A Comprehensive Study on Lathyrus tuberosus L.: Insights into Phytochemical Composition, Antimicrobial Activity, Antioxidant Capacity, Cytotoxic, and Cell Migration Effects

In this study, in vitro antioxidant, antimicrobial, cytotoxic, and cell migration effects of phenolic compounds of Lathyrus tuberosus leaves, known in the Transylvanian ethnomedicine, were investigated. Ultra-high-performance liquid chromatography-tandem mass spectrometry was employed for the analysis of the ethanolic and aqueous extracts. The antimicrobial properties were determined using a conventional microdilution technique. Total antioxidant capacity techniques were used using cell-free methods and cell-based investigations. Cytotoxic effects were conducted on 3T3 mouse fibroblasts and HaCaT human keratinocytes using a multiparametric method, assessing intracellular ATP, total nucleic acid, and protein levels. Cell migration was visualized by phase-contrast microscopy, employing conventional culture inserts to make cell-free areas. Together, 93 polyphenolic and monoterpenoid compounds were characterized, including flavonoid glycosides, lignans, hydroxycinnamic acid, and hydroxybenzoic acid derivatives, as well as iridoids and secoiridoids. The ethanolic extract showed high antioxidant capacity and strong antimicrobial activity against Bacillus subtilis (MIC80 value: 354.37 ± 4.58 µg/mL) and Streptococcus pyogenes (MIC80 value: 488.89 ± 4.75 µg/mL). The abundance of phenolic compounds and the results of biological tests indicate the potential for L. tuberosus to serve as reservoirs of bioactive compounds and to be used in the development of novel nutraceuticals.


Introduction
Throughout the documented ages, plants and plant-based products have played a crucial role in human life, serving both as essential foods and therapeutic agents as well.Since 2000, plant-based compounds have gained considerable popularity as valuable repositories of natural remedies, largely due to the high costs and numerous side effects associated with synthetic medicinal compounds [1].In particular, plant secondary metabolites, including phenolics and flavonoids, exhibit significant biological activities such as antioxidant, antimicrobial, anticancer, and anti-mutagenic properties, all with the slightest detrimental effects on human health compared to their synthetic counterparts [2].As a result, numerous researchers have redirected their attention to characterizing and investigating the biological effects of plants or their secondary metabolites [3,4].Due to intense research activity, investigating untapped wild plants within various folk medicine systems shows high potential as a valuable reservoir of biologically active compounds for the development of drugs and functional foods.Medicinal plants play an important role in ethnomedicine worldwide, as well as in our study carried out in Transylvania, part of Romania.Based on earlier published ethnomedicinal data, including our field studies from 2007 to 2019 in various regions of the country [5][6][7], Lathyrus tuberosus L. (tuberous pea, Fabaceae) was selected in our work for further analysis.L. tuberosus occurs on road edges, crop fields, and dry grasses in Europe, except in the north and extreme south [8,9].
In various regions of Transylvania, it is known under the vernacular names tsunya [10], borsóvirág, vadborsó, unalomvirág [11], borsó viloja [12], disznópityóka [13], juliszta, csunya, and csunyavirág [5,6].Lathyrus species serve dual purposes, serving as both a food source and as traditional medicines.The tuber of the plant was documented in Renaissance herbals in Switzerland [14], while ethnomedicinally, it was used in Romania in love char, for wounds and stomach aches as a bath, also for sweetness, consumed primarily by children [6,[11][12][13]15,16], for digestive problems in Bosnia and Herzegovina [17], and as a snack in Kosovo [18].Furthermore, Lathyrus species play a pivotal role in traditional medicine, exhibiting analgesic properties through the seeds of L. sativus L., with antiinflammatory attributes through aerial components of L. cicero L., and efficacy against rheumatism attributed to the leaves of L. rotundifolius Willd., particularly within the Turkish pharmacological context [19].
Earlier investigations of Lathyrus species have documented the existence of various biologically active compounds, including phenolics, flavonoids, saponins, and polyunsaturated fatty acids [20,21].
To our knowledge, there is no existing study on the biological and chemical profiles of Lathyrus tuberosus in scientific databases.Thus, the purpose of this study was to extract the aerial part of L. tuberosus to assess its antioxidant, antimicrobial, cytotoxic, and cell migration effects, correlating them with its chemical fingerprints by LC-MS.The results obtained may provide valuable information for the formulation of new (traditional) herbal medicines and functional ingredients.In ethnomedicine, various parts of plants are utilized for medicinal purposes.We used only leaves in our experiments because this can offer several advantages, such as easy accessibility and manageability.Additionally, leaves frequently contain biologically active compounds that may exhibit various biological effects.Therefore, the application of leaves can facilitate the easy control of experimental conditions and the elucidation of properties of interest.

Qualitative Analysis of Phenolic Compounds in Plant Extracts with UHPLC-DAD-ESI-MS/MS
Ultra-high-performance liquid chromatography-diode array detection coupled to electrospray ionisation tandem mass spectrometry (UHPLC-DAD-ESI-MS/MS) in negative ionisation mode was used to tentatively identify the phenolic profile of the studied L. tuberosus extracts.93 compounds were characterized by comparing their retention times, UV spectra, and mass spectrometric fragmentation with the literature data.The results are presented in Tables 1 and 2. The UHPLC-UV chromatograms (320 nm) of the extracts are summarised for the ethanolic and aqueous leaf extracts separately in Figures S1 and S2 (Supplementary Materials).

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Flavonol-O-glycosides In line with previous studies, numerous flavonol, flavone, and flavanone derivatives were detected in their glycosidic forms, similar to other species of Lathyrus [21][22][23].In the case of O-glycosides, neutral losses of 132, 162, 146, 204, and 176 Da indicated the cleavage of a pentose, a hexose, a deoxyhexose, an acetylhexose, or a glucuronic acid moiety, respectively [24].Fragment ions of flavonoids generated by the loss of sugar units were observed at m/z 285, 301, and 315, corresponding to deprotonated kaempferol, quercetin, and isorhamnetin aglycones, respectively.In addition to these Y 0  2).Taking into account these, we assumed that compounds 37, 42, and 71 detected at m/z 463, 463, and 505, are two quercetin-3-O-hexoside isomers and quercetin-3-O-acetylhexoside, respectively [21,22,26].Similarly, compound 81, which shows its [M−H] − ion at m/z 431, was tentatively characterized as kaempferol-3-O-deoxyhexoside [21,26,27].Compounds 17, 27, and 35 presented deprotonated molecular ions at m/z 609.Based on the presence of the fragment ion [M−H−Gly (7) ] − or [M−H−146] − at m/z 463, referring to the loss of a deoxyhexose moiety from position C7, components 17 and 27 were established as quercetin-3-O-hexosyl-7-O-deoxyhexoside isomers.On the other hand, the [M−H−Gly (7) ] − ion was missing in the mass spectra of compound 35, therefore, it was characterized as quercetin-3-O-deoxyhexosyl-hexoside [21,26].Compounds 29, 44, and 36 presented fragment ions at m/z 446, 430, and 460 as well as fragment ions at m/z 299, 283, and 283, respectively; therefore, these were characterized as quercetin and kaempferol-3,7-di-O-glycosides [23,25,28].Quercetin-3,7-O-oligoglycosides were also detected using the same interpretation method: quercetin-3-O-deoxyhexosyl-hexosyl-7-O-deoxyhexosyl-hexoside (56) with [M−H] − ion at m/z 917, quercetin-3-O-deoxyhexosyl-7-O-glucuronyl-hexosyl-hexoside (63) at m/z 947 and quercetin-3-O-hexosyl-7-O-hexosyl-hexosyl-deoxyhexoside 41 at m/z 933 [21].Although no ions of the diagnostic product were detectable for compound 34, based on data from the literature, it was assigned as an isomer 41 [21].Compound 26 that displayed the [M−H] − ion at m/z 741 presented the [Y 0 −H] −• product ion corresponding to the radical aglycone at m/z 300.The position of glycosylation could not be indicated by this single product ion.Therefore, 26 was characterized as quercetin-O-deoxyhexosyl-hexosyl-pentoside [23].Compound 31 at m/z 623 presented neutral losses of 146 and 162 Da, and a fragment ion at m/z 315.According to the literature, both the flavonol glycoside isorhamnetin-O-hexosyl-O-deoxyhexoside and the caffeoyl-phenylethanoid-glycoside verbascoside (or acteoside), presenting the same fragment ions, have been described in Lathyrus species [21,22].The neutral loss of 146 atomic-mass unit (amu) indicates the cleavage of a rhamnose moiety for both potential structures.However, the loss of 162 Da could indicate either the elimination of a hexose or that of a caffeoyl moiety.Therefore, the structure of compound 31 could not be established unambiguously; it was presumed either as isorhamnetin-O-hexosyl-Odeoxyhexoside or verbascoside.− ions with high intensity, while the corresponding [M−H] − ions were observed to be much less abundant.As detailed previously, the analysis of the neutral losses yielded by the cleavage of sugar residues from the flavonol-O-glycosides could be employed for their structural characterization.The losses of 132, 162, 146, and 176 Da pointed to the elimination of residues of pentose, hexose, deoxyhexose, and glucuronic acid, respectively.However, neutral losses of 176, 146, and 162 Da could also indicate the cleavage of ferulic acid, coumaric acid, or caffeic acid moieties, respectively.Fortunately, the hydroxycinnamic acid derivatives presented additional characteristic ions that enabled their tentative identification.Fragment ions corresponding to the deprotonated hydroxycinnamic acids were detected at m/z 179, 163, 223, and 193 for caffeic acid, coumaric acid, sinapic acid, and ferulic acid respectively, while additional typical ions at m/z 161, 145, 205, and 175 were generated by cleavage of a H 2 O molecule [24].Furthermore, a neutral loss of 264 Da was also detected in the mass spectra of compounds 72, 88, and 91, indicating the loss of a dihydrophaseoyl moiety.The appearance of esterified kaempferol-O-glycosides with dihydrophaseic acid was previously reported as representative of L. cicera L. seeds [23].We also could deduce the position of the saccharide residues based on the results of Ferreres et al.: only one deoxyhexosyl unit was attached to the C7-OH group, and all other acylated sugars were attached through the C3-OH.
On the basis of these, structures of the acylated quercetin-and kaempferol-O-glycosides could be proposed.The suggested structures of the compounds, together with the interpretation of the product ions that were considered characteristic or diagnostic, are presented in Table 2. Flavonol-O-acyl-glycosides were assigned to four major classes:  [23], the occurrence of flavonoids with molecular masses as high as 1508, 1880, or 1920 Da (compounds 67, 75, and 83) is extremely rare.However, it should also be highlighted that this characterization is rather hypothetical; the unambiguous identification of the constituents was not possible.Compounds 46, 75, and 83 were also detected as doubly charged [M−2H] 2− ions at m/z 917, 939, and 959, respectively.However, except for compound 75, other ions were not detected; therefore, we presumed these were also acylated flavonol-O-oligoglycosides.On the other hand, 75 displayed product ions at m/z 609 and 300, suggesting quercetin aglycone.
Compound 7 was identified as 5-O-caffeoyl-quinic acid based on fragment ion at m/z 191 [39].Likewise, compound 45 was proposed as a 5-O-caffeoyl-quinic acid derivative.Compounds 51, 54, and 65 were characterized as dicaffeoylquinic acid isomers, which could be distinguished based on the relative intensities of their fragment ions [39].For 3,4-Odicaffeoylquinic acid (51), the fragment ion at m/z 173 is the base peak, while the intensity of the secondary fragment ions at m/z 191 and 179 is relatively high.The domination of the fragment ion at m/z 191 indicated 3,5-O-dicaffeoylquinic acid as compound 54, while the base peak at m/z 173 with relatively low intensity secondary peaks referred to 4,5-O-dicaffeoylquinic acid (65) [39].The presence of the latter two isomers was previously reported for L. sativus [22].
In the case of compound 3 at m/z 365, the loss of 162 Da indicated a caffeoyl moiety, while the fragment ions at m/z 203 and 159 referred to a tryptophan moiety, according to Llorent-Martínez et al.Therefore, this compound was tentatively identified as N-caffeoyltryptophan [40].Compound 23 with the [M−H] − ion at m/z 381, suffered the loss of a pentose (m/z 381 249), while the further loss of 88 Da (m/z 249 161) and the fragment ions at m/z 179 and 161 referred to a caffeoylputrescine derivative [41], therefore, 23 was characterized as N-caffeoylputrescine-O-pentoside.According to Gampe et al., non-protein amino acids (e.g., homoproline and homopipecolic acid) are widely distributed in species belonging to the Fabaceae family [42].The appearance of the neurotoxin β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP) in the seeds of L. sativus is also universally known [43].However, hydroxycinnamoyl esters of amino acids, such as N-trans-caffeoyltyramine isolated from Celtis occidentalis L. [44], have not been reported in the genus Lathyrus.
Compound 60 presented a pseudomolecular ion at m/z 503 and fragment ions at m/z 371 [M−H−132] − and 209 [M−H−132−162] − .The neutral losses corresponded to a pentose and a caffeoyl moiety, while the fragment ions at m/z 209 and 161 are characteristic of a tetrahydroxyhexanedioic acid (presumably galactaric acid) and a caffeoyl moiety, respectively.Therefore, compound 60 was identified as caffeoyl-tetrahydroxyhexanedioic acid-O-pentoside [45].Fragment ions of compounds 8, 15, 28, and 33, at m/z 177 and 159 (the latter produced by the cleavage of a H 2 O molecule) indicated the presence of a dimethyl-dihydroxybutanedioic acid (probably dimethyltartaric acid).In the case of Plants 2024, 13, 232 6 of 29 compounds 8, 28, and 33, the neutral loss of 146 Da and the fragment ion at m/z 163 pointed to a coumaroyl moiety, while for compound 15, the loss of 180 Da and the fragment ion at m/z 179 indicated a caffeoyl moiety.Therefore, 28 and 33 were identified as coumaroyl-dimethyl-dihydroxybutanedioic acid isomers, and compound 15 as a caffeoyl derivative [22].Compound 8, showing an additional neutral loss of 162 Da, was characterized as coumaroyl-dimethyl-dihydroxybutanedioic acid-O-hexoside.In the case of compound 6, the neutral loss of 162 Da (a hexose moiety) yielded the [hydroxybenzoic acid−H] − ion at m/z 137; therefore, 6 at m/z 299 was identified as p-hydroxybenzoic acid-O-hexoside [46].Based on the literature, compound 10 was tentatively identified as hydroxybenzyl-O-malic acid (eucomic acid), already reported in legumes [47].

Monoterpene Derivatives
Compound 48 presented a pseudomolecular ion at m/z 523 and fragment ions at m/z 361 (indicating the loss of a caffeoyl moiety) and 161 (characteristic of caffeoyl moiety).Based on the literature, these characteristics suggested the structure of verminoside consisting of the iridoid glucoside catalpol linked to a caffeoyl moiety [52].Constituent 40 that exhibited the [M−H] − ion at m/z 371 was identified as deacetylasperuloside [53].

Determination of Minimum Inhibitory Concentration (MIC 80 )
The impact of the leaf (ethanolic and aqueous) extracts of L. tuberosus on both Grampositive and Gram-negative bacteria was assessed by CLSI M07-A9 (Vol.32, No. 2) guidelines (Table 3).Interestingly, only the ethanolic extract showed an antimicrobial effect, specifically against Bacillus subtilis (MIC 80 value: 354.37 ± 4.58 µg/mL) and Streptococcus pyogenes (MIC 80 value: 488.89 ± 4.75 µg/mL).Neither plant extract (ethanolic and aqueous) had an antibacterial impact on Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus.Erythromycin, as a positive control, has an inhibitory effect on all organisms, as described in Table 3.    Mean ± SD of 5 independent experiments, each in 3 replicates (n = 5 × 3) compared with erythromycin as positive control ( a ); N.D.: not detectable.

Total Antioxidant Capacity (TAC) Assays
To obtain the most reliable results, we determined the antioxidant characteristics of the ethanolic and aqueous extracts of L. tuberosus using approved nonenzymatic chemical methods, including TEAC, DPPH, ECL, and ORAC tests (Figure 1).

Total Antioxidant Capacity (TAC) Assays
To obtain the most reliable results, we determined the antioxidant characteristics of the ethanolic and aqueous extracts of L. tuberosus using approved nonenzymatic chemical methods, including TEAC, DPPH, ECL, and ORAC tests (Figure 1).It is important to note that since the tests are based on different hydrogen atom transfer (HAT) or single electron transfer (SET) mechanisms, their evaluation was also different.Since the DPPH and TEAC tests are based on endpoint measurement and colour change, the higher antioxidant effect will be indicated by a lower IC 50 concentration in these inhibitory activity tests.On the contrary, the ECL and ORAC methods are based on kinetic measurements, and therefore, the most robust antioxidant capacity is evidenced by the highest TE/g (µmol/g) concentration values calculated from the area under the curve.
In general, ethanolic extracts demonstrated stronger antioxidant effectiveness compared to aqueous extracts.

Inhibition of Intracellular ROS Production
The determination of the TAC value by cell-free chemical assays does not fully reflect the behavior of complex plant samples in vivo.Therefore, it is important to evaluate the efficacy of antioxidants under more biologically relevant conditions, for example, by testing compounds in cell-based antioxidant assays [54].
The peroxyl radicals generated by AAPH induced the oxidation of DCFH and DHR in keratinocyte cell cultures [55].Leaf extracts led to a reduction in DCF and derivative rhodamine fluorescence, and the corresponding 50% inhibition values are presented in Table 4.In general, it can be stated that, as in chemical analyses, the ethanolic extract also exhibited stronger intracellular antioxidant properties in the case of DCFH and DHR tests.Both fluorogenic reporter molecules are sensitive to peroxyl radicals, possessing similar oxidation mechanisms.Therefore, it is not surprising that both DHR and DCFH exhibited similar activity in the presence of plant extracts [55].

Cytotoxicity Test
The cytotoxicity data acquired from 3T3 and HaCaT cells is depicted in Figure 2. A general observation indicates that ethanolic extracts of the plant exhibited greater efficacy in dose-dependent reduction of ATP levels, cell numbers, and total protein contents in both cell lines compared to aqueous extracts.The discrepancy between the solvents could not be attributed solely to the ethanol content, as it was capped at 1.5%, which did not significantly affect cell viability.Consequently, the observed toxic effects were attributed to higher concentrations of active ingredients present in ethanolic extracts.
In the 3T3 cell line, there was a more significant decrease in ATP levels, cell count, and protein concentrations compared to the HaCaT cultures.

In Vitro Migration Test
After evaluating the results of compound analyses and antimicrobial, antioxidant, and cytotoxicity data, we opted to employ the ethanolic extracts exclusively in subsequent experiments.This decision was driven by their more abundant ingredient composition and more potent biological effects.Cell migration investigations were conducted using potentially subtoxic concentrations of the ethanolic extracts (at 50, 100, and 200 g/mL), i.e., where fibroblast and keratinocyte cells showed more than 90% viability by our multiparametric plate-reader assay.A general observation indicates that ethanolic extracts of the plant exhibited greater efficacy in dose-dependent reduction of ATP levels, cell numbers, and total protein contents in both cell lines compared to aqueous extracts.The discrepancy between the solvents could not be attributed solely to the ethanol content, as it was capped at 1.5%, which did not significantly affect cell viability.Consequently, the observed toxic effects were attributed to higher concentrations of active ingredients present in ethanolic extracts.
In the 3T3 cell line, there was a more significant decrease in ATP levels, cell count, and protein concentrations compared to the HaCaT cultures.

In Vitro Migration Test
After evaluating the results of compound analyses and antimicrobial, antioxidant, and cytotoxicity data, we opted to employ the ethanolic extracts exclusively in subsequent experiments.This decision was driven by their more abundant ingredient composition and more potent biological effects.Cell migration investigations were conducted using potentially subtoxic concentrations of the ethanolic extracts (at 50, 100, and 200 g/mL), i.e., where fibroblast and keratinocyte cells showed more than 90% viability by our multiparametric plate-reader assay.
In migration assays, it becomes crucial to discern between cell proliferation and migration since cells are typically not synchronized.Some researchers have addressed this by employing mitomycin C, a DNA synthesis inhibitor, for pretreatment.This approach allows monitoring migration during an extended incubation period (48 h), exceeding the cells' generation cycle [56].However, in our experiments, we chose not to utilize this compound due to our limited 24 h incubation time, during which the potential for proliferation was minimal.The HaCaT human keratinocyte cell line exhibits a doubling time of approximately 24 h.In the first passages (2)(3)(4)(5)(6)(7)(8), this time extends to 36.2 ± 1.5 h, while in the later passages (10)(11)(12)(13)(14)(15)(16), it is reduced to 24 ± 0.6 h [57].On the other hand, 3T3 mouse fibroblast cells have a doubling time ranging from 20 to 26 h [58].
The application of PDGF-BB as a positive control at a concentration of 15 ng/mL exhibited a noteworthy stimulatory impact on the migration of both cell lines.Specifically, it resulted in a migration increase of 117.05 ± 6.72% for 3T3 cells and 115.16 ± 8.26% for HaCaT cells compared to untreated cells.In contrast, L. tuberosus demonstrated a mild In migration assays, it becomes crucial to discern between cell proliferation and migration since cells are typically not synchronized.Some researchers have addressed this by employing mitomycin C, a DNA synthesis inhibitor, for pretreatment.This approach allows monitoring migration during an extended incubation period (48 h), exceeding the cells' generation cycle [56].However, in our experiments, we chose not to utilize this compound due to our limited 24 h incubation time, during which the potential for proliferation was minimal.The HaCaT human keratinocyte cell line exhibits a doubling time of approximately 24 h.In the first passages (2)(3)(4)(5)(6)(7)(8), this time extends to 36.2 ± 1.5 h, while in the later passages (10)(11)(12)(13)(14)(15)(16), it is reduced to 24 ± 0.6 h [57].On the other hand, 3T3 mouse fibroblast cells have a doubling time ranging from 20 to 26 h [58].
The application of PDGF-BB as a positive control at a concentration of 15 ng/mL exhibited a noteworthy stimulatory impact on the migration of both cell lines.Specifically, it resulted in a migration increase of 117.05 ± 6.72% for 3T3 cells and 115.16 ± 8.26% for HaCaT cells compared to untreated cells.In contrast, L. tuberosus demonstrated a mild stimulating effect, manifesting itself as an elevation of 102.86 ± 9.84% in 3T3 cells and 108.76 ± 10.92% in HaCaT cells at a concentration of 50 µg/mL, as illustrated in Figure 3.
Plants 2024, 13, x FOR PEER REVIEW 19 stimulating effect, manifesting itself as an elevation of 102.86 ± 9.84% in 3T3 cells 108.76 ± 10.92% in HaCaT cells at a concentration of 50 μg/mL, as illustrated in Figure  derivatives measured in the plant extract increase the migration of keratinocytes and fibroblasts [62].
It was a general observation that the ethanolic extract has stronger biological effectiveness (antioxidant, antimicrobial, cytotoxic properties) compared to aqueous extract, which can be attributed to the solvent used in the extraction process.Ethanol is a more efficient solvent for extracting a wider range of antioxidant and antimicrobial compounds, including phenolic compounds, flavonoids, and other polyphenols.Certain properties of bioactive compounds may be more prevalent or exist in more significant concentrations in the ethanolic extract compared to the aqueous extract.Some compounds may be more polar or lipophilic; in this way, ethanol, being a polar solvent with some lipophilic properties, can effectively dissolve and extract a broader spectrum of these compounds compared to water.In contrast, water may not be as effective in extracting certain types of antioxidants due to its polar nature, which limits its ability to dissolve non-polar compounds.On the other hand, caffeic acid-O-hexoside, some quercetin-O-oligoglycosides, isorhamnetin-Ohexosyl-deoxyhexoside, apigenin-7-O-sinapoyl-pentoside and monoterpene derivatives were present only in the ethanolic extract, which could cause a higher biological effect.
Our findings propose that deep dives into Lathyrus species could pave the way for novel health-promoting agents in the pharmaceutical industries.

Studied Plant Taxa and Plant Extraction
Aerial parts of L. tuberosus were collected in Transylvania, Romania, in July 2020.Samples were dried at room temperature and stored in the dark.The voucher specimen of the plant labelled with a unique code was deposited at the Department of Pharmacognosy, University of Pécs, Pécs, Hungary (Voucher code: HV_10).The plant name follows the terminology of The World Flora Online (WFO, 2023).
For UHLC measurements, the aqueous and 50% (v/v) ethanolic extracts of the leaf were obtained by extracting 3.0 g of leaf powder in 30 mL of distilled water or 50% (v/v) ethanol using an ultrasonic bath (Bandelin Sonorex Digitec DT 1028, Berlin, Germany) three times, 30 min each (nominal ultrasonic power: 300 W, ultrasound frequency: 35 kHz).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).

MS Conditions
Mass spectrometric analyses were performed with a Xevo Q-TOF instrument equipped with an electrospray ionisation 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, collision energy was changed between 10 eV and 45 eV, depending on the structure analyzed.High purity nitrogen was used as the collision gas.Full-scan mass spectra were acquired in the range of m/z 100-2000 in negative ionisation mode.Masslynx 4.1 software was used for data acquisition and qualitative analysis.
The microdilution method was performed in accordance with a previously published protocol with slight modifications [63].In summary, 100 µL of bacterial suspensions (10 5 CFU/mL) in RPMI 1640 media and 100 µL of diluted aqueous or 50% (v/v) ethanolic leaf extracts in media were dispersed in each well of sterile 96-well plates.The negative control consisted of sterile medium, the bacterial growth control involved RPMI 1640 without treatment, and erythromycin served as a positive control.The ethanolic solvent concentration for dilution was capped at 1.0% (v/v) in the wells.Absorbance readings at 595 nm were recorded using a Multiskan EX 355 (Thermo Electron Corporation, Waltham, MA, USA) spectrophotometer after 24 h of incubation at 30 • C. Absorbance values below 20% of bacterial growth controls were designated as MIC 80 .

Oxygen Radical Absorbance Capacity (ORAC) Assay
The ORAC test was implemented following the methodology of Kőszegi et al. without any alterations [64].Serial dilutions were employed as the standard.In summary, 150 µL of working fluorescein solution (400 nM dissolved in 75 mM potassium phosphate buffer, pH 7.5) and 25 µL of blank/standard/plant extract (aqueous/ethanolic) were distributed in each well of standard 96-well plates.The plates underwent a preincubation period of 30 min at 37 • C in the dark.Subsequently, 25 µL of AAPH solution (400 mM dissolved in 75 mM potassium phosphate buffer) was automatically injected and fluorescence intensities were measured in kinetic mode for 80 min at 37 • C, with excitation and emission wavelengths set at 490 and 520 nm, respectively.A plate reader (BioTek Synergy HT, Winooski, VT, USA) was used for measurements.

Enhanced Chemiluminescence (ECL) Assay
This method was carried out according to our previously published study, without any alterations [64].In summary, a premixture of 70 µL of ECL detection reagent (0.15 M boric acid/NaOH, pH 9.6, supplemented with 0.45 mM luminol and 1.8 mM 4-iodophenol) and 200 µL POD enzyme solution (15 µU/mL) was maintained on ice.Trolox dilutions served as the standard.In each well of white optical 96-well plates, 20 µL of Trolox/blank/sample and 270 µL of POD-ECL reagent were introduced.The reaction started with the automated injection of 20 µL ice-cold H 2 O 2 (1.5 mM, 0.1% citric acid).The chemiluminescence signal was observed for 10 min using a plate reader (Biotek Synergy HT) in kinetic analysis mode.The measurement procedure was carried out according to a previously described protocol [65,66], with some modifications.In summary, 50 µL of blank/standard/plant sample dilutions were followed by the addition of 100 µL of DPPH (200 µM, dissolved in 96% ethanol) and 50 µL of acetate buffer (100 mM, pH 5.5) in general microplates.Absorbance changes were monitored at 517 nm using a Perkin Elmer EnSpire Multimode plate reader (Perkin Elmer, Waltham, MA, USA) after 60 min of incubation in the dark at room temperature.The obtained results were compared to serial dilutions of a Trolox standard solution.

Trolox Equivalent Antioxidant Capacity Assay (TEAC)
The TEAC test was conducted with a slight modification of the approach outlined by Re et al. and Stratil et al. [67,68].ABTS •+ was generated by reacting the ABTS stock solution (7 mM of ABTS dissolved in distilled water) with 2.45 mM K 2 S 2 O 8 (final concentration) and diluting the mixture with PBS (pH 7.4) until the absorbance reached 0.70 ± 0.005 at 734 nm.Subsequently, 20 µL aliquots of various concentrations of leaf extracts (ethanolic/aqueous) were allowed to react with 80 µL of ABTS •+ (7 mM), and absorbance readings were recorded at 734 nm using the Perkin Elmer plate reader after a 20-minute incubation in the dark at room temperature.Trolox served as the standard.

Calculation of Total Antioxidant Capacities (TAC)
For the ORAC and ECL assays, the outcomes were determined in terms of Trolox equivalents (TE).In the ORAC method, the net area under the fluorescence curve (netAUC) was calculated by subtracting the AUC of the blank from that of the standard/sample.Then a calibration line was established for the netAUC of Trolox standards.In the ECL technique, the AUC of the emission curves against Trolox standards was utilized to derive the calibration line.In both scenarios, the TE values of the samples were derived from the calibration curves, which were subsequently adjusted by the dilution factor and expressed as µM TE concentration.Ultimately, the total antioxidant capacity (TAC) was normalized to 1 g of initial dry material for each plant sample.
In both the DPPH and TEAC assays, radical scavenging activity was denoted as IC 50 , representing the concentration of plant extract in µg/mL needed to scavenge 50% of the reactions of DPPH or ABTS.This was calculated using a linear regression curve established from the scavenging activities versus the amount of extracts in the samples.Consequently, a lower IC 50 value indicates a higher antioxidant activity in the sample.
Calculating the radical scavenging activity for the leaf extracts, presented as a percentage relative to the blank, was carried out using the following formula: where A 0 is the absorbance of the blank and A 1 is the absorbance of the sample.

Quantification of Intracellular ROS
To evaluate cellular oxidative stress induced by excessive generation of reactive oxygen species (ROS) by AAPH, we employed the DCFH-DA and DHR123 methods [69][70][71].The optimal conditions for conducting the DCFH-DA and DHR123 assays on 96-well culture plates involved a seeding density of 5 × 10 4 cells/mL and an overnight incubation of the cell culture.Following a PBS wash, co-incubation was carried out in Hanks solution (5.5 mM glucose) with 50 µM DCFH-DA or 10 µM DHR123, in the presence of plant extract/quercetin/Trolox on 3T3/HaCaT cells, for 1 h.Upon removal of the treating medium and a subsequent washing with PBS, 1 mM AAPH oxidant was introduced in Hanks glucose.The fluorescence intensity was monitored for 60 min using the Biotek microplate reader at 490/520 nm excitation/emission wavelengths, maintaining a temperature of 37 • C. The determination of radical scavenging activity involved quantifying the IC 50 , which represents the concentration (in µg/mL) of the plant sample required to scavenge 50% of the DCFH or DHR123 fluorescence.This was achieved by linear regression analysis of serial dilutions of leaf extracts.The expression of radical scavenging activity was computed using the provided equation.

Radical scavenging activity(
where AUC 0 refers to the area under the curve values for the blank, while AUC 1 corresponds to the area under the curve values for the sample.Each treatment was subjected to four technical replicates during five independent experiments.

Cytotoxicity Test
We conducted a multiparametric viability assay with one-step extraction, following the methodology outlined in our previously published study, without any modifications [72].The aim was to assess the potential toxicity of 50% (v/v) ethanolic and aqueous leaf extracts. of L. tuberosus.The extracts were tested at concentrations ranging from 100 to 5000 µg/mL (ethanolic) and 1000 to 9000 µg/mL (aqueous).The concentration of ethanolic solvent was capped at 1.5% (v/v) in the wells, a level that does not affect cell viability.In summary, 3T3 and HaCaT cells were exposed to various concentrations of plant extracts for 24 h.Following treatment, ATP levels were measured from cell lysates using the bioluminescence method.The nucleic acid content was analyzed through PI staining, while total levels of intracellular protein were quantified after fluorescent derivatization with fluorescamine.The dose-response curves were calculated from the measured data using OriginLab Pro software (version 2016, OriginLab Corporation, Northampton, MA, USA) after the DoseResp fitting.

In Vitro 'Wound Healing' Assay
To test cellular migration, we used our previous method without any modifications [3].The ethanolic plant extract was tested at subtoxic concentrations of 50, 100, and 200 µg/mL.PDGF-BB served as a positive control at a concentration of 15 ng/mL.Cell migration within the cell-free gap was visualized every 4 h for 24 h by time-lapse imaging in bright field, utilizing phase-contrast microscopy (JuLi Stage Real-Time Cell History Recorder, NanoEn-Tek, Seoul, Republic of Korea).The gap was monitored at an objective magnification of 10×.The closed cell-free area closure rate was determined by quantifying the microphoto density data obtained for each occasion from the same locations using ImageJ 1.x processing software (https://imagej.nih.gov/ij/accessed on 11 December 2023).The closure rate, expressed as a percentage, was calculated using the provided formula.

Closure rate(%) =
Open area 0.h − Open area x.h Open area 0.h × 100 The "open area 0.h" represents the initial cell-free area at the start of the experiment, while the "open area x.h" represents the remaining cell-free space at various time points during sample imaging.Subsequently, closure rate curves were generated, and the area under the curve (AUC) was calculated for each treatment and cell line.The AUC data was then averaged (±SD), and the summarised closure rates for the leaf extracts/PDGF were presented as a percentage (%) relative to the untreated controls.

Statistical Analyses
If applicable, data were presented as a percentage relative to control samples, assumed to be around 100%.In the cytotoxicity test, correlation coefficients for each parameter tested were provided for the dose-response curves.Statistical analysis for the migration assay involved the use of a one-way analysis of variance (ANOVA) test, comparing control and sample data for a specific treatment through SPSS software (IBM, SPSS Statistics, version 22, Armonk, NY, USA).In all cases, the significance level was set at p < 0.05.

Conclusions
Medicinal plants play an important role in ethnomedicine as well as in our research carried out in Transylvania, Romania.In this work, the detailed polyphenolic contents of Lathyrus tuberosus and their related antioxidant, antimicrobial, cytotoxic, and cell migration activities were demonstrated for the first time.Considerable biological abilities of ethanolic and aqueous extract of L. tuberosus are mainly attributed to the high level of polyphenolic substances, such as O-and C-glycosides of flavonoids as well as O-glycosylated lignans, hydroxybenzoic acid derivatives, and monoterpenes.A comprehensive characterisation of 93 compounds was accomplished through UHPLC-MS/MS.Our results provide evidence that the tested plant is capable of directly quenching free radicals to terminate the radical chain reaction, acting as a reducing agent.The ethanolic extract exhibited noteworthy antimicrobial activity by inhibiting the growth of Bacillus subtilis and Streptococcus pyogenes, as demonstrated in the microdulution method.The outcomes highlighted in this paper have inspired our next study for isolation and structure clarification of the active constituents of L. tuberosus.

Figure 1 .
Figure 1.The total antioxidant capacity (TAC) of L. tuberosus was assessed using various spectroscopic methods: TEAC assay (a); DPPH assay (b); ECL assay (c); and ORAC assay (d).IC 50 values (representing the concentration at 50% inhibition) were counted for DPPH and TEAC methods, while TE/g values (Trolox equivalent in µmol per 1 g of initial dry material) were determined for ORAC and ECL tests.The comparison between aqueous and ethanolic extracts was evaluated using a t-probe, and the results are presented as mean ± SD from 5 independent experiments, each performed in 3 replicates (** p < 0.01, *** p < 0.001).

Figure 2 .
Figure 2. Intracellular ATP level, cell number (PI fluorescence), and total protein content of 3T3 and HaCaT cell culture were analyzed with dose-response fitting, illustrating the cytotoxic effects of L. tuberosus on 3T3 (a) and HaCaT cells (b).The data are presented as a percentage of the control, and the results represent the mean ± SD of 5 independent experiments, with n = 5 × 8 replicates for each concentration.Dose-response curves were generated through log10 transformation and nonlinear curve fitting.Correlation coefficients (R 2 ) were calculated for each treatment.

Figure 2 .
Figure 2. Intracellular ATP level, cell number (PI fluorescence), and total protein content of 3T3 and HaCaT cell culture were analyzed with dose-response fitting, illustrating the cytotoxic effects of L. tuberosus on 3T3 (a) and HaCaT cells (b).The data are presented as a percentage of the control, and the results represent the mean ± SD of 5 independent experiments, with n = 5 × 8 replicates for each concentration.Dose-response curves were generated through log10 transformation and nonlinear curve fitting.Correlation coefficients (R 2 ) were calculated for each treatment.

Figure 3 .
Figure 3.A comparative assessment through time-lapse imaging was conducted to analyze wound closure capabilities of PDGF-BB and L. tuberosus extracts on 3T3 monolayer cultures (a) on HaCaT monolayer cultures (b).Cell behavior was observed over a 24-hour period in the abs and presence of plant extract or PDGF, utilizing phase-contrast microscopy.The bar graphs re senting the area under the curve (AUC) were calculated based on cumulative closure rates (C assessed at 4-hour intervals across three different concentrations.Results are presented as me SD from three independent experiments, with n = 3 × 3 replicates (** p < 0.01 compared to the trol).

Figure 3 .
Figure 3.A comparative assessment through time-lapse imaging was conducted to analyze the wound closure capabilities of PDGF-BB and L. tuberosus extracts on 3T3 monolayer cultures (a) and

Table 1 .
LC-MS/MS data and tentative characterization of compounds from the herb of Lathyrus tuberosus.
a Compound numbers and retention times (t R ) refer to UV chromatograms shown in FiguresS1 and S2; b Abbreviations: LtE: Lathyrus tuberosus 50% (v/v) ethanolic extract; LtW: Lathyrus tuberosus aqueous extract; +: present in the extract; -: not present in the extract.

Table 2 .
LC-MS/MS data of flavonol-O-glycosides and acylated flavonol-O-glycosides from the herb of Lathyrus tuberosus.

Table 4 .
The cell-based antioxidant capacity of L. tuberosus extracts was assessed using DCFH-DA and DHR123 on 3T3 and HaCaT cells.The calculation of the 50% inhibitory concentrations was conducted utilizing the equations derived from the inhibitory capacities observed in the serial dilutions of the extracts.