A New Glucosyl Flavone with Inhibitory Activity of Cancer Cell Viability and Other Bioactive Constituents from the Traditional Kurdish Plant Plantago loeflingii L.

A new glucosyl flavone, 5,7,2′,5′-tetrahydroxyflavone 7-O-β-d-glucopyranoside, named loeflingiin, together with apigenin 6-C-glucoside (isovitexin), coumarins citropten and isompinellin, triterpenoids betulin and betulinic acid, and a mixture of phytosterols β-sitosterol, stigmasterol and campesterol were isolated for the first time from the leaves of wild Plantago loeflingii L. (Plantaginaceae) collected in the Iraqi Kurdistan region. The plant is used by local people to treat wounds and as a vulnerary remedy. The structures of isolated compounds were determined by spectroscopic analysis. The activities of isovitexin and loeflingiinon the viability of breast (MCF7), ovarian (BG-1), endometrial (Ishikawa), and mesothelioma (IST-MES1) human cancer cells and two normal cell lines were determined with an MTT assay. Notably, the new 7-O-glucosyl flavone showed effects higher than cisplatin against the Ishikawa and IST-MESI cell lines. The significant biological activities exhibited by all the compounds isolated from P. loeflingii provided scientific evidence to support the use of the plant in the Kurdish traditional medicine.


Introduction
The discovery of the Shanidar IV Neanderthal grave in northern Iraq, and the use of flowers in the burials reveals that herbal medicine was already practiced in the mountains and plains of Kurdistan region of Iraq at least 60,000 years ago [1].Since that time, traditional medicine and plant remedies have been continuously used by nomadic tribes in Kurdish villages and rural areas, especially in the Erbil province [2,3]; moreover, in the main bazars of major towns, such as Erbil and Sulaymaniya, herbalist shops have been trading and selling natural medicinal products to date (Figure 1) [4].As a matter of fact, it has been found that most of the approximately 1500 plants used in Iraq are valued for their medicinal and aromatic properties.Most medicinal plants are collected from wild habitats, but some are also cultivated [5,6].However, despite the great use in Kurdistan of herbal remedies, only a few phytochemical investigations have been carried out so far on the isolation and structure determination of the main secondary metabolites expressed in plants grown in Kurdistan.Even rarer are studies aimed at the determination of the bioactivities of isolated compounds.Indeed, the development in the last century by pharmaceutical companies of drug discovery strategies based on synthetic organic chemistry led to a progressive abandonment of the research of new drugs from natural sources.However, with the failure of most of these approaches, the last decades have seen the resurrection of alternative, plant-based drugs [7], as well as the discovery of new potential applications of natural resources [8].As a result, several plant-originated biomolecules have become economically important commercial products [9].Moreover, many modern drug-discovery programs include a continuous search for alternative natural products, as well as the re-discovery of underutilized plants and the investigation of plants endemic to little-studied geographical regions, such as those growing in biodiversity-rich developing countries.For these reasons, a few years ago, we started a project directed at the scientific validation of Kurdistan traditional plants and the search of new bioactive metabolites produced by them [2,3,10].This work is a continuation of this program.
We drew our attention toward the genus Plantago L. (family Plantaginaceae).This genus encompasses about 270 species of herbs, or rarely subshrubs, that have a terrestrial to an aquatic cosmopolitan distribution, occurring predominantly in temperate and highaltitude tropical regions of the world [11].The genus is characterized by a wide variety of phytochemical components.Iridoid glycosides, flavonoids, phenolic acids, phenylethanoids, terpenoids, vitamins, alkaloids, and saponins are mainly present in the aerial parts, while unsaturated fatty acids and polysaccharides predominate in the seeds.Plantago plants have been used since ancient times as diuretic, anti-inflammatory, and asthmatic drugs in Asia and Europe.The ethnobotanical uses, chemical constituents, and applications of Plantago taxa have been described in a few reviews [12,13], with P. lanceolata, P. major, P. ovata, and P. media having received the greatest attention from scientists [14][15][16][17][18][19].
Sixteen Plantago species are reported to grow in Iraq [20,21], and nine are native to Kurdistan, where they are distributed in different habitats at altitudes between 820 and 1100 m.The list includes Plantago loeflingii L., P. major L., P. lanceolata L., P. ovata Forssk., P. lagopus L., P. cretica L., P. bellardii All., P. afra L., and P. atrata Hoppe.Antioxidant, antiinflammatory, cytotoxic, and wound-healing activities are the most prominent biological properties attributed to parts or tissues of some of these plants [19]; although, in vivo studies are lacking, and most of the compounds responsible for the activities are still unknown.
P. loeflingii is an annual plant and grows primarily in the temperate biome, distributed from the Iberian Peninsula to Algeria-Morocco, Lebanon-Syria, Turkey, Iraq, Iran, As a matter of fact, it has been found that most of the approximately 1500 plants used in Iraq are valued for their medicinal and aromatic properties.Most medicinal plants are collected from wild habitats, but some are also cultivated [5,6].However, despite the great use in Kurdistan of herbal remedies, only a few phytochemical investigations have been carried out so far on the isolation and structure determination of the main secondary metabolites expressed in plants grown in Kurdistan.Even rarer are studies aimed at the determination of the bioactivities of isolated compounds.Indeed, the development in the last century by pharmaceutical companies of drug discovery strategies based on synthetic organic chemistry led to a progressive abandonment of the research of new drugs from natural sources.However, with the failure of most of these approaches, the last decades have seen the resurrection of alternative, plant-based drugs [7], as well as the discovery of new potential applications of natural resources [8].As a result, several plant-originated biomolecules have become economically important commercial products [9].Moreover, many modern drug-discovery programs include a continuous search for alternative natural products, as well as the re-discovery of underutilized plants and the investigation of plants endemic to little-studied geographical regions, such as those growing in biodiversity-rich developing countries.For these reasons, a few years ago, we started a project directed at the scientific validation of Kurdistan traditional plants and the search of new bioactive metabolites produced by them [2,3,10].This work is a continuation of this program.
We drew our attention toward the genus Plantago L. (family Plantaginaceae).This genus encompasses about 270 species of herbs, or rarely subshrubs, that have a terrestrial to an aquatic cosmopolitan distribution, occurring predominantly in temperate and high-altitude tropical regions of the world [11].The genus is characterized by a wide variety of phytochemical components.Iridoid glycosides, flavonoids, phenolic acids, phenylethanoids, terpenoids, vitamins, alkaloids, and saponins are mainly present in the aerial parts, while unsaturated fatty acids and polysaccharides predominate in the seeds.Plantago plants have been used since ancient times as diuretic, anti-inflammatory, and asthmatic drugs in Asia and Europe.The ethnobotanical uses, chemical constituents, and applications of Plantago taxa have been described in a few reviews [12,13], with P. lanceolata, P. major, P. ovata, and P. media having received the greatest attention from scientists [14][15][16][17][18][19].
Sixteen Plantago species are reported to grow in Iraq [20,21], and nine are native to Kurdistan, where they are distributed in different habitats at altitudes between 820 and 1100 m.The list includes Plantago loeflingii L., P. major L., P. lanceolata L., P. ovata Forssk., P. lagopus L., P. cretica L., P. bellardii All., P. afra L., and P. atrata Hoppe.Antioxidant, anti-inflammatory, cytotoxic, and wound-healing activities are the most prominent biological properties attributed to parts or tissues of some of these plants [19]; although, in vivo studies are lacking, and most of the compounds responsible for the activities are still unknown.
P. loeflingii is an annual plant and grows primarily in the temperate biome, distributed from the Iberian Peninsula to Algeria-Morocco, Lebanon-Syria, Turkey, Iraq, Iran, and Pakistan.In the Iraqi Kurdistan region, the plant (Figure 2A) grows in sandy soils and forests, especially in the Zrarati area (Erbil) at altitudes between 887 and 1076 m (Figure 2B).It is commonly used by local communities to heal wounds and as a vulnerary remedy [5]; however, the phytochemical constituents and the evaluation of biological properties of P. loeflingii have not been investigated so far.In this paper, we report, for the first time, the isolation of the main non-volatile constituents from a methanol extract of the aerial parts, including a new O-glucosyl flavone, and the evaluation of the effects of two isolated flavonoids on the viability of four human cancer cell lines and two normal ones.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 1 and Pakistan.In the Iraqi Kurdistan region, the plant (Figure 2A) grows in sandy soils an forests, especially in the Zrarati area (Erbil) at altitudes between 887 and 1076 m (Figur 2B).It is commonly used by local communities to heal wounds and as a vulnerary remed [5]; however, the phytochemical constituents and the evaluation of biological propertie of P. loeflingii have not been investigated so far.In this paper, we report, for the first time the isolation of the main non-volatile constituents from a methanol extract of the aeria parts, including a new O-glucosyl flavone, and the evaluation of the effects of two isolate flavonoids on the viability of four human cancer cell lines and two normal ones.
Compound 1 exhibited UV absorption maxima at 265, 285, and 362 nm and 1 H NMR and 13 C NMR data which were characteristic of a flavone nucleus [22,23].Moreover, the IR band at 1655 cm exhibited a singlet at δ H 6.48 (1H) assigned to H-3 of a flavone and an AB system attributed to two coupled protons (see 1 H-1 H COSY spectrum in Figure S2 in the Supplementary Information) with J = 2.1 Hz which were characteristic of the protons H-6 and H-8 of a 5,7-dihydroxysubstituted flavone [22].The deshielded nature of these two protons in the 1 H NMR spectrum (δ H 6.46 and 6.77) (Figure 4), in addition to the upfield signal of C-7 (δ C 164.4) in the 13 C NMR spectrum of 1 (Figure 5), indicated [23] the attachment of the glucosyl unit to the HO-7 group of the aglycone (cfr.the NMR signals of H-6, H-8 and C-7 for luteolin (10) and luteolin 7-O-β-D-glucopyranoside (11) in Figures 4 and 5, respectively).On the other hand, the β-bond of O-1 ′′ was suggested by the coupling constant of the anomeric H-1 ′′ signal at δ H 5.06 (J 1 ′′ -2 ′′ = 8.0 Hz).Moreover, the HMBC correlation (not shown) of H-1 ′′ with C-7 confirmed the attachment of the glucosyl moiety to the HO-7 of the aglycone.Apart from the signals associated with rings A and C, all the other 1 H and 13 C NMR signals of compound 1 were assigned to the ring B of a flavonoid which, in addition to the C-2-C-1 ′ bond, was substituted by two phenolic OH groups.In fact, the 1 H-NMR and 1 H-1 H COSY spectra (Figures S1 and S2 in the Supplementary Information) showed the signals of an approximate ABX system formed by three aromatic protons resonating at δ H 6.66 (d, J = 8.4 Hz, H-3 ′ ), 7.35 (dd, J = 8.4 and 2.4 Hz, H-4 ′ ), and 7.28 (d, J = 2.4 Hz, H-6 ′ ) (Figure 4), respectively.This spin system was in principle assignable either to the three protons of a 3,4-diphenolic ring B, such as that of luteolin (10) [24] and luteolin 7-O-glucoside (11) [25] (Figure 4), or a 2,4-diphenolic ring B, such as that of norartocarpetin (12) (Figure 4) [26], or a 2,5-diphenolic ring B, such as that of 5,7,2 ′ ,5 ′tetrahydroxyflavone ( 13) [27] (Figure 4).However, comparing the chemical shifts of the ring B protons of compound 1 with those of flavonoids 10-13 (Figure 4), we firmly excluded the 2,4-substitution, as in 12, while the proton patterns of luteolins 10 [24] and 11 [25] were also significantly different from those of compound 1 (Figure 4).Marked differences were also observed between the carbon signals of compounds 1 and 11 [28] in the corresponding 13 C NMR spectra in MeOH-d 4 (Figure 5), except for the ring A carbons.This finding clearly indicated that the patterns of substituents in the ring B of glucosides 1 and 11 were different, which resulted in different electronic environments and thus chemical shifts of the carbons in ring C. Based on these data, the structure 5,7,2 ′ ,5 ′ -tetrahydroxyflavone 7-O-β-D-glucopyranoside was thus assigned to compound 1, named loeflingiin, which, to our knowledge, is a new natural product.Instead, the corresponding aglycone 13 was isolated from Bridelia (Phyllanthaceae) [27], Scutellaria (Labiatae) [29,30], and Euphorbia (Euphorbiaceae) species [31].different, which resulted in different electronic environments and thus chemical shifts of the carbons in ring C. Based on these data, the structure 5,7,2′,5′-tetrahydroxyflavone 7-Oβ-D-glucopyranoside was thus assigned to compound 1, named loeflingiin, which, to our knowledge, is a new natural product.Instead, the corresponding aglycone 13 was isolated from Bridelia (Phyllanthaceae) [27], Scutellaria (Labiatae) [29,30], and Euphorbia (Euphorbiaceae) species [31].different, which resulted in different electronic environments and thus chemical shifts of the carbons in ring C. Based on these data, the structure 5,7,2′,5′-tetrahydroxyflavone 7-Oβ-D-glucopyranoside was thus assigned to compound 1, named loeflingiin, which, to our knowledge, is a new natural product.Instead, the corresponding aglycone 13 was isolated from Bridelia (Phyllanthaceae) [27], Scutellaria (Labiatae) [29,30], and Euphorbia (Euphorbiaceae) species [31].4) is also a quite common plant metabolite, mainly occurring in species belonging to Lamiaceae, Poaceae, Apiaceae, Araliaceae, Zingiberaceae, Rutaceae, Caprifoliaceae, Asteraceae, and Thymelaeaceae families [34].Sterols 5-7 are widely distributed in nature, in many vegetables, fruits, nuts, and seeds, having a major function to maintain the structure and physiology of cell membranes.Betulin (8) and betulinic acid (9) are pentacyclic triterpenoids isolated from the bark and sap of birch trees.They also occur as secondary metabolites in hundreds of different plants ubiquitously distributed across the plant kingdom, including Plantago lanceolata [34].
In conclusion, to the best of our knowledge, this is the first isolation of isovitexin (2), citropten (3), and isopimpinellin (4) from a species belonging to the genus Plantago.

Biological Activities of Isolated Compounds
All the isolated compounds 2-9 have shown various biological activities, which are summarized below.Isovitexin (2), although less studied than vitexin (the 8-C-glucosyl isomer), exerts similar pharmacological effects, partly due to their similar chemical structure [32].Isovitexin exhibited anti-inflammatory and antioxidant activities on lipopolysaccharide-induced acute lung injury by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.It inhibits α-glucosidase, an enzyme that is responsible for the breakdown of carbohydrate to sugar and has anti-tumor and neuroprotective effects.Moreover, by inhibition of xanthine oxidase, it may protect cells from oxidative stress [35].5,7-Dimethoxycoumarin (citropten) (3) showed antioxidant activity, anti-inflammatory activity in colitis, and antiproliferative effects against the A2058 human melanoma cell line [36].The furocoumarin isopimpinellin (4) exhibited moderate cytotoxicity against Colo-205 tumor cells and inhibited the growth of the human bladder carcinoma cell line E-J in vitro.Moreover, it strongly inhibited insulinstimulated lipogenesis, induced hepatic GSTs, and was a potent inhibitor of cytochrome P450 1A1/1B1.Oral administration of isopimpinellin blocked DNA adducts formation and skin tumor initiation by 7,12-dimehylbenz[a]anthracene in SENCAR mice and in mouse mammary glands.Phytosterols 5-7 have been recommended as food additives, having the potential to reduce blood LDL cholesterol and thus the risk of cardiovascular diseases [37] and benign prostatic hyperplasia [38].Stigmasterol (7) has also been shown to exert anti-angiogenic and anti-cancer effects via the downregulation of TNF-α and VEGFR-2 [39].The lupane triterpenoids betulin (8) and betulinic acid (9) exhibited a multitude of biological and pharmacological properties ranging from antimicrobial, antitumor, anti-inflammatory, antimalarial, antiparasitic, and anti-HIV activities and colitis protective effects [40][41][42].Moreover, both substances seem to be promising experimental anti-cancer drugs, and betulin (8) was found to be a potent antimutagenic agent of skin carcinogenesis.In addition, betulinic acid (9) has been reported to reduce oxidative stress and demonstrated an in vivo protective effect on dexamethasone-induced thymocyte apoptosis.Some flavonoids such as quercetin, fisetin, luteolin (10), and luteolin 7-O-glucoside (11) are potent inhibitors of cell proliferation and angiogenesis [43][44][45].
In view of our general interest in biologically active flavonoids [46], we performed a standard MTT assay [47][48][49] to measure the effects of flavonoids loeflingiin (1) and isovitexin (2) on the metabolic activity of human breast (MCF7), ovarian (BG-1), endometrial (Ishikawa), and mesothelioma (IST-MES1) human cancer cells.For comparison, two normal human healthy cells, i.e., peripheral blood mononuclear (PBM) [50] and human umbilical vein endothelial cells (HUVECs) [51], were also included in the test.The MTT assay detects perturbations in mitochondria and their functions, and thus, it is commonly used to measure the metabolic activity of cells, which serves as an indicator of cell proliferation, vitality, and cytotoxicity [52,53].
The choice of the selected cancer cell lines was mainly dictated by the severity and wide spread of certain types of human cancers, such as breast and ovarian cancers which are the most frequent malignancies in females.Clinically relevant cellular models are essential for understanding cancer physiology at advanced stages.MCF-7 is one of the most widely used metastatic breast cancer cell lines in MTT assays [54], as it represents a more prevalent form of human breast adenocarcinoma.It retains several features of differentiated mammary epithelium and is more likely to express both consistently and discordantly expressed genes.The BG-1 human ovarian adenocarcinoma cell line is one of the most widely used cell lines in ovarian cancer research [55] as it is a representative model of the most common ovarian cancers, providing insights into hormone receptor expression and estrogen responsiveness.Ishikawa cells serve as valuable models for investigating the behavior of endometrial adenocarcinoma (uterine cancer) [56], whose exact causes remain unknown, and for evaluating potential therapies.Research involving IST-Mes1 contributes to our understanding of mesothelioma biology and drug sensitivity [57].It is part of the Cancer Dependency Map project and the COSMIC cell lines project, aimed at exploring novel therapeutic approaches to combat this rare but aggressive cancer type that originates from the mesothelium.
The activities of loeflingiin (1) and isovitexin (2) in the MTT assay were compared with cis-diamminedichloroplatinum (II) (cisplatin).This complex is a well-known antitumor drug, commonly used to treat ovarian, triple-negative breast cancer (TNBC), mesothelioma, and endometrial cancer, either alone or in combination with other chemotherapeutic agents, such as doxorubicin or taxol.
The IC 50 values of 1, 2, and cisplatin, i.e., the concentrations of tested compounds which reduced cell viability by 50%, were calculated as the means of three MTT assays for each cell line.Optimized conditions of cell viability assays were used [52,53].The results are reported in Table 1.Notably, the 6-C-glucoside isovitexin (2) was inactive on all the cell lines, while the 7-O-glucoside 1 exhibited good activity against all cancer lines, which was significantly higher than cisplatin against the Ishikawa and BG-1 cells (Table 1).
These results require further studies aimed to elucidate the mechanism underpinning loeflingiin's effects on cancer cells.In fact, it is well known that flavonoids, especially luteolin (10), which is structurally related to 1, inhibit cancer cells' growth by targeting different cellular processes such as apoptosis, cell-cycle progression, angiogenesis, and migration [58][59][60][61].It is thus possible that 1 and cisplatin inhibit cell viability through different mechanisms which would explain the different activities observed in the MTT assays.It would also be important to determine the mechanism for loeflingiin's selective effects on cancerous but not normal cells.Our findings seem to confirm that distinct mechanisms for modulating cellular signaling pathways exist in normal cells and in malignant cancer cells [60,61].

Plant Material
Leaves of P. loeflingii L. were collected in April 2021 during the flowering season, from the Zrarati district, in the Kurdistan region of Iraq; GPS coordinates: longitude: 36 • 23 ′ 38.54 ′′ N; latitude: 44 • 16 ′ 59.53 ′′ E. A voucher specimen (code no.7248) was deposited at the Herbarium of Salahaddin University-Erbil (ESUH).The plant was identified by Dr. A. H. Al-khayyat, Professor of botany at the Salahaddin University-Erbil/Iraq.Fresh leaves were cleaned and air-dried under shade at room temperature (20-25 • C) to a constant weight.After drying, the leaves were finely powdered using a laboratory grinding mill to provide a homogeneous powder which was stored in bottles at room temperature until analysis.

Extraction of Plantago loeflingii Leaves
Powdered dried leaves (100 g) were soaked in n-hexane (3 × 250 mL) in an Erlenmeyer flask with occasional shaking in an ultrasonic bath for 15 min; subsequently, the leaves were left in the same solvent at room temperature for 1 h.The mixture was filtered through a Whatman filter paper, and the solvent was removed in a rotavapor under reduced pressure to give an oily residue (A, 0.61 g).Subsequently, the defatted leaves were placed in a flask and extracted with methanol (250 mL) in an ultrasonic bath for 15 min and then at room temperature for 1 h under continuous stirring.The process was repeated another two times, and the combined methanol extracts were filtered.Evaporation under reduced pressure in a rotavapor at ≤35 • C afforded crude residue B (10.53 g, 10.5% w/w dried leaves).

Chromatographic Separation of Plantago loeflingii Secondary Metabolites
The residue B (10.5 g) was partitioned between distilled water (250 mL) and CH 2 Cl 2 (250 mL) to afford an organic fraction (B1) and an aqueous fraction (B2).The aqueous layer B2 was further partitioned with n-butanol (250 mL) to afford, after evaporation, a n-butanol soluble residue (B3, 5.7 g).The residual aqueous phase was not examined.
Acid hydrolysis of loeflingiin (1).Compound 1 (2 mg) was treated with 10% HC1 (2 mL) in a sealed tube at 100 • C for 4 h.The mixture was extracted with EtOAc, and the aqueous layer was neutralized with Et 3 N and freeze-dried.Glucose was identified in the residue from evaporation of the aqueous layer by TLC on a precoated silica gel plate (Silicagel G-60 F 254 , 0.2 mm, Merck-Sigma Aldrich) using EtOAc/MeOH/H 2 O/AcOH (65:15:15:25 v/v/v/v) as eluent and authentic glucose as a reference compound.The plate was sprayed with a 0.5% soln. of vanillin in H 2 SO 4 /EtOH, 4:1 and heated by a hot gun to give a dark brown spot for glucose.The sign of the rotary power of the residue was positive, consistent with D-glucose.

MTT Assay
The inhibition of cell viability by a sample and cisplatin as a reference compound was evaluated through the reduction of the yellow-colored water-soluble salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by mitochondrial NAD(P)Hdependent dehydrogenase enzymes in metabolically active cells to a purple-blue formazan salt, which has an absorbance maximum near 570 nm [47].Metabolically damaged cells lose the ability to convert MTT into formazan.The color change thus serves as a convenient marker of only the viable cells.The darker the solution is, the greater the number of metabolically active cells is, and the measure of the absorbance can be directly related to the number of viable cells.

Cell Cultures and Reagents
Two human normal cells and four human cancer cell lines were used in the MTT test.MCF7 breast cancer cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)/F-12 and DMEM, respectively, supplemented with heat-inactivated 10% fetal bovine serum (FBS), 100 mg/mL penicillin/streptomycin and 2 mM L-glutamine (Life Technologies and Euroclone S.P.A., Milan, Italy) at 37 • C with 5% CO 2 , 95% air and complete humidity.BG-1 ovarian cancer cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Segrate, Italy) and DMEM medium, respectively, without Phenol Red, supplemented with 10% FBS, 100 mg/mL penicillin/streptomycin and 2 mM L-glutamine (Life technologies).Ishikawa endometrial cancer cells were maintained in Minimum Essential Medium (MEM, Sigma-Aldrich, Milan, Italy) supplemented with 10% FBS, 100 mg/mL penicillin/streptomycin, and 2 mM L-glutamine and 1% non-essential amino acids solution (Life technologies).Mesothelioma cancer cells IST-MES1 were maintained in Ham's F-10 Nutrient Mixture (Thermo Fisher Scientific) supplemented with 20% FBS, 100 mg/mL penicillin/streptomycin. Thawed human normal peripheral blood mononuclear cells (PMBCs) were cultured in RPMI medium in 5% CO 2 at 37 • C [50].Human umbilical vein endothelial cells (HUVECs) were purchased from Merck/Sigma-Aldrich (Milan) and cultured in Endothelial Cell Growth Medium (211-500) (Merck/Sigma-Aldrich) according to the seller protocol.Cancer and PBMC cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), except IST-MES1 cells which were kindly provided by the ICLC (Interlab Cell Line Collection) at Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.The cells were used less than six months after resuscitation.MTT stock solution (5 mg/mL in PBS; Sigma-Aldrich) was stored for less than one month in the dark at 4 • C until use.

Cell Viability Assay
An equal number of cells (around 1 × 10 4 ) were seeded in quadruplicate in 96-well flat-bottom plates in their regular growth medium (100 µL/well) supplemented with 10% FBS and were grown in the dark at 37 • C under a humidified atmosphere of 5%% CO 2 -95% air until 75% confluence.Cells were washed once they had attached; subsequently, they were separately treated for 48 h at 22 • C with 100 µL of loeflingiin (1), isovitexin (2), and cisplatin solutions at eight concentrations (0.1, 1, 5, 10, 25, 50, 100, 125 µM).Stock solutions (20 mM) for cisplatin, used as a positive control, and the two flavonoids were prepared, just before use, using DMSO (Sigma-Aldrich; stored at −78 • C), further diluted with 1X PBS (ThermoFisher Scientific) to the appropriate concentration.The final concentration of DMSO was adjusted at about 0.6% (v/v).Medium was removed;
−1 confirmed the presence of an α,β-unsaturated carbonyl ketone at C-4 in the ring C of a flavonoid molecule.The ESI-MS spectrum (negative ion mode) of 1 revealed a [M-H] − ion peak at m/z 447.33, suggesting a MW = 448 and the formula C 21 H 20 O 11 , based on elemental analysis, and proton and carbon counts in the NMR spectra.The 1 H and 13 C NMR spectra (Figures S1 and S3, respectively, in the Supplementary Information), in addition to the signals associated with a flavonoid aglycone, showed signals for a β-glucosyl moiety.This finding was confirmed by the ion peak [C 15 H 9 O 6 = M-C 6 H 11 O 5 ] − at m/z 285 in the ESI-MS spectrum of compound 1, corresponding to the loss of the sugar unit, and by acid hydrolysis of 1 which afforded D-glucose.The 1 H NMR spectrum of 1

Figure 3 .
Figure 3. Structures of the compounds isolated from P. loeflingii leaves.

Figure 3 .
Figure 3. Structures of the compounds isolated from P. loeflingii leaves.

Table 1 .
Results of an MTT assay of cisplatin and compounds 1 and 2 from Plantago loeflingii.