Phytochemical Investigation of Polyphenols from the Aerial Parts of Tanacetum balsamita Used in Transylvanian Ethnobotany and Parallel Artificial Membrane Permeability Assay

In this study, based on ethnobotanical data recorded in Transylvania, the polyphenolic compounds and the permeability of the aerial part’s extract of Tanacetum balsamita were investigated. Ultrahigh-performance liquid chromatography-tandem mass spectrometry was applied for the analysis of the extracts. Parallel artificial membrane permeability assay (PAMPA) for the gastrointestinal tract and the blood–brain barrier was conducted. In the ethanolic and aqueous extracts of the species traditionally used for wound, furuncle, and liver disorders, 92 polyphenols were characterized (e.g., flavonoid, hydroxycinnamic acid, catechin, dihydroxybenzoyl, lignan derivatives, and a monoterpene) including 54 compounds identified for the first time in the plant. In the PAMPA tests, eight components were shown to be capable of passive diffusion across the studied membranes. These include apigenin and seven methoxylated flavonoid derivatives. Based on these results, methoxylated flavonoids might promote the pharmacological potential of T. balsamita to be applied in the enhancement of novel remedies.


Introduction
Medicinal plants have been investigated for their traditional role since the ancient times worldwide.Traditional data inherited from generation to generation can be collected for their pivotal role for ethnobotanical and ethnomedicinal sciences, which, after comparison with those obtained from relevant sources, can provide new or underrepresented species, used parts, or indications for further analyses [1][2][3].
Our ethnobotanical field surveys started in 2007 in the Úz Valley in Transylvania, a part of Romania, and continued in many regions of the country.Summarizing the earlier published ethnomedicinal data from 2007 to 2019 [4][5][6][7][8], Tanacetum balsamita L. (costmary, Asteraceae) (Figure 1) was selected in our present work for further analyses.Tanacetum genus involving species mentioned in the European, Asian, and African ethnobotany comprises various metabolites, such as terpenes, polyphenols, and polysaccharides [9][10][11][12].The genus has a long history of use in traditional medicine, especially among Greek and early European herbalists.Based on Etymologia botanica, it comes from the Greek tanaos (persistent) and athanasia (immortality).It did not appear in Central Europe until the late 8th or early 9th century; in Capitulare de villis vel curtis imperii or Repertorium Fontium, it is known as tanazita in Medieval Latin [13], referring to the white flowers which retain their color and shape.The first-century Greek physician Dioscorides prescribed it as Pyrethrum tanacetum and P. balsamita, as well as dorycnion, pyrinon, pyrothron, and arnopurites; the Magi call it purites and the Romans salivaris.In this work, the root has been used against phlegm, for toothache boiled with vinegar as a mouthwash, for longlasting chills, and paralysis [14].In Matthioli's works, the plant is described as Tanacetum Athanana [15], applied in wine, beer, vinegar, milk or honey against worms, kidney stones, as an analgesic and diaphoretic drug, for pain and swelling of the legs, and for women against enchanted infatuation as a belief [16].
In Persian ethnomedicine, it has been used for its carminative, cardiotonic, hepatoprotective, antiallergic, sedative, flavoring, and tonic effects, against migraine and dysmenorrhea [50][51][52][53][54][55][56], for diabetes [57], abscess, wound, diabetes mellitus, rheumatism and as an antipyretic agent in Turkey [12,58], for cholecystitis, dyspepsia, inflammation, and Tanacetum genus involving species mentioned in the European, Asian, and African ethnobotany comprises various metabolites, such as terpenes, polyphenols, and polysaccharides [9][10][11][12].The genus has a long history of use in traditional medicine, especially among Greek and early European herbalists.Based on Etymologia botanica, it comes from the Greek tanaos (persistent) and athanasia (immortality).It did not appear in Central Europe until the late 8th or early 9th century; in Capitulare de villis vel curtis imperii or Repertorium Fontium, it is known as tanazita in Medieval Latin [13], referring to the white flowers which retain their color and shape.The first-century Greek physician Dioscorides prescribed it as Pyrethrum tanacetum and P. balsamita, as well as dorycnion, pyrinon, pyrothron, and arnopurites; the Magi call it purites and the Romans salivaris.In this work, the root has been used against phlegm, for toothache boiled with vinegar as a mouthwash, for long-lasting chills, and paralysis [14].In Matthioli's works, the plant is described as Tanacetum Athanana [15], applied in wine, beer, vinegar, milk or honey against worms, kidney stones, as an analgesic and diaphoretic drug, for pain and swelling of the legs, and for women against enchanted infatuation as a belief [16].
The aerial part of T. balsamita var.tanacetoides has been described in the British Pharmacopoeia (1788) for its laxative, diuretic, astringent, antiseptic, and anthelmintic effect [66].Hoppe recommends Flores Tanaceti as a vermifuge and antipyretic drug in veterinary medicine, for rheumatics, for disorders of the nervous system, and as an abortive remedy [67].
The aim of this study was to sum ethnobotanical data on T. balsamita collected in selected regions in Transylvania, Romania.For the identification of polyphenols, the traditionally used aerial part of the plant was studied by ultrahigh-performance liquid chromatography-diode-array detection-electrospray ionization mass spectrometry (UHPLC-DAD-ESI-MS), and the effective permeability of the components in the plant extracts by the parallel artificial membrane permeability assay for the gastrointestinal tract (PAMPA-GI) and the blood-brain barrier (PAMPA-BBB).

Ethnobotanical Data in Selected Areas in Transylvania
The plant is described as vénasszonvirág and vénasszonyvirág (= old women's flower) along the Nagy-Homoród, while bódogasszonlapi and boldogasszonylapi (= glad/blessed women's leaf) in Ghimeş and Úz Valley.The origin of the name vénasszonyvirág was defined: ". ..ami elég szívós, s nincs sok baj vele, s hosszan virágzik".(= . ..which is hard/wiry, it does not cause problems, beeing in flower for a long time).People mentioned its use as an ornamental plant was known for a long time in the studied regions: "Minden háznál vót ezelőtt ilyen virág".(= This plant has been found in all homegardens long ago).In addition, the aerial part was mentioned for wound and liver diseases in the Ghimeş region and for furuncle in the Úz Valley as a decoction.Along the Nagy-Homoród, old women put the fragrant leaves of the plant into the Bible or prayer books during the mass as a refreshing material.

Qualitative Analysis of Phenolic Compounds with UHPLC-DAD-ESI-MS/MS
Ultrahigh-performance liquid chromatography coupled to diode-array detection and electrospray ionization tandem mass spectrometry (UHPLC-DAD-ESI-MS/MS) in negative ionization mode was used to characterize the phenolic profile of the T. balsamita ethanolic and aqueous extracts.UHPLC-DAD chromatograms of the 50% (v/v) ethanol and aqueous extracts (recorded at 330 nm) are presented in Figures 2A and 2B, respectively.A total of 92 compounds were characterized by comparing their retention times, UV spectra, deprotonated molecular ions, and fragment ions generated in their collision-induced dissociation with the literature data; results are shown in Table 1.
Plants 2024, 13, x FOR PEER REVIEW 4 of 22 hard/wiry, it does not cause problems, beeing in flower for a long time).People mentioned its use as an ornamental plant was known for a long time in the studied regions: "Minden háznál vót ezelőtt ilyen virág".(= This plant has been found in all homegardens long ago).
In addition, the aerial part was mentioned for wound and liver diseases in the Ghimeş region and for furuncle in the Úz Valley as a decoction.Along the Nagy-Homoród, old women put the fragrant leaves of the plant into the Bible or prayer books during the mass as a refreshing material.

Qualitative Analysis of Phenolic Compounds with UHPLC-DAD-ESI-MS/MS
Ultrahigh-performance liquid chromatography coupled to diode-array detection and electrospray ionization tandem mass spectrometry (UHPLC-DAD-ESI-MS/MS) in negative ionization mode was used to characterize the phenolic profile of the T. balsamita ethanolic and aqueous extracts.UHPLC-DAD chromatograms of the 50% (v/v) ethanol and aqueous extracts (recorded at 330 nm) are presented in Figures 2A and 2B, respectively.A total of 92 compounds were characterized by comparing their retention times, UV spectra, deprotonated molecular ions, and fragment ions generated in their collision-induced dissociation with the literature data; results are shown in Table 1.The extractable matter content was higher in the hydroethanolic extract (as shown by the remarkably higher peak intensities) and its composition was also greatly different.Caffeoylquinic acids (compounds 9 and 11) and dicaffeoylquinic acids (49, 50, 68) were the predominant constituents, while flavonoid methyl ethers (85, 89, 92) also prevailed.In the aqueous extract, the more polar caffeoyl-O-hexoside (2), dihydroxybenzoyl-O-hexoside (3), apigenin-6,8-di-C-hexoside (13) as well as an apigenin-and some methoxyflavone-Odiglycosides with hexuronic acid moieties (36 and 32, 40, 41) were abundant.Methoxyflavone-O-acylglycosides (70, 75, 81) and flavonoid methyl ethers (87,88) were representative apolar constituents in the aqueous extract.

Flavonoid Derivatives
In accordance with a recent study [94], mainly flavone-O-and C-glycosides were detected in T. balsamita extracts, besides flavonol and chalcone derivatives.During the collision-induced dissociation of flavonoid-O-glycosides, fragment ions corresponding to the deprotonated apigenin, luteolin, and quercetin aglycones generated by the loss of the sugar units were observed at m/z 269, 285, and 301, respectively.Neutral losses deriving from the cleavage of a pentose (132 Da), a hexose (162 Da), a hexuronic acid 176 Da), an acetylhexose (204 Da), or an acetylhexuronose (218 Da) moiety were also characteristic [143].94,118,128,138,139].
Neutral losses of 162, 146, and 176 Da can also point to the cleavage of caffeoyl, coumaroyl, or feruloyl moieties, respectively.However, these hydroxycinnamic acids esterifying flavonoid-glycosides also exhibit characteristic fragment ions contributing to their identification.Fragment ions corresponding to the deprotonated hydroxycinnamic acids were detected at m/z 179, 163, and 193 for caffeic acid, coumaric acid, and ferulic acid, respectively.Ions deriving from the additional cleavage of a H 2 O molecule were also present at m/z 161 for caffeic acid, m/z 145 for coumaric acid, and m/z 205 for sinapic acid.The cleavage of the acylated saccharide moiety, i.e., coumaroylhexose, caffeoylhexose, caffeoylhexuronose, and sinapoylhexose resulted in neutral losses of 308, 324, 338, and 368 Da, respectively [143].Compounds 57, 76, and 74 presented a fragment ion at m/z 145 arising from the neutral loss of 146 Da; thus, these compounds were assumed to be coumaroylhexoside derivatives of luteolin and apigenin.Compounds 28 and 43 showing a neutral loss of 162 Da and a caffeoyl (caffeic acid−H 2 O) fragment ion at m/z 161 were presumed as luteolin-O-hexuronosyl-caffeoylhexuronoside and luteolin-O-hexuronosylcaffeoylhexoside, respectively [113].
Methoxyflavonoids ( (83,85) or a trihydroxy-dimethoxyflavone (89) skeleton.Compounds 82 and 87 with a single loss of 15 Da were characterized as tetrahydroxy-methoxyflavone and trihydroxy-methoxyflavone, respectively [94,140].The structures of glycoside and acylglycoside derivatives of methoxy-flavone were proposed as detailed previously.The neutral losses yielded by the cleavage of sugar residues and cinnamoyl moieties, together with the fragment ions corresponding to the deprotonated and/or dehydrated cinnamic acids were analyzed for the structural characterization of flavonoid methyl ether derivatives [113,129,137,143].
Compounds 4, 9, 11, and 14 were characterized as caffeoylquinic acid isomers, while 22 and 26 were proposed to be feruloylquinic acids.The isomers could be differentiated based on the relative intensities of their fragment ions.In case of 4 and 26, the abundance of the m/z 191 (deprotonated quinic acid) fragment ion and the m/z 179 (deprotonated caffeic acid) or the m/z 193 (deprotonated ferulic acid) secondary fragment ions referred to 3-O-caffeoylquinic acid and 3-O-feruloylquinic acid, respectively.Compounds 9, 14, and 22, presenting the base peak at m/z 191 without any secondary peaks, were identified as 5-O-caffeoylquinic acid isomers and 5-O-feruloylquinic acid.According to Jaiswal et al., the cis isomer of cinnamoylquinic acids is assumed to be the more hydrophobic and is therefore eluted at higher retention times.Thus, 9 was characterized as trans-5-O-caffeoylquinic acid, while (14) as cis-5-O-caffeoylquinic acid.The presence of the fragment ion at m/z 173 indicated a 4-substituted isomer; therefore, 11 was characterized as 4-O-caffeoylquinic acid [108,111,127,133].
Similarly, dicaffeoylquinic acid isomers with [M−H] − ions at m/z 515 could also be distinguished based on their fragment ions.In case of 3,4-O-dicaffeoylquinic acid (49), the fragment ion at m/z 173 was the base peak; however, the intensity of the secondary peaks at m/z 191 and 179 was relatively high.The domination of the fragment ion at m/z 191 indicated the 3,5-O-dicaffeoylquinic acid isomer for 50, while the base peak at m/z 173 with relatively low intensity secondary peaks referred to the 4,5-O-dicaffeoylquinic acid isomer (63).Compound 51 presenting [M−H−H 2 O] − , [quinic acid−H] − and [caffeic acid−H] − at m/z 335, 191, and 179, respectively, pointed to the 1,3-O-dicaffeoylquinic acid isomer [109,133,135,141,142].Additionally, a tri-O-caffeoylquinic acid isomer (84) was also identified in the extracts [111,133,148,149].

Other Constituents
The fragmentation patterns of compounds 3 and 5 indicated the presence of a dihydroxybenzoic acid moiety with its typical fragment ion at m/z 153.The neutral losses of 162 Da and 2 × 132 Da referred to a hexose and two pentose moieties; thus, the compounds were characterized as dihydroxybenzoyl-O-hexoside and dihydroxybenzoyl-di-Opentoside [94,109,112,113].
Compounds 7 and 10 showing the characteristic ions at m/z 305 and 225, as well as m/z 307 and 227 were supposed to be an epigallocatechin/gallocatechin isomer and a hydrated catechin/epicatechin isomer, respectively [114,115].
Compound 18 (m/z 535) was identified as hydroxypinoresinol-O-hexoside.The presence of another lignan was presumed for 73: medioresinol-O-hexoside or eucommin A. However, according to the literature, the dihydrosinapoyl-caffeoyl-O-hexoside structure presenting the same fragment ions may also be probable for 73 [109,113,144].
Compound 19 presented a pseudomolecular ion at m/z 389 and fragment ions at m/z 345, 227, and 209 indicating the loss of a CO (m/z 389 → 345) and a hexose molecule (m/z 389 → 209).Based on the literature, these characteristics suggested the structure of oleoside/secologanside [120,121].

Parallel Artificial Membrane Permeability Assay (PAMPA)
The ability of the compounds in T. balsamita extract (TbE and TbW) to cross biological membranes of the gastrointestinal (GI) tract and the blood-brain barrier (BBB) by passive diffusion was investigated using the PAMPA model [153].The coupling of this assay with UHPLC separation allowed the rapid simultaneous investigation of the membrane permeability of the compounds present in the extract (Figure 3).The PAMPA is considered to be one of the most effective and versatile screening tools for early drug discovery.Due to the artificial nature of the membrane used in the assay, only passive transport mechanisms can occur, unlike in cell-based assays.This is particularly important in the case of plant extracts due to their complexity, as the evaluation of results obtained by different co-existing Plants 2024, 13, 1652 9 of 20 mechanisms (e.g., active transport, or metabolism) can be challenging.Furthermore, no significant differences in effective permeabilities are observed in the PAMPA, whether assessed with single compounds or mixtures [153].
mechanisms can occur, unlike in cell-based assays.This is particularly important in the case of plant extracts due to their complexity, as the evaluation of results obtained by different co-existing mechanisms (e.g., active transport, or metabolism) can be challenging.Furthermore, no significant differences in effective permeabilities are observed in the PAMPA, whether assessed with single compounds or mixtures [153].
In the case of both extracts (TbE and TbW), eight compounds (85)(86)(87)(88)(89)(90)(91)(92) were detected in the acceptor phase of both PAMPA models.Of these, apigenin (86) was identified by comparison of its chromatographic, as well as UV and mass spectrometric behavior to the authentic standard.The remaining seven methoxylated flavonoid derivatives (85, 87-92) were characterized by quadrupole time-of-flight mass spectrometry (QTOF-MS).Based on the chromatographic peak areas (UHPLC-DAD), these compounds were present in higher relative concentrations in the extract prepared with 50% (v/v) ethanol (TbE); therefore, data from this extract were used for the calculation of the logPe values (n = 9), as the larger peak areas help to obtain more accurate results, especially in the case of minor constituents.All of these eight flavonoids exhibited logPe values greater than −6.0 in the PAMPA-BBB studies and greater than −5.0 in the PAMPA-GI experiments (Table 2).In the case of both extracts (TbE and TbW), eight compounds (85-92) were detected in the acceptor phase of both PAMPA models.Of these, apigenin (86) was identified by comparison of its chromatographic, as well as UV and mass spectrometric behavior to the authentic standard.The remaining seven methoxylated flavonoid derivatives (85, 87-92) were characterized by quadrupole time-of-flight mass spectrometry (QTOF-MS).
Based on the chromatographic peak areas (UHPLC-DAD), these compounds were present in higher relative concentrations in the extract prepared with 50% (v/v) ethanol (TbE); therefore, data from this extract were used for the calculation of the logP e values (n = 9), as the larger peak areas help to obtain more accurate results, especially in the case of minor constituents.All of these eight flavonoids exhibited logP e values greater than −6.0 in the PAMPA-BBB studies and greater than −5.0 in the PAMPA-GI experiments (Table 2).Accordingly, these components can be considered as having good membrane permeability [153]; thus, it can be assumed that they are absorbed in the gastrointestinal tract and Plants 2024, 13, 1652 10 of 20 cross the blood-brain barrier by passive diffusion.Although, it must be pointed out that due to the artificial nature of the membrane used in the assays, merely passive transport mechanisms can occur, and active (e.g., efflux) transport of the compounds cannot be studied.
Nevertheless, it is also worth noting that apigenin and the methoxylated flavonoids axillarin, sudachitin, casticin, and nevadensin isolated from a related species, Tanacetum parthenium (L.) Sch.Bip., have already been reported to have good permeability in the PAMPA-BBB model [140].

Discussion
Tanacetum balsamita has been known for centuries in European ethnomedicine and as an ornamental plant.Among the recorded use of the species in the study areas, treatment for wounds was also documented earlier in Transylvania [27,48,49,154], Bucovina [63], and Turkey [12,58], for liver diseases also in Transylvania [47,48] and in Persian traditional medicine [52], while similar to our record as a refreshment, it was found in tonic and flavoring preparations in Iran [52], Lithuania [90], Serbia, and Italy [59,60,64].In the ethnodermatological aspect, our record to treat furuncle was documented as a decoction in Úz Valley.
In search of the potentially bioactive constituents, we tentatively characterized 91 phenolic compounds and a monoterpene in T. balsamita extracts by UHPLC-ESI-MS.In line with the literature data, the most prevalent constituents were flavonoids and cinnamoylquinic acids, with a prominent difference between the composition of the aqueous and the 50% ethanolic extract.The former mainly comprised apigenin-C-glycosides, methoxyflavonedi-O-glycosides, and methoxyflavone-O-acylglycosides, while caffeoylquinic acids and methoxyflavone aglycones were prevailing in the latter.
Besides the phytochemical analyses, we performed PAMPA experiments to assess the capability of the constituents in costmary extracts to cross biological membranes by passive diffusion.According to our results, lipophilic flavonoids are presumably absorbed in the gastrointestinal tract and cross the blood-brain barrier by passive diffusion.Thus, these may contribute to the biological effects of T. balsamita such as the in vivo hepatoprotective activity observed in rats [156], or the sedative [54], and anti-migraine actions [157] reported in ethnobotanical studies.
According to the literature data, T. balsamita extracts characterized by polyphenols such as flavonol glycosides and hydroxycinnamic acid derivatives exerted in vitro antioxidant effects [97,99,158].Extracts of costmary and other Tanacetum species containing caffeoylquinic acids and flavonoids also showed fungistatic and antibacterial effects [99,159,160].However, the results of these in vitro experiments should be assessed carefully.Most of the constituents presumed to be responsible for the antioxidative and antibacterial effects could not permeate through the membrane in our PAMPA studies and, thus, might not be absorbed in the gastrointestinal tract.

Ethnobotanical Survey and Research Areas
The studied settlements and data were selected based on earlier surveys conducted in the period of 2007-2019 in Transylvania, Romania.Lunca de Jos (32  The Hungarian language skills of rural people facilitated the process of semi-structured interviews, complemented with the explanations of local dialects in all villages.Except for Lunca de Jos, villages are not provided by permanent pharmaceutical, medical, and veterinary services.People work mostly in agriculture and livestock farming in a close relationship with nature, involving plants' use from wild habitat or cultivation based on their own experiences and observation.In this study, only the data mentioned on T. balsamita are summarized.The visited informants aged between 62 and 85 years were asked for the vernacular name, cultivation, preparation, and use of the plant.The original quotations were written in italics between inverted commas according to the folk terminology of Csángós and Székelys.

Plant Material and Sample Preparation
Aerial parts of T. balsamita were collected in the EGSC-Melius Medicinal Plant Garden, Pécs, Hungary, in June 2022.The herb was dried at room temperature and stored in the dark until analyses.The voucher specimen of the species labelled with a unique code was deposited at the Department of Pharmacognosy, University of Pécs, Pécs, Hungary (Voucher code: TB_06).The plant name follows the terminology of The World Flora Online (WFO, 2023) [70].
For UHPLC-MS and PAMPA analyses, the aqueous and 50% (v/v) ethanolic extracts were obtained by extracting 3.0 g of herb powder in 30 mL of distilled water (TbW) or 50% (v/v) ethanol (TbE) using an ultrasonic bath (three times, 30 min each) at room temperature.The extracts were distilled to dryness under reduced pressure with a rotary evaporator (Büchi Rotavapor R-200, Flawil, Switzerland) at 45 • C. The residues were dissolved in 20 mL of 70% (v/v) HPLC grade methanol and filtered through Minisart RC 15 0.2 µm syringe filters (Sartorius AG, Goettingen, Germany).For the analysis of the TbW and TbE extracts and the samples from the PAMPA studies, an ultrahigh-performance liquid chromatography-diode-array detection-mass spectrometry (UHPLC-DAD-MS) method was developed.Briefly, an ACQUITY UPLC H-Class PLUS System (Waters Corporation, Milford, MA, USA) hyphenated with a quaternary solvent delivery pump (QSM), an auto-sampler manager (FTN), a column compartment (CM), and a photodiode array (PDA) detector (Waters Corporation, Milford, MA, USA) were employed.The chromatographic separation was performed using an Acquity UPLC BEH C18 (Waters, Dublin, Ireland) (100 mm × 2.1 mm i.d., 1.7 µm) column, with column temperature: 30 • C. The mobile phase consisted of 0.1% formic acid in water (eluent A) and acetonitrile (eluent B).All aqueous solvents were filtered through MF-Millipore (Millipore, Billerica, MA, USA) (0.45 µm, mixed cellulose esters) membrane filters.The following gradient elution was applied at a flow rate of 0.

MS Conditions
Mass spectrometric analyses were performed with a Xevo Q-TOF instrument equipped with an electrospray ionization source (ESI) (Waters Corporation).ESI conditions were as follows: capillary voltage 2.6 kV, sampling cone voltage 40 V, source temperature 120 • C, desolvation temperature 300 • C, desolvation N 2 gas flow 600 L/h.High purity nitrogen was used as collision gas, and the collision energy was changed between 10 eV and 45 eV, depending on the analyzed structure.Full-scan mass spectra were acquired over the range of m/z 100-2000 in negative ionization mode.The Masslynx 4.1 software was used for data acquisition and qualitative analysis.

Parallel Artificial Membrane Permeability Assay (PAMPA)
A parallel artificial membrane permeability assay (PAMPA) was used to determine the effective permeability (Pe) for the components of Tanacetum extracts prepared with water (TbW) and 50% (v/v) aqueous ethanol (TbE).Stock solutions of the extracts (100 mg/mL in DMSO) were diluted with the defined buffer (pH 7.4 for the PAMPA-BBB and pH 6.8 for the PAMPA-GI assays) to obtain the donor solutions (composition: 594.0 µL buffer + 6.0 µL stock solution).The buffers were prepared as follows: pH 6.8: 20.For the PAMPA-BBB test, 5 µL of porcine polar brain lipid extract (PBLE) solution (16.0 mg PBLE + 8.0 mg cholesterol dissolved in 600.0 µL n-dodecane) was applied for each well of the 96-well polycarbonate-based filter donor plates (top plate) (Multiscreen™-IP, MAIPN4510, pore size 0.45 µm; Merck).For the PAMPA-GI assay, the wells of the top plate were coated with 5 µL of the mixture of 16.0 mg phosphatidylcholine and 8.0 mg cholesterol dissolved in 600.0 µL n-dodecane.Some 150.0 µL aliquots of the filtrated donor solutions were placed on the membrane.The 96-well PTFE acceptor plates (bottom plates) (Multiscreen Acceptor Plate, MSSACCEPTOR; Merck) were filled with 300.0 µL buffer solution (0.01 M PBS buffer, pH 7.4).The donor plate was placed upon the acceptor plate, and both plates were incubated together at 37 • C for 4 h in a Heidolph Titramax 1000 Vibrating platform shaker (Heidolph, Schwabach, Germany).
After incubation, sandwich plates were separated, and the concentrations of each compound in the starting donor solution and in the acceptor and donor wells were determined in triplicate by chromatographic peak areas derived from the UHPLC-DAD method described above.UV spectra and chromatograms were recorded at 200-400 nm, and the chromatograms acquired at the UV absorption maxima of each compound were used for data evaluation.The effective permeability and the membrane retention in the PAMPA-BBB and the PAMPA GI experiments were calculated using data from the chromatograms of the TbE extract by Equations ( 1)-( 4), respectively [161]: where Pe is the effective permeability coefficient (cm/s), A is the filter area (0.24 cm 2 ), V D and V A are the volumes in the donor (0.15 cm 3 ) and acceptor phases (0.30 cm 3 ), t is the incubation time (s), τ SS is the time (s) to reach steady-state (240 s), C D (t) is the concentration (mol/cm 3 ) of the compound in the donor phase at time t, and C D (0) is the concentration (mol/cm 3 ) of the compound in the donor phase at time 0. MR is the estimated membrane retention factor (the estimated mole fraction of solute lost to the membrane), and r a is the sink asymmetry ratio (gradient-pH-induced), defined as follows: (3) All experiments were performed in three triplicates on three consecutive days (n = 9); caffeine standard was used as positive, while rutin as negative control.

Conclusions
Ethnomedicinal surveys are of pivotal importance to document and maintain traditional treatments by plants in Transylvania, Romania, and to select species for further analyses.The selected Tanacetum balsamita is applied in recent ethnomedicine in the studied regions in Transylvania nowadays.In our work, polyphenolic compounds of T. balsamita were detected by UHPLC-MS/MS, including 54 constituents identified for the first time in the plant.The PAMPA study of the plant extracts revealed eight compounds with good permeability across the membranes of the gastrointestinal tract and the blood-brain barrier.Based on our recent results and previous data, it can be assumed that methoxylated flavonoids contribute to the pharmacological activity of Tanacetum species.Therefore, further studies investigating the structure, biological activity, and pharmacokinetic properties of T. balsamita flavonoids would be of great interest.

Table 1 .
1. LC-MS/MS data and tentative characterization of compounds from the herb of Tanacetum balsamita.
a Compound numbers and retention times (t R ) refer to UV chromatograms shown in Figure2A,B;b Abbreviations: TbE: T. balsamita 50% (v/v) ethanolic extract; TbW: T. balsamita aqueous extract; +: present in the extract; -: not present in the extract; c Compared to a reference standard; d Reported for the first time in T. balsamita.

Table 2 .
Results of the PAMPA experiments expressed as logP e values (n = 9).