Chemical Constituents and Anticancer Activities of the Extracts from Phlomis × commixta Rech. f. (P. cretica × P. lanata)

The present work is the first report on the ingredients of the P. × commixta hybrid, a plant of the genus Phlomis. So far, thirty substances have been isolated by various chromatographic techniques and identified by spectroscopic methods, such as UV/Vis, NMR, GC-MS and LC-MS. The compounds are classified as flavonoids: naringenin, eriodyctiol, eriodyctiol-7-O-β-D-glucoside, luteolin, luteolin-7-O-β-D-glucoside, apigenin, apigenin-7-O-β-D-glucoside, diosmetin-7-O-β-D-glucoside, quercetin, hesperetin and quercetin-3-O-β-D-glucoside; phenylpropanoids: martynoside, verbascoside, forsythoside B, echinacoside and allysonoside; chromene: 5,7-dihydroxychromone; phenolic acids: caffeic acid, p-hydroxybenzoic acid, chlorogenic acid, chlorogenic acid methyl ester, gallic acid, p-coumaric acid and vanillic acid; aliphatic hydrocarbon: docos-1-ene; steroids: brassicasterol and stigmasterol; a glucoside of allylic alcohol, 3-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranosyl-oct-1-ene-3-ol, was fully characterized as a natural product for the first time. Two tyrosol esters were also isolated: tyrosol lignocerate and tyrosol methyl ether palmitate, the latter one being isolated as a natural product for the first time. Moreover, the biological activities of the extracts from the different polarities of the roots, leaves and flowers were estimated for their cytotoxic potency. All root extracts tested showed a high cytotoxic activity against the Hep2c and RD cell lines.


Introduction
The genus Phlomis L. is one of the largest genera of the subfamily Lamioidae (Lamiaceae), which includes more than 100 species.Northwest Africa, Europe and Asia are the main places where this genus is found, but some species have been identified in southeastern Anatolia and northwestern Iraq [1].Moench, in 1794, pointed out the morphological differences of the genus in two distinct genera, Phlomis and Phlomioides, and divided the genus into two groups distinguished by general differences, such as the chromosomal number (in the Phlomis group, it is generally 2n = 20, while in the Phlomioides group, it is 2n = 22) and the form (species of the Phlomis group are usually shrubs or subshrubs, while the Phlomioides group has mainly herbaceous plants) [2,3].The flowers found in both groups are stalked and radially arranged by the axillary buds, while the lower edge of the flower is trilobate and the middle pod is wider than the other two.There are 12 records of the genus Phlomis in Europe [4], while in Greece, the genus is represented by 9 species.Three species of the genus are found in Crete: Phlomis cretica C. Preslin J. & C. Presl, Phlomis fruticosa L. and Phlomis lanata Willd.Phlomis is characterized as a difficult genus from a taxonomic point of view because it has a high frequency of hybridization and penetration between species [5].The three species found on the island of Crete hybridize in pairs, producing three hybrids: P. × cytherea Rech.f. (P.cretica × P. fruticosa), P. × commixta Rech.f. (P.cretica × P. lanata) and P. × sieberi Vierh.(P.fruticosa × P. lanata).The hybrid P. × commixta has the intermediate morphological characteristics of P. cretica and P. lanata, as described above.The leaves are ovate-elliptic, with a sharply cut or rounded base.The hair on the leaves is dense or whitish.The bracts are linear or narrow lanceolate, with a length of 12-14 mm and a width of 1-3 mm.The width of the inflorescence vertebrae is 15-20 mm.
Over the last few years, there has been a rapid increase in the information available on the structures and pharmacological activities of new compounds isolated and identified from Phlomis species [6][7][8][9][10][11][12][13].
To the best of our knowledge, no phytochemical investigation of the aerial parts and roots of P. × commixta has been previously carried out.The present work on the aerial and root system of the plant resulted in the isolation and identification of a series of components, including two new ones: 3-O-β-(3R)-D-apiofuranosyl-(1→6)-O-β-D-glucopyranosyl-(3S)oct-1-en-3-ol (1) and the tyrosol methyl ether palmitate (2), as well as a third, tyrosol lignocerate (3), that is found for the first time in the genus Phlomis and the family Lamiaceae.The spectroscopic data of compound 3 are reported for the first time.The aim of this paper was to describe the isolation and structural elucidation of the components of the plant, with emphasis on the detailed structural identification of the new compounds, as well as the investigation of the anticancer potential of the plant's extracts.
In the present study, the whole plant of the species Phlomis × commixta was collected, separated (leaves, flowers and roots), dried and extracted with solvents of increasing polarity, and the extracts were assessed for cytotoxicity in Hep2c and RD human cell lines, as well as in the L2OB cell line.

Elucidation of the Compounds
The glucoside bond between β-glucose and C-3 is doubly confirmed: (a) by the HMBC interaction between the anomeric proton H-1′ (δ 4.29 ppm) of glucose and C-3, and b) by the HMBC interaction of the H-3 (δ 4.08 ppm) of alcohol with the C-1 of glucose (δ 103.3).
The sequence of the carbons and the corresponding protons in the glucose molecule is clear, and can be confirmed by the data of the COSY, DEPT, HMBC, HSQC, NOESY and TOCSY spectra in, as well as by the coupling constant values of, the protons (J) in the proton spectrum, many of which are distinct (see Supplementary Data Figures S3-S9).A part of the data is in Table 1.
The peculiarity in the structure of apiose allows for the determination of the sequence of carbons and protons in the molecule.The anomeric proton H-1″ appears at δ 4.99 ppm and is correlated (HSQC) to the anomeric carbon that resonates at 110.8 ppm.
The glucoside bond with glucose C-6′ is confirmed by (a) the NOESY interaction (see Supplementary Data Table S1) of the H-1″ of apiose with glucose H-6′ (3.56/3.92ppm), (b) the interaction of H-1″ with C-6′ (68.4 ppm) in the HMBC spectrum and (c) the interaction of H-6′ glucose with the epimeric C-1″ carbon at δ 110.8 ppm in the same spectrum.
The apiose H-1″ epimeric proton gives a signal at 4.99 ppm, which splits into a doublet, with J = 2.5 Hz, due to the presence of the neighboring H-2″ proton, which appears at 3.88 ppm as a doublet, with J = 2.5 Hz.The carbon C-3″ at 80.6 ppm is easily The HSQC spectrum confirmed the observation by correlation of these two protons to the secondary olefinic carbon at 116.3 ppm.The peak at 5.21 ppm appears as a ddd system, with a trans-vicinal coupling of J 1 = 17.4 Hz, an allylic coupling of J 2 = 1.7 Hz and a geminal coupling of J 3 = 1.1 Hz.The peak at 5.11 ppm also appears as a ddd system, with a cisvicinal coupling of J 1 = 10.4Hz, an allylic coupling of J 2 = 1.7 Hz and a geminal coupling of J 3 = 0.9 Hz.The C-2 olefinic carbon appears at 140.9 ppm, with the corresponding proton at 5.86 ppm.This proton also has the ddd system, with a trans-vicinal coupling of J 1 = 17.4 Hz, a cis-vicinal coupling of J 2 = 10.4Hz and a value of J 3 = 7.1 Hz, typical for the allylic proton.The high chemical shift δ of both the H-3 allylic proton and the C-3 tertiary allylic carbon (4.08 and 83.1 ppm, respectively) is indicative of the fact that the carbon is attached to a heteroatom; it is, in fact, attached to the epimeric oxygen of the glycoside, as will be shown below.The allylic proton is in a "key" position to identify the molecule, as it interacts through the HMBC spectrum with both the C-1 and C-2 olefinic carbons and the neighboring C-4 and C-5 aliphatic secondary carbons at δ 35.8 and 25.7 ppm, respectively (see Supplementary Data Table S1).
The glucoside bond between β-glucose and C-3 is doubly confirmed: (a) by the HMBC interaction between the anomeric proton H-1 ′ (δ 4.29 ppm) of glucose and C-3, and b) by the HMBC interaction of the H-3 (δ 4.08 ppm) of alcohol with the C-1 of glucose (δ 103.3).
The sequence of the carbons and the corresponding protons in the glucose molecule is clear, and can be confirmed by the data of the COSY, DEPT, HMBC, HSQC, NOESY and TOCSY spectra in, as well as by the coupling constant values of, the protons (J) in the proton spectrum, many of which are distinct (see Supplementary Data Figures S3-S9).A part of the data is in Table 1.
The peculiarity in the structure of apiose allows for the determination of the sequence of carbons and protons in the molecule.The anomeric proton H-1 ′′ appears at δ 4.99 ppm and is correlated (HSQC) to the anomeric carbon that resonates at 110.8 ppm.
The glucoside bond with glucose C-6 ′ is confirmed by (a) the NOESY interaction (see Supplementary Data Table S1) of the H-1 ′′ of apiose with glucose H-6 ′ (3.56/3.92ppm), (b) the interaction of H-1 ′′ with C-6 ′ (68.4 ppm) in the HMBC spectrum and (c) the interaction of H-6 ′ glucose with the epimeric C-1 ′′ carbon at δ 110.8 ppm in the same spectrum.
The apiose H-1 ′′ epimeric proton gives a signal at 4.99 ppm, which splits into a doublet, with J = 2.5 Hz, due to the presence of the neighboring H-2 ′′ proton, which appears at 3.88 ppm as a doublet, with J = 2.5 Hz.The carbon C-3 ′′ at 80.6 ppm is easily recognized by 13 C NMR, as it is the only quaternary carbon in the molecule, and therefore does not appear in the DEPT-135.The C4 ′′ (75.0 ppm) is also easily recognizable.This carbon is secondary (DEPT-135) and has two protons at 3.75 and 3.95 ppm, which are diastereotopic, since C-3 ′′ is a stereogenic center.These protons split only with each other, with a typical coupling constant of J = 9.6 Hz.The pack corresponding to C-5 ′′ , with a resonance at 65.7 ppm, carries two protons at 3.57 ppm that appear as a broad singlet in spite of the fact that the neighboring quaternary carbon C3 ′′ is a stereogenic center.Mass spectrometry (ESI-MS) gave negative-ionization fragments, which can be interpreted by the fragmentation process analyzed in Figures S10 and S11   A literature review on this structure showed an initial report with spectroscopic data referring to the structure of 3-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranosyl-(3S)-oct-1-en-3-ol from the plant Clerodendranthus spicatus (Labiateae) [72].
Spectral data in this work were very similar to ours, with small differences in the 13 C-NMR values for the apiose molecule.Based on their literature reference [73], our observations and the data on apiose methyl glycosides [74], we believe that the isolated compound is a 3-S apiose glycoside (Figure 3).The correct apiose structure can be clarified based on the NOESY spectrum, showing the interactions of the protons in space.Indeed, H-1″ interacts strongly with H-5″ and H-4b″ protons, which are "facing down" in a "beta (3R)"-like structure.
This hypothesis was supported by the NOESY data, which further indicated the interaction of the H-5″ proton with the H-4b″ proton, as well as the absence of an interaction between the H-4a″ and H-1″ protons, which are "on opposite sides of the molecule" in a beta (3R) structure.It is, therefore, very likely that the correct structure of the molecule isolated from our group  The correct apiose structure can be clarified based on the NOESY spectrum, showing the interactions of the protons in space.Indeed, H-1 ′′ interacts strongly with H-5 ′′ and H-4b ′′ protons, which are "facing down" in a "beta (3R)"-like structure.
This hypothesis was supported by the NOESY data, which further indicated the interaction of the H-5 ′′ proton with the H-4b ′′ proton, as well as the absence of an interaction between the H-4a ′′ and H-1 ′′ protons, which are "on opposite sides of the molecule" in a beta (3R) structure.It is, therefore, very likely that the correct structure of the molecule isolated from our group In the low-field range, two pairs of orthoconjugated aromatic protons, with peaks at δ 7.08 (J = 8.6 Hz, H-2/H-6) and 6.76 (J = 8.6 Hz, H-3/H-5), typical of an AB system, indicate the presence of a 1,4-substituted aromatic ring.There is also a triplet at 4.23 ppm (J = 7.0 Hz) for the ether moiety carbon (C-8) that split due to the coupling with the neighboring C-7 benzyl hydrogens, which resonate at 2.86 ppm, with a coupling constant of J = 7.0 Hz.Characteristic peaks in the spectrum are the singlet corresponding to the tyrosol methyl ether (C-9) at δ 3.48 and the triplet at δ 2.27, assigned to the methyne protons of the C-2 ′ , split by the neighboring protons (H-3 ′ ) of the side chain, with a coupling constant of J = 7.3 Hz.A broad singlet at δ 1.25 ppm integrates to 22 protons, corresponding to an 11-carbon part of the aliphatic chain, and the triplet at δ 0.87 ppm is assigned to the terminal methyl group of this chain, split by the neighboring (C-15 ′ ) protons, with a coupling constant of J = 7.2 Hz.
In the carbon spectrum, the peak at 173.9 ppm confirmed the presence of the carbonyl carbon C-1 ′ , and a very weak peak at 154.2 ppm was assigned to the quaternary C-4.By combining the NMR, HMBC (Figure 4) and HSQC spectroscopy techniques, it is understood that the peak at 115.2 ppm corresponds to C-3/C-5, since they interact only with the protons of the AB system, and the peak at 130.0 ppm corresponds to C-2/C-6, while the carbon at 50.9 ppm (C-9) corresponds to the methyl of tyrosol ether.The complete spectroscopic dataset for the compound is presented in Table 2 below.
led to the molecular formula C25H42O3, with a molecular ion of m/z (theor.C25H42NaO3; exact mass: 413.3026) (see Supplementary Data the same time, the mass spectrum (ESI-MS, positive ionization) con ion of m/z = 413.6 [M+HΝa] + .
In the low-field range, two pairs of orthoconjugated aromatic p δ 7.08 (J = 8.6 Ηz, Η-2/Η-6) and 6.76 (J = 8.6 Ηz, Η-3/Η-5), typical of an the presence of a 1,4-substituted aromatic ring.There is also a triple Hz) for the ether moiety carbon (C-8) that split due to the coupling C-7 benzyl hydrogens, which resonate at 2.86 ppm, with a coupling c Characteristic peaks in the spectrum are the singlet corresponding ether (C-9) at δ 3.48 and the triplet at δ 2.27, assigned to the methyn split by the neighboring protons (Η-3′) of the side chain, with a cou 7.3 Ηz.A broad singlet at δ 1.25 ppm integrates to 22 protons, cor carbon part of the aliphatic chain, and the triplet at δ 0.87 ppm is ass methyl group of this chain, split by the neighboring (C-15′) proto constant of J = 7.2 Hz.
In the carbon spectrum, the peak at 173.9 ppm confirmed the pre carbon C-1′, and a very weak peak at 154.2 ppm was assigned to th combining the NMR, HMBC (Figure 4) and HSQC spectrosco understood that the peak at 115.2 ppm corresponds to C-3/C-5, sin with the protons of the AB system, and the peak at 130.0 ppm cor while the carbon at 50.9 ppm (C-9) corresponds to the methyl o complete spectroscopic dataset for the compound is presented in Ta   Metabolite 2 is reported for the first time as component of the hybrid or the genus Phlomis.The literature review on this structure showed a report by the Gargouri research team [75], reporting synthetic approaches to lipophilic ester derivatives of tyrosol.The NMR spectroscopic data of the synthetic compound were similar to those reported by our group.Therefore, to the best of our knowledge, metabolite 2 has been isolated as a plant component for the first time.The same group investigated the germicidal activity of tyrosol and synthetic esters against Gram (+) and Gram (−) bacteria.The minimum inhibitory concentration (MIC) against the microorganisms concerned, as well as their antileishmaniasis activity against Leishmania major and L. infantum, were examined.S19-S25).The signal sequence in the 1 H-NMR spectrum showed several similarities to metabolite 2, which indicated that it was an analogue of a tyrosol ester.The 1 H-NMR spectrum showed two pairs of orthoconjugated aromatic protons, with low-field peaks at δ 7.07 (J = 8.5 Hz, H-2) and 6.76 (J = 8.5 Hz, H-3), typical of an AB system, indicating the presence of a 1,4-substituted aromatic ring.There is also a triplet at 4.23 ppm (J = 7.1 Hz) for the protons on the ether-type carbon (C-8), which are split due to the coupling with the adjacent benzylic hydrogens C-7 that resonate at 2.86 ppm, with a coupling constant of J = 7.1 Hz.Characteristic is also the triplet at δ 2.28, corresponding to its methylene proton C-2 ′ , split by neighboring hydrogens (H-3 ′ ) of the side chain, with a coupling constant of J = 7.5 Hz.A broad singlet at δ 1.25 ppm integrates for 36 protons, corresponding to the presence of an aliphatic chain segment comprising 18 methylene moieties, and finally the triplet at δ 0.88 ppm corresponds to the terminal methyl of the aliphatic chain, split by the protons of the neighboring carbon (C-23 ′ ), with a coupling constant of J = 7.2 Hz.
In the 13 C NMR spectrum, the peak at 173.9 ppm confirmed the presence of the carbonyl carbon C-1 ′ , whereas the peak at 154.2 ppm corresponds to the quaternary C-4.The combination of the NMR, HMBC (Figure 5) and HSQC techniques revealed that the peak at 115.2 ppm corresponds to C-3/C-5, since they interact only with the protons of the AB system.From the DEPT-135 range, it is clear that the secondary carbons of the aliphatic chain resonate in the range of 20-35 ppm, and the peak at 14.1 ppm corresponds to the terminal carbon of the chain.The complete spectroscopic data of the compound are presented in Table 3 below.Metabolite 3 was first isolated from the bark of the Buddleja cordata subsp.cordata of the family Loganiaceae [76].It is isolated for the first time regarding both the hybrid and the genus Phlomis in general.This is the second time that this particular metabolite is mentioned in the literature.Compound 3, according to the literature, showed moderate antibacterial activity against Mycobacterium tuberculosis (MIC = 64 µg/mL).Metabolite 3 was first isolated from the bark of the Buddleja cordata subsp.cordata of the family Loganiaceae [76].It is isolated for the first time regarding both the hybrid and the genus Phlomis in general.This is the second time that this particular metabolite is mentioned in the literature.Compound 3, according to the literature, showed moderate antibacterial activity against Mycobacterium tuberculosis (MIC = 64 µg/mL).

Cytotoxicity
The cytotoxic activity of the extracts from the roots, leaves and flowers of the hybrid P. × commixta was assessed using three different cell lines, i.e., Hep2c (human cervix carcinoma), RD (human rhabdomyosarcoma) and L2OB (murine fibroblast).There is a great interest regarding the biological activities of Phlomis spp.according to the literature.The results herein showed that all the samples from the roots expressed higher cytotoxic activity in the case of the Hep2c and RD cell lines, while the opposite was evidenced in the case of the L2OB cell line.The basic criterion for the cytotoxic activity of a plant extract is an IC50 < 30 µg/mL (Table 4).

Chemotaxonomic Significance of the Secondary Metabolites
The genus Phlomis is well known for its richness in flavonoids and phenylpropanoid glycosides.A number of studies reveal that apigenin, luteolin, naringenin and their derivatives are very abundant in Phlomis' polar extracts [12,25].However, while there is only one report of the presence of diosmetin in Phlomis plants (P.fruticosa) [77], there is no one mentioning diosmetin-7-O-β-D-glucoside.It is noteworthy that quercetin is reported as a component only in three species of the genus Phlomis (P.bracteosa [78] and P. elliptica [79], P. sterwartii Hf. [38]).Zhang and Wang [80] initiated a study reporting the detection of phenolic acids in two Chinese Phlomis species.Phenolic acids isolated from Phlomis × commixta (caffeic acid, chlorogenic acid, p-coumaric acid, vanillic acid and gallic acid) have also been detected in acetone and methanol extracts of P. umbrosa and P. megalantha.There are very few references regarding the isolation and detection of caffeic acid (P.stewartii [7] and P. lycunitis [30], P. armeniaca [81]), p-hydroxy benzoic acid (P.stewartii [7] and P. bracteosa [78]) and chlorogenic acid (P.olivieri [82], P. syriaca [83]) in the genus Phlomis.The presence of chlorogenic acid and the methyl ester of an isomer (3-O-caffeoylquinic acid methyl ester) in Phlomis plants has been reported [25]; however, this is the first report on the presence of chlorogenic acid methyl ester in this genus.
Also, this is the first report of docos-1-ene (30) and 5,7-dihydroxy chromone (15) in the genus Phlomis.Stigmasterol is one of the most abundant sterols in the plant kingdom, and has also been isolated from the plants of the genus Phlomis (P.cashmeriana [8], P. bracteosa [84]).Brassicasterol, though, which is also a well-known sterol, is reported for the first time as component in the genus Phlomis.

Anticancer Activity of the Extracts
Cancer is a disease that has a significant global impact on people.To treat and prevent this fatal condition, there is a continuous need for novel therapeutics.Natural substances are gaining attention from scientists because they are considered to have fewer hazardous side-effects than existing treatments, like chemotherapy.In order to create novel medications, secondary metabolites from the plant kingdom have been isolated and assessed for their anticancer properties.
There are many studies regarding the cytotoxic effects of the extracts from the aerial parts of various Phlomis species (Table 5).It must be mentioned that the current study provides the first report on the cytotoxic effects of the extracts from the roots of a Phlomis × commixta Rech.f.Strangely, our literature search did not reveal any reports concerning the evaluation of anticancer activity on the extracts derived from the roots of species belonging to the genus Phlomis.However, there are some studies on the isolation, characterization and antitumor activity of nortriterpenoids from the roots of P. umbrosa var lutibracteata [85], the antitumor activity of phlomiol extracted from the roots of P. younghusbandii [86,87] and phenylethanoid glycosides from the roots of P. umbrosa [88], as well as the chemical constituents in the roots of P. medicinalis and P. younghusbandii [23,89].According to the bibliography, there is a great interest for the antitumor activity exhibited by the extracts of the species belonging to the genus Phlomis.Table 5 describes studies on the antitumor activity of the extracts from Phlomis spp.using in vitro techniques in various human cancer cell lines.In order to compile all this bibliographic data, we used free chemistry databases, such as PubChem, Scopus, Reaxys and Google Scholar.It is interesting that, in all these reports, the plant material was from the aerial or part of the aerial parts (such as the flowers or/and leaves).
According to our results, the plant part that exhibited the highest cytotoxicity was the roots.In terms of the basic criterion for the cytotoxic activity of a plant extract according to American National Cancer Institute (activity < 30 µg/mL), all polar solvent extracts from the roots showed to be active for the human cancer cell lines in doses between 19.61 and 29.77 µg/mL.
Another important observation is that, with the exception of the less-polar extracts from the leaves and the flowers of the plant, the rest of them show a moderate to strong antitumor effect, with IC50 values between 21.84 and 48.29 µg/mL.Moreover, the flowers' extracts showed lower IC50 values than the leaves.Apart from that, the results show that none of the extracts derived after the liquid-liquid extraction of the methanolic extracts from the leaves had a stronger anticancer activity compared to the initial methanolic extracts.This result indicates the possible synergistic activity of the compounds extracted from the leaves.

Plant Material
The plant material of Phlomis × commixta (Lamiaceae) was collected on 04/2012 from the village Tylisos of Heraklion, Crete, in Greece (coordinates: 35 The plant was botanically identified by Prof. Dr. S. Pirintsos, Department of Biology, University of Crete, Greece, and a voucher specimen was deposited in the Department Herbarium.The plant samples were naturally dried (in the shade and in a well-ventilated environment), grinded by a laboratory mill (particle size approx.1 mm) and stored in the darkness at room temperature.

Extraction and Isolation
The dried flower material (115 g) was extracted repeatedly in a Soxhlet apparatus (petroleum ether; dichloromethane; methanol).This process was thrice repeated (3 × 300 mL for 24 h each time), and after filtration and evaporation under reduced pressure, the petroleum extract (1.5 g), the dichloromethane extract (790 mg) and the methanol extract (27.0 g) residues were collected.The dried leaf herbal material (350 g) was extracted repeatedly at room temperature with petroleum ether, dichloromethane and methanol.This process was thrice repeated (3 × 300 mL for 24 h each time), and filtration and evaporation under reduced pressure afforded the petroleum extract (4.9 g), the dichloromethane extract (4.1 g) and the methanol extract (45.6 g) residues.The root systems of the plants were also extracted at room temperature with ethanol for six days under periodic shaking.The extract gave a solid residue weighing 7.2 g.

Component Isolation from the Flowers
The flower methanol extract was evaporated under reduced pressure to yield a crude residue (29.6 g), which was then suspended in 1 L of boiling water and partitioned with Et 2 O, EtOAc and n-BuOH, respectively.The Et 2 O residue (3.1 g) was subjected to column chromatography (C.C.) on a Sephadex LH-20 (4.00 × 40.00 cm) and eluted with a mixture of CH 2 Cl 2 :MeOH (1:1) to obtain fractions A-Q.Fraction D (184.5 mg) was submitted to Sephadex LH-20 separation using a mixture of CH 2 Cl 2 :MeOH (1:2) as an eluent to obtain eight fractions (DA-DS).One of these fractions, DO (4.3 mg), was identified as naringenin.Fraction E (111.2 mg) was submitted to Sephadex LH-20 separation using a mixture of CH 2 Cl 2 :MeOH (1:2) as an eluent to obtain eight fractions (EA-ES).One of these fractions, EM (10.1 mg), was identified again as naringenin.Fraction F (27.1 mg) was submitted to Sephadex LH-20 separation using MeOH as an eluent to obtain eight fractions (FA-FO).From these, fraction FB (5.8 mg) was identified as p-hydroxybenzoic acid and fraction FC (1.2 mg) as 5,7-dihydroxychromone.Fraction G (11.6 mg) was separated on the Sephadex LH-20 using MeOH as an eluent to obtain eight fractions (GA-GO).From these, fractions GH (2.5 mg) and GI (1.1 mg) were identified as naringenin.Fraction H (17.1 mg) was separated on the Sephadex LH-20 using MeOH as an eluent to obtain eight fractions (HA-HC).From these, fraction HC (0.7 mg) was identified as caffeic acid, while fractions HL (1.8 mg) and HM (1.0 mg) were identified as naringenin, fraction HO (1.7 mg) was identified as a mixture of two compounds (eriodyctiol and luteolin) and the HP (1.7 mg) fraction was identified as luteolin.Fraction I (7.8 was submitted to Sephadex LH-20 separation using a mixture of CH 2 Cl 2 :MeOH (1:2) as an eluent to obtain eight fractions (IA-IH).From these, fraction IE (3.2 mg) was identified again as luteolin.Fraction K (24.0 mg) was submitted to Sephadex LH-20 separation using a mixture of CH 2 Cl 2 :MeOH (1:2) as an eluent to obtain eight fractions (KA-KQ).From these, fraction KL (7.5 mg) was identified as a mixture of two compounds (eriodyctiol and luteolin) and fraction KM (1.0 mg) was identified as luteolin.

Cytotoxic Activity of the Studied Extracts
A standard cytotoxic activity test was performed [113] using the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay [114,115].The following cell lines were used (cell lines were donated from the collection of cell lines of the Institute of Virology, Vaccines and Serums "Torlak", Belgrade, Serbia): RD (substrate: MEM Eagle/10% FCS) (cell line derived from human rhabdomyosarcoma), Hep2c (medium: MEM Eagle/5% FCS) (cell line derived from human cervix carcinoma-HeLa derivative) and L2OB (medium: MEM Eagle/10% FCS) (cell line derived from murine fibroblast), against which the activity of the extracts was measured.The results were expressed as IC50 values (µg/mL), a threshold which was defined as the concentration of an agent inhibiting cell survival by 50% in comparison to a vehicle-treated control [116].The experiment was conducted in accordance with the method described by Mašković et al., 2015 [116].

Conclusions
The work presented in this publication is the first report on the isolation and the identification of the components of the Phlomis × commixta hybrid, an endemic plant of Crete, Greece.The component analysis included extraction from the flowers, the leaves and the roots of the plant, the subsequent component isolation using various chromatographic techniques and identification by spectroscopic methods, such as UV/Vis, NMR, GC-MS and LC-MS.
A total of thirty components were isolated, including flavonoids, phenylethanoic glycosides, phenolic acids, tyrosol esters, steroids, a chromone and an aliphatic hydrocarbon.
This report includes a detailed structural elucidation of allylic alcohol glycoside 3-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranosyl-oct-1-ene-3-ol, which was fully characterized as a natural product for the first time, and two tyrosol esters: tyrosol lignocerate and the tyrosol methyl ether palmitate, the former one mentioned only once in the literature as a component in a different species, and the latter one isolated as a natural product for the first time.
All the root extracts tested showed a high cytotoxic activity against the Hep2c and RD cell lines, with the leaf and flower extracts showing a moderate, though distinct, activity.

Figure 1 .
Figure 1.Isolated compounds from the leaves, flowers and roots of Phlomis × commixta Rech.f.

Figure 1 .
Figure 1.Isolated compounds from the leaves, flowers and roots of Phlomis × commixta Rech.f.
This research led to the isolation of two new natural products: 3-O-β-(3R)-D-apiofuranosyl-(1→6)-O-β-D-glucopyranosyl-(3S)-oct-1-en-3-ol (1) and the tyrosol methyl ether palmitate (2).Moreover, this is the first study that fully describes the NMR of tyrosol lignocerate (3).The combination of NMR spectroscopic data with those of the high-resolution mass spectrum (HRMS) data led to the molecular formula C 19 H 34 O 10 , with a molecular ion at m/z = 445.2044[M + HNa] + (theor.C 19 H 34 NaO 10 ; exact mass: 445.2050).At the same time, the mass spectrum (ESI-MS, negative ionization) showed, as the base peak, the fragment m/z = 421.4[MH] − .Characteristic is the cleavage of the glycosidic O-bond, yielding a fragment of m/z = 131.0 of apiose, as well as a fragment of m/z = 289.3[M-Apio-H] − .The next set of data were from the homonuclear 1D NMR and heteronuclear 2D NMR experiments (Figure the fragment m/z = 421.4[MH] − .Characteristic is the cleavage of the glycosidic O-bond, yielding a fragment of m/z = 131.0 of apiose, as well as a fragment of m/z = 289.3[M-Apio-H] − .The next set of data were from the homonuclear 1D NMR and heteronuclear 2D NMR experiments (Figure
in Supplementary Material.The specific rotation value of the molecule was determined as [α] D 20 = −420 (c = 2.0, CH 3 OH).

Figure 4 .
Figure 4. Main correlations of the COSY (highlighted) (a) and HMBC (b) of
* Reported correlation are from the HMBC and HSQC spectra.

Table 4 .
Cytotoxic activity of the extracts from P. × commixta in one murine (L2OB) and two human cell lines (Hep2c, RD) compared to the standard cytotoxic agent cis-diammine dichloroplatinum (cis-DDP).

Table 5 .
Short synopsis of the cytotoxicity of the extracts from Phlomis spp.against human cancer cell lines.