Demirci, Fatih

Hohmann, Judit

Karaca, Nursenem

Kirmizibekmez, Hasan

Kúsz, Norbert

Kirmizibekmez Hasan

Kúsz Norbert

Karaca Nursenem

Demirci Fatih

Hohmann Judit

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  Secondary Metabolites from the Leaves of Digitalis viridiflora  Hasan Kırmızıbekmeza*, Norbert Kúszb, Nursenem Karacac, Fatih Demircid,e and Judit Hohmannb,f  aDepartment of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, TR-34755, Kayisdagi, İstanbul, Turkey  bInstitute of Pharmacognosy, Szeged University, Eötvös u 6, H-6720 Szeged, Hungary  cGraduate School of Health Sciences, Anadolu University, 26470, Eskişehir, Turkey  dDepartment of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir, Turkey  eFaculty of Health Sciences, Anadolu University, 26470, Eskişehir, Turkey   fInterdisciplinary Centre of Natural Products, University of Szeged, Eötvös u. 6., H-6720 Szeged, Hungary  hasankbekmez@yahoo.com   Received: September 11th, 2016; Accepted: November 4th, 2016     A new phenylethanoid glycoside, named digiviridifloroside (1), was isolated from the leaves of Digitalis viridiflora Lindley along with a known phenylethanoid glycoside, calceolarioside A (2), two flavonoid glycosides, scutellarein 7-O-β-D-glucopyranoside (3) and hispidulin 7-O-β-D-glucopyranoside (4), two cleroindicins, cleroindicins B (5) and F (6), a nucleoside, adenosine (7), as well as a mixture of β-glucopyranosyl-(16)-4-O-caffeoyl-/β-glucopyranose and 3,4-dihydroxyphenylethanol. The structure of the new compound was established as 3,4-dihydroxy-β-phenylethoxy-6-O-(E)-feruloyl-β-glucopyranosyl-(16)-4-O-(E)-caffeoyl-β-glucopyranoside (1) based on extensive 1D- and 2D-NMR spectroscopy, as well as HR-ESI-MS. Digiviridifloroside represents a rare type of phenylethanoid glycoside which bears two aromatic acyl units in its structure. In addition to phytochemical studies, the isolates were evaluated for their in vitro antimicrobial activities against three pathogenic bacteria and three yeast strains using a microdilution method. Among the tested compounds, 5 exhibited moderate antibacterial activity against Bacillus cereus NRRLB 3711 with a MIC value of 25 µg/mL, whereas compounds 5 and 6 showed relatively high anticandidal activity against Candida strains with MIC values down to 12.5 µg/mL, in comparison to the standard antimicrobial compounds.  Keywords:  Digitalis viridiflora, Plantaginaceae, Phenylethanoid glycoside, Digiviridifloroside, Antimicrobial activity.    The genus Digitalis (Plantaginaceae) contains biennial or perennial species. It is represented by nine species in the flora of Turkey including D. viridiflora Lindley [1]. Previous studies on the genus showed the presence of a wide range of secondary metabolites including phenylethanoid glycosides, cardiac glycosides, steroidal saponins, pregnane glycosides, cleroindicins, flavonoids and anthraquinones [2-6]. In continuation of our systematic survey on the phytochemical composition of Digitalis species from Turkey, five phenylethanoid glycosides were recently reported from the initial work on D. viridiflora [7]. Further detailed chromatographic studies on the chemical constituents of the leaves of D. viridiflora led to the isolation of one new (1) and one known phenylethanoid glycoside, two flavonoid glycosides, two cleroindicins and a nucleoside (Figure 1). This paper reports the isolation, structure elucidation and antimicrobial activities of these compounds.  Compound 1 was obtained as a yellowish amorphous powder. Its UV and IR spectra were characteristic for a phenylethanoid glycoside.  It possesses a molecular formula of C39H44O19, as determined by the analysis of its HRESIMS (m/z 839.2390 [M + Na]+, calcd for C39H44 NaO19, 839.2375) and 13C NMR data (Table 1).  The 1H NMR spectrum (Table 1) of 1 showed resonances at H 7.55 and 6.25 (each d, J = 15.8 Hz) as an AX system as well as signals at H 7.00 (d, J = 2.0 Hz), 6.86 (dd, J = 8.1, 2.0 Hz) and  6.74 (d, J = 8.1 Hz) as an ABX system attributable to an (E)-caffeoyl moiety. Moreover, the spectrum also contained three aromatic signals as an ABX system at H 6.70 (d, J = 1.9 Hz), 6.67 (d, J = 8.0 Hz), and 6.55 (dd, J = 8.0 and 1.9 Hz), two geminal benzylic methylene signals at H 2.78 (t, J = 8.0 Hz), and two nonequivalent oxymethylene signals H 4.01 (m) and 3.71 arising from a  OOHOHOO OOHHOOHOHHO OOHHOOOHOMeOO1 35''''''1'4' 6'1''6''1'''5'''3'''1''''3''''5''''1OOHOHOO OOHHOOHOHOH2OOOHOHROOHO OHO3   R = H4   R = CH3HOOHOOHHO5O6OOHNNNNNH2OOHOHHO7 Figure 1: Chemical structures of compounds 1-7.  3,4-dihydroxyphenylethanol moiety. Furthermore, the presence of two anomeric signals at H 4.37 (d, J = 7.8 Hz) and 4.35 (d, J = 7.7 Hz) revealed the diglycosidic structure of 1, which was confirmed by the corresponding anomeric carbon resonances at C 105.0 and 104.5 in the 13C NMR spectrum. These findings taken together with 2D NMR experiments (COSY, HSQC and HMBC (Figure 2) revealed the presence of a lugrandoside [2] backbone in 1. However, the 1H NMR spectrum of 1 contained additional signals at H 7.18 (d, J = 2.0 Hz), 7.05 (dd, J = 8.0, 2.0) and 6.80 (d, J = 8.0 Hz) as an ABX type, a pair of trans-coupled AX type signals at H 7.62 and 6.37 (each d, J = 15.9 Hz) and a methoxy signal at H 3.87 (s) suggesting the presence of a (E)-feruloyl unit in the structure of 1.   The deshielded H2-6 (H 4.53 and 4.26) and C-6  (C 64.6)  signals of the terminal -glucopyranose signals indicated that the (E)-feruloyl unit was located at C-6(OH), which was further NPC Natural Product Communications 2017 Vol. 12 No. 1 59 - 61  60  Natural Product Communications Vol. 12 (1) 2017 Kırmızıbekmez et al. Table 1.13C and 1H NMR dataa for digiviridifloroside (1) (CD3OD, 13C 125 MHz; 1H 500 MHz).  Position C (ppm) H (ppm, J in Hz)Aglycone   1 131.6 - 2 117.2 6.70 (d, J = 1.9) 3 146.2 - 4 144.7 - 5 116.6 6.67 (d, J = 8.0) 6 121.5 6.55 (dd, J = 8.0, 1.9)  72.5 4.01 (m) / 3.71†  36.7 2.78 (t, J = 8.0) Glucose   1′ 104.5 4.37 (d, J = 7.8) 2′ 75.4 3.28 † 3′ 75.8 3.62 (t, J = 8.0) 4′ 72.8 4.83 † 5′ 75.1 3.77 (m) 6′ 70.3 3.85 †   3.69 (dd, J = 11.5, 4.7) Glucose   1′′ 105.0 4.35 (d, J = 7.7) 2′′ 74.9 3.23 (t, J = 8.2) 3′′ 77.7 3.35† 4′′′ 77.4 3.33† 5′′ 75.7 3.48 (m) 6′′ 64.6 4.53 (dd, J = 11.7, 1.8)   4.26 (dd, J = 11.7, 5.8) Caffeoyl   1′′′ 127.8 - 2′′′ 115.4 7.00 (d, J = 2.0) 3′′′ 146.9 - 4′′′ 149.8 - 5′′′ 116.5 6.74 (d, J = 8.1) 6′′′ 123.2 6.86 (dd, J = 8.1, 2.0) ′ 114.8 6.25 (d, J = 15.8)  ′ 147.8 7.55 (d, J = 15.8)  C=O 168.7 - Feruloyl   1′′′′ 127.8 - 2′′′′ 111.8 7.18 (d, J = 2.0) 3′′′′ 150.8 - 4′′′′ 149.5 - 5′′′′ 116.8 6.80 (d, J = 8.0) 6′′′′ 124.5 7.05 (dd, J = 8.0, 2.0) ′′ 115.4 6.37 (d, J = 15.9)  ′′ 147.3 7.62 (d, J = 15.9)  C=O 169.2 - OMe 56.6 3.87 s aAssignments are based on COSY, HSQC and HMBC experiments. † Overlapped.  OOHOHOO OOHHOOHOHHO OOHHOOOHOMeOO1  Figure 2: Key HMBC (CH) correlations for 1.  confirmed by the long-range correlation of the carbonyl carbon    (C 169.2) of the (E)-feruloyl unit with H2-6 of the terminal glucopyranose unit in the HMBC spectrum (Figure 2). Based        on these spectroscopic data, the structure of 1 was elucidated as 2-(3,4-dihydroxyphenyl)ethyl-O-6-O-(E)-feruloyl-β-glucopyranosyl- (16)-4-O-(E)-caffeoyl-β-glucopyranoside, and named digiviridifloroside.   The known compounds were characterized as calceolarioside A (2) [8], scutellarein 7-O-β-D-glucopyranoside (3) [9], hispidulin 7-O-β-D-glucopyranoside (4) [10], cleroindicins B (5) and F (6) [11], and adenosine (7) [12] by comparing their spectroscopic data with those published previously. Moreover, a mixture of β-glucopyranosyl-(16)-4-O-caffeoyl-α/β-glucopyranose and 3,4-dihydroxyphenyl-ethanol, which could be an artefact formed during the isolation procedure, was characterized.  To the best of our knowledge, digiviridifloroside (1) is the third example of a rare phenylethanoid glycoside obtained from the genus Digitalis, which contains two aromatic acyl units in its structure; the first two such compounds were reported from D. lanata [4]. In a very recent study by Skhirtladze et al. [13], another new phenylethanoid glycoside esterified with two aromatic acids was reported. Therefore, the occurrence of such rare phenylethanoid glycosides might possess significant chemotaxonomic importance for the genus Digitalis within its new family Plantaginaceae.   The in vitro antimicrobial activities of the isolates (except for the mixture) were evaluated against three pathogenic bacteria (Bacillus cereus NRRLB 3711, Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 6538) and three yeast (Candida albicans ATCC 90028, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258) strains using a microdilution method. Compounds 4 and 5 displayed moderate activity against Bacillus cereus NRRLB 3711 with MIC values 50 and 25 µg/mL respectively, while the rest were inactive against the tested bacteria (Table 2). Compounds 4 - 6 displayed moderate activity against all Candida strains tested with MIC values ranging from 12.5 to 100 µg/mL, being 6 the most potent one against C. parapsilosis ATCC 22019. To the best of our knowledge the antimicrobial activities of the cleroindicins (5 and 6) are being reported for the first time in this study.  Table 2: Antimicrobial activities (MIC, µg/mL) of compounds 1-7.     Comp. B.  cereus NRRLB 3711 P. aeruginosa ATCC 10145 S.  aureus ATCC 6538 C. albicans ATCC 90028 C.  parapsilosis ATCC  22019 C. krusei  ATCC 6258 1 >100 >100 >100 >100 >100 100 2 >100 100 >100 >100 >100 100 3 >100 >100 >100 >100 >100 >100 4 50 >100 >100 50 100 50 5 25 >100 >100 25 50 25 6 >100 >100 >100 >100 12.5 25 7  >100 >100 >100 >100 >100 >100 S1 - - - 0.031 0.062 0.125 S2 0.002 0.062 0.001 - - - S1: Amphotericin B, S2:  Chloramphenicol.  Experimental  General experimental procedures: Optical rotation ([α]D26) was measured on a Perkin-Elmer 341 polarimeter. UV spectra and IR spectra were recorded on a HP Agilent 8453 spectrophotometer and a Perkin-Elmer 2000 FT-IR spectrometer, respectively. NMR experiments were performed on a Bruker Avance DRX 500 instrument. COSY, HSQC and HMBC experiments were run under standard conditions at 300 K, dissolving each sample in 550 µL of 99.8% D CD3OD (VWR) (1H, δ= 3.34 ppm; 13C, δ= 49.0 ppm). A Q Exactive orbitrap from Thermo Scientific with a HESI ion source was used for HRMS analysis. TLC analyses were carried out on silica gel 60 F254 precoated plates (Merck, Darmstadt), and the compounds were stained with 1% vanillin/H2SO4 and heating at 105C. for 1-2 min. For medium-pressure liquid chromatographic (MPLC) separations, Sepacore® Flash Systems X10 / X50 (Büchi) was used with Redi sep columns packed with LiChroprep C18 (13, 43 and 130 g, Teledyne Isco) and SiO2 (40 g, Teledyne Isco). Open column chromatography (CC) was performed using Silica gel 60 (0.063-0.200 mm; Merck, Darmstadt), polyamide and Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA).  Plant material: The leaves of Digitalis viridiflora Lindley were collected from Demirköy, Kırklareli, Turkey, in July 2012 and authenticated by Dr. H. Kırmızıbekmez. A voucher specimen (YEF 12012) has been deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, İstanbul, Turkey. Phytoconstituents from Digitalis viridiflora Natural Product Communications Vol. 12 (1) 2017  61 Extraction and isolation: The air-dried and powdered leaves of D. viridiflora (185 g) were extracted with MeOH (2 L x 2) at 45C for 4 h. The solvent was evaporated in vacuo to afford the crude MeOH extract (45.1 g, yield 24.3%), which was suspended in H2O (100 mL) and partitioned with CHCl3 (100 mL x 3). The H2O sub-extract (35 g) was subjected to a polyamide column (120 g) eluting with a gradient solvent system of H2O/MeOH (100:0 to 0:100) to give 7 main fractions, A-G [7].  Fr. B (12.1 g, eluted with 20% MeOH) was applied to silica gel (150 g) CC eluting with a CH2Cl2/MeOH/H2O gradient (90:10:1 to 50:40:10) to obtain 4 sub-fractions, B1-4.  Purification of sub-fraction B1 (340 mg) by medium pressure liquid chromatography (SiO2, 40 g) eluting with a stepwise CH2Cl2/MeOH gradient (100:0 to 70:30) gave compounds 6 (50 mg) and 5 (7 mg). Repeated chromatography of sub-fraction B4 (610 mg) by C18-medium pressure liquid chromatography (C18-MPLC, 43 g, using a H2O/MeOH gradient, 90:10 to 30:70) and Sephadex LH-20 CC (10 g, MeOH), respectively, gave 7 (2 mg).  Fraction D (656 mg, eluted with 40% MeOH) was submitted to C18-MPLC (130 g) eluting with a H2O/MeOH gradient (85:15 to 35:65) to yield a mixture of β-glucopyranosyl-(16)-4-O-caffeoyl-α/β-glucopyranose and 3,4-dihydroxyphenylethanol (11 mg). Fraction G (380 mg, eluted with 100% MeOH) was applied to Sephadex LH-20 CC (60 g) eluting with MeOH to give sub-fraction G1 as well as 3 (24 mg). Purification of sub-fraction G1 (65 mg) by C18-MPLC (13 g) eluting with H2O/MeOH mixtures (85:15 to 0:100) yielded 2 (4 mg), 4 (4 mg) and 1 (15 mg).  Digiviridifloroside (1)  [α]D26: -59 (c 0.1, MeOH). IR (KBr): 3383, 1698, 1630, 1604, 1515, 1462 cm-1. UV/Vis λmax (MeOH) nm: 219, 288 (sh), 328. HR-MS-ESI: m/z [M + Na+] calcd. for C39H44 NaO19: 839.2375; found: 839.2390. 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz, CD3OD): Table 1. Antimicrobial activity assay: A micro-dilution broth susceptibility assay was used, as previously described [14,15]. All microorganisms were stored at -85°C in 15% glycerol prior to the experiments. The bacteria were refreshed on Mueller Hinton agar (MHA, Mast Diagnostics, U.K.), whereas the Candida strains were refreshed on Potato Dextrose Agar (PDA, Merck) plates at 37°C. Thereafter, the bacterial suspensions were grown overnight in Mueller-Hinton broth (MHB, Merck, Germany) and were standardized to 1 x 108 CFU/mL versus McFarland No: 0.5 in Mueller-Hinton broth (MHB, Merck, Germany), turbidimetrically. Also, Candida strains were inoculated, and standardized in the same way, however in sterile saline (% 0.85) to 5 x 103 CFU/ per well in RPMI medium (Sigma-Aldrich). Stock solutions of the test samples were prepared in dimethylsulfoxide (DMSO). Dilution series were prepared from 0.6-100 µg/mL accordingly in Mueller Hinton Broth (MHB, Merck) for bacteria and RPMI medium for Candida strains in 96-well microtiter plates. Each bacterial (10 μL) and fungal suspension (100 μL) was then added to each well. The last row containing medium with microorganism was used as negative control and medium served as a positive growth control. After incubation at 37°C for 24 h, for staining of viable microorganisms, 0.01% resazurin (20 µL) was added to all of the plates. The first blue well was determined as the minimal inhibitory concentration (MIC, µg/mL). Amphotericin B and chloramphenicol (Sigma, Germany) were used as standard antimicrobial agents at a concentration range of 0.125-64 µg/mL. All experiments were repeated in triplicate and average MICs are given in Table 2.  Supplementary data: HR-MS, 1H and 13C NMR, COSY, HSQC, HMBC spectra of the new compound 1.  Acknowledgments - The authors thank Attila Csorba (Department of Pharmacognosy, University of Szeged) for the HRMS measurements. References [1]  Davis PH. (1978) Digitalis L. In Flora of Turkey and the East Aegean Islands, Vol. 6, Davis PH. (Ed.), University Press, Edinburgh, 680-697.  [2]  Çalış İ,  Taşdemir  D, Sticher O, Nishibe S. (1999) Phenylethanoid glycosides from Digitalis ferruginea subsp. ferruginea (= D. aurea). Chemical and Pharmaceutical Bulletin, 47, 1305-1307. [3]  Kırmızıbekmez H, Taşdemir D, Ersöz T, Ireland CM, Çalış İ. (2002) A new pregnane glycoside and a furostanol glycoside from Digitalis cariensis. Pharmazie, 57, 716–720.  [4]  Kırmızıbekmez H, Celep E, Masullo M, Bassarello C, Yeşilada E, Piacente S. (2009) Phenylethyl glycosides from Digitalis lanata. 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Secondary metabolites from the leaves of Digitalis viridiflora

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Secondary metabolites from the leaves of Digitalis viridiflora

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