chronic myelogenous leukemia K562 cells (11.5% and 14.2% at 48 and 72 h, respectively), with no significant effect towards colorectal adenocarcinoma CaCo-2 and neuroblastoma SH-SY5Y cells.
Once the inhibition of angiogenesis has been recognized to be advantageous for prevention of inflammation and neoplastic growth [33,96], methanolic extracts of T. capitatus (100 µg/mL) were shown to inhibit significantly micro-vessel formation in rat aortic rings [97]. Still, as the authors did not find significant effects against proliferation and migration of the human endothelial EA.hy926 cells after extract exposure, they concluded that the in vivo observed anti-angiogenic effects must occur thorough an indirect mechanisms of action [97].
The activity of enzymes involved in inflammation, such as COX-2 and its product PGE-2 (known as carcinogenesis promoters), and others involved in enhancing detoxification, like as GPx, SOD or catalase [34,35], and cytokines or others immunological mediators, have also been used for evaluation of the antitumoral activity of Thymus extracts [42,59]. With this regard, ethanolic extracts of T. vulgaris (1 and 0.5 mg/mL), with high amounts of rosmarinic acid, were described to induce a significant increase of GPx activity in human HepG2 cells, while SOD activity was significantly lowered with respect to the control [59]. Likewise, hydroalcoholic extracts of T. longicaulis, particularly rich in rosmarinic acid and methylapigenin, exhibited a strong anti-inflammatory and anticancer effectiveness through inhibition of COX-2 gene expression on human leukemia cell line THP-1 macrophages [42]. These results suggest that the downregulation of COX-2 expression/activity and growth inhibition of tumor cells are closely associated, determining the relationships of anti-inflammatory, antioxidant and cytotoxic potential [42,98].
Moreover, a water extract of Thymus pannonicus was demonstrated to cause a significant increase in the activity of xanthine oxidase, glutathione reductase and GPx in Ehrlich ascites carcinoma mice cells (EACs), having the authors concluded that the induction of oxidative stress may play an important role in antitumor properties of the extract [99]. The authors also demonstrated that this Thymus extract caused a strong tumor growth inhibition in mice, which was followed by a high percentage of apoptotic/necrotic cells in a dose dependent manner, as well as significant decrease (96-98%) in the EAC volume [99]. The extract was particularly rich in rosmarinic acid, salvianolic acid H, catechin derivatives and glycosidic derivatives of luteolin and apigenin. On another study, Caproli and coworkers also reported that an ethanolic extract of T. lanceolatus, rich in rosmarinic acid and flavonoids, was an efficient ROS scavenger, exhibiting a good antioxidant activity (since it decreases the ROS generation by about 30% in SH-SY5Y (neuroblastoma) and 70% in CaCo-2 (colorectal adenocarcinoma) and K562 (chronic myelogenous leukemia) cells [94].

Conclusions
The Thymus genus is a rich source of secondary metabolites with claimed health benefits. For the last years, phenolic-rich extracts from distinct Thymus species have been the focus of attention and screened for distinct bioactive properties of interest for application in industrial fields like food, pharmaceutical and cosmetics. This review highlights the biological activities of phenolic-rich extracts from various Thymus species, with emphasis devoted to antioxidant, anti-inflammatory and anticancer properties. Globally, the documented data revealed that Thymus extracts, rich in phenolic acids and flavonoids compounds, may have enormous ability to scavenge radicals and reduce inflammatory events. Importantly, several studies disclose that thyme extracts have the capacity to inhibit diverse proinflammatory mediators, such as cytokines (IL-1β, IL-6, IL-12, INF-γ, TNF-α), enzymes (iNOS, COX-2) and others, which are majorly regulated by the transcriptional factor NF-κB, whose activation is also associated with increased ROS generation. Moreover, there is also a considerable amount of work reflecting the promising cytotoxic activity of some Thymus phenolic-riche extracts on tumor cell lines, as well as their ability as regulators of the tumor cell cycle, leading to programed cell death or inhibiting angiogenesis. Hence, the evidenced health-benefits of Thymus extracts as antioxidant and anti-inflammatory agents corroborate their potential applications for pharmaceutical purposes, including the development of antitumoral compounds.
Regardless that, one must remark that so far, most of the gathered investigation has been based on in vitro experiments whereas there is a huge lack of in vivo studies, in order to be able to confirm the beneficial effects of Thymus polar extracts on human health.

Conflicts of Interest:
The authors declare no conflict of interest.