Ak et al. (2021) performed a study of hexane, hydroethanolic and aqueous extract from T. vulgare aerial parts, stems and flowering heads. They reported the highest total phenolic content (TPC) and total flavonoid content (TFC) in both T. vulgare hydroethanolic extracts from stems and aerial parts (up to 93 mg GAE (gallic acid equivalent)/g dry extract and 53 mg RE (rutin equivalent)/g). TFC up 82 mg RE /g was determined in the methanol-aqueous extract from T. macrophyllum flowering heads (Gevrenova et al. 2020). Extraction methods highly impact the TPC and TFC yielding up to 65.05 mg GAE/g (TPC) by accelerated solvent extraction and 55.40 mg RE/g (TFC) by maceration in the ethanolic extracts from T. parthenium (Zengin et al. 2020). TPC varied in large ranges – the lowest levels were reported for T. vulgare (26.37 mg GAE/g), T. corymbosum (5.90 mg GAE/g) and T. macrophyllum (0.64 mg GAE/g), while it reached up to 221 mg GAE/g in T. vulgare roots (Devrnja et al. 2017; Ivanescu et al. 2018).

In a comparative analysis of TPC in different agrorefinery products from the residue after the hydrodistillation of the essential oil from T. vulgare aerial parts (aqueous extract, acetone balsam and acetone extract), the highest content was found in the aqueous extract (142.30 mg GAE/g dry extract) (Baranauskiene et al. 2014).

Williams et al. (1999a, 1999b) investigated epicuticular (surface) and vacuolar flavonoids in 7 species of genus Tanacetum. The lipophilic compounds consisted mainly of 6-hydroxykaempferol-3,6,4’-trimethylether (89) and quercetagetin-3,6,3’-trimethylether (91), together with 11 other surface flavonoids (Table 1). The methyl ethers of scutellarein (125, 126) and 6-hydroxyluteolin (124) are characteristic of species with corymb inflorescences of flowering heads. Vacuolar flavonoid glycosides are represented by apigenin 7-O-glucoside (98) and luteolin 7-O-glucoside (109), 6-hydroxyluteolin 7-glucoside (119) occurs in T. vulgare and T. pseudoachillea; chrysoeriol 7-glucuronide (118) – in T. parthenium, T. macrophyllum and T. corymbosum. Highly methoxylated flavonoids such as eupatilin (127), eupatorin (127), casticin (130) have been proven in the recent studies (Devrnja et al. 2017; Ivanescu et al. 2018). Methoxylated derivatives of quercetagetin (90–94), 6-hydroxykaempferol (88 and 89), scutellarein (125 and 126) and 6-hydroxyluteolin (124) have been identified in T. macrophyllum flowering heads and leaves (Williams et al. 1999a). T. macrophyllum aerial parts were especially rich in the glucosides and glucuronides of apigenin (98, 99, 102, and 115) and kaempferol (79 and 81)(Venditti et al. 2018).

HPLC-DAD analysis of the major compounds in ethanol-aqueous extracts of T. vulgare and T. balsamita showed 1.37 g/100 g extract and 0.93 g/100 g chlorogenic acid (23), and 3.33 g/100 g and 2.78 g/100 g chicoric acid (69), respectively (Baczek et al. 2017). The total content of the determined phenolic acids (caffeic (16), ferulic (20), chlorogenic (23), rosmarinic (68) and chicoric acids (69)) was over 4 g/100 g extract in both species. The highest content of apigenin 7-O-glucoside (98) (1.10 g/100 g extract) was determined in T. vulgare, and of chrysoeriol (100) (0.63 g/100 g) in T. balsamita.

Mono- and dicaffeoylquinic acids, flavone and flavonol glucosides and glucuronides were identified/annotated in methanolic, hydroalcoholic and aqueous extracts of T. vulgare by UPLC/ESI-QTOF-MS, LC-DAD/ESI-TOF-MS and HPLC-MS (Baranauskiene et al. 2014; Devrnja et al. 2017; Ivanescu et al. 2018) (Table 1). Among the methoxylated flavonoids, wide spread in Asteraceae family, the greatest diversity was found in T. macrophyllum: eupatilin (127) (144.09 µg/g), hispidulin (125) (55.83 µg/g), eupatorin (128) (1.48 µg/g), and acacetin (103), jaceosidine (129) and casticin(130) were below 1 µg/g (Ivanescu et al. 2018). Zengin et al. (2020) determined chlorogenic acid (23) up to 487 mg/kg, quercetin (72) – up to 377 mg/kg and p-hydroxyphenylacetic acid (6) – up to 280 mg/kg in the T. parthenium ethanolic extract obtained by accelerated solvent extraction.

Recently, a hyphenated platform liquid chromatography - high resolution mass spectrometry (LC-HRMS) for annotation and dereplication of acylquinic acids (AQAs), 6-methoxylated flavonoids and sesquiterpene lactones in selected Tanacetum speies was developed (Gevrenova et al. 2020, 2023; Ak et al. 2021). Secondary metabolites were annotated/identified in methanol-aqueous extracts from the flowering heads, leaves and roots of Tanacetum macrophyllum, T. vulgare and T. balsamita. They were profiled and characterized by reverse phase chromatography coupled with hybrid quadrupole-Orbitrap HRMS. The annotation/dereplication was based on the diagnostic fragment ions for each subclass of the compounds. An in-depth depiction of more than 100 secondary metabolites was achieved.

Overall, monoAQA, diAQA and triAQA (23–67) were evidenced in the assayed Tanacetum species (Table 1). Among them, caffeoyl-, feruloyl- and coumaroylquinic acids, dicaffeoylquinic, feruloyl-caffeolylquinic, p-coumaroyl-caffeoylquinic, hydroxydihydrocaffeoyl (HC)-caffeolylquinic and dehydrocaffeoyl (DC)-caffeoylquinic acids were annotated together with tricaffeoylquinic acids and dicaffeoylquinic acid-hexosides. A variety of methoxylated derivatives of scutellarein (125 and 126), quercetagetin (91–94) and 6-hydroxyluteolin (124) was dereplicated.

For the first time, an exceptional variation of AQAs in T. macrophyllum was reported (Gevrenova et al. 2020). Multivariate data analysis (PCA and hierarchical clustering analysis) allowed for the identification of markers able to discriminate the aerial parts and flowering heads from the roots (Gevrenova et al. 2020). Among them, methoxylated flavonoids patuletin (92), eupatorin (128), quercetagetin trimethyl ether (94) and STLs artekalin (243) (eudesmanolide) and artemisiifolin (238) (germacranolide) were dereplicated. In addition, flowering heads are discernable by 4-p-coumaroyl-5-caffeoylquinic acid (54) and 4-caffeoyl-5-p-coumaroylquinic acid (42) (Fig. 1).

Figure 1. 

Structure of some acylquinic acids and flavonoids in Tanacetum species.

PCA and discriminant analyses allowed for the identification of the most discriminant secondary metabolites and bioactivities in T. vulgare flowering heads, stems and aerial parts in terms of extraction solvents (Ak et al. 2021). Among the compounds are jaceosidin (129), kaempferol 3-O-glucoside (78), luteolin 7-O-glucoside (109), naringenin O-hexuronide (151), caffeic acid O-hexoside, caffeoyl-hexose (21), thujone (185 and 186), and caryophyllen/bisabolene (194).

The main feature of genus Tanacetum and the species of tribe Anthemideae is the production of a specific “non-standard” group of monoterpenes, the result of the so-called “middle-to-tail” coupling of the terpene precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate (in contrast to the typical “head-to-tail” condensation) (Abad et al. 1995). A major precursor in this group is chrysanthemic acid. Opening the ring of this compound in various ways leads to the formation of artemisia ketone (189), artemisia alcohol (190), etc. Abad et al. (1995) divided Tanacetum species into two main groups according to essential oil (EO) composition: “monoterpene group (T. vulgare, T. parthenium, T. boarale)” and “sesquiterpene group” (species from Central and Southeast Europe: T. macrophyllum, T. cilicium, T. corymbosum, T. millefolium).

The bitter taste of the aerial parts of Tanacetum species is due to sesquiterpenes (farnesene (196), germacrene D (198), etc.). The EO composition of Tanacetum species and its biological activity is summarized by Kumar and Tyagi (2013). Over 200 compounds have been identified. The main characteristic is the chemovariability in the species and subspecies taxonomic category. The main components are camphor (172), bornyl acetate (180), α-phellandrene (181), chrysanthenyl acetate (187), α-terpinene (163), p-cymene (165), terpinen-4-ol (177), α-terpineol (176), caryophyllene oxide (208), α- and β-thujone (185 and 186), α-pinene (158), camphene (160), β-caryophyllene (194), carvacrol (184) etc. (Table 1) (Kumar and Tyagi 2013).

About 30 chemotypes have been reported for T. vulgare depending on the composition of the essential oil and their main components, as well as for T. balsamita - 4 (Baczek et al. 2017; Devrnja et al. 2017). The most common chemotypes are β-thujone (186), α-thujone (185), camphor (172), trans-chrysanthenyl acetate (187), eucalyptol (173), and artemisia ketone (189). Less common are the chemotypes α-pinene (158), sabinene (161), dihydrocarvone (191), chrysanthenol (182) (Rohloff et al. 2004). In Europe, the most common hemotypes are thujone (185 and 186), camphor (172), chrysantenyl acetate (187), and artemisia ketone (189). Regarding T. balsamita, 4 main chemotypes have been described in the literature survey – carvone (174), camphor (172), camphor (172)+thujone (185 and 186), carvone (174)+thujone (185 and 186) (Baczek et al. 2017). Trans-chrysanthenol (182), β-thujone (186), bornyl acetate (180) and pinocarvone (175) types have also been described.

GC-MS analysis of EM from T. vulgare (aerial parts, origin Serbia) showed a 93% content of oxidized monoterpenes, among which trans-chrysanthenyl acetate (187) (41%), trans-chrysanthenol (182) (12.5%), cis-thujone (185 and 186) (5.3%), camphor (172) (5%), 1,8-cineole (eucalyptol) (173) (3.9%) (Devrnja et al. 2017). According to the classification based on the composition of EM, this type belongs to the chrysantenyl acetate (187) chemotype. For Serbia, the types thujone (185 and 186), chrysanthenyl, chrysanthenyl acetate (187) and camphor (172) have been established. Buckwheat EM yield and composition are affected by genetic variation and/or environmental conditions, e.g., the yield of EM from 40 populations in Norway is varying between 0.35 and 1.90% (Rohloff et al. 2004). The chrysantenyl acetate (187) chemotype is characterized by a good antibacterial effect (Devrnja et al. 2017).

In a comparison of EM between T. vulgare (Poland) and T. balsamita (Turkey), it was found that the differences were mainly quantitative - 96% and 83% oxidized monoterpenes, respectively (Baczek et al. 2017). T. balsamita EM mainly contains β-thujone (186) (84.4%), α-thujone (185) (4.7%), eucalyptol (173) (4.1%), while major components in T. vulgare EM are trans-chrysanthenyl acetate (187) (18.4%), (E)-dihydrocarvone (191) (11%) and artemisia ketone (189) (9.2%), β- and α-thujone (185 and 186) are 14.3% and 0.8%. Of the monoterpenes, α-pinene (158) and sabinene (161) are contained in the EM of T. vulgare, and only in traces in T. balsamita. There is also a difference in sesquiterpenes: 5.1% oxidized sesquiterpenes and 0.4% sesquiterpene hydrocarbons in T. vulgare and only 0.7% oxidized sesquiterpenes in T. balsamita (Baczek et al. 2017). Baranauskiene et al. (2014) identified EM from Lithuania as β-thujone (186) chemotype (86%) together with α-thujone (185) (2.5%) and sabinene (161) (2.5%). The composition of T. macrophyllum EM is presented in Table 1. Radulovic et al. (2010) found eucalyptol (173) (8.6%), camphor (172) (6.4%), γ-eudesmol (214) (6.2%) and isobornyl acetate (9.5%) in the EM from aerial parts of T. macrophyllum. In Turkish populations, the main components are β-eudesmol (209) (21.4%) and cis-chrysanthenol (182) (12%) (Demirci and Baser 2007). Polatoğlu et al. (2013) analyzed EM from flowering heads and leaves separately and identified 64.9% oxidized sesquiterpenes (γ-eudesmol (209), sesquivalandulol (213), and copaborneol (216) in the flowering heads, and 47.2% oxidized monoterpenes (eucalyptol (173), bornyl acetate (180), borneol (178), and β-thujone (186)) in the leaves. A different EM profile from flowering heads and leaves of T. macrophyllum (origin Italy) was found by Venditti et al. (2018). EM from the flowering heads is characterized by 39.4% oxidized monoterpenes and 28% oxidized sesquiterpenes; the individual components are 10-epi-γ-eudesmol (204) (12.5%), camphor (172) (11%), linalool (171) (10.8%) and eucalyptol (173) (8.8%). The leaves EM contains more sesquiterpene hydrocarbons (39.3%) - mainly germacrene D (198) (30.9%) and oxylated monoterpenes (25.4%), of which camphor (172) (11.1%) and eucalyptol (173) (5.5%). Oxidized sesquiterpenes in the leaves were fewer and included 10-epi-γ-eudesmol (209) (3.9%) and caryophyllene oxide (208) (3.4%). Significant amount of p-methyl benzyl alcohol was found in T. macrophyllum EM (Abad et al. 1995). Recently, the dominant compounds of T. parthenium EM were found to be camphor (172) (45.47%), trans-chrisantenyl acetate (187) (21.65%), camphene (160) (9.48%), and cis-isogeraniol (5.42%) (Lechkova et al. 2023), while camphor (172) (25.24%), trans-chrysantenyl acetate (187) (18.35%), cis-verbenol (10.58%), thujone (185) (6.06%), eucaliptol (173) (5.99%), and α-campholenal (5.98%) were the main compounds in T. vulgare (Karcheva-Bahchevanska et al. 2023).

STLs in Tanacetum species distributed in Bulgaria were studied by Todorova and Evstatieva (2001) (Fig. 2) and correspond to their taxonomic position - in two sections of Tanacetum (T. vulgare, T. achilleifolium, T. millefolium) and sect. Pyrethrum (T. corymbosum, T. macrophyllum, T. parthenium). T. achilleifolium and T. millefolium produce only STLs of eudesmanolid type; they are the only Bulgarian species in which 11, 13 dihydroeudesmanolides were determined. Three chemotypes have been established for T. vulgare: with germacranolides, with eudesmanolides and without STLs (Todorova and Evstatieva 2001). Similar results were found for the species from Italy (Rosselli et al. 2012). T. vulgare STLs have α- methylene-γ-lactone residue (Fig. 2) (Abad et al. 1995). T. corymbosum contains lactones from the small group of eudesman-12,8-olides, which are not typical for the genus (Todorova and Evstatieva 2001).

Figure 2. 

Structure of STLs (germacranolides, guaianolides and eudesmanolides) in species of the genus Tanacetum.

T. macrophyllum and T. parthenium have the all three types of STLs, but mostly - guaianolides, which have a variety of O-containing functional groups. Among the STLs in T. macrophyllum, the guaianolides macrotanacin (223) and tanaphyllin (224) (found in large quantities in the species originating in Bulgaria) should be noted. A major STL in T. parthenium is parthenolide (239). Douglanin (258) has been isolated from T. praeteritum, T. argenteum subsp. canum, T. vulgare ssp. siculum (Gören 1995; Gören and Tahtasakal 1997; Rosselli et al. 2012). Ludovicin B (257), A (256) were found in T. praeteritum and T. vulgare, and 1α-hydroxy-1-deoxyarglanin (259)- T. praeteritum (Gören 1995; Rosselli et al. 2012).

Fraisse et al. (2011) established a correlation between the antioxidant activity, the total dihydroxycinnamic derivatives and the total phenols in extracts of Asteraceae species, among which were T. vulgare and T. parthenium. There is a significant relationship between DPPH radical scavenging activity and TPC (R2 = 0.8904), total dihydroxycinnamic derivatives (R2 = 0.8529) and total caffeoyl derivatives (R2 = 0.7172). Thus, the antioxidant activity mainly depends on the main caffeoyl derivatives; the quantity of 3,5-dicaffeoylquinic acid (28) in T. parthenium (30.08 g/kg) was 48.92%. In a comparative study of methanolic extracts of T. vulgare different plant parts, TPC was found from 83.6 to 221.7 mg GAE/g dry weight, with the content decreasing in the order roots>leaves>flowering heads>stems (Devrnja et al. 2017). In the DPPH test, the roots have the highest activity with an IC₅₀ of 44 μg/ml, followed by the flowering heads – 58.3 μg/ml. The Pearson correlation coefficient between the two indicators is R=-0.8 with R2 = 0.64. In the test for reducing power, the root extract was the most active: in a concentration of 0.1 mg/ml – 0.123, 1 mg/ml – 0.903 (Trolox 0.926).

Applying the biorefinery (agrorefinery) concept, Baranauskiene et al. (2014) compared the antioxidant activity of EO and different products of the residue after hydrodistillation (water extract, acetone balsam and acetone extract). The highest TPC in the aqueous extract (142.30 mg GAE/g dry extract) is also consistent with the highest antioxidant activity in the DPPH assay (IC₅₀ 1.33 mg/ml), ABTS (IC₅₀ 1.40 mg /ml), FRAP (546.20 µM Trolox/g extract) and ORAC (11947.5 µM Trolox/g extract). The antioxidant potential of two varieties of Chrysanthemum balsamita var. balsamita and Ch. balsamita var. tanacetoides was established in DPPH assays (IC₅₀ 59.70 and 121.13 µg/ml, respectively), SNPAC (silver nanoparticle antioxidant capacity) (71.44 and 34.25 µMGAE/g, respectively), EPR radical detection (integral intensity 228.04 and 361.50, respectively) (Benedec et al. 2016).

According to Baranauskiene et al. (2014), the antioxidant activity of the water extracts is due to acylquinic acids - mono- and dicaffeoylquinic acids; the effect of 3,5-dicaffeoylquinic acid (28) (Juan-Badaturuge et al. 2009) as well as that of flavonoids (Juan-Badaturuge et al. 2009; Pukalskas et al. 2010; Benedec et al. 2016) are specifically discussed.

The antioxidant potential of Tanacetim species is exclusively associated with the content of phenolic acids. The high antioxidant potential of T. vulgare is due to the high content of caffeic (16), rosmarinic (68) and ferulic (20) acids (Baczek et al. 2017). According to Baranauskiene et al. (2014), mono- and dicaffeylquinic acids are with the most pronounced effect as well as 3,5-dicaffeylquinic acid (28) and flavonoids (substituted with hydroxyl groups in ring B and C) (Juan-Badaturuge et al. 2009).

In the antioxidant assays of T. vulgare extracts, the hydroalcoholic aerial parts extract exhibited the strongest radical scavenging activity (up to 148 mg TE/g and 176 mg TE/g for DPPH and ABTS, respectively) and reducing power (404 and 188 mg TE/g for CUPRAC and FRAP) (Ak et al. 2021). The best metal chelating activity and total antioxidant capacity in phosphomolibdenum assay was found for the infusion stem extract (25 mg EDTAE/g) and hydroalcoholic stem extract (2.07 mg TE/g). Discriminant analysis showed that radical scavenging activity (DPPH and ABTS), reducing power (CUPRAC and FRAP), and methal-chelation ability account mostly for the extract discrimination.

Antioxidant activity of T. parthenium was especially pronounced in the ethanolic extracts obtained by accelerated solvent extraction in terms of radical scavenging activity (Zengin et al. 2020), and notably the reducing power in the FRAP assay (up to 330 mg TE/g). Remarkable radical scavenging activity (300 mg TE/g and 573 mg TE/g for DPPH and ABTS) and reducing power (443 mg TE/g and 277 mg TE/g for CUPRAC and FRAP) was assessed for T. macrophyllum roots and flowering heads, respectively (Gevrenova et al. 2020). Multivariate analysis demonstrated that radical scavenging activity (DPPH and ABTS) and reducing power mostly accounted for the high antioxidant potential of T. macrophyllum. Generally, ABTS hold significance for the separation of the studied T. macrophyllum plant parts. Concerning T. balsamita hydromethanolic extracts, supervised partial least square discriminant analysis (PLC-DA) revealed that radical scavenging activity and reducing power have marked impact on the pronounced antioxidant potential of flower heads (Gevrenova et al. 2023). The same trend was observed for the water extracts from T. poteriifolium aerial parts (Zengin et al. 2019). Overall, the smallest antioxidant activity was evaluated for T. balsamita in comparison with T. vulgare, T. macrophyllum, T. poteriifolium and T. parthenium.

The bio-inhibitory effects of EO can be explained rather by the synergistic effects of their components. The AChE inhibitory potential of some EO terpenes of T. macrophyllum flowering heads and leaves was determined: 1,8-cineole (173) (IC₅₀ = 0.67 mM), camphor (172) (IC₅₀ > 10 mM), germacrene D (198) also has proven inhibitory capacity (Venditti et al. 2018).

On the other hand, the interaction between different terpenes plays an important role, in the light of synergism between monoterpenes and antagonism between monoterpenes and sesquiterpenes. EO of T. macrophyllum flowering heads and leaves had a lower AChE inhibitory effect (IC₅₀ 15.23 and 1049 mg/ml) compared to the ethanolic extracts (IC₅₀ 0.57 mg/ml). This is a 60-fold lower value than that of galantamine (positive control). In many cases, the insecticidal activity of plant extracts is due to AChE inhibitory activity.

In a comparative study of enzyme inhibitory activity of ethanol extracts of T. parthenium obtained with different extraction methods, activity towards α-glucosidase (1.63–1.67 mM ACAE (acarbose)/g extract) and α-amylase (0.51–0.56 mM ACAE/g) was assayed (Zengin et al. 2020). A strong inhibitory effect on glucosidase combined with a moderate one on amylase has a good effect on postprandial hyperglycemia, which is the main cause of complications such as diabetic retinopathy, neuropathy, cardiovascular problems in diabetics. The inhibitory activity on tyrosinase was from 163.67 mg KAE (kojic acid)/g extract to 183.07 mg KAE/g extract obtained by ultrasound and accelerated liquid extraction, respectively. The inhibition of cholinesterases is more pronounced on BСhE (4.63–5.21 mg GALAE (galantamine)/g), and on AChE - 2.84–3.38 mg GALAE/g extract (Zengin et al. 2020). In this regard, increased activity of AChE was found in the early stages of dementia, and of BСhE – in the later stage. Hierarchical clustering combined with PCA showed that ultrasound extraction was the most distinct from the other extraction methods and was associated with lower antioxidant activity in terms of DPPH, ABTS, FRAP, CUPRAC and tyrosinase inhibitory activity, but with pronounced metal-chelating ability and amylase inhibition (Zengin et al. 2020).

T. vulgare flowering heads hydroethanolic extract showed the best AChE and BChE (1.95 and 1.79 mg GAE/g, respectively), and α-glucosidase (10.77 mg ACAE/g) inhibitory activity, while hexane extract inhibited tyrosinase (31.81 mg KAE/g) and α-amylase (0.53 mg ACAE/g) (Ak et al. 2021). In this context the most pronounced AChE inhibitory potential has been found for the T. macrophyllum methanol-aqueous extract (up to 4.46 mg GAE/g) (Gevrenova et al. 2020). It’s worth noting that the T. balsamita methanol-aqueous extracts exerted lipase inhibitory activity up to 8.15 mg OE (orlistat)/g (Gevrenova et al. 2023).

Methoxylated flavonoids isolated from leaves of T. parthenium and/or T. vulgare have been investigated for inhibition of cyclooxygenase and 5-lipoxygenase (Williams et al. 1999a). The T. parthenium flavonoids 6-hydroxykaempferol 3,6-dimethyl ether (88) and santin (6-hydroxykaempferol 3,6,4’-trimethyl ether) (127) have the same enzyme inhibitory activity profile, but santin is more active (IC₅₀ 27 and 58, respectively µM). Quercetagetin 3,6,3’-trimethyl ether (91) was more active against cyclooxygenase (IC₅₀ 22 µM). Nepetin (6-hydroxyluteolin) (124) and jaceosidin (6-hydroxyluteolin 6,3’-dimethyl ether) (129) from T. vulgare leaves inhibited both enzymes but were less active against cyclooxygenase (IC₅₀ 61 and 89 µM, respectively) compared to the corresponding flavonols. Methoxylated flavonoids inhibit the two main pathways of arachidonic acid metabolism – the cyclooxygenase and 5-lipoxygenase pathways.

The anti-inflammatory activity of T. parthenium, T. vulgare, T. niveum, T. ptarmiciflorum was investigated in a human polymorphonuclear leukocyte model (Brown et al. 1997). In the acetone extracts of the leaves and their fractions, the germacranolide parthenolide (239) was found, which is associated with the anti-inflammatory effect of T. parthenium and T. niveum (1.72% and 2.62% parthenolide (239)); The IC₅₀ was 0.79 and 1.32 mg leaf dry weight/ml blood, respectively. This activity is due to inhibition of protein kinase C.

In a comparative study of the antiviral activity of different polarity fractions of methanolic extracts of T. vulgare aerial parts and roots, and compounds of the species on Herpes simplex viruses HSV-1 and HSV-2, it was found that the petroleum ether and ethyl acetate fractions were the most active with a good selective index (Álvarez et al. 2011). The mean effective concentrations (EC₅₀) were 69.9 and 95.7 µg/ml (HSV-1) and 61.6 and 59.4 µg/ml (HSV-1), respectively. The isolated compound with the highest activity was 3,5-dicaffeylquinic acid (28) (EC₅₀ 31.1 and 46.99 µg/ml against HSV-1 and HSV-2, respectively). Among the other compounds, the flavonoid axillarin (90) showed strong activity against HSV-2 - EC50 42.7 µg/ml.

STL parthenolide (239) (germacran type) showed the highest cytotoxicity at 25 µg/ml and had no antiviral activity at a concentration 1.5 times lower than the cytotoxic concentration. Parthenolide (239) was found from 0.33% in the crude extract to 2.7% in the chloroform fraction. Antiviral activity of T. vulgare is mainly associated with 3,5-dicaffeylquinic acid (28). Ethyl acetate extract of T. vulgare and parthenolide (239) were tested for HSV-1 antiviral activity; they have an IC₅₀ of 40 µg/ml and 0.3 µg/ml, respectively (Onozato et al. 2009). Parthenolide (239) exhibits activity on viral replication.

T. vulgare ssp. siculum chloroform extract was analyzed and 5 STLs from the group of eudesmanolides were isolated, which were tested for cytotoxicity against lung carcinoma cell lines A549 and hamster lung fibroblast-like line V79379A. IC₅₀s were 15.3–59.4 µM and 5.0–33.4 µM, respectively (Rosselli et al. 2012). The main STL douglanin (258) shows the highest activity. STL parthenolide (239) is involved in the processes of oxidative stress in prostate carcinoma cells by activating NADPH oxidase - the formation of ROS is the basis of the effects on hepatocellular carcinoma. The compound has anti-angiogenesis and anti-metastatic activity (Shields 2017). Methanol extracts of T. vulgare leaves and flowering heads have a cytotoxic effect on HeLa cell line - IC₅₀ is below 100 μg/mL (Devrnja et al. 2017). The activity is associated with the content of methoxylated flavonoids nepetin (124), hispidulin (125) and eupatilin (127).

Toxicological profile of T. vulgare extracts from different polarity was assayed in the brine shrimp (Artemia salina) lethality test where all of them showed a high degree of toxicity with IC₅₀ < 2 mg/ml (Ak et al. 2021). The cytotoxic effects towards HypoE22 cells was found in the concentration range 50–100 μg/mL.

The bioinformatics analysis through the SwissTargetPrediction platform predicted interaction of flavonoids axillarin (90), quercetagetin -3,6,3’(4’)-trimethyl ester (94) and hesperetin (146) together with quinic (70), chlorogenic (23) and dicaffeoylquinic acid (28), and parthenolide (239) with target proteins (hydrolase), electrochemical transporters and transcription factors involved in neuromodulation and neuroprotection (Ak et al. 2021).

The possible neuroprortective effects of T. vulgare extracts was studied on hypothalamic HypoE22 cells (Ak et al. 2021). At 10 µg/ml (non toxic concentation) hexane extracts reduced the marker of oxidative stress MDA (malonyldialdehyde) and gene expression of TNFα (pro-inflammation cytokine). It’s worth noting that down regulation of the BDNF (brain-derived neurotrophic factor) gene expression was found. On the other hand, the hexane extracts exerted stimulating effects on the norepinephrine levels in HypoE22 cells. All together, the aforementioned effects suggest putative involvement of the T. vulgare hexane extracts in the modulation of the hypothalamic appetite-regulating network. However, the reduced BDNF gene expression could account also for potential neurotoxicity following the administration of T. vulgare herbal extracts.

T. parthenium (feverfew), has been traditionally employed as a phytotherapeutic remedy in the treatment of migraine (Guibot et al. 2017; Moscano et al. 2019). T. parthenium crude extract and STL parthenolide (239) inhibit serotonin and platelet aggregation relevant to migraine (Marles et al. 1992). Aqueous extract of the species has been shown to inhibit prostaglandin biogenesis and gives a very good result in migraine patients without side effects.

The dopaminergic pathways are key targets for novel pharmacological approaches in counteracting migraine attacks. In this context, anti-inflammatory and neuromodulatory effects of a commercial aqueous T. parthenium extract was studied in an ex vivo model of cortical spreading depression (CSD) in mice cortex. It reduced prostaglandin G2 (PGE2) release and IL-1β gene expression in ex-vivo mice cortex, while up-regulation of IL-10 and BDNF gene expressions was observed. The extract could be effective in controlling the inflammatory pathways that occur during CSD. In the concentration range 10–100 μg/mL the extract decreased in concentration dependant manner the extracellular dopamine level, while between 10 and 50 μg/mL it increased the dopamine transporter (DAT) gene expression. In silico interaction between parthenolide (239) and DAT binding site was found.